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Molecular Immunology Jun 2024Endothelial cell injury and dysfunction lead to cholesterol and lipid accumulation and atherosclerotic plaque formation in the arterial wall during atherosclerosis (AS)...
BACKGROUND
Endothelial cell injury and dysfunction lead to cholesterol and lipid accumulation and atherosclerotic plaque formation in the arterial wall during atherosclerosis (AS) progression, Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), a DNA methylation regulator, was strongly upregulated in atherosclerotic plaque lesions in mice. This study aimed to investigate the precise biological functions and regulatory mechanisms of UHRF1 on endothelial dysfunction during AS development.
METHODS
UHRF1 levels in the atherosclerotic plaque tissues and normal arterial intima from AS patients were tested with Western blot analysis and immunohistochemistry assays. Human umbilical vein endothelial cells (HUVECs) were stimulated with oxidized low-density lipoprotein (ox-LDL) to induce an injury model and then transfected with short hairpin RNA targeting UHRF1 (sh-UHRF1). Cell proliferation, migration, apoptosis, the levels of inflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and the protein levels adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were measured. Moreover, co-immunoprecipitation assay was used to determine the interactions between UHRF1 and DNA methyltransferases 1 (DNMT1), As well as mothers against DPP homolog 7 (SMAD7) and yes-associated protein 1 (YAP1). SMAD7 promoter methylation was examined with methylation-specific PCR. In addition, we established an AS mouse model to determine the in vivo effects of UHRF1 on AS progression.
RESULTS
UHRF1 was upregulated in atherosclerotic plaque tissues and ox-LDL-treated HUVECs. UHRF1 knockdown mitigated ox-LDL-induced proliferation and migration inhibition, apoptosis and the production of TNF-α, IL-6, VCAM-1, and ICAM-1 in HUVECs. Mechanistically, UHRF1 promoted DNMT1-mediated SMAD7 promoter methylation and inhibited its expression. SMAD7 knockdown abolished the protective effects of UHRF1 knockdown on ox-LDL-induced HUVEC injury. Moreover, SMAD7 interacted with YAP1 and inhibited YAP1 expression by promoting YAP1 protein ubiquitination-independent degradation in HUVECs. YAP1 overexpression abrogated SMAD7 overexpression-mediated protective effects on ox-LDL-induced HUVEC injury. Finally, UHRF1 knockdown alleviated atherosclerotic plaque deposition and arterial lesions in AS mice.
CONCLUSION
UHRF1 inhibition mitigates vascular endothelial cell injury and ameliorates AS progression in mice by regulating the SMAD7/YAP1 axis.
Topics: Animals; Humans; Male; Mice; Adaptor Proteins, Signal Transducing; Apoptosis; Atherosclerosis; CCAAT-Enhancer-Binding Proteins; Cell Proliferation; Human Umbilical Vein Endothelial Cells; Lipoproteins, LDL; Mice, Inbred C57BL; Plaque, Atherosclerotic; Signal Transduction; Smad7 Protein; Ubiquitin-Protein Ligases; YAP-Signaling Proteins
PubMed: 38657333
DOI: 10.1016/j.molimm.2024.04.001 -
Proceedings of the National Academy of... Apr 2024The S-phase checkpoint involving CHK1 is essential for fork stability in response to fork stalling. PARP1 acts as a sensor of replication stress and is required for CHK1...
The S-phase checkpoint involving CHK1 is essential for fork stability in response to fork stalling. PARP1 acts as a sensor of replication stress and is required for CHK1 activation. However, it is unclear how the activity of PARP1 is regulated. Here, we found that UFMylation is required for the efficient activation of CHK1 by UFMylating PARP1 at K548 during replication stress. Inactivation of UFL1, the E3 enzyme essential for UFMylation, delayed CHK1 activation and inhibits nascent DNA degradation during replication blockage as seen in PARP1-deficient cells. An in vitro study indicated that PARP1 is UFMylated at K548, which enhances its catalytic activity. Correspondingly, a PARP1 UFMylation-deficient mutant (K548R) and pathogenic mutant (F553L) compromised CHK1 activation, the restart of stalled replication forks following replication blockage, and chromosome stability. Defective PARP1 UFMylation also resulted in excessive nascent DNA degradation at stalled replication forks. Finally, we observed that PARP1 UFMylation-deficient knock-in mice exhibited increased sensitivity to replication stress caused by anticancer treatments. Thus, we demonstrate that PARP1 UFMylation promotes CHK1 activation and replication fork stability during replication stress, thus safeguarding genome integrity.
