UBE2N promotes cell viability and glycolysis by promoting Axin1 ubiquitination in prostate cancer cells.

BACKGROUND: Ubiquitin-conjugating enzyme E2 N (UBE2N) is recognized in the progression of some cancers; however, little research has been conducted to describe its role in prostate cancer. The purpose of this paper is to explore the function and mechanism of UBE2N in prostate cancer cells. METHODS: UBE2N expression was detected in Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) data, prostate cancer tissue microarrays, and prostate cancer cell lines, respectively. UBE2N knockdown or overexpression was used to analyze its role in cell viability and glycolysis of prostate cancer cells and tumor growth. XAV939 or Axin1 overexpression was co-treated with UBE2N overexpression to detect the involvement of the Wnt/ß-catenin signaling and Axin1 in the UBE2N function. UBE2N interacting with Axin1 was analyzed by co-immunoprecipitation assay. RESULTS: UBE2N was upregulated in prostate cancer and the UBE2N-high expression correlated with the poor prognosis of prostate cancer. UBE2N knockdown inhibited cell viability and glycolysis in prostate cancer cells and restricted tumor formation in tumor-bearing mice. Wnt/ß-catenin inhibition and Axin1 overexpression reversed the promoting viability and glycolysis function of UBE2N. UBE2N promoted Axin1 ubiquitination and decreased Axin1 protein level.

Swd2/Cps35 determines H3K4 tri-methylation via interactions with Set1 and Rad6.

BACKGROUND: Histone H3K4 tri-methylation (H3K4me3) catalyzed by Set1/COMPASS, is a prominent epigenetic mark found in promoter-proximal regions of actively transcribed genes. H3K4me3 relies on prior monoubiquitination at the histone H2B (H2Bub) by Rad6 and Bre1. Swd2/Cps35, a Set1/COMPASS component, has been proposed as a key player in facilitating H2Bub-dependent H3K4me3. However, a more comprehensive investigation regarding the relationship among Rad6, Swd2, and Set1 is required to further understand the mechanisms and functions of the H3K4 methylation. RESULTS: We investigated the genome-wide occupancy patterns of Rad6, Swd2, and Set1 under various genetic conditions, aiming to clarify the roles of Set1 and Rad6 for occupancy of Swd2. Swd2 peaks appear on both the 5' region and 3' region of genes, which are overlapped with its tightly bound two complexes, Set1 and cleavage and polyadenylation factor (CPF), respectively. In the absence of Rad6/H2Bub, Set1 predominantly localized to the 5' region of genes, while Swd2 lost all the chromatin binding. However, in the absence of Set1, Swd2 occupancy near the 5' region was impaired and rather increased in the 3' region. CONCLUSIONS: This study highlights that the catalytic activity of Rad6 is essential for all the ways of Swd2's binding to the transcribed genes and Set1 redistributes the Swd2 to the 5' region for accomplishments of H3K4me3 in the genome-wide level.

UBE2C promotes the proliferation of acute myeloid leukemia cells through PI3K/AKT activation.

This study aims to investigate the role and mechanism of tubiquitin-conjugating enzyme E2 C (UBE2C) in acute myeloid leukemia (AML). Initially, UBE2C expression in leukemia was analyzed using the Cancer Genome Atlas database. Further, we silenced UBE2C expression using small-hairpin RNA (sh-RNA). UBE2C expression was detected via the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot analysis. Apoptotic events and reactive oxygen species (ROS) levels were detected by flow cytometry. A xenograft model of leukemia cells were established, and the protein levels of UBE2C, KI-67, and cleaved-caspase 3 were detected by immunohistochemistry. We reported an overexpression of UBE2C in leukemia patients and cell lines (HL60, THP-1, U937, and KG-1 cells). Moreover, a high expression level of UBE2C was correlated with a dismal prognosis in AML patients. UBE2C knockdown inhibited the viability and promoted apoptosis in AML cells by regulating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. Furthermore, UBE2C knockdown increased cellular Fe2+ and ROS levels, and enhanced erastin-induced ferroptosis in a proteasome-dependent manner. UBE2C knockdown also suppressed the tumor formation of AML cells in the mouse model. In summary, our findings suggest that UBE2C overexpression promotes the proliferation and inhibits ferroptosis in AML cells by activating the PI3K/AKT pathway.

