Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 9.655
Filtrar
1.
PLoS One ; 15(7): e0235718, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32639976

RESUMO

Sugar alcohols (polyols) are abundant carbohydrates in lichen-forming algae and transported to other lichen symbionts, fungi, and bacteria. Particularly, ribitol is an abundant polyol in the lichen Cetraria sp. Polyols have important physiological roles in lichen symbiosis, but polyol utilization in lichen-associated bacteria has been largely unreported. Herein, we purified and characterized a novel ribitol dehydrogenase (RDH) from a Cetraria sp.-associated bacterium Sphingomonas sp. PAMC 26621 grown on a minimal medium containing D-ribitol (the RDH hereafter referred to as SpRDH). SpRDH is present as a trimer in its native form, and the molecular weight of SpRDH was estimated to be 39 kDa by SDS-PAGE and 117 kDa by gel filtration chromatography. SpRDH converted D-ribitol to D-ribulose using NAD+ as a cofactor. As far as we know, SpRDH is the first RDH belonging to the medium-chain dehydrogenase/reductase family. Multiple sequence alignments indicated that the catalytic amino acid residues of SpRDH consist of Cys37, His65, Glu66, and Glu157, whereas those of short-chain RDHs consist of Ser, Tyr, and Lys. Furthermore, unlike other short-chain RDHs, SpRDH did not require divalent metal ions for its catalytic activity. Despite SpRDH originating from a psychrophilic Arctic bacterium, Sphingomonas sp., it had maximum activity at 60°C and exhibited high thermal stability within the 4-50°C range. Further studies on the structure/function relationship and catalytic mechanism of SpRDH will expand our understanding of its role in lichen symbiosis.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Líquens/microbiologia , Ribitol/metabolismo , Sphingomonas/enzimologia , Desidrogenase do Álcool de Açúcar/isolamento & purificação , Desidrogenase do Álcool de Açúcar/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Homologia de Sequência , Sphingomonas/crescimento & desenvolvimento , Especificidade por Substrato , Desidrogenase do Álcool de Açúcar/genética
2.
PLoS One ; 15(7): e0235746, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32678853

RESUMO

Azole resistant fungal infections remain a health problem for the immune compromised. Current therapies are limited due to rises in new resistance mechanisms. Therefore, it is important to identify new drug targets for drug discovery and novel therapeutics. Arv1 (are1 are2 required for viability 1) function is highly conserved between multiple pathogenic fungal species. Candida albicans (C. albicans) cells lacking CaArv1 are azole hypersusceptible and lack virulence. Saccharomyces cerevisiae (S. cerevisiae) Scarv1 cells are also azole hypersusceptible, a phenotype reversed by expression of CaArv1, indicating conservation in the molecular mechanism for azole susceptibility. To define the relationship between Arv1 function and azole susceptibility, we undertook a structure/function analysis of ScArv1. We identified several conserved amino acids within the ScArv1 homology domain (ScAhd) required for maintaining normal azole susceptibility. Erg11 lanosterol 14-α-demethylase is the rate-limiting enzyme in sterol biosynthesis and is the direct target of azole antifungals, so we used our ScArv1 mutants in order to explore the relationship between ScArv1 and ScErg11. Specific ScArv1 mutants ectopically expressed from a low copy plasmid were unable to restore normal azole susceptibility to Scarv1 cells and had reduced Erg11 protein levels. Erg11 protein stability depended on its ability to form a heterodimeric complex with Arv1. Complex formation was required for maintaining normal azole susceptibility. Scarv1 cells expressing orthologous CaArv1 mutants also had reduced CaErg11 levels, were unable to form a CaArv1-CaErg11 complex, and were azole hypersusceptible. Scarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 could not sustain proper levels of the azole resistant CaErg11Y132F F145L protein. Caarv1/Caarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 were found to lack virulence using a disseminated candidiasis mouse model. Expressing CaErg11Y132F F145L did not reverse the lack of virulence. We hypothesize that the role of Arv1 in Erg11-dependent azole resistance is to stabilize Erg11 protein level. Arv1 inhibition may represent an avenue for treating azole resistance.


