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1.
Shanghai Kou Qiang Yi Xue ; 29(4): 337-342, 2020 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-33089278

RESUMO

PURPOSE: Based on the Cre-Loxp gene knockout system, this study intended to construct tamoxifen-inducible STAT3 conditional knockout mice and verify the knockout efficiency. METHODS: The inducible osteoblasts-specific Stat3 knockout mice Stat3Col1ERT2 were obtained by hybridization through C57 mice of Stat3fl/fl and Col1 creERT2. Bone mesenchymal stem cells(BMSCs) of these mice were isolated and cultured with or without 4-hydroxytamoxin(4-OTH), to verify the effect of Stat3 knockout in vitro by real-time quantitative PCR and Western blotting in the level of mRNA and protein. Meanwhile, wild type and Stat3Col1ERT2 mice were both intraperitoneally injected with tamoxifen, the expression of STAT3 in the maxillary alveolar bone was observed by immunofluorescent staining to confirm the knockout effect in vivo. Statistical analysis was conducted with SPSS 24.0 software package. RESULTS: Real-time quantitative PCR and Western blotting results demonstrated that mRNA(P<0.05) and protein levels of STAT3 were significantly decreased (P<0.05) in BMSCs derived from Stat3Col1ERT2 mice by 4-OHT induced knockout in vitro. Immunofluorescent staining indicated that STAT3 expression was significantly reduced(P<0.05) in osteoblasts of the maxillary alveolar bone in Stat3Col1ERT2 mice. CONCLUSIONS: This study successfully constructed the inducible osteoblasts-specific Stat3 gene knockout mice, which helped investigators control the time and space of gene knockout, therefore providing new insights and guidance for research fields of orthodontic tooth movement, distraction osteogenesis and jaw fractures in the future.


Assuntos
Camundongos Knockout , Osteoblastos , Fator de Transcrição STAT3 , Técnicas de Movimentação Dentária , Animais , Técnicas de Inativação de Genes , Camundongos , RNA Mensageiro
2.
Nat Commun ; 11(1): 4913, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004813

RESUMO

Reprograming of proline metabolism is critical for tumor growth. Here we show that PINCH-1 is highly expressed in lung adenocarcinoma and promotes proline synthesis through regulation of mitochondrial dynamics. Knockout (KO) of PINCH-1 increases dynamin-related protein 1 (DRP1) expression and mitochondrial fragmentation, which suppresses kindlin-2 mitochondrial translocation and interaction with pyrroline-5-carboxylate reductase 1 (PYCR1), resulting in inhibition of proline synthesis and cell proliferation. Depletion of DRP1 reverses PINCH-1 deficiency-induced defects on mitochondrial dynamics, proline synthesis and cell proliferation. Furthermore, overexpression of PYCR1 in PINCH-1 KO cells restores proline synthesis and cell proliferation, and suppresses DRP1 expression and mitochondrial fragmentation. Finally, ablation of PINCH-1 from lung adenocarcinoma in mouse increases DRP1 expression and inhibits PYCR1 expression, proline synthesis, fibrosis and tumor growth. Our results identify a signaling axis consisting of PINCH-1, DRP1 and PYCR1 that regulates mitochondrial dynamics and proline synthesis, and suggest an attractive strategy for alleviation of tumor growth.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma de Pulmão/patologia , Proteínas com Domínio LIM/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Membrana/metabolismo , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Proliferação de Células/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Dinaminas/metabolismo , Feminino , Técnicas de Inativação de Genes , Humanos , Proteínas com Domínio LIM/genética , Pulmão/citologia , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Proteínas Musculares/metabolismo , Proteínas de Neoplasias/metabolismo , Prolina/biossíntese , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirrolina Carboxilato Redutases/metabolismo , Análise de Sobrevida
3.
Nat Commun ; 11(1): 4484, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901027

