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Molecular Cell Apr 2024Cullin-RING ligases (CRLs) ubiquitylate specific substrates selected from other cellular proteins. Substrate discrimination and ubiquitin transferase activity were...
Cullin-RING ligases (CRLs) ubiquitylate specific substrates selected from other cellular proteins. Substrate discrimination and ubiquitin transferase activity were thought to be strictly separated. Substrates are recognized by substrate receptors, such as Fbox or BCbox proteins. Meanwhile, CRLs employ assorted ubiquitin-carrying enzymes (UCEs, which are a collection of E2 and ARIH-family E3s) specialized for either initial substrate ubiquitylation (priming) or forging poly-ubiquitin chains. We discovered specific human CRL-UCE pairings governing substrate priming. The results reveal pairing of CUL2-based CRLs and UBE2R-family UCEs in cells, essential for efficient PROTAC-induced neo-substrate degradation. Despite UBE2R2's intrinsic programming to catalyze poly-ubiquitylation, CUL2 employs this UCE for geometrically precise PROTAC-dependent ubiquitylation of a neo-substrate and for rapid priming of substrates recruited to diverse receptors. Cryo-EM structures illuminate how CUL2-based CRLs engage UBE2R2 to activate substrate ubiquitylation. Thus, pairing with a specific UCE overcomes E2 catalytic limitations to drive substrate ubiquitylation and targeted protein degradation.
Topics: Humans; Ubiquitin-Protein Ligases; Cullin Proteins; Ubiquitination; Ubiquitin; Polyubiquitin; Carrier Proteins
PubMed: 38382526
DOI: 10.1016/j.molcel.2024.01.022 -
ACS Synthetic Biology Mar 2024Commercially synthesized genes are typically made using variations of homology-based cloning techniques, including polymerase cycling assembly from chemically...
Commercially synthesized genes are typically made using variations of homology-based cloning techniques, including polymerase cycling assembly from chemically synthesized microarray-derived oligonucleotides. Here, we apply Data-optimized Assembly Design (DAD) to the synthesis of hundreds of codon-optimized genes in both constitutive and inducible vectors using Golden Gate Assembly. Starting from oligonucleotide pools, we synthesize genes in three simple steps: (1) amplification of parts belonging to individual assemblies in parallel from a single pool; (2) Golden Gate Assembly of parts for each construct; and (3) transformation. We construct genes from receiving DNA to sequence confirmed isolates in as little as 4 days. By leveraging the ligation fidelity afforded by T4 DNA ligase, we expect to be able to construct a larger breadth of sequences not currently supported by homology-based methods, which require stability of extensive single-stranded DNA overhangs.
Topics: Oligonucleotides; Synthetic Biology; DNA; DNA, Single-Stranded; Cloning, Molecular; Genetic Vectors
PubMed: 38377591
DOI: 10.1021/acssynbio.3c00694 -
Nucleic Acids Research Apr 2024Base excision repair (BER) involves the tightly coordinated function of DNA polymerase β (polβ) and DNA ligase I (LIG1) at the downstream steps. Our previous studies...
Base excision repair (BER) involves the tightly coordinated function of DNA polymerase β (polβ) and DNA ligase I (LIG1) at the downstream steps. Our previous studies emphasize that defective substrate-product channeling, from gap filling by polβ to nick sealing by LIG1, can lead to interruptions in repair pathway coordination. Yet, the molecular determinants that dictate accurate BER remains largely unknown. Here, we demonstrate that a lack of gap filling by polβ leads to faulty repair events and the formation of deleterious DNA intermediates. We dissect how ribonucleotide challenge and cancer-associated mutations could adversely impact the ability of polβ to efficiently fill the one nucleotide gap repair intermediate which subsequently results in gap ligation by LIG1, leading to the formation of single-nucleotide deletion products. Moreover, we demonstrate that LIG1 is not capable of discriminating against nick DNA containing a 3'-ribonucleotide, regardless of base-pairing potential or damage. Finally, AP-Endonuclease 1 (APE1) shows distinct substrate specificity for the exonuclease removal of 3'-mismatched bases and ribonucleotides from nick repair intermediate. Overall, our results reveal that unfilled gaps result in impaired coordination between polβ and LIG1, defining a possible type of mutagenic event at the downstream steps where APE1 could provide a proofreading role to maintain BER efficiency.