Assuntos
Candida albicans/patogenicidade , Candidíase/microbiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esterol 14-Desmetilase/metabolismo , Virulência , Sequência de Aminoácidos , Animais , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência , Esterol 14-Desmetilase/genética
3.
Proc Natl Acad Sci U S A ; 117(29): 17195-17203, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32606248

RESUMO

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.


Assuntos
Actinobacteria/genética , Genoma Bacteriano , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/metabolismo , Actinobacteria/metabolismo , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Evolução Molecular , Células HEK293 , Humanos , Modelos Moleculares , Conformação Proteica , Homologia de Sequência , Sirolimo/química , Sirolimo/metabolismo , Bibliotecas de Moléculas Pequenas/química , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
4.
BMC Bioinformatics ; 21(1): 311, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32677889

RESUMO

BACKGROUND: Polyploid organisms such as wheat complicate even the simplest of procedures in molecular biology. Whilst knowledge of genomic sequences in crops is increasing rapidly, the scientific community is still a long way from producing a full pan-genome for every species. Polymerase chain reaction and Sanger sequencing therefore remain widely used as methods for characterizing gene sequences in many varieties of crops. High sequence similarity between genomes in polyploids means that if primers are not homeologue-specific via the incorporation of a SNP at the 3' tail, sequences other than the target sequence will also be amplified. Current consensus for gene cloning in wheat is to manually perform many steps in a long bioinformatics pipeline. RESULTS: Here we present AutoCloner ( www.autocloner.com ), a fully automated pipeline for crop gene cloning that includes a free-to-use web interface for users. AutoCloner takes a sequence of interest from the user and performs a basic local alignment search tool (BLAST) search against the genome assembly for their particular polyploid crop. Homologous sequences are then compiled with the input sequence into a multiple sequence alignment which is mined for single-nucleotide polymorphisms (SNPs). Various combinations of potential primers that cover the entire gene of interest are then created and evaluated by Primer3; the set of primers with the highest score, as well as all possible primers at every SNP location, are then returned to the user for polymerase chain reaction (PCR). We have successfully used AutoCloner to clone various genes of interest in the Apogee wheat variety, which has no current genome sequence. In addition, we have successfully run the pipeline on ~ 80,000 high-confidence gene models from a wheat genome assembly. CONCLUSION: AutoCloner is the first tool to fully-automate primer design for gene cloning in polyploids, where previously the consensus within the wheat community was to perform this process manually. The web interface for AutoCloner provides a simple and effective polyploid primer-design method for gene cloning, with no need for researchers to download software or input any other details other than their sequence of interest.


Assuntos
Clonagem Molecular , Biologia Computacional/métodos , Primers do DNA/metabolismo , Poliploidia , Homologia de Sequência , Software , Triticum/genética , Substituição de Aminoácidos/genética , Sequência de Bases , Primers do DNA/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único/genética
5.
EBioMedicine ; 58: 102890, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32707445