Echinococcus granulosus ubiquitin-conjugating enzymes (E2D2 and E2N) promote the formation of liver fibrosis in TGFß1-induced LX-2 cells.

BACKGROUND: Cystic echinococcosis (CE) is a widespread zoonosis caused by the infection with Echinococcus granulosus sensu lato (E. granulosus s.l.). CE cysts mainly develop in the liver of intermediate hosts, characterized by the fibrotic tissue that separates host organ from parasite. However, precise mechanism underlying the formation of fibrotic tissue in CE remains unclear. METHODS: To investigate the potential impact of ubiquitin-conjugating enzymes on liver fibrosis formation in CE, two members of ubiquitin-conjugating (UBC) enzyme of Echinococcus granulosus (EgE2D2 and EgE2N) were recombinantly expressed in Escherichia coli and analyzed for bioinformatics, immunogenicity, localization, and enzyme activity. In addition, the secretory pathway and their effects on the formation of liver fibrosis were also explored. RESULTS: Both rEgE2D2 and rEgE2N possess intact UBC domains and active sites, exhibiting classical ubiquitin binding activity and strong immunoreactivity. Additionally, EgE2D2 and EgE2N were widely distributed in protoscoleces and germinal layer, with differences observed in their distribution in 25-day strobilated worms. Further, these two enzymes were secreted to the hydatid fluid and CE-infected sheep liver tissues via a non-classical secretory pathway. Notably, TGFß1-induced LX-2 cells exposed to rEgE2D2 and rEgE2N resulted in increasing expression of fibrosis-related genes, enhancing cell proliferation, and facilitating cell migration. CONCLUSIONS: Our findings suggest that EgE2D2 and EgE2N could secrete into the liver and may interact with hepatic stellate cells, thereby promoting the formation of liver fibrosis.

Genome­wide identification and expression analysis of the UBC gene family in wheat (Triticum aestivum L.).

BACKGROUND: Ubiquitination is an important regulatory step of selective protein degradation in the plant UPS (ubiquitin-proteasome system), which is involved in various biological processes in eukaryotes. Ubiquitin-conjugating enzymes play an intermediate role in the process of protein ubiquitination reactions and thus play an essential role in regulating plant growth and response to adverse environmental conditions. However, a genome-wide analysis of the UBC gene family in wheat (Triticum aestivum L.) has not yet been performed. RESULTS: In this study, the number, physiochemical properties, gene structure, collinearity, and phylogenetic relationships of TaUBC family members in wheat were analyzed using bioinformatics methods. The expression pattern of TaUBC genes in different tissues/organs and developmental periods, as well as the transcript levels under abiotic stress treatment, were analyzed using RNA-Seq data and qRT-PCR. Meanwhile, favorable haplotypes of TaUBC25 were investigated based on wheat resequencing data of 681 wheat cultivars from the Wheat Union Database. The analyses identified a total of 93 TaUBC family members containing a UBC domain in wheat genome. These genes were unevenly distributed across 21 chromosomes, and numerous duplication events were observed between gene members. Based on phylogenetic analysis, the TaUBC family was divided into 13 E2 groups and a separate UEV group. We investigated the expression of TaUBC family genes under different tissue/organ and stress conditions by quantitative real-time PCR (qRT-PCR) analysis. The results showed that some TaUBC genes were specifically expressed in certain tissues/organs and that most TaUBC genes responded to NaCl, PEG6000, and ABA treatment with different levels of expression. In addition, we performed association analysis for the two haplotypes based on key agronomic traits such as thousand-kernel weight (TKW), kernel length (KL), kernel weight (KW), and kernel thickness (KT), examining 122 wheat accessions at three environmental sites. The results showed that TaUBC25-Hap II had significantly higher TKW, KL, KW, and KT than TaUBC25-Hap I. The distribution analysis of haplotypes showed that TaUBC25-Hap II was preferred in the natural population of wheat. CONCLUSION: Our results identified 93 members of the TaUBC family in wheat, and several genes involved in grain development and abiotic stress response. Based on the SNPs detected in the TaUBC sequence, two haplotypes, TaUBC25-Hap I and TaUBC25-Hap II, were identified among wheat cultivars, and their potential value for wheat breeding was validated by association analysis. The above results provide a theoretical basis for elucidating the evolutionary relationships of the TaUBC gene family and lay the foundation for studying the functions of family members in the future.

