RING Domain Research Articles

Lack of dominant-negative activity for tumor-related ZNRF3 missense mutations at endogenous levels.

ZNRF3, a negative regulator of β-catenin signaling, removes Wnt receptors from the membrane. Currently, it is unknown which tumor-associated variants can be considered driver mutations and through which mechanisms they contribute to cancer. Here we show that all truncating mutations analyzed at endogenous levels exhibit loss-of-function, with longer variants retaining partial activity. Regarding missense mutations, we show that 27/82 ZNRF3 variants in the RING and R-Spondin domain structures, lead to (partial) loss-of-function/hyperactivation. Mechanistically, defective R-Spondin domain variants appear to undergo endoplasmic-reticulum-associated degradation due to protein misfolding, leading to reduced protein levels. They fail to reach the membrane correctly, which can be partially restored for several variants by culturing cells at 27 °C. Although RING and R-Spondin domain mutations in RNF43/ZNRF3 are often considered to possess dominant-negative oncogene-like activity in cancers, our findings challenge this notion. When representative variants are heterozygously introduced into endogenous ZNRF3, their impact on β-catenin signaling mirrors that of heterozygous knockout, suggesting that the supposed dominant-negative effect is non-existent. In other words, so-called "hyperactivating" ZNRF3/RNF43 mutations behave as classical loss-of-function mutations at endogenous levels.

Characterization of tripartite motif containing 59 (TRIM59) in Epinephelus akaara: Insights into its immune involvement and functional properties in viral pathogenesis, macrophage polarization, and apoptosis regulation

The tripartite motif-containing (TRIM) superfamily is the largest family of RING-type E3 ubiquitin ligases that is conserved across the metazoan kingdom. Previous studies in mammals have demonstrated that TRIM59 possesses ubiquitin-protein ligase activity and acts as a negative regulator of NF-κB signaling. However, TRIM59 has rarely been characterized in fish. This study aimed to characterize TRIM59 from Epinephelus akaara (Eatrim59) and elucidate its structural features, expression patterns, and functional properties in innate immune responses and in the regulation of apoptosis. Eatrim59 is composed of 406 amino acids with a molecular weight of 45.84 kDa and a theoretical isoelectric point of 5.25. It comprises a conserved RING domain, a B-box motif, and a coiled-coil region. Subcellular localization analysis revealed that Eatrim59 was localized in the endoplasmic reticulum. Eatrim59 was ubiquitously expressed in all tissues examined, with the highest relative expression detected in the blood, followed by the brain and spleen. Temporal expression of Eatrim59 was dynamically regulated in response to in vivo immune stimulation by Toll-like receptor ligands and nervous necrosis virus infection. In FHM cells overexpressing Eatrim59, an increase in viral replication was observed upon infection with the Viral hemorrhagic septicemia virus. This phenomenon is attributed to Eatrim59-mediated downregulation of interferon, pro-inflammatory cytokines, and other antiviral pathways. Moreover, macrophages stably overexpressing Eatrim59 exhibited a decrease in nitric oxide production and the formation of a filamentous actin structure upon lipopolysaccharide stimulation, indicating dampened M1 polarization. Furthermore, a decrease in apoptosis was observed in Eatrim59-overexpressing FHM cells under oxidative stress induced by H2O2. In conclusion, these findings demonstrate the multifaceted role of Eatrim59 as a regulator of innate immune response and apoptosis in E. akaara.

IRF2BP2 binds to a conserved RxSVI motif of protein partners and regulates megakaryocytic differentiation

