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1.
Int J Mol Sci ; 22(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34502139

RESUMO

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causative agent of the COVID19 pandemic. The SARS-CoV-2 genome encodes for a small accessory protein termed Orf9b, which targets the mitochondrial outer membrane protein TOM70 in infected cells. TOM70 is involved in a signaling cascade that ultimately leads to the induction of type I interferons (IFN-I). This cascade depends on the recruitment of Hsp90-bound proteins to the N-terminal domain of TOM70. Binding of Orf9b to TOM70 decreases the expression of IFN-I; however, the underlying mechanism remains elusive. We show that the binding of Orf9b to TOM70 inhibits the recruitment of Hsp90 and chaperone-associated proteins. We characterized the binding site of Orf9b within the C-terminal domain of TOM70 and found that a serine in position 53 of Orf9b and a glutamate in position 477 of TOM70 are crucial for the association of both proteins. A phosphomimetic variant Orf9bS53E showed drastically reduced binding to TOM70 and did not inhibit Hsp90 recruitment, suggesting that Orf9b-TOM70 complex formation is regulated by phosphorylation. Eventually, we identified the N-terminal TPR domain of TOM70 as a second binding site for Orf9b, which indicates a so far unobserved contribution of chaperones in the mitochondrial targeting of the viral protein.


Assuntos
COVID-19/transmissão , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , SARS-CoV-2/patogenicidade , Animais , Sítios de Ligação/genética , COVID-19/imunologia , COVID-19/virologia , Chlorocebus aethiops , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/isolamento & purificação , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/isolamento & purificação , Mutação , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica/genética , Ligação Proteica/imunologia , Domínios Proteicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Células Vero
2.
Mol Syst Biol ; 17(9): e10079, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34519429

RESUMO

We modeled 3D structures of all SARS-CoV-2 proteins, generating 2,060 models that span 69% of the viral proteome and provide details not available elsewhere. We found that ˜6% of the proteome mimicked human proteins, while ˜7% was implicated in hijacking mechanisms that reverse post-translational modifications, block host translation, and disable host defenses; a further ˜29% self-assembled into heteromeric states that provided insight into how the viral replication and translation complex forms. To make these 3D models more accessible, we devised a structural coverage map, a novel visualization method to show what is-and is not-known about the 3D structure of the viral proteome. We integrated the coverage map into an accompanying online resource (https://aquaria.ws/covid) that can be used to find and explore models corresponding to the 79 structural states identified in this work. The resulting Aquaria-COVID resource helps scientists use emerging structural data to understand the mechanisms underlying coronavirus infection and draws attention to the 31% of the viral proteome that remains structurally unknown or dark.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Interações Hospedeiro-Patógeno/genética , Processamento de Proteína Pós-Traducional , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Sítios de Ligação , COVID-19/genética , COVID-19/metabolismo , COVID-19/virologia , Biologia Computacional/métodos , Proteínas do Envelope de Coronavírus/química , Proteínas do Envelope de Coronavírus/genética , Proteínas do Envelope de Coronavírus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Humanos , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Modelos Moleculares , Mimetismo Molecular , Neuropilina-1/química , Neuropilina-1/genética , Neuropilina-1/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas/métodos , Multimerização Proteica , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas Viroporinas/química , Proteínas Viroporinas/genética , Proteínas Viroporinas/metabolismo , Replicação Viral
3.
Nat Commun ; 12(1): 4910, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389706

RESUMO

Human pre-mRNA introns vary in size from under fifty to over a million nucleotides. We searched for essential factors involved in the splicing of human short introns by screening siRNAs against 154 human nuclear proteins. The splicing activity was assayed with a model HNRNPH1 pre-mRNA containing short 56-nucleotide intron. We identify a known alternative splicing regulator SPF45 (RBM17) as a constitutive splicing factor that is required to splice out this 56-nt intron. Whole-transcriptome sequencing of SPF45-deficient cells reveals that SPF45 is essential in the efficient splicing of many short introns. To initiate the spliceosome assembly on a short intron with the truncated poly-pyrimidine tract, the U2AF-homology motif (UHM) of SPF45 competes out that of U2AF65 (U2AF2) for binding to the UHM-ligand motif (ULM) of the U2 snRNP protein SF3b155 (SF3B1). We propose that splicing in a distinct subset of human short introns depends on SPF45 but not U2AF heterodimer.


