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Characterization of essential domains in HSD17B13 for cellular localization and enzymatic activity

2020; Elsevier BV; Volume: 61; Issue: 11 Linguagem: Inglês

10.1194/jlr.ra120000907

1539-7262

Yanling Ma, Suman Karki, Philip M. Brown, Dennis D. Lin, Maren C. Podszun, Wenchang Zhou, Olga V. Belyaeva, Natalia Y. Kedishvili, Yaron Rotman,

Diet, Metabolism, and Disease

Human genetic studies recently identified an association of SNPs in the 17-β hydroxysteroid dehydrogenase 13 (HSD17B13) gene with alcoholic and nonalcoholic fatty liver disease development. Mutant HSD17B13 variants devoid of enzymatic function have been demonstrated to be protective from cirrhosis and liver cancer, supporting the development of HSD17B13 as a promising therapeutic target. Previous studies have demonstrated that HSD17B13 is a lipid droplet (LD)-associated protein. However, the critical domains that drive LD targeting or determine the enzymatic activity have yet to be defined. Here we used mutagenesis to generate multiple truncated and point-mutated proteins and were able to demonstrate in vitro that the N-terminal hydrophobic domain, PAT-like domain, and a putative α-helix/β-sheet/α-helix domain in HSD17B13 are all critical for LD targeting. Similarly, we characterized the predicted catalytic, substrate-binding, and homodimer interaction sites and found them to be essential for the enzymatic activity of HSD17B13, in addition to our previous identification of amino acid P260 and cofactor binding site. In conclusion, we identified critical domains and amino acid sites that are essential for the LD localization and protein function of HSD17B13, which may facilitate understanding of its function and targeting of this protein to treat chronic liver diseases. Human genetic studies recently identified an association of SNPs in the 17-β hydroxysteroid dehydrogenase 13 (HSD17B13) gene with alcoholic and nonalcoholic fatty liver disease development. Mutant HSD17B13 variants devoid of enzymatic function have been demonstrated to be protective from cirrhosis and liver cancer, supporting the development of HSD17B13 as a promising therapeutic target. Previous studies have demonstrated that HSD17B13 is a lipid droplet (LD)-associated protein. However, the critical domains that drive LD targeting or determine the enzymatic activity have yet to be defined. Here we used mutagenesis to generate multiple truncated and point-mutated proteins and were able to demonstrate in vitro that the N-terminal hydrophobic domain, PAT-like domain, and a putative α-helix/β-sheet/α-helix domain in HSD17B13 are all critical for LD targeting. Similarly, we characterized the predicted catalytic, substrate-binding, and homodimer interaction sites and found them to be essential for the enzymatic activity of HSD17B13, in addition to our previous identification of amino acid P260 and cofactor binding site. In conclusion, we identified critical domains and amino acid sites that are essential for the LD localization and protein function of HSD17B13, which may facilitate understanding of its function and targeting of this protein to treat chronic liver diseases. Associated with the global epidemic of lifestyle-associated obesity and metabolic syndrome, NAFLD has become a major global health burden and one of the leading causes for end-stage liver disease, hepatocellular carcinoma, and liver transplants (1Younossi Z. Anstee Q.M. Marietti M. Hardy T. Henry L. Eslam M. George J. Bugianesi E. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.Nat. Rev. Gastroenterol. Hepatol. 