Journal: Cancer Biology & Therapy

Article Title: UBE2S promotes gastric cancer progression through RACGAP1 stabilization associated with K292-dependent K63-linked ubiquitination

doi: 10.1080/15384047.2026.2737476

Figure Lengend Snippet: UBE2S-dependent RACGAP1 stabilization is associated with K292-dependent K63-linked ubiquitination and reciprocal changes in K48-linked ubiquitination. (A) qRT-PCR analysis of UBE2S and RACGAP1 mRNA expression in GC cells following stable knockdown of UBE2S ( n = 3 per group). (B–C) Co-IP assays detecting the endogenous association between UBE2S and RACGAP1 in AGS and HGC-27 cells. (D) Protein stability analysis via CHX chase assays at indicated time points in cells overexpressing UBE2S compared to control cells. (E) Western blot analysis of UBE2S and RACGAP1 protein abundance in shCtrl-, shUBE2S-1#-, and shUBE2S-2#-expressing cells treated with vehicle or MG132. (F) Endogenous RACGAP1 was immunoprecipitated from the indicated cells, followed by immunoblotting with K48- and K63-linkage-specific ubiquitin antibodies. Linkage-specific ubiquitination signals were normalized to immunoprecipitated RACGAP1. (G) IP of RACGAP1 followed by immunoblotting with K63-linked and K48-linked ubiquitin-specific antibodies in control or UBE2S-overexpressing cells. (H) Schematic representation of potential lysine (K) ubiquitination sites on the RACGAP1 protein based on PhosphoSitePlus databases. (I) Western blot analysis comparing the protein levels of Flag-tagged wild-type (WT) RACGAP1 and various lysine-to-arginine (K-R) mutants (K122R, K292R, K423R, K490R, K587R) in the presence or absence of UBE2S overexpression. (J) Ubiquitination assay utilizing a K63-only ubiquitin mutant (HA-K63-only-Ub) to detect the K63-linked polyubiquitination levels of RACGAP1-WT versus the K292R mutant under UBE2S overexpression conditions. For panel A, data are presented as the mean ± SD and were analyzed by one-way ANOVA followed by the appropriate post-hoc test.

Article Snippet: For the analysis of endogenous ubiquitin linkage, control and oeUBE2S GC cells were treated with 5 μM MG132 (MCE, Cat No. HY-13259) for 6 h prior to harvesting to block proteasomal degradation.

Techniques: Ubiquitin Proteomics, Quantitative RT-PCR, Analysis, Expressing, Knockdown, Co-Immunoprecipitation Assay, Stability, Control, Western Blot, Quantitative Proteomics, Immunoprecipitation, Over Expression, Mutagenesis

Journal: Journal of Translational Medicine

Article Title: USP22 suppresses trophoblast cell necroptosis in preeclampsia by stabilizing KAT2A-mediated histone acetylation at the SFRP1 promoter

