Diverse structures, functions and uses of FK506 binding proteins

Accepted Manuscript Diverse structures, functions and uses of FK506 binding proteins

Julia Maeve Bonner, Gabrielle L. Boulianne PII: DOI: Reference:

S0898-6568(17)30171-7 doi: 10.1016/j.cellsig.2017.06.013 CLS 8942

To appear in:

Cellular Signalling

Received date: Revised date: Accepted date:

17 March 2017 15 June 2017 20 June 2017

Please cite this article as: Julia Maeve Bonner, Gabrielle L. Boulianne , Diverse structures, functions and uses of FK506 binding proteins, Cellular Signalling (2017), doi: 10.1016/ j.cellsig.2017.06.013

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ACCEPTED MANUSCRIPT Diverse structures, functions and uses of FK506 binding proteins

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Julia Maeve Bonner1,2 & Gabrielle L. Boulianne1,2,*

1. The Hospital for Sick Children, Program in Developmental and Stem Cell Biology,

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Peter Gilgan Center for Research and Learning, 686 Bay Street, Toronto, Ontario

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M5G 0A4, Canada

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M5G0A4, Canada

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2. Department of Molecular Genetics, University of Toronto, Toronto, Ontario

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*Correspondence: [email protected]

Key Words: FK506, FK506 binding protein (FKBP), immunophilin, peptidylprolyl cis/trans isomerase

ACCEPTED MANUSCRIPT Abstract FK506 (Tacrolimus), isolated from Streptomyces tsukubaenis is a powerful immunosuppressant shown to inhibit T cell activation. FK506 mediated immunosuppression requires the formation of a complex between FK506, a FK506 binding protein (FKBP) and

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calcineurin. Numerous FKBPs have been identified in a wide range of species, from single

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celled organisms to humans. FKBPs show peptidylprolyl cis/trans isomerase (PPIase) activity and have been shown to affect a wide range of cellular processes including protein

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folding, receptor signaling and apoptosis. FKBPs also affect numerous biological functions

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in addition to immunosuppression including regulation of cardiac function, neuronal function and development and have been implicated in several diseases including cardiac disease,

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cancer and neurodegenerative diseases such as Alzheimer’s disease. More recently, FKBPs have proven useful as molecular tools for studying protein interactions, localization and

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functions. This review provides an overview of the current state of knowledge of FKBPs and

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their numerous biological functions and uses.

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Contents 1. Introduction

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2. FKBP Structure and Localization

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3. Cellular Functions of FKBPs

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a. FKBPs in development

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b. Stress Response

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c. Cardiac Function

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e. Neuronal Function

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d. Cancer

4. FKBPs as molecular tools

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5. Concluding Remarks

Acknowledgements References

ACCEPTED MANUSCRIPT Introduction Immunophilins are a large family of predominantly cytosolic proteins characterized by their ability to bind to and mediate the effects of the immunosuppressant drugs cyclosporin A (CsA), FK506 or rapamycin [1]. The immunophilins are divided into sub-families based on

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the immunosuppressant they interact with. Immunophilins that preferentially interact with CsA are termed cyclophilins while immunophilins that interact with FK506 or rapamycin are

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termed FK506 binding proteins (FKBPs). Both cyclophilins and FKBPs form a binary

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complex with their respective immunosuppressants to inhibit T-cell activation and

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proliferation. The FKBPs are named for their affinity for FK506, a compound with immunosuppressant properties originally isolated from Streptomyces tsukubaensis [2-4].

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The first member of this family identified was FKBP12, which is a cytosolic protein able to bind to FK506 and rapamycin and mediate the immunosuppressant effects of these drugs [2,

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5 , 6]. FKBP12 consists of a 108-amino acid protein that comprises the core sequence of all

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FKBPs [4, 7]. Since the identification of the original FKBP12, numerous other family members have been identified. It was subsequently shown that FKBP12 belongs to a highly

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conserved family of proteins found in a wide range of organisms [4]. Early analysis in Saccharomyces cerevisiae suggested that FKBPs were dispensable for

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viability despite their high conservation and distribution across all organelles [8]. However, increasing evidence suggests these proteins have evolved to play multiple roles in multicellular models. Functional properties attributed to FKBPs include protein folding [911], chaperone activity [12-15], receptor signaling [16-18], protein trafficking [19], transcription [20], immunosuppression [2, 21-23], and apoptosis [24, 25]. Critical neurotrophic and neuroprotective roles have been documented for FKBPs [26-28], as well as roles in development [29] and the regulation of apoptosis via the interaction of FKBPs with

ACCEPTED MANUSCRIPT their binding partners [26, 28]. Moreover, FKBPs have been associated with a number of disorders and diseases, including cardiovascular disease [30-32], cancer [33-36], and neurodegeneration [37-41]. Together, these studies place FKBPs at central positions in multiple biological pathways and

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processes, and suggest critical roles in several oncogenic and neurodegenerative pathologies. This review aims to summarize the current state of knowledge regarding contribution of

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FKBPs to multicellular development and disease, and discuss the future of FKBP research,

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including potential application to the development of novel therapeutics.