FOXA1/UBE2T Inhibits CD8+T Cell Activity by Inducing Mediates Glycolysis in Lung Adenocarcinoma.

BACKGROUND: Immune escape is a key factor influencing survival rate of lung adenocarcinoma (LUAD) patients, but molecular mechanism of ubiquitin binding enzyme E2T (UBE2T) affecting immune escape of LUAD remains unclear. The objective was to probe role of UBE2T in LUAD. METHODS: Bioinformatics means were adopted for analyzing UBE2T and forkhead box A1 (FOXA1) expression in LUAD tissues, the gene binding sites, the pathway UBE2T regulates, and the correlation between UBE2T and glycolysis genes. Dual luciferase and chromatin immunoprecipitation (ChIP) assays were conducted for validating the binding relationship between the two genes. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot were employed to evaluate UBE2T, FOXA1, and programmed death ligand 1 (PD-L1) levels in cancer cells. MTT assay was conducted for detecting cell viability. Cytotoxicity assay detected CD8+T cell toxicity. Cytokine expression was assayed by enzyme linked immunosorbent assay (ELISA). Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were assayed by extracellular flow analyzer. Glycolytic gene expression was analyzed by qRT-PCR, and glycolysis-related indicators were detected by ELISA. Immunohistochemistry (IHC) detected CD8+T cell infiltration in tumor tissues. RESULTS: FOXA1 and UBE2T were up-regulated in LUAD, and a binding site existed between UBE2T and FOXA1. Overexpressing UBE2T could increase PD-L1 expression and inhibit toxicity of CD8+T cells to LUAD cells. Overexpressing UBE2T repressed CD8+T cell activity in LUAD by activating the glycolysis pathway, and the addition of glycolysis inhibitor 2-deoxy-d-glucose (2-DG) reversed the above results. Mechanistically, FOXA1 promoted the immune escape of LUAD by up-regulating UBE2T and thus mediating glycolysis. In vivo experiments revealed that UBE2T knockdown hindered tumor growth, inhibited PD-L1 expression, and facilitated CD8+T cell infiltration. CONCLUSION: FOXA1 up-regulated the expression of UBE2T, which activated glycolysis, and thus inhibited activity of CD8+T cells, causing immune escape of LUAD.

UBE2L3 expression in human gastric cancer and its clinical significance.

PURPOSE: Gastric cancer (GC) is prevalent as one of the most common malignant tumors globally, with a particularly high incidence in China. The role of UBE2L3 in the initiation and progression of various cancers has been well documented, but its specific significance in GC is not yet fully elucidated. The objective of this study is to examine the expression and importance of UBE2L3 in human gastric cancer tissues. METHODS: Immunohistochemical staining and survival analysis were conducted on 125 cases of GC. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to assess the expression of UBE2L3 in GC cell lines. Cell lines with UBE2L3 knockdown and overexpression were cultured through lentivirus transfection and subsequently assessed using Western blot analysis. The involvement of UBE2L3 in the proliferation, invasion, and apoptosis of GC cells was confirmed through in vitro experiments, and its capacity to facilitate tumor growth was also validated in in vivo studies. RESULTS: The up-regulation of UBE2L3 expression was observed in GC, and its high expression was found to be significantly associated with the degree of differentiation (χ2 = 6.153, P = 0.0131), TNM stage (χ2 = 6.216, P = 0.0447), and poor overall survival. In vitro, UBE2L3 has been shown to enhance functions in GC cell lines, such as promoting proliferation and invasion, and inhibiting apoptosis. In vivo experiments have validated the role of UBE2L3 in promoting tumor growth. CONCLUSIONS: The findings of our study demonstrate the significant involvement of UBE2L3 in the pathogenesis and advancement of gastric cancer, suggesting its potential as a therapeutic target.

