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1.
Science ; 369(6500): 202-207, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32647003

RESUMO

Immunodeficiency often coincides with hyperactive immune disorders such as autoimmunity, lymphoproliferation, or atopy, but this coincidence is rarely understood on a molecular level. We describe five patients from four families with immunodeficiency coupled with atopy, lymphoproliferation, and cytokine overproduction harboring mutations in NCKAP1L, which encodes the hematopoietic-specific HEM1 protein. These mutations cause the loss of the HEM1 protein and the WAVE regulatory complex (WRC) or disrupt binding to the WRC regulator, Arf1, thereby impairing actin polymerization, synapse formation, and immune cell migration. Diminished cortical actin networks caused by WRC loss led to uncontrolled cytokine release and immune hyperresponsiveness. HEM1 loss also blocked mechanistic target of rapamycin complex 2 (mTORC2)-dependent AKT phosphorylation, T cell proliferation, and selected effector functions, leading to immunodeficiency. Thus, the evolutionarily conserved HEM1 protein simultaneously regulates filamentous actin (F-actin) and mTORC2 signaling to achieve equipoise in immune responses.


Assuntos
Actinas/metabolismo , Citocinas/biossíntese , Síndromes de Imunodeficiência/genética , Transtornos Linfoproliferativos/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas de Membrana/fisiologia , Fator 1 de Ribosilação do ADP/metabolismo , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Humanos , Síndromes de Imunodeficiência/imunologia , Transtornos Linfoproliferativos/imunologia , Proteínas de Membrana/genética , Linhagem , Fosforilação , Família de Proteínas da Síndrome de Wiskott-Aldrich/química , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo
2.
Science ; 368(6496): 1205-1210, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32527825

RESUMO

Cell migration is driven by local membrane protrusion through directed polymerization of F-actin at the front. However, F-actin next to the plasma membrane also tethers the membrane and thus resists outgoing protrusions. Here, we developed a fluorescent reporter to monitor changes in the density of membrane-proximal F-actin (MPA) during membrane protrusion and cell migration. Unlike the total F-actin concentration, which was high in the front of migrating cells, MPA density was low in the front and high in the back. Back-to-front MPA density gradients were controlled by higher cofilin-mediated turnover of F-actin in the front. Furthermore, nascent membrane protrusions selectively extended outward from areas where MPA density was reduced. Thus, locally low MPA density directs local membrane protrusions and stabilizes cell polarization during cell migration.


Assuntos
Actinas/metabolismo , Movimento Celular , Extensões da Superfície Celular , Actinas/química , Actinas/genética , Membrana Celular , Polaridade Celular , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos
3.
Arterioscler Thromb Vasc Biol ; 40(6): e166-e179, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32349534

RESUMO

OBJECTIVE: Recent studies suggest that the P2Y12 (P2Y purinoceptor 12) receptor of vascular smooth muscle cells in atherosclerotic plaques aggravates atherosclerosis, and P2Y12 receptor inhibitors such as CDL (clopidogrel) may effectively treat atherosclerosis. It is imperative to identify an effective biomarker for reflecting the P2Y12 receptor expression on vascular smooth muscle cells in plaques. Approach and Results: We found that there was a positive correlation between the level of circulating sLRP1 (soluble low-density lipoprotein receptor-related protein 1) and the number of LRP1+ α-SMA+ (α-smooth muscle actin), P2Y12+, or P2Y12+ LRP1+ cells in plaques from apoE-/- mice fed a high-fat diet. Furthermore, activation of the P2Y12 receptor increased the expression and shedding of LRP1 in vascular smooth muscle cells by inhibiting cAMP (3'-5'-cyclic adenosine monophosphate)/PKA (protein kinase A)/SREBP-2 (sterol regulatory element binding transcription factor 2). Conversely, genetic knockdown or pharmacological inhibition of the P2Y12 receptor had the opposite effects. Additionally, CDL decreased the number of lesional LRP1+ α-SMA+ cells and the levels of circulating sLRP1 by activating cAMP/PKA/SREBP-2 in apoE-/- mice fed a high-fat diet. CONCLUSIONS: Our study suggests that sLRP1 may be a biomarker that reflects the P2Y12 receptor level in plaques and has the potential to be an indicator for administering P2Y12 receptor inhibitors for patients with atherosclerosis.