Assuntos
Íntrons/genética , Fatores de Processamento de RNA/metabolismo , Splicing de RNA , Fator de Processamento U2AF/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Humanos , Modelos Genéticos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismo , Fator de Processamento U2AF/genética
4.
Curr Protoc ; 1(8): e221, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34411463

RESUMO

This paper provides a guideline for optimizing and utilizing Mn2+ Phos-tag gel technology to separate phosphorylated proteins from their unphosphorylated counterparts. It provides key insights into methods for careful sample preparation and experimental directions for determining the appropriate Phos-tag gel compositions and electrophoresis and western blotting conditions. This protocol has been used to successfully resolve proteins extracted from cardiac and skeletal muscles. The guidelines can be extended for optimizing protocols to resolve proteins from other cells or tissue sources. With this, phosphoproteomics and the elucidation of underlying mechanisms of disease progression can be accelerated. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.


Assuntos
Fosfoproteínas , Resinas Acrílicas , Eletroforese em Gel de Poliacrilamida , Humanos , Fosfoproteínas/metabolismo , Fosforilação , Piridinas
5.
Nat Commun ; 12(1): 4664, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34341353

RESUMO

Excessive inflammatory responses induced upon SARS-CoV-2 infection are associated with severe symptoms of COVID-19. Inflammasomes activated in response to SARS-CoV-2 infection are also associated with COVID-19 severity. Here, we show a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation. N protein facilitates maturation of proinflammatory cytokines and induces proinflammatory responses in cultured cells and mice. Mechanistically, N protein interacts directly with NLRP3 protein, promotes the binding of NLRP3 with ASC, and facilitates NLRP3 inflammasome assembly. More importantly, N protein aggravates lung injury, accelerates death in sepsis and acute inflammation mouse models, and promotes IL-1ß and IL-6 activation in mice. Notably, N-induced lung injury and cytokine production are blocked by MCC950 (a specific inhibitor of NLRP3) and Ac-YVAD-cmk (an inhibitor of caspase-1). Therefore, this study reveals a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation and induces excessive inflammatory responses.


Assuntos
COVID-19/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Inflamassomos/metabolismo , Inflamação/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , SARS-CoV-2/metabolismo , Animais , COVID-19/virologia , Células Cultivadas , Citocinas/metabolismo , Células HEK293 , Humanos , Inflamassomos/genética , Lesão Pulmonar/genética , Lesão Pulmonar/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fosfoproteínas/metabolismo , Ligação Proteica , SARS-CoV-2/fisiologia , Células THP-1
6.
Ann Clin Lab Sci ; 51(4): 470-486, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34452885

RESUMO

OBJECTIVE: Epithelium-specific ETS protein 3 (Ese-3) is a member of the ETS family that is associated with tumor progression. However, there is little knowledge about Ese-3 in skin cancer. This study was conducted to explore the effects of Ese-3 on clinical prognosis in skin cancer and the functions of HaCaT cells. MATERIALS AND METHODS: Gene expression and clinical data were collected from The Cancer Genome Atlas (TCGA), The Genotype-Tissue Expression (GTEx), and three GSE datasets (GSE15605, GSE46517, and GSE114445). Comparison of data between groups was performed by Student's t-test and chi square test. Survival analysis was performed using log-rank test. Univariate and multivariate analyses were performed using Cox proportional hazards models. Enrichment analysis was used to predict Ese-3 related functions. Cell proliferation assays, colony formation assays, and flow cytometry were used to assess cell proliferation, while Transwell assays analyzed cell migration and invasion. RESULTS: Compared with normal tissues, the Ese-3 mRNA in cutaneous malignant melanoma (CMM) patients was downregulated (P<0.0001). Ese-3 mRNA was associated with the T stage (χ 2=10.015, P=0.018), clinical stage (χ 2=4.122, P=0.042), and prognosis in CMM patients (P=0.0219) and was an independent prognostic predictor in CMM (HR=1.878, P=0.048). Enrichment analysis showed that differentially expressed proteins were associated with "protein kinase B (AKT) binding." CONCLUSION: Ese-3 inhibited the proliferation, migration, and invasion of HaCaT cells by downregulating PSIP1 and NUCKS1 expression levels to inactivate the phosphorylation of AKT.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/antagonistas & inibidores , Fosfoproteínas/antagonistas & inibidores , Neoplasias Cutâneas/patologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Idoso , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Feminino , Células HaCaT , Humanos , Masculino , Invasividade Neoplásica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Prognóstico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Taxa de Sobrevida , Fatores de Transcrição/genética
7.
Nat Commun ; 12(1): 4568, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315882

