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The Journal of Cell Biology Jul 2024Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol...
Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol as part of the ER-associated degradation (ERAD). Despite several ERAD branches involving different Ub E3 ligases, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. Through a series of multiplex knockdown/knockout experiments with real-time kinetic measurements, we demonstrate that HERC3 operates independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to mediate the retrotranslocation and ERAD of misfolded CFTR. While RNF5/185 participates in the ERAD process of both misfolded ABCB1 and CFTR, HERC3 uniquely promotes CFTR ERAD. In vitro assay revealed that HERC3 directly interacts with the exposed membrane-spanning domains (MSDs) of CFTR but not with the MSDs embedded in liposomes. Therefore, HERC3 could play a role in the quality control of MSDs in the cytoplasm and might be crucial for the ERAD pathway of select membrane proteins.
Topics: Humans; Cystic Fibrosis Transmembrane Conductance Regulator; DNA-Binding Proteins; Endoplasmic Reticulum; Endoplasmic Reticulum-Associated Degradation; Guanine Nucleotide Exchange Factors; HEK293 Cells; HeLa Cells; Membrane Proteins; Protein Binding; Protein Domains; Protein Folding; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 38722278
DOI: 10.1083/jcb.202308003 -
Genes, Chromosomes & Cancer May 2024Pleomorphic rhabdomyosarcoma (PRMS) is a rare and highly aggressive sarcoma, occurring mostly in the deep soft tissues of middle-aged adults and showing a variable...
Pleomorphic rhabdomyosarcoma (PRMS) is a rare and highly aggressive sarcoma, occurring mostly in the deep soft tissues of middle-aged adults and showing a variable degree of skeletal muscle differentiation. The diagnosis is challenging as pathologic features overlap with embryonal rhabdomyosarcoma (ERMS), malignant Triton tumor, and other pleomorphic sarcomas. As recurrent genetic alterations underlying PRMS have not been described to date, ancillary molecular diagnostic testing is not useful in subclassification. Herein, we perform genomic profiling of a well-characterized cohort of 14 PRMS, compared to a control group of 23 ERMS and other pleomorphic sarcomas (undifferentiated pleomorphic sarcoma and pleomorphic liposarcoma) using clinically validated DNA-targeted Next generation sequencing (NGS) panels (MSK-IMPACT). The PRMS cohort included eight males and six females, with a median age of 53 years (range 31-76 years). Despite similar tumor mutation burdens, the genomic landscape of PRMS, with a high frequency of TP53 (79%) and RB1 (43%) alterations, stood in stark contrast to ERMS, with 4% and 0%, respectively. CDKN2A deletions were more common in PRMS (43%), compared to ERMS (13%). In contrast, ERMS harbored somatic driver mutations in the RAS pathway and loss of function mutations in BCOR, which were absent in PRMS. Copy number variations in PRMS showed multiple chromosomal arm-level changes, most commonly gains of chr17p and chr22q and loss of chr6q. Notably, gain of chr8, commonly seen in ERMS (61%) was conspicuously absent in PRMS. The genomic profiles of other pleomorphic sarcomas were overall analogous to PRMS, showing shared alterations in TP53, RB1, and CDKN2A. Overall survival and progression-free survival of PRMS were significantly worse (p < 0.0005) than that of ERMS. Our findings revealed that the molecular landscape of PRMS aligns with other adult pleomorphic sarcomas and is distinct from that of ERMS. Thus, NGS assays may be applied in select challenging cases toward a refined classification. Finally, our data corroborate the inclusion of PRMS in the therapeutic bracket of pleomorphic sarcomas, given that their clinical outcomes are comparable.
Topics: Humans; Male; Female; Adult; Middle Aged; Aged; Rhabdomyosarcoma, Embryonal; Rhabdomyosarcoma; Mutation; High-Throughput Nucleotide Sequencing; Genomics; Biomarkers, Tumor; Retinoblastoma Binding Proteins; Ubiquitin-Protein Ligases
PubMed: 38722224
DOI: 10.1002/gcc.23238 -
Cell Death & Disease May 2024RAD18, an important ubiquitin E3 ligase, plays a dual role in translesion DNA synthesis (TLS) and homologous recombination (HR) repair. However, whether and how the...