Assuntos
Biomarcadores/análise , Expressão Gênica , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/análise , Placa Aterosclerótica/metabolismo , Receptores Purinérgicos P2Y12/genética , Actinas/análise , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Apolipoproteínas E/fisiologia , Clopidogrel/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dieta Hiperlipídica , Técnicas de Silenciamento de Genes , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/química , Músculo Liso Vascular/metabolismo , Placa Aterosclerótica/química , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y12/efeitos dos fármacos , Receptores Purinérgicos P2Y12/fisiologia , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
4.
Life Sci ; 253: 117740, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32376265

RESUMO

AIMS: Annexin A2 (ANXA2) is closely associated with tumor malignancy and its N-terminus includes a vital domain for its function. The aims are to explore the correlation between the sites (Tyr23, Ser1, Ser11 and Ser25) in the domain and its roles. MAIN METHODS: We re-expressed ANXA2 with mutated sites in ANXA2-deleted human colonic adenocarcinoma cell line caco2 (ANXA2-/-caco2). A series of analyses were used to determine the correlation of each site with ANXA2 activation, cell malignancy enhancement and motility-associated microstructural development. Bioinformatics and luciferase reporter assays were employed to validate ANXA2-targeted miRNAs. KEY FINDINGS: The in vitro results showed that all single and multiple mutations of the ANXA2 N-terminal sites inhibited ANXA2 phosphorylation at different levels and subsequently inhibited the proliferation, motility, and polymerization of F-actin and ß-tubulin in caco2 cells. Motility-associated microstructures were significantly remodeled when these sites were mutated. The forced expression of miR-206 significantly suppressed the proliferation, motility and epithelial-mesenchymal transition (EMT) of caco2 cells. The in vivo results showed that all the ANXA2 N-terminal site mutations and forced expression of miR-206 significantly inhibited tumor growth. Overall, this study demonstrated that the sites of the ANXA2 N-terminus, especially Tyr23, play crucial roles in maintaining the high malignancy of colonic adenocarcinoma. Furthermore, miR-206 targets ANXA2 and plays a role as a cancer suppressor in colonic adenocarcinoma. SIGNIFICANCE: Our study provided evidence that further elucidates the molecular mechanism of ANXA2 and its roles in colonic adenocarcinoma and suggested potential targets of ANXA2 for colonic adenocarcinoma therapy by using miR-206 as a novel strategy.


Assuntos
Adenocarcinoma/patologia , Anexina A2/genética , Neoplasias do Colo/patologia , MicroRNAs/genética , Actinas/metabolismo , Adenocarcinoma/genética , Animais , Células CACO-2 , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias do Colo/genética , Transição Epitelial-Mesenquimal/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação/genética , Tubulina (Proteína)/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
PLoS Pathog ; 16(5): e1008489, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365140

RESUMO

Remodelling of the actin cytoskeleton in response to external stimuli is obligatory for many cellular processes in the amoebic cell. A rapid and local rearrangement of the actin cytoskeleton is required for the development of the cellular protrusions during phagocytosis, trogocytosis, migration, and invasion. Here, we demonstrated that EhC2B, a C2 domain-containing protein, is an actin modulator. EhC2B was first identified as an effector of EhRab21 from E. histolytica. In vitro interaction studies including GST pull-down, fluorescence-based assay and ITC also corroborated with our observation. In the amoebic trophozoites, EhC2B accumulates at the pseudopods and the tips of phagocytic cups. FRAP based studies confirmed the recruitment and dynamics of EhC2B at the phagocytic cup. Moreover, we have shown the role of EhC2B in erythrophagocytosis. It is well known that calcium-dependent signal transduction is essential for the cytoskeletal dynamics during phagocytosis in the amoebic parasite. Using liposome pelleting assay, we demonstrated that EhC2B preferentially binds to the phosphatidylserine in the presence of calcium. The EhC2B mutants defective in calcium or lipid-binding failed to localise beneath the plasma membrane. The cells overexpressing these mutants have also shown a significant reduction in erythrophagocytosis. The role of EhC2B in erythrophagocytosis and pseudopod formation was also validated by siRNA-based gene knockdown approach. Finally, with the help of in vitro nucleation assay using fluorescence spectroscopy and total internal reflection fluorescence microscopy, we have established that EhC2B is an actin nucleator. Collectively, based on the results from the study, we propose that EhC2B acts like a molecular bridge which promotes membrane deformation via its actin nucleation activity during the progression of the phagocytic cup in a calcium-dependent manner.