RESUMO

Insulin/IGF-1 Signaling (IIS) is known to constrain longevity by inhibiting the transcription factor FOXO. How phosphorylation mediated by IIS kinases regulates lifespan beyond FOXO remains unclear. Here, we profile IIS-dependent phosphorylation changes in a large-scale quantitative phosphoproteomic analysis of wild-type and three IIS mutant Caenorhabditis elegans strains. We quantify more than 15,000 phosphosites and find that 476 of these are differentially phosphorylated in the long-lived daf-2/insulin receptor mutant. We develop a machine learning-based method to prioritize 25 potential lifespan-related phosphosites. We perform validations to show that AKT-1 pT492 inhibits DAF-16/FOXO and compensates the loss of daf-2 function, that EIF-2α pS49 potently inhibits protein synthesis and daf-2 longevity, and that reduced phosphorylation of multiple germline proteins apparently transmits reduced DAF-2 signaling to the soma. In addition, an analysis of kinases with enriched substrates detects that casein kinase 2 (CK2) subunits negatively regulate lifespan. Our study reveals detailed functional insights into longevity.


Assuntos
Caenorhabditis elegans/fisiologia , Insulina/metabolismo , Longevidade/fisiologia , Transdução de Sinais , Algoritmos , Sequência de Aminoácidos , Animais , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Células Germinativas/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Modelos Biológicos , Mutação/genética , Fosfoproteínas/metabolismo , Fosforilação , Proteoma/metabolismo , Proteômica
8.
Genes Dev ; 35(15-16): 1123-1141, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34301768

RESUMO

Spliceosomal small nuclear RNAs (snRNAs) are modified by small Cajal body (CB)-specific ribonucleoproteins (scaRNPs) to ensure snRNP biogenesis and pre-mRNA splicing. However, the function and subcellular site of snRNA modification are largely unknown. We show that CB localization of the protein Nopp140 is essential for concentration of scaRNPs in that nuclear condensate; and that phosphorylation by casein kinase 2 (CK2) at ∼80 serines targets Nopp140 to CBs. Transiting through CBs, snRNAs are apparently modified by scaRNPs. Indeed, Nopp140 knockdown-mediated release of scaRNPs from CBs severely compromises 2'-O-methylation of spliceosomal snRNAs, identifying CBs as the site of scaRNP catalysis. Additionally, alternative splicing patterns change indicating that these modifications in U1, U2, U5, and U12 snRNAs safeguard splicing fidelity. Given the importance of CK2 in this pathway, compromised splicing could underlie the mode of action of small molecule CK2 inhibitors currently considered for therapy in cholangiocarcinoma, hematological malignancies, and COVID-19.


Assuntos
Células Intersticiais de Cajal/metabolismo , Metilação , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Splicing de RNA , RNA Nuclear Pequeno/metabolismo , COVID-19/tratamento farmacológico , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Colangiocarcinoma/tratamento farmacológico , Neoplasias Hematológicas/tratamento farmacológico , Humanos , Fosforilação , RNA Nuclear Pequeno/química , Ribonucleoproteínas/metabolismo , Spliceossomos/genética
9.
Biochem Biophys Res Commun ; 569: 154-160, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34246830

