RESUMO
The Structural Maintenance of Chromosomes (SMC) protein complexes are DNA-binding molecular machines required to shape chromosomes into functional units and to safeguard the genome through cell division. These ring-shaped multi-subunit protein complexes, which are present in all kingdoms of life, achieve this by organizing chromosomes in three-dimensional space. Mechanistically, the SMC complexes hydrolyze ATP to either stably entrap DNA molecules within their lumen, or rapidly reel DNA into large loops, which allow them to link two stretches of DNA in cis or trans. In this chapter, the canonical structure of the SMC complexes is first introduced, followed by a description of the composition and general functions of the main types of eukaryotic and prokaryotic SMC complexes. Thereafter, the current model for how SMC complexes perform in vitro DNA loop extrusion is presented. Lastly, chromosome loop formation by SMC complexes is introduced, and how the DNA loop extrusion mechanism contributes to chromosome looping by SMC complexes in cells is discussed.
Assuntos
Cromossomos , Cromossomos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , DNA/química , DNA/metabolismo , DNA/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/química , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/químicaRESUMO
Hi-C reads, which represent ligation events between different regions of the genome, must be processed into matrices of interaction frequencies for downstream analysis. Here, I describe a procedure for mapping Hi-C reads to the genome and conversion of mapped reads into the HOMER tag directory format and interaction matrix format for visualization with Juicebox. The method is demonstrated for the mouse composite X chromosome in which reads from the active and inactive X chromosomes are combined after mock DMSO treatment or targeted degradation of cohesin.
Assuntos
Cromossomo X , Animais , Cromossomo X/genética , Camundongos , Software , Coesinas , Mapeamento Cromossômico/métodos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Biologia Computacional/métodosRESUMO
Cohesin is a protein complex that plays a key role in regulating chromosome structure and gene expression. While next-generation sequencing technologies have provided extensive information on various aspects of cohesin, integrating and exploring the vast datasets associated with cohesin are not straightforward. CohesinDB ( https://cohesindb.iqb.u-tokyo.ac.jp ) offers a web-based interface for browsing, searching, analyzing, visualizing, and downloading comprehensive multiomics cohesin information in human cells. In this protocol, we introduce how to utilize CohesinDB to facilitate research on transcriptional regulation and chromatin organization.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Coesinas , Navegador , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Humanos , Software , Biologia Computacional/métodos , Genômica/métodos , Bases de Dados Genéticas , Cromatina/metabolismo , Cromatina/genética , Internet , MultiômicaRESUMO
DNA meiotic recombinase 1 (disrupted meiotic cDNA, Dmc1) protein is homologous to the Escherichia coli RecA protein, was first identified in Saccharomyces cerevisiae. This gene has been well studied as an essential role in meiosis in many species. However, studies on the dmc1 gene in reptiles are limited. In this study, a cDNA fragment of 1,111 bp was obtained from the gonadal tissues of the Chinese soft-shell turtle via RT-PCR, containing a 60 bp 3' UTR, a 22 bp 5' UTR, and an ORF of 1,029 bp encoding 342 amino acids, named Psdmc1. Multiple sequence alignments showed that the deduced protein has high similarity (>95 %) to tetrapod Dmc1 proteins, while being slightly lower (86-88 %) to fish species.Phylogenetic tree analysis showed that PsDmc1 was clustered with the other turtles' Dmc1 and close to the reptiles', but far away from the teleost's. RT-PCR and RT-qPCR analyses showed that the Psdmc1 gene was specifically expressed in the gonads, and much higher in testis than the ovary, especially highest in one year-old testis. In situ hybridization results showed that the Psdmc1 was mainly expressed in the perinuclear cytoplasm of primary and secondary spermatocytes, weakly in spermatogonia of the testes. These results indicated that dmc1 would be majorly involved in the developing testis, and play an essential role in the germ cells' meiosis. The findings of this study will provide a basis for further investigations on the mechanisms behind the germ cells' development and differentiation in Chinese soft-shell turtles, even in the reptiles.