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FKBP structure and subcellular localization

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FKBPs are members of a superfamily of proteins with peptidylprolyl cis/trans isomerase (PPIase) activity [1, 28]. FKBP12 is the archetypical FKBP and is comprised of a single FK506-like binding domain (FKBP_C) that confers PPIase activity [2, 5, 7, 42]. The ligand

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bound and unbound forms of FKBP12 are composed of a 5-stranded -sheet that wraps around an -helix with an overall conical shape and a hydrophobic cleft that binds to ligands

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and PPIase substrates [43-46]. Larger multi-domain FKBPs consist of one to four FKBP_C motifs and one or more functionally independent motifs such as calmodulin-binding (CaM),

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Ca2+ binding EF hand, tetricapeptide repeat (TPR), and DNA binding motifs [4, 26, 47-49]. The variability in domain organization of FKBPs suggests that FKBPs have evolved to fill changing roles within evolving organisms [50]. While FKBPs are not fundamentally required in a single cell context [8], valuable structural and functional knowledge of the family has been obtained using these models. For example, structural analysis of the FKBPs SlyD and trigger factor from Escherichia coli indicated that while PPIase catalytic activity was carried out by the FKBP_C domain, additional chaperone domains that folded

ACCEPTED MANUSCRIPT independently conferred much higher catalysis efficiency [51-53]. It has been shown that FKBP catalysis, unlike that of cyclophilins, exhibits a high level of sequence specificity for the target [6]. Accessory chaperone domains in some FKBPs however, may be able to overcome this inherently high specificity, allowing broader target recognition [54]. Analysis of protein complexes in S. cerevisiae, on the other hand, has suggested that FKBPs, including

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those lacking accessory domains, interact with distinct sets of proteins [55, 56] and have

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seemingly discrete functions [57].

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Like the domain structure of FKBPs, the subcellular localization of FKBPs is highly variable. The monodomain archetype FKBP12 is localized to the cytosol [2, 5]. However, additional

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motifs in larger FKBPs have a range of effects on the localization of these proteins. For

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example, several FKBPs have ER retention signals and show strong ER localization [15, 29, 58-63]. Some FKBPs show nuclear localization [64-69] while other FKBPs translocate

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between subcellular compartments such as the ER and mitochondria [24, 70-72].

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A summary of the domain organization and subcellular localization of human FKBPs is shown in figure 1. The variability in both domain organization and subcellular localization

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contributes to a wide functional variability amongst FKBPs by predisposing the FKBPs to interact with different molecular pathways in the cell. FKBPs have been shown to affect a

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diverse range of cellular functions including protein folding [9-11], chaperone activity [1215], receptor signaling [16-18], protein trafficking [19], transcription [20] and apoptosis [24, 25]. In the subsequent sections, we will summarize the functions of several key FKBPs, with attention to their respective roles in various diseases.

Cellular functions of FKBPs Despite the range of cellular functions that FKBPs are involved in, deletion of all FKBPs as well as all members of another major immunophilin family, cyclophilins in Saccharomyces

ACCEPTED MANUSCRIPT cerevisiae is viable [8]. Both families of proteins are highly conserved in yeast and distributed across all organelles. Interestingly the phenotype of the duodecuple mutant (4 FKBPs and 8 cyclophilins) was comparable to the sum of the phenotypes of the twelve individual mutants suggesting that each immunophilin interacts with a unique set of partners

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to perform specific functions [8]. Receptor signaling

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The cytosolic FKBP12 has been shown to interact with numerous receptor signalling

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pathways in different tissues. FKBP12 was originally identified as the target and mediator of

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the immunosuppressant drugs FK506 and rapamycin [2,5,6]. The binary complex of FKBP12 and FK506 interacts with calcineurin (CaN), a Ca2+ dependent Serine/Threonine

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phosphatase [22, 73] while the FKBP12/rapamycin complex interacts with the target of rapamycin (TOR) [74-76]. CaN dephosphorylates the nuclear factor of activated T-Cells

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(NFAT) leading to nuclear translocation of the transcription factor and initiation of a

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transcription cascade that leads to T-cell activation [77]. Interaction between FKBP12/FK506 and CaN prevents dephosphorylation of NFAT and blocks T-cell activation

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[77]. Interestingly, FKBP12 is not the only FKBP that interacts with CaN. FKBP51 inhibits calcineurin and blocks transcription of Nuclear Factor of Activated T Cells (NFAT) [78, 79].