RESUMO

The SARS-CoV-2 N protein binds several cell host proteins including 14-3-3γ, a well-characterized regulatory protein. However, the biological function of this interaction is not completely understood. We analyzed the variability of ∼90 000 sequences of the SARS-CoV-2 N protein, particularly, its mutations in disordered regions containing binding motifs for 14-3-3 proteins. We studied how these mutations affect the binding energy to 14-3-3γ and found that changes positively affecting the predicted interaction with 14-3-3γ are the most successfully spread, with the highest prevalence in the phylogenetic tree. Although most residues are highly conserved within the 14-3-3 binding site, compensatory mutations to maintain the interaction energy of N-14-3-3γ were found, including half of the current variants of concern and interest. Our results suggest that binding of N to 14-3-3γ is beneficial for the virus, thus targeting this viral-host protein-protein interaction seems an attractive approach to explore antiviral strategies.


Assuntos
Proteínas 14-3-3/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/análise , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Sítios de Ligação , Proteínas do Nucleocapsídeo de Coronavírus/genética , Humanos , Mutação/genética , Fosfoproteínas/análise , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Filogenia , Ligação Proteica
10.
Cell Death Dis ; 12(7): 659, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193827

RESUMO

Cellular stress can lead to several human disease pathologies due to aberrant cell death. The p53 family (tp53, tp63, and tp73) and downstream transcriptional apoptotic target genes (PUMA/BBC3 and NOXA/PMAIP1) have been implicated as mediators of stress signals. To evaluate the importance of key stress response components in vivo, we have generated zebrafish null alleles in puma, noxa, p53, p63, and p73. Utilizing these genetic mutants, we have deciphered that the apoptotic response to genotoxic stress requires p53 and puma, but not p63, p73, or noxa. We also identified a delayed secondary wave of genotoxic stress-induced apoptosis that is p53/puma independent. Contrary to genotoxic stress, ER stress-induced apoptosis requires p63 and puma, but not p53, p73, or noxa. Lastly, the oxidative stress-induced apoptotic response requires p63, and both noxa and puma. Our data also indicate that while the neural tube is poised for apoptosis due to genotoxic stress, the epidermis is poised for apoptosis due to ER and oxidative stress. These data indicate there are convergent as well as unique molecular pathways involved in the different stress responses. The commonality of puma in these stress pathways, and the lack of gross or tumorigenic phenotypes with puma loss suggest that a inhibitor of Puma may have therapeutic application. In addition, we have also generated a knockout of the negative regulator of p53, mdm2 to further evaluate the p53-induced apoptosis. Our data indicate that the p53 null allele completely rescues the mdm2 null lethality, while the puma null completely rescues the mdm2 null apoptosis but only partially rescues the phenotype. Indicating Puma is the key mediator of p53-dependent apoptosis. Interestingly the p53 homozygous null zebrafish develop tumors faster than the previously described p53 homozygous missense mutant zebrafish, suggesting the missense allele may be hypomorphic allele.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Dano ao DNA , Estresse do Retículo Endoplasmático , Estresse Oxidativo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica , Macrolídeos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Tapsigargina/farmacologia , Fatores de Tempo , Transativadores/genética , Transcrição Genética , Proteína Supressora de Tumor p53/genética , Raios X , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
11.
Nat Commun ; 12(1): 4507, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301951

RESUMO

Approximately half of genetic disease-associated mutations cause aberrant splicing. However, a widely applicable therapeutic strategy to splicing diseases is yet to be developed. Here, we analyze the mechanism whereby IKBKAP-familial dysautonomia (FD) exon 20 inclusion is specifically promoted by a small molecule splice modulator, RECTAS, even though IKBKAP-FD exon 20 has a suboptimal 5' splice site due to the IVS20 + 6 T > C mutation. Knockdown experiments reveal that exon 20 inclusion is suppressed in the absence of serine/arginine-rich splicing factor 6 (SRSF6) binding to an intronic splicing enhancer in intron 20. We show that RECTAS directly interacts with CDC-like kinases (CLKs) and enhances SRSF6 phosphorylation. Consistently, exon 20 splicing is bidirectionally manipulated by targeting cellular CLK activity with RECTAS versus CLK inhibitors. The therapeutic potential of RECTAS is validated in multiple FD disease models. Our study indicates that small synthetic molecules affecting phosphorylation state of SRSFs is available as a new therapeutic modality for mechanism-oriented precision medicine of splicing diseases.


