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1.
J Cell Biol ; 223(7)2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38717338

ABSTRACT

Senataxin is an evolutionarily conserved RNA-DNA helicase involved in DNA repair and transcription termination that is associated with human neurodegenerative disorders. Here, we investigated whether Senataxin loss affects protein homeostasis based on previous work showing R-loop-driven accumulation of DNA damage and protein aggregates in human cells. We find that Senataxin loss results in the accumulation of insoluble proteins, including many factors known to be prone to aggregation in neurodegenerative disorders. These aggregates are located primarily in the nucleolus and are promoted by upregulation of non-coding RNAs expressed from the intergenic spacer region of ribosomal DNA. We also map sites of R-loop accumulation in human cells lacking Senataxin and find higher RNA-DNA hybrids within the ribosomal DNA, peri-centromeric regions, and other intergenic sites but not at annotated protein-coding genes. These findings indicate that Senataxin loss affects the solubility of the proteome through the regulation of transcription-dependent lesions in the nucleus and the nucleolus.


Subject(s)
DNA Helicases , Multifunctional Enzymes , RNA Helicases , RNA, Untranslated , Humans , Cell Nucleolus/metabolism , Cell Nucleolus/genetics , DNA Damage , DNA Helicases/metabolism , DNA Helicases/genetics , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Multifunctional Enzymes/metabolism , Multifunctional Enzymes/genetics , Protein Aggregates , Proteostasis , R-Loop Structures/genetics , RNA Helicases/metabolism , RNA Helicases/genetics , RNA, Untranslated/genetics , RNA, Untranslated/metabolism
2.
Front Immunol ; 15: 1358036, 2024.
Article in English | MEDLINE | ID: mdl-38690262

ABSTRACT

Background: It remains unclear whether BPIV3 infection leads to stress granules formation and whether G3BP1 plays a role in this process and in viral replication. This study aims to clarify the association between BPIV3 and stress granules, explore the effect of G3BP1 on BPIV3 replication, and provide significant insights into the mechanisms by which BPIV3 evades the host's antiviral immunity to support its own survival. Methods: Here, we use Immunofluorescence staining to observe the effect of BPIV3 infection on the assembly of stress granules. Meanwhile, the expression changes of eIF2α and G3BP1 were determined. Overexpression or siRNA silencing of intracellular G3BP1 levels was examined for its regulatory control of BPIV3 replication. Results: We identify that the BPIV3 infection elicited phosphorylation of the eIF2α protein. However, it did not induce the assembly of stress granules; rather, it inhibited the formation of stress granules and downregulated the expression of G3BP1. G3BP1 overexpression facilitated the formation of stress granules within cells and hindered viral replication, while G3BP1 knockdown enhanced BPIV3 expression. Conclusion: This study suggest that G3BP1 plays a crucial role in BPIV3 suppressing stress granule formation and viral replication.


Subject(s)
DNA Helicases , Poly-ADP-Ribose Binding Proteins , RNA Helicases , RNA Recognition Motif Proteins , Stress Granules , Virus Replication , Animals , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , RNA Recognition Motif Proteins/metabolism , DNA Helicases/metabolism , DNA Helicases/genetics , RNA Helicases/metabolism , RNA Helicases/genetics , Stress Granules/metabolism , Cattle , Eukaryotic Initiation Factor-2/metabolism , Respirovirus Infections/immunology , Respirovirus Infections/metabolism , Host-Pathogen Interactions/immunology , Phosphorylation , Cell Line , Cytoplasmic Granules/metabolism
3.
Proc Natl Acad Sci U S A ; 121(19): e2317954121, 2024 May 07.
Article in English | MEDLINE | ID: mdl-38683976

ABSTRACT

Several microbial genomes lack textbook-defined essential genes. If an essential gene is absent from a genome, then an evolutionarily independent gene of unknown function complements its function. Here, we identified frequent nonhomologous replacement of an essential component of DNA replication initiation, a replicative helicase loader gene, in Vibrionaceae. Our analysis of Vibrionaceae genomes revealed two genes with unknown function, named vdhL1 and vdhL2, that were substantially enriched in genomes without the known helicase-loader genes. These genes showed no sequence similarities to genes with known function but encoded proteins structurally similar with a viral helicase loader. Analyses of genomic syntenies and coevolution with helicase genes suggested that vdhL1/2 encodes a helicase loader. The in vitro assay showed that Vibrio harveyi VdhL1 and Vibrio ezurae VdhL2 promote the helicase activity of DnaB. Furthermore, molecular phylogenetics suggested that vdhL1/2 were derived from phages and replaced an intrinsic helicase loader gene of Vibrionaceae over 20 times. This high replacement frequency implies the host's advantage in acquiring a viral helicase loader gene.