Assuntos
Gametogênese , Filogenia , Tartarugas , Animais , Feminino , Masculino , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Clonagem Molecular , Gametogênese/genética , Meiose/genética , Ovário/metabolismo , Espermatócitos/metabolismo , Testículo/metabolismo , Tartarugas/genética , Tartarugas/metabolismoRESUMO
Despite novel therapeutic strategies, advanced-stage prostate cancer (PCa) remains highly lethal, pointing out the urgent need for effective therapeutic strategies. While dysregulation of the splicing process is considered a cancer hallmark, the role of certain splicing factors remains unknown in PCa. This study focuses on characterizing the levels and role of SRSF6 in this disease. Comprehensive analyses of SRSF6 alterations (copy number/mRNA/protein) were conducted across eight well-characterized PCa cohorts and the Hi-MYC transgenic model. SRSF6 was up-regulated in PCa samples, correlating with adverse clinical parameters. Functional assays, both in vitro (cell proliferation, migration, colony, and tumorsphere formation) and in vivo (xenograft tumors), demonstrated the impact of SRSF6 modulation on critical cancer hallmarks. Mechanistically, SRSF6 regulates the splicing pattern of the histone-chaperone HIRA, consequently affecting the activity of H3.3 in PCa and breast cancer cell models and disrupting pivotal oncogenic pathways (AR and E2F) in PCa cells. These findings underscore SRSF6 as a promising therapeutic target for PCa/advanced-stage PCa.
Assuntos
Chaperonas de Histonas , Neoplasias da Próstata , Fatores de Processamento de Serina-Arginina , Humanos , Fatores de Processamento de Serina-Arginina/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Animais , Linhagem Celular Tumoral , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/genética , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Regulação Neoplásica da Expressão Gênica , Camundongos , Splicing de RNA , Proliferação de Células , Histonas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , FosfoproteínasRESUMO
Identification of unique essential bacterial genes is important for not only the understanding of their cell biology but also the development of new antimicrobials. Here, we report a previously unrecognized core component of the Acinetobacter baumannii divisome. Our results reveal that the protein, termed Aeg1 interacts with multiple cell division proteins, including FtsN, which is required for components of the divisome to localize to the midcell. We demonstrate that the FtsAE202K and FtsBE65A mutants effectively bypassed the need of Aeg1 by A. baumannii, as did the activation variants FtsWM254I and FtsWS274G. Our results suggest that Aeg1 is a cell division protein that arrives at the division site to initiate cell division by recruiting FtsN, which activates FtsQLB and FtsA to induce the septal peptidoglycan synthase FtsWI. The discovery of the new essential cell division protein has provided a new target for the development of antibacterial agents.
Assuntos
Acinetobacter baumannii , Proteínas de Bactérias , Divisão Celular , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genéticaRESUMO
Severe viral pneumonia can induce rapid expansion of KRT5+ basal-like cells in small airways and alveoli; this forms a scar-like structure that persists in the injured alveoli and impedes normal alveolar epithelium regeneration. In this study, we investigated the mechanism by which viral infection induced this remodeling response. Through comparing different lung-injury models, we demonstrated that infection induced strong IFN-γ signal-stimulated dysplastic KRT5+ cell formation. Inactivation of interferon receptor 1 (Ifngr1) reduced dysplastic cell formation, ameliorated lung fibrosis, and improved lung-function recovery. Mechanistically, IFN-γ regulated dysplastic cell formation via the focal adhesion kinase (FAK)/Yes-associated protein 1 (YAP) pathway. Inhibiting FAK/Src diminished IFN-γ-induced YAP nuclear translocation and dysplastic cell formation. Inhibiting YAP during viral infection prevented dysplastic cell formation, whereas inhibiting YAP in persistent KRT5+ cells led to their conversion into distal club cells. Importantly, human dysplastic cells exhibited elevated FAK and YAP activity, and IFN-γ treatment promoted the transformation of human alveolar progenitor cells into dysplastic cells. These findings uncover the role of infection-induced inflammatory response in alveolar remodeling and may provide potential therapeutic avenues for the treatment of alveolar remodeling in patients with severe viral pneumonia.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Quinase 1 de Adesão Focal , Interferon gama , Alvéolos Pulmonares , Proteínas de Sinalização YAP , Proteínas de Sinalização YAP/metabolismo , Animais , Camundongos , Humanos , Interferon gama/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/virologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Transdução de Sinais , Camundongos Knockout , Inflamação/patologia , Inflamação/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMO
Nucleotide-binding oligomerization domain 2 (NOD2) is an immune sensor crucial for eliciting the innate immune responses. Nevertheless, discrepancies exist regarding the effect of NOD2 on different types of cancer. This study aimed to investigate these function of NOD2 in melanoma and its underlying mechanisms. We have validated the tumor suppressor effect of NOD2 in melanoma. NOD2 inhibited the proliferation of melanoma cells, hindering their migration and invasion while promoting the onset of apoptosis. Our study showed that NOD2 expression is closely related to pyrimidine and folate metabolism. NOD2 inhibits thymidylate synthase (TYMS) expression by promoting K48-type ubiquitination modification of TYMS, thereby decreasing the resistance of melanoma cells to 5-fluorouracil (5-FU) and capecitabine (CAP). TYMS was identified to form a complex with Polo-like Kinase 1 (PLK1) and activate the PLK1 signaling pathway. Furthermore, we revealed that the combination of the PLK1 inhibitor volasertib (BI6727) with 5-FU or CAP had a synergistic effect repressing the proliferation, migration, and autophagy of melanoma cells. Overall, our research highlights the protective role of NOD2 in melanoma and suggests that targeting NOD2 and the TYMS/PLK1 signaling axis is a high-profile therapy that could be a prospect for melanoma treatment.
