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The tubby-like proteins kingdom in animals and plants
Accepted Manuscript The tubby-like proteins kingdom in animals and plants
Meng Wang, Zongchang Xu, Yingzhen Kong PII: DOI: Reference:
S0378-1119(17)30923-X doi:10.1016/j.gene.2017.10.077 GENE 42298
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ACCEPTED MANUSCRIPT The Tubby-like Proteins Kingdom in Animals and Plants
Meng Wanga,b,1, Zongchang Xua,b,1, Yingzhen Konga,*
Key Laboratory for Tobacco Gene Resources, Tobacco Research Institute,
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a
Graduate School of Chinese Academy of Agricultural Science, Beijing
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b
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Chinese Academy of Agricultural Sciences, Qingdao 266101, P.R. China
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100081, China
MW: [email protected]
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ZX: [email protected]
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Email addresses:
These authors contributed equally to this work
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1
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YK: [email protected]
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* Corresponding author; Yingzhen Kong Phone: +86-532-66715890 Fax: +86-532-66715890 Email: [email protected] Tobacco Research Institute, Chinese Academy of Agricultural Sciences, No. 11 Ke Yuan Jing 4th Road, Laoshan District, Qingdao 266101, Shandong, PR China. 1
ACCEPTED MANUSCRIPT Abstract Each gene of the tubby-like family is characterized by a signature of C-terminal tubby domain. The wide spread of this family in plants and animals implies they have an important function in various organisms.
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Even though the tubby-like genes are suggested to be putative
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transcription factors (TFs), how they execute the function as TFs is not
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yet clear. The biological functions of most animal tubby-like genes have been well studied, especially for vertebrate TUB, TULP1 and TULP3, but
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not with TULP2 and TULP4. Plants possess more tubby-like genes than
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animals, but their functions are still elusive except the idea that they are involved in stress responses with indistinct mechanisms. Here we
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reviewed the current knowledge of the versatile functions and roles of the
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tubby-like family members in plants and animals.
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function
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Keywords: tubby-like proteins; subcellular localization; biological
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ACCEPTED MANUSCRIPT 1. Introduction:
The Tubby-like gene family was initially identified in an obesity tubby mouse more than two decades ago (Coleman et al. 1990;
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Noben-Trauth et al. 1996; Kleyn et al. 1996). This tubby mouse in
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Jackson’s laboratory was a C57BL/6J (F125) male breeder, and its
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offspring had a remarkably and slowly weight increase during the age of 12 to 24 weeks. Meanwhile the autosomal recessive mutant “tubby”
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mouse was found to be infertile, insulin resistant, and had hearing loss
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and retinal degeneration (Kleyn et al. 1996). Subsequently, the tubby-like proteins (TULPs) have been identified in various species, including mice,
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human (North et al. 1997), Caenorthabditis elegans (Mukhopadhyay et al.
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2005), Drosophila (Ronshaugen et al. 2002), chicken (Heikenwalder et al. 2001), rat (Figlewicz et al. 2004), Arabidopsis (Lai et al. 2004), rice (Kou
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et al. 2009), poplar (Yang et al. 2008), maize, sorghum (Yulong et al.
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2015), apple tree (Xu et al. 2016), and wheat (Hong et al. 2015). The wide distribution of this protein family in different species suggests that these proteins must have some conserved functions in the organisms. The expansion of the tubby-like family is suggested to be the result of segmental duplication and random translocation and insertion (Yang et al. 2008). The Tubby-like family is characterized by the signature conserved 3
ACCEPTED MANUSCRIPT C-terminal tubby domain which is 55% to 95% identical across species (Ikeda et al. 2002a). This domain consists of a β barrel enclosing a central α helix and binds selectively to specific membrane phosphoinositides (PIPs) (Santagata et al. 2001). Most studies suggested that the C-terminal
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domain might play a major role in the function of tubby-like proteins.
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Recently, the C-terminal tubby domain was suggested to be essential for
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TUB’s proper folding, solubility and subcellular localization (Kim et al. 2017). Unlike the conserved tubby domain, the N-terminal motifs are
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varied in diverse tubby-like proteins of various species, leading to the
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tubby-like proteins dividing into 3 classes (Lai et al. 2012). Besides the unique and conserved tubby domain, class I TULPs are characterized by
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the N-terminal WD40 domain and/or a suppressor of cytokine signaling
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(SOCS) box; class II TULPs do not have any other domain or motif in the N-terminus; class III TULPs all harbor F-box domain. Intriguingly,
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almost all of the plant tubby-like proteins have F-box motif in their
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N-terminus regions. The F-box domain can interact with other domains for recruiting specific proteins and targeting them for ubiquitin mediated proteolysis (Patton et al. 1998; Xiao et al. 2000). We cataloged the various key functional roles of tubby-like proteins from animals and plants in this paper (Table 1).
2. The subcellular localization of tubby-like proteins 4
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Using the structural-based functional analysis, the tubby-like proteins were implicated as TFs (Boggon et al. 1999), and they also were regarded as TFs in Arabidopsis in a review paper (Mitsuda et al. 2009). However,
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the subcellular localization of tubby-like proteins is not limited in the
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nucleus, they have been reported to usually localize in PM/cytoplasmic
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(Mukhopadhyay et al. 2011). Besides PM/cytoplasmic and nucleus, some proteins have other unusual subcellular localizations. For instance,
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TULP3 was reported to be in the tips of the primary cilia (Norman et al.
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2009); all the four TaTULPs localized to the Golgi apparatus (Hong et al. 2015); CaTLP1-YFP fusion protein showed predominant fluorescence
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signals in extracellular matrix of cell walls (Wardhan et al. 2012).
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Additionally, the different domains of tubby-like proteins have different subcellular localization. The full length and C-terminal of most tubby-like
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proteins tend to localize to PM, whereas the N-terminal domain localizes
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to the nucleus (Santagata et al. 2001; He et al. 2000; Reitz et al. 2012; Reitz et al. 2013). Previous studies reported that the conserved C-terminal tubby domain could bind phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) in the PM to form a stable but reversible complex. Mutations of the conserved positively charged phosphate-coordinating residues, K330 (Lysine330) and R332 (Arginine332), could result in their abolishing localization in 5
ACCEPTED MANUSCRIPT the PM (Santagata et al. 2001), such as TUB and TULP1 (Mukhopadhyay et al. 2010). These charged phosphate-coordinating residues are not only conserved among the animal TULPs (Santagata et al. 2001), but also in plants. For example, AtTLP3 also has the conserved PtdIns(4,5)P2
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binding sites K187 and R189, while mutating these sites with Alanine (A)
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disrupted its PM localization (Reitz et al. 2012; Reitz et al. 2013; Bao et
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al. 2014). Amino acid alignment analysis revealed that almost all of the AtTLPs have the conserved PtdIns(4,5)P2 biding site K and R, except
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AtTLP5 with R192K mutation, AtTLP9 with K168R mutation and
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AtTLP8 without these binding sites (Bao et al. 2014). However, the tubby-PtdIns(4,5)P2 complex is reversible. In some
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specific conditions, the tubby-like proteins can translocate into nucleus
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from PM (Figure1). Upon the G-protein signaling pathway, TUB can translocate from PM to the nucleus. The model has been depicted as
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follow: At first, TUBBY resides on the membrane through PtdIns(4,5)P2
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binding to form tubby-PtdIns(4,5)P2 complex. TUBBY can also associate with Gαq (or Gα11), which is the Gαq subset of G-proteins, leading to form a tubby-Gαq/11 complex. Once Gαq/11 activated by G-protein-coupled receptor (GPCR), the activated Gαq/11* will release from the receptor and activate the phospholipase Cβ (PLCβ). However, PLCβ can hydrolyze PtdIns(4,5)P2 into inositol trisphosphate (InsP3) and disrupt the tubby-PtdIns(4,5)P2 complex. This finally results in the dissociation of 6
ACCEPTED MANUSCRIPT tubby from the PM enabling its translocation to the nucleus (Santagata et al. 2001; Carroll et al. 2004). Recently, reports showed that insulin- or leptin-induced
TUB
tyrosine phosphorylation
could
induce the
translocation of TUB from PM into the nucleus, and this process were
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also associated with the hydrolysis role of PtdIns(4,5)P2 upon PLCβ
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(Prada et al. 2013). Further studies reported that tyrosine residue 464
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(Y464) of TUB was an important phosphorylation site by insulin, and mutation Y464A could not be phosphorylated by insulin and failure in
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translocation to the nucleus (Kim et al. 2014b), suggesting that Y464 is
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vital for insulin-induced TUB nuclear translocation. Moreover, abiotic stresses such as mannitol, NaCl and H2O2 treatments can trigger AtTLP3
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detaching from PM to the cytosol (Bao et al. 2014). Similarly, the YFP
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signals of a fused protein CaTLP1-YFP dominantly accumulated in nucleus under dehydration condition, suggesting that CaTLP1 may be
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et al. 2012).
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recruited to the nucleus to transfer signals to respond to stresses (Wardhan
It has been suggested that TUB might also function in a signaling role based on its dual localization, which is reminiscent of TFs like Sterol regulatory element-binding protein (SREBP), Nuclear factor kappa B (NF-kB), Drosophila mothers against decapentaplegics (SMADS), Signal transducers and activators of transcription (STATs) and Nuclear factor-activated T cells (NF-AT). These TFs could transit into the nucleus 7
ACCEPTED MANUSCRIPT in response to particular cell signaling events (Santagata et al. 2001). But little is known about the TF-regulating function of tubby-like proteins. Though OsTLP2 has been shown to be able to bind to the promotor of OsWRKY13 in vitro by electrophoresis mobility shift assay (Cai et al.
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2008), no evidence has been reported that tubby-like proteins could
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regulate TFs in vivo directly. So further studies are needed to elucidate the
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mechanism of how tubby-like genes function as TFs.
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3.1 Trafficking proteins into cilium
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3. The functions of tubby-like proteins in animals
Both mice TUB and TULP3 are involved in trafficking GPCRs into
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cilium in brains neural tubes. The primary cilium is an antenna-like cellular protrusion mediating sensory and neuroendocrine signaling, and
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it is enveloped by a membrane contiguous with the PM. The ciliary membrane is enriched with multiple integral membrane proteins, and
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TULP3 is defined as a general adaptor for trafficking integral membrane proteins to cilia (Badgandi et al. 2017). TULP3 is required for trafficking of
at
least
16
class
A
cilia–targeted
GPCRs
including
Melanin-concentrating hormone receptor (MCHR1) and Somatostatin receptor subtype3 (SSTR3) (Mukhopadhyay et al. 2010), Neuropeptide Y (NPY) receptors (NPY2R) (Loktev et al. 2013), Dopamine receptors 2 (short isoform, D2R short), 1 (D1R) and 5 (D5R), Galanin receptors 2 8
ACCEPTED MANUSCRIPT and 3 (GAL2R and GAL3R), G protein–coupled receptor 161 (GPR161) (Mukhopadhyay et al. 2013) and Orphan receptors GPR19, GPR83 and GPR88, Kisspeptin receptor (KISS1R), Neuropeptide FF receptor 1 (NPFFR1), Purinergic receptor (P2RY1) and the prolactin-releasing
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hormone receptor (PRlHR) (Badgandi et al. 2017). Besides the 16 class A
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cilia–targeted GPCRs, trafficking of fibrocystic ciliary localization
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sequence (CLS) fusions and transient receptor potential (TRP) channel family proteins polycystins PC1/2 (which form PC1/2 complex in the
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endoplasmic reticulum (ER), and are trafficked to cilia in an
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interdependent manner) (Kim et al. 2014a; Gainullin et al. 2015; Cai et al. 2017), are also adapted by TULP3. Specifically, TULP3 determines the
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entry of GPR161 into cilia directly. However, there are also exceptions of
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integral membrane proteins, such as
Smoothened
(SMO) and
cilia-targeted multispan adenylyl cyclases, which are not regulated by
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TULP3 (Mukhopadhyay et al. 2010; Bishop et al. 2007; Choi et al. 2011).
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Badgandi et al proposed a tripartite steps model for TULP3-mediated trafficking integral proteins to cilia which described as follow: firstly TULP3 captured membrane cargo in the PM dependent on PtdIns(4,5)P2, then TULP3-CLS complex (with the cargo) were delivered into the ciliary compartment by TULP3-IFT-A (Intraflagellar transport A) complex, lastly the cargo was released in the PI(4,5)P2-deficient ciliary membrane (Badgandi et al. 2017). This study also showed that the tubby 9
ACCEPTED MANUSCRIPT domain was responsible for proximity between the CLSs and TULP3. Similarly, TUB also can traffic certain rhodopsin family GPCRs to neuronal cilia, including SSTR3, MCHR1, and NPY2R (Sun et al. 2012; Loktev et al. 2013), but not GPR161 or GPR19. However, TULP3 is
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more effective than TUB in binding to the IFT-A core, suggesting that
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TULP3 might be more effective in trafficking GPCRs. However,
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Badgandi et al proposed that low-expressed GPCRs were preferentially trafficked by the high-expressed but poor IFT-A–binding TUB, and the
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highly expressed GPCRs required TULP3 and TUB redundantly for
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trafficking to the compartment (Badgandi et al. 2017). Mice TUB is not only essential for ciliary trafficking GPCRs in brain
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neural tubes, but also in photoreceptors. The photoreceptor outer segment (OS) is a modified cilium (Sun et al. 2012). The proximal end of the OS
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is linked to the cell body (inner segment, IS) via a connecting cilium (CC) which is structurally homologous to the transition zone of primary cilia.
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Sun et al reported that the 7-pass transmembrane GPCRs–rhodopsin and cone opsins, which should normally distribute in the OSs, were ectopically localized in the ISs and cell bodies or the ISs, the perinuclear region and the synaptic terminals in tubby mutant mice (Sun et al. 2012). This phenotype was also observed in the tulp1-/- mice (Hagstrom et al. 1999). The retinal defects of mice tubby mutant were milder than tulp1 mutant although mostly similar (Sun et al. 2012). Another marked feature 10
ACCEPTED MANUSCRIPT was the transient massive extracellular accumulation of rhodopsin-laden vesicles in the inter photoreceptor matrix between the adjacent ISs of both tubby and tulp1 mutant mouse, and the vesicles peaked at 3 weeks of age when rhodopsin should be rapidly synthesized to build up the OSs
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and quickly diminished (Heckenlively et al. 1995; Hagstrom et al. 1999).
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The vesicles accumulation appears to be a distinct ultrastructural feature
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shared by a small group of retinal disease models (Hagstrom et al. 1999). Thus, we can hypothesize that both TUB and TULP1 have an important
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role in rhodopsin trafficking in photoreceptors. Additionally, tubby tulp1
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double mutants have a much more severe retinal phenotype than either mutant alone, and this would appear to suggest that TUB and TULP1 may
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(Hagstrom et al. 2001).