Subject(s)
DNA Helicases , DNA Replication , Phylogeny , Vibrionaceae , Vibrionaceae/genetics , Vibrionaceae/enzymology , DNA Helicases/metabolism , DNA Helicases/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Viral Proteins/genetics , Viral Proteins/metabolism , Bacteriophages/genetics , Bacteriophages/enzymology , Evolution, Molecular , Genome, Bacterial , DnaB Helicases/metabolism , DnaB Helicases/genetics , Vibrio/genetics , Vibrio/enzymology
4.
Zhonghua Yi Xue Za Zhi ; 104(16): 1410-1417, 2024 Apr 23.
Article in Chinese | MEDLINE | ID: mdl-38644292

ABSTRACT

Objective: To investigate the genetic and expression characteristics of transcription factor IIH (TFIIH) in pre-initiationcomplex in prostate cancer (PCa) and its relationship with prostate cancer progression. Methods: Analyzing the expression characteristics and clinical signification of TFIIH subunits about 495 cases of PCa and 52 cases of adjacent cancer in The Cancer Genome Atlas-Prostate adenocarcinoma (TCGA-PRAD) database. PCa microarray chip was used to verify the correlation between the key factor General Transcription Factor IIH Subunit 4 (GTF2H4) in TFIIH and clinical features. Results: The 495 patients with PCa were (61.01±6.82) years old.The mRNA expression of ERCC3、GTF2H4 and MNAT1 were high in PCa tissues with GS≥8(P<0.05). The expression of GTF2H4 and MNAT1 were relevant to the pathological stages(P<0.05). High expression of GTF2H4 has higher biochemical recurrence (BCR) rate in PCa patients(HR=2.47, 95%CI:1.62-3.77, P<0.001), which has better predictive effect of BCR in PCa patients(The 3rd, 5th, and 7th year AUC all>0.7) than other subunits, and it has been verified in four additional databases. Single-factor Cox regression analysis showed that GTF2H4 were risk factors for BCR (HR=2.470, 95%CI:1.620-3.767, P<0.001) and GTF2H5 were protective factors(HR=0.506,95%CI: 0.336-0.762, P=0.001). The results of immunohistochemical staining showed that the protein expression of GTF2H4 was correlated with the clinical features of PCa patients.The differences of the above results were statistically significant. Conclusion: GTF2H4, the key factor of TFIIH, is highly expressed in PCa and indicates a poor prognosis.


Subject(s)
Computational Biology , Prostatic Neoplasms , Humans , Male , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Prostatic Neoplasms/genetics , Prognosis , Middle Aged , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , DNA Helicases/metabolism , DNA Helicases/genetics , Aged , Transcription Factors, TFII/metabolism , Transcription Factors, TFII/genetics
5.
Mol Cell ; 84(9): 1684-1698.e9, 2024 May 02.
Article in English | MEDLINE | ID: mdl-38593805

ABSTRACT

The Bloom syndrome (BLM) helicase is critical for alternative lengthening of telomeres (ALT), a homology-directed repair (HDR)-mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2, commensurate with the appearance of telomere C-strand-specific single-stranded DNA (ssDNA). DNA2 nuclease deficiency increased 5'-flap formation in a BLM-dependent manner, while telomere C-strand, but not G-strand, nicks promoted ALT. These findings define the seminal events in the ALT DNA damage response, linking aberrant telomeric lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers.


Subject(s)
DNA Damage , DNA Replication , RecQ Helicases , Telomere Homeostasis , Telomere , RecQ Helicases/metabolism , RecQ Helicases/genetics , Humans , Telomere/metabolism , Telomere/genetics , DNA, Single-Stranded/metabolism , DNA, Single-Stranded/genetics , X-linked Nuclear Protein/genetics , X-linked Nuclear Protein/metabolism , DNA Helicases/metabolism , DNA Helicases/genetics , Bloom Syndrome/genetics , Bloom Syndrome/metabolism , Bloom Syndrome/enzymology , Bloom Syndrome/pathology , Cell Line, Tumor
6.
PLoS One ; 19(4): e0298575, 2024.
Article in English | MEDLINE | ID: mdl-38593124

