Cross-link MS analysis of human 26S proteasome in complex with RPN13:UCHL5 (without substrate).
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Eclipse
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Shang-Te Danny Hsu
PROVIDER: MSV000092664 | MassIVE | Wed Aug 16 02:42:00 BST 2023
REPOSITORIES: MassIVE
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Project description:Cross-link MS analysis of human 26S proteasome in complex with RPN13:UCHL5 with the added ubiquitinated Sic1PY substrate.
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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
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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.
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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.
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Project description:Proteins play a central role in most biological processes within the cell and deciphering how they interact is key to understand their function. Cross-linking coupled to mass spectrometry is an essential tool for elucidating protein-protein interactions. Despite its importance, we still know surprisingly little about the principles that underlie the process of chemical cross-link formation itself and how it is influenced by different physicochemical factors. To understand the molecular details of cross-link formation, we have set-up a comprehensive kinetic model and carried out simulations of protein cross-linking on large protein complexes. We dissect the contribution on the cross-link yield of parameters such as amino acid reactivity, cross-linker concentration, and hydrolysis rate. Our model can compute cross-link formation based solely on the structure of a protein complex, thereby enabling realistic predictions for a diverse set of systems. We quantitatively show how cross-links and mono-links are in direct competition and how the hydrolysis rate and abundance of cross-linker and proteins directly influence their relative formation. We show how cross-links and mono-links exist in a “all-against-all” competition due to their simultaneous formation, resulting in a non-intuitive network of interdependence. We show that this interdependence is locally confined and mainly limited to direct neighbors or residues in direct vicinity. These results enable us to identify the optimal cross-linker concentration at which the maximal number of cross-links are formed. Taken together, our study establishes a comprehensive kinetic model to quantitatively describe cross-link formation for protein-protein interactions.
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