RESUMO

BACKGROUND: The novel coronavirus (SARS-CoV-2) shares approximately 80% whole genome sequence identity and 66% spike (S) protein identity with that of SARS-CoV. The cross-neutralization between these viruses is currently not well-defined. METHODS: Here, by using the live SARS-CoV-2 virus infection assay as well as HIV-1 based pseudotyped-virus carrying the spike (S) gene of the SARS-CoV-2 (ppSARS-2) and SARS-CoV (ppSARS), we examined whether infections with SARS-CoV and SARS-CoV-2 can induce cross-neutralizing antibodies. FINDINGS: We confirmed that SARS-CoV-2 infects cells via angiotensin converting enzyme 2 (ACE2), the functional receptor for SARS-CoV, and we also found that the recombinant receptor binding domain (RBD) of the S protein of SARS-CoV effectively inhibits ppSARS-2 entry in Huh7.5 cells. However, convalescent sera from SARS-CoV and SARS-CoV-2 patients showed high neutralizing activity only against the homologous virus, with no or limited cross-neutralization activity against the other pseudotyped virus. Similar results were also observed in vaccination studies in mice. INTERPRETATION: Our study demonstrates that although both SARS-CoV and SARS-CoV-2 use ACE2 as a cellular receptor, the neutralization epitopes are not shared by these two closely-related viruses, highlighting challenges towards developing a universal vaccine against SARS-CoV related viruses. FUNDING: This work was supported by the National Key Research and Development Program of China, the National Major Project for Control and Prevention of Infectious Disease in China, and the One Belt and One Road Major Project for infectious diseases.


Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Reações Cruzadas , Vírus da SARS/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos , Betacoronavirus/genética , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Vírus da SARS/genética , Homologia de Sequência , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
6.
Viruses ; 12(6)2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32549200

RESUMO

As more cases of COVID-19 are studied and treated worldwide, it had become apparent that the lethal and most severe cases of pneumonia are due to an out-of-control inflammatory response to the SARS-CoV-2 virus. I explored the putative causes of this specific feature through a detailed genomic comparison with the closest SARS-CoV-2 relatives isolated from bats, as well as previous coronavirus strains responsible for the previous epidemics (SARS-CoV and MERS-CoV). The high variability region of the nsp3 protein was confirmed to exhibit the most variations between closest strains. It was then studied in the context of physiological and molecular data available in the literature. A number of convergent findings suggest de-mono-ADP-ribosylation (de-MARylation) of STAT1 by the SARS-CoV-2 nsp3 as a putative cause of the cytokine storm observed in the most severe cases of COVID-19. This may suggest new therapeutic approaches and help in designing assays to predict the virulence of naturally circulating SARS-like animal coronaviruses.


Assuntos
ADP-Ribosilação/fisiologia , Betacoronavirus/genética , Síndrome da Liberação de Citocina/patologia , Fator de Transcrição STAT1/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos/genética , Infecções por Coronavirus/patologia , Humanos , Inflamação/patologia , Inflamação/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Pandemias , Peptidil Dipeptidase A/biossíntese , Peptidil Dipeptidase A/genética , Pneumonia Viral/patologia , Vírus da SARS/genética , Homologia de Sequência , Proteínas não Estruturais Virais/genética
8.
Proc Natl Acad Sci U S A ; 117(26): 15193-15199, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32522874

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses an immediate, major threat to public health across the globe. Here we report an in-depth molecular analysis to reconstruct the evolutionary origins of the enhanced pathogenicity of SARS-CoV-2 and other coronaviruses that are severe human pathogens. Using integrated comparative genomics and machine learning techniques, we identify key genomic features that differentiate SARS-CoV-2 and the viruses behind the two previous deadly coronavirus outbreaks, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), from less pathogenic coronaviruses. These features include enhancement of the nuclear localization signals in the nucleocapsid protein and distinct inserts in the spike glycoprotein that appear to be associated with high case fatality rate of these coronaviruses as well as the host switch from animals to humans. The identified features could be crucial contributors to coronavirus pathogenicity and possible targets for diagnostics, prognostication, and interventions.


