Corrigendum: Ginsenoside Rg1 ameliorates neuroinflammation via suppression of connexin43 ubiquitination to attenuate depression.

[This corrects the article DOI: 10.3389/fphar.2021.709019.].

TRIM35 Negatively Regulates the cGAS-STING-Mediated Signaling Pathway by Attenuating K63-Linked Ubiquitination of STING.

The cGAS-STING-mediated antiviral response plays an important role in the defense against DNA virus infection. Tripartite motif protein 35 (TRIM35), an E3 ubiquitin ligase, was identified as a positive regulator of RLR-mediated antiviral signaling in our previous study, but the effect of TRIM35 on the cGAS-STING signaling pathway has not been elucidated. Herein, we showed that TRIM35 negatively regulates the cGAS-STING signaling pathway by directly targeting STING. TRIM35 overexpression significantly inhibited the cGAMP-triggered phosphorylation of TBK1 and IRF3, attenuating IFN-ß expression and the downstream antiviral response. Mechanistically, TRIM35 colocalized and directly interacted with STING in the cytoplasm. TRM35 removed K63-linked ubiquitin from STING through the C36 and C44 sites in the RING domain, which impaired the interaction of STING with TBK1 or IKKε. In addition, we demonstrated that the RING domain is a key region for the antiviral effects of TIRM35. These results collectively indicate that TRIM35 negatively regulates type I interferon (IFN-I) production by targeting and deubiquitinating STING. TRIM35 may be a potential therapeutic target for controlling viral infection.

Ubiquitin-modifying enzymes in thyroid cancer：Mechanisms and functions.

Thyroid cancer is the most common malignant tumor of the endocrine system, and evidence suggests that post-translational modifications (PTMs) and epigenetic alterations play an important role in its development. Recently, there has been increasing evidence linking dysregulation of ubiquitinating enzymes and deubiquitinases with thyroid cancer. This review aims to summarize our current understanding of the role of ubiquitination-modifying enzymes in thyroid cancer, including their regulation of oncogenic pathways and oncogenic proteins. The role of ubiquitination-modifying enzymes in thyroid cancer development and progression requires further study, which will provide new insights into thyroid cancer prevention, treatment and the development of novel agents.

Post-translational modifications: The potential ways for killing cancer stem cells.

While strides in cancer treatment continue to advance, the enduring challenges posed by cancer metastasis and recurrence persist as formidable contributors to the elevated mortality rates observed in cancer patients. Among the multifaceted factors implicated in tumor recurrence and metastasis, cancer stem cells (CSCs) emerge as noteworthy entities due to their inherent resistance to conventional therapies and heightened invasive capacities. Characterized by their notable abilities for self-renewal, differentiation, and initiation of tumorigenesis, the eradication of CSCs emerges as a paramount objective. Recent investigations increasingly emphasize the pivotal role of post-translational protein modifications (PTMs) in governing the self-renewal and replication capabilities of CSCs. This review accentuates the critical significance of several prevalent PTMs and the intricate interplay of PTM crosstalk in regulating CSC behavior. Furthermore, it posits that the manipulation of PTMs may offer a novel avenue for targeting and eliminating CSC populations, presenting a compelling perspective on cancer therapeutics with substantial potential for future applications.

Momordica charantia L.-derived exosome-like nanovesicles stabilize p62 expression to ameliorate doxorubicin cardiotoxicity.

