Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells

Murine desnutrin/human ATGL is a triacylglycerol (TAG) hydrolase with a predicted catalytic dyad within an α-β 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 Vmax than the full-length form without changes in Km, 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 α-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 α-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. Murine desnutrin/human ATGL is a triacylglycerol (TAG) hydrolase with a predicted catalytic dyad within an α-β 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 Vmax than the full-length form without changes in Km, 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 α-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 α-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. Triacylglycerol (TAG) serves as the major energy reserve in higher eukaryotic organisms (1Duncan R.E. Ahmadian M. Jaworski K. Sarkadi-Nagy E. Sul H.S. Regulation of lipolysis in adipocytes.Annu. Rev. Nutr. 2007; 27: 79-101Google Scholar). FFAs are mobilized from TAG through the hydrolytic action of lipases to provide substrates for oxidative metabolism in tissues, as well as substrates for synthesis of complex lipids and signaling molecules. Lipolysis, the breakdown of TAG to FFA and glycerol, occurs in an orderly and regulated manner, with different enzymes acting sequentially at each step (1Duncan R.E. Ahmadian M. Jaworski K. Sarkadi-Nagy E. Sul H.S. Regulation of lipolysis in adipocytes.Annu. Rev. Nutr. 2007; 27: 79-101Google Scholar, 2Ahmadian M. Duncan R.E. Jaworski K. Sarkadi-Nagy E. Sul H.S. Triacylglycerol metabolism in adipose tissue.Future Lipidol. 2007; 2: 229-237Google Scholar, 3Jaworski K. Sarkadi-Nagy E. Duncan R.E. Ahmadian M. Sul H.S. Regulation of triglyceride metabolism. IV. Hormonal regulation of lipolysis in adipose tissue.Am. J. Physiol. Gastrointest. Liver Physiol. 2007; 293: G1-G4Google Scholar). Hydrolysis of TAG to diacylglycerol is the first step (4Jenkins C.M. Mancuso D.J. Yan W. Sims H.F. Gibson B. Gross R.W. Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities.J. Biol. Chem. 2004; 279: 48968-48975Google Scholar, 5Villena J.A. Roy S. Sarkadi-Nagy E. Kim K.H. Sul H.S. Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis.J. Biol. Chem. 2004; 279: 47066-47075Google Scholar, 6Zimmermann R. Strauss J.G. Haemmerle G. Schoiswohl G. Birner-Gruenberger R. Riederer M. Lass A. Neuberger G. Eisenhaber F. Hermetter A. et al.Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase.Science. 2004; 306: 1383-1386Google Scholar), and our laboratory, and others, recently identified a novel TAG hydrolase in mice that we called desnutrin (5Villena J.A. Roy S. Sarkadi-Nagy E. Kim K.H. Sul H.S. Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis.J. Biol. Chem. 2004; 279: 47066-47075Google Scholar) [also known as TTS2.2, patatin-like phospholipase A domain containing 2, iPLAζ (4Jenkins C.M. Mancuso D.J. Yan W. Sims H.F. Gibson B. Gross R.W. Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities.J. Biol. Chem. 2004; 279: 48968-48975Google Scholar), or in humans, adipose triglyceride lipase (ATGL) (6Zimmermann R. Strauss J.G. Haemmerle G. Schoiswohl G. Birner-Gruenberger R. Riederer M. Lass A. Neuberger G. Eisenhaber F. Hermetter A. et al.Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase.Science. 2004; 306: 1383-1386Google Scholar)]. Desnutrin is highly expressed in white and brown adipose tissue, where it is a major TAG lipase, but is also found at lower levels in most other tissues where it also plays an important role in TAG hydrolysis (5Villena J.A. Roy S. Sarkadi-Nagy E. Kim K.H. Sul H.S. Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis.J. Biol. Chem. 2004; 279: 47066-47075Google Scholar, 7Haemmerle G. Lass A. Zimmermann R. Gorkiewicz G. Meyer C. Rozman J. Heldmaier G. Maier R. Theussl C. Eder S. et al.Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase.Science. 2006; 312: 734-737Google Scholar, 8Schweiger M. Schreiber R. Haemmerle G. Lass A. Fledelius C. Jacobsen P. Tornqvist H. Zechner R. Zimmermann R. Adipose triglyceride lipase and hormone-sensitive lipase are the major enzymes in adipose tissue triacylglycerol catabolism.J. Biol. Chem. 2006; 281: 40236-40241Google Scholar). Desnutrin contains an N-terminal patatin-like domain, spanning amino acids 8–180, that is characteristic of many plant lipid acyl hydrolases (5Villena J.A. Roy S. Sarkadi-Nagy E. Kim K.H. Sul H.S. Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis.J. Biol. Chem. 2004; 279: 47066-47075Google Scholar, 9Rydel T.J. Williams J.M. Krieger E. Moshiri F. Stallings W.C. Brown S.M. Pershing J.C. Purcell J.P. Alibhai M.F. The crystal structure, mutagenesis, and activity studies reveal that patatin is a lipid acyl hydrolase with a Ser-Asp catalytic dyad.Biochemistry. 