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

• DOI: 10.1194/jlr.ra120000907
• Publication Date: 2020-09-25T01:45:13Z
• Authors: Yanling Ma; Suman Karki; Philip M. Brown; Dennis D. Lin; Maren C. Podszun; Wenchang Zhou; Olga V. Belyaeva; Natalia Y. Kedish...; Yaron Rotman
• Keywords: nonalcoholic fatty liver disease; 0303 health sciences; retinoids; 17-Hydroxysteroid Dehydrogenases; lipid droplets; Liver Diseases; QD415-436; Biochemistry; 3. Good health; Small Molecule Libraries; 03 medical and health sciences; Chronic Disease; Humans; enzyme regulation; protein structure; Cells, Cultured; alcoholic liver 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 enzymatic function have been demonstrated to be protective from cirrhosis cancer, supporting development as a promising therapeutic target. Previous that is lipid droplet (LD)-associated protein. However, critical domains drive LD targeting or determine activity yet defined. Here we used mutagenesis generate multiple truncated point-mutated proteins were able demonstrate vitro N-terminal hydrophobic domain, PAT-like putative α-helix/β-sheet/α-helix domain are all for targeting. Similarly, characterized predicted catalytic, substrate-binding, homodimer interaction sites found them essential HSD17B13, addition our previous identification amino acid P260 cofactor binding site. In conclusion, localization protein which may facilitate understanding its this treat chronic diseases. Associated global epidemic lifestyle-associated obesity metabolic syndrome, NAFLD has become major health burden one leading causes end-stage disease, hepatocellular carcinoma, transplants (1Younossi Z. Anstee Q.M. Marietti M. Hardy T. Henry L. Eslam George J. Bugianesi E. Global NASH: trends, predictions, risk factors prevention.Nat. Rev. Gastroenterol. Hepatol. 2018; 15: 11-20Crossref PubMed Scopus (1530) Google Scholar, 2Pais R. Barritt A.St. Calmus Y. Scatton O. Runge Lebray P. Poynard Ratziu V. Conti F. transplantation: current expected challenges.J. 2016; 65: 1245-1257Abstract Full Text PDF (200) Scholar). Limited treatment options available, stimulating search novel molecular targets suitable pharmacological intervention (3Rotman Sanyal A.J. Current upcoming pharmacotherapy non-alcoholic disease.Gut. 2017; 66: 180-190Crossref (238) One approach discovery these targets, based on known heritability NAFLD, use (4Loomba Schork N. Chen C.H. Bettencourt Bhatt A. Ang B. Nguyen Hernandez C. Richards Salotti et al.Heritability hepatic fibrosis steatosis prospective twin study.Gastroenterology. 2015; 149: 1784-1793Abstract (180) 5Schwimmer J.B. Celedon M.A. Lavine J.E. Salem Campbell N.J. Shiehmorteza Yokoo Chavez Middleton M.S. disease.Gastroenterology. 2009; 136: 1585-1592Abstract (304) 6Browning J.D. Szczepaniak L.S. Dobbins Nuremberg Horton Cohen J.C. Grundy S.M. Hobbs H.H. Prevalence urban population United States: impact ethnicity.Hepatology. 2004; 40: 1387-1395Crossref (2771) Several genes related incidence progressive form NASH using approach, including PNPLA3 (7Romeo S. Kozlitina Xing Pertsemlidis Cox D. Pennacchio L.A. Boerwinkle Genetic variation confers susceptibility disease.Nat. Genet. 2008; 1461-1465Crossref (1942) 8Yuan X. Waterworth Perry J.R. Lim Song K. Chambers Zhang W. Vollenweider Stirnadel H. Johnson al.Population-based genome-wide reveal six loci influencing plasma levels enzymes.Am. Hum. 83: 520-528Abstract (315) 9Sookoian Castano G.O. Burgueno A.L. Gianotti T.F. Rosselli Pirola C.J. A nonsynonymous variant adiponutrin associated severity.J. Lipid Res. 50: 2111-2116Abstract (278) 10Rotman Koh Zmuda J.M. Kleiner D.E. Liang T.J. CRN The variability patatin-like phospholipase domain-containing 3 (PNPLA3) histological severity disease.Hepatology. 