Human acetyl-CoA carboxylase 2 expressed in silkworm Bombyx mori exhibits posttranslational biotinylation and phosphorylation.

In-Wook Hwang,Sungjo Park,Enoch Y Park,Yu Makishima,Andre Terzic,Tatsuya Kato

doi:10.1007/s00253-014-5715-6

Abstract

Biotin-dependent human acetyl-CoA carboxylases (ACCs) are integral in homeostatic lipid metabolism. By securing posttranslational biotinylation, ACCs perform coordinated catalytic functions allosterically regulated by phosphorylation/dephosphorylation and citrate. The production of authentic recombinant ACCs is heeded to provide a reliable tool for molecular studies and drug discovery. Here, we examined whether the human ACC2 (hACC2), an isoform of ACC produced using the silkworm BmNPV bacmid system, is equipped with proper posttranslational modifications to carry out catalytic functions as the silkworm harbors an inherent posttranslational modification machinery. Purified hACC2 possessed genuine biotinylation capacity probed by biotin-specific streptavidin and biotin antibodies. In addition, phosphorylated hACC2 displayed limited catalytic activity whereas dephosphorylated hACC2 revealed an enhanced enzymatic activity. Moreover, hACC2 polymerization, analyzed by native page gel analysis and atomic force microscopy imaging, was allosterically regulated by citrate and the phosphorylation/dephosphorylation modulated citrate-induced hACC2 polymerization process. Thus, the silkworm BmNPV bacmid system provides a reliable eukaryotic protein production platform for structural and functional analysis and therapeutic drug discovery applications implementing suitable posttranslational biotinylation and phosphorylation.

Highlights

Acetyl-CoA carboxylases (ACCs) are biotin-dependent enzymes catalyzing the production of malonyl-CoA from acetyl-CoA, a critical metabolic intermediate in lipid metabolism (Brownsey et al 2006; Kim 1997; Saggerson 2008; Tong 2013; Wakil and Abu-Elheiga 2009)
Human ACC2 is a large polypeptide comprised of a mitochondrial attachment domain, a mitochondrial target sequence domain, a biotin carboxylase domain, a biotin carboxyl carrier protein domain, and a carboxyltransferase domain (Fig. 1a) (Bianchi et al 1990; Tanabe et al 1975; Tong 2013)
The biotin binding residue in human ACC2 has not been clearly identified, yet structural studies using nuclear magnetic resonance suggests that biotin is attached to lysine 929 within a biotin carboxyl carrier protein domain (BCCP) domain (Lee et al 2008)

Summary

Introduction

Acetyl-CoA carboxylases (ACCs) are biotin-dependent enzymes catalyzing the production of malonyl-CoA from acetyl-CoA, a critical metabolic intermediate in lipid metabolism (Brownsey et al 2006; Kim 1997; Saggerson 2008; Tong 2013; Wakil and Abu-Elheiga 2009). ACC1, encoded by ACACA, predominantly exists in the cytosol of lipogenic organs such as adipose tissue and liver where malonyl-CoA functions as a substrate for long chain fatty acids synthesis. ACACB-encoded ACC2 is associated with the outer membrane of mitochondria in oxidative tissues such as the heart, liver, and skeletal muscle where malonyl-CoA is utilized as a negative regulator of fatty acid oxidation. ACC2 knockout demonstrates anti-obesity effects and prevention of cardiac remodeling (Abu-Elheiga et al 2001, 2003; Kolwicz et al 2012). ACC activity regulation has been recognized as an attractive therapeutic target for dysregulated lipid metabolism such as obesity, diabetes, cancer, and cardiovascular disease (Tong and Harwood 2006)

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