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FKBP12 also mediates the immunosuppressant actions of rapamycin, through the TOR signaling pathway [74-76]. TOR is a serine/Threonine kinase that interacts with many proteins to form two distinct complexes, TOR complex 1 (TORC1) and TORC2 with different upstream effectors and downstream targets [80]. TOR phosphorylates ribosomal protein 6 kinase (S6K) and eukaryotic initiation factor 4E binding protein 1 (4EBP1) promoting cell growth and proliferation [81]. The FKBP12-rapamycin binary complex inhibits the kinase activity of TOR blocking cell growth and proliferation [74-76, 82, 83].

ACCEPTED MANUSCRIPT TOR activity is also inhibited by FKBP38, [84, 85], however, while inhibition of TOR by FKBP12 requires rapamycin, FKBP38 appears to be an inherent inhibitor of TOR and antagonizes TOR function in the absence of rapamycin [85]. The interaction between TOR and FKBP38 is antagonized by the small GTPase Rheb, which interacts with FKBP38 preventing it from binding to TOR [85, 86]. The interaction between Rheb and FKBP38 is

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regulated by the availability of growth factors and nutrients such that in the presence of

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growth factors, Rheb prevents the inhibition of TOR by FKBP38 [85].

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FKBP12 has also been shown to interact with membrane associated receptors including the ryanodine receptor (RyR) and the inositol 1,4,5 triphosphate receptor (IP3R) [42]. Upon

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binding their respective ligands, the RyR and IP3R mediate calcium release from the ER or sarcoplasmic reticulum [87-90]. FKBP12 and its paralogue FKPB12.6 bind and modulate the

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gating properties of RyR receptors [91-93], which play crucial roles in Ca2+ regulation and signalling in many major physiological processes. The interaction between FKBP12 and

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RyR receptors is inhibited by FK506 or rapamycin and dissociation of FKBP12 from the RyR

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results in ‘leaky’ channels, depletion of ER calcium stores and a disruption of calcium signaling [87, 90, 94]. FKBP12 has a similar effect on IP3R such that dissociation of

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FKBP12 from IP3R in the presence of FK506 or rapamycin leads to ‘leaky’ receptors and

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depletion of ER calcium stores [16, 91]. CaN has been found in complexes with both FKBP12-RyR and FKBP12-IP3R complexes and may contribute to the FKBP12-dependenat regulation of calcium signaling by modulating the phosphorylation status of the receptors [16, 95]. Other signaling pathways that are modulated by FKBPs include the transforming growth factor TGF-signaling pathway and the sonic hedgehog pathway. FKBP12 binds directly to type I receptors for TGF- (TGF-R1) and blocks their phosphorylation by TGF-

ACCEPTED MANUSCRIPT R2 trapping the receptors in the inactive state [17, 18] and FKBP38 has been shown to be a negative regulator of sonic hedgehog signaling [96, 97]. Protein folding and trafficking Several FKBPs have been shown to act as chaperones that can regulate both protein folding

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and trafficking. A key feature of FKBPs and other immunophilins is their PPIase activity. PPIase domains catalyze the cis/trans isomerization of X-pro bonds, which is a rate limiting

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step in protein folding [98, 99]. However, the chaperone function of FKBPs does not always

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require a functional PPIase domain. For example, the PPIase domain of Arabidopsis

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FKBP42 does not show any significant PPIase activity or affinity for FK506. FKBP42 however, has a strong chaperone activity [100 , 101]. There are several examples of FKBPs

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acting as chaperones including human FKBP38, which acts as a chaperone for the antiapoptotic protein Bcl-2 [13] and the mammalian FKBP51 and FKBP52, which act as co-

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chaperones with Heat shock protein 90 (HSP90) to regulate steroid hormone receptor activity

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[102-106]. Interestingly, FKBP52 and FKBP51 both have two FKBP_C domains, yet only the first domain shows any PPIase activity [107].

