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Conjugation of ubiquitin and other Class 1 ubiquitin-like polypeptides to specific protein targets serves diverse regulatory functions in eukaryotes. The obligatory first step of conjugation requires ATP-coupled activation of the ubiquitin-like protein by members of a superfamily of evolutionarily related enzymes. Kinetic and equilibrium studies of the human ubiquitin-activating enzyme (HsUba1a) reveal that mutations within the ATP·Mg2+ binding site have remarkably pleiotropic effects on the catalytic phenotype of the enzyme. Mutation of Asp576 or Lys528 results in dramatically impaired binding affinities for ATP·Mg2+, a shift from ordered to random addition in co-substrate Binding, and a significantly reduced rate of ternary complex formation that shifts the rate-limiting step to ubiquitin adenylate formation. Mutations at neither position affect the affinity of HsUbc2b binding; however, differences in kcat values determined from ternary complex formation versus HsUbc2b transthiolation suggest that binding of the E2 enhances the rate of bound ubiquitin adenylate formation. These results confirm that Asp576 and Lys528 are important for ATP·Mg2+ binding but are essential catalytic groups for ubiquitin adenylate transition state stabilization. The latter mechanistic effect explicates the observed loss-of-function phenotype associated with mutation of residues paralogous to Asp576 within the activating enzymes for other ubiquitin-like proteins. [ABSTRACT FROM AUTHOR]

Describes the protein interactions within the n-ends ubiquitin ligation pathway. Employment of rate studies as a reporter function to probe protein-protein interactions; Presence of free ubiquitins in cells; Analysis of protein interactions within a ubiquitin ligation pathway.

Current evidence shows that cardiomyocyte apoptosis plays a central role in the pathogenesis of myocardial disease and that reactive oxygen species is critically responsible for mediating cardiomyocyte apoptosis in both ischemia-reperfusion injury and dilated cardiomyopathy. ARC (Apoptosis Repressor with Caspase recruitment domain) is an anti-apoptotic protein that is found abundantly in terminally differentiated cells such as cardiomyocytes. The ARC knock-out mouse developed larger infarct in response to ischemia-reperfusion and transitioned more rapidly and severely to dilated cardiomyopathy following aortic constriction. In addition, ARC protein levels are decreased in human dilated cardiomyopathy and when cardiomyocytes are exposed to oxidative stress in vitro, but the mechanisms regulating ARC protein levels are not known. Here we show that degradation of ARC is dependent on the p53-induced ubiquitin E3 ligase, MDM2. Oxidative stress reduced ARC levels and up-regulated MDM2. MDM2 directly accelerated ARC protein turnover via ubiquitination and proteasomal-dependent degradation. This activity requires a functioning MDM2 ring finger domain because the MDM2C~4A mutant was unable to direct ARC degradation. Furthermore, ARC degradation requires MDM2, because MDM2 knock-out fibroblasts showed defective ARC degradation that could be rescued by MDM2, Proteasomal inhibitors rescued both MDM2 and H2O2-induced degradation of ARC and inhibited cardiomyocyte apoptosis. Dilated cardiomyopathic hearts from mice that have undergone transverse aortic banding have increased MDM2 levels associated with decreased ARC levels. We conclude that MDM2 is a critical regulator of ARC levels in cardiomyocytes. Prevention of MDM2-induced degradation of ARC represents a potential therapeutic target to prevent cardiomyocyte apoptosis. [ABSTRACT FROM AUTHOR]

Efficient induction of apoptosis requires not only the activation of death-promoting proteins but also the inactivation of inhibitors of cell death. ARC (apoptosis repressor with caspase recruitment domain) is an endogenous inhibitor of apoptosis that antagonizes both central apoptosis pathways. Despite its potent inhibition of cell death, cells that express abundant ARC eventually succumb. A possible explanation is that ARC protein levels decrease dramatically in response to death stimuli. The mechanisms that mediate decreases in ARC protein levels during apoptosis and whether these decreases initiate the subsequent cell death are not known. Here we show that endogenous ARC protein levels decrease in response to death stimuli in a variety of cell contexts as well as in a model of myocardial ischemia-reperfusion in intact mice. Decreases in ARC protein levels are not explained by alterations in the abundance of ARC transcripts. Rather, pulse-chase experiments show that decreases in steady state ARC protein levels during apoptosis result from marked destabilization of ARC protein. ARC protein destabilization, in turn, is mediated by the ubiquitin-proteasomal pathway, as mutation of ARC ubiquitin acceptor residues stabilizes ARC protein and preserves its steady state levels during apoptosis. In addition, this degradation-resistant ARC mutant exhibits improved cytoprotection. We conclude that decreases in ARC protein levels in response to death stimuli are mediated by increased ARC protein degradation via the ubiquitin-proteasomal pathway. Moreover, these data demonstrate that decreases in ARC protein levels are a trigger, and not merely a consequence, of the ensuing cell death. [ABSTRACT FROM AUTHOR]