Assuntos
Actinas/metabolismo , Citofagocitose , Entamoeba histolytica/metabolismo , Eritrócitos , Proteínas de Protozoários/metabolismo , Pseudópodes/metabolismo , Actinas/genética , Domínios C2 , Entamoeba histolytica/genética , Humanos , Proteínas de Protozoários/genética , Pseudópodes/genética
6.
F1000Res ; 9: 52, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32419926

RESUMO

A novel coronavirus recently identified in Wuhan, China (2019-nCoV) has expanded the number of highly pathogenic coronaviruses affecting humans. The 2019-nCoV represents a potential epidemic or pandemic threat, which requires a quick response for preparedness against this infection. The present report uses the informational spectrum methodology to identify the possible origin and natural host of the new virus, as well as putative therapeutic and vaccine targets. The performed in silico analysis indicates that the newly emerging 2019-nCoV is closely related to severe acute respiratory syndrome (SARS)-CoV and, to a lesser degree, Middle East respiratory syndrome (MERS)-CoV. Moreover, the well-known SARS-CoV receptor (ACE2) might be a putative receptor for the novel virus as well. Additional results indicated that civets and poultry are potential candidates for the natural reservoir of the 2019-nCoV, and that domain 288-330 of S1 protein from the 2019-nCoV represents promising therapeutic and/or vaccine target.


Assuntos
Betacoronavirus/química , Glicoproteína da Espícula de Coronavírus/química , Tropismo Viral , Actinas/química , Simulação por Computador , Infecções por Coronavirus/tratamento farmacológico , Humanos , Ibuprofeno/uso terapêutico , Coronavírus da Síndrome Respiratória do Oriente Médio , Pandemias , Peptidil Dipeptidase A/química , Pneumonia Viral/tratamento farmacológico , Receptores Virais/química , Vírus da SARS
7.
J Biol Regul Homeost Agents ; 34(1): 57-67, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32466632

RESUMO

Klotho is a putative aging suppressor gene that is primarily expressed in renal tubular epithelial cells. Its expression has been reported to protect against fibrosis in human chronic kidney disease. However, the roles of klotho in epithelial-mesenchymal transition (EMT) and renal fibrosis are yet to be elucidated. The present study aimed to investigate the putative roles of klotho in angiotensin (Ang) II-induced damage of renal tubular epithelial cells. NRK-52E rat cells were treated with various combinations of Ang II, the Ang-converting enzyme inhibitor fosinopril (Fos) and the Ang II receptor antagonist valsartan (Val). The levels of transforming growth factor (TGF)-ß1, soluble klotho, α-smooth muscle actin (α-SMA) and E-cadherin in NRK-52E culture supernatants were measured using enzyme-linked immunosorbent assays. Furthermore, the mRNA and protein expression of TGF-ß1, klotho, α-SMA and E-cadherin was detected using semiquantitative reverse transcription-polymerase chain reaction, immunocytochemistry and Western blot analysis. The results demonstrated that Ang II inhibited the expression of klotho and E-cadherin, while it upregulated the expression of TGF-ß1 and α-SMA, in NRK52E cells. Fos and/or Val were revealed to enhance klotho and E-cadherin expression, and suppress the expression of TGF-ß1 and α-SMA, compared with the Ang II-only group. Furthermore, a positive linear correlation was detected between the expression of klotho and E-cadherin, while negative linear correlations with klotho expression were detected for TGF-ß1 and α-SMA expression. In conclusion, the expression of klotho was demonstrated to be enhanced following treatment with Fos and Val in Ang II-treated NRK-52E cells. The present results indicate that klotho may be involved in the inhibition of Ang II-induced EMT in renal tubular epithelial cells. Therefore, klotho may serve as a protective factor in renal tubulointerstitial fibrosis and aid the treatment of chronic kidney disease (CKD) patients using precision therapy.


Assuntos
Células Epiteliais/citologia , Transição Epitelial-Mesenquimal , Glucuronidase/fisiologia , Sistema Renina-Angiotensina , Actinas/metabolismo , Angiotensina II/farmacologia , Animais , Caderinas/metabolismo , Linhagem Celular , Fibrose , Fosinopril/farmacologia , Humanos , Túbulos Renais/citologia , Ratos , Fator de Crescimento Transformador beta1/metabolismo , Valsartana/farmacologia
8.
PLoS One ; 15(4): e0231174, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267880

RESUMO

As the outermost organ, the skin can be damaged following injuries such as wounds and bacterial or viral infections, and such damage should be rapidly restored to defend the body against physical, chemical, and microbial assaults. However, the wound healing process can be delayed or prolonged by health conditions, including diabetes mellitus, venous stasis disease, ischemia, and even stress. In this study, we developed a vibrational cell culture model and investigated the effects of mechanical vibrations on human keratinocytes. The HaCaT cells were exposed to vibrations at a frequency of 45 Hz with accelerations of 0.8g for 2 h per day. The applied mechanical vibration did not affect cell viability or cell proliferation. Cell migratory activity did increase following exposure to vibration, but the change was not statistically significant. The results of immunostaining (F-actin), western blot (ERK1/2), and RT-qPCR (FGF-2, PDGF-B, HB-EGF, TGF-ß1, EGFR, and KGFR) analyses demonstrated that the applied vibration resulted in rearrangement of the cytoskeleton, leading to activation of ERK1/2, one of the MAPK signaling pathways, and upregulation of the gene expression levels of HB-EGF and EGFR. The results suggest that mechanical vibration may have wound healing potential and could be used as a mechanical energy-based treatment for enhancing wound healing efficiency.


