Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 341
Filtrar
1.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641474

RESUMO

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause severe clinical disease in allograft recipients and infants infected in utero Virus-neutralizing antibodies defined in vitro have been proposed to confer protection against HCMV infection, and the virion envelope glycoprotein B (gB) serves as a major target of neutralizing antibodies. The viral fusion protein gB is nonfusogenic on its own and requires glycoproteins H (gH) and L (gL) for membrane fusion, which is in contrast to requirements of related class III fusion proteins, including vesicular stomatitis virus glycoprotein G (VSV-G) or baculovirus gp64. To explore requirements for gB's fusion activity, we generated a set of chimeras composed of gB and VSV-G or gp64, respectively. These gB chimeras were intrinsically fusion active and led to the formation of multinucleated cell syncytia when expressed in the absence of other viral proteins. Utilizing a panel of virus-neutralizing gB-specific monoclonal antibodies (MAbs), we could demonstrate that syncytium formation of the fusogenic gB/VSV-G chimera can be significantly inhibited by only a subset of neutralizing MAbs which target antigenic domain 5 (AD-5) of gB. This observation argues for differential modes of action of neutralizing anti-gB MAbs and suggests that blocking the membrane fusion function of gB could be one mechanism of antibody-mediated virus neutralization. In addition, our data have important implications for the further understanding of the conformation of gB that promotes membrane fusion as well as the identification of structures in AD-5 that could be targeted by antibodies to block this early step in HCMV infection.IMPORTANCE HCMV is a major global health concern, and antiviral chemotherapy remains problematic due to toxicity of available compounds and the emergence of drug-resistant viruses. Thus, an HCMV vaccine represents a priority for both governmental and pharmaceutical research programs. A major obstacle for the development of a vaccine is a lack of knowledge of the nature and specificities of protective immune responses that should be induced by such a vaccine. Glycoprotein B of HCMV is an important target for neutralizing antibodies and, hence, is often included as a component of intervention strategies. By generation of fusion-active gB chimeras, we were able to identify target structures of neutralizing antibodies that potently block gB-induced membrane fusion. This experimental system provides an approach to screen for antibodies that interfere with gB's fusogenic activity. In summary, our data will likely contribute to both rational vaccine design and the development of antibody-based therapies against HCMV.


Assuntos
Anticorpos Neutralizantes/farmacologia , Citomegalovirus/genética , Proteínas Mutantes Quiméricas/genética , Proteínas do Envelope Viral/genética , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/farmacologia , Sítios de Ligação , Fusão Celular , Linhagem Celular , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/virologia , Expressão Gênica , Células Gigantes/efeitos dos fármacos , Células Gigantes/metabolismo , Células Gigantes/ultraestrutura , Células Gigantes/virologia , Células HEK293 , Humanos , Camundongos , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/metabolismo , Cultura Primária de Células , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/virologia , Vesiculovirus/genética , Vesiculovirus/metabolismo , Proteínas do Envelope Viral/metabolismo
2.
J Leukoc Biol ; 107(6): 1137-1154, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32533638

RESUMO

The chemokine CCL20 is broadly produced by endothelial cells in the liver, the lung, in lymph nodes and mucosal lymphoid tissues, and recruits CCR6 expressing leukocytes, particularly dendritic cells, mature B cells, and subpopulations of T cells. How CCL20 is systemically scavenged is currently unknown. Here, we identify that fluorescently labeled human and mouse CCL20 are efficiently taken-up by the atypical chemokine receptor ACKR4. CCL20 shares ACKR4 with the homeostatic chemokines CCL19, CCL21, and CCL25, although with a lower affinity. We demonstrate that all 4 human chemokines recruit ß-arrestin1 and ß-arrestin2 to human ACKR4. Similarly, mouse CCL19, CCL21, and CCL25 equally activate the human receptor. Interestingly, at the same chemokine concentration, mouse CCL20 did not recruit ß-arrestins to human ACKR4. Further cross-species analysis suggests that human ACKR4 preferentially takes-up human CCL20, whereas mouse ACKR4 similarly internalizes mouse and human CCL20. Furthermore, we engineered a fluorescently labeled chimeric chemokine consisting of the N-terminus of mouse CCL25 and the body of mouse CCL19, termed CCL25_19, which interacts with and is taken-up by human and mouse ACKR4.