Topics: DNA Replication; Animals; Poly (ADP-Ribose) Polymerase-1; Checkpoint Kinase 1; Mice; Humans; DNA Damage; Ubiquitin-Protein Ligases
PubMed: 38657044
DOI: 10.1073/pnas.2322520121 -
Genome Biology Apr 2024The proliferation antigen Ki-67 has been widely used in clinical settings for cancer staging for many years, but investigations on its biological functions have lagged....
BACKGROUND
The proliferation antigen Ki-67 has been widely used in clinical settings for cancer staging for many years, but investigations on its biological functions have lagged. Recently, Ki-67 has been shown to regulate both the composition of the chromosome periphery and chromosome behaviour in mitosis as well as to play a role in heterochromatin organisation and gene transcription. However, how the different roles for Ki-67 across the cell cycle are regulated and coordinated remain poorly understood. The progress towards understanding Ki-67 function have been limited by the tools available to deplete the protein, coupled to its abundance and fluctuation during the cell cycle.
RESULTS
Here, we use a doxycycline-inducible E3 ligase together with an auxin-inducible degron tag to achieve a rapid, acute and homogeneous degradation of Ki-67 in HCT116 cells. This system, coupled with APEX2 proteomics and phospho-proteomics approaches, allows us to show that Ki-67 plays a role during DNA replication. In its absence, DNA replication is severely delayed, the replication machinery is unloaded, causing DNA damage that is not sensed by the canonical pathways and dependent on HUWE1 ligase. This leads to defects in replication and sister chromatids cohesion, but it also triggers an interferon response mediated by the cGAS/STING pathway in all the cell lines tested.
CONCLUSIONS
We unveil a new function of Ki-67 in DNA replication and genome maintenance that is independent of its previously known role in mitosis and gene regulation.
Topics: Humans; DNA Damage; DNA Replication; Genomic Instability; HCT116 Cells; Ki-67 Antigen; Ubiquitin-Protein Ligases
PubMed: 38649976
DOI: 10.1186/s13059-024-03243-5 -
The EMBO Journal Jun 2024Mitochondria are cellular powerhouses that generate energy through the electron transport chain (ETC). The mitochondrial genome (mtDNA) encodes essential ETC proteins in...
Mitochondria are cellular powerhouses that generate energy through the electron transport chain (ETC). The mitochondrial genome (mtDNA) encodes essential ETC proteins in a compartmentalized manner, however, the mechanism underlying metabolic regulation of mtDNA function remains unknown. Here, we report that expression of tricarboxylic acid cycle enzyme succinate-CoA ligase SUCLG1 strongly correlates with ETC genes across various TCGA cancer transcriptomes. Mechanistically, SUCLG1 restricts succinyl-CoA levels to suppress the succinylation of mitochondrial RNA polymerase (POLRMT). Lysine 622 succinylation disrupts the interaction of POLRMT with mtDNA and mitochondrial transcription factors. SUCLG1-mediated POLRMT hyposuccinylation maintains mtDNA transcription, mitochondrial biogenesis, and leukemia cell proliferation. Specifically, leukemia-promoting FMS-like tyrosine kinase 3 (FLT3) mutations modulate nuclear transcription and upregulate SUCLG1 expression to reduce succinyl-CoA and POLRMT succinylation, resulting in enhanced mitobiogenesis. In line, genetic depletion of POLRMT or SUCLG1 significantly delays disease progression in mouse and humanized leukemia models. Importantly, succinyl-CoA level and POLRMT succinylation are downregulated in FLT3-mutated clinical leukemia samples, linking enhanced mitobiogenesis to cancer progression. Together, SUCLG1 connects succinyl-CoA with POLRMT succinylation to modulate mitochondrial function and cancer development.
