Cezary Wojcik

RNA interference of valosin-containing protein (VCP/p97) reveals multiple cellular roles linked to ubiquitin/proteasome-dependent proteolysis

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.

Ubiquitin-Proteasome System and Proteasome Inhibition: New Strategies in Stroke Therapy

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.

Regulation of apoptosis by the ubiquitin and proteasome pathway

Regulated proteolysis plays important roles in cell physiology as well as in pathological conditions. In most of the cases, regulated proteolysis is carried out by the ubiquitin‐ and proteasome‐dependent proteolytic system, which is also in charge of the bulk of cytoplasmic proteolysis. However, apoptosis or the process of programmed cell death is regulated by a different proteolytic system, i.e. by caspases, a family of specialized cysteine proteases. Nevertheless, there is plenty of evidence of a crosstalk between the apoptotic pathways and the ubiquitin and proteasome system, whose function in apoptosis appears to be very complex. Proteasome inhibitors induce apoptosis in multiple cell types, while in other they are relatively harmless or even prevent apoptosis induced by other stimuli. Proteasomes degrade specific proteins during apoptosis, but on the other hand some components of the proteasome system are degraded by caspases. The knowledge about the involvement of the ubiquitin‐ and proteasome‐dependent system in apoptosis is already clinically exploited, since proteasome inhibitors are being tested as experimental drugs in the treatment of cancer and other pathological conditions, where manipulation of apoptosis is desirable.

Aggregate formation in the spinal cord of mutant SOD1 transgenic mice is reversible and mediated by proteasomes.

Cu,Zn superoxide dismutase (SOD1) mutations cause one form of familial amyotrophic lateral sclerosis by a toxic gain of function that may be related to abnormal protein folding and aggregate formation. However, the processing pathways involved in SOD1 aggregate generation within spinal cord remain unclear. We have now developed an experimental system for studying SOD1 aggregate formation and clearance in intact spinal cord tissue. Here we demonstrate that the formation of SOD1‐positive aggregates in G93A SOD1 transgenic mouse spinal cord tissue involves proteasome‐mediated proteolysis. Organotypic spinal cord slices from 9‐day‐old transgenic mice expressing G93A SOD1 develop SOD1 aggregates with proteasome inhibition. In contrast, SOD1 aggregates do not form in spinal cord slices from wild type mice or transgenic mice overexpressing wild type SOD1 following proteasome inhibition. Furthermore, SOD1 aggregate formation within G93A SOD1 spinal cord is both sensitive to small changes in overall proteasome activity and reversible with the restoration of proteasome function. Our results also establish that adult mouse spinal cord exhibits a relative deficiency in proteasome activity compared with non‐CNS tissue that may help explain the propensity of spinal cord to form SOD1‐positive aggregates.

Ubiquitin--more than just a signal for protein degradation.

Abstract Research related to the ubiquitin- and proteasome-dependent protein-degradation pathway is growing exponentially. Top scientists in this field met recently in the green mountains of Vermont. Among the numerous topics discussed at this meeting* were ubiquitin-like proteins, ubiquitin-interacting proteins, non-proteolytic roles of ubiquitin and the regulation and function of proteasomes. (*FASEB Summer Conference: Ubiquitin and Intracellular Protein Degradation; Saxtons River, Vermont, USA; 23–28 June 2001. Organized by George DeMartino and Daniel Finley.)

Proteasome activator subunit PA28α and related Ki antigen (PA28γ) are absent from the nuclear fraction purified by sucrose gradient centrifugation

Abstract The aim of the present work was to attempt to partially purify PA28 (REG) α and γ (Ki antigen) in the nuclear fraction from NT2/D1 cells. Nuclei were isolated by the hypertonic sucrose gradient centrifugation method and fractionated into membrane/nucleoplasmic and chromatin/nucleolar fractions. Western blotting with anti-histone and anti-β-tubulin monoclonal antibodies confirmed the accuracy of the procedure. Proteasomes were present mainly in the cytoplasm but also in the nuclei. Disruption of the nuclear envelope released the proteasomes implying a loose or no binding with the chromatin. PA28α and γ were detected mainly in the cytosol and to a lesser extent in the crude nuclear pellet, however the purified nuclei were devoid of PA28α and γ. This indicates, that only a small fraction of the PA28 activator is present in the nuclei as detected by immunofluorescence or/and it is easily removed during nuclear purification.

VCP – the missing link in protein degradation?