IRF2BP2 is a transcriptional coregulator that plays diverse regulatory roles in various cellular processes in either IRF2-dependent or IRF2-independent manner through interactions with protein partners via its RING domain; however, the underlying molecular mechanisms remain unclear. In this study, we conduct a motif discovery search on the sequences of interacting proteins IRF2 and VGLL4 of IRF2BP2 and identify a conserved RxSVI motif. Biochemical and structural data reveal that the RING domain binds to the motif-containing peptides of IRF2 and VGLL4 with comparable affinities and in a similar manner. The motif-containing peptides tend to form a short loop along with a short β-strand, which facilitates effective recognition and tight binding by the RING domain. Further exploration of this motif in the human proteome identifies the transcription factor ZBTB16 as an interacting protein of IRF2BP2. Biochemical, structural, and cell biological data demonstrate that the RING domain binds to the motif-containing peptide of ZBTB16 in a manner similar to that of IRF2 and VGLL4. Moreover, IRF2BP2 plays a crucial regulatory role in megakaryocytic differentiation through interaction with ZBTB16. These findings elucidate the molecular basis for how IRF2BP2 can engage with different protein partners, thereby exerting diverse regulatory functions in many cellular processes.

RNF38 promotes gilteritinib resistance in acute myeloid leukemia via inducing autophagy by regulating ubiquitination of LMX1A

BackgroundGilteritinib is a commonly used targeted drug for acute myeloid leukemia (AML), but the emergence of gilteritinib resistance greatly reduces the therapeutic effect. RING finger protein 38 (RNF38), a protein with RING Finger domain and E3 ubiquitin ligase activity, has been implicated in tumorigenesis and drug resistance. However, the role and mechanism of RNF38 in the gilteritinib resistance of AML remains unclear.MethodsNormal AML cells were treated with gilteritinib to construct gilteritinib-resistant cells (MV4-11/Gilteritinib and MOLM-13/Gilteritinib). CCK8 assay, TUNEL staining and EdU assay were used to assess gilteritinib resistance, cell apoptosis and proliferation. The protein levels of autophagy-related markers, RNF38 and LIM homeobox transcription factor 1 alpha (LMX1A) were determined by western blot. Also, RNF38 and LMX1A mRNA levels were tested using qRT-PCR. Autophagic flux was assessed using mRFP-GFP-LC3 labeling, and autophagosome numbers was counted under transmission electron microscopy. Co-IP assay was employed to analyze interaction between RNF38 and LMX1A. The effects of LMX1A and RNF38 on AML tumorigenesis were analyzed by in vivo experiments.ResultsIn gilteritinib-resistant AML cells, autophagy-related markers, mRFP-GFP-LC3 signals and autophagosome numbers were significantly enhanced. Autophagy inhibitor 3-MA could suppress gilteritinib resistance in AML cells. RNF38 knockdown inhibited gilteritinib resistance and autophagy in AML cells. Mechanistically, RNF38 reduced LMX1A expression by inducing its ubiquitination. RNF38 overexpression reversed the inhibitory effect of LMX1A on gilteritinib resistance and autophagy in AML cells, as well as AML tumor growth in vivo, while these effects could be abolished by proteasome inhibitor MG132.ConclusionsRNF38 induced autophagy to promote gilteritinib resistance in AML by increasing the ubiquitination of LMX1A.Graphical 1. Autophagy is activated in gilteritinib-resistant AML cells.2. Reduction of autophagy suppresses gilteritinib resistance in AML cells.3. RNF38 knockdown inhibits gilteritinib resistance and autophagy in AML cells.4. RNF38 reduces LMX1A expression via inducing its ubiquitination.

Functional Diversity of A-Type RING Ligases (ATL) Genes: Insights into the Crucial Roles of E3 Ubiquitin Ligases in Plant Biology