UBE2C: A pan-cancer diagnostic and prognostic biomarker revealed through bioinformatics analysis.

BACKGROUND: The diverse and complex attributes of cancer have made it a daunting challenge to overcome globally and remains to endanger human life. Detection of critical cancer-related gene alterations in solid tumor samples better defines patient diagnosis and prognosis, and indicates what targeted therapies must be administered to improve cancer patients' outcome. MATERIALS AND METHODS: To identify genes that have aberrant expression across different cancer types, differential expressed genes were detected within the TCGA datasets. Subsequently, the DEGs common to all pan cancers were determined. Furthermore, various methods were employed to gain genetic alterations, co-expression genes network and protein-protein interaction (PPI) network, pathway enrichment analysis of common genes. Finally, the gene regulatory network was constructed. RESULTS: Intersectional analysis identified UBE2C as a common DEG between all 28 types of studied cancers. Upregulated UBE2C expression was significantly correlated with OS and DFS of 10 and 9 types of cancer patients. Also, UBE2C can be a diagnostic factor in CESC, CHOL, GBM, and UCS with AUC = 100% and diagnose 19 cancer types with AUC ≥90%. A ceRNA network constructed including UBE2C, 41 TFs, 10 shared miRNAs, and 21 circRNAs and 128 lncRNAs. CONCLUSION: In summary, UBE2C can be a theranostic gene, which may serve as a reliable biomarker in diagnosing cancers, improving treatment responses and increasing the overall survival of cancer patients and can be a promising gene to be target by cancer drugs in the future.

A transposon insertion in the promoter of OsUBC12 enhances cold tolerance during japonica rice germination.

Low-temperature germination (LTG) is an important agronomic trait for rice (Oryza sativa). Japonica rice generally has greater capacity for germination at low temperatures than the indica subpopulation. However, the genetic basis and molecular mechanisms underlying this complex trait are poorly understood. Here, we report that OsUBC12, encoding an E2 ubiquitin-conjugating enzyme, increases low-temperature germinability in japonica, owing to a transposon insertion in its promoter enhancing its expression. Natural variation analysis reveals that transposon insertion in the OsUBC12 promoter mainly occurs in the japonica lineage. The variation detected in eight representative two-line male sterile lines suggests the existence of this allele introgression by indica-japonica hybridization breeding, and varieties carrying the japonica OsUBC12 locus (transposon insertion) have higher low-temperature germinability than varieties without the locus. Further molecular analysis shows that OsUBC12 negatively regulate ABA signaling. OsUBC12-regulated seed germination and ABA signaling mainly depend on a conserved active site required for ubiquitin-conjugating enzyme activity. Furthermore, OsUBC12 directly associates with rice SUCROSE NON-FERMENTING 1-RELATED PROTEIN KINASE 1.1 (OsSnRK1.1), promoting its degradation. OsSnRK1.1 inhibits LTG by enhancing ABA signaling and acts downstream of OsUBC12. These findings shed light on the underlying mechanisms of UBC12 regulating LTG and provide genetic reference points for improving LTG in indica rice.

p53 accelerates endothelial cell senescence in diabetic retinopathy by enhancing FoxO3a ubiquitylation and degradation via UBE2L6.

Diabetic retinopathy (DR) is the most common ocular fundus disease in diabetic patients. Chronic hyperglycemia not only promotes the development of diabetes and its complications, but also aggravates the occurrence of senescence. Previous studies have shown that DR is associated with senescence, but the specific mechanism has not been fully elucidated. Here, we first detected the differentially expressed genes (DEGs) and cellular senescence level of db/db mouse retinas by bulk RNA sequencing. Then, we used single-cell sequencing (scRNA-seq) to identify the main cell types in the retina and analyzed the DEGs in each cluster. We demonstrated that p53 expression was significantly increased in retinal endothelial cell cluster of db/db mice. Inhibition of p53 can reduce the expression of SA-ß-Gal and the senescence-associated secretory phenotype (SASP) in HRMECs. Finally, we found that p53 can promote FoxO3a ubiquitination and degradation by increasing the expression of the ubiquitin-conjugating enzyme UBE2L6. Overall, our results demonstrate that p53 can accelerate the senescence process of endothelial cells and aggravate the development of DR. These data reveal new targets and insights that may be used to treat DR.

