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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.
Cells ; 10(7)2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34359932

RESUMO

MicroRNAs (miRNAs) are critical regulators of gene expression that may be used to identify the pathological pathways influenced by disease and cellular interactions. Viral miRNAs (v-miRNAs) encoded by both DNA and RNA viruses induce immune dysregulation, virus production, and disease pathogenesis. Given the absence of effective treatment and the prevalence of highly infective SARS-CoV-2 strains, improved understanding of viral-associated miRNAs could provide novel mechanistic insights into the pathogenesis of COVID-19. In this study, SARS-CoV-2 v-miRNAs were identified by deep sequencing in infected Calu-3 and Vero E6 cell lines. Among the ~0.1% small RNA sequences mapped to the SARS-CoV-2 genome, the top ten SARS-CoV-2 v-miRNAs (including three encoded by the N gene; v-miRNA-N) were selected. After initial screening of conserved v-miRNA-N-28612, which was identified in both SARS-CoV and SARS-CoV-2, its expression was shown to be positively associated with viral load in COVID-19 patients. Further in silico analysis and synthetic-mimic transfection of validated SARS-CoV-2 v-miRNAs revealed novel functional targets and associations with mechanisms of cellular metabolism and biosynthesis. Our findings support the development of v-miRNA-based biomarkers and therapeutic strategies based on improved understanding of the pathophysiology of COVID-19.


Assuntos
COVID-19/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/genética , Redes e Vias Metabólicas , MicroRNAs/genética , RNA Viral/genética , SARS-CoV-2/fisiologia , Animais , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Interações Hospedeiro-Patógeno , Humanos , Fosfoproteínas/genética , SARS-CoV-2/genética , Células Vero
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
5.
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
6.
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
7.
Fish Physiol Biochem ; 47(4): 1299-1311, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34241762

RESUMO

Genistein is an abundant phytoestrogen in soybean. This study aimed to determine the effects of genistein on cholesterol distribution and metabolism in female yellow catfish. Three hundred fish (49.2 ± 1.4 g) were randomly divided into five treatments and received intraperitoneal injections as follows: (1) blank, no injection; (2) control, vehicle only; (3) E2, 17ß-estradiol at 10 µg·g-1 body weight; (4) low genistein doses, genistein at 10 µg·g-1 body weight; (5) high genistein doses, genistein at 100 µg·g-1 body weight. Both high and low genistein doses significantly reduced (p < 0.05) serum TC and LDL-C 24 h after injection. Moreover, the high genistein doses significantly reduced (p < 0.05) serum HDL-C. Both high and low doses of genistein significantly increased (p < 0.05) hepatic TC. Only high genistein doses significantly increased (p < 0.05) ovary TC. In the liver, both high and low genistein doses significantly increased (p < 0.05) protein and mRNA expression of ldlr. Meanwhile, high genistein doses significantly decreased (p < 0.05) mRNA expression of hmgcr. In ovary tissue, high genistein doses significantly decreased (p < 0.05) mRNA expression of cyp11a1. These results suggested that genistein affected the cholesterol distribution in female yellow catfish. Both high and low doses of genistein reduced cholesterol content in blood and increased its content in the liver by increasing the uptake of blood cholesterol. Meanwhile, high genistein doses may inhibit hepatic cholesterol synthesis. Additionally, high genistein doses could increase cholesterol transfer from serum into the ovary and disturb cholesterol conversion to pregnenolone.


Assuntos
Peixes-Gato/metabolismo , Colesterol/metabolismo , Genisteína/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Animais , Peixes-Gato/sangue , Peixes-Gato/genética , Colesterol/sangue , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Feminino , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Injeções Intraperitoneais , Fígado/efeitos dos fármacos , Fígado/metabolismo , Ovário/efeitos dos fármacos , Ovário/metabolismo , Fosfoproteínas/genética , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética
8.
J Med Virol ; 93(10): 5969-5976, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34196423

RESUMO

In-house assays for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), are feasible alternatives, particularly in developing countries. Cycle threshold (Ct ) values obtained by qRT-PCR were compared with clinical and laboratory data from saliva of inpatients with COVID-19 and asymptomatic health workers (AHW) were studied. Saliva specimens from 58 inpatients confirmed by qRT-PCR for SARS-CoV-2 using nasopharyngeal specimens, and 105 AHW were studied by qRT-PCR using three sets of primers for the N (N1, N2, and N3) gene of SARS-CoV-2, according to the CDC Diagnostic Panel protocol, showing a positivity of 88% for inpatients and 8% for AHW. Bivariate analysis revealed an association between Ct < 38.0 values for N2 and mechanical ventilation assistance among patients (p = .013). In addition, values of aspartate-transaminase, lactate dehydrogenase, and ferritin showed significant correlations with Ct values of N1 and N3 genes in inpatients. Therefore, our results show that Ct values correlate with some relevant clinical data for inpatients with COVID-19.


