Proteomics in Ataxia Telangiectasia Fibroblasts Revealed Disease Hallmarks Recovered by ATM Variants.

Ataxia-telangiectasia (A-T) is a multisystemic genetic disorder caused by pathogenic mutations in the ATM gene, with effects extending beyond the canonical DNA damage response. We investigated whether two compact ATM transcript variants, ATM 4-53 (exons 4 and 53 joined) and ATM SINT (an in silico-designed ATM variant), could rescue disease associated with cellular alterations and inform construct selection for future delivery strategies. A-T fibroblasts transduced with either ATM variants or an empty vector were compared with wild-type fibroblasts using complementary label-free and tandem mass tag-based quantitative proteomics. Bioinformatic pathway and network analyses were integrated with biochemical and functional validation assays. A-T fibroblasts displayed coordinated alterations in proteostasis, proteasome function, RNA processing pathways, interferon-associated signaling, extracellular matrix organization, cell migration, and nanomechanical properties. Expression of both ATM variants partially or extensively reversed these abnormalities, restoring protein folding and proteasomal components, reducing protein aggregation, attenuating inflammatory signaling, and normalizing extracellular matrix-related phenotypes. ATM SINT generally produced the broader and more consistent rescue, particularly for proteostasis and RNA processing-associated networks. These findings show that compact ATM variants can recover multiple non canonical ATM dependent cellular functions in A-T fibroblasts and identify ATM SINT as the more effective construct in this model. Proteomic profiling combined with orthogonal functional validation may therefore support rational selection of ATM variants for subsequent preclinical delivery studies.