Assuntos
Queratinócitos/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Estresse Fisiológico , Vibração/efeitos adversos , Cicatrização/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Técnicas de Cultura de Células/métodos , Linhagem Celular , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Receptores ErbB/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Humanos , Regulação para Cima
9.
Artigo em Chinês | MEDLINE | ID: mdl-32306670

RESUMO

Objective: To investigate the role of CD40/CD40L Pathway in the formation of silicosis fibrosis. Methods: Totally 64 inpatients were recruited and assigned to the silicosis group and the control group, 23 in each group. The alveolar lavage fluid was collected from all patients and isolated. The expression of CD40L protein was detected by Flow Cytometry. The level of IL-8、The IL-6、INF-γ and MCP-1 was detected by ELISA. Two groups of BALF were co-cultured with HFL-1 cells, the expression of Collagen I and α-SMA was detected by Immunohistochemistry. Results: Compared with the control group, CD40L was highly expressed on T lymphocyte cells in silicosis group (P<0.05) , and the contents of IL-8、The IL-6、INF-γand MCP-1 in Silicosis group were significantly higher than those in control group (P<0.05) . After co-culture of BALF and HFL-1 cells, the expression levels of Collagen I and α-SMA in Silicosis group were significantly higher than those in control group (P<0.05) . Conclusion: CD40-CD40L cross-linking system can promote the activation of T cells, release inflammatory factors, promote the synthesis of collagen I and α-SMA by fibroblasts, make the lung fibrous tissue proliferate, and lead to the formation of silicosis fibrosis.


Assuntos
Antígenos CD40/imunologia , Ligante de CD40/imunologia , Fibrose Pulmonar/imunologia , Silicose/imunologia , Actinas , Colágeno Tipo I , Células Matadoras Induzidas por Citocinas , Humanos , Pulmão/fisiopatologia , Linfócitos T/imunologia
10.
Proc Natl Acad Sci U S A ; 117(18): 9922-9931, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32312818

RESUMO

The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the protein encoded by the photoreceptor-specific gene C2orf71, which is mutated in inherited retinal dystrophy (RP54). We demonstrate that C2orf71/PCARE (photoreceptor cilium actin regulator) can interact with the Arp2/3 complex activator WASF3, and efficiently recruits it to the primary cilium. Ectopic coexpression of PCARE and WASF3 in ciliated cells results in the remarkable expansion of the ciliary tip. This process was disrupted by small interfering RNA (siRNA)-based down-regulation of an actin regulator, by pharmacological inhibition of actin polymerization, and by the expression of PCARE harboring a retinal dystrophy-associated missense mutation. Using human retinal organoids and mouse retina, we observed that a similar actin dynamics-driven process is operational at the base of the photoreceptor OS where the PCARE module and actin colocalize, but which is abrogated in Pcare -/- mice. The observation that several proteins involved in retinal ciliopathies are translocated to these expansions renders it a potential common denominator in the pathomechanisms of these hereditary disorders. Together, our work suggests that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane at the initiation of new outer segment disk formation.


Assuntos
Cílios/genética , Distrofias de Cones e Bastonetes/genética , Proteínas do Olho/genética , Segmento Externo da Célula Bastonete/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/genética , Animais , Cílios/patologia , Distrofias de Cones e Bastonetes/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Knockout , RNA Interferente Pequeno/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Segmento Externo da Célula Bastonete/patologia
11.
Proc Natl Acad Sci U S A ; 117(18): 10055-10066, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32312822

RESUMO

Synaptic activity in neurons leads to the rapid activation of genes involved in mammalian behavior. ATP-dependent chromatin remodelers such as the BAF complex contribute to these responses and are generally thought to activate transcription. However, the mechanisms keeping such "early activation" genes silent have been a mystery. In the course of investigating Mendelian recessive autism, we identified six families with segregating loss-of-function mutations in the neuronal BAF (nBAF) subunit ACTL6B (originally named BAF53b). Accordingly, ACTL6B was the most significantly mutated gene in the Simons Recessive Autism Cohort. At least 14 subunits of the nBAF complex are mutated in autism, collectively making it a major contributor to autism spectrum disorder (ASD). Patient mutations destabilized ACTL6B protein in neurons and rerouted dendrites to the wrong glomerulus in the fly olfactory system. Humans and mice lacking ACTL6B showed corpus callosum hypoplasia, indicating a conserved role for ACTL6B in facilitating neural connectivity. Actl6b knockout mice on two genetic backgrounds exhibited ASD-related behaviors, including social and memory impairments, repetitive behaviors, and hyperactivity. Surprisingly, mutation of Actl6b relieved repression of early response genes including AP1 transcription factors (Fos, Fosl2, Fosb, and Junb), increased chromatin accessibility at AP1 binding sites, and transcriptional changes in late response genes associated with early response transcription factor activity. ACTL6B loss is thus an important cause of recessive ASD, with impaired neuron-specific chromatin repression indicated as a potential mechanism.


