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. 2019 Sep 20:6:2242-2247.
doi: 10.1016/j.mex.2019.09.021. eCollection 2019.

A simple method to disrupt and restore subunit interaction of acyl-CoA:cholesterol acyltransferase 1

Bryan Neumann  1 , Catherine C Y Chang  1 , Ta-Yuan Chang  1
Affiliations

A simple method to disrupt and restore subunit interaction of acyl-CoA:cholesterol acyltransferase 1

Bryan Neumann et al. MethodsX. .

Abstract

Acyl-CoA:cholestereol acyltransferase 1 (ACAT1) is a two-fold dimer (homotetramer) and has two distinct dimerization domains. One domain is in an alpha-helical rich region near the cytoplasmic N-terminus. The other is proposed to be near the C-terminus where multiple transmembrane domains promote hydrophobic interactions between two ACAT1 subunits. The truncation of the ACAT1 N-terminal dimerization domain, Δ1-65, creates a dimer which is fully enzymatically active. It is currently not known how the C-terminal dimerization domain contributes to ACAT1 enzymatic activity. Here we describe a simple method that dissociates ACAT1 dimers through the addition of the non-ionic detergents Triton X-100 or octyl glucoside which disrupt the C-terminal dimerization domain. We also document the protocols for a method to exchange Triton X-100 with CHAPS to restore C-terminal dimerization of the ACAT1 protein, and an optimized liposomal assay to assess ACAT enzymatic activity. •This method can be applied to dissociate ACAT1 subunits by using Triton X-100 or octyl glucoside.•ACAT1 dimerization can be restored by exchanging Triton X-100 with CHAPS.•The liposomal ACAT activity assay conditions have been optimized.

Keywords: ACAT1; Cholesterol; Detergent; Dissociation of ACAT1 subunits by non-ionic detergents; Enzyme activity; Liposomes; MBOAT; Quaternary structure; SOAT1; Transmembrane; Two-fold dimer.

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Figures

None
Graphical abstract
Fig. 1
Fig. 1
Schematic diagram of the optimized liposomal ACAT activity assay.
See this image and copyright information in PMC

References

    1. Chang C.C., Lee C.Y., Chang E.T., Cruz J.C., Levesque M.C., Chang T.Y. Recombinant acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesicles in a highly cooperative manner. J. Biol. Chem. 1998;273(52):35132–35141. - PubMed
    1. Kaduce T.L., Schmidt R.W., Spector A.A. Acylcoenzyme A:cholesterol acyltransferase activity: solubilization and reconstitution in liposomes. Biochem. Biophys. Res. Commun. 1978;81(2):462–468. - PubMed
    1. Neumann B., Chang C.C.Y., Chang T.Y. Triton X-100 or octyl glucoside inactivates acyl-CoA:cholesterol acyltransferase 1 by dissociating it from a two-fold dimer to a two-fold monomer. Arch. Biochem. Biophys. 2019;671:103–110. - PMC - PubMed
    1. Yu C., Zhang Y., Lu X., Chen J., Chang C.C., Chang T.Y. Role of the N-terminal hydrophilic domain of acyl-coenzyme A:cholesterol acyltransferase 1 on the enzyme’s quaternary structure and catalytic efficiency. Biochemistry. 2002;41(11):3762–3769. - PubMed
    1. Bishop J.E., Hajra A.K. A method for the chemical synthesis of 14C-labeled fatty acyl coenzyme A’s of high specific activity. Anal. Biochem. 1980;106(2):344–350. - PubMed

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