Assuntos
Quimiocina CCL19/metabolismo , Quimiocina CCL20/metabolismo , Quimiocina CCL21/metabolismo , Quimiocinas CC/metabolismo , Receptores CCR/metabolismo , beta-Arrestinas/genética , Sequência de Aminoácidos , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Sítios de Ligação , Linhagem Celular , Quimiocina CCL19/química , Quimiocina CCL19/genética , Quimiocina CCL20/química , Quimiocina CCL20/genética , Quimiocina CCL21/química , Quimiocina CCL21/genética , Quimiocinas CC/química , Quimiocinas CC/genética , Células HEK293 , Células HeLa , Humanos , Ligantes , Camundongos , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Receptores CCR/química , Receptores CCR/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Transfecção , beta-Arrestinas/metabolismo
3.
Neuron ; 107(2): 292-305.e6, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32375063

RESUMO

GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) and lead to the production of aggregating dipeptide repeat proteins (DPRs) via repeat associated non-AUG (RAN) translation. Here, we show the similar intronic GGCCTG HREs that causes spinocerebellar ataxia type 36 (SCA36) is also translated into DPRs, including poly(GP) and poly(PR). We demonstrate that poly(GP) is more abundant in SCA36 compared to c9ALS/FTD patient tissue due to canonical AUG-mediated translation from intron-retained GGCCTG repeat RNAs. However, the frequency of the antisense RAN translation product poly(PR) is comparable between c9ALS/FTD and SCA36 patient samples. Interestingly, in SCA36 patient tissue, poly(GP) exists as a soluble species, and no TDP-43 pathology is present. We show that aggregate-prone chimeric DPR (cDPR) species underlie the divergent DPR pathology between c9ALS/FTD and SCA36. These findings reveal key differences in translation, solubility, and protein aggregation of DPRs between c9ALS/FTD and SCA36.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Dipeptídeos/genética , Demência Frontotemporal/genética , Proteínas Mutantes Quiméricas/genética , Ataxias Espinocerebelares/genética , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Elementos Antissenso (Genética)/genética , Expansão das Repetições de DNA , Feminino , Humanos , Íntrons/genética , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Sequências Repetitivas de Ácido Nucleico
4.
FEBS J ; 287(4): 671-694, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31423733

RESUMO

In eukaryotes, Hsp110s are unambiguous cognates of the Hsp70 chaperones, in primary sequence, domain organization, and structure. Hsp110s function as nucleotide exchange factors (NEFs) for the Hsp70s although their apparent loss of Hsp70-like chaperone activity, nature of interdomain communication, and breadth of domain functions are still puzzling. Here, by combining single-molecule FRET, small angle X-ray scattering measurements (SAXS), and MD simulation, we show that yeast Hsp110, Sse1 lacks canonical Hsp70-like interdomain allostery. However, the protein exhibits unique noncanonical conformational changes within its domains. Sse1 maintains an open-lid substrate-binding domain (SBD) in close contact with its nucleotide-binding domain (NBD), irrespective of its ATP hydrolysis status. To further appreciate such ATP-hydrolysis-independent exhaustive interaction between two domains of Hsp110s, NBD-SBD chimera was constructed between Hsp110 (Sse1) and Hsp70 (Ssa1). In Sse1/Ssa1 chimera, we observed undocking of two domains leading to complete loss of NEF activity of Sse1. Interestingly, chimeric proteins exhibited significantly enhanced ATPase rate of Sse1-NBD compared to wild-type protein, implying that intrinsic ATPase activity of the protein remains mostly repressed. Apart from repressing the high ATPase activity of its NBD, interactions between two domains confer thermal stability to Sse1 and play critical role in the (co)chaperoning function of Sse1 in Ssa1-mediated disaggregation activity. Altogether, Sse1 exhibits a unique interdomain interaction, which is essential for its NEF activity, suppression of high intrinsic ATPase activity, co-chaperoning activity in disaggregase machinery, and stability of the protein.