2003; 42: 6696-6708Google Scholar). Enzymatic activity of desnutrin is predicted to be derived from an S47-D166 catalytic dyad that lies within an α-β hydrolase fold in the patatin-like domain (5Villena J.A. Roy S. Sarkadi-Nagy E. Kim K.H. Sul H.S. Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis.J. Biol. Chem. 2004; 279: 47066-47075Google Scholar, 9Rydel T.J. Williams J.M. Krieger E. Moshiri F. Stallings W.C. Brown S.M. Pershing J.C. Purcell J.P. Alibhai M.F. The crystal structure, mutagenesis, and activity studies reveal that patatin is a lipid acyl hydrolase with a Ser-Asp catalytic dyad.Biochemistry. 2003; 42: 6696-6708Google Scholar). Mutation of the S47 residue to alanine has been shown to result in a complete loss of function in vitro, confirming the predicted serine-esterase activity of this enzyme (10Lake A.C. Sun Y. Li J.L. Kim J.E. Johnson J.W. Li D. Revett T. Shih H.H. Liu W. Paulsen J.E. et al.Expression, regulation, and triglyceride hydrolase activity of Adiponutrin family members.J. Lipid Res. 2005; 46: 2477-2487Google Scholar). However, the requirement of the D166 site has not yet been tested. Four individuals with point mutations in desnutrin/ATGL have been identified, and all developed neutral lipid storage disease with myopathy (NLSDM) (11Akiyama M. Sakai K. Ogawa M. McMillan J.R. Sawamura D. Shimizu H. Novel duplication mutation in the patatin domain of adipose triglyceride lipase (PNPLA2) in neutral lipid storage disease with severe myopathy.Muscle Nerve. 2007; 36: 856-859Google Scholar, 12Fischer J. Lefevre C. Morava E. Mussini J-M. Laforet P. Negre-Salvayre A. Lathrop M. Salvayre R. The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy.Nat. Genet. 2007; 39: 28-30Google Scholar). In one, a duplication mutation within the N-terminal patatin-like domain caused a frameshift after L159 that is predicted to truncate the enzyme at amino acid position 178 as well as to cause a D166R mutation (11Akiyama M. Sakai K. Ogawa M. McMillan J.R. Sawamura D. Shimizu H. Novel duplication mutation in the patatin domain of adipose triglyceride lipase (PNPLA2) in neutral lipid storage disease with severe myopathy.Muscle Nerve. 2007; 36: 856-859Google Scholar). In the other three individuals, mutations occurred distal to the patatin-like domain, causing truncation of the C-terminal region of desnutrin in one or both alleles (12Fischer J. Lefevre C. Morava E. Mussini J-M. Laforet P. Negre-Salvayre A. Lathrop M. Salvayre R. The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy.Nat. Genet. 2007; 39: 28-30Google Scholar). Surprisingly, however, truncated desnutrin/ATGL lacking the C-terminal region has been shown in vitro to have similar or even higher activity than the full-length form (13Kobayashi K. Inoguchi T. Maeda Y. Nakashima N. Kuwano A. Eto E. Ueno N. Sasaki S. Sawada F. Fujii M. et al.The lack of the C-terminal domain of adipose triglyceride lipase causes neutral lipid storage disease through impaired interactions with lipid droplets.J. Clin. Endocrinol. Metab. 2008; 93: 2877-2884Google Scholar, 14Schweiger M. Schoiswohl G. Lass A. Radner F.P.W. Haemmerle G. Malli R. Wolfgang G. Cornaciu I. Oberer M. Salvayre R. et al.The C-terminal region of human adipose triglyceride lipase enzyme activity and lipid droplet Biol. Chem. 2008; Scholar). To identify and functional sites within we activity of a of C-terminal in and C- and N-terminal by in cultured cells and TAG We found that truncation of the C-terminal region of desnutrin increases TAG hydrolase activity in vitro by the apparent Vmax without substrate which we may be to of an interaction of the C-terminal region with the N-terminal region. we also found that C-terminal truncation TAG hydrolase activity in live cells. be in between with and human of that the C-terminal region of desnutrin is for TAG breakdown in may to the of human C-terminal C-terminal region in full-length desnutrin have been as sites that be and Y. S. Y. J. Liu P. Y. A for of without or Res. 2006; Scholar). We investigated loss of phosphorylation at these sites may to impaired activity of C-terminally truncated desnutrin in cells, but found that desnutrin TAG breakdown and localization to lipid were by mutation of these We also the N-terminal region and found that both S47 and D166 catalytic dyad residues within the α-β hydrolase fold are critical for TAG hydrolysis within cells and in vitro. the N-terminal we identified two overlapping a glycine-rich motif and an amphipathic α-helix that are predicted by to be neutral lipid J. and of and human in The region is for both binding and of Scholar, domain of and Scholar). We by that this the amphipathic is critical for TAG acid and were from from were from were from all other were from desnutrin with in the (5Villena J.A. Roy S. Sarkadi-Nagy E. Kim K.H. Sul H.S. Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis.J. Biol. Chem. 2004; 279: 47066-47075Google Scholar) as a for the of desnutrin for by and sites of The truncation by and the with the sites of and the sites of The truncation and the resulting sites of and the sites of The N-terminal truncation by of desnutrin and the resulting the site of The truncation by of desnutrin with the resulting sites of were to point desnutrin in or by the for of for for and for with a of at The resulting with to and from to identify were the to the containing full-length or truncated desnutrin with were by with and cells. to containing were with and cells in cells, in cells the cells and cells were from cells were a from D. of D.J. J. J. of and Lipid Res. 42: Scholar). were in containing with of and of to cells and cells were to as R.E. Sarkadi-Nagy E. Jaworski K. Ahmadian M. Sul H.S. and functional of A2 Biol. Chem. 2008; Scholar). cells were and as R.E. Sarkadi-Nagy E. Jaworski K. Ahmadian M. Sul H.S. and functional of A2 Biol. Chem. 2008; Scholar). cells were in in complete containing acid to lipid droplet were at a of of and for a were in with with with and with were the and a were from cells or mutated desnutrin by in and by at for at were by of containing of in to of substrate containing of from as in with and were to for at and were by the of of acids were with of and in of after for at by in as a of TAG hydrolase activity in at each and from in were to full-length desnutrin levels as by levels of acid were with in to with and to cells, or cells in for to for complex lipids and cells well of acid TAG and of acid the well of acid the of TAG and well of acid were with complete and with full-length or C-terminally truncated adenoviral desnutrin at a of were in complete for an TAG hydrolysis in cells as we have (5Villena J.A. Roy S. Sarkadi-Nagy E. Kim K.H. Sul H.S. Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis.J. Biol. Chem. 2004; 279: 47066-47075Google Scholar). from cells were by the of and and by acid A of lipid and J. Physiol. Scholar), after which lipids were by and by of the by an of of in TAG of and cells and in The were to well and expressed as a of were with and for with and at for at to of in were with glycerol, and and by for to and to and to were for and at first with to or and with or were by cells were with an N-terminal truncation of desnutrin with and a C-terminal truncation of desnutrin with in were at for at and at with to or or with a by of of for were by and with to by and are were by with To identify and critical functional domains, we a of studies with mutated and truncated of desnutrin TAG hydrolysis in live cells. is predicted to TAG hydrolase activity from an S47-D166 catalytic dyad within an α-β hydrolase fold in the N-terminal called the patatin-like domain, for with the plant acyl hydrolase However, with have been found mutations that both within and distal to the patatin-like domain, suggesting the of other in desnutrin for activity in We full-length desnutrin with as well as desnutrin lacking of the C-terminal region. full-length and are shown in with a of The to the truncated that from a mutation in a with (12Fischer J. Lefevre C. Morava E. Mussini J-M. Laforet P. Negre-Salvayre A. Lathrop M. Salvayre R. The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy.Nat. Genet. 2007; 39: 28-30Google Scholar). The to truncate the C-terminal region the between amino acids and of desnutrin (12Fischer J. Lefevre C. Morava E. Mussini J-M. Laforet P. Negre-Salvayre A. Lathrop M. Salvayre R. The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy.Nat. Genet. 2007; 39: 28-30Google Scholar). We first the in vitro catalytic activity of full-length desnutrin as well as the and C-terminal region TAG hydrolase activity of desnutrin/ATGL is measured in vitro, for this enzyme have not been three of desnutrin displayed The for for full-length desnutrin to be In this the for for hormone-sensitive lipase has been to be C. P. S. C. and are the major of of human hormone-sensitive lipase lipid 2008; Scholar). of desnutrin for with is in with the recently function of as a diacylglycerol than a TAG hydrolase G. Zimmermann R. M. Theussl C. G. E. W. Zechner R. lipase in mice causes in adipose tissue, and Biol. Chem. Scholar). three desnutrin had similar that for TAG not with loss of the C-terminal region The apparent however, to higher for of desnutrin than for the full-length catalytic activity This finding is in with a suggesting that the C-terminal region of desnutrin/ATGL a function in vitro