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In addition to chaperone activity, some FKBPs have been shown to play important roles in protein trafficking. For example, the HSP90/FKBP52 complex interacts with dynein-

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dynactin complex and with the glucocorticoid receptor and has been implicated in the retrograde movement of the steroid receptors [19, 67, 68, 108-110]. FKBP52 has also been shown to link the HSP90-human telomerase reverse transcriptase (hTERT) complex to the dynein-dynactin motor, promoting nuclear transport and telomerase activity [111]. Transcriptional control A central component of the immunosuppressive effects of FK506 is the CaN dependent inhibition of interleukin-2 (IL-2) gene expression. T-cell receptor (TCR) stimulation activates

ACCEPTED MANUSCRIPT the phosphatase CaN, which leads to nuclear translocation of the transcription factors nuclear factor of activate T cells (NFAT) and nuclear factor B (NFB) where they mediate the transcription of several lymphokine genes including IL-2 [112, 113]. Early studies on the mechanisms of FK506 mediated immunosuppression found that the FKBP12/FK506 binary complex inhibits the phosphatase activity of CaN thus preventing nuclear translocation of

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NFAT and NFB and transcription of IL-2 [22, 112, 114-118]. More recent work however,

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has shown that different FKBPs may have multiple effects on IL-2 transcription. For

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example, FKBP51 was shown to inhibit NFB transcriptional activity while the homologous FKBP52 has the opposite effect, strongly favoring NFB transcriptional activity [20].

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Interestingly, while the effects of FKBP52 on NFB transcriptional activity were blocked by

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FK506 and inactivation of the PPIase domain, FKBP51-mediated inhibition was not dependent on PPIase activity and was unaffected by FK506 [20].

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FKBP51 and FKBP52 have a similar antagonist effect on steroid receptor signaling. In classical steroid receptor signaling, steroid hormones enter through the plasma membrane and bind to a cytoplasmic receptor such as the glucocorticoid receptor (GR), and the

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hormone/receptor complex is translocated to the nucleus to mediate a transcriptional response

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[119]. As mentioned in the previous section, both FKBP51 and FKBP52 act as co-chaperones with HSP90 to regulate nuclear translocation of steroid hormone receptors [19, 67, 68, 109, 110]. Interestingly, loss of function studies on both FKBP51 and FKBP52 indicate that these homologous proteins have different and often opposing effects on steroid receptor signaling. Specifically, FKBP51 and FKBP52 differentially affect the binding of GR to dynein and nuclear translocation [19, 108].

ACCEPTED MANUSCRIPT In addition to the role of some FKBPs on translocation of specific transcription factors to the nucleus, there is also evidence of FKBPs acting directly within the nucleus to regulate transcription. For example, FKBP25 has a N-terminal hydrophilic helix-loop-helix (HLH) domain capable of binding DNA [65,120,121]. FKBP25 has also been shown to interact with several transcriptional regulators including histone deacetylases HDAC1 and HDAC2 [122],

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HDAC-binding transcriptional regulator [122], nucleolin [64,123] mouse double minute 2

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(MDM2), p53 [124] and Elongation factor 1 [120].

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Apoptosis

Apoptosis describes a distinct form of cell death characterized morphologically by nuclear

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fragmentation and condensation and plasma membrane blebbing leading to the formation of

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apoptotic bodies and biochemically by mitochondrial outer membrane permeabilization and activation of effector caspases 3, 6 and 7 [125, 126]. B-cell lymphoma 2 (Bcl-2) and the homologue Bcl-Xl are anti-apoptotic proteins that regulate the release of cytochrome C from

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the mitochondria, [127,128]. FKBP38 is localized to the outer mitochondrial membrane where it interacts with antiapoptotic protein Bcl-2 recruiting it to the mitochondrial membrane to inhibit apoptosis [24]. Suppression of FKBP38 function alters Bcl-2

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localization and increases apoptosis while overexpression of FKBP38 inhibits apoptosis by

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protecting Bcl-2 from caspase-dependent degradation, [24,129,130]. Interestingly, the interaction between FKBP38 and Bcl-2 is regulated by the small GTPase Rheb providing a link between nutrient status and apoptotic activity [131].

Physiological roles for FKBPs FKBPs contribute to a variety of physiological processes and have been associated with several disorders and diseases, including cardiovascular disease, cancer, and neurodegeneration, as well as complex developmental and behavioral disorders.