2018; 15: 11-20Crossref PubMed Scopus (1530) Google Scholar, 2Pais R. Barritt A.St. Calmus Y. Scatton O. Runge T. Lebray P. Poynard T. Ratziu V. Conti F. NAFLD and liver transplantation: current burden and expected challenges.J. Hepatol. 2016; 65: 1245-1257Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Limited treatment options are available, stimulating the search for novel molecular targets suitable for therapeutic pharmacological intervention (3Rotman Y. Sanyal A.J. 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Sanna S. Kavousi M. Baumeister S.E. et al.Genome-wide association study identifies loci influencing concentrations of liver enzymes in plasma.Nat. Genet. 2011; 43: 1131-1138Crossref PubMed Scopus (367) Google Scholar, 19Abul-Husn N.S. Cheng X. Li A.H. Xin Y. Schurmann C. Stevis P. Liu Y. Kozlitina J. Stender S. Wood G.C. et al.A protein-truncating HSD17B13 variant and protection from chronic liver disease.N. Engl. J. Med. 2018; 378: 1096-1106Crossref PubMed Scopus (284) Google Scholar). Of these, HSD17B13 (17-β hydroxysteroid dehydrogenase 13) represents a likely therapeutic target for the treatment of chronic liver disease with a potential for pharmaceutical intervention. HSD17B13 is a hepatic lipid droplet (LD)-associated enzyme with steroid substrates, bioactive lipids (19Abul-Husn N.S. Cheng X. Li A.H. Xin Y. Schurmann C. Stevis P. Liu Y. Kozlitina J. Stender S. Wood G.C. et al.A protein-truncating HSD17B13 variant and protection from chronic liver disease.N. Engl. J. Med. 2018; 378: 1096-1106Crossref PubMed Scopus (284) Google Scholar), and retinol (17Ma Y. Belyaeva O.V. Brown P.M. Fujita K. Valles K. Karki S. de Boer Y.S. Koh C. Chen Y. Du X. et al.17-Beta hydroxysteroid dehydrogenase 13 is a hepatic retinol dehydrogenase associated with histological features of nonalcoholic fatty liver disease.Hepatology. 2019; 69: 1504-1519Crossref PubMed Scopus (106) Google Scholar) suggested as potential enzymatic substrates. Three independent genetic variants in HSD17B13 were found to confer protection from injury in NASH (17Ma Y. Belyaeva O.V. Brown P.M. Fujita K. Valles K. Karki S. de Boer Y.S. Koh C. Chen Y. Du X. et al.17-Beta hydroxysteroid dehydrogenase 13 is a hepatic retinol dehydrogenase associated with histological features of nonalcoholic fatty liver disease.Hepatology. 2019; 69: 1504-1519Crossref PubMed Scopus (106) Google Scholar, 19Abul-Husn N.S. Cheng X. Li A.H. Xin Y. Schurmann C. Stevis P. Liu Y. Kozlitina J. Stender S. Wood G.C. et al.A protein-truncating HSD17B13 variant and protection from chronic liver disease.N. Engl. J. Med. 2018; 378: 1096-1106Crossref PubMed Scopus (284) Google Scholar, 20Pirola C.J. Garaycoechea M. Flichman D. Arrese M. San Martino J. Gazzi C. Castano G.O. Sookoian S. Splice variant rs72613567 prevents worst histologic outcomes in patients with nonalcoholic fatty liver disease.J. Lipid Res. 2019; 60: 176-185Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), alcoholic liver disease (17Ma Y. Belyaeva O.V. Brown P.M. Fujita K. Valles K. Karki S. de Boer Y.S. Koh C. Chen Y. Du X. et al.17-Beta hydroxysteroid dehydrogenase 13 is a hepatic retinol dehydrogenase associated with histological features of nonalcoholic fatty liver disease.Hepatology. 2019; 69: 1504-1519Crossref PubMed Scopus (106) Google Scholar, 19Abul-Husn N.S. Cheng X. Li A.H. Xin Y. Schurmann C. Stevis P. Liu Y. Kozlitina J. Stender S. Wood G.C. et al.A protein-truncating HSD17B13 variant and protection from chronic liver disease.N. Engl. J. Med. 2018; 378: 1096-1106Crossref PubMed Scopus (284) Google Scholar), and hepatocellular carcinoma (21Stickel F. Lutz P. Buch S. Nischalke H.D. Silva I. Rausch V. Fischer J. Weiss K.H. Gotthardt D. Rosendahl J. et al.Genetic variation in HSD17B13 reduces the risk of developing cirrhosis and hepatocellular carcinoma in alcohol misusers.Hepatology. 