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
It has been discovered that Trichorhinophalangeal Syndrome-1 (TRPS1), a novel member of the GATA transcription factor family, participates in both normal physiological processes and the development of numerous diseases. Recently, TRPS1 has been identified as a new biomarker to aid in cancer diagnosis and is very common in breast cancer (BC), especially in triple-negative breast cancer (TNBC). In this review, we discussed the structure and function of TRPS1 in various normal cells, focused on its role in tumorigenesis and tumor development, and summarize the research status of TRPS1 in the occurrence and development of BC. We also analyzed the potential use of TRPS1 in guiding clinically personalized precision treatment and the development of targeted drugs.
Assuntos
Biomarcadores Tumorais , Neoplasias da Mama , Proteínas de Ligação a DNA , Proteínas Repressoras , Fatores de Transcrição , Humanos , Feminino , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Regulação Neoplásica da Expressão Gênica , Carcinogênese/genética , Carcinogênese/metabolismo , AnimaisRESUMO
To function effectively as an integrated system, the transcriptional and post-transcriptional machineries must communicate through mechanisms that are still poorly understood. Here, we focus on the zinc-finger Sfp1, known to regulate transcription of proliferation-related genes. We show that Sfp1 can regulate transcription either by binding to promoters, like most known transcription activators, or by binding to the transcribed regions (gene bodies), probably via RNA polymerase II (Pol II). We further studied the first mode of Sfp1 activity and found that, following promoter binding, Sfp1 binds to gene bodies and affects Pol II configuration, manifested by dissociation or conformational change of its Rpb4 subunit and increased backtracking. Surprisingly, Sfp1 binds to a subset of mRNAs co-transcriptionally and stabilizes them. The interaction between Sfp1 and its client mRNAs is controlled by their respective promoters and coincides with Sfp1's dissociation from chromatin. Intriguingly, Sfp1 dissociation from the chromatin correlates with the extent of the backtracked Pol II. We propose that, following promoter recruitment, Sfp1 accompanies Pol II and regulates backtracking. The backtracked Pol II is more compatible with Sfp1's relocation to the nascent transcripts, whereupon Sfp1 accompanies these mRNAs to the cytoplasm and regulates their stability. Thus, Sfp1's co-transcriptional binding imprints the mRNA fate, serving as a paradigm for the cross-talk between the synthesis and decay of specific mRNAs, and a paradigm for the dual-role of some zinc-finger proteins. The interplay between Sfp1's two modes of transcription regulation remains to be examined.
The ability to fine-tune the production of proteins in a cell is essential for organisms to exist. An imbalance in protein levels can be the cause of various diseases. Messenger RNA molecules (mRNA) link the genetic information encoded in DNA and the produced proteins. Exactly how much protein is made mostly depends on the amount of mRNA in the cell's cytoplasm. This is controlled by two processes: the synthesis of mRNA (also known as transcription) and mRNA being actively degraded. Although much is known about mechanisms regulating transcription and degradation, how cells detect if they need to degrade mRNA based on the levels of its synthesis and vice versa is poorly understood. In 2013, researchers found that proteins known as 'RNA decay factors' responsible for mRNA degradation are actively moved from the cell's cytoplasm into its nucleus to instruct the transcription machinery to produce more mRNA. Kelbert, Jordán-Pla, de-Miguel-Jiménez et al. including some of the researchers involved in the 2013 work investigated how mRNA synthesis and degradation are coordinated to ensure a proper mRNA level. The researchers used advanced genome engineering methods to carefully manipulate and measure mRNA production and degradation in yeast cells. The experiments revealed that the protein Sfp1 a well-characterized transcription factor for stimulating the synthesis of a specific class of mRNAs inside the nucleus can also prevent the degradation of these mRNAs outside the nucleus. During transcription, Sfp1 bound directly to mRNA. The investigators could manipulate the co-transcriptional binding of Sfp1 to a certain mRNA, thereby changing the mRNA stability in the cytoplasm. This suggests that the ability of Sfp1 to regulate both the production and decay of mRNA is dependent on one another and that transcription can influence the fate of its transcripts. This combined activity can rapidly change mRNA levels in response to changes in the cell's environment. RNA plays a key role in ensuring correct levels of proteins. It can also function as an RNA molecule, independently of its coding capacity. Many cancers and developmental disorders are known to be caused by faulty interactions between transcription factors and nucleic acids. The finding that some transcription factors can directly regulate both mRNA synthesis and its destruction introduces new angles for studying and understanding these diseases.
Assuntos
RNA Polimerase II , RNA Mensageiro , Fatores de Transcrição , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Estabilidade de RNA , Regiões Promotoras Genéticas , Ligação Proteica , Dedos de Zinco , Transcrição Gênica , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Citoplasma/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiaeRESUMO
EVI1 is a zinc finger transcription factor encoded by the MECOM locus and is essential for the development and maintenance of hematopoietic stem cells. However, overexpression of EVI1 in various myeloid malignancies is associated with aggressive clinical behavior and poor outcome. The locus encodes multiple isoforms that are differentially acting and independently regulated. EVI1 interacts with a variety of transcription and epigenetic factors via different domains. It also regulates cell survival, differentiation, and proliferation through a variety of mechanisms, including transcriptional activation and repression, regulation of other transcription factors' activity, and chromatin remodeling. While the mechanism by which 3q26 translocation leads to high EVI1 expression through enhancer hijacking of genes active in myeloid development is now better understood, regulation of EVI1 expression in the absence of chromosomal translocations and in normal hematopoiesis remains unclear. Recent studies have provided insight into the regulatory mechanisms of EVI1 expression and action, which may lead to development of targeted therapies in the near future.
Assuntos
Neoplasias Hematológicas , Hematopoese , Proteína do Locus do Complexo MDS1 e EVI1 , Proteína do Locus do Complexo MDS1 e EVI1/genética , Proteína do Locus do Complexo MDS1 e EVI1/metabolismo , Humanos , Hematopoese/genética , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Animais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proto-Oncogenes/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão GênicaRESUMO
Studies of proteins, found in one of the most stress-resistant animals tardigrade Ramazzottius varieornatus, aim to reveal molecular principles of extreme tolerance to various types of stress and developing applications based on them for medicine, biotechnology, pharmacy, and space research. Tardigrade DNA/RNA-binding damage suppressor protein (Dsup) reduces DNA damage caused by reactive oxygen spices (ROS) produced upon irradiation and oxidative stresses in Dsup-expressing transgenic organisms. This work is focused on the determination of structural features of Dsup protein and Dsup-DNA complex, which refines details of protective mechanism. For the first time, intrinsically disordered nature of Dsup protein with highly flexible structure was experimentally proven and characterized by the combination of small angle X-ray scattering (SAXS) technique, circular dichroism spectroscopy, and computational methods. Low resolution models of Dsup protein and an ensemble of conformations were presented. In addition, we have shown that Dsup forms fuzzy complex with DNA.
Assuntos
DNA , Proteínas Intrinsicamente Desordenadas , Tardígrados , Animais , Tardígrados/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , DNA/metabolismo , DNA/química , Dano ao DNA , Espalhamento a Baixo Ângulo , Dicroísmo Circular , Modelos Moleculares , Ligação Proteica , Difração de Raios X , Conformação Proteica , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genéticaRESUMO
Rho guanine nucleotide exchange factor (RGNEF) is a guanine nucleotide exchange factor (GEF) mainly involved in regulating the activity of Rho-family GTPases. It is a bi-functional protein, acting both as a guanine exchange factor and as an RNA-binding protein. RGNEF is known to act as a destabilizing factor of neurofilament light chain RNA (NEFL) and it could potentially contribute to their sequestration in nuclear cytoplasmic inclusions. Most importantly, RGNEF inclusions in the spinal motor neurons of ALS patients have been shown to co-localize with inclusions of TDP-43, the major well-known RNA-binding protein aggregating in the brain and spinal cord of human patients. Therefore, it can be hypothesized that loss-of-function of both proteins following aggregation may contribute to motor neuron death/survival in ALS patients. To further characterize their relationship, we have compared the transcriptomic profiles of neuronal cells depleted of TDP-43 and RGNEF and show that these two factors predominantly act in an antagonistic manner when regulating the expression of axon guidance genes. From a mechanistic point of view, our experiments show that the effect of these genes on the processivity of long introns can explain their mode of action. Taken together, our results show that loss-of-function of factors co-aggregating with TDP-43 can potentially affect the expression of commonly regulated neuronal genes in a very significant manner, potentially acting as disease modifiers. This finding further highlights that neurodegenerative processes at the RNA level are the result of combinatorial interactions between different RNA-binding factors that can be co-aggregated in neuronal cells. A deeper understanding of these complex scenarios may lead to a better understanding of pathogenic mechanisms occurring in patients, where more than one specific protein may be aggregating in their neurons.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Íntrons , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Animais , Orientação de Axônios/genética , Neurônios Motores/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Regulação da Expressão GênicaRESUMO
Traumatic brain injury (TBI) is an acquired insult to the brain caused by an external mechanical force, potentially resulting in temporary or permanent impairment. Microglia, the resident immune cells of the central nervous system, are activated in response to TBI, participating in tissue repair process. However, the underlying epigenetic mechanisms in microglia during TBI remain poorly understood. ARID1A (AT-Rich Interaction Domain 1 A), a pivotal subunit of the multi-protein SWI/SNF chromatin remodeling complex, has received little attention in microglia, especially in the context of brain injury. In this study, we generated a Arid1a cKO mouse line to investigate the potential roles of ARID1A in microglia in response to TBI. We found that glial scar formation was exacerbated due to increased microglial migration and a heightened inflammatory response in Arid1a cKO mice following TBI. Mechanistically, loss of ARID1A led to an up-regulation of the chemokine CCL5 in microglia upon the injury, while the CCL5-neutralizing antibody reduced migration and inflammatory response of LPS-stimulated Arid1a cKO microglia. Importantly, administration of auraptene (AUR), an inhibitor of CCL5, repressed the microglial migration and inflammatory response, as well as the glial scar formation after TBI. These findings suggest that ARID1A is critical for microglial response to injury and that AUR has a therapeutic potential for the treatment of TBI.
Assuntos
Lesões Encefálicas Traumáticas , Quimiocina CCL5 , Proteínas de Ligação a DNA , Camundongos Knockout , Microglia , Fatores de Transcrição , Animais , Lesões Encefálicas Traumáticas/patologia , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/genética , Microglia/metabolismo , Microglia/patologia , Quimiocina CCL5/metabolismo , Quimiocina CCL5/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Movimento Celular , Cicatriz/patologia , Cicatriz/metabolismo , Camundongos Endogâmicos C57BL , MasculinoRESUMO
BACKGROUND: Cholangiocarcinoma (CCA) is a highly malignant, rapidly progressing tumor of the bile duct. Owing to its chemoresistance, it always has an extremely poor prognosis. Therefore, detailed elucidation of the mechanisms of chemoresistance and identification of therapeutic targets are still needed. METHODS: We analyzed the expression of MBD2 (Methyl-CpG-binding domain 2) in CCA and normal bile duct tissues using the public database and immunohistochemistry (IHC). The roles of MBD2 in CCA cell proliferation, migration, and chemoresistance ability were validated through CCK-8, plate cloning assay, wound healing assays and xenograft mouse model. In addition, we constructed a primary CCA mouse model to further confirm the effect of MBD2. RNA-seq and co-IP-MS were used to identify the mechanisms by how MBD2 leads to chemoresistance. RESULTS: MBD2 was upregulated in CCA. It promoted the proliferation, migration and chemoresistance of CCA cells. Mechanistically, MBD2 directly interacted with WDR5, bound to the promoter of ABCB1, promoted the trimethylation of H3K4 in this region through KMT2A, and activated the expression of ABCB1. Knocking down WDR5 or KMT2A blocked the transcriptional activation of ABCB1 by MBD2. The molecular inhibitor MM-102 targeted the interaction of WDR5 with KMT2A. MM-102 inhibited the expression of ABCB1 in CCA cells and decreased the chemoresistance of CCA to cisplatin. CONCLUSION: MBD2 promotes the progression and chemoresistance of CCA through interactions with WDR5. MM-102 can effectively block this process and increase the sensitivity of CCA to cisplatin.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Proteínas de Ligação a DNA , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Peptídeos e Proteínas de Sinalização Intracelular , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/patologia , Humanos , Animais , Camundongos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/tratamento farmacológico , Proliferação de Células , Linhagem Celular Tumoral , Masculino , Feminino , Regulação Neoplásica da Expressão Gênica , Ensaios Antitumorais Modelo de Xenoenxerto , Movimento Celular , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genéticaRESUMO
Obesity is increasing globally and is closely associated with a range of metabolic disorders, including metabolic associated fatty liver disease, diabetes, and cardiovascular diseases. An effective strategy to combat obesity involves stimulating brown and beige adipocyte thermogenesis, which significantly enhances energy expenditure. Recent research has underscored the vital role of PRDM16 in the development and functionality of thermogenic adipocytes. Consequently, PRDM16 has been identified as a potential therapeutic target for obesity and its related metabolic disorders. This review comprehensively examines various studies that focus on combating obesity by directly targeting PRDM16 in adipose tissue.
Assuntos
Tecido Adiposo , Proteínas de Ligação a DNA , Doenças Metabólicas , Obesidade , Termogênese , Fatores de Transcrição , Humanos , Obesidade/metabolismo , Animais , Doenças Metabólicas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Tecido Adiposo/metabolismo , Metabolismo Energético , Tecido Adiposo Marrom/metabolismoRESUMO
Clonal hematopoiesis of indeterminate potential (CHIP) has emerged as a previously unrecognized, potent, age-related, and common risk factor for atherosclerosis. Somatic mutations in certain known leukemia driver genes give rise to clones of mutant cells in peripheral blood. The increased risk of developing hematologic malignancy does not, on its own, explain excess mortality in individuals with CHIP. Cardiovascular disease accounts for much of this gap. Experimental evidence supports the causality of certain CHIP mutations in accelerated atherosclerosis. CHIP due to mutations in different driver genes varies in their promotion of atherosclerotic events and in the region of augmented atherosclerotic involvement. For example, CHIP due to mutations in DNMT3a appears less atherogenic than CHIP that arises from TET2 or JAK2, forms of CHIP that incite inflammation. The recognition of certain CHIP mutations as promoters of atherosclerotic risk has opened new insights into understanding of the pathophysiology of this disease. The accentuated cardiovascular risk and involvement of distinct pathways of various forms of CHIP also inform novel approaches to allocation of targeted therapies, affording a step toward personalized medicine.
Assuntos
Aterosclerose , Hematopoiese Clonal , DNA Metiltransferase 3A , Dioxigenases , Mutação , Humanos , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Hematopoiese Clonal/genética , DNA Metiltransferase 3A/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Animais , Janus Quinase 2/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fatores de RiscoRESUMO
Nucleotide excision repair (NER) is vital for genome integrity. Yet, our understanding of the complex NER protein machinery remains incomplete. Combining cryo-EM and XL-MS data with AlphaFold2 predictions, we build an integrative model of the NER pre-incision complex(PInC). Here TFIIH serves as a molecular ruler, defining the DNA bubble size and precisely positioning the XPG and XPF nucleases for incision. Using simulations and graph theoretical analyses, we unveil PInC's assembly, global motions, and partitioning into dynamic communities. Remarkably, XPG caps XPD's DNA-binding groove and bridges both junctions of the DNA bubble, suggesting a novel coordination mechanism of PInC's dual incision. XPA rigging interlaces XPF/ERCC1 with RPA, XPD, XPB, and 5' ssDNA, exposing XPA's crucial role in licensing the XPF/ERCC1 incision. Mapping disease mutations onto our models reveals clustering into distinct mechanistic classes, elucidating xeroderma pigmentosum and Cockayne syndrome disease etiology.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA , Endonucleases , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/química , Humanos , Endonucleases/metabolismo , Endonucleases/genética , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/genética , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/química , Microscopia Crioeletrônica , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ligação Proteica , DNA/metabolismo , DNA/química , DNA/genética , Proteína de Replicação A/metabolismo , Proteína de Replicação A/genética , Modelos Moleculares , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Reparo por Excisão , Proteínas NuclearesRESUMO
An RNA interference-based method was proposed to achieve an inducible knockdown of genes essential for cell viability. In the method, a genetic cassette in which a copper ion-dependent inducible metallothionein promoter controls expression of a siRNA precursor is inserted into a genomic pre-integrated transgene by CRIPSR/Cas9 technology. The endogenous siRNA source allows the gene knockdown in cell cultures that are refractory to conventional transfection with exogenous siRNA. The efficiency of the method was demonstrated in Drosophila ovarian somatic cell culture (OSC) for two genes that are essential for oogenesis: Cul3, encoding a component of the multiprotein ubiquitin-ligase complex with versatile functions in proteostasis, and cut, encoding a transcription factor regulating differentiation of ovarian follicular cells.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Técnicas de Silenciamento de Genes , Animais , Drosophila melanogaster/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Proteínas Culina/genética , Proteínas Culina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ovário/metabolismo , Ovário/citologia , Oogênese/genética , Interferência de RNA , Genes Essenciais , Sistemas CRISPR-Cas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
As renal progenitor cells, parietal epithelial cells (PECs) have demonstrated multilineage differentiation potential in response to kidney injury. However, the function of exosomes derived from PECs has not been extensively explored. Immunofluorescent staining of Claudin-1 was used to identify primary PECs isolated from mouse glomeruli. Transmission electron microscopy, nanoparticle tracking analysis, and western blotting were used to characterize the properties of PECs-derived exosomes (PEC-Exo). The therapeutic role of PEC-Exo in tubulointerstitial fibrosis was investigated in the unilateral ureteral obstruction (UUO) mouse model and TGF-ß1-stimulated HK-2 cells. High-throughput miRNA sequencing was employed to profile PEC-Exo miRNAs. One of the most enriched miRNAs in PEC-Exo was knocked down by transfecting miRNA inhibitor, and then we investigated whether this candidate miRNA was involved in PEC-Exo-mediated tubular repair. The primary PECs expressed Claudin-1, PEC-Exo was homing to obstructed kidney, and TGF-ß1 induced HK-2 cells. PEC-Exo significantly alleviated renal inflammation and ameliorated tubular fibrosis both in vivo and in vitro. Mechanistically, let-7b-5p, highly enriched in PEC-Exo, downregulated the protein levels of transforming growth factor beta receptor 1(TGFßR1) and AT-Rich Interaction Domain 3A(ARID3a) in tubular epithelial cells (TECs), leading to the inhibition of p21 and p27 to restoring cell cycle. Furthermore, administration of let-7b-5p agomir mitigated renal fibrosis in vivo. Our findings demonstrated that PEC-derived exosomes significantly repressed the expression of TGFßR1 and ARID3a by delivering let-7b-5p, thereby alleviating renal fibrosis. This study provides novel insights into the role of PEC-Exo in the repair of kidney injury and new ideas for renal fibrosis intervention.
Assuntos
Células Epiteliais , Exossomos , Fibrose , MicroRNAs , Receptor do Fator de Crescimento Transformador beta Tipo I , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Camundongos , Fibrose/metabolismo , Exossomos/metabolismo , Células Epiteliais/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Humanos , Masculino , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Camundongos Endogâmicos C57BL , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Nefropatias/metabolismo , Nefropatias/patologia , Nefropatias/genética , Fator de Crescimento Transformador beta1/metabolismo , Linhagem CelularRESUMO
OBJECTIVE: Hepatocellular carcinoma (HCC) poses a significant challenge to global health. Its pathophysiology involves interconnected processes, including cell proliferation, autophagy, and macrophage polarization. However, the role of Absent in Melanoma 2 (AIM2) in HCC remains elusive. METHODS: The expression of AIM2 in Huh-7 and Hep3B cell lines was manipulated and cell proliferation, autophagy, apoptosis, and migration/invasion, together with the polarization of M2 macrophages, were evaluated. The markers of autophagy pathway, LC3B, Beclin-1, and P62, underwent examination through Western blot analysis. An autophagy inhibitor, 3-MA, was used to measured the role of autophagy in HCC. Finally, the effect of AIM2 overexpression on HCC was further evaluated using a subcutaneous tumor model in nude mice. RESULTS: Our results established that AIM2 overexpression inhibits HCC cell proliferation, migration, and invasion while promoting apoptosis and autophagy. Conversely, knockdown of AIM2 engendered opposite effects. AIM2 overexpression was correlated with reduced M2 macrophage polarization. The autophagy inhibitor substantiated AIM2's role in autophagy and identified its downstream impact on cell proliferation, migration, invasion, and macrophage polarization. In the in vivo model, overexpression of AIM2 led to the inhibition of HCC tumor growth. CONCLUSION: The findings underscore AIM2's crucial function in modulating major biological processes in HCC, pointing to its potential as a therapeutic target. This study inaugurally demonstrated that AIM2 activates autophagy and influences macrophage polarization, playing a role in liver cancer progression.
Assuntos
Autofagia , Carcinoma Hepatocelular , Movimento Celular , Proliferação de Células , Neoplasias Hepáticas , Macrófagos , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Autofagia/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Animais , Humanos , Camundongos , Macrófagos/metabolismo , Macrófagos/imunologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Apoptose/genética , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto , Ativação de Macrófagos/genéticaRESUMO
Neoantigen-based immunotherapy is an attractive potential treatment for previously intractable tumors. To effectively broaden the application of this approach, stringent biomarkers are crucial to identify responsive patients. ARID1A, a frequently mutated subunit of SWI/SNF chromatin remodeling complex, has been reported to determine tumor immunogenicity in some cohorts; however, mutations and deletions of ARID1A are not always linked to clinical responses to immunotherapy. In this study, we investigated immunotherapeutic responses based on ARID1A status in targeted therapy-resistant cancers. Mouse and human BRAFV600E melanomas with or without ARID1A expression were transformed into resistant to vemurafenib, an FDA-approved specific BRAFV600E inhibitor. Anti-PD-1 antibody treatment enhanced antitumor immune responses in vemurafenib-resistant ARID1A-deficient tumors but not in ARID1A-intact tumors or vemurafenib-sensitive ARID1A-deficient tumors. Neoantigens derived from accumulated somatic mutations during vemurafenib resistance were highly expressed in ARID1A-deficient tumors and promoted tumor immunogenicity. Furthermore, the newly generated neoantigens could be utilized as immunotherapeutic targets by vaccines. Finally, targeted therapy resistance-specific neoantigen in experimental human melanoma cells lacking ARID1A were validated to elicit T-cell receptor responses. Collectively, the classification of ARID1A-mutated tumors based on vemurafenib resistance as an additional indicator of immunotherapy response will enable a more accurate prediction to guide cancer treatment. Furthermore, the neoantigens that emerge with therapy resistance can be promising therapeutic targets for refractory tumors. Significance: Chemotherapy resistance promotes the acquisition of immunogenic neoantigens in ARID1A-deficient tumors that confer sensitivity to immune checkpoint blockade and can be utilized for developing antitumor vaccines, providing strategies to improve immunotherapy efficacy.
Assuntos
Antígenos de Neoplasias , Proteínas de Ligação a DNA , Resistencia a Medicamentos Antineoplásicos , Melanoma , Fatores de Transcrição , Vemurafenib , Animais , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Camundongos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Resistencia a Medicamentos Antineoplásicos/imunologia , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/genética , Vemurafenib/farmacologia , Vemurafenib/uso terapêutico , Melanoma/imunologia , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/terapia , Imunoterapia/métodos , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/imunologia , Linhagem Celular Tumoral , Feminino , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Mutação , Terapia de Alvo Molecular/métodos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Mutations occurring in nucleic acids or proteins may affect the binding affinities of protein-nucleic acid interactions. Although many efforts have been devoted to the impact of protein mutations, few computational studies have addressed the effect of nucleic acid mutations and explored whether the identical methodology could be applied to the prediction of binding affinity changes caused by these two mutation types. RESULTS: Here, we developed a generalized algorithm named PNBACE for both DNA and protein mutations. We first demonstrated that DNA mutations could induce varying degrees of changes in binding affinity from multiple perspectives. We then designed a group of energy-based topological features based on different energy networks, which were combined with our previous partition-based energy features to construct individual prediction models through feature selections. Furthermore, we created an ensemble model by integrating the outputs of individual models using a differential evolution algorithm. In addition to predicting the impact of single-point mutations, PNBACE could predict the influence of multiple-point mutations and identify mutations significantly reducing binding affinities. Extensive comparisons indicated that PNBACE largely performed better than existing methods on both regression and classification tasks. CONCLUSIONS: PNBACE is an effective method for estimating the binding affinity changes of protein-nucleic acid complexes induced by DNA or protein mutations, therefore improving our understanding of the interactions between proteins and DNA/RNA.
Assuntos
Algoritmos , DNA , Mutação , Ligação Proteica , DNA/metabolismo , Biologia Computacional/métodos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genéticaRESUMO
BACKGROUND: Breast tumorigenesis is a complex and multistep process accompanied by both genetic and epigenetic dysregulation. In contrast to the extensive studies on DNA epigenetic modifications 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in malignant breast tumors, their roles in the early phases of breast tumorigenesis remain ambiguous. RESULTS: DNA 5hmC and 5mC exhibited a consistent and significant decrease from usual ductal hyperplasia to atypical ductal hyperplasia and subsequently to ductal carcinoma in situ (DCIS). However, 5hmC showed a modest increase in invasive ductal breast cancer compared to DCIS. Genomic analyses showed that the changes in 5hmC and 5mC levels occurred around the transcription start sites (TSSs), and the modification levels were strongly correlated with gene expression levels. Meanwhile, it was found that differentially hydroxymethylated regions (DhMRs) and differentially methylated regions (DMRs) were overlapped in the early phases and accompanied by the enrichment of active histone marks. In addition, TET2-related DNA demethylation was found to be involved in breast tumorigenesis, and four transcription factor binding sites (TFs: ESR1, FOXA1, GATA3, FOS) were enriched in TET2-related DhMRs/DMRs. Intriguingly, we also identified a certain number of common DhMRs between tumor samples and cell-free DNA (cfDNA). CONCLUSIONS: Our study reveals that dynamic changes in DNA 5hmC and 5mC play a vital role in propelling breast tumorigenesis. Both TFs and active histone marks are involved in TET2-related DNA demethylation. Concurrent changes in 5hmC signals in primary breast tumors and cfDNA may play a promising role in breast cancer screening.
Assuntos
5-Metilcitosina , Neoplasias da Mama , Proteínas de Ligação a DNA , Dioxigenases , Proteínas Proto-Oncogênicas , Humanos , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Feminino , Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Carcinogênese/genética , Metilação de DNA/genética , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica , Desmetilação do DNARESUMO
In DNA, methylation at the fifth position of cytosine (5mC) by DNA methyltransferases is essential for eukaryotic gene regulation. Methylation patterns are dynamically controlled by epigenetic machinery. Erasure of 5mC by Fe2+ and 2-ketoglutarate (2KG) dependent dioxygenases in the ten-eleven translocation family (TET1-3), plays a key role in nuclear processes. Through the event of active demethylation, TET proteins iteratively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), each of which has been implicated in numerous diseases when aberrantly generated. A wide range of biochemical assays have been developed to characterize TET activity, many of which require multi-step processing to detect and quantify the 5mC oxidized products. Herein, we describe the development and optimization of a sensitive MALDI mass spectrometry-based technique that directly measures TET activity and eliminates tedious processing steps. Employing optimized assay conditions, we report the steady-state activity of wild type TET2 enzymes to furnish 5hmC, 5fC and 5caC. We next determine IC50 values of several small-molecule inhibitors of TETs. The utility of this assay is further demonstrated by analyzing the activity of V1395A which is an activating mutant of TET2 that primarily generates 5caC. Lastly, we describe the development of a secondary assay that utilizes bisulfite chemistry to further examine the activity of wildtype TET2 and V1395A in a base-resolution manner. The combined results demonstrate that the activity of TET proteins can be gauged, and their products accurately quantified using our methods.