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function synergistically to facilitate rhodopsin trafficking to the OSs
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Because the main defects of mice tulp1-/- mutant are retinal degeneration, the function of TULP1 in polarized vesicular translocation
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of rhodopsin from its site of synthesis in the IS through the CC to its final destination in the OS of the photoreceptor cell has been well studied. Moreover, studies have shown that TULP1 could associate with cellular membranes PIPs and interact with cytoskeleton proteins F-actin, Microtubule-associated protein 1A (MAP1A) and MAP1B, and neuronal-specific GTPase Dynamin-1 (Xi et al. 2005; Xi et al. 2007; Grossman et al. 2014), all of which involving in vesicular transport. PIPs 11
ACCEPTED MANUSCRIPT play a critical role in vesicular protein transport and organization of the cytoskeleton (Roth 2004). Numerous studies implicated that actin could associate with protein movement in vesicle assembly and polarized transport (Xi et al. 2005). Besides stabilizing microtubules, MAP proteins
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also have other functions for trafficking vesicles and organelles (Brenman
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et al. 1998; Thomas et al. 2000). Dynamin-1 is an essential component of
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vesicle formation in receptor-mediated endocytosis, synaptic vesicle recycling, and vesicle trafficking in and out of the trans-Golgi network
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(TGN) (Bliek 1999; Mcniven et al. 2000). Interestingly, the interaction
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between Dynamin-1 and the actin cytoskeleton has also been reported. Dynamin-1 was thought to act as a polymeric contractile scaffold at the
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interface between membranes and actin cytoskeleton to generate vesicles
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at different cellular sites (Orth et al. 2003). These data imply that F-actin, MAP1A, MAP1B and Dynamin-1 may aid help for TULP1-mediated
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rhodopsin vesicular transport. Besides rhodopsin, there were several other
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OS proteins mislocalized in tulp1-/- mice retinas including GC-activating protein 1 (GCAP1), GCAP2, Guanylate cyclase 1 (GC1) and blue cone opsin, which should be restricted to OS whereas had a more extensively localization (Grossman et al. 2011). Based on the analysis of several mouse mutant phenotypes, it has been proposed that GCAP1, GCAP2, GC1 and GC2 are co-transported, and their transport is independent of the rhodopsin trafficking system (Baehr et al. 2007; Karan et al. 2008). So 12
ACCEPTED MANUSCRIPT possibly GC2 is mislocalized in tulp1-/- retinas as well. In addition, in the tulp1-/- retina, the light-dependent translocation of signaling protein arrestin which binds to phosphorylated rhodopsin and plays an important role in the recovery phase of phototransduction was severely impaired
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(Grossman et al. 2011). In the WT (wild type) retina, arrestin was
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localized to the IS, ONL (outer nuclear layer) and OPL (outer plexiform
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layer) in darkness and redistributed to the OS in the light. However, in the tulp1-/- retina, the distribution of arrestin remained confined to the IS,
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ONL and OPL in both the dark and light. Significantly, protein complexes
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that regulate the trafficking of rhodopsin, RAB6, RAB8 and RAB11 were mislocalized in tulp1-/- retinas as well (Grossman et al. 2011). RAB6
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can function in the generation and budding of rhodopsin-laden vesicles
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from the TGN. RAB8 is important for the docking and fusion of rhodopsin-bearing vesicles to the IS PM, in close proximity to the CC.
with
RAB6
(Deretic
2006).
According
to
the
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conjunction
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RAB11 functions in the sorting and budding of vesicles from the TGN in
above-mentioned data, we can propose a simplified and possible pathway for rhodopsin vesicular transport. Considering TULP1 can associate with PIPs, actin and microtubules, the TULP1-Dymanin-1 complex could form a polymeric contractile scaffold at the interface between membranes and cytoskeleton, which can lead the vesicular transport. In the WT IS, rhodopsin molecules are posttranslationally modified in the Golgi 13
ACCEPTED MANUSCRIPT apparatus, and inserted into carrier vesicles generated from the TGN surface. Then TULP1-Dynamin-1 complex leads to the budding of rhodopsin-laden vesicles with the help of RAB6, then RAB11 attends to transport the vesicles through the membranes and cytoskeleton. RAB8 is
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responsible for docking and fusion of vesicles with the IS PM, which is
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necessary for the subsequent transport of rhodopsin through CC to OS
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discs. However, if TULP1 is absent, the cargo vesicles will exocytose through the basal IS PMs (occasional rhodopsin labeling vesicles were
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observed to be connected with the IS PMs (Hagstrom et al. 2001)) and
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then accumulate in the extracellular inter photoreceptor matrix. Besides mice TULPs, Drosophila TULP (dTULP) which is a TUBBY
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homolog in Drosophila genome (Ronshaugen et al. 2002), can also
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mediate trafficking proteins into cilia. The expression of dTULP was confirmed in cilia and cell body of chordotonal neurons of Johnston’s
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organs which is essential for flies to hear. Additionally, several TRP
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channels such as Inactive (IAV), NOMPC and Spacemaker (SPAM) were ectopically localized in cilia of dtulp mutants. TRP channels are essential factors for Drosophila hearing transduction and amplification (Lehnert et al. 2013). The ectopically localization of these TRP channels suggest the extraordinary role of dTULP in mediating hearing in Drosophila. The domains which bind IFT-A and PIP are conserved in dTULP and mice TULP3. The putative IFT- and PIP-binding domains of dTULP have been 14
ACCEPTED MANUSCRIPT suggested to be required for the proper localization of TRPs in cilia. However, the two putative domains perhaps have different roles in the proper IAV distribution in cilia, as the IFT-binding domain is required for the ciliary entry for dTULP, and the PIP-binding domain affects
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recruitment of IAV-containing preciliary vesicles to dTULP. Furthermore,
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unlike the mice TULP3, dTULP ciliary access is not dependent on IFT-A
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as dTULP ciliary trafficking is not affected by the mutation of IFT-A proteins. It has been explained that dTULP facilitated the relay of
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preciliary vesicles to the IFT complex at the base of cilia rather than
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moving together with ciliary membrane proteins into the cilia (Park et al. 2013).
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As known from literature, primary cilia are sensory organelles
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wherein components of numerous signaling pathways concentrate. Their functions include involvement in many aspects such as olfaction,
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photoreception and development, which are mediated by targeting of
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receptors, channels, and their downstream effector proteins into cilia correctly (Berbari et al. 2009). Therefore, the factor-mediated ciliary trafficking has a critical role in ciliary function. TULP3 is defined as a general adaptor for trafficking at least 16 GPCRs, and TUB can also traffic at least 3 GPCRs into cilia. Among them, MCHR1 is involved in the regulation of feeding and energy balance; SSTR3 is important in synaptic plasticity and novelty detection (Mukhopadhyay et al. 2010); 15
ACCEPTED MANUSCRIPT NPY2R is involved in energy homeostasis (Loktev et al. 2013); GPR161 is a negative regulator of Shh signaling. Also, the photoreception-related critical factors rhodopsin, blue cone opsin, arrestin, RAB6, RAB8 and RAB11 are mislocalized in tulp1-/- and/or tub-/- mutants. Furthermore, the
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hearing-related TRP channels IAV, NOMPC and SPAM are mislocalized
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in dtulp mutants. Taken together, we can conclude that trafficking
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proteins into cilia mediated by tubby-like genes play an important role in ciliary function, such as energy balance, development, photoreception
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and hearing.
3.2 TUB has a key role in energy balance
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According to the previous studies, the mutation of TUB gene in mice
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and humans mainly resulted in obesity phenotype (Coleman et al. 1990; Kleyn et al. 1996; Noben-Trauth et al. 1996; Kapeller et al. 1999; Borman
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et al. 2014). Especially for humans, the body composition and
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macronutrient intake of middle-aged women are associated with genetic variation of TUB gene, suggesting that TUB may influence eating behavior (van Vliet-Ostaptchouk et al. 2008). TUB has been shown to have expression in adipocyte (Noben-Trauth et al. 1996), and its expression is regulated developmentally during adipogenic differentiation (Stretton et al. 2009). Furthermore, mutation of TUB-1 which is a TUBBY homolog in C. elegans, resulted in an extended life span and increased fat 16
ACCEPTED MANUSCRIPT deposition (Mukhopadhyay et al. 2005), further supporting the view that tubby-like proteins are involved in energy balance. Additionally, TUB was reported to be regulated by thyroid hormone or insulin (Stretton et al. 2009), another study revealed that TUB involved in insulin’s and leptin’s
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effects on energy balance and glucose metabolism (Prada et al. 2013). As
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described in the literature, leptin and insulin signals controlled energy
anorexigenic
neuropeptides.
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balance in part by regulating the expression of orexigenic and However,
the
leptin/insulin-induced
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increases of proopiomelanocortin (POMC) and thyroid releasing hormone
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(TRH), and the leptin/insulin-induced reduction of melanin-concentrating hormone (MCH) and orexin neuropeptides were blunted when TUB
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antisense oligonucleotide (ASO) was used in the hypothalamus to reduce
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TUB protein expression. Put together, these data suggest that TUB is perhaps essential for regulating the expression of orexigenic and
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anorexigenic neuropeptides in the leptin/insulin signaling pathway.
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Furthermore, the tyrosine residue of TUB could be phosphorylated in response to insulin or insulin-like growth factor (Kapeller et al. 1999). Further studies suggested that both insulin and leptin could take TUB as substrate, and insulin receptor (IR) could utilize its endogenous tyrosine kinase (IRK) activity to induce TUB tyrosine phosphorylation directly; however, leptin receptor (LEPR) could use Janus kinase 2 (JAK2) to induce TUB phosphorylation (Prada et al. 2013). The phosphorylation of 17
ACCEPTED MANUSCRIPT insulin- or leptin-induced TUB tyrosine residue is in a dose-dependent and time-dependent manner, and this phosphorylation could result in nuclear translocation of TUB from the PM. As described in the section of subcellular
localization,
Y464
site
of
TUB
is
important
for
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insulin-induced nuclear translocation. Moreover, TUB has been shown to
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be able to associate with Protein tyrosine phosphatase 1B (PTP1B) which
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may have a role in the dephosphorylation of TUB. These data imply that maybe insulin/leptin and PTP1B function in antagonism to control the
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3.3 Functions in endocytosis
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phosphorylation level of TUB, then to regulate the energy balance.
Recently, a study found that TULP1 colocalized with endocytic
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proteins (dynamin and clathrin heavy chain) and an important signaling phospholipid for initiating endocytosis–PIP2 in the periactive zone where
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has been shown to be a hotpot of endocytic activity of the bovine retina (Wahl et al. 2016). The endocytic proteins (dynamin and clathrin heavy
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chain) were highly enriched in the photoreceptor synapses periactive zone of WT mice at postnatal day (P) 16, whereas these proteins were almost disappeared concomitant with severely defect in periactive zone endocytosis of tulp1-/- mice. Moreover, the TULP1 can directly interact with the synaptic ribbon protein RIBEYE. These novel findings may hint us a new model for TULP1-mediated localization of the endocytic machinery at the periactive zone of ribbon synapses and offer a new 18
ACCEPTED MANUSCRIPT rationale mechanism for vision loss associated with genetic defects in TULP1 (Wahl et al. 2016). Other evidence further verified that the Tubby-like family could mediate the endocytosis. The Drosophila king-tubby (KTUB) gene
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specially localized in the rhabdomere domain of photoreceptors R1 to R6
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of the WT flies reared in dark. When the dark-reared WT flies exposed to
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light for 3 hours, the KTUB protein would move from rhabdomere to cytoplasm concomitant with a significant amount of rhodopsin 1
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(Rh1)-immunopositive large vesicles (RLVs) in the cytoplasm. However
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in the ktub mutant flies, besides the defect of rhabdomere in photoreceptor R1 to R6, the Rh1 also was mislocalized in the
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rhabdomeric domain with only a few RLVs found in the cytoplasm.
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Furthermore, the ktub norpA double mutant could rescue the phenotype of massive internalization of rhodopsin from rhabdomere to cell body in
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norpA mutant for a 12 hours’ light stimulation (Orem et al. 2002). This
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suggests us the KTUB gene may play an important role in mediating rhodopsin endocytosis. And the C-terminus tubby domain is critical for rhodopsin endocytosis (Chen et al. 2012). C.
elegans
TUB-1
gene
can
negatively
regulate
RBG-3
(Mukhopadhyay et al. 2005), and RBG-3 can promote the GTPase hydrolysis of RAB-7 which could control endocytic sorting specifically in the neurons (Deinhardt et al. 2006) to regulate fat storage. From this 19
ACCEPTED MANUSCRIPT data we can propose that TUB-1 gene regulate fat storage through a RAB-7-mediated endocytic mechanism, supporting a more general role of tubby-like family proteins in endocytosis.
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3.4 Keep synapse maintenance or architecture
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Even though TULP1 does not predominantly localize in synaptic
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terminals, it has a role in synapse maintenance or architecture. In mice WT retinas, TULP1 had a diffusely staining in the OPL and colocalized
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with the presynaptic ribbon-associated protein Bassoon (Grossman et al.
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2009). Piccolo and Bassoon proteins were reported to have roles in organization of the photoreceptor synapse, the functioning of the ribbon
D
and the trafficking of vesicles at the synapse (Dick et al. 2003; Dick et al.