Assuntos
Adenosina Trifosfatases/química , Proteínas de Choque Térmico HSP70/química , Proteínas Mutantes Quiméricas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Hidrólise , Simulação de Dinâmica Molecular , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
J Am Soc Nephrol ; 30(12): 2449-2463, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31575699

RESUMO

BACKGROUND: Atypical hemolytic uremic syndrome (HUS) is associated with high recurrence rates after kidney transplant, with devastating outcomes. In late 2011, experts in France recommended the use of highly individualized complement blockade-based prophylaxis with eculizumab to prevent post-transplant atypical HUS recurrence throughout the country. METHODS: To evaluate this strategy's effect on kidney transplant prognosis, we conducted a retrospective multicenter study from a large French nationwide registry, enrolling all adult patients with atypical HUS who had undergone complement analysis and a kidney transplant since January 1, 2007. To assess how atypical HUS epidemiology in France in the eculizumab era evolved, we undertook a population-based cohort study that included all adult patients with atypical HUS (n=397) between 2007 and 2016. RESULTS: The first study included 126 kidney transplants performed in 116 patients, 58.7% and 34.1% of which were considered to be at a high and moderate risk of atypical HUS recurrence, respectively. Eculizumab prophylaxis was used in 52 kidney transplants, including 39 at high risk of recurrence. Atypical HUS recurred after 43 (34.1%) of the transplants; in four cases, patients had received eculizumab prophylaxis and in 39 cases they did not. Use of prophylactic eculizumab was independently associated with a significantly reduced risk of recurrence and with significantly longer graft survival. In the second, population-based cohort study, the proportion of transplant recipients among patients with ESKD and atypical HUS sharply increased between 2012 and 2016, from 46.2% to 72.3%, and showed a close correlation with increasing eculizumab use among the transplant recipients. CONCLUSIONS: Results from this observational study are consistent with benefit from eculizumab prophylaxis based on pretransplant risk stratification and support the need for a rigorous randomized trial.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Síndrome Hemolítico-Urêmica Atípica/tratamento farmacológico , Inativadores do Complemento/uso terapêutico , Transplante de Rim , Adulto , Síndrome Hemolítico-Urêmica Atípica/epidemiologia , Síndrome Hemolítico-Urêmica Atípica/genética , Síndrome Hemolítico-Urêmica Atípica/cirurgia , Proteínas Inativadoras do Complemento C3b/genética , Proteínas do Sistema Complemento/análise , Feminino , França , Sobrevivência de Enxerto/efeitos dos fármacos , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Proteínas Mutantes Quiméricas/genética , Cuidados Pré-Operatórios , Modelos de Riscos Proporcionais , Recidiva , Sistema de Registros , Estudos Retrospectivos , Prevenção Secundária
6.
PLoS One ; 14(10): e0223337, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31577830

RESUMO

BACKGROUND: RNA sequencing has been proposed as a means of increasing diagnostic rates in studies of undiagnosed rare inherited disease. Recent studies have reported diagnostic improvements in the range of 7.5-35% by profiling splicing, gene expression quantification and allele specific expression. To-date however, no study has systematically assessed the presence of gene-fusion transcripts in cases of germline disease. Fusion transcripts are routinely identified in cancer studies and are increasingly recognized as having diagnostic, prognostic or therapeutic relevance. Isolated reports exist of fusion transcripts being detected in cases of developmental and neurological phenotypes, and thus, systematic application of fusion detection to germline conditions may further increase diagnostic rates. However, current fusion detection methods are unsuited to the investigation of germline disease due to performance biases arising from their development using tumor, cell-line or in-silico data. METHODS: We describe a tailored approach to fusion candidate identification and prioritization in a cohort of 47 undiagnosed, suspected inherited disease patients. We modify an existing fusion transcript detection algorithm by eliminating its cell line-derived filtering steps, and instead, prioritize candidates using a custom workflow that integrates genomic and transcriptomic sequence alignment, biological and technical annotations, customized categorization logic, and phenotypic prioritization. RESULTS: We demonstrate that our approach to fusion transcript identification and prioritization detects genuine fusion events excluded by standard analyses and efficiently removes phenotypically unimportant candidates and false positive events, resulting in a reduced candidate list enriched for events with potential phenotypic relevance. We describe the successful genetic resolution of two previously undiagnosed disease cases through the detection of pathogenic fusion transcripts. Furthermore, we report the experimental validation of five additional cases of fusion transcripts with potential phenotypic relevance. CONCLUSIONS: The approach we describe can be implemented to enable the detection of phenotypically relevant fusion transcripts in studies of rare inherited disease. Fusion transcript detection has the potential to increase diagnostic rates in rare inherited disease and should be included in RNA-based analytical pipelines aimed at genetic diagnosis.