Topics: Animals; Humans; Mice; Acyl Coenzyme A; Cell Line, Tumor; Cell Proliferation; Disease Progression; DNA, Mitochondrial; DNA-Directed RNA Polymerases; Leukemia; Mitochondria; Mitochondrial Proteins; Organelle Biogenesis; Succinate-CoA Ligases
PubMed: 38649537
DOI: 10.1038/s44318-024-00101-9 -
The International Journal of... Jun 2024DNA methylation is one of the most important epigenetic mark involved in many physiologic cellular processes and pathologies. During mitosis, the transmission of DNA...
DNA methylation is one of the most important epigenetic mark involved in many physiologic cellular processes and pathologies. During mitosis, the transmission of DNA methylation patterns from a mother to the daughter cells is ensured through the action of the Ubiquitin-like, containing PHD and RING domains, 1/DNA methyltransferase 1 (UHRF1/DNMT1) tandem. UHRF1 is involved in the silencing of many tumor suppressor genes (TSGs) via mechanisms that remain largely to be deciphered. The present study investigated the role and the regulation of UHRF1 poly-ubiquitination induced by thymoquinone, a natural anti-cancer drug, known to enhance or re-activate the expression of TSGs. We found that the auto-ubiquitination of UHRF1, induced by TQ, is mediated by reactive oxygen species, and occurs following DNA damage. We demonstrated that the poly-ubiquitinated form of UHRF1 is K63-linked and can still silence the tumor suppressor gene p16/CDKN2A We further showed that TQ-induced auto-ubiquitination is mediated via the activity of Tip60. Since this latter is known as a nuclear receptor co-factor, we investigated if the glucocorticoid receptor (GR) might be involved in the regulation of UHRF1 ubiquitination. Activation of the GR, with dexamethasone, did not influence auto-ubiquitination of UHRF1. However, we could observe that TQ induced a K48-linked poly-ubiquitination of GR, probably involved in the proteosomal degradation pathway. Mass-spectrometry analysis of FLAG-HA-tagged UHRF1 identified UHRF1 partners involved in DNA repair and showed that TQ increased their association with UHRF1, suggesting that poly-ubiquitination of UHRF1 is involved in the DNA repair process. We propose that poly-ubiquitination of UHRF1 serves as a scaffold to recruit the DNA repair machinery at DNA damage sites.
Topics: Humans; Ubiquitin-Protein Ligases; CCAAT-Enhancer-Binding Proteins; Ubiquitination; Benzoquinones; DNA Repair; Antineoplastic Agents; DNA Damage
PubMed: 38649007
DOI: 10.1016/j.biocel.2024.106582 -
Analytical Chemistry Apr 2024Complex structures and devices, both natural and artificial, can often undergo assembly and disassembly. Assembly and disassembly allow multiple stimuli to initiate, for...
Complex structures and devices, both natural and artificial, can often undergo assembly and disassembly. Assembly and disassembly allow multiple stimuli to initiate, for example, the assembly and disassembly of primary cilia under the control of E3 ubiquitin ligases and deubiquitinases. Although biology relies on such schemes, they are rarely available in materials science. Here, we demonstrate a DNA-functionalized colloidal Au response to endogenous biomarkers to trigger simultaneous assembly and disassembly techniques. Colloidal Au is initially inert because the starting DNA strands are paired and prehybridized. TK1 mRNA competes to bind one of the paired strands and release its complement. The released complement binds to the next colloidal Au to initiate assembly, and APE1 can shear the colloidal Au assembly binding site to initiate disassembly. Our strategy provides temporal and spatial logic control during colloidal Au assembly and disassembly, and this simultaneous assembly and disassembly process can be used for sequential detection and cellular imaging of two biomarkers, effectively reducing signal false-positive results and shortening detection time. This work highlights biomarker-controlled colloidal Au simultaneous assembly and disassembly in ways that are simple and versatile, with the potential to enrich the application scope of DNA nanotechnology and provide an idea for the application of precision medicine testing.
