title: Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells.
abstract: Murine desnutrin/human ATGL is a triacylglycerol (TAG) hydrolase with a predicted catalytic dyad within an alpha-beta hydrolase fold in the N-terminal region. In humans, mutations resulting in C-terminal truncation cause neutral lipid storage disease with myopathy. To identify critical functional domains, we measured TAG breakdown in cultured cells by mutated or truncated desnutrin. In vitro, C-terminally truncated desnutrin displayed an even higher apparent V(max) than the full-length form without changes in K(m), which may be explained by our finding of an interaction between the C- and N-terminal domains. In live cells, however, C-terminally truncated adenoviral desnutrin had lower TAG hydrolase activity. We investigated a role for the phosphorylation of C-terminal S406 and S430 residues but found that these were not necessary for TAG breakdown or lipid droplet localization in cells. The predicted N-terminal active sites, S47 and D166, were both critical for TAG hydrolysis in live cells and in vitro. We also identified two overlapping N-terminal motifs that predict lipid substrate binding domains, a glycine-rich motif (underlined) and an amphipathic alpha-helix (bold) within amino acid residues 10-24 (ISFAGCGFLGVYHIG). G14, F17, L18, and V20, but not G16 and G19, were important for TAG hydrolysis, suggesting a potential role for the amphipathic alpha-helix in TAG binding. This study identifies for the first time critical sites in the N-terminal region of desnutrin and reveals the requirement of the C-terminal region for TAG hydrolysis in cultured cells.
authors.affiliation: Department of Nutritional Science and Toxicology, University of California, Berkeley, CA 94720, USA.
authors.forename: Robin E
authors.initials: RE
authors.lastname: Duncan
authors.forename: Yuhui
authors.initials: Y
authors.lastname: Wang
authors.forename: Maryam
authors.initials: M
authors.lastname: Ahmadian
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lu
authors.forename: Eszter
authors.initials: E
authors.lastname: Sarkadi-Nagy
authors.forename: Hei Sook
authors.initials: HS
authors.lastname: Sul
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D006801:Humans; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D014280:Triglycerides / Q000378:metabolism*; D000217:Acyltransferases
chemical_list: D002265:Carboxylic Ester Hydrolases; D008049:Lipase; D014280:Triglycerides; C494445:PNPLA2 protein, mouse; D000217:Acyltransferases
references.citation: Can J Biochem Physiol. 1959 Aug;37(8):911-7
references.pmid: 13671378
references.citation: Proc Natl Acad Sci U S A. 1992 May 1;89(9):4094-8
references.pmid: 1570336
references.citation: J Proteome Res. 2007 Aug;6(8):3256-65
references.pmid: 17608402
references.citation: Biochemistry. 2003 Jun 10;42(22):6696-708
references.pmid: 12779324
references.citation: Nat Genet. 2007 Jan;39(1):28-30
references.pmid: 17187067
references.citation: Annu Rev Nutr. 2007;27:79-101
references.pmid: 17313320
references.citation: J Lipid Res. 2001 Jun;42(6):910-5
references.pmid: 11369798
references.citation: J Biol Chem. 2004 Nov 19;279(47):48968-75
references.pmid: 15364929
references.citation: Am J Physiol Gastrointest Liver Physiol. 2007 Jul;293(1):G1-4
references.pmid: 17218471
references.citation: Science. 2006 May 5;312(5774):734-7
references.pmid: 16675698
references.citation: J Proteome Res. 2006 Dec;5(12):3446-52
references.pmid: 17137347
references.citation: J Biol Chem. 2006 Dec 29;281(52):40236-41
references.pmid: 17074755
references.citation: J Lipid Res. 2006 Feb;47(2):375-83
references.pmid: 16282638
references.citation: Cell. 1999 Apr 30;97(3):349-60
references.pmid: 10319815
references.citation: J Biol Chem. 2002 Feb 15;277(7):4806-15
references.pmid: 11717312
references.citation: J Clin Endocrinol Metab. 2008 Jul;93(7):2877-84
references.pmid: 18445677
references.citation: J Biol Chem. 2008 Jun 20;283(25):17211-20
references.pmid: 18445597
references.citation: PLoS One. 2008;3(11):e3756
references.pmid: 19018281
references.citation: Future Lipidol. 2007 Apr;2(2):229-237
references.pmid: 19194515
references.citation: FEBS Lett. 1986 Jul 14;203(1):99-103
references.pmid: 2941317
references.citation: J Biol Chem. 2004 Nov 5;279(45):47066-75
references.pmid: 15337759
references.citation: Muscle Nerve. 2007 Dec;36(6):856-9
references.pmid: 17657808
references.citation: FASEB J. 1996 Sep;10(11):1257-69
references.pmid: 8836039
references.citation: J Biol Chem. 2008 Sep 12;283(37):25428-25436
references.pmid: 18614531
references.citation: Science. 2004 Nov 19;306(5700):1383-6
references.pmid: 15550674
references.citation: J Lipid Res. 2005 Nov;46(11):2477-87
references.pmid: 16150821