ABSTRACT

Osteoarthritis (OA) is a widespread chronic, progressive, degenerative joint disease that causes pain and disability. Current treatments for OA have limited effectiveness and new biomarkers need to be identified. Bioinformatics analysis was conducted to explore differentially expressed genes and DNA repair/recombination protein 54 L (RAD54L) was selected. We firstly overexpressed RAD54L in interleukin-1ß (IL-1ß)-induced human articular chondrocytes or in OA rats to investigate its effect on OA. Chondrocyte viability and apoptotic rate were measured by Cell Counting Kit-8 and flow cytometry, respectively. Then we evaluated OA severity in vivo by Hematoxylin-eosin staining and Osteoarthritis Research Society International standards. The expression of inflammatory mediators was tested by enzyme-linked immunosorbent assay. Finally, western blot was performed to determine the relative expression level of hypoxia-inducible factors 1α (HIF-1α) and vascular endothelial growth factor (VEGF). Overexpression of RAD54L promoted cell viability and attenuated apoptosis in IL-1ß-induced human chondrocytes. A lower Osteoarthritis Research Society International score and a remarkable alleviation of chondrocyte disordering and infiltration of inflammatory cells were found in cartilage tissues of OA rats after overexpressing RAD54L. The inflammatory response induced by OA was decreased by RAD54L overexpression in vitro and in vivo. In addition, RAD54L overexpression decreased the relative expression level of HIF-1α and VEGF. Overexpression of RAD54L could attenuate OA by suppressing the HIF-1α/VEGF signaling pathway, indicating that RAD54L may be a potential treatment target for OA.


Subject(s)
Cartilage, Articular , Osteoarthritis , Animals , Humans , Rats , Apoptosis , Cartilage, Articular/metabolism , Chondrocytes/metabolism , DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Interleukin-1beta/metabolism , Osteoarthritis/genetics , Osteoarthritis/metabolism , Signal Transduction , Vascular Endothelial Growth Factor A/metabolism
7.
Nat Commun ; 15(1): 3138, 2024 Apr 11.
Article in English | MEDLINE | ID: mdl-38605034

ABSTRACT

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.


Subject(s)
RNA Splice Sites , Retinitis Pigmentosa , Spliceosomes , Humans , Spliceosomes/genetics , Spliceosomes/metabolism , Proteomics , RNA Splicing/genetics , Alternative Splicing/genetics , RNA, Small Nuclear/genetics , RNA, Small Nuclear/metabolism , RNA, Messenger/metabolism , Mutation , DNA Helicases/metabolism , RNA-Binding Proteins/metabolism
8.
Nat Commun ; 15(1): 2857, 2024 Apr 02.
Article in English | MEDLINE | ID: mdl-38565848

ABSTRACT

PARP2 is a DNA-dependent ADP-ribosyl transferase (ARTs) enzyme with Poly(ADP-ribosyl)ation activity that is triggered by DNA breaks. It plays a role in the Base Excision Repair pathway, where it has overlapping functions with PARP1. However, additional roles for PARP2 have emerged in the response of cells to replication stress. In this study, we demonstrate that PARP2 promotes replication stress-induced telomere fragility and prevents telomere loss following chronic induction of oxidative DNA lesions and BLM helicase depletion. Telomere fragility results from the activity of the break-induced replication pathway (BIR). During this process, PARP2 promotes DNA end resection, strand invasion and BIR-dependent mitotic DNA synthesis by orchestrating POLD3 recruitment and activity. Our study has identified a role for PARP2 in the response to replication stress. This finding may lead to the development of therapeutic approaches that target DNA-dependent ART enzymes, particularly in cancer cells with high levels of replication stress.


Subject(s)
DNA Repair , DNA , Poly (ADP-Ribose) Polymerase-1/genetics , Poly (ADP-Ribose) Polymerase-1/metabolism , DNA/metabolism , DNA Damage , DNA Helicases/genetics , DNA Helicases/metabolism , Telomere/genetics , Telomere/metabolism
9.
Bioorg Med Chem Lett ; 106: 129761, 2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38642810

ABSTRACT

Helicase-primase is an interesting target for the therapy of herpes simplex virus (HSV) infections. Since amenamevir is already approved for varicella-zoster virus (VZV) and HSV in Japan and pritelivir has received breakthrough therapy status for the treatment of acyclovir-resistant HSV infections in immunocompromised patients, the target has sparked interest in me-too approaches. Here, we describe the attempt to improve nervous tissue penetration in Phaeno Therapeutics drug candidate HN0037 to target the latent reservoir of HSV by installing less polar moieties, mainly a difluorophenyl instead of a pyridyl group, and replacing the primary sulfonamide with a methyl sulfoximine moiety. However, all obtained stereoisomers exhibited a weaker inhibitory activity on HSV-1 and HSV-2.