BACKGROUND: Doxorubicin (DOX) is a first-line chemotherapeutic drug for various malignancies that causes cardiotoxicity. Plant-derived exosome-like nanovesicles (P-ELNs) are growing as novel therapeutic agents. Here, we investigated the protective effects in DOX cardiotoxicity of ELNs from Momordica charantia L. (MC-ELNs), a medicinal plant with antioxidant activity. RESULTS: We isolated MC-ELNs using ultracentrifugation and characterized them with canonical mammalian extracellular vesicles features. In vivo studies proved that MC-ELNs ameliorated DOX cardiotoxicity with enhanced cardiac function and myocardial structure. In vitro assays revealed that MC-ELNs promoted cell survival, diminished reactive oxygen species, and protected mitochondrial integrity in DOX-treated H9c2 cells. We found that DOX treatment decreased the protein level of p62 through ubiquitin-dependent degradation pathway in H9c2 and NRVM cells. However, MC-ELNs suppressed DOX-induced p62 ubiquitination degradation, and the recovered p62 bound with Keap1 promoting Nrf2 nuclear translocation and the expressions of downstream gene HO-1. Furthermore, both the knockdown of Nrf2 and the inhibition of p62-Keap1 interaction abrogated the cardioprotective effect of MC-ELNs. CONCLUSIONS: Our findings demonstrated the therapeutic beneficials of MC-ELNs via increasing p62 protein stability, shedding light on preventive approaches for DOX cardiotoxicity.

Secondary interactions in ubiquitin-binding domains achieve linkage or substrate specificity.

Small ubiquitin-binding domains (UBDs) recognize small surface patches on ubiquitin with weak affinity, and it remains a conundrum how specific cellular responses may be achieved. Npl4-type zinc-finger (NZF) domains are ∼30 amino acid, compact UBDs that can provide two ubiquitin-binding interfaces, imposing linkage specificity to explain signaling outcomes. We here comprehensively characterize the linkage preference of human NZF domains. TAB2 prefers Lys6 and Lys63 linkages phosphorylated on Ser65, explaining why TAB2 recognizes depolarized mitochondria. Surprisingly, most NZF domains do not display chain linkage preference, despite conserved, secondary interaction surfaces. This suggests that some NZF domains may specifically bind ubiquitinated substrates by simultaneously recognizing substrate and an attached ubiquitin. We show biochemically and structurally that the NZF1 domain of the E3 ligase HOIPbinds preferentially to site-specifically ubiquitinated forms of NEMO and optineurin. Thus, despite their small size, UBDs may impose signaling specificity via multivalent interactions with ubiquitinated substrates.

Arabidopsis COP1 suppresses root hair development by targeting type I ACS proteins for ubiquitination and degradation.

Root hairs (RHs) are an innovation of vascular plants whose development is coordinated by endogenous and environmental cues, such as ethylene and light conditions. However, the potential crosstalk between ethylene and light conditions in RH development is unclear. We report that Arabidopsis constitutive photomorphogenic 1 (COP1) integrates ethylene and light signaling to mediate RH development. Darkness suppresses RH development largely through COP1. COP1 inhibits both cell fate determination of trichoblast and tip growth of RHs based on pharmacological, genetic, and physiological analyses. Indeed, COP1 interacts with and catalyzes the ubiquitination of ACS2 and ACS6. COP1- or darkness-promoted proteasome-dependent degradation of ACS2/6 leads to a low ethylene level in underground tissues. The negative role of COP1 in RH development by downregulating ethylene signaling may be coordinated with the positive role of COP1 in hypocotyl elongation by upregulating ethylene signaling, providing an evolutionary advantage for seedling fitness.

CYYR1 promotes the degradation of the E3 ubiquitin ligase WWP1 and is associated with favorable prognosis in breast cancer.

Ubiquitination plays a crucial role in cellular homeostasis by regulating the degradation, localization, and activity of proteins, ensuring proper cell function and balance. Among E3 ubiquitin ligases, WWP1 is implicated in cell proliferation, survival and apoptosis. Notably WWP1 is frequently amplified in breast cancer and associated with poor prognosis. Here we identify the protein CYYR1 that had previously no assigned function, as a regulator of WWP1 activity and stability. We show that CYYR1 binds to the WW domains of the E3 ubiquitin ligase WWP1 through its PPxY motifs. This interaction triggers K63-linked auto-ubiquitination and subsequent degradation of WWP1. We furthermore demonstrate that CYYR1 localizes to late endosomal vesicles and directs poly-ubiquitinated WWP1 toward lysosomal degradation through binding to ANKRD13A. Moreover, we found that CYYR1 expression attenuates breast cancer cell growth in anchorage-dependent and independent colony formation assays in a PPxY-dependent manner. Finally, we highlight that CYYR1 expression is significantly decreased in breast cancer and is associated with beneficial clinical outcome. Taken together our study suggests tumor suppressor properties for CYYR1 through regulation of WWP1 auto-ubiquitination and lysosomal degradation.