Recruitment of ubiquitin E2 enzymes is determined jointly by the U-box domains and substrates of E3 ligases.

Ubiquitination is a cascade reaction involving E1, E2, and E3 enzymes. The orthogonal ubiquitin transfer (OUT) method has been previously established to identify potential substrates of E3 ligases. In this study, we verified the ubiquitination of five substrates mediated by the E3 ligases CHIP and E4B. To further explore the activity of U-box domains of E3 ligases, two mutants with the U-box domains interchanged between CHIP and E4B were generated. They exhibited a significantly reduced ubiquitination ability. Additionally, different E3s recruited similar E2 ubiquitin-conjugating enzymes when ubiquitinating the same substrates, highlighting that U-box domains determined the E2 recruitment, while the substrate determined the E2 selectivity. This study reveals the influence of substrates and U-box domains on E2 recruitment, providing a novel perspective on the function of U-box domains of E3 ligases.

Identification and validation of UBE2B as a prognostic biomarker promoting the development of esophageal carcinomas.

Introduction: Ubiquitination is a crucial biological mechanism in humans, essential for regulating vital biological processes, and has been recognized as a promising focus for cancer therapy. Our objective in this research was to discover potential enzymes associated with ubiquitination that may serve as therapeutic targets for individuals with esophageal carcinoma (ESCA). Methods: To identify genes linked to the prognosis of ESCA, we examined mRNA sequencing data from patients with ESCA in the TCGA database. Further investigation into the role of the candidate gene in ESCA was conducted through bioinformatic analyses. Subsequently, we carried out biological assays to assess its impact on ESCA development. Results: Through univariate Cox regression analysis, we identified Ubiquitin Conjugating Enzyme E2 B (UBE2B) as a potential gene associated with the prognosis of ESCA. UBE2B exhibited significant upregulation and was found to be correlated with survival outcomes in ESCA as well as other cancer types. Additionally, UBE2B was observed to be involved in various biological pathways linked to the development of ESCA, including TNF-a signaling via NF-κB, epithelial-mesenchymal transition, inflammatory response, and hypoxia. Moreover, immune-related pathways like B cell activation (GO: 0042113), B cell receptor signaling pathway (GO: 0050853) and B cell mediated immunity (GO:0019724) were also involved. It was found that high expression of UBE2B was correlated with the increase of several kinds of T cells (CD8 T cells, Th1 cells) and macrophages, while effector memory T cell (Tem) and Th17 cells decreased. Furthermore, UBE2B showed potential as a prognostic biomarker for ESCA, displaying high sensitivity and specificity. Notably, proliferation and migration in ESCA cells were effectively suppressed when the expression of UBE2B was knocked down. Conclusions: To summarize, this study has made a discovery regarding the importance of gaining new insights into the role of UBE2B in ESCA. UBE2B might be an oncogene with good ability in predicting and diagnosing ESCA. Consequently, this discovery highlights the feasibility of targeting UBE2B as a viable approach for treating patients with ESCA.

Insights on E1-like enzyme ATG7: functional regulation and relationships with aging-related diseases.

Autophagy is a dynamic self-renovation biological process that maintains cell homeostasis and is responsible for the quality control of proteins, organelles, and energy metabolism. The E1-like ubiquitin-activating enzyme autophagy-related gene 7 (ATG7) is a critical factor that initiates classic autophagy reactions by promoting the formation and extension of autophagosome membranes. Recent studies have identified the key functions of ATG7 in regulating the cell cycle, apoptosis, and metabolism associated with the occurrence and development of multiple diseases. This review summarizes how ATG7 is precisely programmed by genetic, transcriptional, and epigenetic modifications in cells and the relationship between ATG7 and aging-related diseases.