Assuntos
Estudos de Associação Genética , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Predisposição Genética para Doença , Proteínas Mutantes Quiméricas/genética , Doenças Raras/diagnóstico , Doenças Raras/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética/métodos , Marcadores Genéticos , Humanos , Lactente , Padrões de Herança , Masculino , Pessoa de Meia-Idade , Fenótipo , Fluxo de Trabalho , Adulto Jovem
7.
Biochem Biophys Res Commun ; 515(2): 386-393, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31155288

RESUMO

Chickens, one of the most important industrial animals, are a biological animal model. Here we focused on the transient receptor potential vanilloid 1 (TRPV1) to understand the pain system for acidic stimuli in chickens compared with mice. By using a whole-cell patch clamp system, we confirmed that acidic stimuli activate both chicken TRPV1 (cTRPV1) and mouse TRPV1 (mTRPV1), but the peak current of cTRPV1 is lower than that of mTRPV1, and it is difficult to desensitize cTRPV1 with an acidic stimulus compared to mTRPV1. Since the C-terminal of the calmodulin (CaM) binding site in TRPV1 was reported as one of the important structures for TRPV1 desensitization, we made chimeric cTRPV1 in which the CaM binding site of chicken is changed to that of mouse (cTRPV1-mCaM). We also compared the acidic responses of native chicken dorsal root ganglion (DRG) cells with that of mouse DRG cells. The TRPV1-mCaM results showed that the desensitization of mutant cTRPV1 was similar to that of mTRPV1, and that the basal activities of mutant cTRPV1 were significantly higher than those of cTRPV1. It was also difficult to desensitize the chicken DRG cells with an acidic stimulus, unlike the mouse DRG cells. These results suggest that there are differences in the pain transduction systems for acidic stimuli between chickens and mice that are caused by the dysfunction of the C-terminal CaM biding site of cTRPV1. These results imply that chickens repeatedly feel weak pain from an acidic stimulus, without desensitization.


Assuntos
Proteínas Aviárias/metabolismo , Canais de Cátion TRPV/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Proteínas Aviárias/genética , Sítios de Ligação/genética , Calmodulina/metabolismo , Capsaicina/farmacologia , Células Cultivadas , Galinhas , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Técnicas de Patch-Clamp , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade da Espécie , Canais de Cátion TRPV/química , Canais de Cátion TRPV/genética
8.
J Mol Diagn ; 21(5): 924-931, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31229653

RESUMO

Many patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency have CAH-X syndrome, a connective tissue dysplasia consistent with hypermobility-type Ehlers-Danlos syndrome due to a contiguous gene deletion involving the adjacent CYP21A2 and TNXB genes. CAH-X syndrome is caused by carrying CYP21A1P-TNXA/TNXB chimeric genes [CAH-X chimera 1 (CH-1) and chimera 2 (CH-2)] on one or more alleles. Genetic analysis is cumbersome due to pseudogene interference. We developed a PCR-based CAH-X high-throughput screening method to assess the copy numbers of TNXB exons 35 and 40; this method is amenable to either real-time quantitative PCR or droplet digital PCR (ddPCR). The assay was validated in a cohort of 278 subjects from 146 unrelated CAH families. Results were confirmed by a validated Sanger sequencing platform. A total of 44 CAH-X-positive calls were made, with 42 (26 CH-1 and 16 CH-2) confirmed. The assay had 100% sensitivity (42 true/42 positives), 99.2% specificity (234 true/236 negatives), and an overall 99.3% accuracy (276/278). Calls made by real-time quantitative PCR and ddPCR were consistent (100%), and ddPCR offered easier data interpretation. The CAH-X prevalence was 15.6% (21/135 probands), higher than the previously estimated 8.5%, and was particularly high (29.2% or 21/72) in those with a 30-Kb deletion. This assay is suitable for high-throughput CAH-X screening, especially in subjects testing positive for CAH in neonatal screening.


Assuntos
Hiperplasia Suprarrenal Congênita/complicações , Síndrome de Ehlers-Danlos/diagnóstico , Proteínas Mutantes Quiméricas/genética , Mutação , Pseudogenes/genética , Tenascina/genética , Adolescente , Adulto , Criança , Pré-Escolar , Síndrome de Ehlers-Danlos/etiologia , Síndrome de Ehlers-Danlos/genética , Feminino , Deleção de Genes , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Adulto Jovem
9.
J Pharmacol Exp Ther ; 370(3): 703-714, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31010843

RESUMO

With the advancement of medicine, the utility of protein therapeutics is increasing exponentially. However, a significant number of protein therapeutics suffer from grave limitations, which include their subpar pharmacokinetics. In this study, we have reviewed the emerging field of protein chimerization for improving the short circulatory half-life of protein therapeutics. We have discussed various aspects of protein therapeutics aiming at their mechanism of clearance and various approaches used to increase their short circulatory half-life with principal focus on the concept of chimerization. Furthermore, we have comprehensively reviewed various components of chimera, such as half-life extension partners and linkers, their shortcomings, and prospective work to be undertaken for developing effective chimeric protein therapeutics.