Subject(s)
Antiviral Agents , DNA Primase , Sulfonamides , Sulfonamides/chemistry , Sulfonamides/pharmacology , Sulfonamides/chemical synthesis , DNA Primase/antagonists & inhibitors , DNA Primase/metabolism , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/chemical synthesis , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/chemical synthesis , Structure-Activity Relationship , DNA Helicases/antagonists & inhibitors , DNA Helicases/metabolism , Herpesvirus 1, Human/drug effects , Herpesvirus 2, Human/drug effects , Humans , Molecular Structure , Microbial Sensitivity Tests , Dose-Response Relationship, Drug , Imines/chemistry , Imines/pharmacology , Imines/chemical synthesis
10.
Life Sci ; 347: 122605, 2024 Jun 15.
Article in English | MEDLINE | ID: mdl-38642845

ABSTRACT

AIMS: Hepatocellular carcinoma (HCC) is a lead cause of cancer-related deaths. In the present study we investigated the role of Brahma-related gene 1 (BRG1), a chromatin remodeling protein, in HCC the pathogenesis focusing on identifying novel transcription targets. METHODS AND MATERIALS: Hepatocellular carcinogenesis was modeled in mice by diethylnitrosamine (DEN). Cellular transcriptome was evaluated by RNA-seq. RESULTS: Hepatocellular carcinoma was appreciably retarded in BRG1 knockout mice compared to wild type littermates. Transcriptomic analysis identified ATP Binding Cassette Subfamily C Member 3 (ABCC3) as a novel target of BRG1. BRG1 over-expression in BRG1low HCC cells (HEP1) up-regulated whereas BRG1 depletion in BRG1high HCC cells (SNU387) down-regulated ABCC3 expression. Importantly, BRG1 was detected to directly bind to the ABCC3 promoter to activate ABCC3 transcription. BRG1 over-expression in HEP1 cells promoted proliferation and migration, both of which were abrogated by ABCC3 silencing. On the contrary, BRG1 depletion in SNU387 cells decelerated proliferation and migration, both of which were rescued by ABCC3 over-expression. Importantly, high BRG1/ABCC3 expression predicted poor prognosis in HCC patients. Mechanistically, ABCC3 regulated hepatocellular carcinogenesis possibly by influencing lysosomal homeostasis. SIGNIFICANCE: In conclusion, our data suggest that targeting BRG1 and its downstream target ABCC3 can be considered as a reasonable approach for the intervention of hepatocellular carcinoma.


Subject(s)
Carcinogenesis , Carcinoma, Hepatocellular , DNA Helicases , Gene Expression Regulation, Neoplastic , Liver Neoplasms , Mice, Knockout , Multidrug Resistance-Associated Proteins , Nuclear Proteins , Transcription Factors , Animals , DNA Helicases/genetics , DNA Helicases/metabolism , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/metabolism , Mice , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Liver Neoplasms/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Humans , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Carcinogenesis/genetics , Carcinogenesis/metabolism , Cell Proliferation , Male , Cell Line, Tumor , Cell Movement , Diethylnitrosamine/toxicity , Mice, Inbred C57BL
11.
In Vivo ; 38(3): 1260-1265, 2024.
Article in English | MEDLINE | ID: mdl-38688602

