The START-domain proteins in intracellular lipid transport and beyond

Journal Pre-proof The START-domain proteins in intracellular lipid transport and beyond Barbara J. Clark PII:

S0303-7207(20)30004-6

DOI:

https://doi.org/10.1016/j.mce.2020.110704

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MCE 110704

To appear in:

Molecular and Cellular Endocrinology

Received Date: 4 October 2019 Revised Date:

8 January 2020

Accepted Date: 8 January 2020

Please cite this article as: Clark, B.J., The START-domain proteins in intracellular lipid transport and beyond, Molecular and Cellular Endocrinology (2020), doi: https://doi.org/10.1016/j.mce.2020.110704. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

The START-domain proteins in intracellular lipid transport and beyond.

Barbara J. Clark, Ph.D. Department of Biochemistry & Molecular Genetics, University of Louisville School of Medicine, Louisville, KY 40292 [email protected] Short title: The START domain lipid shuttle

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Abstract The Steroidogenic Acute Regulatory Protein-related Lipid Transfer (START) domain is a ~210 amino acid sequence that folds into an α/β helix-grip structure forming a hydrophobic pocket for lipid binding. The helix-grip fold structure defines a large superfamily of proteins, and this review focuses on the mammalian START domain family members that include single START domain proteins with identified ligands, and larger multi-domain proteins that may have novel roles in metabolism. Much of our understanding of the mammalian START domain proteins in lipid transport and changes in metabolism has advanced through studies using knockout mouse models, although for some of these proteins the identity and/or physiological role of ligand binding remains unknown. The findings that helped define START domain lipidbinding specificity, lipid transport, and changes in metabolism are presented to highlight that fundamental questions remain regarding the biological function(s) for START domain-containing proteins. Keywords: START domain, lipid trafficking, metabolism

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1. Introduction 1.1

Non-vesicular lipid trafficking Lipid transport proteins play an important role in non-vesicular trafficking of cholesterol,

phospholipids, and sphingolipids between biological membranes to help maintain the proper cholesterol:phospholipid:sphingolipid distribution [reviewed in [1, 2]].

Cholesterol content is

maintained at relatively low levels within the endoplasmic reticulum (ER) and mitochondrial membranes compared to the plasma membrane (PM) [3]. The source of PM cholesterol is from both de novo synthesis in the ER and cellular uptake of low density lipoprotein (LDL)-derived cholesterol. De novo synthesized cholesterol is rapidly transferred to the PM by non-vesicular trafficking mechanism(s), implicating a role for soluble sterol transport proteins [4]. Receptormediated endocytosis of LDL delivers lipoproteins to the late endosomes/lysosomes where free cholesterol is hydrolyzed from cholesterol esters [5]. the PM or transported to the ER.

The free cholesterol is recycled back to

In the PM, cholesterol can be found clustered with

sphingolipids into detergent-resistant protein-lipid microdomains referred to as lipid rafts [6-8]. Functionally, lipid rafts are proposed to provide an organized membrane region for signaling and other functions [9]. Thus, maintaining proper cholesterol distribution within the cell is important for cholesterol homeostasis and membrane function [4, 10]. Similarly, phospholipid transfer from sites of synthesis to organelle membranes is required for cell growth and signaling. Examples where START proteins, as well as other lipid transport proteins, play an important role in lipid trafficking include: 1) modulating ER membrane free cholesterol levels, 2) transport of cholesterol to mitochondria for production of steroid hormones and bile acids, 3) transfer of sphingolipids from the ER to the Golgi membrane, and 4) transfer of phospholipids from the ER to the PM and mitochondria for cell growth and signaling. There are two major lipid transport protein families: the oxysterol-binding protein (OSBP) family that includes the OSBP-related proteins (ORPs) (reviewed in [11]), and the START-domain family that is the focus of this review.

1.2.

Introduction to the START domain lipid transport protein family: The Steroidogenic Acute Regulatory Protein-related lipid transfer (START) domain is a

region of ~210 amino acids that forms an α/β helix-grip fold with an antiparallel β-sheet structure that forms a U-shaped hydrophobic cleft flanked by amino- and carboxyl-terminal alpha helices [12]. [Figure 1]. (reviewed in [13-15]). The helix-grip fold structure defines a large superfamily of proteins that bind lipids. The START domain is conserved from plants to mammals [16] with sterols, phospholipids, bile acids, and ceramides identified as ligands. Lipid access to the

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binding pocket requires a conformational change involving movement of the C-terminal helix with specificity for lipid binding being driven by residues that form the hydrophobic binding cavity [17, 18]. Membrane interactions via the C-terminal alpha helix and movement of the omega-1 loop promote access to the lipid binding pocket. This superfamily has been referred to as the SRPBCC protein superfamily on NCBI’s Conserved Domain Database (cl14643 NCBI) [19], the START domain superfamily [12, 16], and most recently the StARkin superfamily to broaden the

Figure 1. hSTARD1 START Domain Structure. Ribbon diagram of the crystal structure for human STARD1 START domain reported by Thorsell et al. Image from the RCSB PDB (rcsb.org) of PDB ID 3P0L. N-terminus is shown in blue and C-terminus is shown in red.

family to include the lipid transfer proteins anchored at membrane contact sites (LAM) originally identified in yeast [20]. In brief, the LAM proteins are missed if protein sequence conservation is used to identify a START domain [21]. However, molecular modeling and crystal structures of LAM family identified START-like domains in both yeast and vertebrates [22, 23]. The yeast Lam proteins share common structural domains that include a single carboxy-terminal transmembrane domain that anchors these proteins in the ER, a central START-like domain(s) that bind sterols, and an amino-terminal pleckstrin homology (PH)-like domain (GRAM domain) that binds glycerophospholipids [23]. In mammals, the ASTER-A, -B, -C (Aster for star) proteins encoded by the GRAMD1a,-b,-c

genes are newly described cholesterol binding proteins

involved ER- PM trafficking that have a similar domain structure as the Lam family members [24]. The domain structure of the Lam and Aster proteins supports a mechanism for these proteins to bridge from their ER anchor point to plasma/golgi/mitochondria membrane via a phospholipid - GRAM domain interaction and shuttle sterols between the membranes through the START-like domain [reviewed in [25, 26]. This mechanism is similar to that described below for STARD11/CERT for ceramide transfer between the ER and Golgi.