Assuntos
Proteínas Mutantes Quiméricas/farmacocinética , Proteínas Mutantes Quiméricas/uso terapêutico , Engenharia de Proteínas/métodos , Animais , Humanos , Proteínas Mutantes Quiméricas/genética , Engenharia de Proteínas/tendências
10.
ACS Chem Biol ; 14(4): 696-703, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30921511

RESUMO

Piperazate (Piz) is a nonproteinogenic amino acid noted for its unusual N-N bond motif. Piz is a proline mimic that imparts conformational rigidity to peptides. Consequently, piperazyl molecules are often bioactive and desirable for therapeutic exploration. The in vitro characterization of Kutzneria enzymes KtzI and KtzT recently led to a biosynthetic pathway for Piz. However, Piz anabolism in vivo has remained completely uncharacterized. Herein, we describe the systematic interrogation of actinobacterial Piz metabolism using a combination of bioinformatics, genetics, and select biochemistry. Following studies in Streptomyces flaveolus, Streptomyces lividans, and several environmental Streptomyces isolates, our data suggest that KtzI-type enzymes are conditionally dispensable for Piz production. We also demonstrate the feasibility of Piz monomer production using engineered actinobacteria for the first time. Finally, we show that some actinobacteria employ fused KtzI-KtzT chimeric enzymes to produce Piz. Our findings have implications for future piperazyl drug discovery, pathway engineering, and fine chemical bioproduction.


Assuntos
Aminoácidos/química , Piridazinas/química , Aminoácidos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biologia Computacional , Cinética , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Mutação , Piridazinas/metabolismo , Streptomyces/genética , Streptomyces/isolamento & purificação , Streptomyces/metabolismo
11.
Technol Cancer Res Treat ; 18: 1533033818823029, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30803359

RESUMO

Long noncoding RNAs are capable of regulating gene expression at multiple levels. These RNA molecules are also involved in a variety of physiological and pathological processes. Emerging data demonstrate that a series of differentially expressed long noncoding RNAs are implicated in tumorigenesis. In the present study, we used microarray analysis to identify long noncoding RNAs that are dysregulated in non-small-cell lung cancer when compared to normal lung tissues. Accordingly, we performed quantitative real-time polymerase chain reaction to analyze the levels of long noncoding RNA and the cis target gene. We further found the oncogene property of long noncoding RNA that long noncoding RNA downexpression inhibits non-small-cell lung cancer cells proliferation and migration based on 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide and colony formation assays and wound healing as well as transwell assays. The influence of long noncoding RNA on cell cycle of non-small-cell lung cancer cells is also analyzed by flow cytometry. Among the dysregulated long noncoding RNAs, we identified INS-IGF2 readthrough, transcript variant 1, noncoding RNA (NR_003512.3) is upregulated in non-small-cell lung cancer tissues, the cis gene of which is insulin-like growth factor 2 gene hinted by bioinformatics analysis. We also observed that downregulation of INS-IGF2 readthrough, transcript variant 1, noncoding RNA reduces insulin-like growth factor 2 messenger RNA expression. Furthermore, INS-IGF2 readthrough, transcript variant 1, noncoding RNA downregulation suppresses non-small-cell lung cancer cell proliferation and migration. This downregulation results in a concomitant inhibition of the G1/S transition in non-small-cell lung cancer cells. Our findings suggest that INS-IGF2 readthrough, transcript variant 1, noncoding RNA may be an oncogene involved in the development of lung cancer. Therefore, we speculate that INS-IGF2 readthrough, transcript variant 1, noncoding RNA represents a potential therapeutic target for lung cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular , Proliferação de Células , Fase G1 , Proteínas Mutantes Quiméricas/genética , RNA Longo não Codificante/genética , Fase S , Apoptose , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Estudos de Casos e Controles , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Prognóstico , Células Tumorais Cultivadas
12.
BMC Genomics ; 20(1): 38, 2019 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-30642248

