Project description:Many neurodegenerative disorders are characterized by two pathological hallmarks: progressive loss of neurons and occurrence of inclusion bodies containing ubiquitinated proteins. The mechanisms responsible for these pathological features remain elusive. Inflammation may be critical to neurodegeneration associated with ubiquitin-protein aggregates. We previously showed that prostaglandin J2 (PGJ2), one of the endogenous products of inflammation, induces neuronal death and accumulation of ubiquitinated proteins into distinct aggregates. We now report that temporal microarray analysis of human neuroblastoma SK-N-SH cells treated with PGJ2 revealed changes relevant to neurodegeneration. PGJ2 triggered a “repair” response including increased expression of heat shock, protein folding, stress and cellular defense genes. PGJ2 also decreased expression of DNA repair genes and increased expression of apoptotic genes. Overtime pro-death responses prevailed over pro-survival responses, leading to cellular demise. Furthermore, PGJ2 increased the expression of proteasome and other ubiquitin-proteasome pathway genes. This increase failed to overcome PGJ2-inhibition of 26S proteasome activity. Ubiquitinated proteins are degraded by the 26S proteasome, shown here to be the most active proteasomal form in SK-N-SH cells. We demonstrate that PGJ2 impairs 26S proteasome assembly, which is an ATP-dependent process. Interestingly, PGJ2 is known to perturb mitochondrial function, which could be critical to the observed 26S proteasome disassembly and suggest a crosstalk between mitochondrial and proteasomal impairment. In conclusion neurotoxic products of inflammation, such as PGJ2, may play a role in neurodegenerative disorders associated with the aggregation of ubiquitinated proteins by impairing 26S proteasome activity and inducing a chain of events that culminates in neuronal cell death. Keywords: Drug response time course
Project description:To realize the heterogeneity analysis of the structure and interaction for human 26S proteasomes in the cytoplasm and nucleus, combined with living cell cross-linking and efficient cytoplasm and nucleus separation method, the conformation and interaction resolution of 26S proteasome in the cytoplasm and nucleus was realized. Besides, by ensemble refinement of the interaction conformation of proteasome and ubiquitin with the crosslinking restraints, the transport path of ubiquitin on the proteasome was depicted in the cytoplasm and nucleus. It is of great significance for gaining an intensive understanding of the ubiquitin proteasome system for degrading proteins in the cytoplasm and nucleus.
Project description:A systematic and comparative study of the proteolysis products generated by purified human 26S and 20S proteasome following the in vitro cleavage of non-modified (naked), mono-ubiquitinated and poly-ubiquitinated cyclin B.
Project description:A systematic and comparative study of the proteolysis products generated by purified human 26S and 20S proteasome following the in vitro cleavage of non-modified (naked), mono-ubiquitinated and poly-ubiquitinated cyclin B.
Project description:Most eukaryotic proteins are degraded by the 26S proteasome after modification with a polyubiquitin chain. Substrates lacking unstructured segments cannot be degraded directly and require prior unfolding by the Cdc48 ATPase (p97 or VCP in mammals) in complex with its ubiquitin-binding partner Ufd1-Npl4 (UN). Here, we use purified yeast components to reconstitute Cdc48-dependent degradation of well-folded model substrates by the proteasome. We show that a minimal system consists of the 26S proteasome, the Cdc48-UN ATPase complex, the proteasome cofactor Rad23, and the Cdc48 cofactors Ubx5 and Shp1. Rad23 and Ubx5 stimulate polyubiquitin binding to the 26S proteasome and the Cdc48-UN complex, respectively, allowing these machines to compete for substrates before and after their unfolding. Shp1 stimulates protein unfolding by the Cdc48-UN complex, rather than substrate recruitment. In vivo experiments confirm bidirectional substrate shuttling between the 26S proteasome and Cdc48 ATPase and identify proteins that require both machines for their degradation.
Project description:Proteasomes are a critical component of quality control that regulates turnover of short-lived, unfolded, and misfolded proteins. Proteasome activity has been therapeutically targeted and considered as a treatment option for several chronic lung disorders including pulmonary fibrosis. Though pharmacologic inhibition of proteasome activity effectively prevents the transformation of fibroblasts to myofibroblasts, the effect on alveolar type 2 (AT2) epithelial cells is not clear. To address this knowledge gap, we generated a genetic model in which a proteasome subunit, RPT3, which promotes assembly of active 26S proteasome, was conditionally deleted in AT2 cells of mice. Targeted deletion of RPT3 in AT2 cells resulted in 26S proteasome dysfunction, leading to augmented cell stress and death. Acute loss of AT2 cells resulted in depletion of alveolar surfactant, disruption of the alveolar epithelial barrier and, ultimately, lethal acute respiratory distress syndrome. This study underscores the need for further investigation into the differential effect of proteasome inhibition in lung cell types.
Project description:The conditions for chemical protein-protein cross-linking using the reagents pimelic dihydrazide (PDH) and 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (DMTMM) chloride were optimized using a set of five model proteins and two large complexes, the 20S proteasome and the 70S ribosome. This dataset contains data from the bovine 20S proteasome only.
Project description:RNA quality control (RQC) and post-transcriptional gene silencing (PTGS) target and degrade aberrant endogenous RNAs and foreign RNAs, contributing to homeostasis of cellular RNAs. In plants, RQC and PTGS compete for foreign and selected endogenous RNAs; however, little is known about the mechanism interconnecting the two pathways. Using a reporter system designed for monitoring PTGS, we revealed that the 26S proteasome subunit RPT2a enhances transgene PTGS by promoting the accumulation of transgene-derived short interfering RNAs without affecting their biogenesis. RPT2a physically associated with a subset of RQC components and downregulated the protein level. Overexpression of the RQC components interfered with transgene silencing, and impairment of the RQC machinery reinforced transgene PTGS attenuated by rpt2a. Overall, we demonstrate that the 26S proteasome subunit RPT2a promotes PTGS by repressing the RQC machinery to control foreign RNAs.