Ubiquitination and deubiquitination in cancer: from mechanisms to novel therapeutic approaches.

Ubiquitination, a pivotal posttranslational modification of proteins, plays a fundamental role in regulating protein stability. The dysregulation of ubiquitinating and deubiquitinating enzymes is a common feature in various cancers, underscoring the imperative to investigate ubiquitin ligases and deubiquitinases (DUBs) for insights into oncogenic processes and the development of therapeutic interventions. In this review, we discuss the contributions of the ubiquitin-proteasome system (UPS) in all hallmarks of cancer and progress in drug discovery. We delve into the multiple functions of the UPS in oncology, including its regulation of multiple cancer-associated pathways, its role in metabolic reprogramming, its engagement with tumor immune responses, its function in phenotypic plasticity and polymorphic microbiomes, and other essential cellular functions. Furthermore, we provide a comprehensive overview of novel anticancer strategies that leverage the UPS, including the development and application of proteolysis targeting chimeras (PROTACs) and molecular glues.

Post-Translational Modifications (PTMs) of mutp53 and Epigenetic Changes Induced by mutp53.

Wild-type (wt) p53 and mutant forms (mutp53) play a key but opposite role in carcinogenesis. wtP53 acts as an oncosuppressor, preventing oncogenic transformation, while mutp53, which loses this property, may instead favor this process. This suggests that a better understanding of the mechanisms activating wtp53 while inhibiting mutp53 may help to design more effective anti-cancer treatments. In this review, we examine possible PTMs with which both wt- and mutp53 can be decorated and discuss how their manipulation could represent a possible strategy to control the stability and function of these proteins, focusing in particular on mutp53. The impact of ubiquitination, phosphorylation, acetylation, and methylation of p53, in the context of several solid and hematologic cancers, will be discussed. Finally, we will describe some of the recent studies reporting that wt- and mutp53 may influence the expression and activity of enzymes responsible for epigenetic changes such as acetylation, methylation, and microRNA regulation and the possible consequences of such changes.

CALCR exacerbates renal cell carcinoma progression via stabilizing CD44.

The calcitonin receptor (CALCR) is an essential protein for maintaining calcium homeostasis and has been reported to be upregulated in numerous cancers. However, the molecular role of CALCR in renal cell carcinoma (RCC) is not well understood. In this study, we identified the overexpression of CALCR in RCC using human tissue chip by immunohistochemical (IHC) staining, which was associated with a poor prognosis. Functionally, CALCR depletion inhibited RCC cell proliferation and migration, and induced cell apoptosis and cycle arrest. CALCR is also essential for in vivo tumor formation. Mechanistically, we demonstrated that CALCR could directly bind to CD44, preventing CD44 protein degradation and thereby upregulating CD44 expression. Moreover, a deficiency in CD44 significantly attenuated the promoting role of CALCR on RCC cell proliferation, migration and anti-apoptosis capacities. Collectively, CALCR exacerbates RCC progression via stabilizing CD44, offering a fundamental basis for considering CALCR as a potential therapeutic target for RCC patients.

USP53 Affects the Proliferation and Apoptosis of Breast Cancer Cells by Regulating the Ubiquitination Level of ZMYND11.