AbstractE3 ubiquitin ligases are vital components of the ubiquitin–proteasome system (UPS), responsible for maintaining protein balance and controlling cellular functions. E3 ligases target specific proteins for degradation or modify their activities through ubiquitin attachment. One prominent E3 ligase family is the ATL family, which comprises 100 members in Arabidopsis thaliana and has significantly expanded in plant genomes. All ATLs share a common domain architecture, featuring a transmembrane domain at the amino-terminal region, a distinct RING-H2 finger domain, and the GLD motif. The RING domain facilitates interactions between E3 ligases, E2-conjugating enzymes, and target proteins, enabling the transfer of ubiquitin molecules. The amino-terminal and carboxy-terminal regions introduce sequence diversity and potentially mediate interactions with other components that assist in UPS function or target recognition. ATLs had been classified within groups, each group encompasses specific ATLs with defined roles in various biological processes. For example, group C-ATLs are implicated in drought tolerance, flower development, phosphate homeostasis, and immune signaling. G-ATLs are associated with carbon/nitrogen stress, immune signaling, salt stress, ABA responses, cadmium tolerance, and sugar-mediated plant growth. A-ATLs participate in early elicitor-response, salt and drought responses, and flowering time regulation. Lastly, D-ATLs are involved in the regulation of programmed cell death. This review let perceive ATLs as a cohesive group of E3 ligases, shedding light on their functional diversifity and redundancy, specifically examining their participation in diverse biological processes, explore their evolutionary history shaped by gene duplication events, and appraise their interactions with key proteins and targets of ubiquitination. This comprehensive overview aims to offer insights into the role of ATLs in plant adaptation, defense mechanisms, and stress tolerance, while also underlying molecular and evolutionary mechanisms and regulatory networks that govern these processes.

DNA damage repair factor Rad18 controls virulence partially via transcriptional suppression of genes HWP1 and ECE1 in Candida albicans

ABSTRACT DNA damage repair is a crucial cellular mechanism for rectifying DNA lesions arising during growth and development. Among the various repair pathways, postreplication repair (PRR) plays a pivotal role in resolving single-stranded gaps induced by DNA damage. However, the contribution of PRR to virulence remains elusive in the fungal pathogen Candida albicans (C. albicans). In this study, we investigated the role of Rad18, a critical component of PRR, in DNA damage response and virulence in C. albicans. We observed that deletion of RAD18 in C. albicans resulted in heightened sensitivity to DNA damage stress. Through deletion of specific internal domains coupled with spot assay analysis, we show that the internal RING and SAP domains play essential roles in DNA damage response, whereas the ZNF domain was less important. Surprisingly, the lack of Rad18 in C. albicans resulted in heightened intracellular survival within macrophages and elevated virulence in the Galleria mellonella model. RNAseq analysis revealed that loss of Rad18 upregulated the transcription of genes encoding transporters and oxidoreductases, as well as virulence genes, including HWP1 and ECE1. Suppression of the transcription of these virulence genes in the RAD18 deletion strain by a dCas9-mediated CRISPRi system reversed this increased virulence. Taken together, these data demonstrate that Rad18 plays a significant role in virulence partially through transcriptional suppression of virulence genes HWP1 and ECE1 in C. albicans. Our findings provide valuable insights into the intricate relationship between DNA damage response and virulence in C. albicans.

The E3 ligase TaE3V-B1 ubiquitinates proteins encoded by the vernalization gene TaVRN1 and regulates developmental processes in wheat.

In wheat (Triticum aestivum), early maturity is desired to avoid the hot and dry summer season, especially in view of climate change. Here, we report that TaE3V1, a C3H2C3 RING-type E3 ligase that interacts with TaVRN1, is associated with early development. Aside from its RING domain, TaE3V1 does not harbor any domains that are conserved in other RING-type or other E3 ligase proteins. TaE3V-B1b, encoded by the functional TaE3V1 allele, interacts with and ubiquitinates TaVRN1. In contrast, TaE3V-B1a, encoded by a natural nonfunctional TaE3V1 allele, neither interacts with TaVRN1 nor has E3 ligase activity. TaE3V-B1b activity decreases with plant age under warmer temperatures, but not under the low temperatures required for vernalization. We employed a gene editing method to simultaneously inactivate the three homoeologous TaE3V1 genes to validate their functions. Overall, our results suggest that the naturally mutated and edited TaE3V1 alleles can accelerate wheat development and aid adaptation to warming climates.

Read-write mechanisms of H2A ubiquitination by Polycomb repressive complex 1.