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1.3

The mammalian START domain family The mammalian START domain family has 15 members divided into six subfamilies

based on sequence similarities, common ligand binding, and similar structures. Three subfamilies have established ligands: the cholesterol-specific binding and membrane targeted STARD1/D3 subfamily, the sterol-binding and soluble STARD4 subfamily (composed of STARD4/D5/D6), and the phospholipid/ceramide-binding STARD2 subfamily (composed of STARD2/D7/D10/D11) [Figure 2]. Coupling the START domain with other motifs is a common

Figure 2. The Mammalian START Domain Protein Family. Each START protein subfamily is grouped within an oval and each member of the subfamily is designated by START protein name followed by a common name, if used. The domain structures are depicted with the major lipid that bind indicated for the three subfamilies that have lipid transfer function (STARD1/3, STARD4/5/6, and STARD2/7/10/11). A ? indicates no ligand has been identified for members of the three subfamilies with enzymatic activity (STARD12/13/8, STARD14,15, and STARD9). Abbreviations: StAR, steroidogenic acute regulatory protein; START, StAR-related lipid transfer domain; PC-TP, phosphatidylcholine transfer protein; DLC, deleted in liver cancer; ss, signal sequence; MENTAL, MLN64-N terminal domain; PH, pleckstrin homology domain; FFAT, peptide EFFDAxE; SAM, sterile alpha domain; Hotdog, conserved domain structure for acyl-coenzyme A thioesterase family; KM, kinesin motor domain; FHA, forkhead associated phosphopeptide binding domain.

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theme as START domains in other phyla are found in multi-domain proteins that provide additional functions such as protein localization, enzymatic activity, or signaling [12, 16]. Members of the remaining three subfamilies are multi-domain proteins with possible enzymatic function and no ligand established for the START domain: the RhoGap STARD8/12/13 subfamily, the acyl-CoA thioesterase STARD14/15 subfamily, and the kinesin motor STARD9 subfamily [Figure 2]. Crystal structures for the START domains of human STARD1, STARD5, STARD2/PC-TP, STARD11/CERT, STARD13, STARD14, and murine STARD4 confirm the basic three-dimensional helix-grip fold structure is conserved across the five mammalian subfamilies [18, 27-31].

2.

Cholesterol transfer by mammalian START domain proteins.

2.1

STARD1 STARD1, STARD3, STARD4, STARD5, and STARD6 all have been reported to

influence cholesterol transport and homeostasis. The biological function of STARD1 (StAR hereafter) in regulating the rate-limiting step in steroidogenesis is well established and supported by extensive literature (reviewed in [32, 33]). In brief, the regulated and rate-limiting step in steroidogenesis is the delivery of cholesterol from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM) where the first enzymatic reaction occurs; the conversion of cholesterol to pregnenolone catalyzed by the mitochondrial cytochrome P450 cholesterol side-chain cleavage enzyme located in the IMM. The rapid or acute response of steroidogenic tissues to hormonal stimulation requires StAR synthesis for cholesterol transfer from

the

OMM

to

the

inner

mitochondrial

membrane

(IMM).

StAR,

the

Steroidogenic Acute Regulatory protein, was named for this function in regulating the acute step in hormone-stimulated steroid hormone biosynthesis [34]. StAR protein expression is highest in tissues with high de novo steroidogenic capacity: the three zones of the adrenal cortex, ovarian theca, granulosa, and corpora luteal cells, and testicular Leydig cells [35, 36]. StAR selectively binds cholesterol due to key residues that line the hydrophobic cleft of the START domain [reviewed in [14, 37]]. The initial studies characterizing StAR (known as the 30kDa protein at the time), established its localization to the mitochondria matrix [38-40]. Subsequent cloning and expression studies established that StAR’s structure is composed of a START domain with an amino-terminal classical mitochondrial targeting sequence [34]. Expression of the cDNA in steroidogenic cells or in vitro in the presence of isolated mitochondria followed by Western blot analysis confirmed StAR is synthesized as a larger precursor protein that is processed by mitochondria [34, 41, 42]. Key

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data that support StAR’s biological role in steroidogenesis are from clinical studies that identified mutations in the human STAR gene (STARD1) as the basis for the genetic disorder congenital lipoid adrenal hyperplasia (lipoid CAH). Patients with lipoid CAH have decreased to absent levels of steroid hormones and accumulation of cholesterol in lipid droplets in the steroidogenic cells [41, 43]. Knockout mice lacking StAR expression have a similar phenotype as the human disorder - cholesterol droplets accumulate in the adrenals and gonads and steroid hormone levels are non-detectable [44].

Thus, in the absence of StAR de novo steroid hormone

biosynthesis is blocked at the step of cholesterol trafficking from the OMM to the IMM. While StAR’s biological role in steroidogenesis is established, fundamental questions on the molecular mechanisms for StAR action remain.

StAR’s biological function is typically

measured as an increase in cholesterol metabolism to pregnenolone, which is a read-out for cholesterol transfer to the IMM and the cytochrome P450 cholesterol side-chain cleavage enzyme. Early work showed that expression of a cDNA lacking the N-terminal mitochondrial targeting sequence (N62StAR) was functional in stimulating steroid production in steroidogenic cells or heterologous COS-1 cells [45, 46]. In vitro studies confirmed that purified recombinant N62StAR added to isolated mitochondria was functional. These studies provided evidence that StAR localized to the OMM can promote cholesterol movement from the OMM to the IMM. The proposed cholesterol-binding function for StAR is to drive structural changes in StAR that initiate cholesterol transfer into the mitochondria [47-49]. The current model proposes specific proteinprotein interactions between StAR and the OMM initiate a channel opening for cholesterol transfer [32, 33, 50].

