Pathogenic TRIM74 Mutation Disrupts Protein Homeostasis and Triggers Proteotoxic Neurodegeneration via Structural Destabilization.

Ubiquitin ligases play a critical role in maintaining proteostasis, synaptic function, and neuronal survival, and their dysfunction is increasingly implicated in neurodevelopmental disorders with neurodegenerative features. In this study, we investigate mutation in the ubiquitin ligase gene [a novel homozygous missense variant c.562C > T (p.Pro121Leu)] in a 5-year-old male proband presenting with global developmental delay, hypotonia, seizures, and diffuse cerebral atrophy with mega cisterna magna. Structural and simulation studies revealed that Pro121, located at the start of a β sheet, likely functions as a sheet breaker. Substitution to leucine (P121L) resulted in aberrant beta strand extension, protein destabilization, and increased aggregation propensity. Free energy calculations indicated that all possible substitutions at this position were destabilizing. Multiple in silico prediction tools consistently classified the mutation as damaging or disease-causing. In proband-derived fibroblasts, TRIM74-P121L exhibited significant cytosolic aggregation and elevated Proteostat-positive granules, reflecting proteotoxic stress. Despite comparable transcript and total protein levels, mutant cells showed increased cell death and impaired cell cycle progression. Interaction network and gene ontology analyses revealed that TRIM74 and its partners are involved in ubiquitination, protein quality control, and transcriptional regulation─processes essential to neuronal homeostasis. TRIM74 expression was highest in the cerebellum and medulla, aligning with MRI abnormalities. Together, our findings establish the aberrant functioning of mutant TRIM74 as a pathogenic cause of neurodegenerative neurodevelopmental disorder and highlight the importance of ubiquitin ligases in maintaining neuronal integrity and preventing neurodegeneration.