Epigenetic inheritance of silent chromatin domains is fundamental to cellular memory during embryogenesis, but it must overcome the dilution of repressive histone modifications during DNA replication1. One such modification, histone H2A lysine 119 monoubiquitination (H2AK119Ub), needs to be re-established by the Polycomb repressive complex 1 (PRC1) E3 ligase to restore the silent Polycomb domain2,3. However, the exact mechanism behind this restoration remains unknown. Here, combining cryo-electron microscopy (cryo-EM) and functional approaches, we characterize the read-write mechanism of the non-canonical PRC1-containing RYBP (ncPRC1RYBP). This mechanism, which functions as a positive-feedback loop in epigenetic regulation4,5, emphasizes the pivotal role of ncPRC1RYBP in restoring H2AK119Ub. We observe an asymmetrical binding of ncPRC1RYBP to H2AK119Ub nucleosomes, guided in part by the N-terminal zinc-finger domain of RYBP binding to residual H2AK119Ub on nascent chromatin. This recognition positions the RING domains of RING1B and BMI1 on the unmodified nucleosome side, enabling recruitment of the E2 enzyme to ubiquitinate H2AK119 within the same nucleosome (intra-nucleosome read-write) or across nucleosomes (inter-nucleosome read-write). Collectively, our findings provide key structural and mechanistic insights into the dynamic interplay of epigenetic regulation, highlighting the significance of ncPRC1RYBP in H2AK119Ub restoration to sustain repressive chromatin domains.

Analyses of Off-Target Effects on Cardiac and Skeletal Muscles by Berberine, a Drug Used to Treat Cancers and Induce Weight Loss.

Previous reports from our laboratory describing the formation of myofibrils in cultured embryonic cardiac and skeletal muscle cells have proposed that myofibrillogenesis occurs in three steps of increasing protein organization: beginning with premyofibrils, followed by nascent myofibrils, and ending in mature myofibrils. Inhibitors of the ubiquitin proteasome system (UPS) prevented nascent myofibrils from progressing directly to mature myofibrils in cultured cardiac and skeletal muscle cells, supporting a three-step model of assembly in which some of the proteins in nascent myofibrils are proteolyzed to allow the assembly of mature myofibrils. Application of UPS inhibitors on cultured muscle cells suggests possible explanations for the off-target cardiac and skeletal muscle adverse effects of UPS drugs, which are used on cancer patients. Berberine, a plant derivative, has been used to treat various cancers, including multiple myelomas. In contrast to the use of UPS drugs, success was reported with Berberine in multiple myeloma patients with no off-target effects on their hearts. We have exposed cultured cardiac and skeletal muscle cells to Berberine, a ligase inhibitor of UHRF1 (ubiquitin-like with PHD and RING finger domains). Berberine inhibited myofibril assembly at the nascent myofibril stage in embryonic skeletal muscle cells but had no effect in the assembly of mature myofibrils in embryonic heart cells. RT-PCR experiments demonstrated Berberine inhibition of mRNA for muscle myosin II heavy chains but not for muscle actin mRNA in skeletal muscle cells. Berberine is also being used as a popular weight losing compound, because it is much cheaper and available without a prescription than the semaglutide containing weight losing drugs (Wegovy and Ozempic). In contrast to Berberine, semaglutide had no effects on myofibril assembly in culture assays for both cardiac and skeletal muscle cells. We postulate that analyses of cultured embryonic cardiac and skeletal muscle cells will provide a preclinical assay for the testing of novel cancer drugs with improved outcomes for patients, an important goal for cancer therapeutics.

Identification and characterization of chicken TRIM45 and its role as a negative regulator of ALV-J replication in vitro

