E6AP/UBE3A catalyzes encephalomyocarditis virus 3C protease polyubiquitylation and promotes its concentration reduction in virus-infected cells.

The encephalomyocarditis virus (EMCV) 3C protease (3Cpro) is one of a small number of viral proteins whose concentration is known to be regulated by the cellular ubiquitin-proteasome system. Here we report that the ubiquitin-conjugating enzyme UbcH7/UBE2L3 and the ubiquitin-protein ligase E6AP/UBE3A are components of a previously unknown EMCV 3Cpro-polyubiquitylating pathway. Following the identification of UbcH7/UBE2L3 as a participant in 3Cpro ubiquitylation, we purified a UbcH7-dependent 3Cpro-ubiquitylating activity from mouse cells, which we identified as E6AP. In vitro reconstitution assays demonstrated that E6AP catalyzes the synthesis of 3Cpro-attached Lys48-linked ubiquitin chains, known to be recognized by the 26S proteasome. We found that the 3Cpro accumulates to higher levels in EMCV-infected E6AP knockdown cells than in control cells, indicating a role for E6AP in in vivo 3Cpro concentration regulation. We also discovered that ARIH1 functions with UbcH7 to catalyze EMCV 3Cpro monoubiquitylation, but this activity does not influence the in vivo 3Cpro concentration.

The ubiquitin-protein ligase E6AP/UBE3A supports early encephalomyocarditis virus replication.

Many viruses make use of, and even direct, the ubiquitin-proteasome system to facilitate the generation of a cellular environment favorable for virus replication, while host cells use selected protein ubiquitylation pathways for antiviral defense. Relatively little information has been acquired, however, regarding the extent to which protein ubiquitylation determines the replication success of picornaviruses. Here we report that the ubiquitin-protein ligase E6AP/UBE3A, recently shown to be a participant in encephalomyocarditis virus (EMCV) 3C protease concentration regulation, also facilitates the early stages of EMCV replication, probably by a mechanism that does not involve 3C protease ubiquitylation. Using stably transfected E6AP knockdown cells, we found that reduced E6AP concentration extends the time required for infected cells to undergo the morphological changes caused by virally induced pathogenesis and to begin the production of infectious virions. This lag in virion production is accompanied by a corresponding delay in the appearance of detectable levels of viral proteins and RNA. We also found, by using both immunofluorescence microscopy and cell fractionation, that E6AP is partially redistributed from the nucleus to the cytoplasm in EMCV-infected cells, thereby increasing its availability to participate in cytoplasmic virus replication processes.

Diabetic cardiomyocyte dysfunction and myocyte insulin resistance: role of glucose-induced PKC activity.

Increased protein kinase C (PKC) activity has been implicated in the pathogenesis of a number of diabetic complications, and high concentrations of glucose have been shown to increase PKC activity. The present study was designed to examine the role of PKC in diabetes-induced (and glucose-induced) cardiomyocyte dysfunction and insulin resistance (measured by glucose uptake). Adult rat ventricular myocytes were isolated from nondiabetic and type 1 diabetic animals (4-5 days post-streptozotocin treatment), and maintained overnight, with/without the nonspecific PKC inhibitor chelerythrine (CHEL = 1 microM). Myocyte mechanical properties were evaluated using a video edge-detection system. Basal and insulin-stimulated glucose uptake was measured with [3H]-2-deoxyglucose. Blunted insulin-stimulated glucose uptake was apparent in diabetic myocytes, and both mechanical dysfunctions (e.g., slowed shortening/relengthening) and insulin resistance were maintained in culture, and normalized by CHEL. Cardiomyocytes isolated from nondiabetic animals were cultured in a high concentration of glucose (HG = 25.5 mM) medium, with/without CHEL. HG myocytes exhibited slowed shortening/relengthening and impaired insulin-stimulated glucose uptake compared to myocytes cultured in normal glucose (5.5 mM), and both impairments were prevented by culturing cells in CHEL. Our data support the view that PKC activation contributes to both diabetes-induced abnormal cardiomyocyte mechanics and insulin resistance, and that elevated glucose is sufficient to induce these effects.

