Alteration of mRNA expression in breast cancer cells upon chemical inhibition of the 26S proteasome
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PROVIDER: PRJNA594967 | ENA |
REPOSITORIES: ENA
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Project description:The 26S proteasome regulates degradation of many cellular proteins to maintain cellular homeostasis. Disruption of proteasome activity followed by dysregulation of tumor suppressors and oncogenes is rampant in many cancers, hence chemical inhibitors of the proteasome have utility as cancer therapeutics, although the underlying mechanisms of their effects in the clinic is poorly understood. We have employed whole genome microarray expression profiling as a discovery platform to identify genes and potential signaling pathways that are affected MCF7 breast cancer cells are treated with MG132 a chemical inhibitor of the proteasome. Total RNA was collected from MCF7 breast cancer cells treated with DMSO (vehicle control) and 1 uM MG132 for 4 or 24H . We identified time dependent changes in the expression of genes enriched in p53 and estrogen receptor pathways, two signatures relevant to breast cancer biology.
2020-01-07 | GSE141858 | GEO
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
2006-06-01 | GSE4329 | GEO
Project description:The proteasome is the main proteolytic system for targeted protein degradation in the cell. Its function is fine-tuned according to cellular needs. Inhibition of the respiratory chain impairs proteasome activity, regulation of proteasome function by mitochondrial metabolism, however, is unknown. Here, we demonstrate that mitochondrial dysfunction reduces the assembly and activity of the 26S proteasome. Defects in respiratory chain caused metabolic reprogramming of the Krebs cycle and deficiency in the amino acid aspartate resulting in reduced 26S proteasome function. Aspartate supplementation fully restored assembly and activity of 26S proteasome complexes. This metabolic reprogramming involved sensing of aspartate via the mTORC1 pathway and the mTORC1-dependent transcriptional activation of defined proteasome assembly factors. Metabolic regulation of 26S function was confirmed in patient-derived skin fibroblasts with respiratory dysfunction containing a single mitochondrial mutation. Importantly, treatment of primary human lung fibroblasts with the respiratory chain inhibitor and anti-diabetic drug metformin similarly reduced assembly and activity of 26S proteasome complexes, which was fully reversible and rescued by supplementation of aspartate or pyruvate. Our study uncovers a fundamental novel mechanism of how mitochondrial metabolism adaptively adjusts protein degradation by the proteasome. It thus unravels unexpected consequences of defective mitochondrial metabolism in disease or drug-targeted mitochondrial reprogramming for proteasomal protein degradation in the cell. As metabolic inhibition of proteasome function can be alleviated by treatment with aspartate or pyruvate, our results also have therapeutic implications.
2020-09-18 | PXD019697 | Pride
Project description:The proteasome is the main proteolytic system for targeted protein degradation in the cell. Its function is fine-tuned according to cellular needs. Inhibition of the respiratory chain impairs proteasome activity, regulation of proteasome function by mitochondrial metabolism, however, is unknown. Here, we demonstrate that mitochondrial dysfunction reduces the assembly and activity of the 26S proteasome. Defects in respiratory chain caused metabolic reprogramming of the Krebs cycle and deficiency in the amino acid aspartate resulting in reduced 26S proteasome function. Aspartate supplementation fully restored assembly and activity of 26S proteasome complexes. This metabolic reprogramming involved sensing of aspartate via the mTORC1 pathway and the mTORC1-dependent transcriptional activation of defined proteasome assembly factors. Metabolic regulation of 26S function was confirmed in patient-derived skin fibroblasts with respiratory dysfunction containing a single mitochondrial mutation. Importantly, treatment of primary human lung fibroblasts with the respiratory chain inhibitor and anti-diabetic drug metformin similarly reduced assembly and activity of 26S proteasome complexes, which was fully reversible and rescued by supplementation of aspartate or pyruvate. Our study uncovers a fundamental novel mechanism of how mitochondrial metabolism adaptively adjusts protein degradation by the proteasome. It thus unravels unexpected consequences of defective mitochondrial metabolism in disease or drug-targeted mitochondrial reprogramming for proteasomal protein degradation in the cell. As metabolic inhibition of proteasome function can be alleviated by treatment with aspartate or pyruvate, our results also have therapeutic implications.
2020-11-16 | PXD019695 | Pride
Project description:Microbiota alteration upon blueberry consumption
| PRJNA679320 | ENA
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.
2024-02-23 | PXD042878 | Pride
Project description:The fundamental importance of the 26S proteasome in health and disease suggests that its function must be finely controlled, and yet our knowledge about proteasome regulation remains limited. Posttranslational modifications, especially phosphorylation, of proteasome subunits have been shown to impact proteasome function through different mechanisms, although the vast majority of proteasome phosphorylation events have not been studied. Here, we have characterized 1 of the most frequently detected proteasome phosphosites, namely Ser361 of Rpn1, a base subunit of the 19S regulatory particle. Using a variety of approaches including CRISPR/Cas9-mediated gene editing and quantitative mass spectrometry, we found that loss of Rpn1-S361 phosphorylation reduces proteasome activity, impairs cell proliferation, and causes oxidative stress as well as mitochondrial dysfunction. A screen of the human kinome identified several kinases including PIM1/2/3 that catalyze S361 phosphorylation, while its level is reversibly controlled by the proteasome-resident phosphatase, UBLCP1. Mechanistically, Rpn1-S361 phosphorylation is required for proper assembly of the 26S proteasome, and we have utilized a genetic code expansion system to directly demonstrate that S361-phosphorylated Rpn1 more readily forms a precursor complex with Rpt2, 1 of the first steps of 19S base assembly. These findings have revealed a prevalent and biologically important mechanism governing proteasome formation and function.
2019-12-08 | PXD016643 |
Project description:Upon DNA damage, numerous proteins are targeted for ubiquitin-dependent proteasomal degradation. We show that phosphorylation of Rpn10/PSMD4 (ubiquitin receptor of the proteasome) at Ser266, is required for DNA repair. Here, we performed TurboID proximity labeling for capturing the proteasome substrates under DNA damage. Numerous proteins which involve in DNA damage response, chromosome reorganization cell cycle and histone modification were identified as the potential pronuteasome substrates. We find that DNA damage reduced the overall affinity between substrates and wild-type Rpn10 compared to Rpn10-Ser266 mutant cells. Combining TMT quantitative proteomic studies, we observed the dynamics of more than 2,000 nuclear proteins upon DNA damage and time course release. Most proteins were accumulated in wild-type cells in the time course release, while the Rpn10-Ser266 mutant cells show almost no changes in protein level. These findings reveal an inherent self-limiting mechanism of the proteasome that, by controlling substrate recognition through Rpn10 phosphorylation, fine-tunes protein degradation for optimal responses under stress.
2023-11-28 | PXD047298 |
Project description:Phosphorylation status of yeast 26S proteasome subunits was analyzed by phos-tag diagonal electrophoresis and GeLC-MS analysis.
2021-08-03 | PXD020706 | JPOST Repository
Project description:Phosphorylation status of human 26S proteasome subunits was analyzed by phos-tag diagonal electrophoresis and GeLC-MS analysis.
2021-08-03 | PXD020705 | JPOST Repository