The challenge for the field is that several StAR-interacting complexes at

the OMM have been characterized [51-54], without a consistent complex assembly reported or independent validation. In addition, determining the molecular mechanisms of StAR action will required defining the role of StAR phosphorylation in the OMM protein interactions.

It is

established that S194 (mouse) or S195 (human) is phosphorylated by protein kinase A, and in vitro studies demonstrated that a StAR-S194A phosphomutant was inactive for cholesterol transfer [55-58]. Expression of the StAR-S194A transgene in StAR knockout mice was unable to restore glucocorticoid and testosterone levels and cholesterol droplets accumulated in the adrenal and testis while re-expression of the wild-type StAR protein restored steroid levels [59]. Phosphorylation does not appear to be required for cholesterol binding or StAR import and processing by mitochondria [55-57, 59, 60], yet studies are needed to test whether StAR-S194A phosphomutant disrupts interaction with any of the OMM proteins identified.

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2.2

STARD3 STARD3, also known as MLN64, was identified as an amplified transcript in breast

cancer-derived metastatic axillary lymph nodes (MLN) [61]. It is a multi-domain START protein with amino terminal MENTAL (MLN64 N-terminal) domain followed by a FFAT (diphenylalanine (FF)–acidic track (AT)) motif, and the carboxyl-terminal START domain. The MENTAL domain is composed of four transmembrane helices that localize STARD3 to the late endosomes and orients the START domain towards the cytoplasm. Proteins that contain a FFAT motif attach to the endoplasmic reticulum (ER) through protein-protein interactions with a vesicle-associated membrane protein-associated protein (VAP) [Figure 3]. Overexpression of STARD3 in HeLa cells followed by immunofluorescence demonstrated that mutation of the FFAT motif resulted in loss of STARD3 colocalization with the ER [62]. siRNA silencing of the VAP protein(s) in HeLa cells recapitulated the loss of STARD3 association with the ER. In situ proximity ligation assays to label endogenous STARD3 and VAP-A interactions revealed the possibility for these two proteins to interact and form a late endosome-ER membrane contact site [62].

STARD3

overexpression studies indicate that the presence of STARD3 increases late endosome size, membrane networks, and ER association, suggesting STARD3 facilitates ER to endosome cholesterol movement [63]. Thus STARD3 is localized for potential endosome –ER cholesterol trafficking. While the cholesterol transfer function of STARD3 is characterized, studying the biological function has been challenging. Early work eliminated STARD3 as the acceptor protein for Niemann Pick type C protein (NPC1)-mediated cholesterol efflux from the late endosomes (reviewed in [13]), and these studies are supported by data that show NPC-1 and STARD3 are localized to distinct late endosome compartments indicating these two proteins are not within the same cholesterol trafficking pathway [64]. However, it is not clear that the START domain is important for the cholesterol transfer function of STARD3, or whether the MENTAL domain, which also binds cholesterol, is responsible. STARD3NL, also known as MENTHO for MLN64 N-terminal domain homologue, shares high sequence similarity with the MENTAL domain of STARD3 yet lacks the START domain. STARD3NL is ubiquitously expressed and localized to the late endosomes via the MENTAL domain [65]. Like STARD3, STARD3NL contains a FFAT motif and can form endosome-ER membrane contact sites via VAP interaction. Thus, STARD3 and STARD3NL appear to have redundant functions in promoting ER-endosome membrane contacts. In addition, mice that express a STARD3 form that lacks the START domain are viable and fertile and have no dysregulation of sterol metabolism, even after dietary stress [66]. This finding questions the requirement of the START domain in STARD3-mediated ERendosome cholesterol trafficking (reviewed in [67]).

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Lastly, in human and rodent tissues, MLN64 was detected in placenta and the START domain is processed from the larger protein, providing a possible mechanism for cholesterol transport into mitochondria in this tissue [68, 69]. STARD3 overexpression is capable of enriching cholesterol in the mitochondria [70] or plasma membrane [71], depending on the cell type studied.

Yet placental progesterone synthesis is not ablated in the absence of

STARD3/MLN64 [66, 70] indicating other mechanisms are at play [72, 73]. 2.3

STARD4 STARD4 was identified as a novel cholesterol-regulated gene that was repressed in

mouse liver by a high cholesterol diet [74], while STARD5 and STARD6 were identified through database searches for sequence similarity to STARD4 [74].

STARD4/5/6 contain only the

START domain and the soluble nature of these lipid transporters contributes to dynamic membrane associations. In direct ligand binding assays, purified recombinant STARD4 binds cholesterol with highest affinity [75]. STARD4 has been reported to shuttle cholesterol between the PM and ER, PM and endosome recycling compartment (ERC), and possibly mitochondria [Figure 3]. Immunofluorescent detection of endogenous STARD4 in NIH-3T3 cells and THP-1 macrophages showed co-localization with ER resident proteins calnexin and acyl coenzyme A: cholesteryl acyl transferase (ACAT), respectively [76]. In U2OS osteosarcoma cells STARD4 overexpression enhanced the transport rate of a fluorescent cholesterol analog between cellular membranes of the endosome recycling complex (ERC) and the ER or plasma membranes [77, 78]. Based on the kinetics for cholesterol transport the authors estimate that STARD4 accounts for 33% of the non-vesicular cholesterol trafficking between the ERC and plasma membrane. The mechanism that drives membrane association is not clear, although anionic phospholipid interactions have been proposed to play a role (reviewed in [79]). [78] At the ER membrane the cholesterol transport function for STARD4 is likely to increase cholesterol ester (CE) synthesis and suppress ER cholesterol sensing [78, 80]. Cholesterol is converted to CE by acyl-CoA:cholesterol acyl transferase activity (ACAT) and an increase in ER cholesterol levels activates ACAT leading to increased cholesterol ester (CE) synthesis. CE synthesis was increased by either addition of recombinant, purified STARD4 to isolated microsomes or overexpression of STARD4 in primary hepatocytes or U2OS cells [75, 76, 78]. Conversely, knockdown of STARD4 expression in HepG2 cells resulted in decreased ER cholesterol levels, decreased ACAT1 activity, and decreased cellular CE levels without changing total cholesterol levels [80]. Overexpression of STARD4 in U2OS cells also resulted in suppression of ER cholesterol-sensing by the SCAP-SREBP-2 (Sterol regulatory element-

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binding protein cleavage-activating protein - Sterol regulatory element binding protein-2) complex [81-84]. These data support STARD4 cholesterol transport function can contribute to maintaining proper cholesterol distribution within the cell, and promote CE synthesis for cholesterol delivered to the ER [75, 85]. However, the biological function of STARD4 is not clear.