PT E
2001). But Piccolo and Bassoon proteins were unable to coordinate into the normal horseshoe-shaped ribbon architecture in mice tulp1-/- mutant,
CE
though they were able to arrive at their correct destinations and often in immediate proximity with another (Grossman et al. 2009). In addition,
AC
fewer intact ribbons were present in the P13 tulp1-/- retina (Grossman et al. 2009). Recently, a study reported that the number of synaptic ribbons significantly decreased in tulp1-/- mice at P30 whereas they should increase in the course of postnatal maturation (Wahl et al. 2016). In WT P16 mice retinas, the electroretinogram (ERG) had distinct a-waves and b-waves which indicated the functional invaginating synapses (Keeler et 20
ACCEPTED MANUSCRIPT al. 1928). However, the ERG a-waves generated by depolarizing bipolar cells (DBCs) were reduced in amplitude but retained normal kinetics with a normal leading edge, and the b-waves had a reduced amplitude and a desensitized intensity-response function at P16 and P15 in tulp1-/- mice
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(Grossman et al. 2009; Xi et al. 2007). All of the above data indicate the
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malformation of tulp1-/- photoreceptor synapses. Furthermore, there was a
SC
profound shortening of individual dendrites and a severe attenuation of dendrite branching which indicated the underdeveloped or malformed
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structural platform for relaying signaling from photoreceptors to the inner
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retina in tulp1-/- mice (Grossman et al. 2009). The reduction of the DBC dendritic field in the tulp1-/- retina was suggested to be a developmental
D
consequence of an attenuated trophic and transmitter release that would
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be expected from a presynaptic malformation (Grossman et al. 2009). MAP1A is a genetic modifier of both TULP1 and TUB, it can reduce
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hearing loss in tub-/- mice, retinal degeneration in tub-/- and tulp1-/- mice
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(Ikeda et al. 2002b; Maddox et al. 2012). TULP1 can interact with MAP1A (Grossman et al. 2014). MAP1A is predominantly expressed in the synapses of adult neurons, and it can physically interact with the membrane-associated guanylate kinase family member DLG4 which is crucial in establishing the post synaptic (Brenman et al. 1998; Thomas et al. 2000). TULP1 could interact with neuronal-specific GTPase Dynamin-1 which has a role in PM and vesicle recycling at neuronal 21
ACCEPTED MANUSCRIPT synapses (Xi et al. 2007). Moreover, TUB might also have functions in synaptic architecture, because the TUB’s G-protein receptor signaling function occurs at synaptic junctions and TUB has a high expression in the synaptic junctions formed region (Ikeda et al. 1999; Santagata et al.
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2001; Ikeda et al. 2002b). Given all the findings mentioned above, both
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TULP1 and TUB possibly have functions in synapse maintenance or
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architecture.
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3.5 Stimulate phagocytosis
Phagocytosis for the removal of apoptotic cells or cellular debris, has
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an important role in many biological processes such as development, tissue homeostasis and disease resistance (Erwig et al. 2007). Besides
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D
initiating phagocytosis, phagocytosis ligands are also the key to selecting extracellular cargos for clearance, understanding phagocyte beneficial
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and detrimental functional roles and regulating phagocyte activities with therapeutic potentials (Li 2012). TULP1 was identified as a putative
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phagocytosis ligand, and further experiments proved that TUB and TULP1 could stimulate phagocytosis of retinal pigment epithelium (RPE) cells and apoptotic cells (Caberoy et al. 2010a). Moreover, TUB and TULP1 might function in synergy to stimulate phagocytosis of RPE cells, because the association of TULP1 and TUB has been verified (Caberoy et al. 2010b; Caberoy et al. 2010a) . But how TUB and TULP1 stimulate phagocytosis is needed to be 22
ACCEPTED MANUSCRIPT elucidated. Further studies have proved that TUB and TULP1 are Mer tyrosine kinase (MerTK) ligands and stimulate RPE phagocytosis in a MerTK-dependent manner. MerTK is a well-characterized phagocytic receptor and essential for maintaining retinal homeostasis, and the retinal
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degeneration of MerTK deficient mutant mice is a result of the
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accumulation of unphagocytosed debris and defective RPE phagocytosis
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(Seitz et al. 2007). According to the literature, phagocytosis is initiated by ligands with receptor activation and signaling cascades, followed by
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cytoskeletal rearrangement and engulfment. MerTK activation can lead to
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MerTK autophosphorylation. TUB and TULP1 can not only promote the autophosphorylation of MerTK, they also induce the redistribution of
TUB/TULP1-MerTK
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the
D
non-muscle myosin II-A (NMMII-A) redistribution, which suggests that RPE
phagocytosis
pathway
facilitate
MerTK-dependent cytoskeletal reorganization (Caberoy et al. 2010c).
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Similar to the two well-characterized MerTK ligands, GAS6 and protein
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S, facilitate MerTK-dependent phagocytosis with their N-terminus Gla domain interacting with phosphatidylserine on apoptotic cells and the C-terminal two globular laminin G-like domains (2×LG) binding to MerTK on phagocytes (Lemke et al. 2008; Hafizi S et al. 2006). TUB and TULP1 are also bridging molecules with their N-terminal minimal phagocytosis determinants (MPDs) as MerTK-binding domain and the C-terminal region as phagocytosis prey-binding domain (PPBD) which 23
ACCEPTED MANUSCRIPT through a phosphatidylserine and PtdIns(4,5)P2-independent mechanism (Caberoy et al. 2010c). MPDs are critical for MerTK binding and autophosphorylation,
five
MPDs
of
K/R(X)1–2KKK
in
TULP1
N-terminus have been predicted. The PPBD has been mapped to the
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C-terminal 54 amino acids of TULP1 (Caberoy et al. 2010c). Besides
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RPE and macrophage phagocytosis, TUB also can stimulate microglial
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phagocytosis in BV-2 microglial cell line and primary microglia. Similarly, TUB can activate MerTK and then lead to MerTK
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autophosphorylation, NMMII rearrangement in BV-2 microglial cell line
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(Caberoy et al. 2012). Interestingly, TUB can induce both microglial phagocytosis and MerTK’s phosphorylation in a concentration-dependent
D
manner with minimal and maximal induction concentrations at 0.1 nM
PT E
and 10 nM, respectively. However, the activity of TUB to stimulate microglial phagocytosis will be reduced at a high concentration (200 nM)
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but the phosphorylation of MerTK is slightly affected (Caberoy et al.
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2012). From these data, we can conclude that TUB and TULP1 can stimulate RPE and macrophage phagocytosis, and TUB also can induce microglial phagocytosis (Caberoy et al. 2012; Caberoy et al. 2010c). Even so, TUB and TULP1 are intracellular proteins which should perform intracellular functions. But how intracellular proteins have extracellular functions?
TUB and TULP1 were reported to be secreted
through unconventional pathways, not the classical ER–Golgi pathway, 24
ACCEPTED MANUSCRIPT and this unconventional pathway partially depends on an essential secretory signal in the N-terminus between Asn51 and Arg100 with the PI(4,5)P2-binding activity (Caberoy et al. 2009). TUB and TULP1 may have multiple intracellular and extracellular functions similar to
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GALECTIN-3 (Dumic et al. 2006).
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3.6 The eye diseases of mutation in TULP1 of human
The eyes diseases associated with the mutant TULP1 can be classified
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into five types: autosomal recessive Retinitis Pigmentosa (arRP), retinal
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degeneration (RD), Leber congenital amaurosis (LCA), juvenile onset retinitis pigmentosa (JRP) and rod-cone dystrophy (RCD) (Ullah et al.
D
2016). Until now, a total of 54 different casual mutations harboring amino
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acids substitution, duplication and deletion, aberrant splicing and nonsense mutation in TULP1 have been reported, and each of the
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majority mutations only causes one type of TULP1-caused eye diseases
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(Ullah et al. 2016). However, there are also exceptions such as p(patient).Q301* null mutation in TULP1 can cause LCA and RCD (Li et al. 2009; Khan et al. 2015), p.L461V mutation leads to JRP and LCA diseases (den Hollander et al. 2007). More surprisingly, the substitution of the same amino acid in the fixed position usually cause one disease (p.I459K and p.I459T both caused the same disease arRP (Hagstrom et al. 1998; Wang et al. 2014)), but p.R420S in TULP1 cause RCD (Roosing et 25
ACCEPTED MANUSCRIPT al. 2013), p.R420P cause arRP (Hagstrom et al. 1998). All of the casual mutations in TULP1 patients provide us vital information for therapies to the TULP1 mutations resultant vision impairment. Recently, the mechanism of photoreceptor cell death in four
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TULP1 missense mutations (p.D94Y, p.R420P, p.I459K and p.F491L)
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were implicated (Lobo et al. 2016). Bioinformatics and protein structural
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analysis of the four TULP1 mutations predicted that they all would result in misfolded and/or unstable with altered protein structures and therefore
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likely be pathogenic when expressed. No matter in-vitro or in-vivo,
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mutant TULP1 proteins localized to ER and caused induction of the ER-UPR (unfolded protein response) stress complex. And those mutant
D
TULP1-expressing cells had significantly elevated levels of BiP which
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are ER chaperones. This suggests that the mutant TULP1 proteins can cause ER cellular stress and activation of the UPR complex. The two
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major apoptotic arms of the UPR pathways, PKR-like ER kinase (PERK)
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and Inositol-requiring enzyme 1 (IRE1), were strongly induced in the four mutant TULP1-expressing cells. In addition, C/EBP homologous protein (CHOP), which is downstream pro-apoptotic target of pPERK (phosphorylated PERK) and IRE1, had high levels expression in mutant cell lines. All of these results indicate that long term retention of mutant TULP1 within cells can activate the ER-UPR stress complex, leading to the initiation of apoptosis via the CHOP signaling pathways. 26
ACCEPTED MANUSCRIPT
3.7 A critical repressor of mouse Sonic hedgehog signaling Sonic hedgehog (Shh) acts as signaling molecule critically involved in multiple aspects of embryonic development and developmental
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processes in vertebrates (Martí et al. 2002; Palma et al. 2005; Zhang et al.
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2001). Especially, Shh signaling pathway plays a significant role in
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normal growth and patterning of many vertebrate tissues such as limb and neural tube (McGlinn et al. 2006; Ulloa et al. 2007; Varjosalo et al. 2008).
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But how the Shh signaling works in the growth and patterning of limb
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and neural tube? Previous studies reported that Shh provided positional information for the patterning of neural cell fates in the developing
D
central nervous system and controlled the digit pattern in the developing
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limbs. Almost at the same time, 3 studies identified TULP3 as a critical repressor of Shh signaling pathway (Patterson et al. 2009; Norman et al.
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2009; Cameron et al. 2009). Mutants tulp3 and hitchhiker (a strong
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hypomorph of TULP3) showed defects in spinal neural tube and the developing limbs such as exencephaly, spina bifida, enlarged branchial arches and preaxial polydactyly on both fore- and hind-limbs, which resemble the phenotypes of inappropriate activation of the Shh signaling pathway mutations. PTCH1 and GLI1, which are target genes of Shh signaling pathway, were up-regulated and ectopically expressed in dorsal regions of spinal neural tube in both tulp3-/- and hitchhiker mutant. And 27
ACCEPTED MANUSCRIPT the dorsal-to-ventral patterning defects were evident at stage of Embryo 9.5 in mutants. These data verify that the mutants’ neural tube experience elevated levels of Shh pathway activity. Similarly, the limb buds of tulp3-/- and hitchhiker mutant also exhibited ectopically expression of
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GLI1 and PTCH1 in the anterior regions with anterior–posterior polarity
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perturbed which demonstrated inappropriate activation of the Shh
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pathway and resulted in polydactyly. Furthermore, TULP3 was regarded as genetically downstream gene of SHH and SMO, and genetically
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interacted with GLI3.
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Now, we have to consider the question that how does TULP3 repress the Shh signaling pathway? The primary cilia localization of TULP3 may
D
implicate that TULP3 possibly regulates the hedgehog pathway within
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this structure (Norman et al. 2009). As described in the “trafficking proteins into cilium” section, TULP3 and IFT-A are both negative
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regulators of Shh signaling and are able to traffic GPCRs into cilia
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(Patterson et al. 2009; Norman et al. 2009; Cameron et al. 2009; Qin et al. 2011; Tran et al. 2008; Liem et al. 2012). Maybe one of the GPCRs could have roles in the TULP3/IFT-A-regulated antagonism of Shh signaling. Recently, a report suggested that TULP3 might determine the entry of GPR161 into cilia (Badgandi et al. 2017). Furthermore, GPR161 can increase cAMP, then activate Protein kinase A (PKA), leading to the formation of GLI3R and subsequent repression of Shh target transcription 28
ACCEPTED MANUSCRIPT (Mukhopadhyay et al. 2013). Thus we can hypothesize that the negative Shh regulator GPR161 plays an important role in TULP3-mediated repression of Shh-signaling. However, Shh-dependent loss of GPR161 from cilia not only relies on decreasing entry into cilia, but also on
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increasing removal. The removal of GPR161 out of cilia depends on
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another distinct mechanism. The accumulation and activation of the
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effector SMO within cilia which results from stimulation of Shh signaling, can increase GPR161–β-arrestin binding and lead to the removal of
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GPR161 by clathrin-mediated endocytosis (Pal et al. 2016). Recently,
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reports suggested that the ciliary membrane PIP composition which regulated by INPP5E, could be read out by TULP3 to control ciliary
D
protein localization and enable Shh singaling (Garcia-Gonzalo et al. 2015;
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Chavez et al. 2015). With the existence of INPP5E, PI(4,5)P2 can be restricted at the proximal end of cilia whereas PI(4)P distributed along the
CE
length of cilia. As TULP3 binds to PI(4,5)P2 instead of PI(4)P
AC
(Mukhopadhyay et al. 2010), TULP3 can only be restricted at the tip of the cilia (Badgandi et al. 2017). Once INPP5E inactivated, PI(4,5)P2 accumulate in the cilia with the depletion of PI(4)P, leading to more TULP3 and GPR161 recruited to the cilia and increases of Shh signaling. These data imply that the ciliary membrane PIP composition have a great role for the location of TULP3 and GPR161 in cilia to regulate Shh signaling. 29
ACCEPTED MANUSCRIPT
3.8 TUSP regulates the formation of the 7S SNARE complex Drosophila TUSP (tubby domain superfamily protein) is the fly homologue of mammalian TULP4 (Mukhopadhyay et al. 2011).
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Mutations in vertebrate TULP4 are associated with short stature, cleft lip,
role
for
TUSP
in
the
assembly
of
the
7S
SC
unexpected
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and cleft palate (Allen et al. 2010; Conte et al. 2016). A study revealed an
synaptobrevin-syntaxin-SNAP-25 (SNARE) ternary complex (Yoon et al.
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2017). Mutant tusp flies exhibited a phenotype of temperature-sensitive
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paralysis which could be rescued by ectopic expression of TUSP in the giant fibers (GFs) and peripherally synapsing interneurons of the GF
D
system. Reduction in the assembly of the ternary 7S SNARE complex,
PT E
which is essential for neurotransmitter release, was observed in tusp mutant flies, though the transcription of each gene of the individual
CE
SNARE complex component was not changed. Thus, these data suggest
AC
that TUSP is a novel regulator of neurotransmitter release. Although the regulatory mechanisms such as ubiquitination or SNARE chaperone were suggested, further studies are needed to lift the veil.
4. The functions of tubby-like proteins in plants
Compared with the wide array of cellular functions identified in 30
ACCEPTED MANUSCRIPT animal TULPs, the functions of plant TLPs are relatively elusive. The plant genome harbors more tubby-like genes than that of animals. In wheat, apple tree, Arabidopsis, poplar, sorghum, rice and maize, there are 4, 9, 11, 11, 13, 14 and 15 TLP members, respectively. But limited
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functions of the plant TLPs have been reported so far.
