We have used RNA interference (RNAi) to examine the functional relationship between valosin-containing protein (VCP/p97/Cdc48p/TER94) ATPase and the ubiquitin-proteasome system (UPS) in Drosophila S2 and human HeLa cells. In both cell types, RNAi of VCP (and, to a lesser extent, of certain VCP-interacting proteins) caused significant accumulation of high-molecular-weight conjugates of ubiquitin, an indication of inhibited UPS function. However, decreased VCP levels did not directly inhibit proteasome activity. In HeLa cells, polyubiquitinated proteins accumulated as dispersed aggregates rather than as single aggresomes, even in the presence of proteasome inhibitors, which normally promote aggresome formation. RNAi of VCP caused extensive vacuolization of the cytoplasm, and proteasome inhibitors exaggerated this feature. RNAi of VCP had little effect on S2 cell proliferation but blocked cell-cycle progression and induced mitotic abnormalities and apoptosis in HeLa cells. These results indicate that VCP plays an important general role in mediating the function of the UPS, probably by interacting with potential proteasome substrates before they are degraded by the proteasome.

Valosin-containing protein (VCP; p97; cdc48 in yeast) is a hexameric ATPase of the AAA family (ATPases with multiple cellular activities) involved in multiple cellular functions, including degradation of proteins by the ubiquitin (Ub)–proteasome system (UPS). We examined the consequences of the reduction of VCP levels after RNA interference (RNAi) of VCP. A new stringent method of microarray analysis demonstrated that only four transcripts were nonspecifically affected by RNAi, whereas ∼30 transcripts were affected in response to reduced VCP levels in a sequence-independent manner. These transcripts encoded proteins involved in endoplasmic reticulum (ER) stress, apoptosis, and amino acid starvation. RNAi of VCP promoted the unfolded protein response, without eliciting a cytosolic stress response. RNAi of VCP inhibited the degradation of R-GFP (green fluorescent protein) and Ub-_G76V-GFP, two cytoplasmic reporter proteins degraded by the UPS, and of α chain of the T-cell receptor, an established substrate of the ER-associated degradation (ERAD) pathway. Surprisingly, RNAi of VCP had no detectable effect on the degradation of two other ERAD substrates, α1-antitrypsin and δCD3. These results indicate that VCP is required for maintenance of normal ER structure and function and mediates the degradation of some proteins via the UPS, but is dispensable for the UPS-dependent degradation of some ERAD substrates.

Background and Purpose— Proteasomes are large multicatalytic proteinase complexes that are found in the cytosol and in the nucleus of eukaryotic cells with a central role in cellular protein turnover. The ubiquitin-proteasome system (UPS) has a central role in the selective degradation of intracellular proteins. Among the key proteins whose levels are modulated by the proteasome are those involved in the control of inflammatory processes, cell cycle regulation, and gene expression. There are now overwhelming data suggesting that the UPS contributes to cerebral ischemic injury. Summary of Review— Proteasome inhibition is a potential treatment option for stroke. Thus far, proof of principle has been obtained from studies in several animal models of cerebral ischemia. Treatment with proteasome inhibitors reduces effectively neuronal and astrocytic degeneration, cortical infarct volume, infarct neutrophil infiltration, and NF-κB immunoreactivity with an extension of the neuroprotective effect at least 6 hours after ischemic insult. However, it is clear that the UPS represents a central pathway for the processing and metabolism of multiple proteins with critical roles in cellular function. To avoid eliciting significant side effects associated with complete inhibition of the proteasome and the possible immunosuppressive effects from persistent suppression of NF-κB activation, it is critical that we understand how to partially and temporally attenuate proteasome function to elicit the desired therapeutic effect before any large-scale use in humans. Conclusion— This review highlights the most recent advances in our knowledge on UPS, as well as the early experience of using proteasome inhibition strategies to treat acute stroke.