Mechanistic underpinnings of PRPS deficiency disorders

About This Grant

PROJECT SUMMARY This proposal seeks to understand the mechanistic basis for diseases caused by loss-of-function missense mutations in the gene encoding Phosphoribosyl Pyrophosphate Synthetase 1 (PRPS1). The PRPS enzyme is conserved from bacteria to humans and it catalyzes an essential chokepoint reaction in cellular metabolism that routes sugars into nucleotide production pathways. Previous work in our lab has revealed that the PRPS enzyme operates as a heteromeric biochemical complex comprised of four PRPS paralogs that arose through a series of gene duplications in the Opisthokonta lineage. Our work also demonstrated the vital nature of this PRPS enzyme assembly, as cells exclusively expressing PRPS1 had diminished fitness metabolically characterized by decreased nucleotide production and defective mitochondrial respiration. We hypothesize that alterations in preferential interactions or changes in conformational dynamics between PRPS1 and other members of the PRPS complex produce the panoply of pathophysiological disease manifestations caused by PRPS1 loss-of- function variants. Further, we hypothesize that regardless of the structural basis for PRPS enzyme deficiency, a convergence on a core set of metabolic dysfunctions will be the outcome, which includes diminished nucleotide production, deregulated redox homeostasis, and mitochondrial respiratory defects. We will test these hypotheses in two of the three aims, which will employ a suite of isogenic fibroblast cell lines harboring CRISPR-mediated knockouts of PRPS paralogs, alone or in combination, in all viable configurations. Reconstitution of the PRPS1 KO clones with wild-type or each of the >30 loss-of-function variants will enable comprehensive structural and metabolic characterization of disease-causing variant effects, thereby generating new knowledge regarding fundamental features of enzyme complex organization, activity, and metabolic pathway control. To move beyond simply understanding the mechanisms that contribute to PRPS deficiency disorders, we also aim to develop and perform proof-of-concept testing on enzyme replacement therapeutic strategies that may ultimately be used to treat human patients with these incurable genetic diseases. Collectively, this proposal will generate new insights into the inner workings of the complicated and poorly understood PRPS enzyme complex and its control of cellular metabolic pathways and fluxes. Successful completion of our proposed studies will aid in the understanding, diagnosis, and treatment of PRPS deficiency disorders like Sensorineural Deafness (DFNX1), Charcot-Marie-Tooth disease (CMTX5), and Arts syndrome that are caused by missense loss-of-function mutations in PRPS1.