RAD18, an important ubiquitin E3 ligase, plays a dual role in translesion DNA synthesis (TLS) and homologous recombination (HR) repair. However, whether and how the regulatory mechanism of O-linked N-acetylglucosamine (O-GlcNAc) modification governing RAD18 and its function during these processes remains unknown. Here, we report that human RAD18, can undergo O-GlcNAcylation at Ser130/Ser164/Thr468, which is important for optimal RAD18 accumulation at DNA damage sites. Mechanistically, abrogation of RAD18 O-GlcNAcylation limits CDC7-dependent RAD18 Ser434 phosphorylation, which in turn significantly reduces damage-induced PCNA monoubiquitination, impairs Polη focus formation and enhances UV sensitivity. Moreover, the ubiquitin and RAD51C binding ability of RAD18 at DNA double-strand breaks (DSBs) is O-GlcNAcylation-dependent. O-GlcNAcylated RAD18 promotes the binding of RAD51 to damaged DNA during HR and decreases CPT hypersensitivity. Our findings demonstrate a novel role of RAD18 O-GlcNAcylation in TLS and HR regulation, establishing a new rationale to improve chemotherapeutic treatment.
Topics: Humans; DNA-Binding Proteins; Ubiquitin-Protein Ligases; Recombinational DNA Repair; Acetylglucosamine; Rad51 Recombinase; Proliferating Cell Nuclear Antigen; Phosphorylation; DNA Replication; Ubiquitination; DNA Breaks, Double-Stranded; DNA-Directed DNA Polymerase; Cell Cycle Proteins; DNA Damage; DNA; HEK293 Cells; Ultraviolet Rays; Protein Binding; Glycosylation; Translesion DNA Synthesis
PubMed: 38719812
DOI: 10.1038/s41419-024-06700-y -
The Journal of Biological Chemistry May 2024Base excision repair (BER) requires a tight coordination between the repair enzymes through protein-protein interactions and involves gap filling by DNA polymerase (pol)...
Base excision repair (BER) requires a tight coordination between the repair enzymes through protein-protein interactions and involves gap filling by DNA polymerase (pol) β and subsequent nick sealing by DNA ligase (LIG) 1 or LIGIIIα at the downstream steps. Apurinic/apyrimidinic-endonuclease 1 (APE1), by its exonuclease activity, proofreads 3' mismatches incorporated by polβ during BER. We previously reported that the interruptions in the functional interplay between polβ and the BER ligases result in faulty repair events. Yet, how the protein interactions of LIG1 and LIGIIIα could affect the repair pathway coordination during nick sealing at the final steps remains unknown. Here, we demonstrate that LIGIIIα interacts more tightly with polβ and APE1 than LIG1, and the N-terminal noncatalytic region of LIG1 as well as the catalytic core and BRCT domain of LIGIIIα mediate interactions with both proteins. Our results demonstrated less efficient nick sealing of polβ nucleotide insertion products in the absence of LIGIIIα zinc-finger domain and LIG1 N-terminal region. Furthermore, we showed a coordination between APE1 and LIG1/LIGIIIα during the removal of 3' mismatches from the nick repair intermediate on which both BER ligases can seal noncanonical ends or gap repair intermediate leading to products of single deletion mutagenesis. Overall results demonstrate the importance of functional coordination from gap filling by polβ coupled to nick sealing by LIG1/LIGIIIα in the presence of proofreading by APE1, which is mainly governed by protein-protein interactions and protein-DNA intermediate communications, to maintain repair efficiency at the downstream steps of the BER pathway.
PubMed: 38718860
DOI: 10.1016/j.jbc.2024.107355 -
Revista Da Associacao Medica Brasileira... 2024This prospective study aimed to provide a comprehensive analysis of the methylation status of two pivotal genes, CDKN2A/p16INK4A (cyclin-dependent kinase inhibitor 2A)...
OBJECTIVE
This prospective study aimed to provide a comprehensive analysis of the methylation status of two pivotal genes, CDKN2A/p16INK4A (cyclin-dependent kinase inhibitor 2A) and RB1 (retinoblastoma transcriptional corepressor 1), in breast cancer patients.
METHODS
Samples were obtained from 15 women diagnosed with breast cancer and who underwent a total mastectomy. DNA was extracted from the tumor, non-tumor tissue, and peripheral blood (circulating cell-free DNA). The methylation pattern of cell-free DNA extracted from blood collected on the day of mastectomy was compared with the methylation pattern of cell-free DNA from blood collected 1 year post-surgery. The methylation analysis was carried out by sodium bisulfite conversion and polymerase chain reaction, followed by electrophoresis.
RESULTS
Methylation of CDKN2A/p16INK4A was identified in 13 tumor samples and 12 non-tumor tissue samples. Two patients exhibited CDKN2A/p16INK4A methylation in the cell-free DNA of the first blood collection, while another showed methylation only in the cell-free DNA of the subsequent blood collection. Regarding RB1, 11 tumors and 8 non-tumor tissue samples presented methylation of the gene.
CONCLUSION
This study presents a novel approach for monitoring breast cancer patients through the analysis of cell-free DNA methylation. This analysis can detect changes in methylation patterns before any visible sign of cancer appears in breast tissue and could help predict the recurrence of malignant breast tumors.