Structural basis for RAD18 regulation by MAGEA4 and its implications for RING ubiquitin ligase binding by MAGE family proteins.

MAGEA4 is a cancer-testis antigen primarily expressed in the testes but aberrantly overexpressed in several cancers. MAGEA4 interacts with the RING ubiquitin ligase RAD18 and activates trans-lesion DNA synthesis (TLS), potentially favouring tumour evolution. Here, we employed NMR and AlphaFold2 (AF) to elucidate the interaction mode between RAD18 and MAGEA4, and reveal that the RAD6-binding domain (R6BD) of RAD18 occupies a groove in the C-terminal winged-helix subdomain of MAGEA4. We found that MAGEA4 partially displaces RAD6 from the RAD18 R6BD and inhibits degradative RAD18 autoubiquitination, which could be countered by a competing peptide of the RAD18 R6BD. AlphaFold2 and cross-linking mass spectrometry (XL-MS) also revealed an evolutionary invariant intramolecular interaction between the catalytic RING and the DNA-binding SAP domains of RAD18, which is essential for PCNA mono-ubiquitination. Using interaction proteomics, we found that another Type-I MAGE, MAGE-C2, interacts with the RING ubiquitin ligase TRIM28 in a manner similar to the MAGEA4/RAD18 complex, suggesting that the MAGEA4 peptide-binding groove also serves as a ligase-binding cleft in other type-I MAGEs. Our data provide new insights into the mechanism and regulation of RAD18-mediated PCNA mono-ubiquitination.

Discovery and characterization of noncanonical E2-conjugating enzymes.

E2-conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the attachment of ubiquitin to a substrate. This process, termed ubiquitylation, is required to maintain cellular homeostasis and affects almost all cellular process. By interacting with multiple E3 ligases, E2s dictate the ubiquitylation landscape within the cell. Since its discovery, ubiquitylation has been regarded as a posttranslational modification that specifically targets lysine side chains (canonical ubiquitylation). We used Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry to identify and characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. We used structural modeling and prediction tools to identify the key activity determinants that these E2s use to interact with ubiquitin as well as their substrates. Our results unveil the missing E2s necessary for noncanonical ubiquitylation, underscoring the adaptability and versatility of ubiquitin modifications.

Formation of functional E3 ligase complexes with UBC2 and UEV1 of Leishmania mexicana.

In eukaryotic cells, molecular fate and cellular responses are shaped by multicomponent enzyme systems which reversibly attach ubiquitin and ubiquitin-like modifiers to target proteins. The extent of the ubiquitin proteasome system in Leishmania mexicana and its importance for parasite survival has recently been established through deletion mutagenesis and life-cycle phenotyping studies. The ubiquitin conjugating E2 enzyme UBC2, and the E2 enzyme variant UEV1, with which it forms a stable complex in vitro, were shown to be essential for the differentiation of promastigote parasites to the infectious amastigote form. To investigate further, we used immunoprecipitation of Myc-UBC2 or Myc-UEV1 to identify interacting proteins in L. mexicana promastigotes. The interactome of UBC2 comprises multiple ubiquitin-proteasome components including UEV1 and four RING E3 ligases, as well as potential substrates predicted to have roles in carbohydrate metabolism and intracellular trafficking. The smaller UEV1 interactome comprises six proteins, including UBC2 and shared components of the UBC2 interactome consistent with the presence of intracellular UBC2-UEV1 complexes. Recombinant RING1, RING2 and RING4 E3 ligases were shown to support ubiquitin transfer reactions involving the E1, UBA1a, and UBC2 to available substrate proteins or to unanchored ubiquitin chains. These studies define additional components of a UBC2-dependent ubiquitination pathway shown previously to be essential for promastigote to amastigote differentiation.

Prognostic implication of UBE2C + CD8 + T cell in neoadjuvant immune checkpoint blockade plus chemotherapy for locally advanced esophageal cancer.

