del Cano-Ochoa, F. | bioRxiv Science

bioRxiv2024

Disruption of CAD oligomerization by pathogenic variants

CAD is a multi-enzymatic protein essential for initiating the de novo biosynthesis of pyrimidine nucleotides, forming large hexamers whose structure and function are not fully understood. Defects in CAD result in a severe neurometabolic disorder that is challenging to diagnose. We developed a cellular functional assay to identify defective CAD variants, and in this study, we characterized five pathogenic missense mutations in CADs dihydroorotase (DHO) and aspartate transcarbamylase (ATC) domains. All mutations impaired enzymatic activities, with two notably disrupting the formation of DHO dimers and ATC trimers. Combining crystal structures and AlphaFold predictions, we modeled the hexameric CAD complex, highlighting the central role of the DHO and ATC domains in its assembly. Our findings provide insight into CADs stability, function, and organization, revealing that correct oligomerization of CAD into a supramolecular complex is required for its function in nucleotide synthesis and that mutations affecting this assembly are potentially pathogenic.

biochemistry↗

bioRxiv2022

Functional and structural deficiencies of Gemin5 variants associated with neurological disease

Dysfunction of RNA-binding proteins are often linked to a wide range of human disease in general, and particularly with neurological conditions. Gemin5 is a member of the survival of motor neuron (SMN) complex, a ribosome-binding protein and a translation reprogramming factor. Recently, pathogenic mutations in Gemin5 protein have been reported but the functional consequences of these variants remain elusive. Here we report functional and structural deficiencies associated with compound heterozygosity variants within the Gemin5 gene found in patients with neurodevelopmental disorders. These clinical variants are located in key domains of Gemin5, the tetratricopeptide repeat (TPR)-like dimerization module and the non-canonical RNA-binding site 1 (RBS1). We show that the TPR-like variants disrupt protein dimerization while the RBS1 variant confers protein instability. All mutants are defective in the interaction with protein networks involved in translation and RNA-driven pathways. Importantly, the TPR-like variants fail to associate with native ribosomes, abolishing the cap-dependent and selective translation control of Gemin5, and establishing a functional difference with the wild type protein. Our study provides a molecular basis of disease associated with malfunction of Gemin5 protein.

molecular biology↗