RESUMO

Chronic stress is a key risk factor for mood disorders like depression, but the stress-induced changes in brain circuit function and gene expression underlying depression symptoms are not completely understood, hindering development of novel treatments. Because of its projections to brain regions regulating reward and anxiety, the ventral hippocampus is uniquely poised to translate the experience of stress into altered brain function and pathological mood, though the cellular and molecular mechanisms of this process are not fully understood. Here, we use a novel method of circuit-specific gene editing to show that the transcription factor ΔFosB drives projection-specific activity of ventral hippocampus glutamatergic neurons causing behaviorally diverse responses to stress. We establish molecular, cellular, and circuit-level mechanisms for depression- and anxiety-like behavior in response to stress and use circuit-specific gene expression profiling to uncover novel downstream targets as potential sites of therapeutic intervention in depression.


Assuntos
Aprendizagem da Esquiva/fisiologia , Hipocampo/fisiologia , Proteínas Proto-Oncogênicas c-fos/fisiologia , Animais , Ansiedade/metabolismo , Comportamento Animal/fisiologia , Técnicas de Inativação de Genes , Inativação Gênica , Hipocampo/anatomia & histologia , Hipocampo/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-fos/deficiência , Proteínas Proto-Oncogênicas c-fos/genética , Comportamento Social , Estresse Psicológico
4.
Chem Biol Interact ; 330: 109236, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32866467

RESUMO

A series of novel pyrrolopyrimidine urea derivatives were synthesized and evaluated for their anticancer activity against colon cancer cell lines. Compounds showed the remarkable cytotoxic activity on HCT-116 wt cell line. The most potent compound 4c (IC50 = 0.14 µM) induced apoptosis in HCT-116 wt and HCT-116 p53-/- cell lines. Otherwise, treatment of HCT-116 BAX-/-BAK-/- cells with compound 4c didn't lead to activation of apoptosis, suggesting that compound 4c induces apoptotic cell death by activating BAX/BAK-dependent pathway. Moreover, while the compound 4c increase the activation of caspase-3 and caspase-9 levels in HCT-116 wt and HCT-116 p53-/- cells, caspase-3 or caspase-9 activation was not observed in HCT-116 BAX-/-BAK-/- cells. In addition, compound 4c induced mitochondrial apoptosis in cells grown as oncospheroids, which better mimic the in vivo milieu of tumors. 4c treatment also activated JNK along with inhibition of prosurvival kinases such as Akt and ERK 1/2 in HCT-116 wt and HCT-116 p53 -/- cells as well as in HCT-116 BAX-/-BAK-/- cells. Notably, our results indicated that compound 4c induced mitochondrial apoptosis through activation p53-independent apoptotic signaling pathways.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Pirimidinas/farmacologia , Pirróis/farmacologia , Proteína Supressora de Tumor p53/fisiologia , Caspase 3/genética , Caspase 9/genética , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Técnicas de Inativação de Genes , Células HCT116 , Humanos , Mitocôndrias/metabolismo , Pirimidinas/síntese química , Pirimidinas/química , Pirróis/síntese química , Pirróis/química , Proteína Supressora de Tumor p53/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genética
6.
PLoS Pathog ; 16(9): e1008834, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32956422

