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

Abstract

Triacylglycerol (TAG) serves as the major energy reserve in higher eukaryotic organisms ( 1 ). 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 ( 1-3 ). Hydrolysis of TAG to diacylglycerol is the fi rst step ( 4-6 ), and our laboratory, and others, recently identifi ed a novel TAG hydrolase in mice that we called desnutrin ( 5 ) [also known as TTS2.2, patatin-like phospholipase A domain containing 2, iPLA ( 4 ), or in humans, adipose triglyceride lipase (ATGL) ( 6 )]. 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 ( 5,7,8 ).Desnutrin contains an N-terminal patatin-like domain, spanning amino acids 8-180, that is characteristic of many plant lipid acyl hydrolases ( 5, 9 ). Enzymatic activity of desnutrin is predicted to be derived from an S47-D166 catalytic dyad that lies within an ␣ -␤ hydrolase fold in the patatin-like domain ( 5, 9 ). Mutation of the S47 residue to alanine has been shown to result in a complete loss of function in vitro, confi rming the predicted serine-esterase activity of this enzyme ( 10 ). However, the requirement of the D166 site has not yet been tested. Four individuals with point mutations in desnutrin/ATGL have been identifi ed, and all developed neutral lipid storage disease with myopathy (NLSDM) ( 11,12 ). 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 mutaAbstract Murine desnutrin/human ATGL is a triacylglycerol (TAG) hydrolase with a predicted catalytic dyad within an ␣ -␤ 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 fi nding 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 identifi ed two overlapping N-terminal motifs that predict lipid substrate binding domains, a glycine-rich ...

Pigment Epithelium-derived Factor (PEDF) Prevents Retinal Cell Death via PEDF Receptor (PEDF-R)

“…4) and altering either Ser 47 or Asp 166 abolishes triglyceride lipase activity (44,45). Furthermore, in the present study, the C-terminal truncated variants of PEDF-R demonstrated PLA 2 activity similar to that of full-length PEDF-R.…”

“…we used nanolipoprotein particles to improve the folding of recombinant polypeptides) could explain this discrepancy. Other reports describe the presence of an inhibitory region toward the C-terminal end of PEDF-R (44,47,48). This region does not seem to be inhibitory for PLA 2 activity because the C-terminal truncated variants of this study exhibit PLA 2 activity identical to that of full-length polypeptides.…”

Triacylglycerol lipases and metabolic control: implications for health and disease

“…ATGL activity does not appear to be regulated directly by PKA phosphorylation, although preliminary evidence in mammalian cells indicates that ATGL is phosphorylated by other unknown kinases (84). A phosphoprotein proteomic analysis of purified LDs reported phosphorylation at Ser 404 and Ser 428 of human ATGL (4); however, site-directed mutagenesis of these sites did not affect TG breakdown under basal conditions (20). Hence, the reason for ATGL phosphorylation in cells is currently unresolved, as are the upstream kinases.…”

“…Biochemical evaluation of cell lysates and high-resolution imaging of fluorescence reporters in living cells also show that ATGL resides in the cytoplasm and on lipid droplets (LD) in the basal state (28,84), and cAMP-dependent protein kinase (PKA) activation elicits only minor translocation of ATGL to LDs (28). The COOH-terminal region of ATGL that contains a hydrophobic region is essential for this appropriate cellular localization and association with LDs (45,65), whereas the NH 2 -terminal region (residues 10 -24) appears to be important for TG binding (20). ATGL activity does not appear to be regulated directly by PKA phosphorylation, although preliminary evidence in mammalian cells indicates that ATGL is phosphorylated by other unknown kinases (84).…”

“…The predicted NH 2 -terminal active sites within the ␣/␤ hydrolase fold, S47 and D166, are critical for ATGL-mediated TG hydrolysis ( Fig. 1) (20,48). Biochemical evaluation of cell lysates and high-resolution imaging of fluorescence reporters in living cells also show that ATGL resides in the cytoplasm and on lipid droplets (LD) in the basal state (28,84), and cAMP-dependent protein kinase (PKA) activation elicits only minor translocation of ATGL to LDs (28).…”