Whole body STARD4 knock-out mice have no major liver or plasma lipid changes

compared to wild-type mice [86]. Female STARD4 knockout mice had ~20% lower plasma total cholesterol and cholesterol ester levels relative to the wild-type female mice with no differences in hepatic cholesterol, cholesterol ester, or triacylglycerol levels after 1 week on a high cholesterol diet.

Decreasing hepatic cholesterol levels by inhibiting de novo cholesterol

synthesis had no effect on plasma or liver lipid profiles between the wild-type and STARD4 knock-out mice. Therefore, within the short treatment time (1 week) for this experiment, modest and perhaps sex-dependent changes were reported.

It is likely that other non-vesicular

cholesterol transporters that contribute to 67% of cholesterol trafficking can compensate for the loss of STARD4 under normal, physiological conditions. What remains to be determined is whether the loss of STARD4 affects cholesterol handling under certain stress or chronic disease states. Alternatively, the loss of STARD4 may not be pathogenic, rather aberrant STARD4 overexpression may contribute to lipid dysregulation. The original northern blot analysis for STARD4 showed transcripts are highly expressed in liver and kidney and STARD4 protein expression has been confirmed in mouse liver primary hepatocytes, HepG2 cells, Kupffer cells, and peritoneal macrophages [76]. In macrophages an increase in STARD4 would be predicted to increase cholesterol ester formation and favor foam cell development, an early marker of atherogenesis.

2.4

STARD5 STARD5 membrane associations are reported for Golgi, ER and PM [Figure 3] [87], and

appear to be cell-type and/or condition-dependent. Immunofluorescence and transmission immuno-electronmicroscopy detection of STARD5 in mouse kidney sections showed diffuse cytoplasmic distribution within the proximal tubule cells and association with brush-border (apical) and rough ER membranes and no apparent association with mitochondria or Golgi apparatus [88]. In THP-1 macrophages STARD5 was localized to the perinuclear regions of the cell and co-localized with Golgi but not endosome markers [89].

Filipin staining showed

cholesterol levels were highest within the Golgi, suggesting localization of STARD5 with membranes enriched in free cholesterol. The challenge has been to differentiate whether the co-localization of STARD5 with cholesterol-rich membranes under different experimental

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Figure 3. Summary for non-vesicular lipid trafficking by the START domain proteins. The figure depicts the major START domain – membrane associations for the START proteins with established ligands. The cholesterol-binding START proteins, StAR/STARD1, STARD3, STARD4, and STARD5 are shown at subcellular locations identified by immunohistochemistry or biochemical approaches (see text for details). The ligands are shown by orange (cholesterol), green (phosphatidylcholine), and red (ceramide) circles attached that the START domain. StAR is shown at the mitochondria OMM, the site of function, although the protein is imported and processed by mitochondria. STARD4 association with ACAT and increased CE synthesis is depicted, along with the role in endosome recycling compartment (ERC) cholesterol homeostasis. The ER PM non-vesicular cholesterol trafficking is shown to be STARD4- and STARD5-mediated, although the contribution of each to this process is not known. STARD5’s potential influence on cholesterol efflux to ApoA-1 is shown. ApoA-1 is a component of the high density lipoprotein (HDL) for reverse cholesterol trafficking to the liver. The soluble nature for STARD4 and STARD5 is indicated by unliganded START proteins, however, ligand-binding cannot be excluded. STARD3 is depicted as a bridge between late endosomes and the ER. The two other known lipid-binding START proteins with established chaperone function are shown at their sites of action. STARD7 is localized at the mitochondria and transfers phosphatidylcholine to the inner membrane and STARD11 is depicted as a bridge between Golgi and the ER to shuttle ceramide.

conditions is due to STARD5 shuttling cholesterol to the membrane or STARD5 being “targeted” to membrane(s) enriched in cholesterol [88]. Human STARD5 overexpression in primary rat hepatocytes resulted in increased cellular free cholesterol levels as measured by filipin staining [90]. Cholesterol levels in isolated microsomes were also shown to be increased, suggesting that overexpression of STARD5 increased the ER cholesterol content [90]. However, STARD5 had no direct effect on ACAT activity or cholesterol ester synthesis in THP-1 cells [76], providing evidence that STARD4 and STARD5 have distinct functions. Nevertheless, the absence of STARD5 may indirectly contribute to cellular cholesterol ester synthesis.

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Cholesterol ester

levels were increased in primary peritoneal macrophages isolated from STARD5 knock-out mice (Stard5-/-) compared to wild-type (WT) mice and ACAT activity was increased in isolated microsomes from both macrophages and liver of Stard5-/- mice [91]. Stard5-/- mice also had increased total cholesterol and triacylglycerol levels in liver, and free cholesterol levels were trending toward a decrease in isolated macrophages- consistent with overexpression of STARD5 promoting an increase cellular free cholesterol levels. Isolated macrophages and primary hepatocytes were used to monitor cholesterol trafficking with a focus on measuring the accessible PM cholesterol pool using the fluorescence labeled cholesterol-binding protein ALOD4 and measuring changes in membrane fluidity. The Stard5-/- macrophages had a decrease in PM cholesterol that was associated with an increase in membrane fluidity and lower ApoA-1-dependent cholesterol efflux compared to WT macrophages. These data, combined with studies that demonstrate STARD5-mediated transfer of cholesterol from a donor to an acceptor liposome, support STARD5 may shuttle cholesterol to the PM for efflux by the ABCA-1 transporter.

