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Structure-based design and characterization of Parkin-activating mutations.

ABSTRACT: Autosomal recessive mutations in the Parkin gene cause Parkinson's disease. Parkin encodes an ubiquitin E3 ligase that functions together with the kinase PINK1 in a mitochondrial quality control pathway. Parkin exists in an inactive conformation mediated by autoinhibitory domain interfaces. Thus, Parkin has become a target for the development of therapeutics that activate its ligase activity. Yet, the extent to which different regions of Parkin can be targeted for activation remained unknown. Here, we have used a rational structure-based approach to design new activating mutations in both human and rat Parkin across interdomain interfaces. Out of 31 mutations tested, we identified 11 activating mutations that all cluster near the RING0:RING2 or REP:RING1 interfaces. The activity of these mutants correlates with reduced thermal stability. Furthermore, three mutations V393D, A401D, and W403A rescue a Parkin S65A mutant, defective in mitophagy, in cell-based studies. Overall our data extend previous analysis of Parkin activation mutants and suggests that small molecules that would mimic RING0:RING2 or REP:RING1 destabilisation offer therapeutic potential for Parkinson's disease patients harbouring select Parkin mutations.

SUBMITTER: Stevens MU

PROVIDER: S-EPMC10027901 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Structure-based design and characterization of Parkin-activating mutations.

Stevens Michael U MU Croteau Nathalie N Eldeeb Mohamed A MA Antico Odetta O Zeng Zhi Wei ZW Toth Rachel R Durcan Thomas M TM Springer Wolfdieter W Fon Edward A EA Muqit Miratul Mk MM Trempe Jean-François JF

Life science alliance 20230320 6

Autosomal recessive mutations in the Parkin gene cause Parkinson's disease. Parkin encodes an ubiquitin E3 ligase that functions together with the kinase PINK1 in a mitochondrial quality control pathway. Parkin exists in an inactive conformation mediated by autoinhibitory domain interfaces. Thus, Parkin has become a target for the development of therapeutics that activate its ligase activity. Yet, the extent to which different regions of Parkin can be targeted for activation remained unknown. He ...[more]

PMID: 36941054

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Project description:The importance of glucokinase (GK) in the regulation of insulin secretion has been highlighted by the phenotypes of individuals with activating and inactivating mutations in the glucokinase gene (GCK). Here we report 10 individuals with congenital hyperinsulinism (HI) caused by eight unique activating mutations of GCK. Six are novel and located near previously identified activating mutations sites. The first recognized episode of hypoglycemia in these patients occurred between birth and 24 years, and the severity of the phenotype was also variable. Mutant enzymes were expressed and purified for enzyme kinetics in vitro. Mutant enzymes had low glucose half-saturation concentration values and an increased enzyme activity index compared with wild-type GK. We performed functional evaluation of islets from the pancreata of three children with GCK-HI who required pancreatectomy. Basal insulin secretion in perifused GCK-HI islets was normal, and the response to glyburide was preserved. However, the threshold for glucose-stimulated insulin secretion in perifused glucokinase hyperinsulinism (GCK-HI) islets was decreased, and glucagon secretion was greatly suppressed. Our evaluation of novel GCK disease-associated mutations revealed that the detrimental effects of these mutations on glucose homeostasis can be attributed not only to a lowering of the glucose threshold of insulin secretion but also to a decreased counterregulatory glucagon secretory response.Article highlightsOur evaluation of six novel and two previously published activating GCK mutations revealed that the detrimental effects of these mutations on glucose homeostasis can be attributed not only to a lowering of the glucose threshold of insulin secretion but also to a decreased counterregulatory glucagon secretory response. These studies provide insights into the pathophysiology of GCK-hyperinsulinism and the dual role of glucokinase in β-cells and α-cells to regulate glucose homeostasis.

Dataset Information

Structure-based design and characterization of Parkin-activating mutations.

Publications

Structure-based design and characterization of Parkin-activating mutations.

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