RESUMO

BACKGROUND: The process of gene fusion involves the formation of a single chimeric gene from multiple complete or partial gene sequences. Gene fusion is recognized as an important mechanism by which genes and their protein products can evolve new functions. The presence-absence of gene fusions can also be useful characters for inferring evolutionary relationships between organisms. RESULTS: Here we show that the nuclear genomes of two unrelated single-celled algae, the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans, possess an unexpected diversity of genes for ubiquitin fusion proteins, including novel arrangements in which ubiquitin occupies amino-terminal, carboxyl-terminal, and internal positions relative to its fusion partners. We explore the evolution of the ubiquitin multigene family in both genomes, and show that both algae possess a gene encoding an ubiquitin-nickel superoxide dismutase fusion protein (Ubiq-NiSOD) that is widely but patchily distributed across the eukaryotic tree of life - almost exclusively in phototrophs. CONCLUSION: Our results suggest that ubiquitin fusion proteins are more common than currently appreciated; because of its small size, the ubiquitin coding region can go undetected when gene predictions are carried out in an automated fashion. The punctate distribution of the Ubiq-NiSOD fusion across the eukaryotic tree could serve as a beacon for the spread of plastids from eukaryote to eukaryote by secondary and/or tertiary endosymbiosis.


Assuntos
Cercozoários/genética , Criptófitas/genética , Fusão Gênica , Proteínas Mutantes Quiméricas/genética , Ubiquitinas/classificação , Ubiquitinas/genética , Evolução Molecular , Filogenia , Simbiose
13.
Breast Cancer ; 26(3): 305-316, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30446971

RESUMO

BACKGROUND: This study aimed to identify the differentially expressed genes (DEGs) and the typical fusion genes in different types of breast cancers using RNA-seq. METHODS: GSE52643 was downloaded from Gene Expression Omnibus, which included 1 normal sample (MCF10A) and 7 breast cancer samples (BT-474, BT-20, MCF7, MDA-MB-231, MDA-MB-468, T47D, and ZR-75-1). The transcript abundance and the DEGs screening were performed by Cufflinks. The functional and pathway enrichment was analyzed by Gostats. SnowShoes-FTD was applied to identify the fusion genes. RESULTS: We screened 430, 445, 397, 417, 369, 557, and 375 DEGs in BT-474, BT-20, MCF7, DA-MB-231, MDA-MB-468, T47D, and ZR-75-1, respectively, compared with MCF10A. DEGs in each comparison group (such as CD40 and CDH1) were significantly enriched in the functions of cell adhesion and extracellular matrix organization and pathways of CAMs and ECM receptor interaction. UCP2 was a common DEG in the 7 comparison groups. SFRP1 and MMP7 were significantly enriched in wnt/-catenin signaling pathway in MDA-MB-231. FAS was significantly enriched in autoimmune thyroid disease pathway in BT-474. Besides, we screened 96 fusion genes, such as ESR1-C6orf97 in ZR-75-1, COBRA1-C9orf167 in BT-20, and VAPB-IKZF3 and ACACA-STAC2 in BT-474. CONCLUSIONS: The DEGs such as SFRP1, MMP7, CDH1, FAS, and UCP2 might be the potential biomarkers in breast cancer. Furthermore, some pivotal fusion genes like ESR1-C6orf97 with COBRA1-C9orf167 and VAPB-IKZF3 with ACACA-STAC2 were found in Luminal A and Luminal B breast cancer, respectively.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Proteínas Mutantes Quiméricas/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Biologia Computacional , Bases de Dados Genéticas , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Humanos , Proteínas Mutantes Quiméricas/metabolismo , Transdução de Sinais
14.
Nucleic Acids Res ; 47(D1): D994-D1004, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30407583