Topics: DNA Polymerase beta; DNA Ligase ATP; DNA Repair; Humans; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA; DNA Damage; DNA Ligases; Excision Repair
PubMed: 38366780
DOI: 10.1093/nar/gkae104 -
Microbiology Spectrum Apr 2024(), the causative agent of Rocky Mountain spotted fever (RMSF), is the most pathogenic member among spp. Previous studies have shown that tripartite motif-containing...
UNLABELLED
(), the causative agent of Rocky Mountain spotted fever (RMSF), is the most pathogenic member among spp. Previous studies have shown that tripartite motif-containing 56 (TRIM56) E3 ligase-induced ubiquitination of STING is important for cytosolic DNA sensing and type I interferon production to induce anti-DNA viral immunity, but whether it affects intracellular replication of remains uncharacterized. Here, we investigated the effect of TRIM56 on HeLa and THP-1 cells infected with . We found that the expression of TRIM56 was upregulated in the -infected cells, and the overexpression of TRIM56 inhibited the intracellular replication of , while replication was enhanced in the TRIM56-silenced host cells with the reduced phosphorylation of IRF3 and STING and the increased production of interferon-β. In addition, the mutation of the TRIM56 E3 ligase catalytic site impairs the inhibitory function against in HeLa cells. Altogether, our study discovers that TRIM56 is a host restriction factor of by regulating the cGAS-STING-mediated signaling pathway. This study gives new evidence for the role of TRIM56 in the innate immune response against intracellular bacterial infection and provides new therapeutic targets for RMSF.
IMPORTANCE
Given that () is the most pathogenic member within the genus and serves as the causative agent of Rocky Mountain spotted fever, there is a growing need to explore host targets. In this study, we examined the impact of host TRIM56 on infection in HeLa and THP-1 cells. We observed a significant upregulation of TRIM56 expression in -infected cells. Remarkably, the overexpression of TRIM56 inhibited the intracellular replication of , while silencing TRIM56 enhanced bacterial replication accompanied by reduced phosphorylation of IRF3 and STING, along with increased interferon-β production. Notably, the mutation of the TRIM56's E3 ligase catalytic site did not impede replication in HeLa cells. Collectively, our findings provide novel insights into the role of TRIM56 as a host restriction factor against through the modulation of the cGAS-STING signaling pathway.
Topics: Humans; Rickettsia rickettsii; Rocky Mountain Spotted Fever; Interferon Type I; HeLa Cells; Ubiquitin-Protein Ligases; Interferon-beta; Nucleotidyltransferases; Tripartite Motif Proteins
PubMed: 38358243
DOI: 10.1128/spectrum.03695-23 -
Frontiers in Immunology 2024Tripartite-motif 56 (TRIM56) is a member of the TRIM family, and was shown to be an interferon-inducible E3 ubiquitin ligase that can be overexpressed upon stimulation...
Tripartite-motif 56 (TRIM56) is a member of the TRIM family, and was shown to be an interferon-inducible E3 ubiquitin ligase that can be overexpressed upon stimulation with double-stranded DNA to regulate stimulator of interferon genes (STING) to produce type I interferon and thus mediate innate immune responses. Its role in tumors remains unclear. In this study, we investigated the relationship between the expression of the TRIM56 gene and its prognostic value in pan-cancer, identifying TRIM56 expression as an adverse prognostic factor in glioma patients. Therefore, glioma was selected as the primary focus of our investigation. We explored the differential expression of TRIM56 in various glioma subtypes and verified its role as an independent prognostic factor in gliomas. Our research revealed that TRIM56 is associated with malignant biological behaviors in gliomas, such as proliferation, migration, and invasion. Additionally, it can mediate M2 polarization of macrophages in gliomas. The results were validated and . Furthermore, we utilized single-cell analysis to investigate the impact of TRIM56 expression on cell communication between glioma cells and non-tumor cells. We constructed a multi-gene signature based on cell markers of tumor cells with high TRIM56 expression to enhance the prediction of cancer patient prognosis. In conclusion, our study demonstrates that TRIM56 serves as a reliable immune-related prognostic biomarker in glioma.