SC
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4.1 TLPs perhaps involved in male gametophyte development To search the male gametophyte development related genes, 74
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Arabidopsis T-DNA insert lines of 49 genes in 21 TF families have been
MA
collected (Renak et al. 2012). The fully open flowers grains in developmental stage 14 of these mutants were analyzed by microscopy.
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Among these mutants, attlp6 mutant plants produced 5% two-celled
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pollen, 15% misarranged male germ unit (MGU) pollen and 5–10% aborted grains. The attlp7 mutant plants showed 5–10% two-celled
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phenotype and 20–25% pollen with misarranged MGU (Renak et al. 2012). Based on the Arabidopsis eFP Browser 2.0 database
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(http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi), AtTLP6, AtTLP7 and AtTLP2 are predominantly expressed in pollen grains. Previous studies reported that AtTLP6 can interact with ASK11 and ASK13; AtTLP7 also can interact with ASK1 in yeast two hybrid analysis (Bao et al. 2014). ASK1 gene is essential for male meiosis (Yang et al. 1999). qRT-PCR results showed that ASK11 and ASK13 had a higher expression in 31
ACCEPTED MANUSCRIPT inflorescence than other tissues, and the in situ-hybridization results also indicated that the expression of ASK1, ASK2 and ASK11 could be detected in pollen grains (Zhao et al. 2003). All of this data imply that the
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4.2 TLPs involving in multifarious stress response
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tubby-like proteins might have functions in pollen grains.
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4.2.1. Biotic stress response
The OsTLPs can respond to biotic stress such as pathogen. The
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transcription of all of the 14 OsTLPs could be induced by pathogen
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inoculation, suggesting that they all may be involved in the interaction of rice-Xoo pathogen (Kou et al. 2009). It has been reported that
D
OsWRKY13 can regulate genes which act both upstream and
PT E
downstream of salicylic acid (SA) to function as regulators for rice resistance to bacterial blight and fungal blast diseases. Over-expression of
CE
OsWRKY13 could enhance the resistance to bacterial blight and fungal
AC
blast, on the contrary, the low-expression of OsWRKY13 enabled rice to be more susceptible to pathogen infection (Qiu et al. 2009). Furthermore, OsTLP2 could bind to the PRE4 elements in the promotor of OsWRKY13, suggesting that OsTLP2 can regulate the expression of OsWRKY13 (Cai et al. 2008). Even though this binding hasn’t been verified in vivo, the expression level of OsTLP2 has been shown to be induced approximately 11-fold after pathogen inoculation, further supporting that OsTLP2 may 32
ACCEPTED MANUSCRIPT be involved in defense responses against pathogen invasion (Cai et al. 2008). 4.2.2. Abiotic stresses responses
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AtTLP3 and AtTLP9 can function redundantly in the ABA signaling
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pathway during seed germination and early seedling development (Lai et
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al. 2004; Bao et al. 2014). Single mutants attlp3 or attlp9, and double mutants attlp3attlp9 were all insensitive to ABA during seed germination.
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When these mutants were cultured on MS media containing ABA, the
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germination frequency of mutants were higher than WT. The germination frequency of double mutants was higher than either the single mutant,
D
suggesting that attlp3atltp9 double mutant plants are more tolerant to
PT E
ABA. Furthermore, attlp3atltp9 double mutant plants are more insensitive to mannitol than the attlp9 mutants. Taken together, the above
CE
findings imply the redundant function of AtTLPs in abiotic stress signaling. However, the AtTLPs have no redundant function in
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contributing to Arabidopsis colonization by Piriformospora indica, as individual AtTLP protein contributes to the colonization success (Reitz et al. 2012; Reitz et al. 2013). P. indica is able to colonize the roots of multiple plant species and confer its hosts various beneficial traits such as increased growth, higher seed yield and enhanced tolerance to biotic and abiotic stresses (Qiang et al. 2012). All attlps mutants except attlp4 33
ACCEPTED MANUSCRIPT (pseudogene) and attlp6, had an impact on colonization of Arabidopsis roots by P. indica, and most of the mutants exhibited delayed colonization phenotypes (Reitz et al. 2013; Reitz et al. 2012), suggesting AtTLPs have
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roles in P. indica colonization. Dehydration or water-deficient, are the most common environmental
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stresses for plants to be exposed. A tubby-like protein CaTLP1 which was
SC
highly up-regulated under dehydration was identified based on the
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extracellular matrix proteome analysis of chickpea under dehydration (Bhushan et al. 2007), suggesting CaTLP1 perhaps has a significant role
MA
in drought stress. Subsequently, the transcript level of CaTLP1 of 3-weeks old chickpea seedlings in response to multiple stresses and
PT E
D
phytohormone treatments including dehydration, high concentration NaCl, ABA, jasmonic acid (JA) and SA treatments, were measured by Northern
CE
blot. However, only dehydration, high concentration NaCl and ABA treatments had a great effect on the transcription of CaTLP1. There was
AC
very little change in accumulation of CaTLP1 transcript in JA and SA treatments (Wardhan et al. 2012). As JA and SA are essential for pathogen- and wound-signaling and reported to mimic patho-stress response in plants (Clarke et al. 2000), CaTLP1 possibly has little or no role in patho-stress signaling. Moreover, overexpression of CaTLP1 conferred the transgenic tobacco plants increased root and leaf development, net photosynthesis, plant biomass, and improved tolerance 34
ACCEPTED MANUSCRIPT to dehydration, salinity and oxidative stresses. Recently, a study reported that the gene MdTLP7 of apple tree was up-regulated 512-fold in the leaves under the cold stress (Du et al. 2015). Transformation of MdTLP7 in E. coli could improve the tolerance to
PT
NaCl, KCl, chilling and heat shock in bacteria. Upon analysis of deletion
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of MdTLP7 amino acids, the 120–310 residues which form the 8 β strands
SC
of β barrel of the tubby domain were suggested to play crucial roles in stress tolerance (Du et al. 2014). To investigate the potential functions of
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MdTLPs under abiotic stress conditions, apple seedlings were exposed to
MA
various stresses including PEG, H2O2, exogenous ABA, and cold stress (Xu et al. 2016). Under PEG treatment, six MdTLPs (MdTLP1-5, 9) were
D
up-regulated significantly, while the remaining MdTLPs (MdTLP6, 7, 8)
PT E
were down-regulated in leaves. However, five MdTLPs (MdTLP3, 4, 7-9) were up-regulated significantly by PEG treatment in roots. In response to
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H2O2 or ABA treatment, MdTLP1-5 were significantly up-regulated in
AC
leaves, while the expression of MdTLP4, 6, 8, 9 showed a significant increase in roots. Under cold stress, almost all MdTLPs showed significantly up-regulated transcript levels in roots and leaves. Especially, the expression of MdTLP4 had significant changes in response to all the four stresses (PEG, H2O2, exogenous ABA, and cold stress treatment), suggesting MdTLP4 perhaps is the most important gene for stresses tolerance in the TLPs family of Malus domestica. 35
ACCEPTED MANUSCRIPT Soil salinity can aggravate the hyper-osmotic and hyper-ionic conditions, leading to considerable reduction of food production. From the microarray data in the NCBI database, TUBBY and ERF1 were identified as candidates for the response to salinity in Medicago (M.)
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truncatula. Furthermore, salinity could increase the level of expression of
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TLP gene in roots and leaves in M. truncatula , M. polymorpha and M.
stresses tolerance in different plants.
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laciniata (Gharaghani et al. 2015), suggesting the universal function of
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Four tubby-like genes have been identified in wheat and named as
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TaTULP1-4 (Hong et al. 2015). Gene expression of TaTULP1, TaTULP3 and TaTULP4 could be induced by salt stress, cold stress and MeJA
D
treatments, whereas the TaTULP2 transcript was repressed by these
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treatments. In addition, transcripts of all TaTULPs except TaTULP3 were also induced by ABA treatment.
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Analysis of the 15 ZmTLPs’ promoters cis-element showed that all
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ZmTLP genes contain a putative ABA responsive element (ABRE), low dehydration-responsive element (DRE), and temperature-responsive element (LTRE) (Yulong et al. 2015). Consistent with this prediction, the transcription of ZmTLP3, 4, 5, 6, 8, 9, and 12 were up-regulated by ABA treatment; and the transcription of ZmTLP3, 5, 8, 9, and 12 were induced greatly under heat treatment; the expression of ZmTLP2, 3, 4, and 11 were induced by PEG treatment; however, only the alteration of 36
ACCEPTED MANUSCRIPT ZmTLP8’s expression was observed under NaCl treatment (Yulong et al. 2015).
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5. Concluding remarks
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The tubby-like genes are regarded as putative TFs by structural-based
SC
functional analysis. But they usually locate in PM instead of the nucleus, as the tubby domain can bind PtdIns(4,5)P2. Even so, the tubby-like
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proteins can translocate into the nucleus in some conditions, such as
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G-proteins signaling, insulin- or leptin-induced phosphorylation and stress responses. Among them, the two process which are G-proteins
D
signaling and insulin/leptin-induced phosphorylation rely on PLCβ
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mediated hydrolysis of PtdIns(4,5)P2. But whether stress responses induced nucleus translocation also depend on PLCβ mediated hydrolysis
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of PtdIns(4,5)P2 is not yet clear. Furthermore, more studies are needed to
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explore what functions execute by those the tubby-like genes after translocating into nucleus. The functions of animal tubby-like genes have been well studied. They involve in trafficking proteins into cilia and are essential for ciliary function including energy balance, development, photoreception and hearing. Furthermore, they have functions in endocytosis, phagocytosis, keeping synapse maintenance or architecture, repressing Shh signaling. 37
ACCEPTED MANUSCRIPT Although the phenotype defects of these genes mutants have been well described, the mechanism of how they function is still elusive. Future studies should not lose sight of the mechanism of how they function, as it will aid help for therapies for retinal defects, hearing loss, obesity in these
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genes mutations.
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Even though the plant tubby-like genes have more members than
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animals, their functions have not been well elucidated. Maybe the functions of animal tubby-like genes can give us enlightenments. Until
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now, the plant tubby-like proteins only have been identified involving in
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multifarious stress responses and male gametophyte development. Further studies are needed to clarify the functions of these tubby-like genes in
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plants.
Conflicts of interest
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The authors declare that they have no conflicts of interest.
Acknowledgements This work was supported by the National Natural Science Foundation of China (3147029, 31670302 and 2015BAD15B03) and the Elite Youth Program of CAAS (to YK).
38
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ACCEPTED MANUSCRIPT Figure legends Fig. 1: Schematic model of how Tubby-like proteins binding to PM and detaching from PM to nucleus. (1) The conserved residues K (lysine) and R (Arginine) of tubby domain are charging
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for binding PtdIns(4,5)P2 in the PM. They can form a stable but reversible
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tubby-PtdIns(4,5)P2 complex. (2) Through the G-protein signaling, the tubby-like
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proteins can translocate into nucleus from PM. At first, the conserved residues K and R of tubby domain bind to PtdIns(4,5)P2 which is localized to PM. Also, Tubby-like
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proteins can associate with Gαq to form tubby-Gαq complex. Once the Gαq activated
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by GPCRs, the activated Gαq* can be released from the receptor and activate PLCβ. PLCβ then hydrolyzes PtdIns(4,5)P2 into InsP3, so the tubby-PtdIns(4,5)P2 complex
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can be disrupted and result in the translocation of Tubby-like proteins into the nucleus.
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(3) Phosphorylated Tubby-like proteins can translocate into nucleus. Insulin can utilize tyrosine kinase IRK and JAK2 to induce the phosphorylation of TUB Y464
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directly. And the translocation of phosphorylated Tubby-like proteins are also
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associated with the hydrolysis of PtdIns(4,5)P2 upon PLCβ. (4) In some stress conditions such as mannitol treatment, salinity and dehydration, the plants TLPs can also translocate into nucleus from PM.
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ACCEPTED MANUSCRIPT
Table 1: The gene function annotation of tubby-like family members derived from animals and plants Gene Name
Function Annotation
Mice
TUB,TULP3
trafficking GPCRs into cilium in brains neural tubes keep energy balance
Mice
endocytosis
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TULP1
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Mice,Human TUB
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Species
ciliary trafficking GPCRs in photoreceptors, keep synapse maintenance TUB,TULP1
or architecture, stimulate phagocytosis
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Mice
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repress mouse sonic hedgehog signaling, a transcriptional master regulator TULP3
in pancreatic ductal adenocarcinoma
Human
TULP1
can cause arRP, RD, LCA, JRP and RCD eye diseases
Drosophila
dTULP
regulate TRP channels localization in cilia
Drosophila
TUSP
Arabidopsis
AtTLP2, AtTLP6, AtTLP7
involving in male gametophyte development
Arabidopsis
AtTLP3, AtTLP9
involving in ABA stress responses
Rice
OsTLPs
Chickpea
CaTLP1
Apple tree
MdTLPs
involving in PEG, H2O2, exogenous ABA, and cold stresses responses
Wheat
TaTULPs
involving in cold, salinity, MeJA, and ABA stresses responses
Maize
ZmTLPs
involving in high temperature, salinity and ABA stresses responses
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D
Mice
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regulate the formation of the 7S SNARE complex
involving in defence responses against pathogen invasion involving in dehydration, salinity, ABA and oxidative stresses responses
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Fig. 1
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SC
RI
PT
ACCEPTED MANUSCRIPT
56
ACCEPTED MANUSCRIPT Highlights:
The first time to elaborate functions of tubby-like genes in animals and plants.
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The tubby-like proteins exhibit versatile functions in animal and plants.
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Three detailed ways of tubby-like proteins moving into nucleus were declarative.
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Meng Wang, Zongchang Xu, Yingzhen Kong PII: DOI: Reference:
S0378-1119(17)30923-X doi:10.1016/j.gene.2017.10.077 GENE 42298
To appear in:
Gene
Received date: Revised date: Accepted date:
14 May 2017 15 August 2017 27 October 2017
Please cite this article as: Meng Wang, Zongchang Xu, Yingzhen Kong , The tubbylike proteins kingdom in animals and plants. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/j.gene.2017.10.077
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ACCEPTED MANUSCRIPT The Tubby-like Proteins Kingdom in Animals and Plants
Meng Wanga,b,1, Zongchang Xua,b,1, Yingzhen Konga,*
Key Laboratory for Tobacco Gene Resources, Tobacco Research Institute,
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a
Graduate School of Chinese Academy of Agricultural Science, Beijing
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b
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Chinese Academy of Agricultural Sciences, Qingdao 266101, P.R. China
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100081, China
MW: [email protected]
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ZX: [email protected]
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Email addresses:
These authors contributed equally to this work
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1
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YK: [email protected]
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* Corresponding author; Yingzhen Kong Phone: +86-532-66715890 Fax: +86-532-66715890 Email: [email protected] Tobacco Research Institute, Chinese Academy of Agricultural Sciences, No. 11 Ke Yuan Jing 4th Road, Laoshan District, Qingdao 266101, Shandong, PR China. 1
ACCEPTED MANUSCRIPT Abstract Each gene of the tubby-like family is characterized by a signature of C-terminal tubby domain. The wide spread of this family in plants and animals implies they have an important function in various organisms.