ABSTRACT

BACKGROUND/AIM: Endometrial cancer (EC) is the predominant malignancy among gynecologic cancers and ranks fourth among all types of cancer. Recently, researchers have focused on the development of new prognostic biomarkers. Subunits of the SWI/SNF protein complex, like the ARID1 and BRG1, have been associated with the development of endometrial cancer. The present study aimed to evaluate the expression patterns of ARID1A and BRG1 in a collection of endometrioid adenocarcinomas of the uterus using immunohistochemistry. PATIENTS AND METHODS: The study comprised a total of thirty-three individuals diagnosed with stage I endometrioid endometrial cancer, treated with radical hysterectomy. The histological material was then examined to assess the cytoplasmic and nuclear expression of the proteins. RESULTS: ARID1A exhibited expression in both the cytoplasm and nucleus of cancer cells, whereas BRG1 was mainly expressed in the nuclei. In addition, ARID1A exhibited a notable decrease in expression in grade 3 histology, with no significant correlation with the depth of myometrial invasion. The reduced expression was highly related to tumor expansion into the endocervix. The findings demonstrated a total absence of ARID1A expression in 27% of endometrioid carcinomas, with a significant reduction in expression in an additional 51% of cancer cells. These findings align with the most recent published data. In contrast, in the current study, BRG1 was rarely down-regulated and was extensively expressed in the majority of endometrioid carcinomas, preventing the possibility of statistical analysis. CONCLUSION: In summary, ARID1A expression loss can be used as a biomarker to guide post-operative therapy; however, further investigation is needed, especially for early-stage endometrial cancer.


Subject(s)
Biomarkers, Tumor , DNA Helicases , DNA-Binding Proteins , Endometrial Neoplasms , Immunohistochemistry , Nuclear Proteins , Transcription Factors , Humans , Female , Endometrial Neoplasms/pathology , Endometrial Neoplasms/metabolism , Endometrial Neoplasms/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Middle Aged , Aged , Biomarkers, Tumor/metabolism , Neoplasm Staging , Prognosis , Gene Expression Regulation, Neoplastic , Carcinoma, Endometrioid/pathology , Carcinoma, Endometrioid/metabolism , Carcinoma, Endometrioid/genetics , Adult , Neoplasm Grading
12.
J Cell Biol ; 223(7)2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38652118

ABSTRACT

Chromatin-remodeling protein BRG1/SMARCA4 is pivotal for establishing oligodendrocyte (OL) lineage identity. However, its functions for oligodendrocyte-precursor cell (OPC) differentiation within the postnatal brain and during remyelination remain elusive. Here, we demonstrate that Brg1 loss profoundly impairs OPC differentiation in the brain with a comparatively lesser effect in the spinal cord. Moreover, BRG1 is critical for OPC remyelination after injury. Integrative transcriptomic/genomic profiling reveals that BRG1 exhibits a dual role by promoting OPC differentiation networks while repressing OL-inhibitory cues and proneuronal programs. Furthermore, we find that BRG1 interacts with EED/PRC2 polycomb-repressive-complexes to enhance H3K27me3-mediated repression at gene loci associated with OL-differentiation inhibition and neurogenesis. Notably, BRG1 depletion decreases H3K27me3 deposition, leading to the upregulation of BMP/WNT signaling and proneurogenic genes, which suppresses OL programs. Thus, our findings reveal a hitherto unexplored spatiotemporal-specific role of BRG1 for OPC differentiation in the developing CNS and underscore a new insight into BRG1/PRC2-mediated epigenetic regulation that promotes and safeguards OL lineage commitment and differentiation.


Subject(s)
Cell Differentiation , DNA Helicases , Oligodendroglia , Polycomb Repressive Complex 2 , Animals , Mice , DNA Helicases/metabolism , DNA Helicases/genetics , Epigenesis, Genetic , Histones/metabolism , Histones/genetics , Mice, Inbred C57BL , Neurogenesis/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Oligodendrocyte Precursor Cells/metabolism , Oligodendroglia/metabolism , Polycomb Repressive Complex 2/metabolism , Polycomb Repressive Complex 2/genetics , Remyelination , Transcription Factors/metabolism , Transcription Factors/genetics
13.
Expert Opin Ther Pat ; 34(3): 159-169, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38578210

ABSTRACT

INTRODUCTION: The multi-subunit SWI/SNF chromatin remodeling complex is a key epigenetic regulator for many cellular processes, and several subunits are found to be mutated in human cancers. The inactivating mutations of SMARCA4, the ATPase subunit of the complex, result in cellular dependency on the paralog SMARCA2 for survival. This observed synthetic lethal relationship posits targeting SMARCA2 in SMARCA4-deficient settings as an attractive therapeutic target in oncology. AREAS COVERED: This review covers patent literature disclosed during the 2019-30 June 2023 period which claim ATPase inhibitors and PROTAC degraders that bind to the ATPase domain of SMARCA2 and/or SMARCA4. A total of 16 documents from 6 applicants are presented. EXPERT OPINION: The demonstration of cellular dependence on SMARCA2 ATPase activity in SMARCA4-deficient settings has prompted substantial research toward SMARCA2-targeting therapies. Although selectively targeting the ATPase domain of SMARCA2 is viewed as challenging, several ATPase inhibitor scaffolds have been disclosed within the last five years. Most early compounds are weakly selective, but these efforts have culminated in the first dual SMARCA2/SMARCA4 ATPase inhibitor to enter clinical trials. Data from the ongoing clinical trials, as well as continued advancement of SMARCA2-selective ATPase inhibitors, are anticipated to significantly impact the field of therapies, targeting SMARCA4-deficient tumors.