doi: 10.1186/s12967-026-08513-3

Figure Lengend Snippet: USP22 stabilizes KAT2A protein through deubiquitination in trophoblast cells. ( A-B ) CHX chase assay of KAT2A protein stability in HTR-8/SVneo cells. Panel A (CHX alone) demonstrated that hypoxia accelerates KAT2A degradation, whereas USP22 overexpression stabilizes KAT2A under hypoxic conditions. Panel B (CHX + MG132) revealed that KAT2A degradation is proteasome-dependent, and USP22 overexpression delays this proteasomal degradation. Time points: 0, 2, 4, and 6 h. ( n = 3 independent biological replicates, each with technical triplicates); Two-way ANOVA followed by Tukey’s post hoc test. ( C ) Co-IP analysis of KAT2A ubiquitination. Cell lysates from NC, OE-NC, and OE-USP22 groups were immunoprecipitated with anti-UB antibody and immunoblotted for KAT2A. Left: total input (UB, KAT2A, β-actin); Right: anti-UB IP probed for UB and KAT2A (IgG control). Densitometric quantification showed USP22 overexpression significantly reduced total KAT2A ubiquitination versus NC and OE-NC. ( n = 3 independent biological replicates, each with technical triplicates); one-way ANOVA followed by Tukey’s post hoc test, **** P < 0.0001 vs. OE-NC; ns vs. NC. ( D ) USP22 C185A mutant expression validation by qRT-PCR and Western blotting confirmed significant mRNA and protein elevation versus OE-NC. ( n = 3 independent biological replicates, each with technical triplicates); unpaired Student’s t-test, *** P = 0.002 and **** P < 0.0001 vs. OE-NC. ( E ) Co-IP analysis of K48- and K63-linked ubiquitination of KAT2A. USP22 knockdown cells transfected with OE-NC, OE-USP22 WT, or OE-USP22 C185A under hypoxia were IP’d with anti-USP22 and IB’d for K48-Ub and K63-Ub. OE-USP22 WT markedly reduced K48-Ub in Input/IP without affecting K63-Ub ( P > 0.05). OE-USP22 C185A led to significant K48-Ub accumulation. ( n = 3 independent biological replicates, each with technical triplicates); one-way ANOVA followed by Tukey’s post hoc test, **** P < 0.0001 vs. OE-NC (WT), *** P = 0.003 vs. OE-NC (C185A), #### P < 0.0001 vs. OE-USP22 WT. ( F ) Flow cytometry analysis of hypoxia-induced cell death in USP22-knockdown cells reconstituted with USP22 WT or C185A mutant. Wild-type USP22 significantly reduced total apoptosis rates and Annexin V+/PI+ cell proportions compared to the catalytically inactive C185A mutant. ( n = 3 independent biological replicates, each with technical triplicates); unpaired Student’s t-test, * P = 0.0145 vs. OE-USP22 WT. All data are mean ± SD from three independent experiments unless otherwise specified. Abbreviations (new to this figure): CHX, cycloheximide; MG132, carbobenzoxy-Leu-Leu-leucinal (proteasome inhibitor); UB, ubiquitin; K48-Ub, K48-linked polyubiquitin chain; K63-Ub, K63-linked polyubiquitin chain; IP, immunoprecipitation; IB, immunoblot; WT, wild-type. Abbreviations shared with Figs. , and are as previously defined

Article Snippet: Lysates were subsequently immunoprecipitated with an anti-KAT2A antibody, followed by immunoblotting with linkage-specific antibodies against K48-linked ubiquitin (K48-Ub; A3606, Abclonal) and K63-linked ubiquitin (K63-Ub; A18164, Abclonal).

Techniques: Over Expression, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Immunoprecipitation, Control, Mutagenesis, Expressing, Biomarker Discovery, Quantitative RT-PCR, Western Blot, Knockdown, Transfection, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: USP22 suppresses trophoblast cell necroptosis in preeclampsia by stabilizing KAT2A-mediated histone acetylation at the SFRP1 promoter

doi: 10.1186/s12967-026-08513-3

Figure Lengend Snippet: Schematic model of the USP22-KAT2A-SFRP1 epigenetic regulatory axis in trophoblast necroptosis during PE. Left Panel (USP22-high Protective Pathway) : Under physiological conditions, high USP22 expression maintains trophoblast survival via a protective epigenetic cascade. USP22 deubiquitinase activity removes ubiquitin (Ub) from KAT2A, preventing its proteasomal degradation and maintaining KAT2A stability. Stabilized KAT2A translocates to the nucleus, catalyzing histone acetylation (Ac) at the SFRP1 promoter. This epigenetic activation enhances SFRP1 mRNA transcription and protein expression. Elevated SFRP1 suppresses RIPK1 and RIPK3 kinase activation, inhibiting MLKL-mediated necroptosis and promoting trophoblast survival. Right Panel (USP22-low Pathological Pathway) : In PE, reduced USP22 expression disrupts the protective axis, leading to trophoblast necroptosis. Decreased USP22 deubiquitinase activity increases KAT2A K48-linked ubiquitination and subsequent proteasomal degradation. Loss of KAT2A reduces histone H3K9 and H3K27 acetylation at the SFRP1 promoter, suppressing transcription and decreasing SFRP1 expression. Reduced SFRP1 fails to inhibit RIPK1/RIPK3 kinase activity, resulting in necrosome assembly and MLKL-mediated plasma membrane disruption. This ultimately leads to trophoblast necroptosis, contributing to PE pathogenesis via impaired placental development and function. Abbreviations: USP22, ubiquitin-specific peptidase 22; KAT2A, lysine acetyltransferase 2 A; SFRP1, secreted frizzled-related protein 1; RIPK1, receptor-interacting serine/threonine-protein kinase 1; RIPK3, receptor-interacting serine/threonine-protein kinase 3; MLKL, mixed lineage kinase domain-like pseudokinase; Ub, ubiquitin; Ac, acetylation; PE, preeclampsia

