Item Details

title

A COMPARATIVE ANALYSIS OF INTRACELLULAR CHOLESTEROL ESTERIFYING ENZYMES IN MAMMALS

author

Das, Akash

abstract

In higher eukaryotes, intracellular cholesterol esterification is catalyzed by two isoforms of acyl-CoA:cholesterol O-acyltransferase (ACAT) enzymes. Functional studies indicate that selective inhibition of ACAT2 prevents progression of atherosclerosis whereas deletion of ACAT1 is deleterious in mice. Due to high sequence similarity, it is difficult to find an isozyme specific inhibitor. Hence, detailed study is required to identify unique structural features of ACAT2 that can be utilized to make ACAT2-specific drugs. In this thesis, three independent studies are presented where the two ACAT enzymes were compared side by side to identify unique isozyme-specific properties. In the first study, putative active site residues of ACAT proteins were identified using chemical modification and site directed mutagenesis approaches. A conserved region within the protein was defined as the putative active site domain based on highly conserved sequence similarities from yeast to humans. Since 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 serine, histidine and aspartic acid residues. Results showed that in ACAT1, S456, H460 and D400 are essential for activity of the enzyme. In contrast, in ACAT2, only the analogous H438 was identified to be necessary for enzymatic activity suggesting that the residues required for ACAT activity may be different between the two ACAT proteins. In the second project, the attributes of ACAT2 that lead to pyripyropene A (PPPA) inhibition was determined. PPPA is the only inhibitor as yet identified that has more than 2000 fold higher specificity for ACAT2 than ACAT1. By making chimeric proteins using both ACAT1 and 2 sequences, three residues, located within the fifth transmembrane domain of ACAT2, were identified as the PPPA interaction site. PPPA interaction is non-covalent and does not alter the oligomeric structure of the protein. In the final project, the effect of protein stability on ACAT activity was investigated. Results showed that in vitro ACAT2 activity is always higher than ACAT1 probably due to high abundance of ACAT2 enzyme protein in the assay system since it is intrinsically a more stable protein than ACAT1. Together all of these studies have greatly enhanced our understanding about structure/function relationships of the two distinct yet highly similar ACAT proteins.

subject

Biochemistry

Molecular Biology

contributor

Shelness, Greg (committee chair)

Rudel, Greg (committee member)

Lyles, Doug (committee member)

McPhail, Linda (committee member)

Lowther, Todd (committee member)

date

2009-08-07T13:09:18Z (accessioned)

2010-06-18T18:58:57Z (accessioned)

2009-08-07T13:09:18Z (available)

2010-06-18T18:58:57Z (available)

2009-08-07T13:09:18Z (issued)

degree

Biochemistry & Molecular Biology (discipline)

identifier

http://hdl.handle.net/10339/14810 (uri)

language

en_US (iso)

publisher

Wake Forest University

rights

Release the entire work immediately for access worldwide. (accessRights)

type

Dissertation