Defective and abnormally folded proteins that are present within the lumen of the endoplasmic reticulum (ER) or associated with its membranes are rapidly degraded. This process is called ER-associated degradation (ERAD). ERAD is not solely a protein quality-control pathway, but it also involves the degradation of short-lived ER proteins such as fatty acid desaturase and HMG-CoA reductase. Curiously, ERAD does not take place within ER lumen but involves protein export to the cytosol, ubiquitination and degradation by 26S proteasomes. Until now, it was not clear how ER protein export is coupled to 26S proteasome targeting.This mystery is now at least partially solved. Several independent groups of researchers have shown that the missing link between the ER and the proteasome is a 97-kDa ATPase called VCP (valosin-containing protein, p97 or CDC48) [1.xThe AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Ye, Y et al. Nature. 2001; 414: 652–656Crossref | PubMed | Scopus (673)See all References, 2.xAAA-ATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation. Rabinovich, E et al. Mol. Cell. Biol. 2002; 22: 626–634Crossref | PubMed | Scopus (374)See all References, 3.xProtein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48. Jarosch, E et al. Nat. Cell Biol. 2002; 4: 134–139Crossref | PubMed | Scopus (358)See all References, 4.xRole of the ubiquitin-selective CDC48 (UFD1/NPL4) chaperone (segregase) in ERAD of OLE1 and other substrates. Braun, S et al. EMBO J. 2002; 21: 615–621Crossref | PubMed | Scopus (243)See all References]. VCP is a ubiquituous enzyme, a member of the AAA family (‘ATPases with multiple cellular activities’), that is involved in a plethora of distinct functions such as membrane fusion, nuclear envelope reconstruction, post-mitotic Golgi reassembly and ubiquitin-dependent degradation. VCP forms a homohexamer that binds to several different ancillary proteins. VCP complex binds to polyubiquitin chains and has a chaperone activity that facilitates its ‘segregase’ function – the ability to untether ubiquitinated proteins from their binding partners. VCP in complex with Ufd1/Npl4 is therefore able to extract polyubiquitinated proteins associated with the translocon complex, removing them from ER to the cytoplasm. While some extracted proteins can be released into the cytosol, most of them are probably delivered to the 26S proteasome for degradation.These data establish VCP as a new component of the ubiquitin–proteasome system. It is not clear whether proteasome activity is also required for protein extraction from the ER. It is tempting to speculate that ERAD might be mediated by a macromolecular assembly involving direct interactions between the translocon, VCP, 26S proteasome and, possibly, specific ubiquitinating enzymes. Taking into account the importance of ERAD in various pathological processes, the detailed elucidation of its mechanisms could be of great practical value.

Two blades of a sword: Degradation coupled to deubiquitination

Usually, before being degraded by 26S proteasomes, proteins are first tagged with a polyubiquitin (poly-Ub) chain. However, before they reach this protease assembly, their poly-Ub chain can be trimmed and even completely removed by multiple deubiquitinating enzymes (DUBs). The DUB, UCH37, is one of the subunits that forms the 26S proteasome. It removes ubiquitin (Ub) moieties distal from the protein to be degraded.Now, Tingting Yao and Robert Cohen describe a second DUB activity of the 26S proteasome, which they call ‘cryptic DUB’ [1xA cryptic protease couples deubiquitination and degradation by the proteasome. Yao, T. and Cohen, R.E. Nature. 2002; 419: 403–407Crossref | PubMed | Scopus (433)See all References[1]. They have engineered an artificial substrate of the 26S proteasome, which consists of an N-terminal Ub fused to the unfolded ovomucoid first domain. The degradation of this substrate is linked to the release of free Ub even in the presence of Ub-aldehyde, a potent inhibitor of known DUBs. When the C-terminal Glycine of Ub is replaced by Valine, the release of Ub stops and the degradation of the whole protein continues, but at a much slower rate.This principle has been confirmed with other substrates. Analysis of genomic data combined with yeast genetics reveals that this cryptic DUB activity can be attributed to a 26S proteasome subunit called Rpn11. Rpn11 forms part of the lid of the PA700 complex (19S cap) of the 26S proteasome. In contrast to the other DUBs, which are Cys proteases, Rpn11 is probably a Zn-metalloproteinase. Moreover, Rpn11 is essential for growth, whereas other DUBs are often redundant. This finding is in agreement with the dogma that eukaryotic cells can not function properly without a functional Ub- and proteasome-system of protein degradation.Deubiquitination has therefore found its place as a necessary step in protein degradation. After the ubiquitinated protein is recognized and bound by several subunits of the 26S proteasome cap, the protein is unfolded and fed into the central channel of the 20S core proteasome. This is coupled with removal of the poly-Ub chain by Rpn11, which enables its release and the recycling of Ub. In the absence of Rpn11-associated DUB activity, Ub is unfolded, translocated and degraded.As Ub is a very stable thermodynamically protein, its unfolding becomes a limiting step in proteolysis, slowing it down beyond the efficiency required for normal rates of protein degradation. In contrast to Rpn11, UCH37 remains a non-essential DUB that is probably only associated with the editing function.