Article Snippet: Lysates were subsequently immunoprecipitated with an anti-KAT2A antibody, followed by immunoblotting with linkage-specific antibodies against K48-linked ubiquitin (K48-Ub; A3606, Abclonal) and K63-linked ubiquitin (K63-Ub; A18164, Abclonal).

Techniques: Expressing, Activity Assay, Ubiquitin Proteomics, Activation Assay, Clinical Proteomics, Membrane, Disruption

Journal: Advanced Science

Article Title: RNF138‐Mediated Ubiquitination and Degradation of NS5 Restricts Tick‐Borne Encephalitis Virus Infection

doi: 10.1002/advs.75991

Figure Lengend Snippet: RNF138 Promotes K48‐linked Polyubiquitination of NS5 at K372, K462, and K470. (A) NS5 is preferentially modified by K48‐linked polyubiquitin chains. HEK293T cells expressing HA‐NS5 with Ub‐WT‐Flag, Ub‐K48‐Flag, or Ub‐K63‐Flag were treated with MG132 (10 µ m , 12 h). HA‐IP followed by IB reveals robust K48‐linked polyubiquitination of NS5. (B) RNF138 overexpression enhances NS5 polyubiquitination. HEK293T cells co‐expressing HA‐NS5 and Myc‐RNF138 were treated with MG132 and subjected to HA‐IP and anti‐Ub IB. (C) RNF138 knockdown reduces NS5 polyubiquitination. Stable RNF138‐silenced cells expressing HA‐NS5 were treated with MG132 and analyzed by HA‐IP and anti‐Ub IB. (D) RNF138 directly catalyzes K48‐linked ubiquitination of the TBEV NS5 RdRp domain in vitro. Purified E1, E2, His‐RNF138‐WT or His‐RNF138‐C18A/C54A, GST‐tagged TBEV NS5 RdRp, K48‐linked ubiquitin, and ATP were incubated as indicated. Reaction products were analyzed by IB using an anti‐K48‐linkage‐specific antibody. (E‐F) Ubiquitin‐remnant profiling identifies candidate ubiquitination sites on NS5. (E) Workflow: HEK293T cells expressing Flag‐NS5 and 6×His‐ubiquitin were treated with MG132. Flag‐IP samples were competitively eluted and analyzed by LC‐MS/MS (Created with BioRender.com; agreement number: SA291RDS2B). (F) Ten lysine (K) residues were initially identified as candidate ubiquitination sites. (G) Mutational analysis identifies RNF138‐responsive ubiquitination sites. Cells co‐expressing Myc‐RNF138, Ub‐K48‐Flag, and HA‐NS5 (WT or individual K‐to‐R mutants) were subjected to HA‐IP and IB. (H) Representative LC‐MS/MS spectra confirming site‐specific ubiquitination at K372, K462, and K470. The spectra display K‐ε‐GG‐modified peptides. (I) The triple‐lysine mutant (3KR) abolishes RNF138‐mediated K48‐linked polyubiquitination of NS5. HEK293T cells expressing Myc‐RNF138, Ub‐K48‐Flag, and HA‐NS5 (WT or 3KR) were treated with MG132 and analyzed by HA‐IP and IB. (J) Structural mapping of NS5 ubiquitination sites. The AlphaFold3‐predicted NS5 RdRp structure is shown with K372, K462, and K470 highlighted in red. Unless stated otherwise, MG132 treatment = 10 µ m , 12 h. All data shown are representative of at least three independent experiments.

Article Snippet: After incubation at 37°C for 2 h, the reaction products were subjected to IB with an anti‐K48‐linkage‐specific pAb (ABclonal, A3606, China) to detect K48‐linked ubiquitination.

Techniques: Modification, Expressing, Over Expression, Knockdown, Ubiquitin Proteomics, In Vitro, Purification, Incubation, Liquid Chromatography with Mass Spectroscopy, Mutagenesis