2010; 52: 894-903Crossref (344) 11Speliotes E.K. Butler J.L. Palmer C.D. Voight B.F. GIANT Consortium MIGen Hirschhorn J.N. specifically confer increased histologic but not 904-912Crossref (271) 12Valenti Al-Serri Daly A.K. Galmozzi Rametta Dongiovanni Nobili Mozzi Roviaro G. Vanni al.Homozygosity phospholipase-3/adiponutrin I148M polymorphism influences patients 51: 1209-1217Crossref (458) Scholar), TM6SF2 (13Holmen O.L. Fan Hovelson D.H. Schmidt E.M. Zhou Guo Langhammer Løchen M-L. al.Systematic evaluation coding identifies candidate causal total cholesterol myocardial infarction risk.Nat. 2014; 46: 345-351Crossref (191) 14Kozlitina Smagris Stender Nordestgaard B.G. Tybjaerg-Hansen Vogt Exome-wide study 352-356Crossref (617) 15Mahdessian Taxiarchis Popov Silveira Franco-Cereceda Hamsten Eriksson van't Hooft regulator fat metabolism triglyceride secretion content.Proc. Natl. Acad. Sci. USA. 111: 8913-8918Crossref (192) MBOAT7 (16Buch Stickel Trepo Way Herrmann Nischalke H.D. Brosch Rosendahl Berg Ridinger al.A confirms alcohol-related cirrhosis.Nat. 47: 1443-1448Crossref (272) discovered (17Ma Belyaeva O.V. Brown P.M. Fujita Valles Karki de Boer Y.S. Du al.17-Beta retinol features 2019; 69: 1504-1519Crossref (106) 18Chambers Sehmi Li Wass M.N. Van der Harst Holm Sanna Kavousi Baumeister S.E. al.Genome-wide concentrations enzymes plasma.Nat. 2011; 43: 1131-1138Crossref (367) 19Abul-Husn N.S. Cheng A.H. Xin Schurmann Stevis Liu Wood G.C. protein-truncating protection disease.N. Engl. Med. 378: 1096-1106Crossref (284) Of these, (17-β 13) represents likely target potential pharmaceutical intervention. enzyme steroid substrates, bioactive lipids (19Abul-Husn Scholar) suggested substrates. Three independent injury 20Pirola Garaycoechea Flichman Arrese San Martino Gazzi Sookoian Splice rs72613567 prevents worst outcomes disease.J. 60: 176-185Abstract (57) carcinoma (21Stickel Lutz Buch Silva I. Rausch Fischer Weiss K.H. Gotthardt al.Genetic reduces developing alcohol misusers.Hepatology. 2020; 72: 88-102Crossref (36) 22Yang Nahon Cao Q. Moreno Letouze Imbeaud Bayard Gustot Deviere 17-Beta-hydroxysteroid protects 70: 231-240PubMed We others splice-site SNP leads formation two splicing (HSD17B13-G insertion HSD17B13-exon 6 deletion) 23Ma Rotman Letter Editor: does splice actually yield new type alternative splicing?.Hepatology. 71: 1885-1886Crossref (1) Scholar); rs62305723 encodes proline serine mutation at (AA) position 260 rs143404524 premature truncation (24Kozlitina Blacks Hispanics.N. 379: 1876-1877Crossref (19) All three products devoid, of, activity, confirming importance HSD17B13. Collectively, data also suggest can modulated by large truncations deletions, well single double AA mutations, implying possibility modulating therapeutically, either interfering expression (i.e., antisense oligonucleotides) inhibiting directly small synthetic compounds. Excess cellular esterified neutral stored LDs, main storage reservoirs energy membrane components (25Thiam A.R. Farese Jr., R.V. Walther T.C. biophysics cell biology droplets.Nat. Mol. Cell Biol. 2013; 14: 775-786Crossref (511) LD-associated proteins, play pivotal roles regulation, embeded adherent phospholipid monolayer, includes PC, PE, PI, lyso-PC, lyso-PE (26Fujimoto Parton R.G. Not just fat: structure droplet.Cold Spring Harb. Perspect. 3: a004838Crossref (280) MS proteomic analyses hundreds couple dozen confirmed resident Two types signals proposed directing surface: amphipathic α-helices hairpins 27Kory Targeting mechanisms droplets.Trends 26: 535-546Abstract (150) PAT family encompasses most widely studied (perilipin, ADRP, TIP47, S3-12) (28Bickel P.E. Tansey J.T. Welte ancient regulate stores.Biochim. Biophys. Acta. 1791: 419-440Crossref (488) share conserved their (29Miura Gan J.W. Brzostowski Parisi M.J. Schultz Londos Oliver Kimmel Functional conservation among Perilipin, TIP47 (PAT)-related mammals, Drosophila, Dictyostelium.J. Chem. 2002; 277: 32253-32257Abstract (298) 30Ohsaki Maeda Tauchi-Sato Fujimoto Recruitment droplets controlled cleft.Biochem. Commun. 2006; 347: 279-287Crossref (49) 31Horiguchi Araki Motojima 17beta-Hydroxysteroid liver-specific droplet-associated protein.Biochem. 370: 235-238Crossref (44) 32Su Wang Jia Wu Tian Xu al.Comparative reveals 17beta-HSD13 pathogenic disease.Proc. 11437-11442Crossref (100) N-terminus stability, LD, other correct trafficking LDs fully Pharmacological efforts silence siRNA initiated companies (33Friedman S.L. Neuschwander-Tetri B.A. Rinella Mechanisms strategies.Nat. 24: 908-922Crossref (863) beyond naturally occurring mutants still unknown. study, aimed identify AAs studying proteins. Our work design molecule inhibitors lead better considered disease. Hydropathy analysis was performed online TMHMM server (https://services.healthtech.dtu.dk/service.php?TMHMM-2.0) (34Sonnhammer E.L. von Heijne Krogh hidden Markov model predicting transmembrane helices sequences.Proc. Int. Conf. Intell. Syst. 