Assuntos
Betacoronavirus/genética , Evolução Molecular , Genoma Viral , Proteínas do Nucleocapsídeo/genética , Glicoproteína da Espícula de Coronavírus/genética , Animais , Betacoronavirus/classificação , Betacoronavirus/patogenicidade , Especificidade de Hospedeiro , Humanos , Aprendizado de Máquina , Coronavírus da Síndrome Respiratória do Oriente Médio/classificação , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Mutagênese Insercional , Sinais de Localização Nuclear/genética , Proteínas do Nucleocapsídeo/química , Filogenia , Homologia de Sequência , Glicoproteína da Espícula de Coronavírus/química , Virulência/genética
9.
Gene ; 754: 144857, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32512159

RESUMO

Ribosome Inactivating Proteins (RIPs) are RNA N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of the 28S rRNA. The occurrence of RIP genes has been described in a wide range of plant taxa, as well as in several species of bacteria and fungi. A remarkable case is the presence of these genes in metazoans belonging to the Culicinae subfamily. We reported that these genes are derived from a single horizontal gene transfer event, most likely from a bacterial donor species. Moreover, we have shown evidence that mosquito RIP genes are evolving under purifying selection, suggesting that these toxins have acquired a functional role in these organisms. In the present work, we characterized the intra-specific sequence variability of Aedes aegypti RIP genes (RIPAe1, RIPAe2, and RIPAe3) and tested their expression at the mRNA level. Our results show that RIPAe2 and RIPAe3 are transcribed and polyadenylated, and their expression levels are modulated across the developmental stages. Varibility among genes was observed, including the existence of null alleles for RIPAe1 and RIPAe2, with variants showing partial deletions. These results further support the existence of a physiological function for these foreign genes in mosquitoes. The possible nature of this functionality is discussed.


Assuntos
Aedes/genética , Inibidores da Síntese de Proteínas/metabolismo , Proteínas Inativadoras de Ribossomos/metabolismo , Ribossomos/metabolismo , Toxinas Biológicas/metabolismo , Aedes/fisiologia , Animais , Sequência de Bases , Proteínas Inativadoras de Ribossomos/genética , Homologia de Sequência , Toxinas Biológicas/genética
10.
Gene ; 754: 144861, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32531454

RESUMO

Alu sequences are the most abundant repetitive elements in the human genome, and have proliferated to more than one million copies in the human genome. Primate-specific Alu sequences account for ~10% of the human genome, and their spread within the genome has the potential to generate new exons. The new exons produced by Alu elements appear in various primate genes, and their functions have been elucidated. Here, we identified a new exon in the insulin-like 3 gene (INSL3), which evolved ~50 million years ago, and led to a splicing variant with 31 extra amino acid residues in addition to the original 95 nucleotides (NTs) of INSL3. The Alu-INSL3 isoform underwent diverse changes during primate evolution; we identified that human Alu-INSL3 might be on its way to functionality and has potential to antagonize LGR8-INSL3 function. Therefore, the present study is designed to provide an example of the evolutionary trajectory of a variant peptide hormone antagonist that caused by the insertion of an Alu element in primates.


Assuntos
Evolução Molecular , Insulina/genética , Primatas/genética , Proteínas/genética , Processamento de RNA/genética , Elementos Alu , Sequência de Aminoácidos , Animais , Sequência de Bases , Humanos , Isoformas de Proteínas , Homologia de Sequência
11.
Gene ; 754: 144853, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32531459

RESUMO

The freshwater mussel Hyriopsis cumingii, is the most important species for pearl culture in China. At present, the mechanisms underlying sex differentiation and determination remain unclear in this species. Herein the open reading frame (ORF) of Foxl2 from H. cumingii (Hc-Foxl2) was cloned, and Hc-Foxl2 expression levels in six tissues (the gonad, gill, adductor muscle, foot, mantle, and kidney) were determined. Further, we performed quantitative real-time PCR to compare expressions levels between 1 and 8 months of age and 1-, 2-, and 3-year-old H. cumingii. The localization of Hc-Foxl2 expression in the ovary was analyzed by in situ hybridization, and its function was explored using RNA interference. We found that the ORF region of Hc-Foxl2 was 1215 bp in length, encoded 404 amino acids, and contained conserved FH domains. Hc-Foxl2 was expressed in the male and female tissues, with the expression levels being significantly higher in the ovary than in the testis. In 1-8-month-old H. cumingii, Hc-Foxl2 was expressed at the highest level at 5 months of age, and the gonads began to differentiate at the same time. Moreover, in 1-, 2-, and 3-year-old individuals, Hc-Foxl2 expression levels in the ovaries gradually decreased, but they were higher than those in the testis. Strong hybridization signals for Hc-Foxl2 were detected on the oocyte membrane in 3-year-old female mussels. We also performed double-stranded RNA (dsRNA) interference experiments using three dsRNA strands, which were injected into 5-month-old H. cumingii; the interference effects were the best at 12 h and 48 h post-injection. After interference with Hc-Foxl2, the expression levels of Wnt4, which has an antagonistic relationship with Foxl2 during ovarian development, were slightly increased. Thus, we speculate that Hc-Foxl2 is a female-related gene in H. cumingii and that it is involved in sex differentiation and ovarian development.