BACKGROUND: Immune checkpoint blockers (ICBs) plus chemotherapy as neoadjuvant therapy for patients with esophageal cancer (EC) has gained substantial attention. This study aimed to investigate the early and mid-term outcome of neoadjuvant ICBs plus chemotherapy and discover immune-associated predictors of major pathological response (MPR) for locally advanced EC. METHOD: Patients with locally advanced EC who received neoadjuvant ICBs plus chemotherapy were retrospectively included between June 2019 to December 2021. Conjoint analysis of Bulk-RNA seq (GSE165252) and scRNA seq (GSE188900) were used to investigate potential prognostic factors and immunological mechanisms, then multiplexed immunofluorescence was applied to validate. RESULTS: 76 patients were included. A total of 21 (27.6 %) patients achieved MPR, with 13 (17.1 %) attaining a pathological complete response. Over a median follow-up of 1.8 years, 6 (7.9 %) patients died and 21 (27.6 %) experienced disease recurrence within 0.6 to 2.1 years after surgery. The overall survival rate and recurrence-free survival rate were 93.3 + 2.9 % and 84.8 + 4.2 % at 12 months, 90.8 + 3.7 % and 67.1 + 6.4 % at 24 months, and 90.8 + 3.7 % and 62.9 + 7.2 % at 36 months, respectively. Patients achieving MPR had a significantly lower risk of recurrence compared to non-responders (9.5 % vs 34.5 %, P = 0.017). Analysis of bulk-RNA seq and scRNA-seq revealed that UBE2C and UBE2C + CD8 + T cells were adverse prognostic factors. Immunohistochemistry demonstrated that the non-MPR group had a higher infiltration of UBE2C + immune cells than MPR group after neoadjuvant treatment. Multiplexed immunofluorescence confirmed that infiltrating UBE2C + CD8 + T cells in MPR group were significantly fewer than non-MPR group after neoadjuvant treatment, indicating their poor prognostic role for EC. CONCLUSIONS: Neoadjuvant ICBs plus chemotherapy shows promising efficacy in locally advanced EC, with MPR being a significant predictor of lower recurrence risk. Immunological analyses identified UBE2C + CD8 + T cells as adverse prognostic factors, suggesting their potential as biomarkers for patient stratification and treatment response.

Discovery of WS-384, a first-in-class dual LSD1 and DCN1-UBC12 protein-protein interaction inhibitor for the treatment of non-small cell lung cancer.

Abnormally high expression of lysine-specific demethylase 1 A (LSD1) and DCN1 plays a vital role in the occurrence, development, and poor prognosis of non-small cell lung cancer (NSCLC). Accumulating evidence has shown that the development of small-molecule inhibitors dually targeting LSD1 and the DCN1-UBC12 interaction probably have therapeutic promise for cancer therapy. This work reported that WS-384 dually targeted LSD1 and DCN1-UBC12 interactions and evaluated its antitumor effects in vitro and in vivo. Specifically, WS-384 inhibited A549 and H1975 cells viability and decreased colony formation and EdU incorporation. WS-384 could also trigger cell cycle arrest, DNA damage, and apoptosis. Moreover, WS-384 significantly decreased tumor weight and volume in A549 xenograft mice. Mechanistically, WS-384 increased the gene and protein level of p21 by suppressing the neddylation of cullin 1 and decreasing H3K4 demethylation at the CDKN1A promoter. The synergetic upregulation of p21 contributed to cell cycle arrest and the proapoptotic effect of WS-384 in NSCLC cells. Taken together, our proof of concept studies demonstrated the therapeutic potential of dual inhibition of LSD1 and the DCN1-UBC12 interaction for the treatment of NSCLC. WS-384 could be used as a lead compound to develop new dual LSD1/DCN1 inhibitors for the treatment of human diseases in which LSD1 and DCN1 are dysregulated.

UBE2J1 promotes ALV-A proviral DNA synthesis through the STAT3/IRF1 signaling pathway.

