13 research outputs found
A Novel Conserved Isoform of the Ubiquitin Ligase UFD2a/UBE4B Is Expressed Exclusively in Mature Striated Muscle Cells
Yeast Ufd2p was the first identified E4 multiubiquitin chain assembly factor. Its vertebrate homologues later referred to as UFD2a, UBE4B or E4B were also shown to have E3 ubiquitin ligase activity. UFD2a function in the brain has been well established in vivo, and in vitro studies have shown that its activity is essential for proper condensation and segregation of chromosomes during mitosis. Here we show that 2 alternative splice forms of UFD2a, UFD2a-7 and -7/7a, are expressed sequentially during myoblast differentiation of C2C12 cell cultures and during cardiotoxin-induced regeneration of skeletal muscle in mice. UFD2a-7 contains an alternate exon 7, and UFD2a-7/7a, the larger of the 2 isoforms, contains an additional novel exon 7a. Analysis of protein or mRNA expression in mice and zebrafish revealed that a similar pattern of isoform switching occurs during developmental myogenesis of cardiac and skeletal muscle. In vertebrates (humans, rodents, zebrafish), UFD2a-7/7a is expressed only in mature striated muscle. This unique tissue specificity is further validated by the conserved presence of 2 muscle-specific splicing regulatory motifs located in the 3′ introns of exons 7 and 7a. UFD2a interacts with VCP/p97, an AAA-type ATPase implicated in processes whose functions appear to be regulated, in part, through their interaction with one or more of 15 previously identified cofactors. UFD2a-7/7a did not interact with VCP/p97 in yeast 2-hybrid experiments, which may allow the ATPase to bind cofactors that facilitate its muscle-specific functions. We conclude that the regulated expression of these UFD2a isoforms most likely imparts divergent functions that are important for myogenisis
Oxygen-induced maturation of SOD1: a key role for disulfide formation by the copper chaperone CCS
The antioxidant enzyme Cu,Zn-superoxide dismutase (SOD1) has the distinction of being one of the most abundant disulfide-containing protein known in the eukaryotic cytosol; however, neither catalytic nor physiological roles for the conserved disulfide are known. Here we show that the disulfide status of Saccharomyces cerevisiae SOD1 significantly affects the monomer–dimer equilibrium, the interaction with the copper chaperone CCS, and the activity of the enzyme itself. Disulfide formation in SOD1 by O(2) is slow but is greatly accelerated by the Cu-bound form of CCS (Cu-CCS) in vivo and in vitro even in the presence of excess reductants; once formed, this disulfide is kinetically stable. Biochemical assays reveal that Cu-CCS facilitates Cys oxidation and disulfide isomerization in the stepwise conversion of the immature form of the enzyme to the active state. The immature form of SOD1 is most susceptible to oxidative insult and to aggregation reminiscent of that observed in amyotrophic lateral sclerosis. Thus Cu-CCS mediation of correct disulfide formation in SOD1 is important for regulation of enzyme activity and for prevention of misfolding or aggregation
Mitochondrial DNA damage associated with lipid peroxidation of the mitochondrial membrane induced by Fe2+-citrate
Iron imbalance/accumulation has been implicated in oxidative injury associated with many degenerative diseases such as hereditary hemochromatosis, beta-thalassemia, and Friedreich's ataxia. Mitochondria are particularly sensitive to iron-induced oxidative stress - high loads of iron cause extensive lipid peroxidation and membrane permeabilization in isolated mitochondria. Here we detected and characterized mitochondrial DNA damage in isolated rat liver mitochondria exposed to a Fe2+-citrate complex, a small molecular weight complex. Intense DNA fragmentation was induced after the incubation of mitochondria with the iron complex. The detection of 3' phosphoglycolate ends at the mtDNA strand breaks by a 32P-postlabeling assay, suggested the involvement of hydroxyl radical in the DNA fragmentation induced by Fe2+-citrate. Increased levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine also suggested that Fe2+-citrate-induced oxidative stress causes mitochondrial DNA damage. In conclusion, our results show that iron-mediated lipid peroxidation was associated with intense mtDNA damage derived from the direct attack of reactive oxygen species.<br>DesequilĂbrio/acĂşmulo de ferro tem sido implicado em injĂşria oxidativa associada a diversas doenças degenerativas tais como, hemocromatose hereditária, beta-talassemia e ataxia de Friedreich. As mitocĂ´ndrias sĂŁo particularmente sensĂveis a estresse oxidativo induzido por ferro - um carregamento alto de ferro em mitocĂ´ndrias isoladas pode causar uma extensiva peroxidação lipĂdica e a permeabilização de membrana. Nesse estudo, nĂłs detectamos e caracterizamos danos do DNA mitocondrial em mitocĂ´ndrias isoladas de fĂgado de rato, expostas ao complexo Fe2+-citrato, um dos complexos de baixo peso molecular. A intensa fragmentação do DNA foi induzida apĂłs a incubação das mitocĂ´ndrias com o complexo de ferro. A detecção de finais 3' de fosfoglicolato nas quebras de fitas de DNA mitocondrial pelo ensaio 32P-postlabeling sugere um envolvimento de radicais hidroxila na fragmentação do DNA induzido por complexo Fe2+-citrato. Os nĂveis elevados de 8-oxo-7,8-diidro-2'-desoxiguanosina tambĂ©m sugerem que o estresse oxidativo induzido por Fe2+-citrato causa danos no DNA mitocondrial. Em conclusĂŁo, nossos resultados mostram que a peroxidação lipĂdica mediada por ferro esteve associada com severos danos do DNA mitocondrial derivados de ataque direto das espĂ©cies reativas de oxigĂŞnio