Assuntos
Processamento Alternativo/genética , Disautonomia Familiar/genética , Mutação , Fatores de Elongação da Transcrição/genética , Processamento Alternativo/efeitos dos fármacos , Animais , Células Cultivadas , Modelos Animais de Doenças , Disautonomia Familiar/tratamento farmacológico , Disautonomia Familiar/metabolismo , Elementos Facilitadores Genéticos/genética , Éxons/genética , Células HeLa , Humanos , Íntrons/genética , Camundongos Transgênicos , Estrutura Molecular , Fosfoproteínas/metabolismo , Ligação Proteica/efeitos dos fármacos , Sítios de Splice de RNA/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Elongação da Transcrição/metabolismo
12.
Toxicology ; 458: 152836, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34147545

RESUMO

China's clean energy and resources are mainly located in the west and north while electric load center is concentrated in the middle and east. Thus, these resources and energy need to be converted into electrical energy in situ and transported to electric load center through ultra-high voltage direct current (UHVDC) transmissions. China has built 25,000 km UHVDC transmission lines of 800 kV and 1100 kV, near which the impact of electric field on health has attracted public attention. Previous studies showed that time-varying electromagnetic field exposure could disturb testosterone secretion. To study the effect of non-time-varying electric field caused by direct current transmission lines on testosterone synthesis, male ICR mice were continually (24 h/d) exposed to static electric field of 56.3 ± 1.4 kV/m. Results showed that on the 3rd day of exposure and on the 7th day after ceasing the exposure of 28 d, serum testosterone level and testicular oxidative stress indicators didn't change significantly. On the 28th day of exposure, serum testosterone levels, testicular glutathione peroxidase (GSH-Px) activity, the mRNA and protein levels of testicular StAR, PBR, CYP11A1 decreased significantly, and testicular malondialdehyde (MDA) content increased significantly. Meanwhile, electron-dense edges and vacuolation appeared in lipid droplets of Leydig cells. The gap between inner mitochondrial membrane (IMM) and outer mitochondrial membrane (OMM) enlarged, which would cause the swelling of mitochondria, the rupture and deficiency of mitochondrial membranes. Analysis showed that testicular oxidative stress could induce the damage of mitochondrial structure in Leydig cells, which would decrease the rate of cholesterol transport from cytoplasm to mitochondria. Since cholesterol is the necessary precursor of testosterone synthesis, testosterone synthesis was inhibited. The decrease of the mRNA and protein expression levels of StAR and PBR in testes could diminish the cholesterol transported from OMM to IMM. The decrease of the mRNA and protein expression levels of CYP11A1 could reduce the pregnenolone required in testosterone synthesis and inhibit testosterone synthesis consequently.


Assuntos
Campos Eletromagnéticos , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/efeitos da radiação , Testosterona/biossíntese , Animais , Antioxidantes/metabolismo , Colesterol/metabolismo , Citoplasma/metabolismo , Citoplasma/efeitos da radiação , Glutationa Peroxidase/metabolismo , Células Intersticiais do Testículo/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos ICR , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/efeitos da radiação , Dilatação Mitocondrial/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Fosfoproteínas/metabolismo , Testosterona/sangue , Vacúolos/efeitos da radiação , Vacúolos/ultraestrutura
13.
J Biol Chem ; 297(1): 100880, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34144036

RESUMO

More than half a century ago, reversible protein phosphorylation was linked to mitochondrial metabolism through the regulation of pyruvate dehydrogenase. Since this discovery, the number of identified mitochondrial protein phosphorylation sites has increased by orders of magnitude, driven largely by technological advances in mass spectrometry-based phosphoproteomics. However, the majority of these modifications remain uncharacterized, rendering their function and relevance unclear. Nonetheless, recent studies have shown that disruption of resident mitochondrial protein phosphatases causes substantial metabolic dysfunction across organisms, suggesting that proper management of mitochondrial phosphorylation is vital for organellar and organismal homeostasis. While these data suggest that phosphorylation within mitochondria is of critical importance, significant gaps remain in our knowledge of how these modifications influence organellar function. Here, we curate publicly available datasets to map the extent of protein phosphorylation within mammalian mitochondria and to highlight the known functions of mitochondrial-resident phosphatases. We further propose models by which phosphorylation may affect mitochondrial enzyme activities, protein import and processing, and overall organellar homeostasis.