Breast cancer is the most common female malignancy worldwide. Ubiquitin-specific peptidase 53 (USP53) has been shown to exert cancer-suppressing functions in several solid tumors, but its role and the underlying mechanism in breast cancer has not been clearly elucidated. Therefore, we have carried out a series of detailed studies on this matter at the levels of bioinformatics, clinical tissue, cell function and animal model. We found that USP53 expression was downregulated in breast cancer specimens and was negatively correlated with the clinical stages. Gain- and loss-of-function experiments demonstrated USP53 inhibited proliferation, clonogenesis, cell cycle and xenograft growth, as well as induced apoptosis and mitochondrial damage of breast cancer cells. Co-immunoprecipitation data suggested that USP53 interacted with zinc finger MYND-type containing 11 (ZMYND11), and catalyzed its deubiquitination and stabilization. The 33-50 amino acid Cys-box domain was key for USP53 enzyme activity, but not essential for its binding with ZMYND11. The rescue experiments revealed that the anti-tumor role of USP53 in breast cancer cells was at least partially mediated by ZMYND11. Both USP53 and ZMYND11 were prognostic protective factors for breast cancer. USP53-ZMYND11 axis may be a good potential biomarker or therapeutic target for breast cancer, which can provide novel insights into the diagnosis, treatment and prognosis.

USP8 promotes the tumorigenesis of intrahepatic cholangiocarcinoma via stabilizing OGT.

Ubiquitination was considered to be a crucial factor in intrahepatic cholangiocarcinoma (iCCA) development. Herein, we identified Ubiquitin-specific peptidase 8 (USP8) as a key regulator for promoting the tumorigenesis of iCCA cell via stabilizing OGT. USP8 was overexpressed in human tumor tissues and correlated with worse survival. Moreover, the mass spectrometry and co-immunoprecipitation analysis indicated that USP8 interacted with OGT. USP8 worked as a bona fide deubiquitylase of OGT. It stabilized OGT in a deubiquitylation activity-dependent manner. Meanwhile, DUB-IN3, the USP8 inhibitor, could also restrain the malignancy of intrahepatic cholangiocarcinoma. In addition, USP8 depletion promoted the response of iCCA to pemigatinib. In conclusion, our findings pointed to a previously undocumented catalytic role for USP8 as a deubiquitinating enzyme of OGT. The USP8-OGT axis could be a potential target for iCCA therapy.

Ubiquitination contributes to the regulation of GDP-mannose pyrophosphorylase B activity.

GDP-mannose pyrophosphorylase B (GMPPB) loss-of-function is associated with muscular dystrophy and variable additional neurological symptoms. GMPPB facilitates the catalytic conversion of mannose-1-phosphate and GTP to GDP-mannose, which serves as a mannose donor for glycosylation. The activity of GMPPB is regulated by its non-catalytic paralogue GMPPA, which can bind GDP-mannose and interact with GMPPB, thereby acting as an allosteric feedback inhibitor of GMPPB. Using pulldown, immunoprecipitation, turnover experiments as well as immunolabeling and enzyme activity assays, we provide first direct evidence that GMPPB activity is regulated by ubiquitination. We further show that the E3 ubiquitin ligase TRIM67 interacts with GMPPB and that knockdown of TRM67 reduces ubiquitination of GMPPB, thus reflecting a candidate E3 ligase for the ubiquitination of GMPPB. While the inhibition of GMPPB ubiquitination decreases its enzymatic activity, its ubiquitination neither affects its interaction with GMPPA nor its turnover. Taken together, we show that the ubiquitination of GMPPB represents another level of regulation of GDP-mannose supply.

Lens autophagy protein ATG16L1: a potential target for cataract treatment.

