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DJ-1 protects cells by catalyzing the hydrolysis of cyclic 3-phosphoglyceric anhydride - a highly reactive glycolytic byproduct

ABSTRACT: Mutations in the human protein DJ-1/PARK7 are found to be involved in familial Parkinson’s disease. However, the molecular mechanism of the protein remains unclear. Lately, DJ-1 was found to prevent the acylation of amino groups in proteins. The process of acylation requires a cyclic 3-phosphoglyceric anhydride (cPGA). The cPGA has been shown to be an intermediate product of 1,3-biphosphoglycerate and to be destroyed by DJ-1’s hydrolase activity. In our study, we synthesized and characterized cPGA. Furthermore, we demonstrated an endogenous catalytic hydrolase activity of DJ-1 towards cPGA in crude protein extracts of human HCT116 cells. The addition of synthesized cPGA to DJ-1 knockout HCT116 cytoplasmic cell lysate caused a dramatic increase in phosphoglycerate-modified proteins. The submitted results contain proteomic analysis of phosphoglycerate modifications on lysine residues in wild type and DJ-1 knockout cytoplasmic proteins of HCT116 cells when cPGA was added. Our study confirms that DJ-1 prevents the formation of phosphoglycerate modifications on amino groups of proteins.

ORGANISM(S): Homo Sapiens

SUBMITTER: Darkhan Utepbergenov

PROVIDER: PXD043887 | iProX | Wed Jul 19 00:00:00 BST 2023

REPOSITORIES: iProX

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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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DJ-1 protects cells by catalyzing the hydrolysis of cyclic 3-phosphoglyceric anhydride - a highly reactive glycolytic byproduct

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