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.

To investigate how deubiquitinase OTU domain-containing protein 3 (OTUD3) suppresses the progression of hepatocellular carcinoma via gut-liver axis metabolic remodeling and microbiome dynamics.

A total of 24 male 2-week-old littermate C57BL/6J mice (12 wild-type and 12 Otud3^-/-) were divided into two differential genotype groups before 6 mice from each group were randomly chosen to receive intraperitoneal injections of N-nitrosodiethylamine (DEN) for hepatocellular carcinoma (HCC) induction. The mice were divided into four groups (n=6/group): Otud3^+/+ control (WT CON), Otud3^-/- control (KO CON), Otud3^+/+ DEN-induced HCC (WT DEN), and Otud3^-/- DEN-induced HCC (KO DEN). At 40 weeks of age, liver tissues were collected for metabolomic profiling, and fecal samples were obtained for 16S rRNA sequencing.

Multivariate analyses, including principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), sparse partial least squares-discriminant analysis (sPLS-DA), and orthogonal partial least squares-discriminant analysis (OrthoPLS-DA), demonstrated complete intergroup separability. Fifty-four differential metabolites were identified between the WT DEN and KO DEN groups through metabolomic profiling, with gut-liver axis-associated pathways such as cholesterol metabolism and fatty acid biosynthesis revealed by KEGG pathway analysis. Microbiome analysis indicated an upregulation of Bacteroides at the genus level in the KO DEN group compared to WT DEN. Pearson correlation analysis highlighted amino acids and derivatives as predominant metabolite classes and revealed Bacteroidetes and Firmicutesas the dominant gut microbial phyla.

OTUD3 suppresses HCC progression by modulating gut-liver axis metabolism, potentially mediated by elevated betaine and increased abundance of Odoribacter, Alistipes, and Lachnoclostridium.

To investigate mutations in OTU domain - containing protein 3 (OTUD3) and their functional mechanisms in the initiation and progression of colorectal cancer (CRC).

Gene expression profiling interactive analysis (GEPIA2) and the human protein atlas database (THPA) were used to analyze gene transcription and protein expressions. Samples from 32 patients with CRC were collected to identify OTUD3 mutants. Based on the information about mutation sitesof OTUD3 in an existing database, a plasmid vector containing the OTUD3 gene mutant was constructed. Plasmid vectors containing the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and the OTUD3 gene mutant were co-transfected into HCT116 cells. Western blotting, half-life, immunoprecipitation, ubiquitination, and hybrid algorithm molecular docking (H-DOCK) assays were employed to find out whether and why the OTUD3 mutants affected PTEN protein levels. Functional alterations in CRC cells after OTUD3 mutation were verified by CCK-8 cell proliferation, transwell cell invasion, scratch, and clonal formation assays.

OTUD3 mutations were highly frequent in CRC. OTUD3 mutants R178W and N321S resulted in the loss of function of the stable PTEN protein, leading to enhanced proliferation, invasion, migration, and survival of CRC.

In CRC, OTUD3 mutation reduces the ability to stabilize PTEN and promotes the occurrence and development of CRC.