RESUMO

Despite the widespread use of anti-retroviral therapy, human immunodeficiency virus (HIV) still persists in an infected cell reservoir that harbors transcriptionally silent yet replication-competent proviruses. While significant progress has been made in understanding how the HIV reservoir is established, transcription repression mechanisms that are enforced on the integrated viral promoter have not been fully revealed. In this study, we performed a whole-genome CRISPR knockout screen in HIV infected T cells to identify host genes that potentially promote HIV latency. Of several top candidates, the KRAB-containing zinc finger protein, ZNF304, was identified as the top hit. ZNF304 silences HIV gene transcription through associating with TRIM28 and recruiting to the viral promoter heterochromatin-inducing methyltransferases, including the polycomb repression complex (PRC) and SETB1. Depletion of ZNF304 expression reduced levels of H3K9me3, H3K27me3 and H2AK119ub repressive histone marks on the HIV promoter as well as SETB1 and TRIM28, ultimately enhancing HIV gene transcription. Significantly, ZNF304 also promoted HIV latency, as its depletion delayed the entry of HIV infected cells into latency. In primary CD4+ cells, ectopic expression of ZNF304 silenced viral transcription. We conclude that by associating with TRIM28 and recruiting host transcriptional repressive complexes, SETB1 and PRC, to the HIV promoter, ZNF304 silences HIV gene transcription and promotes viral latency.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Regulação Viral da Expressão Gênica , Inativação Gênica , HIV-1/fisiologia , Proteínas Repressoras , Fatores de Transcrição , Transcrição Genética , Latência Viral , Linfócitos T CD4-Positivos/virologia , Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Estudo de Associação Genômica Ampla , Humanos , Células Jurkat , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismo
7.
Nat Commun ; 11(1): 4818, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968060

RESUMO

Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. We show that T-Plastin widens and lengthens protrusions and is specifically enriched in active protrusions where F-actin is devoid of non-muscle myosin II activity. Together, our study uncovers critical roles of the actin bundler T-Plastin to promote protrusions and migration when adhesion is spatially-gapped.


Assuntos
Movimento Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Sistemas CRISPR-Cas , Adesão Celular , Linhagem Celular , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Técnicas de Inativação de Genes , Humanos , Cinética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/ultraestrutura , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/ultraestrutura , Miosinas/metabolismo , Pseudópodes/metabolismo , Receptor EphB2
8.
Nat Commun ; 11(1): 4813, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968076

RESUMO

Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria; however, resistance threatens to undermine global control efforts. To broadly explore artemisinin susceptibility in apicomplexan parasites, we employ genome-scale CRISPR screens recently developed for Toxoplasma gondii to discover sensitizing and desensitizing mutations. Using a sublethal concentration of dihydroartemisinin (DHA), we uncover the putative transporter Tmem14c whose disruption increases DHA susceptibility. Screens performed under high doses of DHA provide evidence that mitochondrial metabolism can modulate resistance. We show that disrupting a top candidate from the screens, the mitochondrial protease DegP2, lowers porphyrin levels and decreases DHA susceptibility, without significantly altering parasite fitness in culture. Deleting the homologous gene in P. falciparum, PfDegP, similarly lowers heme levels and DHA susceptibility. These results expose the vulnerability of heme metabolism to genetic perturbations that can lead to increased survival in the presence of DHA.


Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Resistência a Medicamentos/genética , Testes Genéticos/métodos , Heme/genética , Heme/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Técnicas de Inativação de Genes , Humanos , Malária Falciparum/tratamento farmacológico , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Toxoplasma/efeitos dos fármacos , Toxoplasma/genética
9.
Nat Commun ; 11(1): 4820, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973160

RESUMO

Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.


Assuntos
Tirosina-tRNA Ligase/genética , Tirosina-tRNA Ligase/metabolismo , Tirosina/análogos & derivados , Tirosina/genética , Tirosina/metabolismo , Animais , Sistemas CRISPR-Cas , Quimiocinas/metabolismo , Cristalografia por Raios X , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Tirosina-tRNA Ligase/química
10.
Nat Commun ; 11(1): 4837, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973183

RESUMO

ATP synthesis and thermogenesis are two critical outputs of mitochondrial respiration. How these outputs are regulated to balance the cellular requirement for energy and heat is largely unknown. Here we show that major facilitator superfamily domain containing 7C (MFSD7C) uncouples mitochondrial respiration to switch ATP synthesis to thermogenesis in response to heme. When heme levels are low, MSFD7C promotes ATP synthesis by interacting with components of the electron transport chain (ETC) complexes III, IV, and V, and destabilizing sarcoendoplasmic reticulum Ca2+-ATPase 2b (SERCA2b). Upon heme binding to the N-terminal domain, MFSD7C dissociates from ETC components and SERCA2b, resulting in SERCA2b stabilization and thermogenesis. The heme-regulated switch between ATP synthesis and thermogenesis enables cells to match outputs of mitochondrial respiration to their metabolic state and nutrient supply, and represents a cell intrinsic mechanism to regulate mitochondrial energy metabolism.