Assuntos
Proteínas Mitocondriais/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Animais , Humanos , Proteínas Mitocondriais/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/genética , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteoma/genética
14.
Mol Cell ; 81(15): 3096-3109.e8, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34146481

RESUMO

Transcription by RNA polymerase II (RNA Pol II) relies on the elongation factors PAF1 complex (PAF), RTF1, and SPT6. Here, we use rapid factor depletion and multi-omics analysis to investigate how these elongation factors influence RNA Pol II elongation activity in human cells. Whereas depletion of PAF subunits PAF1 and CTR9 has little effect on cellular RNA synthesis, depletion of RTF1 or SPT6 strongly compromises RNA Pol II activity, albeit in fundamentally different ways. RTF1 depletion decreases RNA Pol II velocity, whereas SPT6 depletion impairs RNA Pol II progression through nucleosomes. These results show that distinct elongation factors stimulate either RNA Pol II velocity or RNA Pol II progression through chromatin in vivo. Further analysis provides evidence for two distinct barriers to early elongation: the promoter-proximal pause site and the +1 nucleosome. It emerges that the first barrier enables loading of elongation factors that are required to overcome the second and subsequent barriers to transcription.


Assuntos
RNA Polimerase II/metabolismo , RNA/biossíntese , Fatores de Transcrição/metabolismo , Humanos , Células K562 , Nucleossomos/genética , Nucleossomos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Polimerase II/genética , Fatores de Transcrição/genética
15.
J Biol Chem ; 297(1): 100907, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34166681

RESUMO

Endosomal signaling downstream of G-protein-coupled receptors (GPCRs) has emerged as a novel paradigm with important pharmacological and physiological implications. However, our knowledge of the functional consequences of intracellular signaling is incomplete. To begin to address this gap, we combined an optogenetic approach for site-specific generation of the prototypical second messenger generated by active GPCRs, cyclic AMP (cAMP), with unbiased mass-spectrometry-based analysis of the phosphoproteome. We identified 218 unique, high-confidence sites whose phosphorylation is either increased or decreased in response to cAMP elevation. We next determined that the same amount of cAMP produced from the endosomal membrane led to more robust changes in phosphorylation than the plasma membrane. Remarkably, this was true for the entire repertoire of 218 identified targets and irrespective of their annotated subcellular localizations (endosome, cell surface, nucleus, cytosol). Furthermore, we identified a particularly strong endosome bias for a subset of proteins that are dephosphorylated in response to cAMP. Through bioinformatics analysis, we established these targets as putative substrates for protein phosphatase 2A (PP2A), and we propose compartmentalized activation of PP2A by cAMP-responsive kinases as the likely underlying mechanism. Altogether, our study extends the concept that endosomal signaling is a significant functional contributor to cellular responsiveness to cAMP by establishing a unique role for localized cAMP production in defining categorically distinct phosphoresponses.


Assuntos
AMP Cíclico/metabolismo , Endossomos/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Animais , Células HEK293 , Humanos , Fosfoproteínas/química , Fosforilação , Domínios Proteicos , Proteína Fosfatase 2/metabolismo , Proteoma/química
16.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066551