M. Schoiswohl G. Lass A. Radner F.P.W. Haemmerle G. Malli R. Wolfgang G. Cornaciu I. Oberer M. Salvayre R. et al.The C-terminal region of human adipose triglyceride lipase enzyme activity and lipid droplet Biol. Chem. 2008; Scholar). To the C-terminal region may to catalytic activity of the N-terminal we and these domains. as we were to an interaction between the C-terminal region and the N-terminal region This finding that the function of the C-terminal domain in vitro from the of important sites in the catalytic N-terminal patatin domain through the in vitro activity for desnutrin C-terminal in individuals with similar impaired function in we measured TAG hydrolase activity of C-terminal in live cells. cells or were with that complex and with with of adenoviral desnutrin. were for a to for expression and breakdown of We found that in cells, full-length breakdown of TAG by to TAG hydrolase activity by desnutrin in this desnutrin of the C-terminal however, TAG hydrolase activity in cells in cells has that loss of the C-terminal region of human ATGL localization of this to lipid M. Schoiswohl G. Lass A. Radner F.P.W. Haemmerle G. Malli R. Wolfgang G. Cornaciu I. Oberer M. Salvayre R. et al.The C-terminal region of human adipose triglyceride lipase enzyme activity and lipid droplet Biol. Chem. 2008; Scholar), which may of the impaired TAG breakdown by truncated desnutrin in cultured cells. However, our that breakdown of impaired in cells with to suggesting that this truncated desnutrin may TAG breakdown than lack activity. by that the C-terminal region of desnutrin may with interaction with M. Schoiswohl G. Lass A. Radner F.P.W. Haemmerle G. Malli R. Wolfgang G. Cornaciu I. Oberer M. Salvayre R. et al.The C-terminal region of human adipose triglyceride lipase enzyme activity and lipid droplet Biol. Chem. 2008; Scholar). may in a in cells by interactions with or other yet to be identified which are truncated desnutrin lacking the C-terminal region may other lipases from the lipid droplet but to interactions for activity at the lipid droplet In in cells, both and displayed a to this activity to that of full-length The of activity by truncated desnutrin in but not in may to the finding that individuals with truncated desnutrin in are not TAG in other tissues, as and (12Fischer J. Lefevre C. Morava E. Mussini J-M. Laforet P. Negre-Salvayre A. Lathrop M. Salvayre R. The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy.Nat. Genet. 2007; 39: 28-30Google Scholar). we have by that of higher by desnutrin expressed in is the C-terminal region is not This that the C-terminal domain of desnutrin may be in the of and that impaired interactions may also at in to TAG hydrolase activity of truncated desnutrin in cultured cells. studies that the N-terminal region contains all residues necessary for catalytic activity in vitro, the C-terminal region is critical for TAG breakdown in cells. The distal C-terminal region of human ATGL contains two identified residues and that be Y. S. Y. J. Liu P. Y. A for of without or Res. 2006; Scholar), both of which within motifs that are in desnutrin. The of these sites, however, are both and lack these it is that loss of critical at these sites may to loss of TAG hydrolase activity of truncated desnutrin in cells. We investigated the of and mutations in full-length desnutrin localization to the lipid droplet or TAG hydrolase activity in cells. found to the and of both cells and In and of desnutrin and were lipid that phosphorylation at these sites is not critical for localization We the of these mutations TAG breakdown in live cells cells with had TAG breakdown to cells. and TAG breakdown similar to that phosphorylation at these sites is not necessary for TAG breakdown and that loss of phosphorylation at these sites not impaired TAG breakdown by C-terminally truncated desnutrin. is that of the complete function and requirement of the C-terminal region of desnutrin/ATGL for TAG hydrolase activity in live cells an important in the C-terminal of desnutrin and human ATGL is by our We found that TAG hydrolase activity of the truncation impaired in cells, the of the has been shown to be to TAG hydrolase activity of C-terminally truncated human ATGL (13Kobayashi K. Inoguchi T. Maeda Y. Nakashima N. Kuwano A. Eto E. Ueno N. Sasaki S. Sawada F. Fujii M. et al.The lack of the C-terminal domain of adipose triglyceride lipase causes neutral lipid storage disease through impaired interactions with lipid droplets.J. Clin. Endocrinol. Metab. 