Assuntos
Trifosfato de Adenosina/metabolismo , Heme/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Receptores Virais/metabolismo , Termogênese/fisiologia , Animais , Deficiência de Citocromo-c Oxidase , Complexo III da Cadeia de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons , Metabolismo Energético/fisiologia , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Domínios Proteicos , Receptores Virais/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Células THP-1
11.
Proc Natl Acad Sci U S A ; 117(37): 23182-23190, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32873645

RESUMO

Enzyme turnover numbers (k cats) are essential for a quantitative understanding of cells. Because k cats are traditionally measured in low-throughput assays, they can be inconsistent, labor-intensive to obtain, and can miss in vivo effects. We use a data-driven approach to estimate in vivo k cats using metabolic specialist Escherichia coli strains that resulted from gene knockouts in central metabolism followed by metabolic optimization via laboratory evolution. By combining absolute proteomics with fluxomics data, we find that in vivo k cats are robust against genetic perturbations, suggesting that metabolic adaptation to gene loss is mostly achieved through other mechanisms, like gene-regulatory changes. Combining machine learning and genome-scale metabolic models, we show that the obtained in vivo k cats predict unseen proteomics data with much higher precision than in vitro k cats. The results demonstrate that in vivo k cats can solve the problem of inconsistent and low-coverage parameterizations of genome-scale cellular models.


Assuntos
Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Técnicas de Inativação de Genes/métodos , Genoma/genética , Cinética , Aprendizado de Máquina , Modelos Biológicos , Proteômica/métodos
12.
Nat Commun ; 11(1): 4687, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948771

RESUMO

Chemical biology strategies for directly perturbing protein homeostasis including the degradation tag (dTAG) system provide temporal advantages over genetic approaches and improved selectivity over small molecule inhibitors. We describe dTAGV-1, an exclusively selective VHL-recruiting dTAG molecule, to rapidly degrade FKBP12F36V-tagged proteins. dTAGV-1 overcomes a limitation of previously reported CRBN-recruiting dTAG molecules to degrade recalcitrant oncogenes, supports combination degrader studies and facilitates investigations of protein function in cells and mice.


Assuntos
Peptídeo Hidrolases/metabolismo , Proteínas/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Animais , Feminino , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Modelos Animais , Proteômica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/metabolismo , Proteínas de Ligação a Tacrolimo , Proteína Supressora de Tumor Von Hippel-Lindau/genética
13.
Sci Adv ; 6(33): eabb7238, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32851183

RESUMO

Cigarette smoking, the leading cause of chronic obstructive pulmonary disease (COPD), has been implicated as a risk factor for severe disease in patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we show that mice with lung epithelial cell-specific loss of function of Miz1, which we identified as a negative regulator of nuclear factor κB (NF-κB) signaling, spontaneously develop progressive age-related changes resembling COPD. Furthermore, loss of Miz1 up-regulates the expression of Ace2, the receptor for SARS-CoV-2. Concomitant partial loss of NF-κB/RelA prevented the development of COPD-like phenotype in Miz1-deficient mice. Miz1 protein levels are reduced in the lungs from patients with COPD, and in the lungs of mice exposed to chronic cigarette smoke. Our data suggest that Miz1 down-regulation-induced sustained activation of NF-κB-dependent inflammation in the lung epithelium is sufficient to induce progressive lung and airway destruction that recapitulates features of COPD, with implications for COVID-19.