Assuntos
Bivalves/metabolismo , Proteína Forkhead Box L2/metabolismo , Diferenciação Sexual , Sequência de Aminoácidos , Animais , Sequência de Bases , Bivalves/genética , Clonagem Molecular , Proteína Forkhead Box L2/genética , Água Doce , Regulação da Expressão Gênica , Filogenia , Homologia de Sequência
12.
PLoS One ; 15(5): e0233301, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469926

RESUMO

Bacterial spot is a destructive disease of tomato in Florida that prior to the early 1990s was caused by Xanthomonas euvesicatoria. X. perforans was first identified in Florida in 1991 and by 2006 was the only xanthomonad associated with bacterial spot disease in tomato. The ability of an X. perforans strain to outcompete X. euvesicatoria both in vitro and in vivo was at least in part associated with the production of three bacteriocins designated Bcn-A, Bcn-B, and Bcn-C. The objective of this study was to characterize the genetic determinants of these bacteriocins. Bcn-A activity was confined to one locus consisting of five ORFs of which three (ORFA, ORF2 and ORF4) were required for bacteriocin activity. The fifth ORF is predicted to encode an immunity protein to Bcn-A based on in vitro and in vivo assays. The first ORF encodes Bcn-A, a 1,398 amino acid protein, which bioinformatic analysis predicts to be a member of the RHS family of toxins. Based on results of homology modeling, we hypothesize that the amino terminus of Bcn-A interacts with a protein in the outer membrane of X. euvesicatoria. The carboxy terminus of the protein may interact with an as yet unknown protein(s) and puncture the X. euvesicatoria membrane, thereby delivering the accessory proteins into the target and causing cell death. Bcn-A appears to be activated upon secretion based on cell fractionation assays. The other two loci were each shown to be single ORFs encoding Bcn-B and Bcn-C. Both gene products possess homology toward known proteases. Proteinase activity for both Bcn-B and Bcn-C was confirmed using a milk agar assay. Bcn-B is predicted to be an ArgC-like serine protease, which was confirmed by PMSF inhibition of proteolytic activity, whereas Bcn-C has greater than 50% amino acid sequence identity to two zinc metalloproteases.


Assuntos
Proteínas de Bactérias/genética , Bacteriocinas/genética , Loci Gênicos , Lycopersicon esculentum/microbiologia , Doenças das Plantas/microbiologia , Xanthomonas/crescimento & desenvolvimento , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Bacteriocinas/biossíntese , Homologia de Sequência , Xanthomonas/classificação , Xanthomonas/genética , Xanthomonas/metabolismo
13.
PLoS One ; 15(5): e0231296, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32407378