ABSTRACT Avian leukosis virus subgroup J (ALV-J) is an alpharetrovirus that infects chickens, causing immunosuppression and a decrease in production performance, leading to substantial economic losses in the poultry industry. ALV-J is also well-known for its oncogenic properties, inducing tumours such as myelomas and haemangiomas in infected chickens. TRIM45 has been identified as a potential tumour suppressor; however, the relationship between TRIM45 expression and ALV-J infection remains to be elucidated. This study aimed to dissect the molecular characteristics of the chicken TRIM45 gene and its modulation during ALV-J infection, as well as its influence on viral replication. We found that the chicken TRIM45 RING domain is significantly different from that of humans and other mammals. TRIM45 is expressed in all chicken tissues, with the highest levels in the heart. Subcellular localization studies indicated a cytoplasmic distribution of TRIM45, forming aggregates within cells. Our findings demonstrate that ALV-J infection significantly upregulates TRIM45 expression in DF-1 cells. To assess the functional role of TRIM45 in ALV-J replication, we employed both gene silencing and overexpression strategies. Strikingly, the overexpression of TRIM45, including a mutant lacking the RING domain, was found to markedly suppress ALV-J replication. In contrast, TRIM45 knockdown via siRNA resulted in an enhanced viral replication, highlighting the importance of TRIM45 limiting ALV-J replication. Mechanistically, overexpression of TRIM45 induces apoptosis in infected cells, independent of its RING domain function. In conclusion, our study demonstrates that chicken TRIM45 acts as a negative regulator of ALV-J replication in vitro by promoting apoptosis in infected cells. RESEARCH HIGHLIGHTS Chicken TRIM45 RING domain and protein localization significantly differ from humans. TRIM45 negatively regulates ALV-J replication in vitro. TRIM45 inhibits ALV-J replication by inducing apoptosis in infected cells.

Functional role of the c-terminal domains of the msl2 protein of drosophila melanogaster

Dosage compensation complex, consisting of five proteins and two non-coding RNAs roX, specifically binds to the X chromosome in males, providing a higher level of gene expression, which is necessary to compensate for the monosomy of the sex chromosome in male Drosophila compared to two X chromosomes in females. The MSL2 protein contains an N-terminal RING domain, which acts as an E3 ligase in the ubiquitination of proteins and is the only subunit of the complex that is expressed only in males. The functional role of two C-terminal domains of the MSL2 protein, enriched with proline (P-domain) and basic amino acids (B-domain), was investigated. As a result, it was shown that the B-domain destabilizes the MSL2 protein, which is associated with the presence of two lysines whose ubiquitination is under the control of the RING domain of MSL2. The unstructured proline-rich domain stimulates transcription of the roX2 gene, which is necessary for the effective formation of the dosage compensation complex.

Chaperonin‐containing TCP1 subunit 6A inhibition via TRIM21‐mediated K48‐linked ubiquitination suppresses triple‐negative breast cancer progression through the AKT signalling pathway

AbstractBackgroundTriple‐negative breast cancer (TNBC) is distinguished by a significant likelihood of distant recurrence and an unfavourable prognosis. However, the underlying molecules and mechanisms have not been fully elucidated.MethodsWe investigated the expression profile and clinical relevance of chaperonin‐containing TCP1 subunit 6A (CCT6A) in TNBC. We performed cell function assays on TNBC cells with CCT6A knockdown or overexpression. To further explore the mechanism of action of CCT6A, RNA sequencing and co‐immunoprecipitation–mass spectrometry analyses were utilized. Rescue and ubiquitination assays evaluated the impact of TRIM21‐mediated CCT6A ubiquitination and degradation on TNBC progression in vitro and in vivo. Finally, we studied the potential of Ipatasertib, a pharmacological AKT inhibitor, and/or anti‐PD1 therapy in inhibiting TNBC progression.ResultsElevated CCT6A expression in TNBC patients was associated with an adverse prognosis and lymph node metastasis. Mechanistically, CCT6A facilitated cell migration, invasion, epithelial‐mesenchymal transition and proliferation by activating the phosphatidylinositol 3‐kinase (PI3K)/AKT pathway. The TRIM21 RING domain is an E3 ligase, facilitating the K48‐linked ubiquitination‐mediated degradation of CCT6A, thereby impeding TNBC progression. Moreover, in the tumour tissues of the CCT6A‐overexpressing mice, the quantity of CD8+ T cells and the concentration of secreted interferon‐gamma were decreased, whereas in the group double‐overexpression of CCT6A and TRIM21, they were elevated; the opposite was observed in the knockdown and double‐knockdown groups. Ipatasertib demonstrated enhanced efficacy in inhibiting cell proliferation, invasion and migration in TNBC cells ectopically expressing CCT6A. When Ipatasertib and anti‐PD1 therapies were combined, both the tumour volume and mass exhibited a notable reduction, while the expression of CD45+CD8+ T cells increased, and that of CD45+CD4+CTLA4+ and CD45+CD4+PD1+ T cells decreased.ConclusionsOur findings indicate that TRIM21 inhibits TNBC progression by facilitating the K48‐linked ubiquitination‐mediated degradation of CCT6A via the PI3K/AKT signalling pathway. This highlights the potential of Ipatasertib and/or anti‐PD1 as therapeutic strategies, particularly for TNBC patients overexpressing CCT6A.Key points Chaperonin TCP1 subunit 6A (CCT6A) plays an oncogenic role in triple‐negative breast cancer (TNBC) through the AKT signaling pathway. TRIM21 facilitated K48‐linked ubiquitination‐mediated degradation of CCT6A, thereby impeding TNBC progression. Our study collectively underscores the potential of Ipatasertib in conjunction with anti‐PD1 therapy as a promising strategy to counteract CCT6A/AKT hyperactivity‐driven TNBC progression.