2020; 72: 88-102Crossref PubMed Scopus (36) Google Scholar, 22Yang J. Trepo E. Nahon P. Cao Q. Moreno C. Letouze E. Imbeaud S. Bayard Q. Gustot T. Deviere J. et al.A 17-Beta-hydroxysteroid dehydrogenase 13 variant protects from hepatocellular carcinoma development in alcoholic liver disease.Hepatology. 2019; 70: 231-240PubMed Google Scholar). We and others identified a splice-site SNP rs72613567 that leads to the formation of two novel splicing variants (HSD17B13-G insertion and HSD17B13-exon 6 deletion) (17Ma Y. Belyaeva O.V. Brown P.M. Fujita K. Valles K. Karki S. de Boer Y.S. Koh C. Chen Y. Du X. et al.17-Beta hydroxysteroid dehydrogenase 13 is a hepatic retinol dehydrogenase associated with histological features of nonalcoholic fatty liver disease.Hepatology. 2019; 69: 1504-1519Crossref PubMed Scopus (106) Google Scholar, 19Abul-Husn N.S. Cheng X. Li A.H. Xin Y. Schurmann C. Stevis P. Liu Y. Kozlitina J. Stender S. Wood G.C. et al.A protein-truncating HSD17B13 variant and protection from chronic liver disease.N. Engl. J. Med. 2018; 378: 1096-1106Crossref PubMed Scopus (284) Google Scholar, 23Ma Y. Brown P.M. Rotman Y. Letter to the Editor: does the HSD17B13 rs72613567 splice variant actually yield a new type of alternative splicing?.Hepatology. 2020; 71: 1885-1886Crossref PubMed Scopus (1) Google Scholar); the nonsynonymous SNP rs62305723 encodes a proline to serine mutation at amino acid (AA) position 260 (17Ma Y. Belyaeva O.V. Brown P.M. Fujita K. Valles K. Karki S. de Boer Y.S. Koh C. Chen Y. Du X. et al.17-Beta hydroxysteroid dehydrogenase 13 is a hepatic retinol dehydrogenase associated with histological features of nonalcoholic fatty liver disease.Hepatology. 2019; 69: 1504-1519Crossref PubMed Scopus (106) Google Scholar); and the rs143404524 SNP leads to premature truncation (24Kozlitina J. Stender S. Hobbs H.H. Cohen J.C. HSD17B13 and chronic liver disease in Blacks and Hispanics.N. Engl. J. Med. 2018; 379: 1876-1877Crossref PubMed Scopus (19) Google Scholar). All three protective variants generate protein products that are devoid, or predicted to be devoid of, enzymatic activity, confirming the importance of understanding the enzymatic function of HSD17B13. Collectively, these data also suggest that HSD17B13 activity can be modulated by large domain truncations and deletions, as well as by single or double critical AA mutations, implying the possibility of modulating the enzymatic activity therapeutically, either by interfering with gene expression (i.e., by using antisense oligonucleotides) or by inhibiting activity directly using small synthetic compounds. Excess cellular lipids are esterified to neutral lipids and stored in LDs, which function as main storage reservoirs of metabolic energy and membrane lipid components (25Thiam A.R. Farese Jr., R.V. Walther T.C. The biophysics and cell biology of lipid droplets.Nat. Rev. Mol. Cell Biol. 2013; 14: 775-786Crossref PubMed Scopus (511) Google Scholar). LD-associated proteins, which play pivotal roles in lipid metabolism regulation, are embeded in or adherent to the phospholipid monolayer, which includes PC, PE, PI, lyso-PC, and lyso-PE (26Fujimoto T. Parton R.G. Not just fat: the structure and function of the lipid droplet.Cold Spring Harb. Perspect. Biol. 2011; 3: a004838Crossref PubMed Scopus (280) Google Scholar). MS proteomic analyses have identified hundreds of LD-associated