RESUMO

Phytophthora sansomeana infects soybean and causes root rot. It was recently separated from the species complex P. megasperma sensu lato. In this study, we sequenced and annotated its complete mitochondrial genome and compared it to that of nine other Phytophthora species. The genome was assembled into a circular molecule of 39,618 bp with a 22.03% G+C content. Forty-two protein coding genes, 25 tRNA genes and two rRNA genes were annotated in this genome. The protein coding genes include 14 genes in the respiratory complexes, four ATP synthase genes, 16 ribosomal proteins genes, a tatC translocase gene, six conserved ORFs and a unique orf402. The tRNA genes encode tRNAs for 19 amino acids. Comparison among mitochondrial genomes of 10 Phytophthora species revealed three inversions, each covering multiple genes. These genomes were conserved in gene content with few exceptions. A 3' truncated atp9 gene was found in P. nicotianae. All 10 Phytophthora species, as well as other oomycetes and stramenopiles, lacked tRNA genes for threonine in their mitochondria. Phylogenomic analysis using the mitochondrial genomes supported or enhanced previous findings of the phylogeny of Phytophthora spp.


Assuntos
DNA Mitocondrial/genética , Evolução Molecular , Genoma Mitocondrial , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Phytophthora/classificação , Phytophthora/genética , Sequência de Aminoácidos , Filogenia , Homologia de Sequência , Especificidade da Espécie
14.
Mol Cell ; 78(4): 700-713.e7, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32289254

RESUMO

Impairment of ribosome function activates the MAPKKK ZAK, leading to activation of mitogen-activated protein (MAP) kinases p38 and JNK and inflammatory signaling. The mechanistic basis for activation of this ribotoxic stress response (RSR) remains completely obscure. We show that the long isoform of ZAK (ZAKα) directly associates with ribosomes by inserting its flexible C terminus into the ribosomal intersubunit space. Here, ZAKα binds helix 14 of 18S ribosomal RNA (rRNA). An adjacent domain in ZAKα also probes the ribosome, and together, these sensor domains are critically required for RSR activation after inhibition of both the E-site, the peptidyl transferase center (PTC), and ribotoxin action. Finally, we show that ablation of the RSR response leads to organismal phenotypes and decreased lifespan in the nematode Caenorhabditis elegans (C. elegans). Our findings yield mechanistic insight into how cells detect ribotoxic stress and provide experimental in vivo evidence for its physiological importance.


Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , MAP Quinase Quinase Quinases/metabolismo , Peptidil Transferases/metabolismo , RNA Ribossômico 18S/metabolismo , Ribossomos/metabolismo , Estresse Fisiológico , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Ativação Enzimática , Células HeLa , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/genética , Conformação Proteica , Domínios Proteicos , RNA Ribossômico 18S/genética , Homologia de Sequência , Transdução de Sinais
15.
Proc Natl Acad Sci U S A ; 117(19): 10313-10321, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32341169

RESUMO

The H+/Ca2+ (calcium ion) antiporter (CAX) plays an important role in maintaining cellular Ca2+ homeostasis in bacteria, yeast, and plants by promoting Ca2+ efflux across the cell membranes. However, how CAX facilitates Ca2+ balance in response to dynamic cytosolic Ca2+ perturbations is unknown. Here, we identified a type of Ca2+ "mini-sensor" in YfkE, a bacterial CAX homolog from Bacillus subtilis. The mini-sensor is formed by six tandem carboxylate residues within the transmembrane (TM)5-6 loop on the intracellular membrane surface. Ca2+ binding to the mini-sensor triggers the transition of the transport mode of YfkE from a high-affinity to a low-affinity state. Molecular dynamics simulation and fluorescence resonance energy transfer analysis suggest that Ca2+ binding to the mini-sensor causes an adjacent segment, namely, the exchanger inhibitory peptide (XIP), to move toward the Ca2+ translocation pathway to interact with TM2a in an inward-open cavity. The specific interaction was demonstrated with a synthetic peptide of the XIP, which inhibits YfkE transport and interrupts conformational changes mediated by the mini-sensor. By comparing the apo and Ca2+-bound CAX structures, we propose the following Ca2+ transport regulatory mechanism of YfkE: Ca2+ binding to the mini-sensor induces allosteric conformational changes in the Ca2+ translocation pathway via the XIP, resulting in a rearrangement of the Ca2+-binding transport site in the midmembrane. Since the Ca2+ mini-sensor and XIP sequences are also identified in other CAX homologs and/or Ca2+ transporters, including the mammalian Na+/Ca2+ exchanger (NCX), our study provides a regulatory mechanism for the Ca2+/cation transporter superfamily.