Topics: Adult; Aged; Female; Humans; Middle Aged; Biomarkers, Tumor; Breast Neoplasms; Cell-Free Nucleic Acids; Cyclin-Dependent Kinase Inhibitor p16; DNA Methylation; Mastectomy; Polymerase Chain Reaction; Prospective Studies; Retinoblastoma Binding Proteins; Ubiquitin-Protein Ligases
PubMed: 38716944
DOI: 10.1590/1806-9282.20231358 -
International Immunopharmacology Jun 2024Asthma is a prevalent chronic respiratory disease, yet understanding its ecology and pathogenesis remains a challenge. Trim27, a ubiquitination ligase belonging to the...
Asthma is a prevalent chronic respiratory disease, yet understanding its ecology and pathogenesis remains a challenge. Trim27, a ubiquitination ligase belonging to the TRIM (tripartite motif-containing) family, has been implicated in regulating multiple pathophysiological processes such as inflammation, oxidative stress, apoptosis, and cell proliferation. However, the role of Trim27 in asthma has not been investigated. Our study found that Trim27 expression significantly increases in the airway epithelium of asthmatic mice. Knockdown of Trim27 expression effectively relieved ovalbumin (OVA)-induced airway hyperresponsiveness (AHR) and lung tissue histopathological changes. Moreover, Trim27 knockdown exhibited a significant reduction in airway inflammation and oxidative stress in asthmatic mice, and in vitro analysis confirmed the favorable effect of Trim27 deletion on inflammation and oxidative stress in mouse airway epithelial cells. Furthermore, our study revealed that deletion of Trim27 in MLE12 cells significantly decreased NLRP3 inflammasome activation, as evidenced by reduced expression of NLRP3, ASC, and pro-IL-1β mRNA. This downregulation was reversed when Trim27, but not its mutant lacking ubiquitination ligase activity, was replenished in these cells. Consistent with these findings, protein levels of NLRP3, pro-caspase-1, pro-IL-1β, cleaved-caspase-1, and cleaved-IL-1β were higher in Trim27-replenished cells compared to cells expressing Trim27C/A. Functionally, the downregulation of IL-1β and IL-18 levels induced by Trim27 deletion was rescued by replenishing Trim27. Overall, our findings provide evidence that Trim27 contributes to airway inflammation and oxidative stress in asthmatic mice via NLRP3 inflammasome activation, providing crucial insights into potential therapeutic interventions targeting Trim27 as a way to treat asthma.
Topics: Animals; Asthma; NLR Family, Pyrin Domain-Containing 3 Protein; Oxidative Stress; Inflammasomes; Mice; Mice, Inbred BALB C; Ovalbumin; Ubiquitin-Protein Ligases; Lung; Cell Line; Female; Disease Models, Animal; Inflammation; Humans; Mice, Inbred C57BL; Tripartite Motif Proteins; DNA-Binding Proteins
PubMed: 38713938
DOI: 10.1016/j.intimp.2024.112199 -
Biochimica Et Biophysica Acta.... Jun 2024Human DNA polymerase ι (Polι) belongs to the Y-family of specialized DNA polymerases engaged in the DNA damage tolerance pathway of translesion DNA synthesis that is...
Human DNA polymerase ι (Polι) belongs to the Y-family of specialized DNA polymerases engaged in the DNA damage tolerance pathway of translesion DNA synthesis that is crucial to the maintenance of genome integrity. The extreme infidelity of Polι and the fact that both its up- and down-regulation correlate with various cancers indicate that Polι expression and access to the replication fork should be strictly controlled. Here, we identify RNF2, an E3 ubiquitin ligase, as a new interacting partner of Polι that is responsible for Polι stabilization in vivo. Interestingly, while we report that RNF2 does not directly ubiquitinate Polι, inhibition of the E3 ubiquitin ligase activity of RNF2 affects the cellular level of Polι thereby protecting it from destabilization. Additionally, we indicate that this mechanism is more general, as DNA polymerase η, another Y-family polymerase and the closest paralogue of Polι, share similar features.
Topics: Humans; Ubiquitin-Protein Ligases; DNA-Directed DNA Polymerase; DNA Polymerase iota; Ubiquitination; DNA Replication; DNA Damage; HEK293 Cells; Enzyme Stability; Polycomb Repressive Complex 1
PubMed: 38705361
DOI: 10.1016/j.bbamcr.2024.119743 -
Molecular Cell May 2024In a recent publication in Nature, Xu et al. discovered a role of CRL5-SPSB3 ubiquitin ligase in promoting ubiquitination and degradation of nuclear cGAS, which...