Cauchy’s Residue Theorem and Its Application

One of the key theorems in complex analysis, Cauchy’s Residue Theorem, which refers to the integral value of an analytic function along any simple closed contour surrounding an isolated singularity in a certain ring domain divided by 2πi, will greatly simplify the process of computing integrals on contour surrounding singularities. In the field of basic mathematics, the Cauchy’ Residue Theorem plays a key role in the integral calculation of analytic functions, nonuniform complex functions and the argument principle, which establishes a connection between a curve’s winding number and the quantity of zeros and poles inside the curve. Furthermore, Residue Theorem can combine with various subjects, including electromagnetism. This paper gives an overview of the Cauchy’s Residue Theorem and its application. Firstly, it talks about the definition and proof of the Cauchy’ Residue Theorem, along with the definition of residue. Then two examples of using Residue Theorem to integrate functions will be given. Finally, this paper involves application of Residue Theorem in trigonometric sum identities and a remark on the method.

Ubiquitin-Specific Protease 15 Plays an Important Role in Controlling the Tolerance to Salt, Drought and Abscisic Acid in Arabidopsis thaliana.

Ubiquitin-specific proteases (UBPs), the largest subfamily of deubiquitinating enzymes (DUBs), are critical for plant growth and development as well as abiotic-stress responses. In this study, we discovered that the expression of the ubiquitin-specific protease 15 (UBP15) gene of the gene ubiquitin-specific protease 15 (UBP15) was induced by salt, mannitol and abscisic acid (ABA) treatments. Further research revealed that UBP15 is involved in modulation of salt, drought tolerance and ABA signaling during seed germination, early seedling development, post-germination root growth or adult-plant stage. Enrichment analysis showed that many genes related to abiotic stresses and metabolic pathways were altered in the ubp15-1 mutant. Through the joint analysis of the quantitative real-time polymerase chain reaction (qRT-PCR) and differentially-expressed gene relationship network, we found that UBP15 may mainly regulate salt-stress tolerance by modulating the dwarf and delayed flowering 1 (DDF1) pathway through a cascade reaction. In the regulation of drought-stress responses, ring domain ligase1 (RGLG1) may be a direct substrate of UBP15. Moreover, we cannot exclude the possibility that UBP15 acts in a feed-forward loop mechanism in the regulation of drought-stress responses via ethylene response factor 53 (ERF53) and its ubiquitin (Ub) ligase RGLG1. In ABA signal transduction, UBP15 may play a role in at least three aspects of the ABA signaling pathway: ABA synthesis, stomatal closure regulated by ABA signaling, and transcription factors in the ABA pathway. Taken together, our results suggest that UBP15 is involved in salt, osmotic, and drought-stress tolerance and the ABA signaling pathway by directly regulating the stability of key substrates or indirectly affecting the expression of genes related to abiotic stresses in Arabidopsis thaliana. Our research provides new germplasm resources for stress-resistant crops cultivation. These results demonstrate that UBP15 is a key regulator of salt, drought and ABA tolerance in Arabidopsis.

