Akash Das

Wake Forest University, Winston-Salem, North Carolina, United States

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Publications (3)13.42 Total impact

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    Akash Das · Matthew A Davis · Lawrence L Rudel ·
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    ABSTRACT: In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.
    The Journal of Lipid Research 09/2008; 49(8):1770-81. DOI:10.1194/jlr.M800131-JLR200 · 4.42 Impact Factor
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    ABSTRACT: Targeted deletion of acyl-CoA:cholesterol acyltransferase 2 (ACAT2) (A2), especially in the liver, protects hyperlipidemic mice from diet-induced hypercholesterolemia and atherosclerosis, whereas the deletion of ACAT1 (A1) is not as effective, suggesting ACAT2 may be the more appropriate target for treatment of atherosclerosis. Among the numerous ACAT inhibitors known, pyripyropene A (PPPA) is the only compound that has high selectivity (>2000-fold) for inhibition of ACAT2 compared with ACAT1. In the present study we sought to determine the PPPA interaction site of ACAT2. To achieve this goal we made several chimeric proteins where parts of ACAT2 were replaced by the analogous region of ACAT1. Differences in the amino acid sequence and the membrane topology were utilized to design the chimeras. Among chimeras, A2:1-428/A1:444-550 had 50% reduced PPPA selectivity, whereas C-terminal-truncated ACAT2 mutant A2:1-504 (C-terminal last 22 amino acids were deleted) remained selectively inhibited, indicating the PPPA-sensitive site is located within a region between amino acids 440 and 504. Three additional chimeras within this region helped narrow down the PPPA-sensitive site to a region containing amino acids 480-504, representing the fifth putative transmembrane domain of ACAT2. Subsequently, for this region we made single amino acid mutants where each amino acid in ACAT2 was individually changed to its ACAT1 counterpart. Mutation of Q492L, V493L, S494A resulted in only 30, 50, and 70% inhibition of the activity by PPPA, respectively (as opposed to greater than 95% with the wild type enzyme), suggesting these three residues are responsible for the selective inhibition by PPPA of ACAT2. Additionally, we found that PPPA non-covalently interacts with ACAT2 apparently without altering the oligomeric structure of the protein. The present study provides the first evidence for a unique motif in ACAT2 that can be utilized for making an ACAT2-specific drug.
    Journal of Biological Chemistry 05/2008; 283(16):10453-60. DOI:10.1074/jbc.M709460200 · 4.57 Impact Factor
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    ABSTRACT: Comparative Gene Identification-58 (CGI-58) is a member of the alpha/beta-hydrolase family of proteins. Mutations in the human CGI-58 gene are associated with Chanarin-Dorfman syndrome, a rare autosomal recessive genetic disease in which excessive triglyceride (TG) accumulation occurs in multiple tissues. In this study, we investigated the role of CGI-58 in cellular lipid metabolism in several cell models and discovered a role for CGI-58 in promoting the packaging of cytoplasmic TG into secreted lipoprotein particles in hepatoma cells. Using both gain-of-function and loss-of-function approaches, we demonstrate that CGI-58 facilitates the depletion of cellular TG stores without altering cellular cholesterol or phospholipid accumulation. This depletion of cellular TG is attributable solely to augmented hydrolysis, whereas TG synthesis was not affected by CGI-58. Furthermore, CGI-58-mediated TG hydrolysis can be completely inhibited by the known lipase inhibitors diethylumbelliferyl phosphate and diethyl-p-nitrophenyl phosphate, but not by p-chloro-mercuribenzoate. Intriguingly, CGI-58-driven TG hydrolysis was coupled to increases in both fatty acid oxidation and secretion of TG. Collectively, this study reveals a role for CGI-58 in coupling lipolytic degradation of cytoplasmic TG to oxidation and packaging into TG-rich lipoproteins for secretion in hepatoma cells.
    The Journal of Lipid Research 11/2007; 48(10):2295-305. DOI:10.1194/jlr.M700279-JLR200 · 4.42 Impact Factor

Publication Stats

63 Citations
13.42 Total Impact Points


  • 2008
    • Wake Forest University
      • Department of Biochemistry
      Winston-Salem, North Carolina, United States
  • 2007-2008
    • Wake Forest School of Medicine
      • Department of Biochemistry
      Winston-Salem, North Carolina, United States