Assuntos
Proteínas de Ciclo Celular , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Melanoma , Proteína Adaptadora de Sinalização NOD2 , Quinase 1 Polo-Like , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Transdução de Sinais , Timidilato Sintase , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Melanoma/tratamento farmacológico , Melanoma/patologia , Melanoma/metabolismo , Melanoma/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linhagem Celular Tumoral , Timidilato Sintase/metabolismo , Timidilato Sintase/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteína Adaptadora de Sinalização NOD2/genética , Proliferação de Células/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Fluoruracila/farmacologia , Apoptose/efeitos dos fármacos , Pteridinas/farmacologia , Animais , Camundongos , Ubiquitinação/efeitos dos fármacos , Autofagia/efeitos dos fármacosRESUMO
BACKGROUND: iPSC reprogramming technology exhibits significant promise in the realms of clinical therapeutics, disease modeling, pharmaceutical drug discovery, and various other applications. However, the extensive utilization of this technology has encountered impediments in the form of inefficiency, prolonged procedures, and ambiguous biological processes. Consequently, in order to improve this technology, it is of great significance to delve into the underlying mechanisms involved in iPSC reprogramming. The BET protein BRD4 plays a crucial role in the late stage of reprogramming; however, its precise function in the early stage remains unclear. RESULTS: Our study aims to investigate BRD4's role in the early stages of iPSC reprogramming. Our investigation reveals that early inhibition of BRD4 substantially enhances iPSC reprogramming, whereas its implementation during the middle-late stage impedes the process. During the reprogramming, ribosome DNA expression initially increases before decreasing and then gradually recovers. Early inhibition of BRD4 improved the decline and restoration of rDNA expression in the early and middle-late stages, respectively. Additionally, we uncovered the mechanism of BRD4's regulation of rDNA transcription throughout reprogramming. Specifically, BRD4 interacts with UBF and co-localizes to both the rDNA promoter and enhancer regions. Ultimately, BRD4 facilitates rDNA transcription by promoting the enrichment of histone H3 lysine 27 acetylation in the surrounding chromatin. Moreover, we also discovered that early inhibition of BRD4 facilitates cells' transition out of the somatic cell state and activate pluripotent genes. CONCLUSIONS: In conclusion, our results demonstrate that early inhibition of BRD4 promotes sequential dynamic expression of rDNA, which improves iPSC reprogramming efficiency.