Depressed PKA activity contributes to impaired SERCA function and is linked to the pathogenesis of glucose-induced cardiomyopathy.

We have previously described a cardiomyopathy induced by culturing ventricular myocytes from normal adult rats in a medium containing high concentrations of glucose, which recapitulates cellular changes associated with early onset diabetic cardiomyopathy. This investigation was designed to evaluate cellular mechanisms that could contribute to slowed cytosolic Ca(2+) removal and myocyte relaxation in glucose-induced cardiomyopathy. Isolated ventricular myocytes were cultured overnight in medium containing normal glucose (n=5.5mM) or high glucose (HG=25.5mM). Cytosolic Ca(2+) removal was monitored with fluo-3 and myocyte mechanics with video-edge detection. Electrically stimulated Ca(2+) transients were prolonged in HG cells (A(T/PK)=215+/-7ms, n=41) compared to N myocytes (A(T/PK)=173+/-5ms, n=34). By pharmacological and ionic manipulations, Ca(2+) removal attributable to SERCA was slower in the HG group (A(D/PK)=290+/-17ms,n =41) compared to N (A(D/PK)=219+/-10, n=34), whereas NCX function was similar in both groups of cells. Total PKA activity was depressed in HG myocytes by 56% compared to N cells. beta-adrenergic receptor stimulation with ISO (10(-7)M) normalized myocyte relaxation, Ca(2+) transients and PKA activity in HG myocytes. Furthermore, inhibition of PKA with H89 (10(-5)M) depressed peak fractional shortening (PS) and slowed relengthening (A(R/PK)) to a greater extent in N (-50% for PS and 92% for A(R/PK)) than in HG cells (-25% for PS and 48% A(R/PK)). Depressed cytosolic Ca(2+) removal was not, however, associated with changes in basal levels of phosphorylated PLB, nor levels of SERCA, NCX or PLB proteins. We conclude that cellular mechanisms associated with the early onset glucose-induced cardiomyocyte dysfunction involves alterations in Ca(2+) regulation, which may be a common manifestation of other forms of cardiomyopathies.

Sucrose-induced cardiomyocyte dysfunction is both preventable and reversible with clinically relevant treatments.

We recently identified cardiomyocyte dysfunction in the early stage of type 2 diabetes (i.e., diet-induced insulin resistance). The present investigation was designed to determine whether a variety of clinically relevant interventions are sufficient to prevent and reverse cardiomyocyte dysfunction in sucrose (SU)-fed insulin-resistant rats. Subsets of animals were allowed to exercise (free access to wheel attached to cage) or were treated with bezafibrate in drinking water to determine whether these interventions would prevent the adverse effects of SU feeding on cardiomyocyte function. After 6-8 wk on diet and treatment, animals were surgically prepared to assess whole body insulin sensitivity (intravenous glucose tolerance test), and isolated ventricular myocyte mechanics were evaluated (video edge recording). SU feeding produced hyperinsulinemia and hypertriglyceridemia, with euglycemia, and induced characteristic whole body insulin resistance. Both exercise and bezafibrate treatment prevented these metabolic abnormalities. Ventricular myocyte shortening and relengthening were slower in SU-fed rats (42-63%) compared with starch (ST)-fed controls, and exercise or bezafibrate completely prevented cardiomyocyte dysfunction in SU-fed rats. In separate cohorts of animals, after 5 wk of SU feeding, animals were either switched back to an ST diet or given menhaden oil for an additional 7-9 wk to determine whether the cardiomyocyte dysfunction was reversible. Both interventions have previously been shown to have favorable metabolic effects, and both improved myocyte mechanics, but only the ST diet reversed all indications of cardiomyocyte dysfunction induced by SU feeding. Thus phenotypic changes in cardiomyocyte mechanics associated with early stages of type 2 diabetes were found to be both preventable and reversible with clinically relevant treatments, suggesting that the cellular processes contributing to this dysfunction are modifiable.