RESUMO

G-quadruplexes (G4s) are non-canonical structures formed by guanine-rich sequences of DNA or RNA that have attracted increased attention as anticancer agents. This systematic study aimed to investigate the anticancer potential of five G4-forming, sequence-related DNA molecules in terms of their thermodynamic and structural properties, biostability and cellular uptake. The antiproliferative studies revealed that less thermodynamically stable G4s with three G-tetrads in the core and longer loops are more predisposed to effectively inhibit cancer cell growth. By contrast, highly structured G4s with an extended core containing four G-tetrads and longer loops are characterized by more efficient cellular uptake and improved biostability. Various analyses have indicated that the G4 structural elements are intrinsic to the biological activity of these molecules. Importantly, the structural requirements are different for efficient cancer cell line inhibition and favorable G4 cellular uptake. Thus, the ultimate antiproliferative potential of G4s is a net result of the specific balance among the structural features that are favorable for efficient uptake and those that increase the inhibitory activity of the studied molecules. Understanding the G4 structural features and their role in the biological activity of G-rich molecules might facilitate the development of novel, more potent G4-based therapeutics with unprecedented anticancer properties.


Assuntos
Quadruplex G , Oligonucleotídeos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Endocitose/efeitos dos fármacos , Meia-Vida , Humanos , Oligonucleotídeos/sangue , Fosfoproteínas/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Termodinâmica
17.
Cancer Sci ; 112(9): 3711-3721, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34107118

RESUMO

Antimitotic drugs such as vinca alkaloids and taxanes cause mitotic cell death after prolonged mitotic arrest. However, a fraction of cells escape from mitotic arrest by undergoing mitotic slippage, which is related to resistance to antimitotic drugs. Tipping the balance to mitotic cell death thus can be a way to overcome the drug resistance. Here we found that depletion of a mitotic regulator, CHAMP1 (chromosome alignment-maintaining phosphoprotein, CAMP), accelerates the timing of mitotic cell death after mitotic arrest. Live cell imaging revealed that CHAMP1-depleted cells died earlier than mock-treated cells in the presence of antimitotic drugs that resulted in the reduction of cells undergoing mitotic slippage. Depletion CHAMP1 reduces the expression of antiapoptotic Bcl-2 family proteins, especially Mcl-1. We found that CHAMP1 maintains Mcl-1 expression both at protein and mRNA levels independently of the cell cycle. At the protein level, CHAMP1 maintains Mcl-1 stability by suppressing proteasome-dependent degradation. Depletion of CHAMP1 reduces cell viability, and exhibits synergistic effects with antimitotic drugs. Our data suggest that CHAMP1 plays a role in the maintenance of Mcl-1 expression, implying that CHAMP1 can be a target to overcome the resistance to antimitotic drugs.


Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transdução de Sinais/genética , Células A549 , Animais , Antimitóticos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitose/efeitos dos fármacos , Mitose/genética , Neoplasias/genética , Neoplasias/patologia , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Transfecção , Carga Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
18.
J Virol ; 95(13): e0026621, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34110264

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic. While previous studies have shown that several SARS-CoV-2 proteins can antagonize the interferon (IFN) response, some of the mechanisms by which they do so are not well understood. In this study, we describe two novel mechanisms by which SARS-CoV-2 blocks the IFN pathway. Type I IFNs and IFN-stimulated genes (ISGs) were poorly induced during SARS-CoV-2 infection, and once infection was established, cells were highly resistant to ectopic induction of IFNs and ISGs. Levels of two key IFN signaling pathway components, Tyk2 and STAT2, were significantly lower in SARS-CoV-2-infected cells. Expression of nonstructural protein 1 (NSP1) or nucleocapsid in the absence of other viral proteins was sufficient to block IFN induction, but only NSP1 was able to inhibit IFN signaling. Mapping studies suggest that NSP1 prevents IFN induction in part by blocking IRF3 phosphorylation. In addition, NSP1-induced depletion of Tyk2 and STAT2 dampened ISG induction. Together, our data provide new insights into how SARS-CoV-2 successfully evades the IFN system to establish infection. IMPORTANCE SARS-CoV-2 is the causative agent of COVID-19, a serious disease that can have a myriad of symptoms from loss of taste and smell to pneumonia and hypercoagulation. The rapid spread of SARS-CoV-2 can be attributed in part to asymptomatic transmission, where infected individuals shed large amounts of virus before the onset of disease. This is likely due to the ability of SARS-CoV-2 to effectively suppress the innate immune system, including the IFN response. Indeed, we show that the IFN response is efficiently blocked during SARS-CoV-2 infection, a process that is mediated in large part by nonstructural protein 1 and nucleocapsid. Our study provides new insights on how SARS-CoV-2 evades the IFN response to successfully establish infection. These findings should be considered for the development and administration of therapeutics against SARS-CoV-2.