Topics: Humans; Prognosis; Interferons; Glioma; Biomarkers; Single-Cell Analysis; Tripartite Motif Proteins; Ubiquitin-Protein Ligases
PubMed: 38348047
DOI: 10.3389/fimmu.2024.1327898 -
Nature Communications Feb 2024Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that...
Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that cannot be immediately re-ligated are modified by NHEJ processing enzymes, including error-prone polymerases and nucleases, to enable ligation. However, DSB ends that are initially compatible for re-ligation are typically joined without end processing. As both ligation and end processing occur in the short-range (SR) synaptic complex that closely aligns DNA ends, it remains unclear how ligation of compatible ends is prioritized over end processing. In this study, we identify structural interactions of the NHEJ-specific DNA Ligase IV (Lig4) within the SR complex that prioritize ligation and promote NHEJ fidelity. Mutational analysis demonstrates that Lig4 must bind DNA ends to form the SR complex. Furthermore, single-molecule experiments show that a single Lig4 binds both DNA ends at the instant of SR synapsis. Thus, Lig4 is poised to ligate compatible ends upon initial formation of the SR complex before error-prone processing. Our results provide a molecular basis for the fidelity of NHEJ.
Topics: DNA Ligase ATP; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Repair; DNA Ligases; DNA
PubMed: 38341432
DOI: 10.1038/s41467-024-45553-z -
DNA Repair Mar 2024Eukaryotic genome stability is maintained by a complex and diverse set of molecular processes. One class of enzymes that promotes proper DNA repair, replication and cell...
Eukaryotic genome stability is maintained by a complex and diverse set of molecular processes. One class of enzymes that promotes proper DNA repair, replication and cell cycle progression comprises small ubiquitin-like modifier (SUMO)-targeted E3 ligases, or STUbLs. Previously, we reported a role for the budding yeast STUbL synthetically lethal with sgs1 (Slx) 5/8 in preventing G/M-phase arrest in a minichromosome maintenance protein 10 (Mcm10)-deficient model of replication stress. Here, we extend these studies to human cells, examining the requirement for the human STUbL RING finger protein 4 (RNF4) in MCM10 mutant cancer cells. We find that MCM10 and RNF4 independently promote origin firing but regulate DNA synthesis epistatically and, unlike in yeast, the negative genetic interaction between RNF4 and MCM10 causes cells to accumulate in G-phase. When MCM10 is deficient, RNF4 prevents excessive DNA under-replication at hard-to-replicate regions that results in large DNA copy number alterations and severely reduced viability. Overall, our findings highlight that STUbLs participate in species-specific mechanisms to maintain genome stability, and that human RNF4 is required for origin activation in the presence of chronic replication stress.
Topics: Humans; Genomic Instability; DNA Repair; DNA Replication; Mitosis; Saccharomyces cerevisiae; Nuclear Proteins; Transcription Factors
PubMed: 38340377
DOI: 10.1016/j.dnarep.2024.103646 -
ESC Heart Failure Jun 2024Polyglucosan body myopathy 1 (PGBM1) is a type of glycogen storage disease where polyglucosan accumulation leads to cardiomyopathy and skeletal muscle myopathy. Variants...
AIMS
Polyglucosan body myopathy 1 (PGBM1) is a type of glycogen storage disease where polyglucosan accumulation leads to cardiomyopathy and skeletal muscle myopathy. Variants of RBCK1 is related with PGBM1. We present a newly discovered pathogenic RBCK1 variant resulting in dilated cardiomyopathy (DCM) and a comprehensive literature review.
