To profile ubiquitination in cysteinyl aspartate-specific proteinase-2(CASP2) deficient cells under heat shock and investigate the role of CASP2 in stress response.

Ubiquitination levels in subcellular fractions of control and CASP2 knockout (KO) cells were detected via Western blotting. After 2 hours of heat shock treatment, Soluble Ⅱ and Pellet fractions were collected from both control and CASP2 KO cells for ubiquitinome analysis. Anti-di-glycine remnant (K-ε-GG) antibody-based proteomic analysis was performed to identify differentially ubiquitinated proteins and associated key signaling pathways. Proteins that displayed significantly upregulated ubiquitination in CASP2 KO cells under heat shock were subjected to His-tag pull-down assays to find out whether CASP2 regulated the ubiquitination of these proteins.

Under heat shock, CASP2 KO cells displayed significantly higher accumulation of overloaded ubiquitinated conjugates in the Pellet fraction compared to controls. Ubiquitinomics analysis revealed substantial alterations in protein ubiquitination patterns following CASP2 KO. One hundred proteins exhibited significantly elevated ubiquitination levels while 36 proteins had their ubiquitination reduced relative to controls. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that hyper-ubiquitinated proteins were primarily associated with Huntington disease, Alzheimer disease, bile secretion, carbon metabolism and autophagy. His-tag pull-down assays combined with Western blotting revealed increased ubiquitination of nicotinamide adenine dinucleotide reduced-ubiquinone oxidoreductase 1 beta subcomplex subunit 3 (NDUFB3) and autophagy-related protein 9A (ATG9A) in CASP2 KO cells under heat shock.

Overloaded ubiquitinated conjugates are accumulated due to CASP2 deficiency during heat shock. CASP2 modulates ubiquitination levels through multiple signaling pathways.