Assuntos
Transtorno do Espectro Autista/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Hipocampo/patologia , Actinas/genética , Trifosfato de Adenosina/genética , Animais , Transtorno do Espectro Autista/patologia , Comportamento Animal/fisiologia , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Pareamento Cromossômico/genética , Pareamento Cromossômico/fisiologia , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Dendritos/genética , Dendritos/fisiologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Knockout , Mutação/genética , Neurônios/metabolismo , Neurônios/patologia , Fatores de Transcrição/genética
12.
PLoS One ; 15(4): e0232116, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32343719

RESUMO

Myosin XXI (Myo21) is a novel class of myosin present in all kinetoplastid parasites, such as Trypanosoma and Leishmania. This protein in Leishmania promastigotes is predominantly localized to the proximal region of the flagellum, and is involved in the flagellum assembly, cell motility and intracellular vesicle transport. As Myo21 contains two ubiquitin associated (UBA)-like domains (UBLD) in its amino acid sequence, we considered it of interest to analyze the role of these domains in the intracellular distribution and functions of this protein in Leishmania cells. In this context, we created green fluorescent protein (GFP)-conjugates of Myo21 constructs lacking one of the two UBLDs at a time or both the UBLDs as well as GFP-conjugates of only the two UBLDs and Myo21 tail lacking the two UBLDs and separately expressed them in the Leishmania cells. Our results show that unlike Myo21-GFP, Myo21-GFP constructs lacking either one or both the UBLDs failed to concentrate and co-distribute with actin in the proximal region of the flagellum. Nevertheless, the GFP conjugate of only the two UBLDs was found to predominantly localize to the flagellum base. Additionally, the cells that expressed only one or both the UBLDs-deleted Myo21-GFP constructs possessed shorter flagellum and displayed slower motility, compared to Myo21-GFP expressing cells. Further, the intracellular vesicle transport and cell growth were severely impaired in the cells that expressed both the UBLDs deleted Myo21-GFP construct, but in contrast, virtually no effect was observed on the intracellular vesicle transport and growth in the cells that expressed single UBLD deleted mutant proteins. Moreover, the observed slower growth of both the UBLDs-deleted Myo21-GFP expressing cells was primarily due to delayed G2/M phase caused by aberrant nuclear and daughter cell segregation during their cell division process. These results taken together clearly reveal that the presence of UBLDs in Myo21 are essentially required for its predominant localization to the flagellum base, and perhaps also in its involvement in the flagellum assembly and cell division. Possible role of UBLDs in involvement of Myo21 during Leishmania flagellum assembly and cell cycle is discussed.


Assuntos
Flagelos/metabolismo , Leishmania donovani/fisiologia , Miosinas/química , Miosinas/metabolismo , Actinas/metabolismo , Ciclo Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/metabolismo , Miosinas/genética , Domínios Proteicos , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Vesículas Transportadoras/metabolismo , Ubiquitina/metabolismo
13.
PLoS One ; 15(4): e0231905, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32315372

RESUMO

Myocardial fibrosis is often associated with cardiac hypertrophy; indeed, fibrosis is one of the most critical factors affecting prognosis. We aimed to identify the molecules involved in promoting fibrosis under hypertrophic stimuli. We previously established a rat model of cardiac hypertrophy by pulmonary artery banding, in which approximately half of the animals developed fibrosis in the right ventricle. Here, we first comprehensively analyzed mRNA expression in the right ventricle with or without fibrosis in pulmonary artery banding model rats by DNA microarray analysis (GSE141650 at NCBI GEO). The expression levels of 19 genes were up-regulated more than 1.5-fold in fibrotic hearts compared with non-fibrotic hearts. Among them, fibrosis growth factor (FGF) 23 showed one of the biggest increases in expression. Real-time PCR analysis also revealed that, among the FGF receptor (FGFR) family, FGFR1 was highly expressed in fibrotic hearts. We then found that FGF23 was expressed predominantly in cardiomyocytes, while FGFR1 was predominantly expressed in fibroblasts in the rat ventricle. Next, we added FGF23 and transforming growth factor (TGF)-ß1 (10-50 ng/mL of each) to isolated fibroblasts from normal adult rat ventricles and cultured them for three days. While FGF23 itself did not directly affect the expression levels of any fibrosis-related mRNAs, FGF23 enhanced the effect of TGF-ß1 on increasing the expression levels of α-smooth muscle actin (α-SMA) mRNA. This increase in xx-SMA mRNA levels due to the combination of TGF-ß1 and FGF23 was attenuated by the inhibition of FGFR1 or the knockdown of FGFR1 in fibroblasts. Thus, FGF23 synergistically promoted the activation of fibroblasts with TGF-ß1, transforming fibroblasts into myofibroblasts via FGFR1. Thus, we identified FGF23 as a paracrine factor secreted from cardiomyocytes to promote cardiac fibrosis under conditions in which TGF-ß1 is activated. FGF23 could be a possible target to prevent fibrosis following myocardial hypertrophy.