METHODS AND RESULTS
Whole-exome sequencing (WES) was utilized to detect genetic variations in a 7-year-old girl considered the proband. Sanger sequencing was performed to validate the variant in the patient and all the available family members, whether affected or unaffected. The variant's pathogenicity was assessed by conducting a cosegregation analysis within the family with in silico predictive software. WES showed that the proband's RBCK1 gene contained a missense likely pathogenic homozygous nucleotide variant, c.598_599insT: p.His200LeufsTer14 (NM_001323956.1), in exon 8. The computational analysis supported the variant's pathogenicity. The variant was identified in a heterozygous form among all the healthy members of the family. Variants with changes in N-terminal part of the protein were more likely to manifest immunodeficiency and auto-inflammation than those with C-terminal protein modifications according to prior variations of RBCK1 reported in the literature.
CONCLUSIONS
Our study offers novel findings indicating an RBCK1 variant in individuals of Iranian ancestry presenting with DCM leading to heart transplantation and myopathy without immunodeficiency or auto-inflammation.
Topics: Humans; Female; Cardiomyopathy, Dilated; Child; Muscle Weakness; Pedigree; Homozygote; Exome Sequencing; Transcription Factors; DNA; Ubiquitin-Protein Ligases
PubMed: 38329383
DOI: 10.1002/ehf2.14702 -
Nucleic Acids Research Apr 2024CtIP initiates DNA end resection and mediates homologous recombination (HR) repair. However, the underlying mechanisms of CtIP regulation and how the control of its...
CtIP initiates DNA end resection and mediates homologous recombination (HR) repair. However, the underlying mechanisms of CtIP regulation and how the control of its regulation affects DNA repair remain incompletely characterized. In this study, NUDT16 loss decreases CtIP protein levels and impairs CtIP recruitment to double-strand breaks (DSBs). Furthermore, overexpression of a catalytically inactive NUDT16 mutant is unable to rescue decreased CtIP protein and impaired CtIP recruitment to DSBs. In addition, we identified a novel posttranslational modification of CtIP by ADP-ribosylation that is targeted by a PAR-binding E3 ubiquitin ligase, RNF146, leading to CtIP ubiquitination and degradation. These data suggest that the hydrolase activity of NUDT16 plays a major role in controlling CtIP protein levels. Notably, ADP-ribosylation of CtIP is required for its interaction with NUDT16, its localization at DSBs, and for HR repair. Interestingly, NUDT16 can also be ADP-ribosylated. The ADP-ribosylated NUDT16 is critical for CtIP protein stability, CtIP recruitment to DSBs, and HR repair in response to DNA damage. In summary, we demonstrate that NUDT16 and its PARylation regulate CtIP stability and CtIP recruitment to DSBs, providing new insights into our understanding of the regulation of CtIP-mediated DNA end resection in the HR repair pathway.
Topics: Humans; ADP-Ribosylation; Carrier Proteins; DNA Breaks, Double-Stranded; Endodeoxyribonucleases; HEK293 Cells; Nuclear Proteins; Protein Processing, Post-Translational; Pyrophosphatases; Recombinational DNA Repair; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 38324469
DOI: 10.1093/nar/gkae064 -
Nature Structural & Molecular Biology Mar 2024During transcription-coupled DNA repair (TCR), RNA polymerase II (Pol II) transitions from a transcriptionally active state to an arrested state that allows for removal...
During transcription-coupled DNA repair (TCR), RNA polymerase II (Pol II) transitions from a transcriptionally active state to an arrested state that allows for removal of DNA lesions. This transition requires site-specific ubiquitylation of Pol II by the CRL4 ubiquitin ligase, a process that is facilitated by ELOF1 in an unknown way. Using cryogenic electron microscopy, biochemical assays and cell biology approaches, we found that ELOF1 serves as an adaptor to stably position UVSSA and CRL4 on arrested Pol II, leading to ligase neddylation and activation of Pol II ubiquitylation. In the presence of ELOF1, a transcription factor IIS (TFIIS)-like element in UVSSA gets ordered and extends through the Pol II pore, thus preventing reactivation of Pol II by TFIIS. Our results provide the structural basis for Pol II ubiquitylation and inactivation in TCR.
Topics: RNA Polymerase II; Transcription, Genetic; Excision Repair; DNA Repair; DNA; Ubiquitination; Ligases; Receptors, Antigen, T-Cell
PubMed: 38316879
DOI: 10.1038/s41594-023-01207-0