Careful phenotyping analysis of the Stard5-/- mice with measured serum

lipoproprotein levels is needed to support whether the cholesterol efflux changes measured in isolated cells has a biological read-out. STARD5 - membrane interactions may be driven by a redistribution of cholesterol within the cell upon ER stress. STARD5 mRNA expression is induced by agents that induce ER stress, such as thapsigargin, tunicamycin, or cellular cholesterol-loading [74, 85]. Endogenous levels of STARD5 mRNA were increased in NIH-3T3, 3T3-L1, and HK-2 cells after 18- 24 hour thapsigargin treatment, and in mouse macrophages after cholesterol-loading, conditions shown to activate the ER stress response [85, 88]. Thapsagargin-treated primary hepatocytes had increased PM accessible cholesterol, a response that was absent in hepatocytes from the Stard5-/- mice, suggesting that PM cholesterol distribution under ER stress requires STARD5. Chronic ER stress and inflammation are underlying metabolic disorders in many disease states including type II diabetes, non-alcoholic fatty liver disease, and cancer [92, 93]. While ER stress induces STARD5 in isolated cell cultures, it will be important to show in vivo that STARD5 protein expression is increased by ER stress activators, and/or under disease states. One report on changes in STARD5 expression in a disease state showed Stard5 steady-state mRNA and STARD5 protein levels were significantly increased, as were free cholesterol levels, in the kidneys of the OVE diabetic mouse model compared with wild-type control mice [88]. Cholesterol accumulation in the ER is known to promote ER stress; however, the significance of the association between elevated renal cholesterol, ER stress, and STARD5 in diabetic kidney remains to be determined [88].

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In direct ligand binding assays, cholesterol was the preferred ligand for purified recombinant STARD5, with the bile acids cholic acid reported not to be effective in a competitive binding assay [90]. In contrast, studies using nuclear magnetic resonance (NMR), circular dichroism (CD) and isothermal titration calorimetry to measure ligand-dependent changes in STARD5 structure and stability reported bile acids are the preferred ligand(s) [94-96]. In these studies, cholesterol didn’t generate a chemical shift by NMR or change the thermal CD spectra of STARD5, indicating no direct binding of the sterol. Both studies used recombinant human STARD5 and while it is not clear why there is such marked discrepancy, independent assessment of an indirect (competitive binding assay) vs direct (CD,ITC) approach for testing cholesterol and bile acid binding may resolve this issue. Given that chemical chaperones, including the bile acid tauroursodeoxycholic acid, are proven agents that restore insulin sensitivity in ob/ob mice and repress ER stress response in cultured cells [97, 98], it will be important to experimentally test the biological significance of a STARD5-bile acid interaction. The primary bile acid chenodeoxycholic acid (CDCA) is a ligand for the farnesoid X receptor (FXR), a nuclear receptor that controls bile acid homeostasis as whole body insulin responses. STARD5 and FXR have similar binding affinities for CDCA. Given that CDCA and synthetic FXR agonists improve glucose metabolism and insulin sensitivity in diet-induced obesity/insulin resistant rodent models [99, 100], potential modulation of FXR function by STARD5 would influence many processes. As an ER stress response gene, STARD5 would be predicted to either help resolve the ER stress or promote apoptosis and future studies that define whether STARD5 contributes to either side of the stress response may give greater insight into function. Employing a knock-out mouse model should help characterize phenotypic differences between WT and Stard5-/- mice under different stressor conditions, as described for other START proteins below. Given the high levels of STARD5 in macrophages, a proposed biological role in atherogenic progression could be compared between Stard5-/-and WT mice, perhaps with STARD4 overexpression. It is likely both STARD4 and STARD5 share cell-type expression pattern for liver parenchymal and nonparenchymal cells, along with the common ligand cholesterol. Therefore, future studies on tissue- and cell-specific expression patterns as well as developmental expression patterns for STARD5 and STARD4 may help to further differentiate lipid transport and biological functions as well as identify potential redundant functions. It will be important to validate the antibody used by demonstrating responsiveness to regulation (SREBP-2 for STARD4 and ER stress for STARD5) and/or by loss of detection due to knock-down of the target. Low levels of STARD5 were detected in isolated hepatocytes from WT mice but not the

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Stard5-/- mice. This finding is in contrast to a previous report by the same group that showed no STARD5 expression in isolated hepatocytes from WT mice, and expression was restricted to Kupffer cells and other immune cells. [89, 91]. It may be that the lack of detection was due to the source (human and rat v mouse primary hepatocytes), the cell stress levels, or the antibodies used, but this example highlights the necessity for antibody validation. Of note, STARD5 expression in HepG2 cell lysates is used to validate specificity for some commercial STARD5 antibodies.

2.5

STARD6 STARD6 has StAR-like lipid transport activity and can facilitate cholesterol delivery to

mitochondria in overexpression system [101, 102]. However, ligand specificity appears to be cholesterol metabolites, e.g., pregnenolone and testosterone [103] (reviewed in [96]. Although the in vivo biological function is not established, the tissue-specific expression patterns help link ligand binding to a function. In pig ovary STARD6 is highly expressed in the corpus luteum, with low levels in granulosa and theca cells and oocytes [102]. This sets an association between high progesterone synthesis and STARD6 expression. In testes, STARD6 is predominant in the nuclei of germ cells, the site for testosterone-driven spermatogenesis [74, 101, 104]. Outside of steroidogenic tissues STARD6 was detected in rat brain [105] and more studies focused on expression in human neurological tissue seems warranted based on the report that STARD6 SNP rs10164112 is associated with increased susceptibility to Alzheimer’s disease in a cohort that also carries the ApoE4 risk allele [106, 107].

3.

Phospholipid and ceramide transport by START domain proteins Members of the STARD2 subfamily are the other mammalian START proteins with

established ligands.

STARD2/7/10/11 bind phospholipids and sphingolipids, and our

understanding of the biological function of these lipid transporters has increased significantly over the past decade due in large part to studies with genetic knock-out mouse models.