Assuntos
Teste de Ácido Nucleico para COVID-19/estatística & dados numéricos , COVID-19/diagnóstico , Pessoal de Saúde/estatística & dados numéricos , Pacientes Internados/estatística & dados numéricos , Adulto , Idoso , Infecções Assintomáticas , Biomarcadores/sangue , Proteínas do Nucleocapsídeo de Coronavírus/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/genética , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Índice de Gravidade de Doença
9.
Eur J Med Genet ; 64(9): 104291, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34303877

RESUMO

Aicardi-Goutieres Syndrome (AGS) is a heterogeneous genetic syndrome, manifesting early as encephalopathy and is associated with abnormal neurologic findings, hepatosplenomegaly, elevated liver enzymes, thrombocytopenia and intracranial calcification. The most severe neonatal type, AGS1, is caused by biallelic disease-causing variants in TREX1. In this study, we describe four patients with TREX1-related AGS1 whose phenotype overlaps with intra-uterine infections and neonatal lupus. Exome sequencing identified a previously reported TREX1 variant, c.223dup (NM_016381.5; p. Glu75GlyfsTer82) in all the four patients belonging to the Indian subcontinent. The functional consequence of the disease-causing variant was predicted by using a new combination of bioinformatics softwares. The recurrence of this pathogenic variant indicates a possible founder effect in TREX1 for AGS1 in this population. The phenotypic variability in those with this founder mutation can mimic intrauterine infections and neonatal lupus, thereby leading to misdiagnosis warranting a targeted genetic testing approach to be a part of the diagnostic workup to obtain a definite, early and cost-effective diagnosis in patients from Indian subcontinent with early onset encephalopathy.


Assuntos
Doenças Autoimunes do Sistema Nervoso/genética , Exodesoxirribonucleases/genética , Malformações do Sistema Nervoso/genética , Fenótipo , Fosfoproteínas/genética , Doenças Autoimunes do Sistema Nervoso/epidemiologia , Doenças Autoimunes do Sistema Nervoso/patologia , Exodesoxirribonucleases/química , Feminino , Efeito Fundador , Frequência do Gene , Humanos , Índia , Lactente , Masculino , Mutação , Malformações do Sistema Nervoso/epidemiologia , Malformações do Sistema Nervoso/patologia , Fosfoproteínas/química , Domínios Proteicos
10.
Arch Virol ; 166(9): 2529-2540, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34251549

RESUMO

RT-qPCR detection of SARS-CoV-2 RNA still represents the method of reference to diagnose and monitor COVID-19. From the onset of the pandemic, however, doubts have been expressed concerning the sensitivity of this molecular diagnosis method. Droplet digital PCR (ddPCR) is a third-generation PCR technique that is particularly adapted to detecting low-abundance targets. We developed two-color ddPCR assays for the detection of four different regions of SARS-CoV-2 RNA, including non-structural (IP4-RdRP, helicase) and structural (E, N) protein-encoding sequences. We observed that N or E subgenomic RNAs are generally more abundant than IP4 and helicase RNA sequences in cells infected in vitro, suggesting that detection of the N gene, coding for the most abundant subgenomic RNA of SARS-CoV-2, increases the sensitivity of detection during the highly replicative phase of infection. We investigated 208 nasopharyngeal swabs sampled in March-April 2020 in different hospitals of Greater Paris. We found that 8.6% of informative samples (n = 16/185, P < 0.0001) initially scored as "non-positive" (undetermined or negative) by RT-qPCR were positive for SARS-CoV-2 RNA by ddPCR. Our work confirms that the use of ddPCR modestly, but significantly, increases the proportion of upper airway samples testing positive in the framework of first-line diagnosis of a French population.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , RNA Viral/genética , SARS-CoV-2/genética , Proteínas Virais/genética , COVID-19/epidemiologia , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/instrumentação , Cor , Proteínas do Envelope de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/genética , França/epidemiologia , Expressão Gênica , Humanos , Limite de Detecção , Nasofaringe/virologia , Fosfoproteínas/genética , RNA Helicases/genética , RNA Polimerase Dependente de RNA/genética , Carga Viral
11.
Arch Virol ; 166(9): 2551-2561, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34259914