Assuntos
Reprogramação Celular , DNA Ribossômico , Células-Tronco Pluripotentes Induzidas , Fatores de Transcrição , Células-Tronco Pluripotentes Induzidas/metabolismo , Reprogramação Celular/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , DNA Ribossômico/genética , Animais , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Camundongos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas que Contêm BromodomínioRESUMO
Microtubule-based vesicle trafficking usually relies upon kinesin and dynein motors and few reports describe microtubule polymerisation driving directional vesicle trafficking. Here we show that Arabidopsis END BINDING1b (EB1b), a microtubule plus-end binding protein, directly interacts with SYP121, a SNARE protein that mediates the trafficking of the K+ channel KAT1 and its distribution to the plasma membrane (PM) in Arabidopsis guard cells. Knockout of AtEB1b and its homologous proteins results in a modest but significant change in the distribution of KAT1 and SYP121 in guard cells and consequently delays light-induced stomatal opening. Live-cell imaging reveals that a portion of SYP121-associated endomembrane compartments co-localise with AtEB1b at the growing ends of microtubules, trafficking along with the growth of microtubules for targeting to the PM. Our study reveals a mechanism of vesicle trafficking driven by microtubule growth, which is involved in the redistribution of PM proteins to modulate guard cell movement.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Membrana Celular , Proteínas Associadas aos Microtúbulos , Microtúbulos , Estômatos de Plantas , Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Microtúbulos/metabolismo , Estômatos de Plantas/metabolismo , Estômatos de Plantas/fisiologia , Membrana Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Transporte Proteico , Katanina/metabolismo , Katanina/genética , Movimento Celular , Proteínas de Ciclo CelularRESUMO
Bruton tyrosine kinase inhibitor (BTKi)â¯combined with rituximab-based chemotherapy benefits diffuse large B-cell lymphoma (DLBCL) patients. However, drug resistance is the major cause of relapse and death of DLBCL. In this study, we conducted a comprehensive analysis BTKi-resistance related genes (BRRGs) and established a 10-gene (CARD16, TRIP13, PSRC1, CASP1, PLBD1, CARD6, CAPG, CACNA1A, CDH15, and NDUFA4) signature for early identifying high-risk DLBCL patients. The resistance scores based on the BRRGs signature were associated with prognosis. Furthermore, we developed a nomogram incorporating the BRRGs signature, which demonstrated excellent performance in predicting the prognosis of DLBCL patients. Notably, tumor immune microenvironment, biological pathways, and chemotherapy sensitivity were different between high- and low-resistance score groups. Additionally, we identified TRIP13 as a key gene in our model. TRIP13 was found to be overexpressed in DLBCL and BTKi-resistant DLBCL cell lines, knocking down TRIP13 suppresses cell proliferation, promotes cell apoptosis, and enhances the apoptosis effect of BTKi on DLBCL cells by regulating the Wnt/ß-catenin pathway. In conclusion, our study presents a novel BRRGs signature that could serve as a promising prognostic marker in DLBCL, and TRIP13 might be a potential therapeutic target for resistant DLBCL.
Assuntos
Tirosina Quinase da Agamaglobulinemia , Resistencia a Medicamentos Antineoplásicos , Linfoma Difuso de Grandes Células B , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/genética , Tirosina Quinase da Agamaglobulinemia/metabolismo , Prognóstico , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/efeitos dos fármacos , Feminino , Masculino , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ATPases Associadas a Diversas Atividades CelularesRESUMO
Small molecule inhibitors targeting the bromodomain and extra-terminal domain (BET) family proteins have emerged as a promising class of anti-cancer drugs. Nevertheless, the clinical advancement of these agents has been significantly hampered by challenges related to their potency, oral bioavailability, or toxicity. In this study, virtual screening approaches were employed to discover novel inhibitors of the bromodomain-containing protein 4 (BRD4) by analyzing their comparable chemical structural features to established BRD4 inhibitors. Several of these compounds exhibited inhibitory effects on BRD4 activity ranging from 60 % to 70 % at 100 µM concentrations, while one compound also exhibited an 84 % inhibition of Sirtuin 2 (SIRT2) activity. Furthermore, a subset of structurally diverse compounds from the BRD4 inhibitors was selected to investigate their anti-cancer properties in both 2D and 3D cell cultures. These compounds exhibited varying effects on cell numbers depending on the specific cell line, and some of them induced cell cycle arrest in the G0/G1 phase in breast cancer (MDA-MB-231) cells. Moreover, all the compounds studied reduced the sizes of spheroids, and the most potent compound exhibited a 90 % decrease in growth at a concentration of 10 µM in T47D cells. These compounds hold potential as epigenetic regulators for future studies.
Assuntos
Antineoplásicos , Neoplasias da Mama , Fatores de Transcrição , Feminino , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Proteínas que Contêm Bromodomínio , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Estrutura Molecular , Domínios Proteicos/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Flavonoides/química , Flavonoides/farmacologiaRESUMO
Chromatin remodelling complexes (CRC) are ATP-dependent molecular machines important for the dynamic organization of nucleosomes along eukaryotic DNA. CRCs SWI/SNF, RSC and INO80 can move positioned nucleosomes in promoter DNA, leading to nucleosome-depleted regions which facilitate access of general transcription factors. This function is strongly supported by transcriptional activators being able to interact with subunits of various CRCs. In this work we show that SWI/SNF subunits Swi1, Swi2, Snf5 and Snf6 can bind to activation domains of Ino2 required for expression of phospholipid biosynthetic genes in yeast. We identify an activator binding domain (ABD) of ATPase Swi2 and show that this ABD is functionally dispensable, presumably because ABDs of other SWI/SNF subunits can compensate for the loss. In contrast, mutational characterization of the ABD of the Swi2-related ATPase Sth1 revealed that some conserved basic and hydrophobic amino acids within this domain are essential for the function of Sth1. While ABDs of Swi2 and Sth1 define separate functional protein domains, mapping of an ABD within ATPase Ino80 showed co-localization with its HSA domain also required for binding actin-related proteins. Comparative interaction studies finally demonstrated that several unrelated activators each exhibit a specific binding pattern with ABDs of Swi2, Sth1 and Ino80.
Assuntos
Adenosina Trifosfatases , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA , Ligação Proteica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fatores de Transcrição , Ativação Transcricional , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação Fúngica da Expressão Gênica , Domínios Proteicos , Proteínas Nucleares , Proteínas de Ciclo Celular , Fatores de Transcrição Hélice-Alça-Hélice BásicosRESUMO
Beyond its essential roles in ensuring faithful chromosome segregation and genomic stability, the human Smc5/6 complex acts as an antiviral factor. It binds to and impedes the transcription of extrachromosomal DNA templates; an ability which is lost upon integration of the DNA into the chromosome. How the complex distinguishes among different DNA templates is unknown. Here we show that, in human cells, Smc5/6 preferentially binds to circular rather than linear extrachromosomal DNA. We further demonstrate that the transcriptional process, per se, and particularly the accumulation of DNA secondary structures known to be substrates for topoisomerases, is responsible for Smc5/6 recruitment. More specifically, we find that in vivo Smc5/6 binds to positively supercoiled DNA. Those findings, in conjunction with our genome-wide Smc5/6 binding analysis showing that Smc5/6 localizes at few but highly transcribed chromosome loci, not only unveil a previously unforeseen role of Smc5/6 in DNA topology management during transcription but highlight the significance of sensing DNA topology as an antiviral defense mechanism.
Assuntos
Proteínas de Ciclo Celular , DNA Super-Helicoidal , Transcrição Gênica , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , DNA Super-Helicoidal/metabolismo , DNA Super-Helicoidal/genética , Ligação Proteica , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , DNA/metabolismo , DNA/genética , Conformação de Ácido Nucleico , DNA Circular/metabolismo , DNA Circular/genéticaRESUMO
Recent advancements have elucidated the multifaceted roles of the Schlafen (SLFN) family, including SLFN5, SLFN11, SLFN12, SLFN13, and SLFN14, which are implicated in immunological responses. However, little is known about the roles of this gene family in relation to malignancy development. The current study aimed to explore the diagnostic and prognostic potential of Schlafen family genes in colorectal adenocarcinoma (COAD) through bioinformatics analysis. Leveraging advanced bioinformatics tools of bulk RNA-sequencing and single-cell sequencing, we conducted in-depth analyses of gene expressions, functional enrichment, and survival patterns of patients with colorectal cancer compared to normal tissue. Among Schlafen family genes, the transcription levels of SLFN5 in COAD tissues were significantly elevated and correlated with poor survival outcomes. Furthermore, SLFN5 regulated the immune response via Janus kinase (JAK)/signal transduction and activator of transcription (STAT)/interferon (IFN)-alpha/beta signaling. These chemokines in inflammation are associated with diabetes and metabolism, suggesting their involvement in altered cellular energetics for COAD progress. In addition, an immune cell deconvolution analysis indicated a correlation between SLFN5 expression and immune-related cell populations, such as regulatory T cells (Tregs). These findings highlighted the potential clinical significance of SLFN5 in COAD and provided insights into its involvement in the tumor microenvironment and immune regulation. Meanwhile, the drug discovery data of SFLN5 with potential targeted small molecules suggested its therapeutic potential for COAD. Collectively, the current research demonstrated that SFLN5 play crucial roles in tumor development and serve as a prospective biomarker for COAD.
Assuntos
Neoplasias Colorretais , Regulação Neoplásica da Expressão Gênica , Análise de Célula Única , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Análise de Célula Única/métodos , Prognóstico , Biomarcadores Tumorais/genética , Biologia Computacional/métodos , Análise de Sequência de RNA , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Adenocarcinoma/mortalidade , Perfilação da Expressão Gênica , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Proteínas de Ciclo CelularRESUMO
Excessive proinflammatory cytokine release induced by pyroptosis plays a vital role in intestinal mucosal inflammation in ulcerative colitis (UC). Several pyroptosis-related factors are regulated by the centrosome. Pericentriolar material 1 (PCM1) is a primary component of centriolar satellites that is present as cytoplasmic granules around the centrosome. Our previous study revealed that PCM1 was highly expressed in UC patients, but the role of PCM1 in UC remains unknown. This study aimed to elucidate the role of PCM1 in the development of UC, especially the mechanism in pyroptosis process of UC. Clinical mucosal sample and dextran sulfate sodium (DSS)-induced colitis mouse were used to reveal the association between PCM1 and intestinal inflammation. Intestinal epithelial cell-specific PCM1-knockout mice were constructed to determine the role of PCM1 in colitis. Finally, PCM1 RNA interference and overexpression assays in THP1 cells were employed to study the molecular mechanisms of PCM1 in inflammatory responses and pyroptosis. We found that PCM1 expression was upregulated in the colonic mucosa of UC patients and positively correlated with inflammatory indicators. PCM1 expression was elevated in DSS-induced colitis mice and was reduced after methylprednisolone treatment. In the DSS colitis model, intestinal-specific PCM1-knockout mice exhibited milder intestinal inflammation and lower pyroptosis levels than wild-type mice. In cell level, PCM1 exerted a proinflammatory effect by activating the NLRP3 inflammasome and triggering subsequent gasdermin D-mediated pyroptosis to release IL-1ß and IL-18. In conclusion, PCM1 mediates activation of the NLRP3 inflammasome and gasdermin D-dependent pyroptosis, ultimately accelerating intestinal inflammation in UC. These findings revealed a previously unknown role of PCM1 in initiating intestinal mucosal inflammation and pyroptosis in UC, and this factor is expected to be a regulator in the complex inflammatory network of UC.
Assuntos
Colite Ulcerativa , Peptídeos e Proteínas de Sinalização Intracelular , Macrófagos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas de Ligação a Fosfato , Piroptose , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Piroptose/fisiologia , Colite Ulcerativa/metabolismo , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/patologia , Camundongos , Humanos , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Ligação a Fosfato/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Inflamação/metabolismo , Inflamação/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Feminino , Sulfato de Dextrana/toxicidade , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , GasderminasRESUMO
Faithful chromosome segregation requires biorientation, where the pair of kinetochores on the chromosome establish bipolar microtubule attachment. The integrity of the kinetochore, a macromolecular complex built on centromeric DNA, is required for biorientation, but components sufficient for biorientation remain unknown. Here, we show that tethering the outer kinetochore heterodimer NDC80-NUF2 to the surface of apolar microbeads establishes their biorientation-like state in mouse cells. NDC80-NUF2 microbeads align at the spindle equator and self-correct alignment errors. The alignment is associated with stable bipolar microtubule attachment and is independent of the outer kinetochore proteins SPC24-SPC25, KNL1, the Mis12 complex, inner kinetochore proteins, and Aurora. Larger microbeads align more rapidly, suggesting a size-dependent biorientation mechanism. This study demonstrates a biohybrid kinetochore design for synthetic biorientation of microscale particles in cells.