RESUMO

Gene fusion is one of the hallmarks of cancer genome via chromosomal rearrangement initiated by DNA double-strand breakage. To date, many fusion genes (FGs) have been established as important biomarkers and therapeutic targets in multiple cancer types. To better understand the function of FGs in cancer types and to promote the discovery of clinically relevant FGs, we built FusionGDB (Fusion Gene annotation DataBase) available at https://ccsm.uth.edu/FusionGDB. We collected 48 117 FGs across pan-cancer from three representative fusion gene resources: the improved database of chimeric transcripts and RNA-seq data (ChiTaRS 3.1), an integrative resource for cancer-associated transcript fusions (TumorFusions), and The Cancer Genome Atlas (TCGA) fusions by Gao et al. For these ∼48K FGs, we performed functional annotations including gene assessment across pan-cancer fusion genes, open reading frame (ORF) assignment, and retention search of 39 protein features based on gene structures of multiple isoforms with different breakpoints. We also provided the fusion transcript and amino acid sequences according to multiple breakpoints and transcript isoforms. Our analyses identified 331, 303 and 667 in-frame FGs with retaining kinase, DNA-binding, and epigenetic factor domains, respectively, as well as 976 FGs lost protein-protein interaction. FusionGDB provides six categories of annotations: FusionGeneSummary, FusionProtFeature, FusionGeneSequence, FusionGenePPI, RelatedDrug and RelatedDisease.


Assuntos
Bases de Dados Genéticas , Fusão Gênica , Proteínas Mutantes Quiméricas/genética , Neoplasias/genética , Sequência de Aminoácidos , Anotação de Sequência Molecular , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/metabolismo , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/genética , Fases de Leitura Aberta , Mapeamento de Interação de Proteínas , Interface Usuário-Computador
15.
Nat Commun ; 9(1): 4856, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30451839

RESUMO

The development of robust, versatile and accurate toolsets is critical to facilitate therapeutic genome editing applications. Here we establish RNA-programmable Cas9-Cas9 chimeras, in single- and dual-nuclease formats, as versatile genome engineering systems. In both of these formats, Cas9-Cas9 fusions display an expanded targeting repertoire and achieve highly specific genome editing. Dual-nuclease Cas9-Cas9 chimeras have distinct advantages over monomeric Cas9s including higher target site activity and the generation of predictable precise deletion products between their target sites. At a therapeutically relevant site within the BCL11A erythroid enhancer, Cas9-Cas9 nucleases produced precise deletions that comprised up to 97% of all sequence alterations. Thus Cas9-Cas9 chimeras represent an important tool that could be particularly valuable for therapeutic genome editing applications where a precise cleavage position and defined sequence end products are desirable.


Assuntos
Proteínas de Bactérias/genética , Sequência de Bases , Sistemas CRISPR-Cas , Endonucleases/genética , Edição de Genes/métodos , Proteínas Mutantes Quiméricas/genética , Deleção de Sequência , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Endonucleases/metabolismo , Engenharia Genética , Genoma Humano , Células HEK293 , Humanos , Células Jurkat , Células K562 , Proteínas Mutantes Quiméricas/metabolismo , Neisseria meningitidis/enzimologia , Neisseria meningitidis/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética
16.
Nat Commun ; 9(1): 4879, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30451858

RESUMO

Kainate-type glutamate receptors play critical roles in excitatory synaptic transmission and synaptic plasticity in the brain. GluK1 and GluK2 possess fundamentally different capabilities in surface trafficking as well as synaptic targeting in hippocampal CA1 neurons. Here we find that the excitatory postsynaptic currents (EPSCs) are significantly increased by the chimeric GluK1(SPGluK2) receptor, in which the signal peptide of GluK1 is replaced with that of GluK2. Coexpression of GluK1 signal peptide completely suppresses the gain in trafficking ability of GluK1(SPGluK2), indicating that the signal peptide represses receptor trafficking in a trans manner. Furthermore, we demonstrate that the signal peptide directly interacts with the amino-terminal domain (ATD) to inhibit the synaptic and surface expression of GluK1. Thus, we have uncovered a trafficking mechanism for kainate receptors and propose that the cleaved signal peptide behaves as a ligand of GluK1, through binding with the ATD, to repress forward trafficking of the receptor.


Assuntos
Região CA1 Hipocampal/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Sinais Direcionadores de Proteínas/genética , Receptores de Ácido Caínico/metabolismo , Transmissão Sináptica/fisiologia , Animais , Animais Recém-Nascidos , Sítios de Ligação , Região CA1 Hipocampal/citologia , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Hemaglutininas/genética , Hemaglutininas/metabolismo , Humanos , Microtomia , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Plasticidade Neuronal , Técnicas de Cultura de Órgãos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos , Receptores de Ácido Caínico/química , Receptores de Ácido Caínico/genética , Sinapses/metabolismo , Sinapses/ultraestrutura
17.
Plant Sci ; 274: 2-7, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30080604