ACCEPTED MANUSCRIPT FKBPs in Development FKBPs have been shown to play numerous roles in development in a wide range of tissues and organisms. Double and triple mutants for the ER resident FKBPs fkb3, -4, and -5 in Caenorhabditis elegans, develop normally at permissive temperatures, but display cold-

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sensitive larval lethality at low temperatures, establishing that secretory pathway FKBPs are collectively essential for normal development in nematodes [132]. Similarly, mutations in the

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Arabidopsis thaliana FKBP PAS1 (closest human homologue FKBP51/52) lead to

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deregulated control of cell proliferation and an inability to survive under normal growth

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conditions [133, 134], indicating a role for PAS1 in plant cell regulation and development. Similarly, important roles have been demonstrated for multiple FKBPs in several other

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multicellular contexts. In Drosophila melanogaster, the FKBP shutdown (homologous to human FKBP6) was demonstrated to be essential for the normal function of germline stem

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cells and later stages of oogenesis [135,136]. The ER resident mammalian FKBP65 was

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shown in mouse and human tissue to regulate lung development and response to lung injury in a manner that suggests a distinct set of developmentally regulated protein ligands [137].

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Several groups have since identified the matrix collagens and tropoelastins as among these important targets of FKBP65 activity [138-140], suggesting a critical role in matrix protein

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maturation.

FKBPs have also been implicated in Notch signalling dependent developmental processes. Notch signalling is a key regulator of cell fate specification [141]. Analysis of FKBP12 in mice revealed that FKBP12 is a negative modulator of Notch signalling and that FKBP12 deletion in endothelial cells led to abnormal cardiac development [30]. Previous work from our lab identified another ER resident member of the FKBP family, FKBP14, as a genetic interactor with components of the Notch pathway [29, 142]. Specifically, we showed that

ACCEPTED MANUSCRIPT Presenilin-dependent -secretase cleavage, a process required for Notch signalling, is reduced in FKBP14 mutants, establishing that FKBP14 is required in multicellular development at least in part, as a regulator of Notch signalling via Presenilin and the -secretase complex [29].

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Stress response

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FKBPs play an important role in facilitating cellular and organismal responses to changes in

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environmental conditions. One particularly well studied example is the role of FKBP51 and FKBP52 in regulation of the HPA axis and stress induced changes in the activity of the HPA

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axis. The HPA axis is stimulated under conditions of stress, resulting in the release of corticotropin releasing hormone (CRH) from the hypothalamus, which stimulates release of

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adrenocorticotropic hormone (ACTH) from the pituitary gland, triggering corticosteroid release from the adrenal gland. Corticosteroids such as glucocorticoid and mineralocorticoid

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cross the blood-brain barrier to act on their respective receptors in different regions of the

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brain [143, 144]. Corticosteroids acting on the glucocorticoid receptor (GR) form a negative feedback loop for the HPA axis and defects in GR signaling may contribute to altered

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reactivity of the HPA axis [143]. Changes in HPA axis activity affect energy storage and output, emotional state and immune function. Inadequate, excessive or prolonged activation

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of the HPA axis is associated with risk of major depression disorders (MDD), bipolar disorder, post-traumatic stress disorder (PTSD), schizophrenia and anxiety disorders [144]. As discussed earlier, FKBP51 and FKBP52 are HSP90 co-chaperones that facilitate the nuclear translocation and transcriptional activity of steroid hormone receptors (SHR) including GR, [19, 67, 68, 109, 110]. FKBP52 and FKBP51 have antagonistic effects on SHR, with FKBP52 acting as a positive regulator of SHR activity [103, 104, 145] and FKBP51 acting as a negative regulator [103, 146].

ACCEPTED MANUSCRIPT Several studies in mice and humans indicate that the negative regulator FKBP51 plays a central role in stress adaptation. For example, expression of GR and FKBP51 in mice is altered following exposure to chronic stress [147]. Furthermore, while deletion of the FKBP5 gene (FKBP51) in mice did not show any phenotypes under basal, non-stressed conditions, FKBP5 null mice showed improved behavioral responses under a variety of

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different stresses [148-154] indicating a central role for FKBP51 in the regulation of the HPA

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axis activity.

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Polymorphisms in FKBP5 have also been associated with alterations in HPA axis activity in humans and a number of behavioral responses to stress [155]. For example, FKBP5

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polymorphisms have been associated with the onset and recurrence of depression and altered

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responses to anti-depressant treatment [156-158]. FKBP5 polymorphisms have also been associated with the severity and prognosis of peritraumatic stress dissociation and PTSD

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[159, 160, 161, 162, 163, 164, 165]. Genetic associations and post-mortem analysis have also

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revealed a link between FKBP51 and GR levels and vulnerability to suicide [166, 167]. Together, these studies demonstrate that stress disorder outcomes are highly sensitive to

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FKBP51 activity making FKBP51 inhibition an attractive therapeutic approach to stress management. A key requirement for the success of this approach however, is the