Topics: Humans; DNA; Biomarkers; RNA, Messenger; Colloids; Gold; Gold Colloid; DNA-(Apurinic or Apyrimidinic Site) Lyase; Thymidine Kinase
PubMed: 38639728
DOI: 10.1021/acs.analchem.3c05765 -
Biochemical and Biophysical Research... Jun 2024Over the past decades, cancer stem cells (CSCs) have emerged as a critical subset of tumor cells associated with tumor recurrence and resistance to chemotherapy....
Over the past decades, cancer stem cells (CSCs) have emerged as a critical subset of tumor cells associated with tumor recurrence and resistance to chemotherapy. Understanding the mechanisms underlying CSC-mediated chemoresistance is imperative for improving cancer therapy outcomes. This study delves into the regulatory role of NEIL1, a DNA glycosylase, in chemoresistance in ovarian CSCs. We first observed a decreased expression of NEIL1 in ovarian CSCs, suggesting its potential involvement in CSC regulation. Using pan-cancer analysis, we confirmed the diminished NEIL1 expression in ovarian tumors compared to normal tissues. Furthermore, NEIL1 downregulation correlated with an increase in stemness markers and enrichment of CSCs, highlighting its role in modulating CSC phenotype. Further mechanistic investigation revealed an inverse correlation between NEIL1 and RAD18 expression in ovarian CSCs. NEIL1 depletion led to heightened RAD18 expression, promoting chemoresistance possibly via enhancing Translesion DNA Synthesis (TLS)-mediated DNA lesion bypass. Moreover, dowregulation of NEIL1 results in reduced DNA damage accumulation and suppressed apoptosis in ovarian cancer. Overall, our findings unveil a novel mechanism involving NEIL1 and RAD18 in regulating chemoresistance in ovarian CSCs. Targeting this NEIL1-RAD18 axis may offer promising therapeutic strategies for combating chemoresistance and improving ovarian cancer treatment outcomes.
Topics: Humans; Female; Ovarian Neoplasms; Drug Resistance, Neoplasm; Neoplastic Stem Cells; Up-Regulation; DNA Glycosylases; Cell Line, Tumor; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Ubiquitin-Protein Ligases; DNA Damage; Apoptosis
PubMed: 38636303
DOI: 10.1016/j.bbrc.2024.149907 -
Histochemistry and Cell Biology Jun 2024Lung adenocarcinoma (LUAD) is a subtype of lung cancer with high incidence and mortality globally. Emerging evidence suggests that circular RNAs (circRNAs) exert...
Lung adenocarcinoma (LUAD) is a subtype of lung cancer with high incidence and mortality globally. Emerging evidence suggests that circular RNAs (circRNAs) exert critical functions in human cancers, including LUAD. CircRNA_100549 (circ_100549) has been reported to be significantly upregulated in non-small cell lung cancer (NSCLC) samples, while its role in modulating LUAD progression remains to be explored. The current study aims at investigating the functional roles of circ_100549 in LUAD and its downstream molecular mechanism. First, we found that the expression of circ_100549 was higher in LUAD cell lines. Loss-of-function assays verified that depletion of circ_100549 repressed LUAD cell proliferation but accelerated cell apoptosis. Furthermore, in vivo experiments demonstrated that silencing of circ_100549 suppressed tumor growth. Subsequently, based on database analysis, we carried out a series of experiments to explore the mechanisms and effects of circ_100549 underlying LUAD progression, including RNA-binding protein immunoprecipitation (RIP), RNA/DNA pull-down, luciferase reporter, and chromatin immunoprecipitation (ChIP) assays. The results indicated that circ_100549 serves as a ceRNA by sponging miR-95-5p to upregulate BPTF expression, thus upregulating BIRC6 expression at a transcriptional level in LUAD. In summary, our study demonstrated that circ_100549 facilitates LUAD progression by upregulating BIRC6 expression.