Assuntos
Células Epiteliais/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Pulmão/metabolismo , Peptidil Dipeptidase A/metabolismo , Fenótipo , Proteínas Inibidoras de STAT Ativados/genética , Doença Pulmonar Obstrutiva Crônica/genética , Ubiquitina-Proteína Ligases/genética , Regulação para Cima/genética , Animais , Betacoronavirus , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pandemias , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Proteínas Inibidoras de STAT Ativados/metabolismo , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Transdução de Sinais/genética , Fumar/efeitos adversos , Fator de Transcrição RelA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
14.
Plant Mol Biol ; 104(3): 297-307, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32748081

RESUMO

KEY MESSAGE: We have developed multiplex genome editing toolkits for citrus that significantly improve citrus genome editing efficacy. CRISPR/Cas systems have been engineered for genome editing in many organisms, including plants. However, the gene editing efficiency in citrus via CRISPR technology remains too low to be implemented for genetic improvement in practice. Moreover, it is very difficult to obtain homozygous or biallelic knockout mutants in citrus. Here, we have developed multiplex genome editing toolkits for citrus including PEG-mediated protoplast transformation, a GFP reporter system that allows the rapid assessment of CRISPR constructs, citrus U6 promoters with improved efficacy, and tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 44.4% and a homozygous mutation rate of 11.1%, representing a significant improvement in citrus genome editing efficacy. In addition, our study lays the foundation for nontransgenic genome editing of citrus.


Assuntos
Citrus/genética , Edição de Genes/métodos , Genoma de Planta/genética , Homozigoto , Mutação , Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Genes de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Protoplastos , RNA Guia/genética , RNA de Transferência/genética
15.
Sheng Wu Gong Cheng Xue Bao ; 36(7): 1386-1394, 2020 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-32748596

RESUMO

We used CRISPR/Cas9 to delete plin1 of 3T3-L1 preadipocyte, to observe its effect on lipolysis in adipocytes and to explore regulatory pathways. We cultured 3T3-L1 preadipocytes, and the plin1 knockout vectors were transfected by electroporation. Puromycin culture was used to screen successfully transfected adipocytes, and survival rates were observed after transfection. The optimized "cocktail" method was used to differentiate 3T3-L1 preadipocytes. The glycerol and triglyceride contents were determined by enzymatic methods. The changes in lipid droplet form and size were observed by Oil red O staining. The protein expression of PLIN1, PPARγ, Fsp27, and lipases was measured by Western blotting. RT-PCR was used to measure the expression of PLIN1 and lipases mRNA. After the adipocytes in the control group were induced to differentiate, the quantity of tiny lipid droplets was decreased, and the quantity of unilocular lipid droplets was increased and arranged in a circle around the nucleus. Compared with the control group, the volume of unilocular lipid droplets decreased, and the quantity of tiny lipid droplets increased after induction of adipocytes in the knockout group. The expression of PLIN1 mRNA and protein in the adipocytes was significantly inhibited (P<0.05); glycerol levels increased significantly (0.098 4±0.007 6), TG levels decreased significantly (0.031 0±0.005 3); mRNA and protein expression of HSL and ATGL increased (P<0.05); PPARγ and Fsp27 expression unchanged in adipocytes. The above results indicate that the knockout of plin1 enhances the lipolysis of 3T3-L1 adipocytes by exposing lipids in lipid droplets and up-regulating lipases effects.