proteins, including a couple of dozen proteins confirmed to be LD resident proteins (25Thiam A.R. Farese Jr., R.V. Walther T.C. The biophysics and cell biology of lipid droplets.Nat. Rev. Mol. Cell Biol. 2013; 14: 775-786Crossref PubMed Scopus (511) Google Scholar). Two types of LD targeting signals have been proposed for directing proteins to the LD surface: amphipathic α-helices and hydrophobic hairpins (25Thiam A.R. Farese Jr., R.V. Walther T.C. The biophysics and cell biology of lipid droplets.Nat. Rev. Mol. Cell Biol. 2013; 14: 775-786Crossref PubMed Scopus (511) Google Scholar, 27Kory N. Farese Jr., R.V. Walther T.C. Targeting fat: mechanisms of protein localization to lipid droplets.Trends Cell Biol. 2016; 26: 535-546Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The PAT protein family encompasses the most widely studied LD-associated proteins (perilipin, ADRP, TIP47, and S3-12) (28Bickel P.E. Tansey J.T. Welte M.A. PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores.Biochim. Biophys. Acta. 2009; 1791: 419-440Crossref PubMed Scopus (488) Google Scholar) that share conserved PAT domains that are essential for their LD targeting (29Miura S. Gan J.W. Brzostowski J. Parisi M.J. Schultz C.J. Londos C. Oliver B. Kimmel A.R. Functional conservation for lipid storage droplet association among Perilipin, ADRP, and TIP47 (PAT)-related proteins in mammals, Drosophila, and Dictyostelium.J. Biol. Chem. 2002; 277: 32253-32257Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 30Ohsaki Y. Maeda T. Maeda M. Tauchi-Sato K. Fujimoto T. Recruitment of TIP47 to lipid droplets is controlled by the putative hydrophobic cleft.Biochem. Biophys. Res. Commun. 2006; 347: 279-287Crossref PubMed Scopus (49) Google Scholar). HSD17B13 is an LD-associated protein (17Ma Y. Belyaeva O.V. Brown P.M. Fujita K. Valles K. Karki S. de Boer Y.S. Koh C. Chen Y. Du X. et al.17-Beta hydroxysteroid dehydrogenase 13 is a hepatic retinol dehydrogenase associated with histological features of nonalcoholic fatty liver disease.Hepatology. 2019; 69: 1504-1519Crossref PubMed Scopus (106) Google Scholar, 19Abul-Husn N.S. Cheng X. Li A.H. Xin Y. Schurmann C. Stevis P. Liu Y. Kozlitina J. Stender S. Wood G.C. et al.A protein-truncating HSD17B13 variant and protection from chronic liver disease.N. Engl. J. Med. 2018; 378: 1096-1106Crossref PubMed Scopus (284) Google Scholar, 31Horiguchi Y. Araki M. Motojima K. 17beta-Hydroxysteroid dehydrogenase type 13 is a liver-specific lipid droplet-associated protein.Biochem. Biophys. Res. Commun. 2008; 370: 235-238Crossref PubMed Scopus (44) Google Scholar, 32Su W. Wang Y. Jia X. Wu W. Li L. Tian X. Li S. Wang C. Xu H. Cao J. et al.Comparative proteomic study reveals 17beta-HSD13 as a pathogenic protein in nonalcoholic fatty liver disease.Proc. Natl. Acad. Sci. USA. 2014; 111: 11437-11442Crossref PubMed Scopus (100) Google Scholar), and we identified a PAT-like domain at its N-terminus that is essential for its stability, targeting to LD, and enzymatic function (17Ma Y. Belyaeva O.V. Brown P.M. Fujita K. Valles K. Karki S. de Boer Y.S. Koh C. Chen Y. Du X. et al.17-Beta hydroxysteroid dehydrogenase 13 is a hepatic retinol dehydrogenase associated with histological features of nonalcoholic fatty liver disease.Hepatology. 