Assuntos
Proteínas de Ciclo Celular , Segregação de Cromossomos , Cinetocoros , Microesferas , Proteínas Associadas aos Microtúbulos , Microtúbulos , Fuso Acromático , Animais , Camundongos , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/genética , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Fuso Acromático/metabolismoRESUMO
Progressive supranuclear palsy (PSP) is an incurable neurodegenerative disease characterized by 4-repeat (0N/4R)-Tau protein accumulation in CNS neurons. We generated transgenic zebrafish expressing human 0N/4R-Tau to investigate PSP pathophysiology. Tau zebrafish replicated multiple features of PSP, including: decreased survival; hypokinesia; impaired optokinetic responses; neurodegeneration; neuroinflammation; synapse loss; and Tau hyperphosphorylation, misfolding, mislocalization, insolubility, truncation, and oligomerization. Using automated assays, we screened 147 small molecules for activity in rescuing neurological deficits in Tau zebrafish. (+)JQ1, a bromodomain inhibitor, improved hypokinesia, survival, microgliosis, and brain synapse elimination. A heterozygous brd4+/- mutant reducing expression of the bromodomain protein Brd4 similarly rescued these phenotypes. Microglial phagocytosis of synaptic material was decreased by (+)JQ1 in both Tau zebrafish and rat primary cortical cultures. Microglia in human PSP brains expressed Brd4. Our findings implicate Brd4 as a regulator of microglial synaptic elimination in tauopathy and provide an unbiased approach for identifying mechanisms and therapeutic targets in PSP.
Assuntos
Animais Geneticamente Modificados , Modelos Animais de Doenças , Microglia , Paralisia Supranuclear Progressiva , Sinapses , Fatores de Transcrição , Peixe-Zebra , Proteínas tau , Animais , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas tau/metabolismo , Proteínas tau/genética , Microglia/metabolismo , Microglia/patologia , Sinapses/metabolismo , Paralisia Supranuclear Progressiva/metabolismo , Paralisia Supranuclear Progressiva/genética , Paralisia Supranuclear Progressiva/patologia , Azepinas/farmacologia , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Triazóis/farmacologia , Ratos , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Encéfalo/metabolismo , Encéfalo/patologia , Fagocitose , Neurônios/metabolismo , Proteínas que Contêm Bromodomínio , Proteínas de Ciclo CelularRESUMO
The complexity of branching and curvilinear morphology of a complete mitochondrial network within each cell is challenging to analyze and quantify. To address this challenge, we developed an image analysis technique using persistent homology with a multiparameter filtration framework, combining image processing techniques in mathematical morphology. We show that such filtrations contain both topological and geometric information about complex cellular organelle structures, which allows a software program to extract meaningful features. Using this information, we also develop a connectivity index that describes the morphology of the branching patterns. As proof of concept, we utilize this approach to study how mitochondrial networks are altered by genetic changes in the Optineurin gene. Mutations in the autophagy gene Optineurin (OPTN) are associated with primary open-angle glaucoma (POAG), amyotrophic lateral sclerosis (ALS), and Paget's disease of the bone, but the pathophysiological mechanism is unclear. We utilized the proposed mathematical morphology-based multiparameter filtration and persistent homology approach to analyze and quantitatively compare how changes in the OPTN gene alter mitochondrial structures from their normal interconnected, tubular morphology into scattered, fragmented pieces.
Assuntos
Processamento de Imagem Assistida por Computador , Proteínas de Membrana Transportadoras , Mitocôndrias , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fator de Transcrição TFIIIA/genética , Fator de Transcrição TFIIIA/metabolismo , Mutação , SoftwareRESUMO
The lymphatic system consists of a vessel network lined by specialized lymphatic endothelial cells (LECs) that are responsible for tissue fluid homeostasis and immune cell trafficking. The mechanisms for organ-specific LEC responses to environmental cues are not well understood. We found robust lymphangiogenesis during influenza A virus infection in the adult mouse lung. We show that the number of LECs increases twofold at 7 days post-influenza infection (dpi) and threefold at 21 dpi, and that lymphangiogenesis is preceded by lymphatic dilation. We also show that the expanded lymphatic network enhances fluid drainage to mediastinal lymph nodes. Using EdU labeling, we found that a significantly higher number of pulmonary LECs are proliferating at 7 dpi compared to LECs in homeostatic conditions. Lineage tracing during influenza indicates that new pulmonary LECs are derived from preexisting LECs rather than non-LEC progenitors. Lastly, using a conditional LEC-specific YAP/TAZ knockout model, we established that lymphangiogenesis, fluid transport and the immune response to influenza are independent of YAP/TAZ activity in LECs. These findings were unexpected, as they indicate that YAP/TAZ signaling is not crucial for these processes.