The ribosome-associated quality control factor TCF25 imposes K48 specificity on Listerin-mediated ubiquitination of nascent chains by binding and specifically orienting the acceptor ubiquitin.

Polypeptides arising from interrupted translation undergo proteasomal degradation by the ribosome-associated quality control (RQC) pathway. The ASC-1 complex splits stalled ribosomes into 40S subunits and nascent chain-tRNA-associated 60S subunits (60S RNCs). 60S RNCs associate with NEMF that promotes recruitment of the RING-type E3 ubiquitin (Ub) ligase Listerin (Ltn1 in yeast), which ubiquitinates nascent chains. RING-type E3s mediate the transfer of Ub directly from the E2~Ub conjugate, implying that the specificity of Ub linkage is determined by the given E2. Listerin is most efficient when it is paired with promiscuous Ube2D E2s. We previously found that TCF25 (Rqc1 in yeast) can impose K48-specificity on Listerin paired with Ube2D E2s. To determine the mechanism of TCF25's action, we combined functional biochemical studies and AlphaFold3 modeling and now report that TCF25 specifically interacts with the RING domain of Listerin and the acceptor ubiquitin (UbA) and imposes K48-specificity by orienting UbA such that its K48 is directly positioned to attack the thioester bond of the Ube2D1~Ub conjugate. We also found that TCF25 itself undergoes K48-specific ubiquitination by Listerin suggesting a mechanism for the reported upregulation of Rqc1 in the absence of Ltn1 and the observed degradation of TCF25 by the proteasome in vivo.

Genome-wide identification of the TRAF gene family in humpback grouper (Cromileptes altivelis) and analysis of their expression in response to Vibrio harveyi challenge

TRAF (Tumor necrosis factor receptor-associated factor) proteins are key mediators of signal transduction in cell signaling and immune regulation within the toll-like receptor (TLR) and tumor necrosis factor (TNFR) superfamily. Despite the importance of TRAF genes in teleost innate immunity, study on their functions in C. altivelis is limited. This study utilized bioinformatics methods to identify and named eight TRAF genes (CaTRAF2a, CaTRAF2a-like, CaTRAF2b, CaTRAF3, CaTRAF4a, CaTRAF5, CaTRAF6 and CaTRAF7) in C. altivelis. Phylogenetic, syntenic and molecular evolution revealed that all CaTRAF members were evolutionarily conserved in teleost. Domain analysis indicated the presence of a conserved N-terminal RING finger domain in all CaTRAF proteins. Most CaTRAF proteins also featured a MATH domain at the C-terminal, with the exception of CaTRAF7 which contained seven repeat WD40 domains. In addition, qRT-PCR was used to detect the expression patterns of nine different tissues and eight different embryonic development stages of healthy fish, and it was found that there were spatial and tissue specificities among the members. HE staining revealed evident pathological lesions in the tissues post V. harveyi infection. Atrophy and significant bending of the gill lamellae were observed in the gills, while irregular cell shapes, increased fat vacuoles, and enlarged cell volume were noted in the liver. Intestinal tissues displayed thickening of the muscle layer, elongation of intestinal villi, and increased folds. Moreover, the expression of TRAF gene changed significantly after V. harveyi infection. These results would help to clarify the molecular role of CaTRAF gene in the regulation of immune and inflammatory responses in C. altivelis.

Functional Analysis of BARD1 Missense Variants on Homology-Directed Repair in Ovarian and Breast Cancers.