3.1

STARD2 STARD2, commonly referred to as the phosphatidylcholine transfer protein (PC-TP),

binds phosphatidylcholine (PC), and biological functions have been linked to both controlling lipid droplet size under conditions of limiting phosphatidylcholine levels in fatty liver disease [108], and to glucose and lipid metabolism (reviewed in [109]). In brief, the Pctp-/- mice have no significant deficiency in membrane, lung surfactant, or bile PC levels, which were the predicted

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outcomes for the study based on the PC transfer function of STARD2 [110]. However, compared to their wild-type cohorts, Pctp-/- mice have increased insulin sensitivity marked, in part, by a decrease in gluconeogenesis and lower fasting serum glucose levels [111].

A

proposed mechanism for the altered metabolism is through a STARD2/PC-TP-Them2 interaction [109, 111-115]. Them2 is a member of the thioesterase superfamily (member 2) and is encoded by acyl-CoA thioesterase 13 gene (ACOT13). Them2 associates with mitochondria and hydrolyzes fatty-acyl-CoAs to free fatty acids and CoA. The fate of the free fatty acid released by Them2 action is dependent upon the nutritional state; under fasting conditions Them2

activity is

associated

with

increased

fatty acid

β-oxidation

and

increased

gluconeogenesis while in mouse models of diet-induced obesity Them2 activity is associated with increased phospholipid synthesis, ER stress, and insulin resistance [116, 117]. STARD2/PC-TP and Them2 interactions were shown by co-immunoprecipitation from liver extracts and by a yeast-two hybrid screen and GST-pull down assays [112]. Them2 activity in an in vitro enzyme assay was enhanced by the addition of purified His-STARD2/PC-TP [112], and the protein-protein interaction was increased in liver from fasted mice compared to control mice, suggesting STARD2/PC-TP may enhance Them2 activity leading to enhanced fatty acid β-oxidation during fasting [118]. The significance of a STARD2/PC-TP-Them2 protein-protein

interaction is that other START family members, including mammalian STARD14 and STARD15, contain thioesterase domains (discussed below).

However, an open question

remains whether the START domain or PC binding is required for protein interaction with Them2. Treatment of wild-type mice with a STARD2/PC-TP small molecular inhibitor (compound A1) that displaces PC binding attenuated high fat diet-induced increase in serum glucose levels [113].

These data suggest that blocking PC binding results in a similar

metabolic profile as loss of STARD2 in the pctp-/- mice. It remains to be determined whether compound A1 affects STARD2/PC-TP -Them2 interactions. Independent of Them2 interactions, STARD2/PC-TP may function as a PC transporter to help control lipid droplet size under conditions of limiting phosphatidylcholine levels in fatty liver disease [108]. 3.2

STARD7 STARD7 also binds PC and its PC transport function is critical for maintaining

mitochondrial PC levels. This function of STARD7 helps to maintain mitochondria membrane integrity and mitochondrial function [119, 120]. Like STARD1, STARD7 has an amino terminal mitochondrial signal sequence, so function is linked to localization. However, unlike STARD1, cell fractionation studies revealed the processed STARD7 is localized in both the cytosolic and

15

mitochondrial fractions [120-122]. Two isoforms for STARD7 have been reported; a longer form (STARD7-I) that contains an amino terminal mitochondrial signal sequence and a shorter isoform (STARD7-II) with a molecular weight consistent with cleavage of the amino terminal signal sequence (reviewed in [123]).

Experimental data support that STARD7-I can be

processed to STARD7-II, likely by mitochondrial proteases that cleave the targeting sequence, but that the protein remains anchored to the outer mitochondrial membrane via a single-pass transmembrane domain that orients the START domain towards the cytoplasm [121], an orientation that would facilitate PC transfer between the ER and mitochondria. Although the soluble START domain of STARD7 is active in PC transfer from donor to acceptor liposomes [121, 124], mitochondrial association is required for function [Figure 3].

Re-expression of

STARD7 in STARD7 depleted cells restores PC levels in mitochondria only when the transmembrane region is present. This region contains a cleavage site for the inner membrane mitochondrial protease presenilin-associated rhomboid-like (PARL), and PARL-dependent processing contributes to the mitochondrial sorting for STARD7 [121, 122, 124]. The mitochondrial integrity function was demonstrated by studies that showed BEAS-2B human bronchial epithelial cells and murine lung epithelial cells had enlarged mitochondria with less cristae, decreased respiration rates, and increased ROS levels when STARD7 was deleted [125, 126]. Similar results in STARD7-silenced mouse HEPA-1 or human HepG2 liver cell lines provided strong support for STARD7 maintaining mitochondrial PC levels [119, 127]. Further, the decrease in mitochondrial PC was due to loss from the inner membrane [91, 92].

A

proposed physiological role for STARD7 is to help maintain the PC supply for mitochondrial biogenesis in proliferating cells and contribute to epithelial barrier function in the lung [126, 128]. This may expand to cancer cell proliferation as STARD7 is expressed at relatively high levels in lung, colon, and liver cancer cell lines [128]. The original studies characterized STARD7 in placental JEG-3 choriocarcinoma cells as a PC binding START protein [128]. A physiological role for STARD7 in the placenta is supported by studies that show siRNA knockdown of STARD7 in JEG-3 cells promotes differentiation into syncytiotrophoblasts while reducing cell proliferation and migration. Again, these data would be consistent with a role in mitochondrial biogenesis.

Lastly, STARD7 binds ceramide within the same apparent pocket as

phosphatidylcholine binds, although there was no in vitro transfer activity for ceramide between liposomes [124]. The significance of this finding is not clear, but ceramide binding has been suggested to regulate the PC transfer function, which could indirectly affect mitochondrial function.