Assuntos
Sistemas CRISPR-Cas , Lipólise , Perilipina-1 , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Técnicas de Inativação de Genes , Lipase/metabolismo , Lipólise/genética , Camundongos , Perilipina-1/genética , Perilipina-1/metabolismo
16.
Nat Commun ; 11(1): 4150, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811819

RESUMO

The systemic decline in autophagic activity with age impairs homeostasis in several tissues, leading to age-related diseases. A mechanistic understanding of adipocyte dysfunction with age could help to prevent age-related metabolic disorders, but the role of autophagy in aged adipocytes remains unclear. Here we show that, in contrast to other tissues, aged adipocytes upregulate autophagy due to a decline in the levels of Rubicon, a negative regulator of autophagy. Rubicon knockout in adipocytes causes fat atrophy and hepatic lipid accumulation due to reductions in the expression of adipogenic genes, which can be recovered by activation of PPARγ. SRC-1 and TIF2, coactivators of PPARγ, are degraded by autophagy in a manner that depends on their binding to GABARAP family proteins, and are significantly downregulated in Rubicon-ablated or aged adipocytes. Hence, we propose that age-dependent decline in adipose Rubicon exacerbates metabolic disorders by promoting excess autophagic degradation of SRC-1 and TIF2.


Assuntos
Adipócitos/metabolismo , Envelhecimento/fisiologia , Autofagia/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Doenças Metabólicas/metabolismo , Adipócitos/patologia , Adipogenia/genética , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Adiposidade/genética , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Técnicas de Inativação de Genes , Glucose/genética , Glucose/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Coativador 1 de Receptor Nuclear/metabolismo , Coativador 2 de Receptor Nuclear/metabolismo , PPAR gama/metabolismo
17.
Nat Commun ; 11(1): 4332, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859902

RESUMO

The group of enteroviruses contains many important pathogens for humans, including poliovirus, coxsackievirus, rhinovirus, as well as newly emerging global health threats such as EV-A71 and EV-D68. Here, we describe an unbiased, system-wide and time-resolved analysis of the proteome and phosphoproteome of human cells infected with coxsackievirus B3. Of the ~3,200 proteins quantified throughout the time course, a large amount (~25%) shows a significant change, with the majority being downregulated. We find ~85% of the detected phosphosites to be significantly regulated, implying that most changes occur at the post-translational level. Kinase-motif analysis reveals temporal activation patterns of certain protein kinases, with several CDKs/MAPKs immediately active upon the infection, and basophilic kinases, ATM, and ATR engaging later. Through bioinformatics analysis and dedicated experiments, we identify mTORC1 signalling as a major regulation network during enterovirus infection. We demonstrate that inhibition of mTORC1 activates TFEB, which increases expression of lysosomal and autophagosomal genes, and that TFEB activation facilitates the release of virions in extracellular vesicles via secretory autophagy. Our study provides a rich framework for a system-level understanding of enterovirus-induced perturbations at the protein and signalling pathway levels, forming a base for the development of pharmacological inhibitors to treat enterovirus infections.


Assuntos
Infecções por Coxsackievirus/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Proteoma/análise , Animais , Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular , Sobrevivência Celular , Enterovirus/fisiologia , Enterovirus Humano B/fisiologia , Técnicas de Inativação de Genes , Células HeLa , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Fosforilação , Transdução de Sinais , Proteínas Virais/metabolismo
18.
Nat Commun ; 11(1): 4321, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859904

RESUMO

Bacterial colonization of the human intestine requires firm adhesion of bacteria to insoluble substrates under hydrodynamic flow. Here we report the molecular mechanism behind an ultrastable protein complex responsible for resisting shear forces and adhering bacteria to cellulose fibers in the human gut. Using single-molecule force spectroscopy (SMFS), single-molecule FRET (smFRET), and molecular dynamics (MD) simulations, we resolve two binding modes and three unbinding reaction pathways of a mechanically ultrastable R. champanellensis (Rc) Dockerin:Cohesin (Doc:Coh) complex. The complex assembles in two discrete binding modes with significantly different mechanical properties, with one breaking at ~500 pN and the other at ~200 pN at loading rates from 1-100 nN s-1. A neighboring X-module domain allosterically regulates the binding interaction and inhibits one of the low-force pathways at high loading rates, giving rise to a catch bonding mechanism that manifests under force ramp protocols. Multi-state Monte Carlo simulations show strong agreement with experimental results, validating the proposed kinetic scheme. These results explain mechanistically how gut microbes regulate cell adhesion strength at high shear stress through intricate molecular mechanisms including dual-binding modes, mechanical allostery and catch bonds.