RESUMO

The aim of this study was to identify and validate a sensitive, high-throughput, and cost-effective SARS-CoV-2 real-time RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a university surveillance program. We conducted a side-by-side clinical evaluation of a newly developed SARS-CoV-2 multiplex assay (EZ-SARS-CoV-2 Real-Time RT-PCR) with the commercial TaqPath COVID-19 Combo Kit, which has an Emergency Use Authorization from the FDA. The EZ-SARS-CoV-2 RT-PCR incorporates two assays targeting the SARS-CoV-2 N gene, an internal control targeting the human RNase P gene, and a PCR inhibition control in a single reaction. Nasopharyngeal (NP) and anterior nares (AN) swabs were tested as individuals and pools with both assays and in the ABI 7500 Fast and the QuantStudio 5 detection platforms. The analytical sensitivity of the EZ-SARS-CoV-2 RT-PCR assay was 250 copies/ml or approximately 1.75 genome copy equivalents per reaction. The clinical performance of the EZ-SARS-CoV-2 assay was evaluated using NP and AN samples tested in other laboratories. The diagnostic sensitivity of the assay ranged between 94 and 96% across the detection platforms, and the diagnostic specificity was 94.06%. The positive predictive value was 94%, and the negative predictive value ranged from 94 to 96%. Pooling five NP or AN specimens yielded 93% diagnostic sensitivity. The overall agreement between these SARS-CoV-2 RT-PCR assays was high, supported by a Cohen's kappa value of 0.93. The EZ-SARS-CoV-2 RT-PCR assay performance attributes of high sensitivity and specificity with AN sample matrix and pooled upper respiratory samples support its use in a high-throughput surveillance testing program.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/genética , Reação em Cadeia da Polimerase Multiplex/métodos , RNA Viral/genética , SARS-CoV-2/genética , COVID-19/epidemiologia , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/economia , Teste de Ácido Nucleico para COVID-19/instrumentação , Monitoramento Epidemiológico , Expressão Gênica , Humanos , Reação em Cadeia da Polimerase Multiplex/economia , Reação em Cadeia da Polimerase Multiplex/instrumentação , Cavidade Nasal/virologia , Nasofaringe/virologia , Fosfoproteínas/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Carga Viral
12.
Proc Natl Acad Sci U S A ; 118(29)2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34292871

RESUMO

Understanding the trends in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution is paramount to control the COVID-19 pandemic. We analyzed more than 300,000 high-quality genome sequences of SARS-CoV-2 variants available as of January 2021. The results show that the ongoing evolution of SARS-CoV-2 during the pandemic is characterized primarily by purifying selection, but a small set of sites appear to evolve under positive selection. The receptor-binding domain of the spike protein and the region of the nucleocapsid protein associated with nuclear localization signals (NLS) are enriched with positively selected amino acid replacements. These replacements form a strongly connected network of apparent epistatic interactions and are signatures of major partitions in the SARS-CoV-2 phylogeny. Virus diversity within each geographic region has been steadily growing for the entirety of the pandemic, but analysis of the phylogenetic distances between pairs of regions reveals four distinct periods based on global partitioning of the tree and the emergence of key mutations. The initial period of rapid diversification into region-specific phylogenies that ended in February 2020 was followed by a major extinction event and global homogenization concomitant with the spread of D614G in the spike protein, ending in March 2020. The NLS-associated variants across multiple partitions rose to global prominence in March to July, during a period of stasis in terms of interregional diversity. Finally, beginning in July 2020, multiple mutations, some of which have since been demonstrated to enable antibody evasion, began to emerge associated with ongoing regional diversification, which might be indicative of speciation.


Assuntos
Adaptação Fisiológica/genética , Evolução Molecular , SARS-CoV-2/genética , Substituição de Aminoácidos , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/prevenção & controle , COVID-19/virologia , Teste para COVID-19 , Proteínas do Nucleocapsídeo de Coronavírus/genética , Epistasia Genética , Genoma Viral/genética , Humanos , Evasão da Resposta Imune/genética , Mutação , Sinais de Localização Nuclear/genética , Fosfoproteínas/genética , Filogenia , Domínios e Motivos de Interação entre Proteínas/genética , SARS-CoV-2/classificação , Seleção Genética , Glicoproteína da Espícula de Coronavírus/genética , Vacinação
13.
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
14.
Nat Microbiol ; 6(8): 1094-1101, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34163035