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Even though the tubby-like genes are suggested to be putative
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transcription factors (TFs), how they execute the function as TFs is not
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yet clear. The biological functions of most animal tubby-like genes have been well studied, especially for vertebrate TUB, TULP1 and TULP3, but
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not with TULP2 and TULP4. Plants possess more tubby-like genes than
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animals, but their functions are still elusive except the idea that they are involved in stress responses with indistinct mechanisms. Here we
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reviewed the current knowledge of the versatile functions and roles of the
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tubby-like family members in plants and animals.
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function
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Keywords: tubby-like proteins; subcellular localization; biological
2
ACCEPTED MANUSCRIPT 1. Introduction:
The Tubby-like gene family was initially identified in an obesity tubby mouse more than two decades ago (Coleman et al. 1990;
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Noben-Trauth et al. 1996; Kleyn et al. 1996). This tubby mouse in
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Jackson’s laboratory was a C57BL/6J (F125) male breeder, and its
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offspring had a remarkably and slowly weight increase during the age of 12 to 24 weeks. Meanwhile the autosomal recessive mutant “tubby”
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mouse was found to be infertile, insulin resistant, and had hearing loss
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and retinal degeneration (Kleyn et al. 1996). Subsequently, the tubby-like proteins (TULPs) have been identified in various species, including mice,
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human (North et al. 1997), Caenorthabditis elegans (Mukhopadhyay et al.
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2005), Drosophila (Ronshaugen et al. 2002), chicken (Heikenwalder et al. 2001), rat (Figlewicz et al. 2004), Arabidopsis (Lai et al. 2004), rice (Kou
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et al. 2009), poplar (Yang et al. 2008), maize, sorghum (Yulong et al.
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2015), apple tree (Xu et al. 2016), and wheat (Hong et al. 2015). The wide distribution of this protein family in different species suggests that these proteins must have some conserved functions in the organisms. The expansion of the tubby-like family is suggested to be the result of segmental duplication and random translocation and insertion (Yang et al. 2008). The Tubby-like family is characterized by the signature conserved 3
ACCEPTED MANUSCRIPT C-terminal tubby domain which is 55% to 95% identical across species (Ikeda et al. 2002a). This domain consists of a β barrel enclosing a central α helix and binds selectively to specific membrane phosphoinositides (PIPs) (Santagata et al. 2001). Most studies suggested that the C-terminal
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domain might play a major role in the function of tubby-like proteins.
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Recently, the C-terminal tubby domain was suggested to be essential for
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TUB’s proper folding, solubility and subcellular localization (Kim et al. 2017). Unlike the conserved tubby domain, the N-terminal motifs are
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varied in diverse tubby-like proteins of various species, leading to the
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tubby-like proteins dividing into 3 classes (Lai et al. 2012). Besides the unique and conserved tubby domain, class I TULPs are characterized by
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the N-terminal WD40 domain and/or a suppressor of cytokine signaling
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(SOCS) box; class II TULPs do not have any other domain or motif in the N-terminus; class III TULPs all harbor F-box domain. Intriguingly,
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almost all of the plant tubby-like proteins have F-box motif in their
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N-terminus regions. The F-box domain can interact with other domains for recruiting specific proteins and targeting them for ubiquitin mediated proteolysis (Patton et al. 1998; Xiao et al. 2000). We cataloged the various key functional roles of tubby-like proteins from animals and plants in this paper (Table 1).
2. The subcellular localization of tubby-like proteins 4
ACCEPTED MANUSCRIPT
Using the structural-based functional analysis, the tubby-like proteins were implicated as TFs (Boggon et al. 1999), and they also were regarded as TFs in Arabidopsis in a review paper (Mitsuda et al. 2009). However,
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the subcellular localization of tubby-like proteins is not limited in the
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nucleus, they have been reported to usually localize in PM/cytoplasmic
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(Mukhopadhyay et al. 2011). Besides PM/cytoplasmic and nucleus, some proteins have other unusual subcellular localizations. For instance,
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TULP3 was reported to be in the tips of the primary cilia (Norman et al.
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2009); all the four TaTULPs localized to the Golgi apparatus (Hong et al. 2015); CaTLP1-YFP fusion protein showed predominant fluorescence
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signals in extracellular matrix of cell walls (Wardhan et al. 2012).
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Additionally, the different domains of tubby-like proteins have different subcellular localization. The full length and C-terminal of most tubby-like
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proteins tend to localize to PM, whereas the N-terminal domain localizes
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to the nucleus (Santagata et al. 2001; He et al. 2000; Reitz et al. 2012; Reitz et al. 2013). Previous studies reported that the conserved C-terminal tubby domain could bind phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) in the PM to form a stable but reversible complex. Mutations of the conserved positively charged phosphate-coordinating residues, K330 (Lysine330) and R332 (Arginine332), could result in their abolishing localization in 5
ACCEPTED MANUSCRIPT the PM (Santagata et al. 2001), such as TUB and TULP1 (Mukhopadhyay et al. 2010). These charged phosphate-coordinating residues are not only conserved among the animal TULPs (Santagata et al. 2001), but also in plants. For example, AtTLP3 also has the conserved PtdIns(4,5)P2
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binding sites K187 and R189, while mutating these sites with Alanine (A)
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disrupted its PM localization (Reitz et al. 2012; Reitz et al. 2013; Bao et
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al. 2014). Amino acid alignment analysis revealed that almost all of the AtTLPs have the conserved PtdIns(4,5)P2 biding site K and R, except
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AtTLP5 with R192K mutation, AtTLP9 with K168R mutation and
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AtTLP8 without these binding sites (Bao et al. 2014). However, the tubby-PtdIns(4,5)P2 complex is reversible. In some
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specific conditions, the tubby-like proteins can translocate into nucleus
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from PM (Figure1). Upon the G-protein signaling pathway, TUB can translocate from PM to the nucleus. The model has been depicted as
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follow: At first, TUBBY resides on the membrane through PtdIns(4,5)P2
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binding to form tubby-PtdIns(4,5)P2 complex. TUBBY can also associate with Gαq (or Gα11), which is the Gαq subset of G-proteins, leading to form a tubby-Gαq/11 complex. Once Gαq/11 activated by G-protein-coupled receptor (GPCR), the activated Gαq/11* will release from the receptor and activate the phospholipase Cβ (PLCβ). However, PLCβ can hydrolyze PtdIns(4,5)P2 into inositol trisphosphate (InsP3) and disrupt the tubby-PtdIns(4,5)P2 complex. This finally results in the dissociation of 6
ACCEPTED MANUSCRIPT tubby from the PM enabling its translocation to the nucleus (Santagata et al. 2001; Carroll et al. 2004). Recently, reports showed that insulin- or leptin-induced
TUB
tyrosine phosphorylation
could
induce the
translocation of TUB from PM into the nucleus, and this process were
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also associated with the hydrolysis role of PtdIns(4,5)P2 upon PLCβ
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(Prada et al. 2013). Further studies reported that tyrosine residue 464
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(Y464) of TUB was an important phosphorylation site by insulin, and mutation Y464A could not be phosphorylated by insulin and failure in
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translocation to the nucleus (Kim et al. 2014b), suggesting that Y464 is
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vital for insulin-induced TUB nuclear translocation. Moreover, abiotic stresses such as mannitol, NaCl and H2O2 treatments can trigger AtTLP3
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detaching from PM to the cytosol (Bao et al. 2014). Similarly, the YFP
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signals of a fused protein CaTLP1-YFP dominantly accumulated in nucleus under dehydration condition, suggesting that CaTLP1 may be
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et al. 2012).
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recruited to the nucleus to transfer signals to respond to stresses (Wardhan
It has been suggested that TUB might also function in a signaling role based on its dual localization, which is reminiscent of TFs like Sterol regulatory element-binding protein (SREBP), Nuclear factor kappa B (NF-kB), Drosophila mothers against decapentaplegics (SMADS), Signal transducers and activators of transcription (STATs) and Nuclear factor-activated T cells (NF-AT). These TFs could transit into the nucleus 7
ACCEPTED MANUSCRIPT in response to particular cell signaling events (Santagata et al. 2001). But little is known about the TF-regulating function of tubby-like proteins. Though OsTLP2 has been shown to be able to bind to the promotor of OsWRKY13 in vitro by electrophoresis mobility shift assay (Cai et al.
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2008), no evidence has been reported that tubby-like proteins could
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regulate TFs in vivo directly. So further studies are needed to elucidate the
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mechanism of how tubby-like genes function as TFs.
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3.1 Trafficking proteins into cilium
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3. The functions of tubby-like proteins in animals
Both mice TUB and TULP3 are involved in trafficking GPCRs into
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cilium in brains neural tubes. The primary cilium is an antenna-like cellular protrusion mediating sensory and neuroendocrine signaling, and
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it is enveloped by a membrane contiguous with the PM. The ciliary membrane is enriched with multiple integral membrane proteins, and
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TULP3 is defined as a general adaptor for trafficking integral membrane proteins to cilia (Badgandi et al. 2017). TULP3 is required for trafficking of
at
least
16
class
A
cilia–targeted
GPCRs
including
Melanin-concentrating hormone receptor (MCHR1) and Somatostatin receptor subtype3 (SSTR3) (Mukhopadhyay et al. 2010), Neuropeptide Y (NPY) receptors (NPY2R) (Loktev et al. 2013), Dopamine receptors 2 (short isoform, D2R short), 1 (D1R) and 5 (D5R), Galanin receptors 2 8
ACCEPTED MANUSCRIPT and 3 (GAL2R and GAL3R), G protein–coupled receptor 161 (GPR161) (Mukhopadhyay et al. 2013) and Orphan receptors GPR19, GPR83 and GPR88, Kisspeptin receptor (KISS1R), Neuropeptide FF receptor 1 (NPFFR1), Purinergic receptor (P2RY1) and the prolactin-releasing
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hormone receptor (PRlHR) (Badgandi et al. 2017). Besides the 16 class A
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cilia–targeted GPCRs, trafficking of fibrocystic ciliary localization
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sequence (CLS) fusions and transient receptor potential (TRP) channel family proteins polycystins PC1/2 (which form PC1/2 complex in the
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endoplasmic reticulum (ER), and are trafficked to cilia in an
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interdependent manner) (Kim et al. 2014a; Gainullin et al. 2015; Cai et al. 2017), are also adapted by TULP3. Specifically, TULP3 determines the
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entry of GPR161 into cilia directly. However, there are also exceptions of
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integral membrane proteins, such as
Smoothened
(SMO) and
cilia-targeted multispan adenylyl cyclases, which are not regulated by
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TULP3 (Mukhopadhyay et al. 2010; Bishop et al. 2007; Choi et al. 2011).
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Badgandi et al proposed a tripartite steps model for TULP3-mediated trafficking integral proteins to cilia which described as follow: firstly TULP3 captured membrane cargo in the PM dependent on PtdIns(4,5)P2, then TULP3-CLS complex (with the cargo) were delivered into the ciliary compartment by TULP3-IFT-A (Intraflagellar transport A) complex, lastly the cargo was released in the PI(4,5)P2-deficient ciliary membrane (Badgandi et al. 2017). This study also showed that the tubby 9
ACCEPTED MANUSCRIPT domain was responsible for proximity between the CLSs and TULP3. Similarly, TUB also can traffic certain rhodopsin family GPCRs to neuronal cilia, including SSTR3, MCHR1, and NPY2R (Sun et al. 2012; Loktev et al. 2013), but not GPR161 or GPR19. However, TULP3 is
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more effective than TUB in binding to the IFT-A core, suggesting that
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TULP3 might be more effective in trafficking GPCRs. However,
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Badgandi et al proposed that low-expressed GPCRs were preferentially trafficked by the high-expressed but poor IFT-A–binding TUB, and the
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highly expressed GPCRs required TULP3 and TUB redundantly for
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trafficking to the compartment (Badgandi et al. 2017). Mice TUB is not only essential for ciliary trafficking GPCRs in brain
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neural tubes, but also in photoreceptors. The photoreceptor outer segment (OS) is a modified cilium (Sun et al. 2012). The proximal end of the OS
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is linked to the cell body (inner segment, IS) via a connecting cilium (CC) which is structurally homologous to the transition zone of primary cilia.
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Sun et al reported that the 7-pass transmembrane GPCRs–rhodopsin and cone opsins, which should normally distribute in the OSs, were ectopically localized in the ISs and cell bodies or the ISs, the perinuclear region and the synaptic terminals in tubby mutant mice (Sun et al. 2012). This phenotype was also observed in the tulp1-/- mice (Hagstrom et al. 1999). The retinal defects of mice tubby mutant were milder than tulp1 mutant although mostly similar (Sun et al. 2012). Another marked feature 10
ACCEPTED MANUSCRIPT was the transient massive extracellular accumulation of rhodopsin-laden vesicles in the inter photoreceptor matrix between the adjacent ISs of both tubby and tulp1 mutant mouse, and the vesicles peaked at 3 weeks of age when rhodopsin should be rapidly synthesized to build up the OSs
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and quickly diminished (Heckenlively et al. 1995; Hagstrom et al. 1999).
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The vesicles accumulation appears to be a distinct ultrastructural feature
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shared by a small group of retinal disease models (Hagstrom et al. 1999). Thus, we can hypothesize that both TUB and TULP1 have an important
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role in rhodopsin trafficking in photoreceptors. Additionally, tubby tulp1
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double mutants have a much more severe retinal phenotype than either mutant alone, and this would appear to suggest that TUB and TULP1 may
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(Hagstrom et al. 2001).