Rationale: Cataract is the leading cause of blindness and low vision worldwide, yet its pathological mechanism is not fully understood. Although macroautophagy/autophagy is recognized as essential for lens homeostasis and has shown potential in alleviating cataracts, its precise mechanism remains unclear. Uncovering the molecular details of autophagy in the lens could provide targeted therapeutic interventions alongside surgery. Methods: We monitored autophagic activities in the lens and identified the key autophagy protein ATG16L1 by immunofluorescence staining, Western blotting, and transmission electron microscopy. The regulatory mechanism of ATG16L1 ubiquitination was analyzed by co-immunoprecipitation and Western blotting. We used the crystal structure of E3 ligase gigaxonin and conducted the docking screening of a chemical library. The effect of the identified compound riboflavin was tested in vitro in cells and in vivo animal models. Results: We used HLE cells and connexin 50 (cx50)-deficient cataract zebrafish model and confirmed that ATG16L1 was crucial for lens autophagy. Stabilizing ATG16L1 by attenuating its ubiquitination-dependent degradation could promote autophagy activity and relieve cataract phenotype in cx50-deficient zebrafish. Mechanistically, the interaction between E3 ligase gigaxonin and ATG16L1 was weakened during this process. Leveraging these mechanisms, we identified riboflavin, an E3 ubiquitin ligase-targeting drug, which suppressed ATG16L1 ubiquitination, promoted autophagy, and ultimately alleviated the cataract phenotype in autophagy-related models. Conclusions: Our study identified an unrecognized mechanism of cataractogenesis involving ATG16L1 ubiquitination in autophagy regulation, offering new insights for treating cataracts.

OsPUB9 Gene Edited by CRISPR/Cas9 Enhanced Resistance to Bacterial Leaf Blight in Rice (Oryza sativa L.).

Ubiquitination plays a crucial role in regulating signal pathways during the post-translation stage of protein synthesis in response to various environmental stresses. E3 ubiquitin ligase has been discovered to ultimately control various intracellular activities by imparting specificity to proteins to be degraded. This study was conducted to confirm biological and genetic functions of the U-box type E3 ubiquitin ligase (PUB) gene against biotic stress in rice (Oryza sativa L.). OsPUB9 gene-specific sgRNA were designed and transformants were developed through Agrobacterium-mediated transformation. Deep sequencing using callus was performed to confirm the mutation type of T0 plants, and a total of three steps were performed to select null individuals without T-DNA insertion. In the case of the OsPUB9 gene-edited line, a one bp insertion was generated by gene editing, and it was confirmed that early stop codon and multiple open reading frame (ORF) sites were created by inserting thymine. It is presumed that ubiquitination function also changed according to the change in protein structure of U-box E3 ubiquitin ligase. The OsPUB9 gene-edited null lines were inoculated with bacterial leaf blight, and finally confirmed to have a resistance phenotype similar to Jinbaek, a bacterial blight-resistant cultivar. Therefore, it is assumed that the amino acid sequence derived from the OsPUB9 gene is greatly changed, resulting in a loss of the original protein functions related to biological mechanisms. Comprehensively, it was confirmed that resistance to bacterial leaf blight stress was enhanced when a mutation occurred at a specific site of the OsPUB9 gene.

Molecular mechanism of TRIM32 in antiviral immunity in rainbow trout (Oncorhynchus mykiss).

TRIM family proteins are widely found in multicellular organisms and are involved in a wide range of life activities, and also act as crucial regulators in the antiviral natural immune response. This study aimed to reveal the molecular mechanism of rainbow trout TRIM protein in the anti-IHNV process. The results demonstrated that 99.1% homology between the rainbow trout and the chinook salmon (Oncorhynchus tshawytscha) TRIM32. When rainbow trout were infected with IHNV, the TRIM32 was highly expressed in the gill, spleen, kidney and blood. Meanwhile, rainbow trout TRIM32 has E3 ubiquitin ligase activity and undergoes K29-linked polyubiquitination modifications dependent on the RING structural domain was determined by immunoprecipitation. TRIM32 could interact with the NV protein of IHNV and degrade NV protein through the ubiquitin-proteasome pathway, and was also able to activate NF-κB transcription, thereby inhibiting the replication of IHNV. Moreover, the results of the animal studies showed that the survival rate of rainbow trout overexpressing TRIM32 was 70.2% which was significantly higher than that of the control group, and stimulating the body to produce high levels of IgM when the host was infected with the virus. In addition, TRIM32 can activate the NF-κB signalling pathway and participate in the antiviral natural immune response. The results of this study will help us to understand the molecular mechanism of TRIM protein resistance in rainbow trout, and provide new ideas for disease resistance breeding, vaccine development and immune formulation development in rainbow trout.