Dipeptides: rulers of the N-end rule

There are many different E1–E2–E3 cascades leading to the ubiquitination and proteasome-dependent proteolysis of specific proteins. The specificity of each cascade is given by the ubiquitin ligase (E3), and to a certain extent by the ubiquitin-conjugating enzyme (E2), while the ubiquitin-activating enzyme (E1) is unspecific, common to all pathways. One of the best-studied ubiquitination pathways is the N-end rule, which involves the selective degradation of proteins carrying ‘destabilizing’ amino acids at their amino terminus. The N-end rule pathway was discovered through the degradation of engineered proteins with different N-terminal amino acids. In Saccharomyces cerevisiae, the E3 responsible for their ubiquitination was shown to be Ubr1p. The only known physiological function of Ubr1p is the regulation of dipeptide uptake, through degradation of Cup9p, a transcriptional repressor of the di-/tri-peptide transporter Ptr2p. Curiously, the Cup9p degradation signal is not an N-terminal amino acid but is a sequence located in the C-terminal half of the molecule.In a recent paper published in Nature1xPeptides accelerate their uptake by activating a ubiquitin-dependent proteolytic pathway. Turner, G.C. et al. Nature. 2000; 405: 579–583Crossref | PubMed | Scopus (133)See all References1, it is shown that Cup9p degradation is allosterically activated by dipeptides with destabilizing N-termini, resulting in a positive-feedback loop. Imported dipeptides bind to Ubr1p and accelerate the Ubr1p-dependent degradation of Cup9p, which in turn de-represses Ptr2p expression and increases the capacity of the cell to import dipeptides.The importance of this work resides in the fact that, for the first time, it is shown that the activity of an E3 might be regulated by external signals through allosteric activation by a small compound. It remains to be proven whether the N-end rule degradation pathway in higher eukaryotes is also regulated in the same way and whether other E3 enzymes can be regulated like Ubr1p by other low-molecular-mass compounds. It is tempting to speculate that small compounds regulating the activity of different E3 enzymes can be valuable for research and also as therapeutic tools.

Ubiquitin: no longer the chosen one

Since the discovery that proteins can be modified by the covalent attachment of ubiquitin, other ubiquitin-like proteins (ULPs) have been described. ULPs can also be used for posttranslational modifications of substrate proteins in a process similar to ubiquitination. However, ubiquitin was considered unique as only ubiquitin is able to form chains comprising multiple moieties, and polyubiquitin chains linked by Lys48 target proteins for degradation by the 26S proteasome. Mono-ubiquitination, as well as modification by the other ULPs, was considered to serve other, non-proteolytic, roles. Now, this simplistic point of view has been challenged by Yeh and colleagues [1xTargeting of NEDD8 and its conjugates for proteasomal degradation by NUB1. Kamitani, T. et al. J. Biol. Chem. 2001; 276: 46655–46660Crossref | PubMed | Scopus (93)See all References[1].Covalent modification by a ULP called NEDD8 (i.e. neddylation) was shown to target a specific set of proteins for degradation by the 26S proteasome. Neddylated proteins bind to an adaptor protein called NUB1. NUB1 possesses a proteasome-interacting motif known as a ubiquitin-like domain and therefore specifically binds to the ubiquitin-binding component of the 26S proteasome (the S5a subunit of the 19S cap). Neddylated proteins have been found associated with the 26S proteasome in vivo. NUB1 downregulates the expression of NEDD8 and its conjugates, but this effect can be prevented by the use of proteasome inhibitors. Altogether, these data suggest that neddylated proteins are targeted to the 26S proteasome for destruction.Although many proteins are neddylated, the only known ones are cullins. NEDD8 itself – unlike ubiquitin – appears to be degraded by the proteasomes.These findings are important because they show that ubiquitination is no longer the only method of protein tagging for proteasome-dependent destruction. Moreover, through neddylation of the cullins, various crucial pathways might be controlled. For example, neddylation of cullin1, a component of the ubiquitin-ligase SCF complex, could regulate the NF-κB signaling pathway and the cell cycle by indirectly controlling the ubiquitination and subsequent degradation of IκBα, cyclins and cyclin-dependent kinase inhibitors.