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function synergistically to facilitate rhodopsin trafficking to the OSs
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Because the main defects of mice tulp1-/- mutant are retinal degeneration, the function of TULP1 in polarized vesicular translocation
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of rhodopsin from its site of synthesis in the IS through the CC to its final destination in the OS of the photoreceptor cell has been well studied. Moreover, studies have shown that TULP1 could associate with cellular membranes PIPs and interact with cytoskeleton proteins F-actin, Microtubule-associated protein 1A (MAP1A) and MAP1B, and neuronal-specific GTPase Dynamin-1 (Xi et al. 2005; Xi et al. 2007; Grossman et al. 2014), all of which involving in vesicular transport. PIPs 11
ACCEPTED MANUSCRIPT play a critical role in vesicular protein transport and organization of the cytoskeleton (Roth 2004). Numerous studies implicated that actin could associate with protein movement in vesicle assembly and polarized transport (Xi et al. 2005). Besides stabilizing microtubules, MAP proteins
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also have other functions for trafficking vesicles and organelles (Brenman
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et al. 1998; Thomas et al. 2000). Dynamin-1 is an essential component of
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vesicle formation in receptor-mediated endocytosis, synaptic vesicle recycling, and vesicle trafficking in and out of the trans-Golgi network
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(TGN) (Bliek 1999; Mcniven et al. 2000). Interestingly, the interaction
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between Dynamin-1 and the actin cytoskeleton has also been reported. Dynamin-1 was thought to act as a polymeric contractile scaffold at the
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interface between membranes and actin cytoskeleton to generate vesicles
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at different cellular sites (Orth et al. 2003). These data imply that F-actin, MAP1A, MAP1B and Dynamin-1 may aid help for TULP1-mediated
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rhodopsin vesicular transport. Besides rhodopsin, there were several other
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OS proteins mislocalized in tulp1-/- mice retinas including GC-activating protein 1 (GCAP1), GCAP2, Guanylate cyclase 1 (GC1) and blue cone opsin, which should be restricted to OS whereas had a more extensively localization (Grossman et al. 2011). Based on the analysis of several mouse mutant phenotypes, it has been proposed that GCAP1, GCAP2, GC1 and GC2 are co-transported, and their transport is independent of the rhodopsin trafficking system (Baehr et al. 2007; Karan et al. 2008). So 12
ACCEPTED MANUSCRIPT possibly GC2 is mislocalized in tulp1-/- retinas as well. In addition, in the tulp1-/- retina, the light-dependent translocation of signaling protein arrestin which binds to phosphorylated rhodopsin and plays an important role in the recovery phase of phototransduction was severely impaired
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(Grossman et al. 2011). In the WT (wild type) retina, arrestin was
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localized to the IS, ONL (outer nuclear layer) and OPL (outer plexiform
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layer) in darkness and redistributed to the OS in the light. However, in the tulp1-/- retina, the distribution of arrestin remained confined to the IS,
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ONL and OPL in both the dark and light. Significantly, protein complexes
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that regulate the trafficking of rhodopsin, RAB6, RAB8 and RAB11 were mislocalized in tulp1-/- retinas as well (Grossman et al. 2011). RAB6
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can function in the generation and budding of rhodopsin-laden vesicles
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from the TGN. RAB8 is important for the docking and fusion of rhodopsin-bearing vesicles to the IS PM, in close proximity to the CC.
with
RAB6
(Deretic
2006).
According
to
the
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conjunction
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RAB11 functions in the sorting and budding of vesicles from the TGN in
above-mentioned data, we can propose a simplified and possible pathway for rhodopsin vesicular transport. Considering TULP1 can associate with PIPs, actin and microtubules, the TULP1-Dymanin-1 complex could form a polymeric contractile scaffold at the interface between membranes and cytoskeleton, which can lead the vesicular transport. In the WT IS, rhodopsin molecules are posttranslationally modified in the Golgi 13
ACCEPTED MANUSCRIPT apparatus, and inserted into carrier vesicles generated from the TGN surface. Then TULP1-Dynamin-1 complex leads to the budding of rhodopsin-laden vesicles with the help of RAB6, then RAB11 attends to transport the vesicles through the membranes and cytoskeleton. RAB8 is
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responsible for docking and fusion of vesicles with the IS PM, which is
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necessary for the subsequent transport of rhodopsin through CC to OS
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discs. However, if TULP1 is absent, the cargo vesicles will exocytose through the basal IS PMs (occasional rhodopsin labeling vesicles were
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observed to be connected with the IS PMs (Hagstrom et al. 2001)) and
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then accumulate in the extracellular inter photoreceptor matrix. Besides mice TULPs, Drosophila TULP (dTULP) which is a TUBBY
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homolog in Drosophila genome (Ronshaugen et al. 2002), can also
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mediate trafficking proteins into cilia. The expression of dTULP was confirmed in cilia and cell body of chordotonal neurons of Johnston’s
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organs which is essential for flies to hear. Additionally, several TRP
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channels such as Inactive (IAV), NOMPC and Spacemaker (SPAM) were ectopically localized in cilia of dtulp mutants. TRP channels are essential factors for Drosophila hearing transduction and amplification (Lehnert et al. 2013). The ectopically localization of these TRP channels suggest the extraordinary role of dTULP in mediating hearing in Drosophila. The domains which bind IFT-A and PIP are conserved in dTULP and mice TULP3. The putative IFT- and PIP-binding domains of dTULP have been 14
ACCEPTED MANUSCRIPT suggested to be required for the proper localization of TRPs in cilia. However, the two putative domains perhaps have different roles in the proper IAV distribution in cilia, as the IFT-binding domain is required for the ciliary entry for dTULP, and the PIP-binding domain affects
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recruitment of IAV-containing preciliary vesicles to dTULP. Furthermore,
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unlike the mice TULP3, dTULP ciliary access is not dependent on IFT-A
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as dTULP ciliary trafficking is not affected by the mutation of IFT-A proteins. It has been explained that dTULP facilitated the relay of
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preciliary vesicles to the IFT complex at the base of cilia rather than
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moving together with ciliary membrane proteins into the cilia (Park et al. 2013).
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As known from literature, primary cilia are sensory organelles
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wherein components of numerous signaling pathways concentrate. Their functions include involvement in many aspects such as olfaction,
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photoreception and development, which are mediated by targeting of
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receptors, channels, and their downstream effector proteins into cilia correctly (Berbari et al. 2009). Therefore, the factor-mediated ciliary trafficking has a critical role in ciliary function. TULP3 is defined as a general adaptor for trafficking at least 16 GPCRs, and TUB can also traffic at least 3 GPCRs into cilia. Among them, MCHR1 is involved in the regulation of feeding and energy balance; SSTR3 is important in synaptic plasticity and novelty detection (Mukhopadhyay et al. 2010); 15
ACCEPTED MANUSCRIPT NPY2R is involved in energy homeostasis (Loktev et al. 2013); GPR161 is a negative regulator of Shh signaling. Also, the photoreception-related critical factors rhodopsin, blue cone opsin, arrestin, RAB6, RAB8 and RAB11 are mislocalized in tulp1-/- and/or tub-/- mutants. Furthermore, the
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hearing-related TRP channels IAV, NOMPC and SPAM are mislocalized
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in dtulp mutants. Taken together, we can conclude that trafficking
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proteins into cilia mediated by tubby-like genes play an important role in ciliary function, such as energy balance, development, photoreception
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and hearing.
3.2 TUB has a key role in energy balance
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According to the previous studies, the mutation of TUB gene in mice
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and humans mainly resulted in obesity phenotype (Coleman et al. 1990; Kleyn et al. 1996; Noben-Trauth et al. 1996; Kapeller et al. 1999; Borman
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et al. 2014). Especially for humans, the body composition and
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macronutrient intake of middle-aged women are associated with genetic variation of TUB gene, suggesting that TUB may influence eating behavior (van Vliet-Ostaptchouk et al. 2008). TUB has been shown to have expression in adipocyte (Noben-Trauth et al. 1996), and its expression is regulated developmentally during adipogenic differentiation (Stretton et al. 2009). Furthermore, mutation of TUB-1 which is a TUBBY homolog in C. elegans, resulted in an extended life span and increased fat 16
ACCEPTED MANUSCRIPT deposition (Mukhopadhyay et al. 2005), further supporting the view that tubby-like proteins are involved in energy balance. Additionally, TUB was reported to be regulated by thyroid hormone or insulin (Stretton et al. 2009), another study revealed that TUB involved in insulin’s and leptin’s
PT
effects on energy balance and glucose metabolism (Prada et al. 2013). As
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described in the literature, leptin and insulin signals controlled energy
anorexigenic
neuropeptides.
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balance in part by regulating the expression of orexigenic and However,
the
leptin/insulin-induced
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increases of proopiomelanocortin (POMC) and thyroid releasing hormone
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(TRH), and the leptin/insulin-induced reduction of melanin-concentrating hormone (MCH) and orexin neuropeptides were blunted when TUB
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antisense oligonucleotide (ASO) was used in the hypothalamus to reduce
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TUB protein expression. Put together, these data suggest that TUB is perhaps essential for regulating the expression of orexigenic and
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anorexigenic neuropeptides in the leptin/insulin signaling pathway.
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Furthermore, the tyrosine residue of TUB could be phosphorylated in response to insulin or insulin-like growth factor (Kapeller et al. 1999). Further studies suggested that both insulin and leptin could take TUB as substrate, and insulin receptor (IR) could utilize its endogenous tyrosine kinase (IRK) activity to induce TUB tyrosine phosphorylation directly; however, leptin receptor (LEPR) could use Janus kinase 2 (JAK2) to induce TUB phosphorylation (Prada et al. 2013). The phosphorylation of 17
ACCEPTED MANUSCRIPT insulin- or leptin-induced TUB tyrosine residue is in a dose-dependent and time-dependent manner, and this phosphorylation could result in nuclear translocation of TUB from the PM. As described in the section of subcellular
localization,
Y464
site
of
TUB
is
important
for
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insulin-induced nuclear translocation. Moreover, TUB has been shown to
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be able to associate with Protein tyrosine phosphatase 1B (PTP1B) which
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may have a role in the dephosphorylation of TUB. These data imply that maybe insulin/leptin and PTP1B function in antagonism to control the
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3.3 Functions in endocytosis
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phosphorylation level of TUB, then to regulate the energy balance.
Recently, a study found that TULP1 colocalized with endocytic
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proteins (dynamin and clathrin heavy chain) and an important signaling phospholipid for initiating endocytosis–PIP2 in the periactive zone where
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has been shown to be a hotpot of endocytic activity of the bovine retina (Wahl et al. 2016). The endocytic proteins (dynamin and clathrin heavy
AC
chain) were highly enriched in the photoreceptor synapses periactive zone of WT mice at postnatal day (P) 16, whereas these proteins were almost disappeared concomitant with severely defect in periactive zone endocytosis of tulp1-/- mice. Moreover, the TULP1 can directly interact with the synaptic ribbon protein RIBEYE. These novel findings may hint us a new model for TULP1-mediated localization of the endocytic machinery at the periactive zone of ribbon synapses and offer a new 18
ACCEPTED MANUSCRIPT rationale mechanism for vision loss associated with genetic defects in TULP1 (Wahl et al. 2016). Other evidence further verified that the Tubby-like family could mediate the endocytosis. The Drosophila king-tubby (KTUB) gene
PT
specially localized in the rhabdomere domain of photoreceptors R1 to R6
RI
of the WT flies reared in dark. When the dark-reared WT flies exposed to
SC
light for 3 hours, the KTUB protein would move from rhabdomere to cytoplasm concomitant with a significant amount of rhodopsin 1
NU
(Rh1)-immunopositive large vesicles (RLVs) in the cytoplasm. However
MA
in the ktub mutant flies, besides the defect of rhabdomere in photoreceptor R1 to R6, the Rh1 also was mislocalized in the
D
rhabdomeric domain with only a few RLVs found in the cytoplasm.
PT E
Furthermore, the ktub norpA double mutant could rescue the phenotype of massive internalization of rhodopsin from rhabdomere to cell body in
CE
norpA mutant for a 12 hours’ light stimulation (Orem et al. 2002). This
AC
suggests us the KTUB gene may play an important role in mediating rhodopsin endocytosis. And the C-terminus tubby domain is critical for rhodopsin endocytosis (Chen et al. 2012). C.
elegans
TUB-1
gene
can
negatively
regulate
RBG-3
(Mukhopadhyay et al. 2005), and RBG-3 can promote the GTPase hydrolysis of RAB-7 which could control endocytic sorting specifically in the neurons (Deinhardt et al. 2006) to regulate fat storage. From this 19
ACCEPTED MANUSCRIPT data we can propose that TUB-1 gene regulate fat storage through a RAB-7-mediated endocytic mechanism, supporting a more general role of tubby-like family proteins in endocytosis.
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3.4 Keep synapse maintenance or architecture
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Even though TULP1 does not predominantly localize in synaptic
SC
terminals, it has a role in synapse maintenance or architecture. In mice WT retinas, TULP1 had a diffusely staining in the OPL and colocalized
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with the presynaptic ribbon-associated protein Bassoon (Grossman et al.
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2009). Piccolo and Bassoon proteins were reported to have roles in organization of the photoreceptor synapse, the functioning of the ribbon
D
and the trafficking of vesicles at the synapse (Dick et al. 2003; Dick et al.
PT E
2001). But Piccolo and Bassoon proteins were unable to coordinate into the normal horseshoe-shaped ribbon architecture in mice tulp1-/- mutant,
CE
though they were able to arrive at their correct destinations and often in immediate proximity with another (Grossman et al. 2009). In addition,
AC
fewer intact ribbons were present in the P13 tulp1-/- retina (Grossman et al. 2009). Recently, a study reported that the number of synaptic ribbons significantly decreased in tulp1-/- mice at P30 whereas they should increase in the course of postnatal maturation (Wahl et al. 2016). In WT P16 mice retinas, the electroretinogram (ERG) had distinct a-waves and b-waves which indicated the functional invaginating synapses (Keeler et 20
ACCEPTED MANUSCRIPT al. 1928). However, the ERG a-waves generated by depolarizing bipolar cells (DBCs) were reduced in amplitude but retained normal kinetics with a normal leading edge, and the b-waves had a reduced amplitude and a desensitized intensity-response function at P16 and P15 in tulp1-/- mice
PT
(Grossman et al. 2009; Xi et al. 2007). All of the above data indicate the
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malformation of tulp1-/- photoreceptor synapses. Furthermore, there was a
SC
profound shortening of individual dendrites and a severe attenuation of dendrite branching which indicated the underdeveloped or malformed
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structural platform for relaying signaling from photoreceptors to the inner
MA
retina in tulp1-/- mice (Grossman et al. 2009). The reduction of the DBC dendritic field in the tulp1-/- retina was suggested to be a developmental
D
consequence of an attenuated trophic and transmitter release that would
PT E
be expected from a presynaptic malformation (Grossman et al. 2009). MAP1A is a genetic modifier of both TULP1 and TUB, it can reduce
CE
hearing loss in tub-/- mice, retinal degeneration in tub-/- and tulp1-/- mice
AC
(Ikeda et al. 2002b; Maddox et al. 2012). TULP1 can interact with MAP1A (Grossman et al. 2014). MAP1A is predominantly expressed in the synapses of adult neurons, and it can physically interact with the membrane-associated guanylate kinase family member DLG4 which is crucial in establishing the post synaptic (Brenman et al. 1998; Thomas et al. 2000). TULP1 could interact with neuronal-specific GTPase Dynamin-1 which has a role in PM and vesicle recycling at neuronal 21
ACCEPTED MANUSCRIPT synapses (Xi et al. 2007). Moreover, TUB might also have functions in synaptic architecture, because the TUB’s G-protein receptor signaling function occurs at synaptic junctions and TUB has a high expression in the synaptic junctions formed region (Ikeda et al. 1999; Santagata et al.