Subject(s)
Antineoplastic Agents , DNA Helicases , Molecular Targeted Therapy , Neoplasms , Nuclear Proteins , Patents as Topic , Transcription Factors , Humans , Transcription Factors/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Neoplasms/drug therapy , Neoplasms/pathology , Neoplasms/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Antineoplastic Agents/pharmacology , DNA Helicases/metabolism , DNA Helicases/antagonists & inhibitors , DNA Helicases/genetics , Animals , Synthetic Lethal Mutations , Mutation , Adenosine Triphosphatases/metabolism
14.
Vet Microbiol ; 293: 110070, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38593624

ABSTRACT

Stress granules (SGs), the main component is GTPase-activating protein-binding protein 1 (G3BP1), which are assembled during viral infection and function to sequester host and viral mRNAs and proteins, are part of the antiviral responses. In this study, we found that porcine deltacoronavirus (PDCoV) infection induced stable formation of robust SGs in cells through a PERK (protein kinase R-like endoplasmic reticulum kinase)-dependent mechanism. Overexpression of SGs marker proteins G3BP1 significantly reduced PDCoV replication in vitro, while inhibition of endogenous G3BP1 enhanced PDCoV replication. Moreover, PDCoV infected LLC-PK1 cells raise the phosphorylation level of G3BP1. By overexpression of the G3BP1 phosphorylated protein or the G3BP1 dephosphorylated protein, we found that phosphorylation of G3BP1 is involved in the regulation of PDCoV-induced inflammatory response. Taken together, our study presents a vital aspect of the host innate response to invading pathogens and reveals attractive host targets for antiviral target.


Subject(s)
DNA Helicases , Inflammation , Poly-ADP-Ribose Binding Proteins , RNA Helicases , RNA Recognition Motif Proteins , Animals , Swine , RNA Recognition Motif Proteins/genetics , RNA Recognition Motif Proteins/metabolism , Phosphorylation , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , RNA Helicases/metabolism , RNA Helicases/genetics , DNA Helicases/metabolism , DNA Helicases/genetics , Virus Replication , Coronavirus/immunology , Coronavirus/physiology , Cell Line , Swine Diseases/virology , Swine Diseases/immunology , Swine Diseases/genetics , Immunity, Innate
15.
Cell Signal ; 119: 111150, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38552892

ABSTRACT

BACKGROUND: Dilated cardiomyopathy (DCM) and coronary heart disease (CHD) stand as two of the foremost causes of mortality. However, the comprehensive comprehension of the regulatory mechanisms governing DCM and CHD remains limited, particularly from the vantage point of single-cell transcriptional analysis. METHOD: We used the GSE121893 dataset from the GEO database, analyzing single-cell expressions with tools like DropletUtils, Seurat, and Monocle. We also utilized the GSVA package for comparing gene roles in DCM and CHD, Finally, we conducted qRT-PCR and Western blot analyses to measure the expression levels of SMARCA4, Col1A1, Col3A1 and α-SMA, and the role of SMARCA4 on fibroblasts were explored by EdU and Transwell assay. RESULTS: Our analysis identified six cell types in heart tissue, with fibroblasts showing the most interaction with other cells. DEGs in fibroblasts were linked to muscle development and morphogenesis. Pseudotime analysis revealed the dynamics of fibroblast changes in both the normal and disease groups and many transcription factors (TFs) potentially involved in this process. Among these TFs, SMARCA4 which was translated into protein BRG1, showed the most significantly difference. In vivo experiments have demonstrated that SMARCA4 indeed promoted fibroblasts proliferation and migration. CONCLUSION: This study provides a clearer understanding of cell-type dynamics in heart diseases, emphasizing the role of fibroblasts and the significance of SMARCA4 in their function. Our results offer insights into the cellular mechanisms underlying DCM and CHD, potentially guiding future therapeutic strategies.


