Ubiquitin (Ub) is a small protein which occurs in all eukaryotic cells. The biochemical function of Ub was first linked with protein turnover, but it has later been shown that Ub plays an important role in the control of many processes such as cell-cycle progression, transcriptional regulation and receptor down-regulation. A cascade of ubiquitin ligases conjugates monoubiquitin or, more commonly, ubiquitin chains to proteins. These molecules can then be removed by different kinds of deubiquitinating enzymes (DUBs). The development of ubiquitin-derived thiol-reactive probes, which bind covalently to the catalytic cysteine of certain types of DUBs, has made it possible to analyze the composition of DUBs in different types of tissues and cells. The desired probe can be added to the tissue/cell lysate and allowed to react with the different DUBs before the modified enzymes are separated by SDS-Page and visualized by western blotting.My supervisor and co-workers had been working with the HAUbVME probe before I joined them in the Intracellular Signaling Laboratory, Imperial College. They had discovered two DUBs (termed 50 kD and 60 kD), which where present in proliferating CD4+ T-cells but not in non-proliferating T-cells. They had a theory that one of these proteins corresponded to DUB-2 which is a T-cell specific DUB involved in proliferation and differentiation of lymphocytes. By using a probe-binding technique and western blotting for DUB-2 sequences we found that neither of these two enzymes were DUB-2. An experiment on human umbilical vein endothelial cells (HUVEC) showed that neither the 50 kD nor 60 kD DUBs of interest were found in endothelial cells. We concluded that it is likely that these DUBs are T-cell specific.The existence of nuclear-specific DUBs has never been shown before. We ran a probe-binding reaction on nuclear extracts from cells synchronized in different stages of the cell cycle and found two DUBs in the nuclear lysates which were not present in the cytosolic control sample. We also discovered a DUB that was restricted to the G1 phase of the cell cycle. This DUB was also found in the cytosolic lysate. Otherwise there did not seem to be any obvious differences in the levels and types of DUBs in the different stages of the eukaryotic cell cycle.An investigation of the levels of active DUBs in inflammatory bowel disease (IBD) showed that FAM is dysregulated in intermediate colitis (IC). The levels of ubiquitinated proteins were not altered in IBD compared to healthy controls.We also used the probe-binding technique to analyze tissues obtained from patients with ischemic heart disease (IHD), dilated cardiomyopathy (DCM) and transplant donors. We compared the levels of several ubiquitin proteases among these tissues by western blotting and found several interesting differences. HAUSP and USP14 were completely missing in the IHD samples, and the levels of USP15, UCH37 and UCH-L3 were clearly decreased. The level of UCH-L1 was normal. Some of the control samples also showed a decrease in the levels of DUBs, but we believe this was caused by ischemic conditions developed during transplantation or transportation. A western blot for Ub showed that the levels of ubiquitinated proteins were higher for the DCM samples then for the IHD samples and controls, which was consistent with what was found in the literature. To recreate the decreased levels of active DUBs found in the IHD samples we grew HUVEC in an environment with only 1% oxygen. 96 hours of hypoxia was necessary to cause decreased levels of DUBs, though the decrease was not as profound as for the IHD samples. A western blot for Ub showed that more than 16 hours of hypoxia leads to a decreased level of ubiquitinated proteins in HUVEC. This result was unexpected as one would think that decreased levels of USP14 and UCH37, which are both involved in proteasome function, would lead to increased levels of ubiquitinated proteins.The transcription factor NF-κB is tightly regulated by the ubiquitin proteasome system. It is also known that IL-1 induces the translocation of NF-κB from the cytoplasm to the nucleus. We decided to investigate whether changes in the ubiquitin-proteasome system seen in hypoxia correlate with perturbations in NF-κB translocation. We grew duplicate set of HUVEC on coverslips in an environment with only 1% oxygen. Each duplicate dish received IL-1 for the final 30 minutes of the incubation. Hypoxia did not inhibit the translocation of NF-κB in response to IL-1, however it turned out that hypoxia inhibits the constitutive translocation of NF-κB.