In a recent publication in Nature, Xu et al. discovered a role of CRL5-SPSB3 ubiquitin ligase in promoting ubiquitination and degradation of nuclear cGAS, which prevents aberrant cGAS activation by genomic DNA and contributes to the maintenance of immune homeostasis.
Topics: Nucleotidyltransferases; Humans; Homeostasis; Ubiquitination; Ubiquitin-Protein Ligases; Cell Nucleus; Proteolysis; Animals
PubMed: 38701740
DOI: 10.1016/j.molcel.2024.04.003 -
Biomolecular NMR Assignments Jun 2024The BRCA1 carboxyl-terminal (BRCT) domain, an evolutionarily conserved structural motif, is ubiquitous in a multitude of proteins spanning prokaryotic and eukaryotic...
The BRCA1 carboxyl-terminal (BRCT) domain, an evolutionarily conserved structural motif, is ubiquitous in a multitude of proteins spanning prokaryotic and eukaryotic organisms. In Mycobacterium tuberculosis (Mtb), BRCT domain plays a pivotal role in the catalytic activity of the NAD+-dependent DNA ligase (LigA). LigA is pivotal in DNA replication, catalyzing the formation of phosphodiester bonds in Okazaki fragments and repairing single-strand breaks in damaged DNA, essential for the survival of Mtb. Structural and functional aspects of LigA unveil its character as a highly modular protein, undergoing substantial conformational changes during its catalytic cycle. Although the BRCT domain of Mtb LigA plays an essential role in DNA binding and protein-protein interactions, the precise mechanism of action remains poorly understood. Unravelling the structure of the BRCT domain holds the promise of advancing our understanding of this pivotal domain. Additionally, it will facilitate further exploration of the protein-protein interactions and enhance our understanding of inter domain interactions within LigA, specifically between BRCT and the Adenylation domain. In this study, we demonstrate the overexpression of the BRCT domain of Mtb LigA and conduct its analysis using solution NMR spectroscopy, revealing a well-folded structure and we present the nearly complete chemical shift assignments of both backbone and sidechains. In addition, a secondary structure prediction by TALOS N predicts BRCT consisting of 3 α-helices and 4 β-sheets, closely resembling the typical structural topology of most BRCT domains.
Topics: Mycobacterium tuberculosis; Protein Domains; Nuclear Magnetic Resonance, Biomolecular; Protein Structure, Secondary; DNA Ligase ATP; DNA Ligases
PubMed: 38689205
DOI: 10.1007/s12104-024-10175-5 -
Journal of Experimental & Clinical... Apr 2024TP53, the most frequently mutated gene in human cancers, orchestrates a complex transcriptional program crucial for cancer prevention. While certain TP53-dependent genes...
BACKGROUND
TP53, the most frequently mutated gene in human cancers, orchestrates a complex transcriptional program crucial for cancer prevention. While certain TP53-dependent genes have been extensively studied, others, like the recently identified RNF144B, remained poorly understood. This E3 ubiquitin ligase has shown potent tumor suppressor activity in murine Eμ Myc-driven lymphoma, emphasizing its significance in the TP53 network. However, little is known about its targets and its role in cancer development, requiring further exploration. In this work, we investigate RNF144B's impact on tumor suppression beyond the hematopoietic compartment in human cancers.
METHODS
Employing TP53 wild-type cells, we generated models lacking RNF144B in both non-transformed and cancerous cells of human and mouse origin. By using proteomics, transcriptomics, and functional analysis, we assessed RNF144B's impact in cellular proliferation and transformation. Through in vitro and in vivo experiments, we explored proliferation, DNA repair, cell cycle control, mitotic progression, and treatment resistance. Findings were contrasted with clinical datasets and bioinformatics analysis.
RESULTS
Our research underscores RNF144B's pivotal role as a tumor suppressor, particularly in lung adenocarcinoma. In both human and mouse oncogene-expressing cells, RNF144B deficiency heightened cellular proliferation and transformation. Proteomic and transcriptomic analysis revealed RNF144B's novel function in mediating protein degradation associated with cell cycle progression, DNA damage response and genomic stability. RNF144B deficiency induced chromosomal instability, mitotic defects, and correlated with elevated aneuploidy and worse prognosis in human tumors. Furthermore, RNF144B-deficient lung adenocarcinoma cells exhibited resistance to cell cycle inhibitors that induce chromosomal instability.
CONCLUSIONS
Supported by clinical data, our study suggests that RNF144B plays a pivotal role in maintaining genomic stability during tumor suppression.
Topics: Animals; Humans; Mice; Cell Line, Tumor; Cell Proliferation; Genomic Instability; Lung Neoplasms; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases
PubMed: 38685100
DOI: 10.1186/s13046-024-03045-4