1998; 6: 175-182PubMed ProtScale (https://web.expasy.org/protscale) (35Wilkins M.R. Gasteiger Bairoch Sanchez Williams K.L. Appel R.D. Hochstrasser D.F. Protein tools ExPASy server.Methods 1999; 112: 531-552PubMed default settings. HepG2 HEK293 cells cultured DMEM (Corning) supplemented 10% FBS (Sigma-Aldrich) under 5% CO2 37°C. Primary human hepatocytes (Gibco) plated polylysine coated 4-well chamber slide William's E medium FBS. Oleate palmitate solubilized PBS solution heating 55°C 65°C, respectively. Solubilized acids conjugated acid-free-BSA culture stock solution. induced adding into 48 h final concentration 200 µM oleate unless otherwise indicated. Transfections stably expressing HSD17B13-GFP carried out Lipofectamine 3000 (Thermo Fisher Scientific). To homodimerization, grown 6-well dishes transfected 2 µg HSD17B13-FLAG plasmid DNA 10 µl per well. Forty-eight hours after transfection, lysed 500 lysis buffer [1% Triton X-100, 50 mM Tris (pH 8), 150 NaCl] min ice collected centrifugation 13,000 g 30 4°C. full-length (RG213132) exon-2-deleted (Δ71-106, B) HSD17B13-variant B-GFP (RG227799) obtained OriGene. (VB150430-10020) designed constructed VectorBuilder Inc. (Cyagen Biosciences, Santa Clara, CA). Q5® Site-Directed Mutagenesis Kit (NEB, E0554S) mutant plasmids primers (supplemental Table S1). templates assay, respectively, Sanger sequencing. selection point prediction relevant NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) relying similarity short-chain dehydrogenase/reductases (SDRs). Cells seeded NuncTM Lab-TekTM II Chambered Coverglass Scientific) wild-type Reagent following manufacturer's instructions. targeting, added transfection induce LDs. After fixed 4% paraformaldehyde (Electron Microscopy Science) counterstained 1 µg/ml Hoechst nuclei LipidTox (1:500; Thermo colocalization B (HSD17B13-B, Δ71-106) ER, cotransfected SEC61β-GFP (ER marker protein; gift Alexandre Toulmay) HSD17B13-B-Flag. permeabilized 0.3% Triton-X, 3% BSA, normal goat serum (Vector Laboratories) PBS. Immunofluorescence staining HSD17B13-B-FLAG incubating FLAG M2 antibody (F3165; Sigma-Aldrich) room temperature h. Alexa Fluor 568 anti-mouse secondary washing. stain nuclei. apoptosis-inducing factor (AIF), mitochondria, mitochondrial transfection. Antibodies against AIF (5318; Signaling Technology) (F3165) primary incubation. 647 anti-rabbit antibodies recognize rabbit anti-AIF mouse anti-FLAG antibodies, Cellular fluorescence images taken confocal microscopy (Zeiss LSM 700). Enzymatic measured (RDH) assay previously described Briefly, day before being transiently triplicate mutant, empty vector plasmids. All-trans-retinol (Toronto Research Chemicals) 5 ethanol, ethanol ≤0.5% (v/v), medium, incubated 8 Retinoids extracted twice equal volume hexane separated normal-phase HPLC Spherisorb S3W column (4.6 × 100 mm) (Waters Corp.). Retinaldehyde retinoic normalized amount shown relative vector. Wild-type positive control, constructs tested same round experiments sharing control. Aliquots suspensions quantification Western blot analysis. G Dynabeads (Invitrogen) (clone F1804) Sigma-Aldrich anti-turboGFP OTI2H8) Origene overnight rotation For each pulldown, bead slurry. Lysates antibody-conjugated beads temperature. Bound washed eluted SDS containing sample guidelines. Proteins 15% precast PAGE gels (Bio-Rad) transferred PVDF membranes (Invitrogen). blocked nonfat milk Tris-buffered saline 0.05% Tween-20 Membranes polyclonal anti-HSD17B13 (1:2000) (TA350064) conjunction HRP-conjugated (GE Healthcare) Pierce enhanced chemiluminescent substrate detection HRP Differences between Student's t-test (GraphPad Prism version 8). (variant A) targeted when lipid-loaded (Fig. 1A). reduction stability loss 22–28 (Δ22–28) 71–106 B, exon skipping) 1, Fig. 2A). N terminus hydropathy S1) thus could serve anchor LDs; 30–300 reside outside surface. extended detail delineate characteristics sequences surprisingly, fragment only (N1-21) absence 1), suggesting sufficient Interestingly, (AAs 22–28) generates peptide (N1-28) localizes despite 1). test whether necessary further generated 4–16 (Δ4–16) and, expected, without sequence Δ4–16 nonrandom pattern distribution, it localized close proximity mitochondria S2). Thus, indicates both targeting.Fig. 2Identification A: wild-type, (Δ71-106), treated C-terminall