New horizons in the mechanisms and therapeutic strategies for PD-L1 protein degradation in cancer.

Programmed death-ligand 1 (PD-L1) has become a crucial focus in cancer immunotherapy considering it is found in many different cells. Cancer cells enhance the suppressive impact of programmed death receptor 1 (PD-1) through elevating PD-L1 expression, which allows them to escape immune detection. Although there have been significant improvements, the effectiveness of anti-PD-1/PD-L1 treatment is still limited to a specific group of patients. An important advancement in cancer immunotherapy involves improving the PD-L1 protein degradation. This review thoroughly examined the processes by which PD-L1 breaks down, including the intracellular pathways of ubiquitination-proteasome and autophagy-lysosome. In addition, the analysis revealed changes that affect PD-L1 stability, such as phosphorylation and glycosylation. The significant consequences of these procedures on cancer immunotherapy and their potential role in innovative therapeutic approaches are emphasised. Our future efforts will focus on understanding new ways in which PD-L1 degradation is controlled and developing innovative treatments, such as proteolysis-targeting chimeras designed specifically to degrade PD-L1. It is crucial to have a thorough comprehension of these pathways in order to improve cancer immunotherapy strategies and hopefully improve therapeutic effectiveness.

TRIM50 inhibits glycolysis and the malignant progression of gastric cancer by ubiquitinating PGK1.

Ubiquitination plays a pivotal regulatory role in tumor progression. Among the components of the ubiquitin-proteasome system (UPS), ubiquitin-protein ligase E3 has emerged as a key molecule. Nevertheless, the biological functions of E3 ubiquitin ligases and their potential mechanisms orchestrating glycolysis in gastric cancer (GC) remain to be elucidated. In this study, we conducted a comprehensive transcriptomic analysis to identify the core E3 ubiquitin ligases in GC, followed by extensive validation of the expression patterns and clinical significance of Tripartite motif-containing 50 (TRIM50) both in vitro and in vivo. Remarkably, we found that TRIM50 was downregulated in GC tissues, associated with malignant progression and poor patient survival. Functionally, overexpression of TRIM50 suppressed GC cell proliferation and indirectly mitigated the invasion and migration of GC cells by inhibiting the M2 polarization of tumor-associated macrophages (TAMs). Mechanistically, TRIM50 inhibited the glycolytic pathway by ubiquitinating Phosphoglycerate Kinase 1 (PGK1), thereby directly suppressing GC cell proliferation. Simultaneously, the reduction in lactate led to diminished M2 polarization of TAMs, indirectly inhibiting the invasion and migration of GC cells. Notably, the downregulation of TRIM50 in GC was mediated by the METTL3/YTHDF2 axis in an m6A-dependent manner. In our study, we definitively identified TRIM50 as a tumor suppressor gene (TSG) that effectively inhibits glycolysis and the malignant progression of GC by ubiquitinating PGK1, thus offering novel insights and promising targets for the diagnosis and treatment of GC.

UBE2D1 promotes glioblastoma proliferation by modulating p21 ubiquitination.

Glioblastoma (GBM) cells exhibit aberrant proliferative abilities and resistance to conventional therapies. However, the mechanisms underlying these malignant phenotypes are poorly understood. In this study, we identified ubiquitin-conjugating enzyme E2D1 (UBE2D1) as a crucial stimulator of GBM development. It is highly expressed in GBM and closely associated with poor prognosis in patients with GBM. UBE2D1 knockdown inhibits GBM cell growth and leads to G1 cell cycle arrest. Mechanistically, UBCH5A binds to p21 at the protein level and induces the ubiquitination and degradation of p21. This negative regulation is mediated by STUB1. Our findings are the first to identify UBE2D1 as a key driver of GBM growth and provide a potential target for improving prognosis and therapy.