Assuntos
Antiporters/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Citoplasma/metabolismo , Escherichia coli/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Sequência de Aminoácidos , Antiporters/genética , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Escherichia coli/genética , Mutação , Conformação Proteica , Homologia de Sequência , Trocador de Sódio e Cálcio/genética
16.
Cell Host Microbe ; 27(4): 671-680.e2, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: covidwho-8785

RESUMO

Effective countermeasures against the recent emergence and rapid expansion of the 2019 novel coronavirus (SARS-CoV-2) require the development of data and tools to understand and monitor its spread and immune responses to it. However, little information is available about the targets of immune responses to SARS-CoV-2. We used the Immune Epitope Database and Analysis Resource (IEDB) to catalog available data related to other coronaviruses. This includes SARS-CoV, which has high sequence similarity to SARS-CoV-2 and is the best-characterized coronavirus in terms of epitope responses. We identified multiple specific regions in SARS-CoV-2 that have high homology to the SARS-CoV virus. Parallel bioinformatic predictions identified a priori potential B and T cell epitopes for SARS-CoV-2. The independent identification of the same regions using two approaches reflects the high probability that these regions are promising targets for immune recognition of SARS-CoV-2. These predictions can facilitate effective vaccine design against this virus of high priority.


Assuntos
Betacoronavirus/genética , Betacoronavirus/imunologia , Biologia Computacional , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Bases de Dados de Proteínas , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Humanos , Pandemias , Homologia de Sequência
17.
Proc Natl Acad Sci U S A ; 117(14): 7776-7781, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32193351

RESUMO

The Saccharomyces cerevisiae protein Ddi1 and its homologs in higher eukaryotes have been proposed to serve as shuttling factors that deliver ubiquitinated substrates to the proteasome. Although Ddi1 contains both ubiquitin-interacting UBA and proteasome-interacting UBL domains, the UBL domain is atypical, as it binds ubiquitin. Furthermore, unlike other shuttling factors, Ddi1 and its homologs contain a conserved helical domain (helical domain of Ddi1, HDD) and a retroviral-like protease (RVP) domain. The RVP domain is probably responsible for cleavage of the precursor of the transcription factor Nrf1 in higher eukaryotes, which results in the up-regulation of proteasomal subunit genes. However, enzymatic activity of the RVP domain has not yet been demonstrated, and the function of Ddi1 remains poorly understood. Here, we show that Ddi1 is a ubiquitin-dependent protease, which cleaves substrate proteins only when they are tagged with long ubiquitin chains (longer than about eight ubiquitins). The RVP domain is inactive in isolation, in contrast to its retroviral counterpart. Proteolytic activity of Ddi1 requires the HDD domain and is stimulated by the UBL domain, which mediates high-affinity interaction with the polyubiquitin chain. Compromising the activity of Ddi1 in yeast cells results in the accumulation of polyubiquitinated proteins. Aside from the proteasome, Ddi1 is the only known endoprotease that acts on polyubiquitinated substrates. Ddi1 and its homologs likely cleave polyubiquitinated substrates under conditions where proteasome function is compromised.