Assuntos
Fatores de Crescimento de Fibroblastos/farmacologia , Cardiopatias/patologia , Fator de Crescimento Transformador beta1/farmacologia , Regulação para Cima/efeitos dos fármacos , Actinas/genética , Actinas/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibrose , Cardiopatias/metabolismo , Masculino , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Pirróis/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo
14.
PLoS One ; 15(4): e0227393, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32236096

RESUMO

BACKGROUND: TBX5 is a transcription factor that has an important role in development of heart. TBX5 variants in the region encoding the T-box domain have been shown to cause cardiac defects, such as atrial septal defect or ventricular septal defect, while TBX5 variants have also been identified in a few cardiomyopathy patients and considered causative. We identified a TBX5 variant (c.791G>A, p.Arg264Lys), that is over-represented in cardiomyopathy patients. This variant is located outside of the T-box domain, and its pathogenicity has not been confirmed by functional analyses. OBJECTIVE: To investigate whether the TBX5 R264K is deleterious and could contribute to the pathogenesis of cardiomyopathy. METHODS AND RESULTS: We developed mice expressing Tbx5 R264K. Mice homozygous for this variant displayed compensated dilated cardiomyopathy; mild decreased fractional shortening, dilatation of the left ventricle, left ventricular wall thinning and increased heart weight without major heart structural disorders. There was no difference in activation of the ANF promotor, a transcriptional target of Tbx5, compared to wild-type. However, analysis of RNA isolated from left ventricular samples showed significant increases in the expression of Acta1 in left ventricle with concomitant increases in the protein level of ACTA1. CONCLUSIONS: Mice homozygous for Tbx5 R264K showed compensated dilated cardiomyopathy. Thus, TBX5 R264K may have a significant pathogenic role in some cardiomyopathy patients independently of T-box domain pathway.


Assuntos
Cardiomiopatia Dilatada/genética , Ventrículos do Coração/patologia , Miocárdio Ventricular não Compactado Isolado/genética , Proteínas com Domínio T/genética , Actinas/metabolismo , Animais , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/patologia , Criança , Modelos Animais de Doenças , Ecocardiografia , Feminino , Técnicas de Introdução de Genes , Testes Genéticos , Células HEK293 , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/crescimento & desenvolvimento , Heterozigoto , Humanos , Lactente , Recém-Nascido , Miocárdio Ventricular não Compactado Isolado/diagnóstico , Masculino , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único
15.
PLoS One ; 15(4): e0230818, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32315311

RESUMO

The microgravity conditions of prolonged spaceflight are known to result in skeletal muscle atrophy that leads to diminished functional performance. To assess if inhibition of the growth factor myostatin has potential to reverse these effects, mice were treated with a myostatin antibody while housed on the International Space Station. Grip strength of ground control mice increased 3.1% compared to baseline values over the 6 weeks of the study, whereas grip strength measured for the first time in space showed flight animals to be -7.8% decreased in strength compared to baseline values. Control mice in space exhibited, compared to ground-based controls, a smaller increase in DEXA-measured muscle mass (+3.9% vs +5.6% respectively) although the difference was not significant. All individual flight limb muscles analyzed (except for the EDL) weighed significantly less than their ground counterparts at the study end (range -4.4% to -28.4%). Treatment with myostatin antibody YN41 was able to prevent many of these space-induced muscle changes. YN41 was able to block the reduction in muscle grip strength caused by spaceflight and was able to significantly increase the weight of all muscles of flight mice (apart from the EDL). Muscles of YN41-treated flight mice weighed as much as muscles from Ground IgG mice, with the exception of the soleus, demonstrating the ability to prevent spaceflight-induced atrophy. Muscle gene expression analysis demonstrated significant effects of microgravity and myostatin inhibition on many genes. Gamt and Actc1 gene expression was modulated by microgravity and YN41 in opposing directions. Myostatin inhibition did not overcome the significant reduction of microgravity on femoral BMD nor did it increase femoral or vertebral BMD in ground control mice. In summary, myostatin inhibition may be an effective countermeasure to detrimental consequences of skeletal muscle under microgravity conditions.