2019; 69: 1504-1519Crossref PubMed Scopus (106) Google Scholar). However, other domains in HSD17B13 that are essential for correct trafficking to LDs have not yet been fully defined. Pharmacological efforts to silence HSD17B13 by siRNA have been initiated by pharmaceutical companies (33Friedman S.L. Neuschwander-Tetri B.A. Rinella M. Sanyal A.J. Mechanisms of NAFLD development and therapeutic strategies.Nat. Med. 2018; 24: 908-922Crossref PubMed Scopus (863) Google Scholar). However, beyond the naturally occurring mutants and cofactor binding sites that we identified (17Ma Y. Belyaeva O.V. Brown P.M. Fujita K. Valles K. Karki S. de Boer Y.S. Koh C. Chen Y. Du X. et al.17-Beta hydroxysteroid dehydrogenase 13 is a hepatic retinol dehydrogenase associated with histological features of nonalcoholic fatty liver disease.Hepatology. 2019; 69: 1504-1519Crossref PubMed Scopus (106) Google Scholar), other sites or domains critical for its enzymatic activity and targeting are still unknown. In the current study, we aimed to identify critical domains and AAs that are essential for LD targeting and the enzymatic activity of HSD17B13 by studying multiple truncated and point-mutated proteins. Our work may facilitate the design of small molecule inhibitors and lead to a better understanding of the protein function that is essential for HSD17B13 to be considered a therapeutic target in chronic liver disease. Hydropathy analysis of HSD17B13 was performed using the online TMHMM server (https://services.healthtech.dtu.dk/service.php?TMHMM-2.0) (34Sonnhammer E.L. von Heijne G. Krogh A. A hidden Markov model for predicting transmembrane helices in protein sequences.Proc. Int. Conf. Intell. Syst. Mol. Biol. 1998; 6: 175-182PubMed Google Scholar) and ProtScale (https://web.expasy.org/protscale) (35Wilkins M.R. Gasteiger E. Bairoch A. Sanchez J.C. Williams K.L. Appel R.D. Hochstrasser D.F. Protein identification and analysis tools in the ExPASy server.Methods Mol. Biol. 1999; 112: 531-552PubMed Google Scholar) with default settings. HepG2 and HEK293 cells were cultured in DMEM (Corning) supplemented with 10% FBS (Sigma-Aldrich) under 5% CO2 at 37°C. Primary human hepatocytes (Gibco) were plated on a polylysine (Sigma-Aldrich) coated 4-well chamber slide in William's E medium (Gibco) supplemented with 10% FBS. Oleate and palmitate were solubilized in a PBS (Corning) solution by heating to 55°C and 65°C, respectively. Solubilized fatty acids were conjugated with 10% fatty acid-free-BSA in culture medium to generate fatty acid stock solution. LDs were induced by adding fatty acid stock solution into culture medium for 48 h with a final concentration of 200 µM oleate and 200 µM palmitate unless otherwise indicated. Transfections of HepG2 cells stably expressing HSD17B13-GFP (17Ma Y. Belyaeva O.V. Brown P.M. Fujita K. Valles K. Karki S. de Boer Y.S. Koh C. Chen Y. Du X. et al.17-Beta hydroxysteroid dehydrogenase 13 is a hepatic retinol dehydrogenase associated with histological features of nonalcoholic fatty liver disease.Hepatology. 2019; 69: 1504-1519Crossref PubMed Scopus (106) Google Scholar) were carried out using Lipofectamine 3000 (Thermo Fisher Scientific). To study homodimerization, cells were grown in 6-well cell culture dishes and transfected using 2 µg HSD17B13-FLAG plasmid DNA and 10 µl Lipofectamine 3000 per well. Forty-eight hours after transfection, cells were lysed in 500 µl lysis buffer [1% Triton X-100, 50 mM Tris (pH 8), 150 mM NaCl] for 10 min on ice and collected after centrifugation at 13,000 g for 30 min at 4°C. The full-length protein HSD17B13-GFP (RG213132) and the exon-2-deleted variant (Δ71-106, variant B) HSD17B13-variant B-GFP (RG227799) were obtained from OriGene. HSD17B13-FLAG (VB150430-10020) was designed and constructed by VectorBuilder Inc. (Cyagen Biosciences, Santa Clara, CA). The Q5® Site-Directed Mutagenesis Kit (NEB, E0554S) was used to generate mutant HSD17B13 plasmids with designed mutagenesis primers (supplemental Table S1). HSD17B13-GFP and HSD17B13-FLAG plasmids were used as mutagenesis templates for the cellular localization study and for the enzymatic assay, respectively, unless otherwise indicated. Mutant plasmids were confirmed by Sanger sequencing. The selection of the sites for point mutation was based on a prediction of sites relevant to activity using the NCBI conserved domain search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and relying on similarity with other short-chain dehydrogenase/reductases (SDRs). Cells were seeded in a NuncTM Lab-TekTM II Chambered Coverglass (Thermo Fisher Scientific) and transfected with wild-type or mutant plasmids using Lipofectamine 3000 Reagent (Thermo Fisher Scientific) following the manufacturer's instructions. To study LD targeting, fatty acids were added 48 h after the transfection to induce LDs. After 48 h of fatty acid treatment cells were fixed in 4% paraformaldehyde (Electron Microscopy Science) for 10 min and counterstained with 1 µg/ml Hoechst (Thermo Fisher Scientific) for nuclei and LipidTox (1:500; Thermo Fisher Scientific) for LDs. To study the colocalization of HSD17B13 naturally occurring variant B (HSD17B13-B, Δ71-106) with the ER, cells were cotransfected with SEC61β-GFP (ER marker protein; gift from Alexandre Toulmay) and HSD17B13-B-Flag. Cells were fixed in 4% paraformaldehyde and permeabilized in 0.3% Triton-X, 3% BSA, and 10% normal goat serum (Vector Laboratories) in PBS. Immunofluorescence staining of HSD17B13-B-FLAG was performed by incubating cells with FLAG M2 antibody (F3165; Sigma-Aldrich) at room temperature for 1 h. Alexa Fluor 568 goat anti-mouse secondary antibody was used after washing. Hoechst was used to stain nuclei. Immunofluorescence staining of apoptosis-inducing factor (AIF), a marker for mitochondria, and HSD17B13 were performed to demonstrate mitochondrial targeting of mutant HSD17B13 after transfection. Antibodies against AIF (5318; Cell Signaling Technology) and FLAG (F3165) were used for primary incubation. Alexa Fluor 647 goat anti-rabbit and Alexa Fluor 568 goat anti-mouse secondary antibodies were used to recognize rabbit anti-AIF and mouse anti-FLAG antibodies, respectively. Cellular fluorescence images were taken by confocal microscopy (Zeiss LSM 700). Enzymatic activity was measured using the retinol dehydrogenase (RDH) activity assay as previously described (17Ma Y. Belyaeva O.V. Brown P.M. Fujita K. Valles K. Karki S. de Boer Y.S. Koh C. Chen Y. Du X. et al.17-Beta hydroxysteroid dehydrogenase 13 is a hepatic retinol dehydrogenase associated with histological features of nonalcoholic fatty liver disease.Hepatology. 2019; 69: 1504-1519Crossref PubMed Scopus (106) Google Scholar, 27Kory N. Farese Jr., R.V. Walther T.C. Targeting fat: mechanisms of protein localization to lipid droplets.Trends Cell Biol. 2016; 26: 535-546Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Briefly, HEK293 cells were seeded 1 day before being transiently transfected in triplicate with HSD17B13, HSD17B13 mutant, or empty vector plasmids. All-trans-retinol (Toronto Research Chemicals) at 2 or 5 µM in ethanol, with a final ethanol concentration ≤0.5% (v/v), was added to the culture medium, and cells were incubated for 6 or 8 h. Retinoids were extracted twice by equal volume of ethanol and double volume of hexane and were separated by normal-phase HPLC with a Spherisorb S3W column (4.6 × 100 mm) (Waters Corp.). Retinaldehyde and retinoic acid levels were normalized per total protein amount and are shown relative to empty vector. Wild-type HSD17B13 was used as a positive control, with