Handling of human short-chain acyl-CoA dehydrogenase (SCAD) variant proteins in transgenic mice Peter M. Kragh a , Christina B. Pedersen b , Stinne P. Schmidt b , Vibeke S. Winter b , Ga ´bor Vajta c , Niels Gregersen b , Lars Bolund a , Thomas J. Corydon a, * a Institute of Human Genetics, University of Aarhus, DK-8000 Aarhus C, Denmark b Research Unit for Molecular Medicine, Aarhus University Hospital, Skejby Sygehus, DK-8200 Aarhus N, Denmark c Section of Population Genetics and Embryology, Department of Genetics and Biotechnology, University of Aarhus, DK-8830 Tjele, Denmark Received 12 March 2007; accepted 12 March 2007 Available online 25 April 2007 Abstract To investigate the in vivo handling of human short-chain acyl-CoA dehydrogenase (SCAD) variant proteins, three transgenic mouse lines were produced by pronuclear injection of cDNA encoding the wild-type, hSCAD-wt, and two disease causing folding variants hSCAD-319C > T and hSCAD-625G > A. The transgenic mice were mated with an SCAD-deficient mouse strain (BALB/cByJ) and, in the second generation, three mouse lines were obtained without endogenous SCAD expression but harboring hSCAD-wt, hSCAD- 319C > T, and hSCAD-625G > A transgenes, respectively. All three lines had expression of the transgene at the RNA level in liver, mus- cle or brain tissues. Expression at the protein level was detected only in the brain tissue of hSCAD-wt mice, but here it was significantly higher than the level of endogenous SCAD protein in control mouse brains—in correlation with expression at the RNA level. The results may indicate that the two hSCAD folding variants are degraded by the mouse mitochondrial protein quality control system. Indeed, pulse-chase studies with isolated mitochondria revealed that soluble variant hSCAD protein was rapidly eliminated. This is in agreement with the fact that no disease phenotype developed for any of the lines transgenic for the hSCAD folding variants. The indicated remark- able efficiency of the mouse protein quality control system in the degradation of SCAD folding variants should be further substantiated and investigated, since it might indicate ways to prevent disease-causing effects. Ó 2007 Elsevier Inc. All rights reserved. Keywords: SCAD deficiency; ROSA26 promoter; Transgenic model; Mitochondrial degradation; Multifactorial disease; Pronuclear injection; Mouse model; In vivo expression; ROSA26; b-Oxidation; Folding variants; Degradation; Accumulation; Susceptibility gene Introduction Protein misfolding diseases are a group of genetic dis- eases where the folding process of a protein is disturbed [1]. Mutations causing these diseases are mainly missense mutations, short in-frame deletions and insertions. Such mutations may increase the proportion of mutant polypep- tide present in non-functional conformations that are more susceptible to degradation or aggregation than the func- tional conformation. During the folding process of newly synthesized polypeptides cellular factors safeguard the pro- tein chains until they find their right conformation and sort out the polypeptides with an insufficient ability to fold. Such factors are collectively referred to as the ‘‘protein quality control system’’ and comprise molecular chaper- ones, proteases, as well as other factors. A common aspect of chaperones is that they interact with proteins containing a non-native conformation and orchestrate their folding process. In contrast, proteases eliminate improper con- formers by rapid degradation. These systems may have a decisive role in determining the fate and final level of both functional and non-fuctional mutant protein [2]. Recent studies have demonstrated the presence of a specialized mitochondrial protein quality control system that triggers 1096-7192/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2007.03.005 * Corresponding author. Fax: +45 8612 3173. E-mail address: tjc@humgen.au.dk (T.J. Corydon). www.elsevier.com/locate/ymgme Molecular Genetics and Metabolism 91 (2007) 128–137