The ubiquitin-binding enzyme E2J1 is located on the endoplasmic reticulum membrane. It plays a role in transport throughout the process of ubiquitination. In mammals, UBE2J1 can promote RNA virus replication. However, the biological function of chicken UBE2J1 is unclear. In this study, chicken UBE2J1 was cloned for the first time, and UBE2J1 overexpression and shRNA knockdown plasmids were constructed. In chicken embryo fibroblasts, overexpression of UBE2J1 promoted the replication of subtype A avian leukosis virus, while knockdown of UBE2J1 inhibited the replication of ALV-A virus. In addition, we divided virus replication into virus adsorption and invasion into DF-1 cells, synthesis of proviral DNA, and release of viral particles. UBE2J1 promoted the replication of ALV-A virus by promoting the synthesis of proviral DNA. This result was caused by UBE2J1 inhibiting the production of interferon by inhibiting the STAT3/IRF1 pathway. We mutated ser at position 184 of UBE2J1 to Gly and found that this site plays a role as the phosphorylation site of UBE2J1. We confirmed that UBE2J1 promotes ALV-A replication in chicken embryo fibroblasts by inhibiting the STAT3/IRF1 pathway. This study provides new ideas and insights into ubiquitin-related proteins and antiviral immunity.

MX1 and UBE2L6 are potential metaflammation gene targets in both diabetes and atherosclerosis.

Background: The coexistence of diabetes mellitus (DM) and atherosclerosis (AS) is widespread, although the explicit metabolism and metabolism-associated molecular patterns (MAMPs) responsible for the correlation are still unclear. Methods: Twenty-four genetically wild-type male Ba-Ma mini pigs were randomly divided into five groups distinguished by different combinations of 90 mg/kg streptozotocin (STZ) intravenous injection and high-cholesterol/lipid (HC) or high-lipid (HL) diet feeding for 9 months in total. Pigs in the STZ+HC and STZ+HL groups were injected with STZ first and then fed the HC or HL diet for 9 months. In contrast, pigs in the HC+STZ and HL+STZ groups were fed the HC or HL diet for 9 months and injected with STZ at 3 months. The controls were only fed a regular diet for 9 months. The blood glucose and abdominal aortic plaque observed through oil red O staining were used as evaluation indicators for successful modelling of DM and AS. A microarray gene expression analysis of all subjects was performed. Results: Atherosclerotic lesions were observed only in the HC+STZ and STZ+HC groups. A total of 103 differentially expressed genes (DEGs) were identified as common between them. The most significantly enriched pathways of 103 common DEGs were influenza A, hepatitis C, and measles. The global and internal protein-protein interaction (PPI) networks of the 103 common DEGs consisted of 648 and 14 nodes, respectively. The top 10 hub proteins, namely, ISG15, IRG6, IRF7, IFIT3, MX1, UBE2L6, DDX58, IFIT2, USP18, and IFI44L, drive aspects of DM and AS. MX1 and UBE2L6 were the intersection of internal and global PPI networks. The expression of MX1 and UBE2L6 was 507.22 ± 342.56 and 96.99 ± 49.92 in the HC+STZ group, respectively, which was significantly higher than others and may be linked to the severity of hyperglycaemia-related atherosclerosis. Further PPI network analysis of calcium/micronutrients, including MX1 and UBE2L6, consisted of 58 and 18 nodes, respectively. The most significantly enriched KEGG pathways were glutathione metabolism, pyrimidine metabolism, purine metabolism, and metabolic pathways. Conclusions: The global and internal PPI network of the 103 common DEGs consisted of 648 and 14 nodes, respectively. The intersection of the nodes of internal and global PPI networks was MX1 and UBE2L6, suggesting their key role in the comorbidity mechanism of DM and AS. This inference was partly verified by the overexpression of MX1 and UBE2L6 in the HC+STZ group but not others. Further calcium- and micronutrient-related enriched KEGG pathway analysis supported that MX1 and UBE2L6 may affect the inflammatory response through micronutrient metabolic pathways, conceptually named metaflammation. Collectively, MX1 and UBE2L6 may be potential common biomarkers for DM and AS that may reveal metaflammatory aspects of the pathological process, although proper validation is still needed to determine their contribution to the detailed mechanism.