Topics: Humans; RNA, Circular; Lung Neoplasms; Up-Regulation; Adenocarcinoma of Lung; Mice; Inhibitor of Apoptosis Proteins; Cell Proliferation; Apoptosis; Mice, Nude; Animals; Disease Progression; Ubiquitin-Protein Ligases; Mice, Inbred BALB C
PubMed: 38613646
DOI: 10.1007/s00418-024-02275-z -
Nature Cell Biology May 2024DNA-protein crosslinks (DPCs) arise from enzymatic intermediates, metabolism or chemicals like chemotherapeutics. DPCs are highly cytotoxic as they impede DNA-based...
DNA-protein crosslinks (DPCs) arise from enzymatic intermediates, metabolism or chemicals like chemotherapeutics. DPCs are highly cytotoxic as they impede DNA-based processes such as replication, which is counteracted through proteolysis-mediated DPC removal by spartan (SPRTN) or the proteasome. However, whether DPCs affect transcription and how transcription-blocking DPCs are repaired remains largely unknown. Here we show that DPCs severely impede RNA polymerase II-mediated transcription and are preferentially repaired in active genes by transcription-coupled DPC (TC-DPC) repair. TC-DPC repair is initiated by recruiting the transcription-coupled nucleotide excision repair (TC-NER) factors CSB and CSA to DPC-stalled RNA polymerase II. CSA and CSB are indispensable for TC-DPC repair; however, the downstream TC-NER factors UVSSA and XPA are not, a result indicative of a non-canonical TC-NER mechanism. TC-DPC repair functions independently of SPRTN but is mediated by the ubiquitin ligase CRL4 and the proteasome. Thus, DPCs in genes are preferentially repaired in a transcription-coupled manner to facilitate unperturbed transcription.
Topics: DNA Repair; DNA Repair Enzymes; Humans; Transcription, Genetic; Poly-ADP-Ribose Binding Proteins; DNA Helicases; RNA Polymerase II; Proteolysis; Ubiquitin-Protein Ligases; DNA-Binding Proteins; DNA; HEK293 Cells; Transcription Factors; DNA Damage; Proteasome Endopeptidase Complex; Carrier Proteins; Receptors, Interleukin-17
PubMed: 38600236
DOI: 10.1038/s41556-024-01394-y -
International Journal of Antimicrobial... Jun 2024Vancomycin is frequently used as a last line of defence against infections due to multidrug-resistant Staphylococcus aureus (S. aureus). A recent finding described the...
BACKGROUND
Vancomycin is frequently used as a last line of defence against infections due to multidrug-resistant Staphylococcus aureus (S. aureus). A recent finding described the acquisition of vancomycin-resistant S. aureus strains by the integration of an enterococcal plasmid containing the vanA operon into the S. aureus chromosome via homologous recombination involving a specific integration site called locus L2.
METHODS
To characterise all mechanisms of acquisition of vanA, this study analysed the 15 706 S. aureus genomes to look for vanA and described its genetic environment.
RESULTS
A complete vanA operon was found in 25 S. aureus strains isolated from 12 patients, including nine co-isolated with vancomycin-resistant Enterococcus strains. VanA was found within transposon Tn1546-like elements on 17 plasmids and eight chromosomes. VanA might be acquired through conjugation of enterococcal and staphylococcal plasmids, transposition of Tn1546 carrying vanA and plasmid integration into the chromosome. Further, L2 was detected in 2087 genomes (13.3%) of S. aureus strains across different continents. Six potential chromosomal hotspots for integration of the entire vanA-containing enterococcal plasmid were identified by homologous recombination via L2.
CONCLUSIONS
These findings suggest that the recently described scenario in a New York patient could be reproduced anywhere. Surveillance of this possibility is mandatory, especially in patients with vancomycin-resistant Enterococcus infection or colonisation.
Topics: Humans; Plasmids; Operon; Vancomycin Resistance; Staphylococcus aureus; DNA Transposable Elements; Bacterial Proteins; Carbon-Oxygen Ligases; Genome, Bacterial; Staphylococcal Infections; Anti-Bacterial Agents; Vancomycin
PubMed: 38599552
DOI: 10.1016/j.ijantimicag.2024.107154