2008; 93: 2877-2884Google Scholar). in of the C-terminal domain to be critical for activity in cells. In this have also functional between the C-terminal of desnutrin and human ATGL in vitro M. Schoiswohl G. Lass A. Radner F.P.W. Haemmerle G. Malli R. Wolfgang G. Cornaciu I. Oberer M. Salvayre R. et al.The C-terminal region of human adipose triglyceride lipase enzyme activity and lipid droplet Biol. Chem. 2008; Scholar). et that of the C-terminal region of human ATGL with desnutrin in vitro activity of the of the C-terminal region of desnutrin with human ATGL activity M. Schoiswohl G. Lass A. Radner F.P.W. Haemmerle G. Malli R. Wolfgang G. Cornaciu I. Oberer M. Salvayre R. et al.The C-terminal region of human adipose triglyceride lipase enzyme activity and lipid droplet Biol. Chem. 2008; Scholar). The C-terminal region of human the a of with desnutrin. of the spanning amino acids and and the amino acid residues at the of the human may provide the of the C-terminal the in vitro studies that the N-terminal region contains all residues for catalytic activity of we investigated the of predicted functional motifs in this domain for TAG breakdown in cells. We first the requirement of the N-terminal region for of desnutrin the lipid Lipid are complex of a lipid in TAG with a as well as R. M. Y. G. Y. Liu P. activity of lipid phosphorylation and Res. 2007; Scholar). of desnutrin to the of lipid complex and interactions for of the lipase to this as well as for to of residues to TAG substrate in the the domain for lipid droplet localization in cells may be from the catalytic for TAG and mutations in domain may desnutrin activity To the catalytic N-terminal domain is for lipid droplet we cells with desnutrin lacking the N-terminal region and found that it and lipid in cells The C-terminal domain is for lipid droplet the N-terminal region and patatin-like domain contains the TAG substrate binding and hydrolysis sites that are critical for catalytic activity but are not necessary for lipid droplet We the requirement of predicted functional amino acids for TAG hydrolysis by desnutrin in cells. has requirement of S47 for activity in vitro (10Lake A.C. Sun Y. Li J.L. Kim J.E. Johnson J.W. Li D. Revett T. Shih H.H. Liu W. Paulsen J.E. et al.Expression, regulation, and triglyceride hydrolase activity of Adiponutrin family members.J. Lipid Res. 2005; 46: 2477-2487Google Scholar), the D166 residue has not been has the of site been in live cells. we that both sites are critical for TAG hydrolysis in cells and in vitro of desnutrin and human ATGL that two motifs in the N-terminal region are highly The first is a glycine-rich to amino acids This motif of residues in the where a and the is domain of and Scholar). This has been identified in as a binding motif The binding a of binding J. Scholar), but it is also found in A2 where it the domain of and Scholar, A. J. H. M. J. of human phospholipase A2 reveals a novel and catalytic Scholar). We found that impaired TAG breakdown in cells to activity however, and similar TAG breakdown to that these sites are not critical in cells. In vitro, of the sites to be for activity suggesting that the residue may an role in TAG hydrolysis in cells, by in interactions with other The motif of the to amino acids that is a of the where residue and a residue This motif is predicted to form an amphipathic α-helix in binding of lipid substrates and is found in a of enzymes with in lipid TAG which is a TAG lipase J. and of and human in The region is for both binding and of Scholar, M. D. R. Mutation of but not or in the lipid binding domain the activity of triacylglycerol Lipid Res. 2006; Scholar). that the at position is critical to TAG breakdown by desnutrin in cells, of TAG breakdown to levels the other or mutations TAG breakdown to of desnutrin levels of alanine were for these in vitro our an important function for the N-terminal region of desnutrin in TAG hydrolysis, and with other lipid enzymes that this region may have a potential role in binding TAG In we to critical and residues of desnutrin for TAG hydrolysis in cells. We that the C-terminal region of desnutrin is for TAG breakdown in live cells but not for hydrolase activity in vitro, where lacking the C-terminal region a similar but apparent Vmax to the full-length we found binding of the C-terminal region of desnutrin to the N-terminal which the role of the C-terminal domain activity in vitro. of phosphorylation at S406 or S430 not localization or TAG hydrolysis by desnutrin and not to activity of C-terminal in cells. point we the critical of D166 in the catalytic dyad of desnutrin as well as the of both S47 and D166 sites to TAG hydrolysis by desnutrin in cells. mutation of amino acids in the N-terminal region also the of important residues in an amphipathic α-helix that may form a neutral lipid We for with adipose triglyceride lipase neutral lipid storage disease with myopathy triacylglycerol

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