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identification of FKBP51 specific ligands. The ligand binding sites of FKBP51 and FKBP52 are highly homologous [168, 169] and inhibition of FKBP52 has many unwelcome consequences including infertility, increased susceptibility to high fat diets and increased sensitivity to certain stresses [104-106, 170, 171]. Recent work however, has identified many FKBP51 selective ligands [172, 173 , 174 , 175]. The SAFit2 ligand has been shown to mimic the effects of FKBP51 knockout mice on HPA axis regulation, reduce anxiety like

ACCEPTED MANUSCRIPT behaviour and demonstrates anti-depressant like properties [173, 174] suggesting that these ligands may be a promising treatment option for depression and other stress related disorders. Cardiac function Mammalian FKBP12 and its paralogue FKPB12.6 bind and modulate the gating properties of

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the calcium release channels inositol 1,4,5-trisphosphate (IP3) and Ryanodine (Ry) receptors

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[91-93], which play crucial roles in Ca2+ regulation and signalling in many major physiological processes. For instance, the cardiac ryanodine receptor (RyR2), is central to

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Ca2+ induced Ca2+ release from the sarcoplasmic reticulum (SR) during excitation-contraction

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in cardiac muscle [176-178]. FKBP12 can activate RyR2 channels and this activation is antagonized by FKBP12.6 [179], suggesting dual regulation by these FKBPs may be

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important to normal heart function.

Aberrant FKBP12.6-RyR2 receptor interactions have been proposed to be the underlying

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cause of channel dysfunction in acquired and inherited cardiac disease [92]. RyR2 is the channel required for excitation-contraction coupling in the heart, and binding of FKBP12.6 stabilizes and regulates channel function [179, 180] [181]. Defective regulation of RyR2 can

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result in the dissociation of FKBP12.6 from the complex [32]. In failing hearts, dissociation

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of FKBP12.6 from RyR2 leads to pathological consequences, including potentially triggering fatal cardiac arrhythmias [32]. FKBP12.6 null mice are more susceptible to cardiac arrhythmias [182, 183] while overexpression of FKBP12.6 in mouse cardiac myocytes prevents triggered ventricular tachycardia in normal hearts in stress conditions [31]. Cancer Several FKBPs have also been implicated in cancer etiology and chemoresistance, notably the heat shock protein (HSP) 90 co-chaperones FKBP51 and FKBP52. In prostate cancer cell

ACCEPTED MANUSCRIPT lines, increased levels of FKBP51 and FKBP52 were observed, along with an inhibitory effect of FK506 on androgen-dependent cell growth [184]. Several studies have shown marked changes in the expression levels of FKBP51 and FKBP52 in certain cancers including breast cancer, hepatocellular cancer, oral Squamous cell carcinoma and oesophageal adenocarcinoma indicating that these genes may be used as biomarkers for the

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of cancer cells to chemotherapy [34, 188] and radiation [189].

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prognosis of these cancers [33, 36, 185-187]. FKBP51 was also found to affect the response

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The effects of FKBP51 and FKBP52 on the prognosis of different cancers is mediated through a variety of interaction partners. For example, FKBP52 has been shown to act

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synergistically to regulate androgen receptor signalling in prostate cancer and polycystic

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ovary syndrome [190, 191]. FKBP51 regulates cell motility and invasion in melanomas through an interaction with the Rho-GTPase activating proteins deleted in liver cancer 1

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(DLC1) and DLC2 [192]. FKBP51 also promotes melanoma cell migration by increasing

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TGF- signaling and inhibits apoptosis through the activation of NFB in melanoma cells [189, 193, 194] .

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FKBP38 has also been implicated in cancer progression. FKBP38 interacts with the antiapoptotic protein Bcl-2 and protects it from degradation [24, 129] and suppression of

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FKBP38 destabilizes Bcl-2 and contributes to tumorigenesis and chemoresistance [13, 129]. FKBP38 may also contribute to cancer development through regulation of the TOR pathway. As previously mentioned, the TOR pathway controls cell growth and proliferation [85]. Bcl2 has been shown to be a positive regulator of TOR signaling and the interaction between the two pathways is mediated by FKBP38 [195] .