RESUMO

Knowledge on the subcellular localization of target proteins in a plant mutant background is important for revealing the function of the genes investigated. However, in Arabidopsis and rice, mutant lethality is one major barrier to such studies. Here we describe an optimized bombardment-mediated transient expression approach for studying subcellular protein localization in Arabidopsis seedling of lethal mutants. The whole experiment comprises four stages: cultivation and preparation of plants, coating gold particles with plasmid DNA, delivery of DNA into plants via bombardment, plant incubation and gene expression analysis which include localization and dynamics, co-localization comparison with reporter proteins and functional analysis. The entire process takes about 3-10 days from plant cultivation to protein detection. It has a high efficiency and the results are reproducible. Additionally, this protocol is applicable for the transient expression of chimeric fluorescent fusion proteins in juvenile rice seedlings and leaf sheaths, saving time dramatically in comparison of generating transgenic rice plant.


Assuntos
Arabidopsis/metabolismo , Biolística/métodos , Proteínas de Plantas/metabolismo , Plântula/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas/genética , Genes Letais/genética , Microscopia Confocal , Proteínas Mutantes Quiméricas/genética , Mutação/genética , Frações Subcelulares/metabolismo
18.
19.
Sci China Life Sci ; 61(8): 954-965, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29705873

RESUMO

TRIM5α restricts retroviruses in a species-specific manner. Cyclophilin A was independently retrotransposed into the TRIM5 loci in different species, leading to the generation of antiviral TRIM5-cyclophilin A (TRIMCyp) proteins. Previously, we found that assam macaques express a TRIMCyp chimera (amTRIMCyp), along with a TRIM5α allelic protein (amTRIM5α). Herein, we investigated the antiviral activity of amTRIMCyp and amTRIM5α individually, as well as their interaction and joint effects. amTRIMCyp showed a divergent restriction pattern from amTRIM5α. Although both proteins potently restricted the replication of HIV-1, only amTRIM5α inhibited N-MLV. Remarkably, cellular anti-HIV-1 activity increased when amTRIMCyp and amTRIM5α were coexpressed, indicating a synergistic block of HIV-1 replication. Consistently, PMBCs from heterozygous amTRIM5α/TRIMCyp showed stronger resistance to HIV-1 infection than those from amTRIM5α/TRIM5α homozygotes. The anti-HIV-1 synergistic effect was dependent on the amTRIMCyp-amTRIM5α interaction. In contrast, amTRIMCyp completely abrogated the anti-N-MLV activity mediated by amTRIM5α, showing a dominant-negative effect, indicating that the generation of amTRIMCyp was involved in the trade-off between divergent restriction activities. Our results provide a new paradigm to study functional trade-offs mediated by allelic proteins, a theoretical basis for utilizing animal models with various TRIM5 alleles, as well as novel HIV-1 gene therapy strategies.


Assuntos
HIV-1/imunologia , Vírus da Leucemia Murina/imunologia , Macaca/imunologia , Proteínas Mutantes Quiméricas/imunologia , Infecções por Retroviridae/imunologia , Animais , Gatos , Linhagem Celular , Ciclofilina A/genética , Ciclofilina A/imunologia , Ciclofilina A/metabolismo , Expressão Gênica/imunologia , Células HEK293 , HIV-1/fisiologia , Humanos , Vírus da Leucemia Murina/fisiologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Macaca/virologia , Camundongos , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Proteínas/genética , Proteínas/imunologia , Proteínas/metabolismo , Interferência de RNA/imunologia , Infecções por Retroviridae/virologia , Ubiquitina-Proteína Ligases
20.
Science ; 359(6382): 1361-1365, 2018 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-29567707

RESUMO

Adoptive T cell transfer (ACT) is a new area of transfusion medicine involving the infusion of lymphocytes to mediate antitumor, antiviral, or anti-inflammatory effects. The field has rapidly advanced from a promising form of immuno-oncology in preclinical models to the recent commercial approvals of chimeric antigen receptor (CAR) T cells to treat leukemia and lymphoma. This Review describes opportunities and challenges for entering mainstream oncology that presently face the CAR T field, with a focus on the challenges that have emerged over the past several years.


Assuntos
Engenharia Celular/métodos , Imunoterapia Adotiva/métodos , Proteínas Mutantes Quiméricas/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Linfócitos T/transplante , Terapia Baseada em Transplante de Células e Tecidos , Ensaios Clínicos como Assunto , Engenharia Genética , Humanos , Proteínas Mutantes Quiméricas/genética , Receptores de Antígenos de Linfócitos T/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA