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1.
Gene ; 807: 145961, 2022 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-34530088

RESUMO

Vibrio parahaemolyticus produces two types of IV pili: mannose-sensitive haemagglutinin type IV pili (MSHA) and chitin-regulated pili (ChiRP). Both of them are required for biofilm formation and the pathogen persistence in hosts. However, there are few reports on the regulation of their expression. In the present study, we showed that the master quorum sensing (QS) regulators AphA and OpaR oppositely regulated the transcription of mshA1 encoding the pilin of MSHA pilus in V. parahaemolyticus. At low cell density (LCD), AphA indirectly repressed mshA1 transcription. In contrast, at high cell density (HCD), OpaR bound to the regulatory DNA region of mshA1 to activate its transcription. Oppositely regulation of mshA1 by AphA and OpaR led to a gradual increase in the expression level of mshA1 from LCD to HCD. Thus, regulation of type IV pili production was one of the mechanisms that V. parahaemolyticus adopted to control biofilm formation.


Assuntos
Proteínas de Fímbrias/genética , Percepção de Quorum/genética , Vibrio/genética , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Contagem de Células , Proteínas de Fímbrias/metabolismo , Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/genética , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Vibrio/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismo
2.
Int J Mol Sci ; 22(19)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34639201

RESUMO

The arsenic acid-resistant (ArsR) family transcriptional regulators are widely distributed in microorganisms, including in the facultative intracellular pathogen Brucella spp. ArsR proteins are implicated in numerous biological processes. However, the specific roles of ArsR family members in Brucella remain obscure. Here, we show that ArsR6 (BSS2_RS07325) is required for Brucella survival both under heat, oxidative, and osmotic stress and in a murine infection model in vivo. RNA-seq and ChIP-seq reveal that 34 potential target genes for ArsR6 protein were identified, among which eight genes were up-regulated and 26 genes were down-regulated, including outer membrane protein 25D (Omp25D). ArsR6 autoregulates its own expression to maintain bacterial intracellular Cu/Ni homeostasis to benefit bacterial survival in hostile environments. Moreover, ArsR6 also regulates the production of virulence factor Omp25D, which is important for the survival of Brucella under stress conditions. Significantly, Omp25D deletion strain attenuated in a murine infection model in vivo. Altogether, our findings reveal a unique mechanism in which the ArsR family member ArsR6 autoregulates its expression and also modulates Omp25D expression to maintain metal ion homeostasis and virulence in Brucella.


Assuntos
Proteínas de Bactérias/metabolismo , Brucella/crescimento & desenvolvimento , Brucelose/microbiologia , Regulação Bacteriana da Expressão Gênica , Transativadores/metabolismo , Fatores de Virulência/metabolismo , Virulência , Animais , Brucella/genética , Brucella/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Transativadores/genética , Fatores de Virulência/genética
3.
FASEB J ; 35(11): e21950, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34613638

RESUMO

Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders characterized by relapsing intestinal inflammation, but many details of pathogenesis remain to be fully unraveled. Glucocorticoid (GC)-induced leucine zipper (GILZ) is a mediator of the anti-inflammatory effects of GCs, the most powerful drugs for IBD treatment, but they cause several unwanted side effects. The fusion protein TAT-GILZ has been successfully used in some pre-clinical models of inflammatory and autoimmune diseases. To test the efficacy of TAT-GILZ for treating dextran sulfate sodium (DSS)-induced colitis and explore its impact on the gut microbiome, colitis was induced by DSS in C57BL/6J mice and treated with TAT-GILZ or dexamethasone. Various hallmarks of colitis were analyzed, including disease activity index, gut permeability, and expression of pro-inflammatory cytokines and tight junction proteins. TAT-GILZ treatment showed a therapeutic effect when administered after the onset of colitis. Its efficacy was associated with improved gut permeability, as evidenced by zonula occludens-1 and CD74 upregulation in inflamed colonic tissue. TAT-GILZ also ameliorated the changes in the gut microbiota induced by the DSS, thus potentially providing an optimal environment for colonization of the mucosa surface by beneficial bacteria. Overall, our results demonstrated for the first time that TAT-GILZ treatment proved effective after disease onset allowing restoration of gut permeability, a key pathogenic feature of colitis. Additionally, TAT-GILZ restored gut dysbiosis, thereby contributing to healing mechanisms. Interestingly, we found unprecedented effects of exogenous GILZ that did not overlap with those of GCs.


Assuntos
Colite/induzido quimicamente , Colite/tratamento farmacológico , Sulfato de Dextrana/efeitos adversos , Mucosa Intestinal/metabolismo , Permeabilidade/efeitos dos fármacos , Proteínas Recombinantes de Fusão/administração & dosagem , Fatores de Transcrição/administração & dosagem , Animais , Anti-Inflamatórios/administração & dosagem , Antígenos de Diferenciação de Linfócitos B/metabolismo , Colite/metabolismo , Citocinas/metabolismo , Dexametasona/administração & dosagem , Modelos Animais de Doenças , Antígenos de Histocompatibilidade Classe II/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Transativadores/genética , Fatores de Transcrição/genética , Resultado do Tratamento , Regulação para Cima/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismo
4.
Nat Commun ; 12(1): 5931, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34635673

RESUMO

The chromatin remodeler RSF1 enriched at mitotic centromeres is essential for proper chromosome alignment and segregation and underlying mechanisms remain to be disclosed. We here show that PLK1 recruitment by RSF1 at centromeres creates an activating phosphorylation on Thr236 in the activation loop of Aurora B and this is indispensable for the Aurora B activation. In structural modeling the phosphorylated Thr236 enhances the base catalysis by Asp200 nearby, facilitating the Thr232 autophosphorylation. Accordingly, RSF1-PLK1 is central for Aurora B-mediated microtubule destabilization in error correction. However, under full microtubule-kinetochore attachment RSF1-PLK1 positions at kinetochores, halts activating Aurora B and phosphorylates BubR1, regardless of tension. Spatial movement of RSF1-PLK1 to kinetochores is triggered by Aurora B-mediated phosphorylation of centromeric histone H3 on Ser28. We propose a regulatory RSF1-PLK1 axis that spatiotemporally controls on/off switch on Aurora B. This feedback circuit among RSF1-PLK1-Aurora B may coordinate dynamic microtubule-kinetochore attachment in early mitosis when full tension yet to be generated.


Assuntos
Aurora Quinase B/genética , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos , Mitose , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/genética , Transativadores/genética , Ácido Aspártico/metabolismo , Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteínas Nucleares/deficiência , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Serina/metabolismo , Transativadores/deficiência
5.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 39(5): 493-500, 2021 Oct 01.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-34636195

RESUMO

YAP/TAZ are wild over-activated in head and neck squamous cell carcinoma (HNSCC) with high potential as a direct therapy target for HNSCC treatments. However, the efforts on the directly targeting-YAP/TAZ therapies over the past decade, have very limited impacts, mainly caused by: 1. There is still none effective and specific YAP/TAZ inhibitor with clinical potential; 2. YAP/TAZ might not be directly targeted, because of their multiple important biological functions, such as: regulation of cell proliferation and survival, stem cell maintain, regulation of organ development, organ size control, and tissue regeneration. Interestingly, the over-activation of YAP/TAZ in HNSCC mainly be regulated by upstream abnormal molecular or biological events, instead of genes alteration of YAP/TAZ. Therefore, exploring the alternative molecular events regulating YAP/TAZ activation and molecular mechanism in HNSCC might help to uncover novel indirect targets of YAP/TAZ therapies for HNSCC prevention and treatment.


Assuntos
Neoplasias de Cabeça e Pescoço , Fosfoproteínas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Humanos , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Fatores de Transcrição
6.
Int J Mol Sci ; 22(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34638909

RESUMO

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.


Assuntos
Adenoviridae/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , Vírus da Hepatite B/genética , RNA Guia/genética , Transativadores/genética , Proteínas Virais Reguladoras e Acessórias/genética , Adenoviridae/fisiologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Vetores Genéticos/genética , Células HEK293 , Células Hep G2 , Vírus da Hepatite B/metabolismo , Hepatite B Crônica/genética , Hepatite B Crônica/terapia , Hepatite B Crônica/virologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/virologia , RNA Guia/metabolismo , Transativadores/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Replicação Viral/genética
7.
Molecules ; 26(19)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34641320

RESUMO

The nucleoid-associated protein GapR found in Caulobacter crescentus is crucial for DNA replication, transcription, and cell division. Associated with overtwisted DNA in front of replication forks and the 3' end of highly-expressed genes, GapR can stimulate gyrase and topo IV to relax (+) supercoils, thus facilitating the movement of the replication and transcription machines. GapR forms a dimer-of-dimers structure in solution that can exist in either an open or a closed conformation. It initially binds DNA through the open conformation and then undergoes structural rearrangement to form a closed tetramer, with DNA wrapped in the central channel. Here, we show that the DNA binding domain of GapR (residues 1-72, GapRΔC17) exists as a dimer in solution and adopts the same fold as the two dimer units in the full-length tetrameric protein. It binds DNA at the minor groove and reads the spatial distribution of DNA phosphate groups through a lysine/arginine network, with a preference towards AT-rich overtwisted DNA. These findings indicate that the dimer unit of GapR has an intrinsic DNA binding preference. Thus, at the initial binding step, the open tetramer of GapR with two relatively independent dimer units can be more efficiently recruited to overtwisted regions.


Assuntos
Caulobacter crescentus/metabolismo , DNA Bacteriano/metabolismo , Transativadores/química , Transativadores/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Caulobacter crescentus/química , Caulobacter crescentus/genética , Cristalografia por Raios X , DNA Bacteriano/química , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Transativadores/genética
8.
J Biomed Sci ; 28(1): 67, 2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34615538

RESUMO

BACKGROUND: Acute liver failure (ALF) is a syndrome of severe hepatocyte injury with high rate of mortality. Hepatitis B virus (HBV) infection is the major cause of ALF worldwide, however, the underlying mechanism by which HBV infection leads to ALF has not been fully disclosed. METHODS: D-GalN-induced hepatocyte injury model and LPS/D-GalN-induced ALF mice model were used to investigate the effects of HBV X protein (HBx) in vitro and in vivo, respectively. Cell viability and the levels of Glutathione (GSH), malondialdehyde (MDA) and iron were measured using commercial kits. The expression of ferroptosis-related molecules were detected by qRT-PCR and western blotting. Epigenetic modification and protein interaction were detected by chromatin immunoprecipitation (ChIP) assay and co-immunoprecipitation (co-IP), respectively. Mouse liver function was assessed by measuring aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The histological changes in liver tissues were monitored by hematoxylin and eosin (H&E) staining, and SLC7A11 immunoreactivity was assessed by immunohistochemistry (IHC) analysis. RESULTS: D-GalN triggered ferroptosis in primary hepatocytes. HBx potentiated D-GalN-induced hepatotoxicity and ferroptosis in vitro, and it suppressed SLC7A11 expression through H3K27me3 modification by EZH2. In addition, EZH2 inhibition or SLC7A11 overexpression attenuated the effects of HBx on D-GalN-induced ferroptosis in primary hepatocytes. The ferroptosis inhibitor ferrostatin-1 (Fer-1) protected against ALF and ferroptosis in vivo. By contrast, HBx exacerbates LPS/D-GalN-induced ALF and ferroptosis in HBx transgenic (HBx-Tg) mice. CONCLUSION: HBx facilitates ferroptosis in ALF via EZH2/H3K27me3-mediated SLC7A11 suppression.


Assuntos
Ferroptose/fisiologia , Falência Hepática Aguda/virologia , Transativadores/genética , Proteínas Virais Reguladoras e Acessórias/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL
9.
J Gen Virol ; 102(10)2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34661519

RESUMO

Ubiquitin and ubiquitin-like protein modification play important roles in modulating the functions of viral proteins in many viruses. Here we demonstrate that hepatitis B virus (HBV) X protein (HBx) is modified by ISG15, which is a type I IFN-inducible, ubiquitin-like protein; this modification is called ISGylation. Immunoblot analyses revealed that HBx proteins derived from four different HBV genotypes accepted ISGylation in cultured cells. Site-directed mutagenesis revealed that three lysine residues (K91, K95 and K140) on the HBx protein, which are well conserved among all the HBV genotypes, are involved in acceptance of ISGylation. Using expression plasmids encoding three known E3 ligases involved in the ISGylation to different substrates, we found that HERC5 functions as an E3 ligase for HBx-ISGylation. Treatment with type I and type III IFNs resulted in the limited suppression of HBV replication in Hep38.7-Tet cells. When cells were treated with IFN-α, silencing of ISG15 resulted in a marked reduction of HBV replication in Hep38.7-Tet cells, suggesting a role of ISG15 in the resistance to IFN-α. In contrast, the silencing of USP18 (an ISG15 de-conjugating enzyme) increased the HBV replication in Hep38.7-Tet cells. Taken together, these results suggest that the HERC5-mediated ISGylation of HBx protein confers pro-viral functions on HBV replication and participates in the resistance to IFN-α-mediated antiviral activity.


Assuntos
Citocinas/metabolismo , Vírus da Hepatite B/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transativadores/metabolismo , Ubiquitinas/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Replicação Viral , Linhagem Celular , Farmacorresistência Viral , Vírus da Hepatite B/genética , Humanos , Interferon-alfa/farmacologia , Interferon beta/farmacologia , Interferons/farmacologia , Transativadores/química , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais Reguladoras e Acessórias/química
10.
Chin J Nat Med ; 19(10): 750-757, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34688465

RESUMO

Tripterygium wilfordii multiglycoside (GTW) is a commonly used compound for the treatment of rheumatoid arthritis (RA) and immune diseases in clinical practice. However, it can induce liver injury and the mechanism of hepatotoxicity is still not clear. This study was designed to investigate GTW-induced hepatotoxicity in zebrafish larvae and explore the mechanism involved. The 72 hpf (hours post fertilization) zebrafish larvae were administered with different concentrations of GTW for three days and their mortality, malformation rate, morphological changes in the liver, transaminase levels, and histopathological changes in the liver of zebrafish larvae were detected. The reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the levels of microRNA-122 (miR-122) and genes related to inflammation, apoptosis, cell proliferation and liver function. The results showed that GTW increased the mortality of zebrafish larvae, while significant malformations and liver damage occurred. The main manifestations were elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), significant liver atrophy, vacuoles in liver tissue, sparse cytoplasm, and unclear hepatocyte contours. RT-PCR results showed that the expression of miR-122 significantly decreased by GTW; the mRNA levels of inflammation-related genes il1ß, il6, tnfα, il10, cox2 and ptges significantly increased; the mRNA level of tgfß significantly decreased; the mRNA levels of apoptosis-related genes, caspase-8 and caspase-9, significantly increased; the mRNA level of bcl2 significantly decreased; the mRNA levels of cell proliferation-related genes, top2α and uhrf1, significantly reduced; the mRNA levels of liver function-related genes, alr and cyp3c1, significantly increased; and the mRNA level of cyp3a65 significantly decreased. In zebrafish, GTW can cause increased inflammation, enhanced apoptosis, decreased cell proliferation, and abnormal expression of liver function-related genes, leading to abnormal liver structure and function and resulting in hepatotoxicity.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Tripterygium , Animais , Apoptose , Doença Hepática Induzida por Substâncias e Drogas/genética , Inflamação/induzido quimicamente , Inflamação/genética , Transativadores , Peixe-Zebra/genética , Proteínas de Peixe-Zebra
11.
Int J Mol Sci ; 22(19)2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34638756

RESUMO

As sessile organisms, the precise development phase transitions are very important for the success of plant adaptability, survival and reproduction. The transition from juvenile to the adult phase-referred to as the vegetative phase change-is significantly influenced by numbers of endogenous and environmental signals. Here, we showed that brassinosteroid (BR), a major growth-promoting steroid hormone, positively regulates the vegetative phase change in Arabidopsis thaliana. The BR-deficient mutant det2-1 and BR-insensitive mutant bri1-301 displayed the increased ratio of leaf width to length and reduced blade base angle. The plant specific transcription factors SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) are key masters for the vegetative phase transition in plants. The expression levels of SPL9, SPL10 and SPL15 were significantly induced by BR treatment, but reduced in bri1-116 mutant compared to wild-type plants. The gain-of-function pSPL9:rSPL9 transgenic plants displayed the BR hypersensitivity on hypocotyl elongation and partially suppressed the delayed vegetative phase change of det2-1 and bri1-301. Furthermore, we showed that BRASSINAZOLE-RESISTANT 1 (BZR1), the master transcription factor of BR signaling pathway, interacted with SPL9 to cooperatively regulate the expression of downstream genes. Our findings reveal an important role for BRs in promoting vegetative phase transition through regulating the activity of SPL9 at transcriptional and post-transcriptional levels.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação , Transdução de Sinais , Transativadores/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Transativadores/genética
12.
Biophys J ; 120(20): 4360-4377, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34509508

RESUMO

Membrane binding and unbinding dynamics play a crucial role in the biological activity of several nonintegral membrane proteins, which have to be recruited to the membrane to perform their functions. By localizing to the membrane, these proteins are able to induce downstream signal amplification in their respective signaling pathways. Here, we present a 3D computational approach using reaction-diffusion equations to investigate the relation between membrane localization of focal adhesion kinase (FAK), Ras homolog family member A (RhoA), and signal amplification of the YAP/TAZ signaling pathway. Our results show that the theoretical scenarios in which FAK is membrane bound yield robust and amplified YAP/TAZ nuclear translocation signals. Moreover, we predict that the amount of YAP/TAZ nuclear translocation increases with cell spreading, confirming the experimental findings in the literature. In summary, our in silico predictions show that when the cell membrane interaction area with the underlying substrate increases, for example, through cell spreading, this leads to more encounters between membrane-bound signaling partners and downstream signal amplification. Because membrane activation is a motif common to many signaling pathways, this study has important implications for understanding the design principles of signaling networks.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Fatores de Transcrição , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal , Fosfoproteínas/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/metabolismo
13.
Biomaterials ; 277: 121097, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34481290

RESUMO

Skeletal muscle tissue is mechanically dynamic with changes in stiffness influencing function, maintenance, and regeneration. We modeled skeletal muscle mechanical changes in culture with dynamically stiffening hydrogels demonstrating that the chaperone protein BAG3 transduces matrix stiffness by redistributing YAP and TAZ subcellular localization in muscle progenitor cells. BAG3 depletion increases cytoplasmic retention of YAP and TAZ, desensitizing myoblasts to changes in hydrogel elastic moduli. Upon differentiation, muscle progenitors depleted of BAG3 formed enlarged, round myotubes lacking the typical cylindrical morphology. The aberrant morphology is dependent on YAP/TAZ signaling, which was sequestered in the cytoplasm in BAG3-depleted myotubes but predominately nuclear in cylindrical myotubes of control cells. Control progenitor cells induced to differentiate on soft (E' = 4 and 12 kPa) hydrogels formed circular myotubes similar to those observed in BAG3-depleted cells. Inhibition of the Hippo pathway partially restored myotube morphologies, permitting nuclear translocation of YAP and TAZ in BAG3-depleted myogenic progenitors. Thus, BAG3 is a critical mediator of dynamic stiffness changes in muscle tissue, coupling mechanical alterations to intracellular signals and inducing changes in gene expression that influence muscle progenitor cell morphology and differentiation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Mecanotransdução Celular , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo
14.
Chem Commun (Camb) ; 57(75): 9594-9597, 2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34546238

RESUMO

Dynamic changes in intron sequences, with their loss and gain, are poorly detected due to the limited methods for the non-invasive monitoring of the pre-mRNA splicing process. Here, we describe the design of a two-step transcriptional activation (TSTA) reporter for the real-time imaging of the splicing process in living subjects. By taking advantage of the strong transactivating properties of the GAL4-VP16 fusion protein, which can target upstream activation sequence (UAS) elements to boost subsequent firefly luciferase reporter gene expression, we successfully and consistently detected the dynamic pre-mRNA splicing activity in response to exogenous splicing modulators in living cells and animals. Our findings provide a valuable tool for the high-throughput screening of splicing modulators, which could speed up the development of new drugs for the treatment of disordered splicing diseases.


Assuntos
RNA Mensageiro/genética , Transativadores/genética , Humanos , Splicing de RNA/genética , RNA Mensageiro/metabolismo , Transativadores/metabolismo
15.
Medicina (Kaunas) ; 57(9)2021 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-34577857

RESUMO

Breast cancer (BC) is one of the most prevalent malignancies among females worldwide. Globally, distant metastases were reported to be responsible for a large proportion of breast cancer-related deaths. The metastasis-associated colon cancer-1 (MACC1) gene was reported as a reliable biomarker for early detection of metastasis and prediction of prognosis of breast cancer. This study investigated the prognostic significance of MACC1 in breast cancer in relation to the clinicopathologic characteristics and patients' survival. Furthermore, the possible correlation between MACC1 expression and the different immune cells in the tumor microenvironment was explored. MACC1 mRNA was identified using quantitative reverse transcription polymerase chain reaction in 120 breast cancer specimens and adjacent non-cancerous tissues. MACC1 mRNA expression was significantly higher in the cancerous relative to the non-cancerous tissues (p < 0.001). High MACC1 expression was significantly associated with poor prognostic parameters, such as larger tumor size, grade III tumors, positive nodal metastasis, lymphovascular invasion, stage III tumors, and elevated Ki-67 expression. Higher MACC1 mRNA levels were positively correlated with CD163+ tumor-associated macrophages (r = 0.614, p < 0.001), and were negatively correlated with CD56+ natural killer cells (r = -0.398, p < 0.001) and CD8+ cytotoxic T lymphocytes (r = -0.323, p < 0.001). MACC1 expression was associated with poor patient overall survival (OS) and progression-free survival (PFS) (p < 0.001). Multivariate analysis suggested that MACC1 expression and the presence of lymphovascular invasion could be independent prognostic indicators for breast cancer (p = 0.015 and 0.042, respectively). In conclusion, MACC1 is highly expressed in cancerous tissues and is significantly related to poor prognostic factors, overall survival, and progression-free survival. MACC1 may influence infiltration of the immune cells in the tumor microenvironment, enhance immune escape of tumor cells, and may serve as a reliable independent prognostic factor for breast cancer.


Assuntos
Neoplasias da Mama , Neoplasias do Colo , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Feminino , Humanos , Prognóstico , Transativadores , Fatores de Transcrição/genética , Microambiente Tumoral
16.
Curr Microbiol ; 78(11): 3936-3944, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34522977

RESUMO

Our study attempted to explore the mechanism underlying the role of LuxR family transcriptional regulator abaR in biofilm formation by Acinetobacter baumannii. The abaR gene was knocked out in ATCC 17978 strain using homologous recombination method. The growth curve and biofilm formation were measured in the wild type and abaR gene knockdown strains. Transcriptome sequencing was performed in the wild type and abaR gene knockdown strains following 8 h of culture. The growth curve in the abaR gene knockdown strain was similar to that of the wild-type strain. Biofilm formation significantly declined in the abaR gene knockdown strain at 8 and 48 h after culture. A total of 137 differentially expressed genes (DEGs) were obtained including 20 downregulated DEGs and 117 upregulated DEGs. Genes with differential expression were closely related to viral procapsid maturation (GO:0046797), acetoin catabolism (GO:0045150), carbon metabolism (ko01200), and the glycolysis/gluconeogenesis (ko00010)-related pathways. The results of the eight verified expression DEGs were consistent with the results predicted by bioinformatics. AbaR gene knockdown significantly affected biofilm formation by A. baumannii ATCC 17978 strain. The glycolysis/gluconeogenesis pathways were significantly dysregulated and induced by abaR gene knockdown in A. baumannii.


Assuntos
Acinetobacter baumannii , Proteínas Repressoras/fisiologia , Transativadores/fisiologia , Acinetobacter baumannii/genética , Biofilmes , Proteínas Repressoras/genética , Transativadores/genética
17.
Appl Microbiol Biotechnol ; 105(20): 7801-7811, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34581846

RESUMO

Quorum sensing is a mechanism that facilitates cell-to-cell communication. Through signal molecular density for signal recognition, which leads to the regulation of some physiological and biochemical functions. Gluconacetobacter xylinus CGMCC 2955, which produces bacterial cellulose (BC), synthesizes the LuxR protein belonging to the LuxI/LuxR type QS system. Here, a luxR overexpression vector was transformed into G. xylinus CGMCC 2955. The overexpression of luxR increased the yield of BC by 15.6% after 16 days static culture and reduced the cell density by 15.5% after 120-h-agitated culture. The glucose was used up by G. xylinus-pMV24-luxR at 72-h-agitated fermentation, which 12 h earlier than the wild-type (WT). The total N-acylhomoserine lactones (AHL) content of the luxR-overexpressing strain and the WT strain attained 1367.9 ± 57.86 mg/L and 842.9 ± 54.22 mg/L, respectively. The C12-HSL and C14-HSL contents of G. xylinus-pMV24-luxR were 202 ± 21.66 mg/L and 409.6 ± 0.91 mg/L, which were significantly lower than that of WT. In contrast, C6-HSL showed opposite results. The difference of AHL content proved that overexpression of luxR improved the binding of AHL and showed preference for some specific AHL. The metabolic results demonstrated that upon glucose exhaustion, the consumption of gluconic acid was promoted by luxR overexpression, and the content of D- ( +)-trehalose, an antiretrograde metabolite, increased significantly. KEY POINTS: • The overexpression of luxR increased the yield of bacterial cellulose • The content of signal molecules was significantly different • Differential metabolites were involved in multiple metabolic pathways.


Assuntos
Gluconacetobacter xylinus , Percepção de Quorum , Acil-Butirolactonas , Proteínas de Bactérias/genética , Celulose , Gluconacetobacter xylinus/genética , Transativadores/genética
18.
Nat Commun ; 12(1): 5208, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471108

RESUMO

Post-transcriptional gene regulation in T cells is dynamic and complex as targeted transcripts respond to various factors. This is evident for the Icos mRNA encoding an essential costimulatory receptor that is regulated by several RNA-binding proteins (RBP), including Roquin-1 and Roquin-2. Here, we identify a core RBPome of 798 mouse and 801 human T cell proteins by utilizing global RNA interactome capture (RNA-IC) and orthogonal organic phase separation (OOPS). The RBPome includes Stat1, Stat4 and Vav1 proteins suggesting unexpected functions for these transcription factors and signal transducers. Based on proximity to Roquin-1, we select ~50 RBPs for testing coregulation of Roquin-1/2 targets by induced expression in wild-type or Roquin-1/2-deficient T cells. Besides Roquin-independent contributions from Rbms1 and Cpeb4 we also show Roquin-1/2-dependent and target-specific coregulation of Icos by Celf1 and Igf2bp3. Connecting the cellular RBPome in a post-transcriptional context, we find contributions from multiple RBPs to the prototypic regulation of mRNA targets by individual trans-acting factors.


Assuntos
RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Células HEK293 , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Camundongos , Proteínas Proto-Oncogênicas c-vav , Fator de Transcrição STAT1 , Fator de Transcrição STAT4 , Transdução de Sinais , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/genética
19.
Vet Res ; 52(1): 114, 2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34479647

RESUMO

Biofilm formation is a significant virulence factor in Staphylococcus (S.) aureus strains causing subclinical mastitis in dairy cows. A role of environmental signals and communication systems in biofilm development, such as the agr system in S. aureus, is suggested. In the context of multispecies biofilm communities, the presence of non-aureus staphylococci (NAS) might influence S. aureus colonization of the bovine mammary gland, yet, such interspecies interactions have been poorly studied. We determined whether 34 S. chromogenes, 11 S. epidermidis, and 14 S. simulans isolates originating from bovine milk samples and teat apices (TA) were able to affect biofilm formation and dispersion of S. aureus, and if so, how isolate traits such as the capacity to regulate the S. aureus agr quorum sensing system are determinants in this process. The capacity of an agr-positive S. aureus strain to form biofilm was increased more in the presence of S. chromogenes than in the presence of S. simulans and S. epidermidis isolates and in the presence of NAS isolates that do not harbor biofilm related genes. On the other hand, biofilm dispersion of this particular S. aureus strain was suppressed by NAS as a group, an effect that was more pronounced by isolates from TA. Furthermore, the observed effects on biofilm formation and dispersion of the agr-positive S. aureus strain as well as of an agr-negative S. aureus strain did not depend on the capacity of NAS to repress the agr system.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Doenças dos Bovinos/microbiologia , Percepção de Quorum , Infecções Estafilocócicas/veterinária , Staphylococcus/fisiologia , Transativadores/metabolismo , Animais , Bovinos , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia
20.
Elife ; 102021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34499028

RESUMO

The ubiquitous Ca2+ sensor calmodulin (CaM) binds and regulates many proteins, including ion channels, CaM kinases, and calcineurin, according to Ca2+-CaM levels. What regulates neuronal CaM levels, is, however, unclear. CaM-binding transcription activators (CAMTAs) are ancient proteins expressed broadly in nervous systems and whose loss confers pleiotropic behavioral defects in flies, mice, and humans. Using Caenorhabditis elegans and Drosophila, we show that CAMTAs control neuronal CaM levels. The behavioral and neuronal Ca2+ signaling defects in mutants lacking camt-1, the sole C. elegans CAMTA, can be rescued by supplementing neuronal CaM. CAMT-1 binds multiple sites in the CaM promoter and deleting these sites phenocopies camt-1. Our data suggest CAMTAs mediate a conserved and general mechanism that controls neuronal CaM levels, thereby regulating Ca2+ signaling, physiology, and behavior.


Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Proteínas de Drosophila/metabolismo , Neurônios/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Calcineurina/metabolismo , Cálcio/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Edição de Genes , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Ligação Proteica , Transdução de Sinais , Transativadores/genética , Transcriptoma
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