ACCEPTED MANUSCRIPT Neuronal function Roles in neurodegeneration and recovery have been suggested for several FKBPs. FKBPs are enriched in neuronal tissue, and their expression is elevated after nerve injury [28]. Furthermore, FK506 has been shown to have neuroprotective effects and induce neuronal

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regeneration [69, 196-199], suggesting significant roles for FKBPs in neural growth, injury, and regeneration. Neurotrophic effects were also observed in FKBP12 deficient neurons

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suggesting that other FKBPs may be involved [200]. Studies with target specific FKBP

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inhibitors have shown the inhibition of FKBP38 in a rat model of cerebral ischemia led to reduced neural damage and increased neuroregeneration [201] while inhibition of FKBP51

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enhanced neurite outgrowth in cultured neurons [173]. Interestingly, recent evidence has

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shown that FK506 binds the V-ATPase catalytic subunit A and induces autophagy in neuronal cells, suggesting that at least some of the neuroprotective effects of FK506 may be

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mediated independently of FKBPs [202].

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FKBPs have also been implicated in neurodegenerative diseases such as Alzheimer’s Disease (AD) and Parkinson’s Disease (PD). Expression of FKBP12 is increased in the brains of

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patients with Parkinson’s Disease (PD), Alzheimer’s Disease (AD), and dementia with Lewy bodies, colocalizing with -synuclein [37] and neurofibrillary tangles [203]. FKBP12 also

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binds the intracellular domain of amyloid precursor protein (APP) and this interaction can be disrupted by FK506 [39]. Moreover, FKBP12 regulates the localization and processing of APP [40], potentially contributing to AD pathology. FKBP51 and FKBP52 expression has also been associated with AD progression. For example, higher levels of FKBP51 expression were shown to promote neurotoxic tau accumulation [204]. FKBP52 binds specifically to tau in rat brains via the FK binding domain, inhibiting the production of tau microtubules which further contributes to a neuroprotective role [38, 205]. In Drosophila, FKBP52 also binds

ACCEPTED MANUSCRIPT APP and overexpression reduces the toxicity associated with transgenic expression of A fragments [41]. The macromolecular complex formed between Presenilins, FKBP38 and Bcl2 has also been suggested to play a role in neurodegeneration. Mutations in PS associated with familial AD enhance the pro-apoptotic activity of FKBP38-mediated mitochondrial

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targeting of Bcl-2, potentially contributing to familial AD pathology [25]. As discussed earlier, FKBP51 and FKBP52 play a role in regulating the HPA axis and the

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stress response and polymorphisms in FKBP51 have been associated with a number of

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neurological conditions including depression [156-158], PTSD [159, 160, 161, 162, 163, 164, 165], increased vulnerability to suicide [166, 167] and addiction [206-208]. FKBPs have also

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been associated with clinically and genetically heterogeneous diseases such as Ehlers-Danlos

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Syndrome (EDS). An autosomal recessive extreme variant of this disorder, which shares features with congenital muscular dystrophy, has been associated with a frameshift in

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FKBP14 [11]. Individuals with FKBP14-associated EDS show abnormal distribution and

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assembly of extracellular matrix components, especially type I and III collagens and fibronectin [11], suggesting that like FKBP65, FKBP14 may play a role in the maturation of extracellular matrix components. It is unknown whether the phenotype of EDS is related to

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the PS and -secretase associated defects described in FKBP14 mutant Drosophila [29].

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FKBPs as Molecular Tools Finally, FKBPs have recently emerged as novel molecular tools. Banaszynski and colleagues developed a rapid, reversible method to regulate the stability of specific proteins in mammalian cells by taking advantage of FKBP12 mutants that are rapidly and constitutively degraded (Figure 2) [209]. Fusing these “destabilization domains” to proteins of interest targets them for degradation. However, in the presence of a synthetic ligand (Sheild1), the

ACCEPTED MANUSCRIPT proteins are ‘shielded’ from degradation [209]. This method has been successfully adapted in multiple systems [210-214]. FKBPs have also been developed as a tool for studying kinase dependent signalling events. The catalytic domain of a protein kinase of interest can be inactivated by inserting a truncated fragment of human FKBP12 (aa 22-108) into the catalytic domain of the kinase [215]. The

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catalytic activity of the kinase can then be restored upon addition of rapamycin or analogues

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of rapamycin. This tool was shown to allow precise temporal control of kinase activity [215]

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and was used to study the role of Src signalling in prostate cancer [216].