16

3.3

STARD10 Recombinant purified STARD10 can function in in vitro to transfer PC and PE between

donor and acceptor reconstituted lipid vesicles with PC being the preferred ligand [129]. STARD10 was identified as an overexpressed gene in ErbB2/HER2/neu breast tumors [130], and the loss of STARD10 was independently associated with increased metastatic disease and lower disease-free survival outcomes. These data suggested the loss of STAR10, independent of Her2 status, can identify a specific subgroup of patients at high risk [131, 132]. Like many of the mammalian START domain proteins, STARD10 likely functions in tissues beyond the original description. STARD10 knock-out mice (Stard10-/-) were generated to test a role for STARD10 in the liver and lung [110, 133]. Characterization of male Stard10-/- mice revealed no changes in PC levels in the liver and bile compared to the wild-type mice, and the Stard10-/mice have normal lung function, again supporting potential redundant functions for START proteins [133, 134]. However, after a high fat diet the Stard10-/- mice had lower cholesterol, triacylglycerol, conjugated and non-conjugated deoxycholic acid levels in the liver while taurine conjugated CDCA levels were elevated in both bile and liver. These changes in bile acid metabolism suggested changes in bile acid membrane transporters that control bile acids movement in the enterohepatic circulation.

A key piece of data demonstrated siRNA

knockdown and transient overexpression of STARD10 in Hepa-1 cells attenuates and enhances, respectively, synthetic ligand-activated peroxisome proliferator-activated receptoralpha (PPARα)- mediated changes in gene expression [133]. PPARα activates select genes involved in bile acid metabolism and the loss of STARD10 did not alter PPARα expression. The biliary phenotype of the Stard10-/- mice on a high fat diet was consistent with loss of PPARα function on select gene targets. The question remains whether the STARD10 - PPARα link requires the PC transport function of STARD5. To address this question, future studies will need to consider the potential post-translational control of STARD10 function. Casein kinase II phosphorylates Ser284 of STARD10 and decreases the in vitro lipid transport activity that is proposed to be due to a decrease in membrane association [135]. One approach would be to test the effect of STARD10 phosphomutant(s) on PC binding, altered bile acid metabolism, or anti-apoptotic actions reported for breast cancer is one app.

3.3

STARD11 STARD11 is also known as the ceramide transport protein (CERT) and is one of the best

characterized START proteins for function. A detailed review including the proposed model for ceramide transfer by STARD11/CERT has been recently published [136] and only key features

17

that distinguish this START protein is briefly summarized herein.

The mechanism of

STARD11/CERT lipid transport function is linked to the protein domains involved in protein-lipid and protein-protein interactions.

The amino terminal pleckstrin homology domain (PH) of

STARD11 binds to phosphoinositides, specifically phosphatidylinositol 4-phosphate (PI4P) in the Golgi membrane, while the FFAT motif interacts with the ER resident protein VAP. STARD11/CERT phosphorylation is proposed to maintain the protein in a folded, inactive form with dephosphorylation resulting in a conformational change that exposes the PH and FFAT domains for membrane interactions. These interactions then provide a bridge between the ER and Golgi, and brings the C-terminal START domain in close proximity to both membranes to facilitate ceramide extraction from the ER and delivery to the Golgi [Figure 3]. STARD11/CERT is an essential factor in development, as deletion of CERT in either Drosophila or mice is lethal [137, 138]. As ceramide is the backbone for sphingolipids, the essential function of STARD11/CERT is likely linked, in part, to sphingomyelin synthesis that occurs in the transGolgi [139].

4.

START proteins associated with enzymatic function

4.1

STARD8/12/13: deleted in liver cancer (DLC) START proteins STARD8/12/13 are best known as the deleted in liver cancer (DLC) family of proteins

due to the original identification for DLC1/STARD12 being located in a genomic region that is associated with loss of heterozygosity in several cancers [140]. All members of this subfamily (STARD12/DLC-1, STARD13/DLC-2 and STARD8/DLC-3) suppress cell growth and promote apoptosis when overexpressed in cancer cell lines, supporting a biological role in tumor suppression [141, 142].

A multi-domain structure for these proteins include a sterile alpha

motif, a serine–rich region, a RhoGAP domain, and a START domain [143] [Figure 2]. Most studies have focused on the subcellular localization of the protein and the Rho-GAP function in cytoskeletal remodeling and cell polarization in cancer [143-146] (reviewed in [147, 148]). Herein the focus is on the limited studies that have addressed a potential role of the START domain for cellular localization and the tumor suppressor function. Rat STARD12/DLC-1 was reported to co-localize with caveolin-1 (CAV-1) at cell membranes and deletion of either the START domain or RhoGAP domain reduced this interaction [145]. Finer mapping identified regions within the START domain that disrupted protein-protein interactions between STARD12/DLC-1 and CAV-1. Disruption of this region of the START domain also blocked the tumor suppressor function of STARD12 but had no effect on the Rho-GAP function [149].

18

These data suggest that membrane localization and Rho signaling is not mediating the tumor suppressor function for STARD12/DLC-1. Since the START domain mutant would likely disrupt ligand binding, the data indicate that ligand binding to the START domain is not likely required for STARD12/DLC-1 -CAV-1 interactions or Rho-GAP function. However, this needs to be experimentally determined. Perhaps the START domain interacts with another PM protein or the membrane bilayer. Furthermore, STARD8 and STARD13 have different interacting proteins and functions and there are no reports on the role of the START domain contributing to function. The crystal structure for STARD13/DLC-2 was used to predict a charged lipid as the ligand [18]. Therefore, more directed studies using START domain deletions are needed to test STARD8/13 function, and identifying the ligand should help defining potential function of the START domain for this subfamily. 4.2.

STARD14/15: the acyl-coenzyme A thioesterase (ACOT) START proteins. STARD14 and STARD15 are classified as type II acyl-coenzyme A thioesterase

(ACOT) enzymes due to their ACOT activity and presence of the “hotdog domains” that promote dimerization and oligomerization

of the enzyme [116]. ACOTs belong to the thioesterase

protein family that hydrolyze the thioester bond of fatty acyl-CoAs to generate free fatty acids and coenzyme A.

The presence of the C-terminal START domain structure for

STARD14/ACOT11 and STARD15/ACOT12 makes these ACOT proteins unique and places them in the START domain family [Figure 2]. However, neither the role of the START domain nor the ligand that binds to the START domain for these two enzymes are known. STARD15/ACOT12 is a cytosolic acetyl-CoA thioesterase that is highly expressed in liver [150152]. A truncated ACOT12 that lacks the START domain efficiently binds acetyl-CoA but has reduced enzymatic activity, which was attributed to decreased oligomerization of the enzyme [153]. One proposal is the START domain promotes structural changes to optimize oligomer formation for enhanced ACOT activity.