Assuntos
Força Muscular/genética , Músculo Esquelético/fisiologia , Atrofia Muscular/genética , Miostatina/genética , Actinas/genética , Animais , Extremidades/fisiologia , Fêmur/fisiologia , Expressão Gênica/genética , Guanidinoacetato N-Metiltransferase/genética , Imunoglobulina G/genética , Camundongos , Camundongos Endogâmicos BALB C , Força Muscular/fisiologia , Atrofia Muscular/fisiopatologia , Voo Espacial/métodos , Ausência de Peso
16.
Medicine (Baltimore) ; 99(14): e19628, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32243390

RESUMO

BACKGROUND: Prostate cancer (PCa) is one of the leading causes of cancer-related death. In the present research, we adopted a comprehensive bioinformatics method to identify some biomarkers associated with the tumor progression and prognosis of PCa. METHODS: Differentially expressed genes (DEGs) analysis and weighted gene co-expression network analysis (WGCNA) were applied for exploring gene modules correlative with tumor progression and prognosis of PCa. Clinically Significant Modules were distinguished, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to Annotation, Visualization and Integrated Discovery (DAVID). Protein-protein interaction (PPI) networks were used in selecting potential hub genes. RNA-Seq data and clinical materials of prostate cancer from The Cancer Genome Atlas (TCGA) database were used for the identification and validation of hub genes. The significance of these genes was confirmed via survival analysis and immunohistochemistry. RESULTS: 2688 DEGs were filtered. Weighted gene co-expression network was constructed, and DEGs were divided into 6 modules. Two modules were selected as hub modules which were highly associated with the tumor grades. Functional enrichment analysis was performed on genes in hub modules. Thirteen hub genes in these hub modules were identified through PPT networks. Based on TCGA data, 4 of them (CCNB1, TTK, CNN1, and ACTG2) were correlated with prognosis. The protein levels of CCNB1, TTK, and ACTG2 had a degree of differences between tumor tissues and normal tissues. CONCLUSION: Four hub genes were identified as candidate biomarkers and potential therapeutic targets for further studies of exploring molecular mechanisms and individual therapy on PCa.


Assuntos
Biomarcadores Tumorais/análise , Redes Reguladoras de Genes/genética , Neoplasias da Próstata/genética , Actinas/análise , Proteínas de Ligação ao Cálcio/análise , Proteínas de Ciclo Celular/análise , Biologia Computacional/métodos , Ciclina B1/análise , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Humanos , Masculino , Proteínas dos Microfilamentos/análise , Gradação de Tumores , Prognóstico , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/análise , Proteínas Tirosina Quinases/análise
18.
Chem Biol Interact ; 323: 109063, 2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-32224134

RESUMO

Exposure to TiO2 NPs induces several cellular alterations after NPs uptake including disruption of cytoskeleton that is crucial for lung physiology but is not considered as a footprint of cell damage. We aimed to investigate cytoskeleton disturbances and the impact on cell migration induced by an acute TiO2 NPs exposure (24 h) and the recovery capability after 6 days of NPs-free treatment, which allowed investigating if cytoskeleton damage was reversible. Exposure to TiO2 NPs (10 µg/cm2) for 24 h induced a decrease 20.2% and 25.1% in tubulin and actin polymerization. Exposure to TiO2 NPs (10 µg/cm2) for 24 h followed by 6 days of NPs-free had a decrease of 26.6% and 21.3% in tubulin and actin polymerization, respectively. The sustained exposure for 7 days to 1 µg/cm2 and 10 µg/cm2 induced a decrease of 22.4% and 30.7% of tubulin polymerization respectively, and 28.7% and 46.2% in actin polymerization. In addition, 24 h followed 6 days of NPs-free exposure of TiO2 NPs (1 µg/cm2 and 10 µg/cm2) decreased cell migration 40.7% and 59.2%, respectively. Cells exposed (10 µg/cm2) for 7 days had a decrease of 65.5% in cell migration. Ki67, protein surfactant B (SFTPB) and matrix metalloprotease 2 (MMP2) were analyzed as genes related to lung epithelial function. The results showed a 20% of Ki67 upregulation in cells exposed for 24 h to 10 µg/cm2 TiO2 NPs while a downregulation of 20% and 25.8% in cells exposed to 1 µg/cm2 and 10 µg/cm2 for 24 h followed by 6 days of NPs-free exposure. Exposure to 1 µg/cm2 and 10 µg/cm2 for 24 h and 7 days upregulates SFTPB expression in 53% and 59% respectively, MMP2 expression remain unchanged. In conclusion, exposure of TiO2 NPs affected cytoskeleton of lung epithelial cells irreversibly but this damage was not cumulative.


