Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells[S]
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,...
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| Format: | Article |
| Language: | English |
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Elsevier
2010-02-01
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| Series: | Journal of Lipid Research |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227520305356 |
| _version_ | 1818740365370327040 |
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| author | Robin E. Duncan Yuhui Wang Maryam Ahmadian Jennifer Lu Eszter Sarkadi-Nagy Hei Sook Sul |
| author_facet | Robin E. Duncan Yuhui Wang Maryam Ahmadian Jennifer Lu Eszter Sarkadi-Nagy Hei Sook Sul |
| author_sort | Robin E. Duncan |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-18T01:39:34Z |
| format | Article |
| id | doaj.art-f4a66ff2d0ea45d0ab08f6626bbc2da1 |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-18T01:39:34Z |
| publishDate | 2010-02-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-f4a66ff2d0ea45d0ab08f6626bbc2da12022-12-21T21:25:22ZengElsevierJournal of Lipid Research0022-22752010-02-01512309317Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells[S]Robin E. Duncan0Yuhui Wang1Maryam Ahmadian2Jennifer Lu3Eszter Sarkadi-Nagy4Hei Sook Sul5Department of Nutritional ScienceToxicology, University of California, Berkeley, CA 94720Department of Nutritional ScienceToxicology, University of California, Berkeley, CA 94720Department of Nutritional ScienceToxicology, University of California, Berkeley, CA 94720Department of Nutritional ScienceToxicology, University of California, Berkeley, CA 94720Department of Nutritional ScienceToxicology, University of California, Berkeley, CA 94720To whom correspondence should be addressed; Department of Nutritional ScienceToxicology, University of California, Berkeley, CA 94720Murine 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.http://www.sciencedirect.com/science/article/pii/S0022227520305356adipose triglyceride lipasemutantlipid substrate binding domainC-terminal regionN-terminal regionneutral lipid storage disease with myopathy |
| spellingShingle | Robin E. Duncan Yuhui Wang Maryam Ahmadian Jennifer Lu Eszter Sarkadi-Nagy Hei Sook Sul Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells[S] Journal of Lipid Research adipose triglyceride lipase mutant lipid substrate binding domain C-terminal region N-terminal region neutral lipid storage disease with myopathy |
| title | Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells[S] |
| title_full | Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells[S] |
| title_fullStr | Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells[S] |
| title_full_unstemmed | Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells[S] |
| title_short | Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells[S] |
| title_sort | characterization of desnutrin functional domains critical residues for triacylglycerol hydrolysis in cultured cells s |
| topic | adipose triglyceride lipase mutant lipid substrate binding domain C-terminal region N-terminal region neutral lipid storage disease with myopathy |
| url | http://www.sciencedirect.com/science/article/pii/S0022227520305356 |
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