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2001; Ikeda et al. 2002b). Given all the findings mentioned above, both
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TULP1 and TUB possibly have functions in synapse maintenance or
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architecture.
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3.5 Stimulate phagocytosis
Phagocytosis for the removal of apoptotic cells or cellular debris, has
MA
an important role in many biological processes such as development, tissue homeostasis and disease resistance (Erwig et al. 2007). Besides
PT E
D
initiating phagocytosis, phagocytosis ligands are also the key to selecting extracellular cargos for clearance, understanding phagocyte beneficial
CE
and detrimental functional roles and regulating phagocyte activities with therapeutic potentials (Li 2012). TULP1 was identified as a putative
AC
phagocytosis ligand, and further experiments proved that TUB and TULP1 could stimulate phagocytosis of retinal pigment epithelium (RPE) cells and apoptotic cells (Caberoy et al. 2010a). Moreover, TUB and TULP1 might function in synergy to stimulate phagocytosis of RPE cells, because the association of TULP1 and TUB has been verified (Caberoy et al. 2010b; Caberoy et al. 2010a) . But how TUB and TULP1 stimulate phagocytosis is needed to be 22
ACCEPTED MANUSCRIPT elucidated. Further studies have proved that TUB and TULP1 are Mer tyrosine kinase (MerTK) ligands and stimulate RPE phagocytosis in a MerTK-dependent manner. MerTK is a well-characterized phagocytic receptor and essential for maintaining retinal homeostasis, and the retinal
PT
degeneration of MerTK deficient mutant mice is a result of the
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accumulation of unphagocytosed debris and defective RPE phagocytosis
SC
(Seitz et al. 2007). According to the literature, phagocytosis is initiated by ligands with receptor activation and signaling cascades, followed by
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cytoskeletal rearrangement and engulfment. MerTK activation can lead to
MA
MerTK autophosphorylation. TUB and TULP1 can not only promote the autophosphorylation of MerTK, they also induce the redistribution of
TUB/TULP1-MerTK
PT E
the
D
non-muscle myosin II-A (NMMII-A) redistribution, which suggests that RPE
phagocytosis
pathway
facilitate
MerTK-dependent cytoskeletal reorganization (Caberoy et al. 2010c).
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Similar to the two well-characterized MerTK ligands, GAS6 and protein
AC
S, facilitate MerTK-dependent phagocytosis with their N-terminus Gla domain interacting with phosphatidylserine on apoptotic cells and the C-terminal two globular laminin G-like domains (2×LG) binding to MerTK on phagocytes (Lemke et al. 2008; Hafizi S et al. 2006). TUB and TULP1 are also bridging molecules with their N-terminal minimal phagocytosis determinants (MPDs) as MerTK-binding domain and the C-terminal region as phagocytosis prey-binding domain (PPBD) which 23
ACCEPTED MANUSCRIPT through a phosphatidylserine and PtdIns(4,5)P2-independent mechanism (Caberoy et al. 2010c). MPDs are critical for MerTK binding and autophosphorylation,
five
MPDs
of
K/R(X)1–2KKK
in
TULP1
N-terminus have been predicted. The PPBD has been mapped to the
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C-terminal 54 amino acids of TULP1 (Caberoy et al. 2010c). Besides
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RPE and macrophage phagocytosis, TUB also can stimulate microglial
SC
phagocytosis in BV-2 microglial cell line and primary microglia. Similarly, TUB can activate MerTK and then lead to MerTK
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autophosphorylation, NMMII rearrangement in BV-2 microglial cell line
MA
(Caberoy et al. 2012). Interestingly, TUB can induce both microglial phagocytosis and MerTK’s phosphorylation in a concentration-dependent
D
manner with minimal and maximal induction concentrations at 0.1 nM
PT E
and 10 nM, respectively. However, the activity of TUB to stimulate microglial phagocytosis will be reduced at a high concentration (200 nM)
CE
but the phosphorylation of MerTK is slightly affected (Caberoy et al.
AC
2012). From these data, we can conclude that TUB and TULP1 can stimulate RPE and macrophage phagocytosis, and TUB also can induce microglial phagocytosis (Caberoy et al. 2012; Caberoy et al. 2010c). Even so, TUB and TULP1 are intracellular proteins which should perform intracellular functions. But how intracellular proteins have extracellular functions?
TUB and TULP1 were reported to be secreted
through unconventional pathways, not the classical ER–Golgi pathway, 24
ACCEPTED MANUSCRIPT and this unconventional pathway partially depends on an essential secretory signal in the N-terminus between Asn51 and Arg100 with the PI(4,5)P2-binding activity (Caberoy et al. 2009). TUB and TULP1 may have multiple intracellular and extracellular functions similar to
RI
PT
GALECTIN-3 (Dumic et al. 2006).
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3.6 The eye diseases of mutation in TULP1 of human
The eyes diseases associated with the mutant TULP1 can be classified
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into five types: autosomal recessive Retinitis Pigmentosa (arRP), retinal
MA
degeneration (RD), Leber congenital amaurosis (LCA), juvenile onset retinitis pigmentosa (JRP) and rod-cone dystrophy (RCD) (Ullah et al.
D
2016). Until now, a total of 54 different casual mutations harboring amino
PT E
acids substitution, duplication and deletion, aberrant splicing and nonsense mutation in TULP1 have been reported, and each of the
CE
majority mutations only causes one type of TULP1-caused eye diseases
AC
(Ullah et al. 2016). However, there are also exceptions such as p(patient).Q301* null mutation in TULP1 can cause LCA and RCD (Li et al. 2009; Khan et al. 2015), p.L461V mutation leads to JRP and LCA diseases (den Hollander et al. 2007). More surprisingly, the substitution of the same amino acid in the fixed position usually cause one disease (p.I459K and p.I459T both caused the same disease arRP (Hagstrom et al. 1998; Wang et al. 2014)), but p.R420S in TULP1 cause RCD (Roosing et 25
ACCEPTED MANUSCRIPT al. 2013), p.R420P cause arRP (Hagstrom et al. 1998). All of the casual mutations in TULP1 patients provide us vital information for therapies to the TULP1 mutations resultant vision impairment. Recently, the mechanism of photoreceptor cell death in four
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TULP1 missense mutations (p.D94Y, p.R420P, p.I459K and p.F491L)
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were implicated (Lobo et al. 2016). Bioinformatics and protein structural
SC
analysis of the four TULP1 mutations predicted that they all would result in misfolded and/or unstable with altered protein structures and therefore
NU
likely be pathogenic when expressed. No matter in-vitro or in-vivo,
MA
mutant TULP1 proteins localized to ER and caused induction of the ER-UPR (unfolded protein response) stress complex. And those mutant
D
TULP1-expressing cells had significantly elevated levels of BiP which
PT E
are ER chaperones. This suggests that the mutant TULP1 proteins can cause ER cellular stress and activation of the UPR complex. The two
CE
major apoptotic arms of the UPR pathways, PKR-like ER kinase (PERK)
AC
and Inositol-requiring enzyme 1 (IRE1), were strongly induced in the four mutant TULP1-expressing cells. In addition, C/EBP homologous protein (CHOP), which is downstream pro-apoptotic target of pPERK (phosphorylated PERK) and IRE1, had high levels expression in mutant cell lines. All of these results indicate that long term retention of mutant TULP1 within cells can activate the ER-UPR stress complex, leading to the initiation of apoptosis via the CHOP signaling pathways. 26
ACCEPTED MANUSCRIPT
3.7 A critical repressor of mouse Sonic hedgehog signaling Sonic hedgehog (Shh) acts as signaling molecule critically involved in multiple aspects of embryonic development and developmental
PT
processes in vertebrates (Martí et al. 2002; Palma et al. 2005; Zhang et al.
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2001). Especially, Shh signaling pathway plays a significant role in
SC
normal growth and patterning of many vertebrate tissues such as limb and neural tube (McGlinn et al. 2006; Ulloa et al. 2007; Varjosalo et al. 2008).
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But how the Shh signaling works in the growth and patterning of limb
MA
and neural tube? Previous studies reported that Shh provided positional information for the patterning of neural cell fates in the developing
D
central nervous system and controlled the digit pattern in the developing
PT E
limbs. Almost at the same time, 3 studies identified TULP3 as a critical repressor of Shh signaling pathway (Patterson et al. 2009; Norman et al.
CE
2009; Cameron et al. 2009). Mutants tulp3 and hitchhiker (a strong
AC
hypomorph of TULP3) showed defects in spinal neural tube and the developing limbs such as exencephaly, spina bifida, enlarged branchial arches and preaxial polydactyly on both fore- and hind-limbs, which resemble the phenotypes of inappropriate activation of the Shh signaling pathway mutations. PTCH1 and GLI1, which are target genes of Shh signaling pathway, were up-regulated and ectopically expressed in dorsal regions of spinal neural tube in both tulp3-/- and hitchhiker mutant. And 27
ACCEPTED MANUSCRIPT the dorsal-to-ventral patterning defects were evident at stage of Embryo 9.5 in mutants. These data verify that the mutants’ neural tube experience elevated levels of Shh pathway activity. Similarly, the limb buds of tulp3-/- and hitchhiker mutant also exhibited ectopically expression of
PT
GLI1 and PTCH1 in the anterior regions with anterior–posterior polarity
RI
perturbed which demonstrated inappropriate activation of the Shh
SC
pathway and resulted in polydactyly. Furthermore, TULP3 was regarded as genetically downstream gene of SHH and SMO, and genetically
NU
interacted with GLI3.
MA
Now, we have to consider the question that how does TULP3 repress the Shh signaling pathway? The primary cilia localization of TULP3 may
D
implicate that TULP3 possibly regulates the hedgehog pathway within
PT E
this structure (Norman et al. 2009). As described in the “trafficking proteins into cilium” section, TULP3 and IFT-A are both negative
CE
regulators of Shh signaling and are able to traffic GPCRs into cilia
AC
(Patterson et al. 2009; Norman et al. 2009; Cameron et al. 2009; Qin et al. 2011; Tran et al. 2008; Liem et al. 2012). Maybe one of the GPCRs could have roles in the TULP3/IFT-A-regulated antagonism of Shh signaling. Recently, a report suggested that TULP3 might determine the entry of GPR161 into cilia (Badgandi et al. 2017). Furthermore, GPR161 can increase cAMP, then activate Protein kinase A (PKA), leading to the formation of GLI3R and subsequent repression of Shh target transcription 28
ACCEPTED MANUSCRIPT (Mukhopadhyay et al. 2013). Thus we can hypothesize that the negative Shh regulator GPR161 plays an important role in TULP3-mediated repression of Shh-signaling. However, Shh-dependent loss of GPR161 from cilia not only relies on decreasing entry into cilia, but also on
PT
increasing removal. The removal of GPR161 out of cilia depends on
RI
another distinct mechanism. The accumulation and activation of the
SC
effector SMO within cilia which results from stimulation of Shh signaling, can increase GPR161–β-arrestin binding and lead to the removal of
NU
GPR161 by clathrin-mediated endocytosis (Pal et al. 2016). Recently,
MA
reports suggested that the ciliary membrane PIP composition which regulated by INPP5E, could be read out by TULP3 to control ciliary
D
protein localization and enable Shh singaling (Garcia-Gonzalo et al. 2015;
PT E
Chavez et al. 2015). With the existence of INPP5E, PI(4,5)P2 can be restricted at the proximal end of cilia whereas PI(4)P distributed along the
CE
length of cilia. As TULP3 binds to PI(4,5)P2 instead of PI(4)P
AC
(Mukhopadhyay et al. 2010), TULP3 can only be restricted at the tip of the cilia (Badgandi et al. 2017). Once INPP5E inactivated, PI(4,5)P2 accumulate in the cilia with the depletion of PI(4)P, leading to more TULP3 and GPR161 recruited to the cilia and increases of Shh signaling. These data imply that the ciliary membrane PIP composition have a great role for the location of TULP3 and GPR161 in cilia to regulate Shh signaling. 29
ACCEPTED MANUSCRIPT
3.8 TUSP regulates the formation of the 7S SNARE complex Drosophila TUSP (tubby domain superfamily protein) is the fly homologue of mammalian TULP4 (Mukhopadhyay et al. 2011).
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Mutations in vertebrate TULP4 are associated with short stature, cleft lip,
role
for
TUSP
in
the
assembly
of
the
7S
SC
unexpected
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and cleft palate (Allen et al. 2010; Conte et al. 2016). A study revealed an
synaptobrevin-syntaxin-SNAP-25 (SNARE) ternary complex (Yoon et al.
NU
2017). Mutant tusp flies exhibited a phenotype of temperature-sensitive
MA
paralysis which could be rescued by ectopic expression of TUSP in the giant fibers (GFs) and peripherally synapsing interneurons of the GF
D
system. Reduction in the assembly of the ternary 7S SNARE complex,
PT E
which is essential for neurotransmitter release, was observed in tusp mutant flies, though the transcription of each gene of the individual
CE
SNARE complex component was not changed. Thus, these data suggest
AC
that TUSP is a novel regulator of neurotransmitter release. Although the regulatory mechanisms such as ubiquitination or SNARE chaperone were suggested, further studies are needed to lift the veil.
4. The functions of tubby-like proteins in plants
Compared with the wide array of cellular functions identified in 30
ACCEPTED MANUSCRIPT animal TULPs, the functions of plant TLPs are relatively elusive. The plant genome harbors more tubby-like genes than that of animals. In wheat, apple tree, Arabidopsis, poplar, sorghum, rice and maize, there are 4, 9, 11, 11, 13, 14 and 15 TLP members, respectively. But limited
PT
functions of the plant TLPs have been reported so far.
SC
RI
4.1 TLPs perhaps involved in male gametophyte development To search the male gametophyte development related genes, 74
NU
Arabidopsis T-DNA insert lines of 49 genes in 21 TF families have been
MA
collected (Renak et al. 2012). The fully open flowers grains in developmental stage 14 of these mutants were analyzed by microscopy.