RESUMO

Here, we report SARS-CoV-2 genomic surveillance from March 2020 until January 2021 in Uganda, a landlocked East African country with a population of approximately 40 million people. We report 322 full SARS-CoV-2 genomes from 39,424 reported SARS-CoV-2 infections, thus representing 0.8% of the reported cases. Phylogenetic analyses of these sequences revealed the emergence of lineage A.23.1 from lineage A.23. Lineage A.23.1 represented 88% of the genomes observed in December 2020, then 100% of the genomes observed in January 2021. The A.23.1 lineage was also reported in 26 other countries. Although the precise changes in A.23.1 differ from those reported in the first three SARS-CoV-2 variants of concern (VOCs), the A.23.1 spike-protein-coding region has changes similar to VOCs including a change at position 613, a change in the furin cleavage site that extends the basic amino acid motif and multiple changes in the immunogenic N-terminal domain. In addition, the A.23.1 lineage has changes in non-spike proteins including nsp6, ORF8 and ORF9 that are also altered in other VOCs. The clinical impact of the A.23.1 variant is not yet clear and it has not been designated as a VOC. However, our findings of emergence and spread of this variant indicate that careful monitoring of this variant, together with assessment of the consequences of the spike protein changes for COVID-19 vaccine performance, are advisable.


Assuntos
COVID-19/epidemiologia , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Motivos de Aminoácidos , Proteínas do Nucleocapsídeo de Coronavírus/genética , Variação Genética/genética , Genoma Viral/genética , Humanos , Fosfoproteínas/genética , Filogenia , Uganda/epidemiologia , Proteínas Virais/genética
15.
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
16.
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
17.
J Virol Methods ; 295: 114215, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34166701

RESUMO

BACKGROUND: This study aimed to evaluate the impact of four different reverse transcription quantitative PCR (RT-qPCR) master mixes on the performance of SARS-CoV-2 diagnostic PCRs using three primer/probe assays targeting the N gene (A, B and C). The dynamic range and lowest detected quantity was determined using a SARS-CoV-2 partial N gene RNA transcript dilution series (100,000-1 copy/µl) and verified using 72 nose and throat swabs, 29 of which tested positive for SARS-CoV-2 RNA. RESULTS: Assay C consistently detected the lowest quantity of partial N gene RNA transcript with all mastermixes. The Takara One Step PrimeScript™ III RT-PCR Kit mastermix enabled all primer pairs to detect the entire dynamic range evaluated, with the Qiagen Quantifast and Thermofisher TaqPath 1-Step kits also performing well. Sequences from all three primer/probe sets tested in this study (assay A, B and C) have 100 % homology to ≥97 % of the of SARS-CoV-2 sequences available up to 31st December 2020 (n = 291,483 sequences). CONCLUSIONS: This work demonstrates that specific assays (in this case assay C) can perform well in terms of dynamic range and lowest detected quantity regardless of the mastermix used. However we also show that, by choosing the most appropriate mastermix, poorer performing primer pairs are also able to detect all of the template dilutions investigated. This work increases the potential options when choosing assays for SARS-CoV-2 diagnosis and provides solutions to enable them to work with optimal analytical sensitivity.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Proteínas do Nucleocapsídeo de Coronavírus/genética , SARS-CoV-2/isolamento & purificação , COVID-19/diagnóstico , Teste de Ácido Nucleico para COVID-19/instrumentação , Primers do DNA/genética , Humanos , Nariz/virologia , Faringe/virologia , Fosfoproteínas/genética , RNA Viral/genética , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , Sensibilidade e Especificidade , Homologia de Sequência do Ácido Nucleico
18.
Comput Biol Chem ; 93: 107533, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34166886