The rapamycin dependent interaction between FKBP12 and TOR has also been adapted into

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a useful and widespread tool for studying protein interactions and movements. In this system, the FKBP binding domain (FKBD) of FKBP12 is genetically fused to one protein of

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interest (POI), and the FKBP-rapamycin binding domain of TOR (FRB) is fused to a second

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POI. In the presence of rapamycin or an analogue of rapamycin, the FKBD and FRB domains interact bringing the two POIs together [reviewed in 217]. Applications of this

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technique range from monitoring protein-protein interactions or localization, regulation of gene expression or protein function [218], induced translocation [219-221], and precise

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modulation of cell signalling. This approach has also been used to examine the molecular machinery that regulates vesicle movement. By tagging candidate proteins with the FRB and specific motor proteins with the FKBD, several studies have shown that different trafficking proteins associate with specific vesicle populations and regulate specific directions of vesicle movement [222-224]. Inducible FRB-FKBP heterodimerization has also been used to specifically target proteins of interest to specific regions of the cell and to inactivate proteins by sequestering them away

ACCEPTED MANUSCRIPT from their normal site of action. For example, sequestering nuclear proteins in the cytoplasm prevents nuclear signalling [225, 226], while targeting clathrin coated vesicle proteins to the mitochondria blocks endocytosis [221]. A recent study used heterodimerization of FKBP12FRB to mediate nuclear localization of Bcr-Abl in chronic myeloid leukemia (CML) cells leading to apoptosis of the CML cells, providing a potentially new therapy for treatment of

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Tyrosine Kinase Inhibitor resistant CML [227].

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The FKB-FRB dimerization system can also be used to study and regulate cell signalling

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[228-230]. One recent study used FKB-FRB dimerization to create a fluorescent marker for manipulating cholesterol in mammalian cells [231]. Another recent study used the system to

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develop a tool for modulating G-protein coupled receptor signalling at specific subcellular

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locations and demonstrated that localization of the  subunit to different subcellular locations affects apical versus basolateral cargo sorting from the Golgi [232]. The system has also been used to rapidly and specifically label cell surface proteins with recombinant FKBP-

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fused fluorescent proteins. This method offers advantages over antibody labeling, including high specificity and smaller size of FKBP-fused fluorescent proteins [233].

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Concluding Remarks

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FKBPs are a diverse family of proteins that play important roles in numerous cellular processes including protein folding and trafficking, chaperone activity and affect a wide range of biological functions including immune function, cardiac function and neural development. Given the wide range processes affected by FKBPs, it is not surprising that they have been implicated in several diseases including heart disease, neurodegeneration and cancer. FKBPs have also been used as molecular tools to reveal detailed aspects of protein function. With these advances in our understanding of the diverse roles of FKBPs and their

ACCEPTED MANUSCRIPT utility as molecular tools, FKBPs are garnering a lot of interest as potential therapeutic targets.

Acknowledgements

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We thank Dr. D. Knight for helpful discussions and critical comments on the manuscript.

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This work was supported by grants to G.L.B. from the Canadian Institutes of Health Research

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(PJT153063) and the Natural Sciences and Engineering Council of Canada (RGPIN 0522914). G.L.B. is the recipient of a Tier 1 Canada Research Chair in Molecular and

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Developmental Neurobiology.

ACCEPTED MANUSCRIPT Figure Legends

Figure 1: Schematic of human FKBPs

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Schematic of the domains and localizations of the human FKBP protein repertoire. The

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smallest members of the family (FKBP12s) are monodomain proteins consisting of a single

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FK506-like binding domain (FKBP_C), where larger multidomain FKBPs consist of one to four FKBP_Cs together with diverse motifs such as EF hand and Ca 2+ binding,

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tetricapeptide repeat (TPR), and DNA binding motifs. Major regions of each FKBP are shown. FKBPs have been identified in several subcellular compartments and organelles,

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including cytoplasm, ER, nucleus and mitochondria. Specific retention signals are indicated

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for the ER resident FKBPs. Where known, associated disease conditions are listed.

Figure 2: FKBPs as molecular tools

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A. A FKBP degradation domain (DD) is genetically fused to a protein of interest (POI).

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Shld1 protects the POI from DD mediated proteolysis. B. The DD of FKBP is genetically fused to the PiggyBac transposon induction system (PBase). Addition of the ligand (Shld1) protects the DD-PBase fusion from proteolysis and mediates gene transcription. In the absence of ligand, the DD-PBase is constitutively degraded. C. One POI is linked to the ligand binding domain of FKBP12 (FKBD) and a second POI is linked to the FKBPrapamycin binding domain of TOR (FRB). In the presence of rapamycin or a rapamycin analog, the FKBD and FRB bind each other bringing the two POIs together. The FKBD and FRB may also be tagged with fluorescent markers (stars) to visualize localization.

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Fig. 2

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FKBPs affect a wide range of cellular processes

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FKBPs play a key role in development and are implicated in several diseases

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FKBPs can be used as molecular tools to study protein function

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