Assuntos
Aderência Bacteriana/fisiologia , Microbioma Gastrointestinal/fisiologia , Fenômenos Mecânicos , Fenômenos Físicos , Bactérias , Aderência Bacteriana/genética , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Trato Gastrointestinal/microbiologia , Técnicas de Inativação de Genes , Humanos , Cinética , Simulação de Dinâmica Molecular , Método de Monte Carlo , Ligação Proteica , Conformação Proteica , Imagem Individual de Molécula , Estresse Mecânico
19.
Nature ; 584(7822): 589-594, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32814899

RESUMO

The inner surfaces of the human heart are covered by a complex network of muscular strands that is thought to be a remnant of embryonic development1,2. The function of these trabeculae in adults and their genetic architecture are unknown. Here we performed a genome-wide association study to investigate image-derived phenotypes of trabeculae using the fractal analysis of trabecular morphology in 18,096 participants of the UK Biobank. We identified 16 significant loci that contain genes associated with haemodynamic phenotypes and regulation of cytoskeletal arborization3,4. Using biomechanical simulations and observational data from human participants, we demonstrate that trabecular morphology is an important determinant of cardiac performance. Through genetic association studies with cardiac disease phenotypes and Mendelian randomization, we find a causal relationship between trabecular morphology and risk of cardiovascular disease. These findings suggest a previously unknown role for myocardial trabeculae in the function of the adult heart, identify conserved pathways that regulate structural complexity and reveal the influence of the myocardial trabeculae on susceptibility to cardiovascular disease.


Assuntos
Doenças Cardiovasculares/genética , Fractais , Predisposição Genética para Doença , Coração/anatomia & histologia , Coração/fisiologia , Miocárdio/metabolismo , Adulto , Idoso , Animais , Doenças Cardiovasculares/fisiopatologia , Citoesqueleto/genética , Citoesqueleto/fisiologia , Técnicas de Inativação de Genes , Loci Gênicos/genética , Estudo de Associação Genômica Ampla , Coração/embriologia , Hemodinâmica , Humanos , Pessoa de Meia-Idade , Miocárdio/citologia , Oryzias/embriologia , Oryzias/genética , Fenótipo
20.
Science ; 369(6503): 524-530, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32732418

RESUMO

RNA molecules are frequently modified with a terminal 2',3'-cyclic phosphate group as a result of endonuclease cleavage, exonuclease trimming, or de novo synthesis. During pre-transfer RNA (tRNA) and unconventional messenger RNA (mRNA) splicing, 2',3'-cyclic phosphates are substrates of the tRNA ligase complex, and their removal is critical for recycling of tRNAs upon ribosome stalling. We identified the predicted deadenylase angel homolog 2 (ANGEL2) as a human phosphatase that converts 2',3'-cyclic phosphates into 2',3'-OH nucleotides. We analyzed ANGEL2's substrate preference, structure, and reaction mechanism. Perturbing ANGEL2 expression affected the efficiency of pre-tRNA processing, X-box-binding protein 1 (XBP1) mRNA splicing during the unfolded protein response, and tRNA nucleotidyltransferase 1 (TRNT1)-mediated CCA addition onto tRNAs. Our results indicate that ANGEL2 is involved in RNA pathways that rely on the ligation or hydrolysis of 2',3'-cyclic phosphates.


Assuntos
Nucleotidases/química , Ribonucleases/química , Cristalografia por Raios X , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Nucleotidases/genética , Estrutura Secundária de Proteína , Precursores de RNA , Processamento de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleases/genética , Especificidade por Substrato , Proteína 1 de Ligação a X-Box/genética
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