RESUMO

Coronavirus disease 2019 (COVID-19) is the newly emerging viral disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The epidemic sparked in December 2019 at Wuhan city, China that causes a large global outbreak and a major public health catastrophe. Till now, more than 129 million positive cases have been reported in which more than 2.81 million were dead, surveyed by Johns Hopkins University, USA. The diverse symptoms of COVID-19 and an increased number of positive cases throughout the world hypothesize that this virus assembles more variants that are preventing the pursuit of its adequate treatment as well as the development of the vaccine. In this study, 715 SARS-CoV-2 genomes were retrieved from the gisaid and NCBI viral resources involving 39 countries and 164 different types of variants were identified based on 108 Single Nucleotide Polymorphisms (SNPs) in which the ancestral type of SARS-CoV-2 was found as the most frequent and the most prevalent in China. Moreover, variant type A104 was identified as the most frequent in the USA and A52 in Japan. The study also recognized the most common SNPs such as 241, 3037, 8782, 11083, 14408, 23403, and 28144 as well as variants regarding base-pair, C > T. A total of 65 non-synonymous SNPs were recognized which were mostly located in nucleocapsid phosphoprotein, Non-structural protein 3(Nsp3), and spike glycoprotein encoding gene. Molecular divergence analysis revealed that this virus was phylogenetically related to Yunnan 2013 bat strain. This study indicates SARS-CoV-2 frequently alters their genetic material, which mostly affects the nucleocapsid phosphoprotein, and spike glycoprotein-encoding gene and makes it very challenging to develop SARS-Cov-2 vaccine and antibody-mediated rapid diagnostic kit.


Assuntos
COVID-19/virologia , Genoma Viral , SARS-CoV-2/genética , COVID-19/epidemiologia , Proteínas do Nucleocapsídeo de Coronavírus/genética , Surtos de Doenças , Evolução Molecular , Genômica , Fosfoproteínas/genética , Filogenia , Polimorfismo de Nucleotídeo Único , Glicoproteína da Espícula de Coronavírus/genética
19.
Comput Biol Chem ; 93: 107532, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34171504

RESUMO

Zoonotic Novel coronavirus disease 2019 (COVID-19) is highly pathogenic and transmissible considered as emerging pandemic disease. The virus belongs from a large virus Coronaviridae family affect respiratory tract of animal and human likely originated from bat and homology to SARA-CoV and MERS-CoV. The virus consists of single-stranded positive genomic RNA coated by nucleocapsid protein. The rate of mutation in any virulence gene may influence the phenomenon of host radiation. We have studied the molecular evolution of selected virulence genes (HA, N, RdRP and S) of novel COVID-19. We used a site-specific comparison of synonymous (silent) and non-synonymous (amino acid altering) nucleotide substitutions. Maximum Likelihood genealogies based on differential gamma distribution rates were used for the analysis of null and alternate hypothesis. The null hypothesis was found more suitable for the analysis using Likelihood Ratio Test (LRT) method, confirming higher rate of substitution. The analysis revealed that RdRP gene had the fastest rate evolution followed by HA gene. We have also reported the new motifs for different virulence genes, which are further useful to design new detection and diagnosis kit for COVID -19.


Assuntos
Proteínas do Nucleocapsídeo de Coronavírus/genética , RNA-Polimerase RNA-Dependente de Coronavírus/genética , Hemaglutininas/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Virulência/genética , Substituição de Aminoácidos , Sequência de Bases , Evolução Molecular , Genes Virais , Fosfoproteínas/genética , SARS-CoV-2/patogenicidade
20.
Clin Exp Immunol ; 205(3): 363-378, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34061349

RESUMO

Since December 2019, Coronavirus disease-19 (COVID-19) has spread rapidly throughout the world, leading to a global effort to develop vaccines and treatments. Despite extensive progress, there remains a need for treatments to bolster the immune responses in infected immunocompromised individuals, such as cancer patients who recently underwent a haematopoietic stem cell transplantation. Immunological protection against COVID-19 is mediated by both short-lived neutralizing antibodies and long-lasting virus-reactive T cells. Therefore, we propose that T cell therapy may augment efficacy of current treatments. For the greatest efficacy with minimal adverse effects, it is important that any cellular therapy is designed to be as specific and directed as possible. Here, we identify T cells from COVID-19 patients with a potentially protective response to two major antigens of the SARS-CoV-2 virus, Spike and Nucleocapsid protein. By generating clones of highly virus-reactive CD4+ T cells, we were able to confirm a set of nine immunodominant epitopes and characterize T cell responses against these. Accordingly, the sensitivity of T cell clones for their specific epitope, as well as the extent and focus of their cytokine response was examined. Moreover, using an advanced T cell receptor (TCR) sequencing approach, we determined the paired TCR-αß sequences of clones of interest. While these data on a limited population require further expansion for universal application, the results presented here form a crucial first step towards TCR-transgenic CD4+ T cell therapy of COVID-19.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , COVID-19/imunologia , COVID-19/terapia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , COVID-19/virologia , Células Clonais/imunologia , Células Clonais/virologia , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/genética , Citocinas/biossíntese , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Imunização Passiva , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
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