Women with germline BRCA1 mutations face an increased risk of developing breast and ovarian cancers. BARD1 (BRCA1 associated RING domain 1) is an essential heterodimeric partner of BRCA1, and mutations in BARD1 are also associated with these cancers. While BARD1 mutations are recognized for their cancer susceptibility, the exact roles of numerous BARD1 missense mutations remain unclear. In this study, we conducted functional assays to assess the homology-directed DNA repair (HDR) activity of all BARD1 missense substitutions identified in 55 breast and ovarian cancer samples, using the real-world data from the COSMIC and cBioPortal databases. Seven BARD1 variants (V85M, P187A, G491R, R565C, P669L, T719R, and Q730L) were confirmed to impair DNA damage repair. Furthermore, cells harboring these BARD1 variants exhibited increased sensitivity to the chemotherapeutic drugs, cisplatin, and olaparib, compared to cells expressing wild-type BARD1. These findings collectively suggest that these seven missense BARD1 variants are likely pathogenic and may respond well to cisplatin-olaparib combination therapy. This study not only enhances our understanding of BARD1's role in DNA damage repair but also offers valuable insights into predicting therapy responses in patients with specific BARD1 missense mutations.

Mesoscale regulation of MTOCs by the E3 ligase TRIM37.

Centrosomes ensure accurate chromosome segregation during cell division. Although the regulation of centrosome number is well-established, less is known about the suppression of non-centrosomal MTOCs (ncMTOCs). The E3 ligase TRIM37, implicated in Mulibrey nanism and 17q23-amplified cancers, has emerged as a key regulator of both centrosomes and ncMTOCs. Yet, the mechanism by which TRIM37 achieves enzymatic activation to target these mesoscale structures had remained unknown. Here, we elucidate TRIM37's activation process, beginning with TRAF domain-directed substrate recognition, progressing through B-box domain-mediated oligomerization, and culminating in RING domain dimerization. Using optogenetics, we demonstrate that TRIM37's E3 activity is directly coupled to the assembly state of its substrates, activating only when centrosomal proteins cluster into higher-order assemblies resembling MTOCs. This regulatory framework provides a mechanistic basis for understanding TRIM37-driven pathologies and, by echoing TRIM5's restriction of the HIV capsid, unveils a conserved activation blueprint among TRIM proteins for controlling mesoscale assembly turnover.

DTX3L ubiquitin ligase ubiquitinates single-stranded nucleic acids.

Ubiquitination typically involves covalent linking of ubiquitin (Ub) to a lysine residue on a protein substrate. Recently, new facets of this process have emerged, including Ub modification of non-proteinaceous substrates like ADP-ribose by the DELTEX E3 ligase family. Here, we show that the DELTEX family member DTX3L expands this non-proteinaceous substrate repertoire to include single-stranded DNA and RNA. Although the N-terminal region of DTX3L contains single-stranded nucleic acid binding domains and motifs, the minimal catalytically competent fragment comprises the C-terminal RING and DTC domains (RD). DTX3L-RD catalyses ubiquitination of the 3'-end of single-stranded DNA and RNA, as well as double-stranded DNA with a 3' overhang of two or more nucleotides. This modification is reversibly cleaved by deubiquitinases. NMR and biochemical analyses reveal that the DTC domain binds single-stranded DNA and facilitates the catalysis of Ub transfer from RING-bound E2-conjugated Ub. Our study unveils the direct ubiquitination of nucleic acids by DTX3L, laying the groundwork for understanding its functional implications.

DTX3L ubiquitin ligase ubiquitinates single-stranded nucleic acids

Ubiquitination typically involves covalent linking of ubiquitin (Ub) to a lysine residue on a protein substrate. Recently, new facets of this process have emerged, including Ub modification of non-proteinaceous substrates like ADP-ribose by the DELTEX E3 ligase family. Here, we show that the DELTEX family member DTX3L expands this non-proteinaceous substrate repertoire to include single-stranded DNA and RNA. Although the N-terminal region of DTX3L contains single-stranded nucleic acid binding domains and motifs, the minimal catalytically competent fragment comprises the C-terminal RING and DTC domains (RD). DTX3L-RD catalyses ubiquitination of the 3’-end of single-stranded DNA and RNA, as well as double-stranded DNA with a 3’ overhang of two or more nucleotides. This modification is reversibly cleaved by deubiquitinases. NMR and biochemical analyses reveal that the DTC domain binds single-stranded DNA and facilitates the catalysis of Ub transfer from RING-bound E2-conjugated Ub. Our study unveils the direct ubiquitination of nucleic acids by DTX3L, laying the groundwork for understanding its functional implications.