multiple constructs tested in the same round of experiments sharing the positive control. Aliquots of cell suspensions were taken for protein quantification and Western blot analysis. Protein G Dynabeads (Invitrogen) were incubated with anti-FLAG antibody (clone F1804) from Sigma-Aldrich or anti-turboGFP (clone OTI2H8) antibody from Origene overnight with rotation at 4°C. For each pulldown, 3 µg of antibody were incubated with 50 µl of bead slurry. Lysates of transfected HepG2 were incubated with antibody-conjugated beads with rotation for 1 h at room temperature. Bound proteins were washed and eluted with SDS containing sample buffer per the manufacturer's guidelines. Proteins were separated in 4% to 15% precast PAGE gels (Bio-Rad) and transferred to PVDF membranes (Invitrogen). The membranes were blocked with a 5% nonfat milk in Tris-buffered saline containing 0.05% Tween-20 for 1 h. Membranes were incubated with polyclonal anti-HSD17B13 antibody (1:2000) from Origene (TA350064) in conjunction with an HRP-conjugated secondary antibody (GE Healthcare) and Pierce enhanced chemiluminescent substrate for the detection of HRP (Thermo Fisher Scientific). Differences between wild-type HSD17B13 and empty vector or HSD17B13 mutants were tested using Student's t-test (GraphPad Prism version 8). The full-length HSD17B13 protein (variant A) is targeted to LDs when cells are lipid-loaded (Fig. 1A). We previously described the reduction of protein stability and LD targeting with the loss of the PAT-like N-terminal AAs 22–28 (Δ22–28) or with the loss of AAs 71–106 (variant B, a naturally occurring variant with exon 2 skipping) (17Ma Y. Belyaeva O.V. Brown P.M. Fujita K. Valles K. Karki S. de Boer Y.S. Koh C. Chen Y. Du X. et al.17-Beta hydroxysteroid dehydrogenase 13 is a hepatic retinol dehydrogenase associated with histological features of nonalcoholic fatty liver disease.Hepatology. 2019; 69: 1504-1519Crossref PubMed Scopus (106) Google Scholar) (Fig. 1, Fig. 2A). The N terminus of HSD17B13 is predicted by hydropathy analysis to be a putative transmembrane domain (supplemental Fig. S1) and thus could serve to anchor the protein to LDs; AAs 30–300 are likely to reside on the outside membrane surface. We thus extended our study in detail to delineate the characteristics of the N-terminal sequences that are critical for LD targeting. Not surprisingly, a fragment containing only the hydrophobic domain (N1-21) of HSD17B13 was not targeted to LDs in the absence of AAs 22–28 and AAs 71–106 (Fig. 1), suggesting that the hydrophobic domain is not sufficient to drive protein LD targeting. Interestingly, the addition of the PAT-like domain (AAs 22–28) to the hydrophobic domain generates a peptide fragment (N1-28) of HSD17B13 that localizes to LDs despite the absence of AAs 71–106 (Fig. 1). To test whether the hydrophobic domain is necessary for HSD17B13 to target LDs, we further generated an HSD17B13 devoid of the hydrophobic AAs 4–16 (Δ4–16) and, as expected, found that without this hydrophobic sequence HSD17B13 does not target to LDs (Fig. 1A). Interestingly, Δ4–16 has a nonrandom pattern of cellular distribution, and we found it to be localized in close proximity to mitochondria (supplemental Fig. S2). Thus, our data indicates that at the N terminus of HSD17B13, both AAs 4–16 and AAs 22–28 are necessary for LD targeting.Fig. 2Identification of domains critical for LD targeting of HSD17B13. A: HepG2 cells were transiently transfected with HSD17B13 wild-type, the naturally occurring variant B (Δ71-106), or mutant plasmids and treated with fatty acids to induce LDs. Proteins were C-terminall