D
Among these mutants, attlp6 mutant plants produced 5% two-celled
PT E
pollen, 15% misarranged male germ unit (MGU) pollen and 5–10% aborted grains. The attlp7 mutant plants showed 5–10% two-celled
CE
phenotype and 20–25% pollen with misarranged MGU (Renak et al. 2012). Based on the Arabidopsis eFP Browser 2.0 database
AC
(http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi), AtTLP6, AtTLP7 and AtTLP2 are predominantly expressed in pollen grains. Previous studies reported that AtTLP6 can interact with ASK11 and ASK13; AtTLP7 also can interact with ASK1 in yeast two hybrid analysis (Bao et al. 2014). ASK1 gene is essential for male meiosis (Yang et al. 1999). qRT-PCR results showed that ASK11 and ASK13 had a higher expression in 31
ACCEPTED MANUSCRIPT inflorescence than other tissues, and the in situ-hybridization results also indicated that the expression of ASK1, ASK2 and ASK11 could be detected in pollen grains (Zhao et al. 2003). All of this data imply that the
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4.2 TLPs involving in multifarious stress response
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tubby-like proteins might have functions in pollen grains.
SC
4.2.1. Biotic stress response
The OsTLPs can respond to biotic stress such as pathogen. The
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transcription of all of the 14 OsTLPs could be induced by pathogen
MA
inoculation, suggesting that they all may be involved in the interaction of rice-Xoo pathogen (Kou et al. 2009). It has been reported that
D
OsWRKY13 can regulate genes which act both upstream and
PT E
downstream of salicylic acid (SA) to function as regulators for rice resistance to bacterial blight and fungal blast diseases. Over-expression of
CE
OsWRKY13 could enhance the resistance to bacterial blight and fungal
AC
blast, on the contrary, the low-expression of OsWRKY13 enabled rice to be more susceptible to pathogen infection (Qiu et al. 2009). Furthermore, OsTLP2 could bind to the PRE4 elements in the promotor of OsWRKY13, suggesting that OsTLP2 can regulate the expression of OsWRKY13 (Cai et al. 2008). Even though this binding hasn’t been verified in vivo, the expression level of OsTLP2 has been shown to be induced approximately 11-fold after pathogen inoculation, further supporting that OsTLP2 may 32
ACCEPTED MANUSCRIPT be involved in defense responses against pathogen invasion (Cai et al. 2008). 4.2.2. Abiotic stresses responses
PT
AtTLP3 and AtTLP9 can function redundantly in the ABA signaling
RI
pathway during seed germination and early seedling development (Lai et
SC
al. 2004; Bao et al. 2014). Single mutants attlp3 or attlp9, and double mutants attlp3attlp9 were all insensitive to ABA during seed germination.
NU
When these mutants were cultured on MS media containing ABA, the
MA
germination frequency of mutants were higher than WT. The germination frequency of double mutants was higher than either the single mutant,
D
suggesting that attlp3atltp9 double mutant plants are more tolerant to
PT E
ABA. Furthermore, attlp3atltp9 double mutant plants are more insensitive to mannitol than the attlp9 mutants. Taken together, the above
CE
findings imply the redundant function of AtTLPs in abiotic stress signaling. However, the AtTLPs have no redundant function in
AC
contributing to Arabidopsis colonization by Piriformospora indica, as individual AtTLP protein contributes to the colonization success (Reitz et al. 2012; Reitz et al. 2013). P. indica is able to colonize the roots of multiple plant species and confer its hosts various beneficial traits such as increased growth, higher seed yield and enhanced tolerance to biotic and abiotic stresses (Qiang et al. 2012). All attlps mutants except attlp4 33
ACCEPTED MANUSCRIPT (pseudogene) and attlp6, had an impact on colonization of Arabidopsis roots by P. indica, and most of the mutants exhibited delayed colonization phenotypes (Reitz et al. 2013; Reitz et al. 2012), suggesting AtTLPs have
PT
roles in P. indica colonization. Dehydration or water-deficient, are the most common environmental
RI
stresses for plants to be exposed. A tubby-like protein CaTLP1 which was
SC
highly up-regulated under dehydration was identified based on the
NU
extracellular matrix proteome analysis of chickpea under dehydration (Bhushan et al. 2007), suggesting CaTLP1 perhaps has a significant role
MA
in drought stress. Subsequently, the transcript level of CaTLP1 of 3-weeks old chickpea seedlings in response to multiple stresses and
PT E
D
phytohormone treatments including dehydration, high concentration NaCl, ABA, jasmonic acid (JA) and SA treatments, were measured by Northern
CE
blot. However, only dehydration, high concentration NaCl and ABA treatments had a great effect on the transcription of CaTLP1. There was
AC
very little change in accumulation of CaTLP1 transcript in JA and SA treatments (Wardhan et al. 2012). As JA and SA are essential for pathogen- and wound-signaling and reported to mimic patho-stress response in plants (Clarke et al. 2000), CaTLP1 possibly has little or no role in patho-stress signaling. Moreover, overexpression of CaTLP1 conferred the transgenic tobacco plants increased root and leaf development, net photosynthesis, plant biomass, and improved tolerance 34
ACCEPTED MANUSCRIPT to dehydration, salinity and oxidative stresses. Recently, a study reported that the gene MdTLP7 of apple tree was up-regulated 512-fold in the leaves under the cold stress (Du et al. 2015). Transformation of MdTLP7 in E. coli could improve the tolerance to
PT
NaCl, KCl, chilling and heat shock in bacteria. Upon analysis of deletion
RI
of MdTLP7 amino acids, the 120–310 residues which form the 8 β strands
SC
of β barrel of the tubby domain were suggested to play crucial roles in stress tolerance (Du et al. 2014). To investigate the potential functions of
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MdTLPs under abiotic stress conditions, apple seedlings were exposed to
MA
various stresses including PEG, H2O2, exogenous ABA, and cold stress (Xu et al. 2016). Under PEG treatment, six MdTLPs (MdTLP1-5, 9) were
D
up-regulated significantly, while the remaining MdTLPs (MdTLP6, 7, 8)
PT E
were down-regulated in leaves. However, five MdTLPs (MdTLP3, 4, 7-9) were up-regulated significantly by PEG treatment in roots. In response to
CE
H2O2 or ABA treatment, MdTLP1-5 were significantly up-regulated in
AC
leaves, while the expression of MdTLP4, 6, 8, 9 showed a significant increase in roots. Under cold stress, almost all MdTLPs showed significantly up-regulated transcript levels in roots and leaves. Especially, the expression of MdTLP4 had significant changes in response to all the four stresses (PEG, H2O2, exogenous ABA, and cold stress treatment), suggesting MdTLP4 perhaps is the most important gene for stresses tolerance in the TLPs family of Malus domestica. 35
ACCEPTED MANUSCRIPT Soil salinity can aggravate the hyper-osmotic and hyper-ionic conditions, leading to considerable reduction of food production. From the microarray data in the NCBI database, TUBBY and ERF1 were identified as candidates for the response to salinity in Medicago (M.)
PT
truncatula. Furthermore, salinity could increase the level of expression of
RI
TLP gene in roots and leaves in M. truncatula , M. polymorpha and M.
stresses tolerance in different plants.
SC
laciniata (Gharaghani et al. 2015), suggesting the universal function of
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Four tubby-like genes have been identified in wheat and named as
MA
TaTULP1-4 (Hong et al. 2015). Gene expression of TaTULP1, TaTULP3 and TaTULP4 could be induced by salt stress, cold stress and MeJA
D
treatments, whereas the TaTULP2 transcript was repressed by these
PT E
treatments. In addition, transcripts of all TaTULPs except TaTULP3 were also induced by ABA treatment.
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Analysis of the 15 ZmTLPs’ promoters cis-element showed that all
AC
ZmTLP genes contain a putative ABA responsive element (ABRE), low dehydration-responsive element (DRE), and temperature-responsive element (LTRE) (Yulong et al. 2015). Consistent with this prediction, the transcription of ZmTLP3, 4, 5, 6, 8, 9, and 12 were up-regulated by ABA treatment; and the transcription of ZmTLP3, 5, 8, 9, and 12 were induced greatly under heat treatment; the expression of ZmTLP2, 3, 4, and 11 were induced by PEG treatment; however, only the alteration of 36
ACCEPTED MANUSCRIPT ZmTLP8’s expression was observed under NaCl treatment (Yulong et al. 2015).
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5. Concluding remarks
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The tubby-like genes are regarded as putative TFs by structural-based
SC
functional analysis. But they usually locate in PM instead of the nucleus, as the tubby domain can bind PtdIns(4,5)P2. Even so, the tubby-like
NU
proteins can translocate into the nucleus in some conditions, such as
MA
G-proteins signaling, insulin- or leptin-induced phosphorylation and stress responses. Among them, the two process which are G-proteins
D
signaling and insulin/leptin-induced phosphorylation rely on PLCβ
PT E
mediated hydrolysis of PtdIns(4,5)P2. But whether stress responses induced nucleus translocation also depend on PLCβ mediated hydrolysis
CE
of PtdIns(4,5)P2 is not yet clear. Furthermore, more studies are needed to
AC
explore what functions execute by those the tubby-like genes after translocating into nucleus. The functions of animal tubby-like genes have been well studied. They involve in trafficking proteins into cilia and are essential for ciliary function including energy balance, development, photoreception and hearing. Furthermore, they have functions in endocytosis, phagocytosis, keeping synapse maintenance or architecture, repressing Shh signaling. 37
ACCEPTED MANUSCRIPT Although the phenotype defects of these genes mutants have been well described, the mechanism of how they function is still elusive. Future studies should not lose sight of the mechanism of how they function, as it will aid help for therapies for retinal defects, hearing loss, obesity in these
PT
genes mutations.
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Even though the plant tubby-like genes have more members than
SC
animals, their functions have not been well elucidated. Maybe the functions of animal tubby-like genes can give us enlightenments. Until
NU
now, the plant tubby-like proteins only have been identified involving in
MA
multifarious stress responses and male gametophyte development. Further studies are needed to clarify the functions of these tubby-like genes in
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D
plants.
Conflicts of interest
AC
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The authors declare that they have no conflicts of interest.
Acknowledgements This work was supported by the National Natural Science Foundation of China (3147029, 31670302 and 2015BAD15B03) and the Elite Youth Program of CAAS (to YK).
38
ACCEPTED MANUSCRIPT References Allen, H.L., Estrada, K., Lettre, G., Berndt, S.I., Weedon, M.N. et al. 2010. Hundreds of variants clustered in genomic loci and biological pathways affect human height. Nature 467(7317): 832-838.
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molecular evolutionary analysis of tubby-like protein family in Arabidopsis, rice, and poplar.
Yoon, E.J., Jeong, Y.T., Lee, J.E., Moon, S.J., Kim, C.H. 2017. Tubby domain superfamily protein is
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required for the formation of the 7S SNARE complex in Drosophila. Biochem Biophys Res
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Commun 482(4): 814-820. Yulong, C., Wei, D., Baoming, S., Yang, Z., Qing, M. 2015. Genome-wide identification and comparative analysis of the TUBBY-like protein gene family in maize. Genes Genom 38(1): 25-36. Zhang, X.M., Ramalho-Santos, M., McMahon, A.P. 2001. Smoothened mutants reveal redundant roles for shh and ihh signaling including regulation of L/R asymmetry by the mouse node. Cell 105(6): 781-792. 52
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Zhao, D., Ni, W., Feng, B., Han, T., Petrasek, M.G. et al. 2003. Members of the Arabidopsis-SKP1-like gene family exhibit a variety of expression patterns and may play diverse roles in Arabidopsis.
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Plant Physiol 133(1): 203-217.
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ACCEPTED MANUSCRIPT Figure legends Fig. 1: Schematic model of how Tubby-like proteins binding to PM and detaching from PM to nucleus. (1) The conserved residues K (lysine) and R (Arginine) of tubby domain are charging
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for binding PtdIns(4,5)P2 in the PM. They can form a stable but reversible
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tubby-PtdIns(4,5)P2 complex. (2) Through the G-protein signaling, the tubby-like
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proteins can translocate into nucleus from PM. At first, the conserved residues K and R of tubby domain bind to PtdIns(4,5)P2 which is localized to PM. Also, Tubby-like
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proteins can associate with Gαq to form tubby-Gαq complex. Once the Gαq activated
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by GPCRs, the activated Gαq* can be released from the receptor and activate PLCβ. PLCβ then hydrolyzes PtdIns(4,5)P2 into InsP3, so the tubby-PtdIns(4,5)P2 complex
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can be disrupted and result in the translocation of Tubby-like proteins into the nucleus.
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(3) Phosphorylated Tubby-like proteins can translocate into nucleus. Insulin can utilize tyrosine kinase IRK and JAK2 to induce the phosphorylation of TUB Y464
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directly. And the translocation of phosphorylated Tubby-like proteins are also
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associated with the hydrolysis of PtdIns(4,5)P2 upon PLCβ. (4) In some stress conditions such as mannitol treatment, salinity and dehydration, the plants TLPs can also translocate into nucleus from PM.
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Table 1: The gene function annotation of tubby-like family members derived from animals and plants Gene Name
Function Annotation
Mice
TUB,TULP3
trafficking GPCRs into cilium in brains neural tubes keep energy balance
Mice
endocytosis
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TULP1
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Mice,Human TUB
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ciliary trafficking GPCRs in photoreceptors, keep synapse maintenance TUB,TULP1
or architecture, stimulate phagocytosis
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Mice
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repress mouse sonic hedgehog signaling, a transcriptional master regulator TULP3
in pancreatic ductal adenocarcinoma
Human
TULP1
can cause arRP, RD, LCA, JRP and RCD eye diseases
Drosophila
dTULP
regulate TRP channels localization in cilia
Drosophila
TUSP
Arabidopsis
AtTLP2, AtTLP6, AtTLP7
involving in male gametophyte development
Arabidopsis
AtTLP3, AtTLP9
involving in ABA stress responses
Rice
OsTLPs
Chickpea
CaTLP1
Apple tree
MdTLPs
involving in PEG, H2O2, exogenous ABA, and cold stresses responses
Wheat
TaTULPs
involving in cold, salinity, MeJA, and ABA stresses responses
Maize
ZmTLPs
involving in high temperature, salinity and ABA stresses responses
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Mice
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regulate the formation of the 7S SNARE complex
involving in defence responses against pathogen invasion involving in dehydration, salinity, ABA and oxidative stresses responses
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ACCEPTED MANUSCRIPT Highlights:
The first time to elaborate functions of tubby-like genes in animals and plants.
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The tubby-like proteins exhibit versatile functions in animal and plants.
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Three detailed ways of tubby-like proteins moving into nucleus were declarative.
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