Subject(s)
Cardiomyopathy, Dilated , DNA Helicases , Nuclear Proteins , Single-Cell Analysis , Transcription Factors , Transcription Factors/metabolism , DNA Helicases/metabolism , DNA Helicases/genetics , Humans , Cardiomyopathy, Dilated/genetics , Cardiomyopathy, Dilated/metabolism , Cardiomyopathy, Dilated/pathology , Animals , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Fibroblasts/metabolism , Coronary Disease/metabolism , Coronary Disease/genetics , Coronary Disease/pathology , Mice , Cell Proliferation
16.
Nat Commun ; 15(1): 2163, 2024 Mar 09.
Article in English | MEDLINE | ID: mdl-38461299

ABSTRACT

Recent development of new immune checkpoint inhibitors has been particularly successfully in cancer treatment, but still the majority patients fail to benefit. Converting resistant tumors to immunotherapy sensitive will provide a significant improvement in patient outcome. Here we identify Mi-2ß as a key melanoma-intrinsic effector regulating the adaptive anti-tumor immune response. Studies in genetically engineered mouse melanoma models indicate that loss of Mi-2ß rescues the immune response to immunotherapy in vivo. Mechanistically, ATAC-seq analysis shows that Mi-2ß controls the accessibility of IFN-γ-stimulated genes (ISGs). Mi-2ß binds to EZH2 and promotes K510 methylation of EZH2, subsequently activating the trimethylation of H3K27 to inhibit the transcription of ISGs. Finally, we develop an Mi-2ß-targeted inhibitor, Z36-MP5, which reduces Mi-2ß ATPase activity and reactivates ISG transcription. Consequently, Z36-MP5 induces a response to immune checkpoint inhibitors in otherwise resistant melanoma models. Our work provides a potential therapeutic strategy to convert immunotherapy resistant melanomas to sensitive ones.


Subject(s)
DNA Helicases , Enhancer of Zeste Homolog 2 Protein , Immune Evasion , Melanoma , Animals , Humans , Mice , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Immune Evasion/genetics , Melanoma/drug therapy , Methylation , DNA Helicases/genetics , DNA Helicases/metabolism
17.
Int J Mol Sci ; 25(6)2024 Mar 15.
Article in English | MEDLINE | ID: mdl-38542333

ABSTRACT

DNA Damage Tolerance (DDT) mechanisms allow cells to bypass lesions in the DNA during replication. This allows the cells to progress normally through the cell cycle in the face of abnormalities in their DNA. PCNA, a homotrimeric sliding clamp complex, plays a central role in the coordination of various processes during DNA replication, including the choice of mechanism used during DNA damage bypass. Mono-or poly-ubiquitination of PCNA facilitates an error-prone or an error-free bypass mechanism, respectively. In contrast, SUMOylation recruits the Srs2 helicase, which prevents local homologous recombination. The Elg1 RFC-like complex plays an important role in unloading PCNA from the chromatin. We analyze the interaction of mutations that destabilize PCNA with mutations in the Elg1 clamp unloader and the Srs2 helicase. Our results suggest that, in addition to its role as a coordinator of bypass mechanisms, the very presence of PCNA on the chromatin prevents homologous recombination, even in the absence of the Srs2 helicase. Thus, PCNA unloading seems to be a pre-requisite for recombinational repair.


Subject(s)
Saccharomyces cerevisiae Proteins , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , DNA Damage , DNA Helicases/genetics , DNA Helicases/metabolism , Homologous Recombination , DNA Replication , DNA/genetics , DNA/metabolism , Chromatin/genetics , Chromatin/metabolism , Carrier Proteins/metabolism
18.
Bioorg Med Chem Lett ; 104: 129711, 2024 May 15.
Article in English | MEDLINE | ID: mdl-38521175

ABSTRACT

WRN helicase is a critical protein involved in maintaining genomic stability, utilizing ATP hydrolysis to dissolve DNA secondary structures. It has been identified as a promising synthetic lethal target for microsatellite instable (MSI) cancers. However, few WRN helicase inhibitors have been discovered, and their potential binding sites remain unexplored. In this study, we analyzed potential binding sites for WRN inhibitors and focused on the ATP-binding site for screening new inhibitors. Through molecular dynamics-enhanced virtual screening, we identified two compounds, h6 and h15, which effectively inhibited WRN's helicase and ATPase activity in vitro. Importantly, these compounds selectively targeted WRN's ATPase activity, setting them apart from other non-homologous proteins with ATPase activity. In comparison to the homologous protein BLM, h6 exhibits some degree of selectivity towards WRN. We also investigated the binding mode of these compounds to WRN's ATP-binding sites. These findings offer a promising strategy for discovering new WRN inhibitors and present two novel scaffolds, which might be potential for the development of MSI cancer treatment.