Assuntos
Citoesqueleto/patologia , Células Epiteliais/patologia , Pulmão/patologia , Nanopartículas/toxicidade , Titânio/toxicidade , Células A549 , Actinas/metabolismo , Movimento Celular/efeitos dos fármacos , Tamanho Celular , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Endocitose , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Antígeno Ki-67/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Nanopartículas/ultraestrutura , Polimerização , Precursores de Proteínas/metabolismo , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Tubulina (Proteína)/metabolismo
19.
Gene ; 744: 144635, 2020 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-32244053

RESUMO

Spermatogenesis is a highly complex physiological process which contains spermatogonia proliferation, spermatocyte meiosis and spermatid morphogenesis. In the past decade, actin binding proteins and signaling pathways which are critical for regulating the actin cytoskeleton in testis had been found. In this review, we summarized 5 actin-binding proteins that have been proven to play important roles in the seminiferous epithelium. Lack of them perturbs spermatids polarity and the transport of spermatids. The loss of Arp2/3 complex, Formin1, Eps8, Palladin and Plastin3 cause sperm release failure suggesting their irreplaceable role in spermatogenesis. Actin regulation relies on multiple signal pathways. The PI3K/Akt signaling pathway positively regulate the mTOR pathway to promote actin reorganization in seminiferous epithelium. Conversely, TSC1/TSC2 complex, the upstream of mTOR, is activated by the LKB1/AMPK pathway to inhibit cell proliferation, differentiation and migration. The increasing researches focus on the function of actin binding proteins (ABPs), however, their collaborative regulation of actin patterns and potential regulatory signaling networks remains unclear. We reviewed ABPs that play important roles in mammalian spermatogenesis and signal pathways involved in the regulation of microfilaments. We suggest that more relevant studies should be performed in the future.


Assuntos
Citoesqueleto de Actina/metabolismo , Espermatogênese , Proteínas Quinases Ativadas por AMP/metabolismo , Actinas/metabolismo , Animais , Humanos , Masculino , Camundongos , Proteínas dos Microfilamentos/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais
20.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(1): 20-25, 2020 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-32314720

RESUMO

Objective To investigate the role of CXCR2 in the cerebral endothelial activation and migration of neutrophils into the brain in septic encephalopathy (SE) induced by lipopolysaccharide (LPS). Methods C57BL/6J mice and CXCR2-knockout mice were randomly divided into a normal control group, a wildtype mice group with LPS treatment and CXCR2-knockout mice group with LPS treatment. Mouse SE models were induced by intraperitoneal LPS injection. Naphthol AS-D chloroacetate histochemical staining of the brain section was performed to quantitate the neutrophils infiltrating into the cerebral cortex. TNF-α and CXCL1 concentrations in the brain and plasma were determined by ELISA. After the stimulation of LPS (1 µg/mL) and TNF-α (200 ng/mL), the levels of CXCR2 protein in the primary mouse brain microvascular endothelial cells isolated from the cerebral cortex were detected by Western blotting. The levels of F-actin and vascular cell adhesion molecule 1 (VCAM-1) protein in the primary mouse brain microvascular endothelial cells stimulated by CXCL1 (100 ng/mL) were detected by Western blotting. Results After intraperitoneal LPS injection, there was a significant increase in the level of TNF-α in the brain and plasma and there was also an evident increase in the level of CXCL1 in the brain of wild type mice (C57BL/6J mice). And intraperitoneal LPS injection caused increased neutrophil infiltration into the cerebral cortex in the wild type mice (C57BL/6J mice). But CXCR2-knockout mice displayed evidently reduced neutrophil infiltration into the cerebral cortex compared with the wildtype mice. In vitro LPS and TNF-α upregulated the expression of CXCR2 in the primary brain microvascular endothelial cells. CXCL1 increased remarkably the expression of endothelial F-actin and VCAM-1. Conclusion In the SE model, CXCR2 participates in the cerebral endothelial activation and neutrophil migration into the brain.


Assuntos
Encefalopatias/metabolismo , Movimento Celular , Células Endoteliais/citologia , Neutrófilos/citologia , Receptores de Interleucina-8B/metabolismo , Sepse/metabolismo , Actinas/metabolismo , Animais , Encefalopatias/patologia , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Distribuição Aleatória , Sepse/patologia , Molécula 1 de Adesão de Célula Vascular/metabolismo
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