Assuntos
Interferon Tipo I/antagonistas & inibidores , SARS-CoV-2/metabolismo , Transdução de Sinais , Proteínas não Estruturais Virais/metabolismo , Animais , COVID-19/imunologia , COVID-19/virologia , Chlorocebus aethiops , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Células HEK293 , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Fosfoproteínas/metabolismo , SARS-CoV-2/patogenicidade , Fator de Transcrição STAT2/metabolismo , TYK2 Quinase/metabolismo , Células Vero
19.
Biochim Biophys Acta Proteins Proteom ; 1869(9): 140683, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34119693

RESUMO

Myocardial infarction and subsequent therapeutic interventions activate numerous intracellular cascades in every constituent cell type of the heart. Endothelial cells produce several protective compounds in response to therapeutic ultrasound, under both normoxic and ischemic conditions. How endothelial cells sense ultrasound and convert it to a beneficial biological response is not known. We adopted a global, unbiased phosphoproteomics approach aimed at understanding how endothelial cells respond to ultrasound. Here, we use primary cardiac endothelial cells to explore the cellular signaling events underlying the response to ischemia-like cellular injury and ultrasound exposure in vitro. Enriched phosphopeptides were analyzed with a high mass accuracy liquid chromatrography (LC) - tandem mass spectrometry (MS/MS) proteomic platform, yielding multiple alterations in both total protein levels and phosphorylation events in response to ischemic injury and ultrasound. Application of pathway algorithms reveals numerous protein networks recruited in response to ultrasound including those regulating RNA splicing, cell-cell interactions and cytoskeletal organization. Our dataset also permits the informatic prediction of potential kinases responsible for the modifications detected. Taken together, our findings begin to reveal the endothelial proteomic response to ultrasound and suggest potential targets for future studies of the protective effects of ultrasound in the ischemic heart.


Assuntos
Endocárdio/metabolismo , Isquemia Miocárdica/fisiopatologia , Fosfoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida/métodos , Endocárdio/fisiologia , Células Endoteliais/metabolismo , Coração/diagnóstico por imagem , Isquemia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Cultura Primária de Células , Proteoma/metabolismo , Proteômica/métodos , Transdução de Sinais , Espectrometria de Massas em Tandem/métodos , Terapia por Ultrassom/métodos
20.
Nat Commun ; 12(1): 3919, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168128

RESUMO

The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD1-10) subtypes and Smoothened (SMO), remains one of the most enigmatic GPCR families. While SMO relies on cholesterol binding to the 7TM core of the receptor to activate downstream signaling, underlying details of receptor activation remain obscure for FZDs. Here, we aimed to investigate the activation mechanisms of class F receptors utilizing a computational biology approach and mutational analysis of receptor function in combination with ligand binding and downstream signaling assays in living cells. Our results indicate that FZDs differ substantially from SMO in receptor activation-associated conformational changes. SMO manifests a preference for a straight TM6 in both ligand binding and functional readouts. Similar to the majority of GPCRs, FZDs present with a kinked TM6 upon activation owing to the presence of residue P6.43. Functional comparison of FZD and FZD P6.43F mutants in different assay formats monitoring ligand binding, G protein activation, DVL2 recruitment and TOPflash activity, however, underlines further the functional diversity among FZDs and not only between FZDs and SMO.


Assuntos
Receptores Frizzled/química , Receptores Frizzled/metabolismo , Receptor Smoothened/química , Sítios de Ligação , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Compostos de Boro/química , Microscopia Crioeletrônica , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptores Frizzled/genética , Humanos , Simulação de Dinâmica Molecular , Mutação , Fosfoproteínas/metabolismo , Conformação Proteica , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Alcaloides de Veratrum/química , Alcaloides de Veratrum/metabolismo
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