Subject(s)
DNA Helicases , Molecular Dynamics Simulation , Werner Syndrome Helicase/genetics , Werner Syndrome Helicase/metabolism , DNA Helicases/chemistry , DNA Helicases/metabolism , DNA/chemistry , Binding Sites , Adenosine Triphosphate , RecQ Helicases/metabolism
19.
PLoS Biol ; 22(3): e3002552, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38502677

ABSTRACT

Impediments in replication fork progression cause genomic instability, mutagenesis, and severe pathologies. At stalled forks, RPA-coated single-stranded DNA (ssDNA) activates the ATR kinase and directs fork remodeling, 2 key early events of the replication stress response. RFWD3, a recently described Fanconi anemia (FA) ubiquitin ligase, associates with RPA and promotes its ubiquitylation, facilitating late steps of homologous recombination (HR). Intriguingly, RFWD3 also regulates fork progression, restart and stability via poorly understood mechanisms. Here, we used proteomics to identify putative RFWD3 substrates during replication stress in human cells. We show that RFWD3 interacts with and ubiquitylates the SMARCAL1 DNA translocase directly in vitro and following DNA damage in vivo. SMARCAL1 ubiquitylation does not trigger its subsequent proteasomal degradation but instead disengages it from RPA thereby regulating its function at replication forks. Proper regulation of SMARCAL1 by RFWD3 at stalled forks protects them from excessive MUS81-mediated cleavage in response to UV irradiation, thereby limiting DNA replication stress. Collectively, our results identify RFWD3-mediated SMARCAL1 ubiquitylation as a novel mechanism that modulates fork remodeling to avoid genome instability triggered by aberrant fork processing.


Subject(s)
DNA Replication , DNA, Single-Stranded , Humans , DNA, Single-Stranded/genetics , DNA Replication/genetics , Replication Protein A/genetics , Replication Protein A/metabolism , Protein Binding , Ubiquitination , DNA Damage , Genomic Instability , DNA Helicases/genetics , DNA Helicases/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
20.
PLoS One ; 19(3): e0300255, 2024.
Article in English | MEDLINE | ID: mdl-38512854

ABSTRACT

Chromodomain helicase DNA binding domain (CHD) proteins, including CHD7 and CHD8, remodel chromatin to enable transcriptional programs. Both proteins are important for proper neural development as heterozygous mutations in Chd7 and Chd8 are causative for CHARGE syndrome and correlated with autism spectrum disorders, respectively. Their roles in mature neurons are poorly understood despite influencing the expression of genes required for cell adhesion, neurotransmission, and synaptic plasticity. The Drosophila homolog of CHD7 and CHD8, Kismet (Kis), promotes neurotransmission, endocytosis, and larval locomotion. Endocytosis is essential in neurons for replenishing synaptic vesicles, maintaining protein localization, and preserving the size and composition of the presynaptic membrane. Several forms of endocytosis have been identified including clathrin-mediated endocytosis, which is coupled with neural activity and is the most prevalent form of synaptic endocytosis, and activity-dependent bulk endocytosis, which occurs during periods of intense stimulation. Kis modulates the expression of gene products involved in endocytosis including promoting shaggy/GSK3ß expression while restricting PI3K92E. kis mutants electrophysiologically phenocopy a liquid facets mutant in response to paradigms that induce clathrin-mediated endocytosis and activity-dependent bulk endocytosis. Further, kis mutants do not show further reductions in endocytosis when activity-dependent bulk endocytosis or clathrin-mediated endocytosis are pharmacologically inhibited. We find that Kis is important in postsynaptic muscle for proper endocytosis but the ATPase domain of Kis is dispensable for endocytosis. Collectively, our data indicate that Kis promotes both clathrin-mediated endocytosis and activity-dependent bulk endocytosis possibly by promoting transcription of several endocytic genes and maintaining the size of the synaptic vesicle pool.


Subject(s)
Chromatin , Clathrin , Animals , Clathrin/metabolism , Chromatin Assembly and Disassembly , Synaptic Transmission/physiology , Drosophila/metabolism , Endocytosis/genetics , DNA Helicases/genetics , DNA Helicases/metabolism
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