Assuntos
5-Metilcitosina , Proteínas de Ligação a DNA , Dioxigenases , Proteínas Proto-Oncogênicas , Dioxigenases/metabolismo , Dioxigenases/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análise , 5-Metilcitosina/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Ensaios Enzimáticos/métodos , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/química , Metilação de DNA , Citosina/análogos & derivados , Citosina/análise , Citosina/metabolismo , Citosina/química , OxirreduçãoRESUMO
OBJECTIVES: Alcohol and its metabolites, such as acetaldehyde, induced hepatic mitochondrial dysfunction play a pathological role in the development of alcohol-related liver disease (ALD). METHODS: In this study, we investigated the potential of nobiletin (NOB), a polymethoxylated flavone, to counter alcohol-induced mitochondrial dysfunction and liver injury. RESULTS: Our findings demonstrate that NOB administration markedly attenuated alcohol-induced hepatic steatosis, endoplasmic reticulum stress, inflammation, and tissue damage in mice. NOB reversed hepatic mitochondrial dysfunction and oxidative stress in both alcohol-fed mice and acetaldehyde-treated hepatocytes. Mechanistically, NOB restored the reduction of hepatic mitochondrial transcription factor A (TFAM) at both mRNA and protein levels. Notably, the protective effects of NOB against acetaldehyde-induced mitochondrial dysfunction and cell death were abolished in hepatocytes lacking Tfam. Furthermore, NOB administration reinstated the levels of hepatocellular NRF1, a key transcriptional regulator of TFAM, which were decreased by alcohol and acetaldehyde exposure. Consistent with these findings, hepatocyte-specific overexpression of Nrf1 protected against alcohol-induced hepatic Tfam reduction, mitochondrial dysfunction, oxidative stress, and liver injury. CONCLUSIONS: Our study elucidates the involvement of the NRF1-TFAM signaling pathway in the protective mechanism of NOB against chronic-plus-binge alcohol consumption-induced mitochondrial dysfunction and liver injury, suggesting NOB supplementation as a potential therapeutic strategy for ALD.
Assuntos
Flavonas , Transdução de Sinais , Animais , Camundongos , Flavonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Masculino , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Estresse Oxidativo/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Fígado/efeitos dos fármacos , Fígado/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Etanol/toxicidade , Etanol/efeitos adversos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/tratamento farmacológico , Hepatopatias Alcoólicas/prevenção & controle , Hepatopatias Alcoólicas/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Fator 1 Nuclear Respiratório/metabolismo , Fator 1 Nuclear Respiratório/genética , Substâncias Protetoras/farmacologia , Fator 1 Relacionado a NF-E2/metabolismo , Fator 1 Relacionado a NF-E2/genética , Proteínas de Grupo de Alta MobilidadeRESUMO
OBJECTIVE: This study aims to explore the impact of Nesfatin-1 on type 2 diabetic erectile dysfunction (T2DMED) and its underlying mechanism in regulating the phenotypic switching of corpus cavernosum smooth muscle cells (CCSMCs). METHODS: Twenty-four 4-week-old male C57 wild-type mice were randomly assigned to the control group, model group, and Nesfatin-1 treatment group. Monitoring included body weight, blood glucose levels, and penile cavernous pressure (ICP). Histochemistry and Western blot analyses were conducted to assess the expressions of α-SMA, OPN, and factors related to the PI3K/AKT/mTOR signaling pathway. CCSMCs were categorized into the control group, high glucose and high oleic acid group (GO group), Nesfatin-1 treatment group (GO+N group), sildenafil positive control group (GO+S group), and PI3K inhibitor group (GO+N+E group). Changes in phenotypic markers, cell morphology, and the PI3K/AKT/mTOR signaling pathway were observed in each group. RESULTS: (1) Nesfatin-1 significantly ameliorated the body size, body weight, blood glucose, glucose tolerance, and insulin resistance in T2DMED mice. (2) Following Nesfatin-1 treatment, the ICP/MSBP ratio and the peak of the ICP curve demonstrated a significant increase. (3) Nesfatin-1 significantly enhanced smooth muscle and reduced collagen fibers in the corpus cavernosum. (4) Nesfatin-1 notably increased α-SMA expression and decreased OPN expression in CCSMCs. (5) Nesfatin-1 elevated PI3K, p-AKT/AKT, and p-mTOR/mTOR levels in penile cavernous tissue. CONCLUSIONS: Nesfatin-1 not only effectively improves body weight and blood glucose levels in diabetic mice but also enhances erectile function and regulates the phenotypic switching of corpus cavernosum smooth muscle. The potential mechanism involves Nesfatin-1 activating the PI3K/AKT/mTOR signaling pathway to induce the conversion of CCSMCs to a contractile phenotype.