STARD14/ACOT11 (also referred to as Them1 or

mBFIT) was identified as a cold-induced gene in brown adipose tissue and its expression is correlated with increased metabolic activity in BAT [154].

The association with increased

metabolic activity is supported by studies using STARD14/ACOT11 knock-out mice (Them1-/mice) that were shown to have increased metabolic activity in brown fat, and increased resistance to high-fat diet-induced insulin resistance, lipid accumulation in liver, ER stress, and inflammation [155]. A proposed biological role for STARD14/ACOT11 is to blunt BAT energy expenditure to conserve calories. However, under nutritional excess the ACOT activity of STARD14 contributes to elevated free fatty acid pool that promotes ER stress, inflammation,

19

and insulin resistance that is associated with obesity [155, 156]. The crystal structure for the START domain of STARD14/ACOT11 predicts a fatty acid would fill the ligand-binding cavity [18], yet again, studies directed at identifying the ligand(s) remain to be conducted.

The

-/-

apparent similarities for some of the metabolic phenotypes for the STARD2/PC-TP (Pctp ) and STARD14/ACOT11 (Them1-/-) knock-out mice point to a possible common biological role for these two START domain proteins controlling fatty-acyl-CoA levels and thus the levels of free fatty acids.

Combining a START domain and ACOT domain into one protein in

STARD14/ACOT11 (Them1) may serve the same enzymatic function as the proposed interaction between STARD2/PC-TP with Them2.

4.3: STARD9; a kinesin motor protein Lastly, STARD9 was identified as a mitotic microtubule co-purifying protein and has been shown to function as a kinesin motor protein [157]. The multidomain structure from amino to carboxy termini is composed of a kinesin motor domain (KM), a forkhead associated phosphopeptide binding domain (FHA), and the START domain [Figure 2]. siRNA-mediated knock down of STARD9 in several cancer cell lines resulted in increased mitotic arrest and increased apoptosis, along with increased sensitivity to antimitotic cancer drugs [157]. However, the biological functions attributed so far to STARD9 may not require the START domain. Overexpressing only the motor domain of STARD9 was sufficient to rescue the mitotic defects in STAR9-depleted HeLa or HCT116 cells [158, 159]. This is another example for a START protein being proposed as a target, in this case for cancer treatment, without the biological role for the START domain established.

Determining the ligand for the START

domain and its impact on STARD9 function may reveal novel functions.

5.

Summary The mammalian START domain protein family is classified as a lipid transfer protein

family based on the conserved START domain structure, yet the biological function is established for only a few family members. StAR/STARD1, STARD7, and STARD11 biological roles in cholesterol, phosphatidindylcholine, and ceramide membrane transfer, respectively, are well characterized. A common theme is they all contain targeting sequences that direct them to the membrane for the site of action, and they are all essential proteins. Insights from knockout mouse models demonstrate that whole body knockout of STARD1, STARD7, or STARD11 is lethal, supporting their respective fundamental roles in steroidogenesis, mitochondrial membrane structure and function, and sphingolipid synthesis. Other START proteins with

20

established ligands include the sterol binding STARD3, STARD4, STARD5, STARD6, and the phospholipid binding STARD2 and STARD10. These proteins have established lipid transfer function based on

overexpression and knockdown studies in cell culture systems that

demonstrated an association between START protein expression and altered membrane cholesterol or phospholipid content and potential directional sterol trafficking between membranes. However, linking the lipid trafficking function to a biological function has been challenging as no major effects on cellular or serum lipid levels, or membrane phospholipid levels were reported for knockout mouse models for STARD3, STARD4, STARD2, or STARD10. STARD5 knockout mice present with changes in macrophage and liver cellular lipid levels, yet further analysis is needed to elucidate a biological role. Novel insights into metabolic functions for STARD2 were revealed with the loss of STARD2 linked to improved insulin sensitivity.

Despite having established ligands and relatively well described lipid transport

and/or biological functions, there remain several fundamental questions for these START proteins.

What is the molecular mechanism for cholesterol transport across mitochondrial

membranes for steroid hormone synthesis, and how specifically does StAR trigger this transfer? Does STARD3 provide unidirectional transport between the ER-endosome membranes? Are there cell-specific expression profiles and potential unique or redundant lipid transfer functions for the soluble sterol-binding STARD4 and STARD5 in disease states that involve dyslipidemia, inflammation and ER stress? What is the biological significance for STARD5 binding bile acids? Is the START domain and/or PC binding required for STARD2 interaction with Them2 or STARD10 action on bile acid metabolism?

Exposure of the knockout mouse models to

environmental or dietary stressors may be required to uncover potential biological functions. The START protein family members without established ligands share the common theme that they are multidomain proteins associated with enzymatic function. However, a lipid transfer function is an open question. The deleted in liver cancer subfamily, STARD8/12/13, function as tumor suppressors when localized to the PM. The tumor suppressor function appears to be independent of the RhoGAP activity for STARD12, so testing whether the START domain and ligand binding is required for PM localization would begin to address the function of this domain. Similarly, for the thioesterase START protein subfamily members STARD14/15 and for STARD9 the question of whether lipid binding within the START domain affects the enzymatic function associated with these proteins requires direct testing. Identifying the START domain lipids for these two subfamilies would facilitate this line of investigation. Given the START domains for the sterol-binding family members associate with cellular membranes and that this association

21

is facilitated by ligand binding, a similar function for ligand binding to the START domain for the multi-domain START proteins would be predicted. Declaration of Interest: The author has no conflict of interest.

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Acknowledgments:

The author thanks her colleagues and the many investigators who

contribute to this area of research. References

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MCE-D-19-00612_Highlights •

Members of the START lipid transfer proteins family have diverse biological and lipid transfer functions.

•

This review highlights the challenges of linking the lipid trafficking function of a START proteins to the biological function.

•

Insights from knockout mouse models on START protein lipid transport and biological roles in metabolism are presented.

Barbara J. Clark, Ph.D. [email protected]