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Mitochondrial regulation of the 26S proteasome

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Christoph Mayr

LAB HEAD: Silke Meiners

PROVIDER: PXD019697 | Pride | 2020-09-18

REPOSITORIES: Pride

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Project description:Early-onset Parkinson's disease can be caused by mutations in the gene PARK7. Many functions have been proposed for PARK7, but in vivo evidence is largely lacking or conflicting with enzymological data. Here, we provide unequivocal evidence that PARK7 and its orthologues can prevent damage of proteins and metabolites inflicted by the glycolytic intermediate 1,3-bisphosphoglycerate, without changing the abundance of this metabolite. In the absence of PARK7, several glycerate-modified metabolites, as well as numerous glycerate- or phosphoglycerate-modified proteins accumulate in human cell lines, mouse brain and flies. This gives an explanation for the pleiotropic effects of PARK7, indicates that spontaneous metabolic damage contributes to the development of hereditary Parkinson's disease, and opens new roads for future therapeutic approaches. Submitted here are the proteomics analyses, where we identify and quantify glyceroyl- and phosphoglyceroyl-modifications of lysine residues in 5 different experimental models. 1. The human colorectal cancer cell line HCT116 was analyzed in four different conditions: a. Wild type cells transduced with an empty recombinant empty lentivirus b. PARK7 knockout cells transduced with an empty recombinant empty lentivirus c. PARK7 knockout cells transduced with a recombinant lentivirus driving expression of PARK7. 2. Brain samples from wild type and PARK7 knockout mice. 3. Red blood cell lysates from wild type and PARK7 knockout mice 4. Whole body extracts from wild type and DJ1beta knockout Drosophila melanogaster 5. In vitro reactions where 1,3-bisphosphoglycerate was produced either by GAPDH or PGK, in the presence and absence of PARK7. Samples were a. Enzymes in the absence of any cofactors (i.e. no 1,3-BPG production) b. Production via GAPDH (using glyceraldehyde 3-phosphate, phosphate and NAD+ as substrates) in the presence or absence of PARK7 c. Production via PGK (using 3-phosphoglycerate and ATP as substrates) in the presence or absence of PARK7.

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Mitochondrial regulation of the 26S proteasome

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