Assuntos
Chaperonas Moleculares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ubiquitina/genética , Proteínas de Transporte Vesicular/genética , Poliubiquitina/genética , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica/genética , Domínios Proteicos/genética , Homologia de Sequência
18.
J Mol Biol ; 432(7): 2289-2303, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32112804

RESUMO

It is becoming increasingly recognised that disordered proteins may be fuzzy, in that they can exhibit a wide variety of binding modes. In addition to the well-known process of folding upon binding (disorder-to-order transition), many examples are emerging of interacting proteins that remain disordered in their bound states (disorder-to-disorder transitions). Furthermore, disordered proteins may populate ordered and disordered states to different extents depending on their partners (context-dependent binding). Here we assemble three datasets comprising disorder-to-order, context-dependent, and disorder-to-disorder transitions of 828 protein regions represented in 2157 complexes and elucidate the sequence-determinants of the different interaction modes. We found that fuzzy interactions originate from local sequence compositions that promote the sampling of a wide range of different structures. Based on this observation, we developed the FuzPred method (http://protdyn-fuzpred.org) of predicting the binding modes of disordered proteins based on their amino acid sequences, without specifying their partners. We thus illustrate how the amino acid sequences of proteins can encode a wide range of conformational changes upon binding, including transitions from disordered to ordered and from disordered to disordered states.


Assuntos
Bases de Dados de Proteínas , Lógica Fuzzy , Proteínas Intrinsicamente Desordenadas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Análise de Sequência de Proteína/métodos , Algoritmos , Sequência de Aminoácidos , Humanos , Proteínas Intrinsicamente Desordenadas/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Homologia de Sequência
19.
Proc Natl Acad Sci U S A ; 117(15): 8633-8638, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32220960

RESUMO

To adapt to habitat temperature, vertebrates have developed sophisticated physiological and ecological mechanisms through evolution. Transient receptor potential melastatin 8 (TRPM8) serves as the primary sensor for cold. However, how cold activates TRPM8 and how this sensor is tuned for thermal adaptation remain largely unknown. Here we established a molecular framework of how cold is sensed in TRPM8 with a combination of patch-clamp recording, unnatural amino acid imaging, and structural modeling. We first observed that the maximum cold activation of TRPM8 in eight different vertebrates (i.e., African elephant and emperor penguin) with distinct side-chain hydrophobicity (SCH) in the pore domain (PD) is tuned to match their habitat temperature. We further showed that altering SCH for residues in the PD with solvent-accessibility changes leads to specific tuning of the cold response in TRPM8. We also observed that knockin mice expressing the penguin's TRPM8 exhibited remarkable tolerance to cold. Together, our findings suggest a paradigm of thermal adaptation in vertebrates, where the evolutionary tuning of the cold activation in the TRPM8 ion channel through altering SCH and solvent accessibility in its PD largely contributes to the setting of the cold-sensitive/tolerant phenotype.


Assuntos
Adaptação Fisiológica , Temperatura Baixa , Elefantes/fisiologia , Ativação do Canal Iônico , Spheniscidae/fisiologia , Canais de Cátion TRPM/metabolismo , Sequência de Aminoácidos , Animais , Homologia de Sequência , Canais de Cátion TRPM/genética
20.
Cell Host Microbe ; 27(4): 671-680.e2, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32183941

RESUMO

Effective countermeasures against the recent emergence and rapid expansion of the 2019 novel coronavirus (SARS-CoV-2) require the development of data and tools to understand and monitor its spread and immune responses to it. However, little information is available about the targets of immune responses to SARS-CoV-2. We used the Immune Epitope Database and Analysis Resource (IEDB) to catalog available data related to other coronaviruses. This includes SARS-CoV, which has high sequence similarity to SARS-CoV-2 and is the best-characterized coronavirus in terms of epitope responses. We identified multiple specific regions in SARS-CoV-2 that have high homology to the SARS-CoV virus. Parallel bioinformatic predictions identified a priori potential B and T cell epitopes for SARS-CoV-2. The independent identification of the same regions using two approaches reflects the high probability that these regions are promising targets for immune recognition of SARS-CoV-2. These predictions can facilitate effective vaccine design against this virus of high priority.


Assuntos
Betacoronavirus/genética , Betacoronavirus/imunologia , Biologia Computacional , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Bases de Dados de Proteínas , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Humanos , Pandemias , Homologia de Sequência
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA