Overexpression of centromere-localized IFIT3 impairs mitochondrial function and disrupts cellular proliferation and...

Down syndrome (DS, trisomy 21) is the most prevalent human autosomal aneuploidy, driven by non-disjunction of chromosome 21 during maternal gametogenesis. Centromere-kinetochore complexes mediate accurate chromosome segregation, and centromeric protein dysregulation is linked to chromosome instability; nevertheless, how perturbations of centromere-proximal proteins drive cellular pathogenesis in DS remain poorly defined.

We applied CENPA-GFP-APEX2 proximity labeling coupled with LC-MS/MS quantitative proteomics to systematically profile CENPA-proximal proteins in euploid normal amniocytes (NACs) and trisomy 21 DS-derived amniocytes (DSACs). Differentially abundant candidate proteins (DAPs) were biochemically validated by Western blot, followed by bioinformatic enrichment analyses. The top upregulated candidate Interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) was overexpressed in human induced pluripotent stem cells (hiPSCs) to dissect its functional impacts on DS-relevant cellular phenotypes. RNA-seq transcriptome profiling was conducted to uncover signaling and metabolic pathways perturbed by IFIT3 overexpression, and mitochondrial functional assays were performed to validate bioinformatic predictions.

We quantified 999 total CENPA-proximal proteins via label-free proteomics, identifying 32 DAPs in DSACs relative to NACs (19 upregulated, 13 downregulated). Enrichment analyses revealed robust dysregulation of interferon response and ribosomal protein networks in DSACs, with IFIT3 exhibiting the most significant upregulation. Lentiviral IFIT3-OE in pluripotent hiPSCs triggered G1-phase cell cycle arrest, suppressed cell proliferation, and elevated apoptotic rates. Transcriptome-wide RNA-seq demonstrated strong transcriptional repression of oxidative phosphorylation and mitochondrial ATP biogenesis gene sets, accompanied by direct functional deficits including reduced mitochondrial membrane potential and lowered intracellular ATP levels.

This study identifies IFIT3 as a highly dysregulated CENPA-proximal candidate protein in fetal DS amniocytes. Gain-of-function assays in hiPSCs confirm that elevated IFIT3 expression is sufficient to disrupt mitochondrial homeostasis and recapitulate core DS cellular defects. Our work provides a novel proteomic framework linking centromere-proximal signaling to mitochondrial dysfunction in trisomy 21, and highlights IFIT3 as a candidate mediator of DS pathogenesis.