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1.
Mol Cell ; 80(1): 72-86.e7, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32910895

RESUMO

Membrane protein biogenesis faces the challenge of chaperoning hydrophobic transmembrane helices for faithful membrane insertion. The guided entry of tail-anchored proteins (GET) pathway targets and inserts tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane with an insertase (yeast Get1/Get2 or mammalian WRB/CAML) that captures the TA from a cytoplasmic chaperone (Get3 or TRC40, respectively). Here, we present cryo-electron microscopy reconstructions, native mass spectrometry, and structure-based mutagenesis of human WRB/CAML/TRC40 and yeast Get1/Get2/Get3 complexes. Get3 binding to the membrane insertase supports heterotetramer formation, and phosphatidylinositol binding at the heterotetramer interface stabilizes the insertase for efficient TA insertion in vivo. We identify a Get2/CAML cytoplasmic helix that forms a "gating" interaction with Get3/TRC40 important for TA insertion. Structural homology with YidC and the ER membrane protein complex (EMC) implicates an evolutionarily conserved insertion mechanism for divergent substrates utilizing a hydrophilic groove. Thus, we provide a detailed structural and mechanistic framework to understand TA membrane insertion.


Assuntos
Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Complexos Multiproteicos/metabolismo , Linhagem Celular , Sequência Conservada , Evolução Molecular , Humanos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Fosfatidilinositóis/metabolismo , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
DNA Cell Biol ; 39(9): 1606-1620, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32749870

RESUMO

The sugars will eventually be exported transporters (SWEET) gene family is a glycoprotein gene family that can regulate the transport of sugar in plants and plays an important role in plant growth and development, as well as in response to environmental stress. In this study, Kentucky bluegrass (cv. Baron) seedlings were grown in various treatments, including heavy metal cadmium, salt, drought, cold, and heat stress for 6 h, 24 h, 48 h, and 7 day. The relative expression of the identified PpSWEET genes in Kentucky bluegrass was measured. The results showed there were a total of 13 SWEET genes, which could be divided into four clades by phylogenetic analysis. Most PpSWEET genes are alkali proteins with seven transmembrane helices. Moreover, almost all PpSWEET proteins possess similar conserved motifs and active sites. In addition, an analysis of the relative expression of PpSWEET genes under various stress treatments indicated that PpSWEET12 and PpSWEET15 had very high expression under the five types of stress, meaning they can be used as important candidate genes for studying responses to environmental stresses of turfgrass. Furthermore, certain genes only showed changes in expression under one or two specific stress treatments. This study provides important insight into the SWEET gene family in Kentucky bluegrass and its functional roles in responses to various environmental stresses.


Assuntos
Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Plantas/genética , Poa/genética , Estresse Fisiológico , Sequência Conservada , Regulação da Expressão Gênica de Plantas , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Poa/classificação , Poa/metabolismo , Domínios Proteicos
3.
Gene ; 760: 145020, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32755656

RESUMO

Conserved sequences across species have always provided valuable insights to improve our understanding on the human genome's entity and the interplay among different loci. Lymphoma/leukemia related factor (LRF) is encoded by ZBTB7A gene and belongs to an evolutionarily conserved family of transcription factors, implicated in vital cellular functions. The present data, demonstrating the wide-spread and the high overlap of the LRF/ZBTB7A recognition sites with genomic segments identified as CpG islands in the human genome, suggest that its binding capacity strongly depends on a specific sequence-encoded feature within CpGs. We have previously shown that de-methylation of the CpG island 326 lying in the ZBTB7A gene promoter is associated with impaired pharmacological induction of fetal hemoglobin in ß-type hemoglobinopathies patients. Within this context we aimed to investigate the extent of the LRF/ZBTB7A conservation among primates and mouse genome, focusing our interest also on the CpG island flanking the gene's promoter region, in an effort to further establish its epigenetic regulatory role in human hematopoiesis and pharmacological involvement in hematopoietic disorders. Comparative analysis of the human ZBTB7A nucleotide and amino acid sequences and orthologous sequences among non-human primates and mouse, exhibited high conservation scores. Pathway analysis, clearly indicated that LRF/ZBTB7A influences conserved cellular processes. These data in conjunction with the high levels of expression foremost in hematopoietic tissues, highlighted LRF/ZBTB7A as an essential factor operating indisputably during hematopoiesis.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Doenças Hematológicas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Sequência Conservada/genética , Ilhas de CpG/genética , Bases de Dados Genéticas , Hemoglobina Fetal/genética , Hematopoese/genética , Humanos , Camundongos , Primatas/genética , Regiões Promotoras Genéticas/genética
4.
PLoS Genet ; 16(8): e1008691, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32764743

RESUMO

Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, reduced fertility, and randomization of the left/right body axis. It is caused by defects of motile cilia and sperm flagella. We screened a cohort of affected individuals that lack an obvious axonemal defect for pathogenic variants using whole exome capture, next generation sequencing, and bioinformatic analysis assuming an autosomal recessive trait. We identified one subject with an apparently homozygous nonsense variant [(c.1762C>T), p.(Arg588*)] in the uncharacterized CFAP57 gene. Interestingly, the variant results in the skipping of exon 11 (58 amino acids), which may be due to disruption of an exonic splicing enhancer. In normal human nasal epithelial cells, CFAP57 localizes throughout the ciliary axoneme. Nasal cells from the PCD patient express a shorter, mutant version of CFAP57 and the protein is not incorporated into the axoneme. The missing 58 amino acids include portions of WD repeats that may be important for loading onto the intraflagellar transport (IFT) complexes for transport or docking onto the axoneme. A reduced beat frequency and an alteration in ciliary waveform was observed. Knockdown of CFAP57 in human tracheobronchial epithelial cells (hTECs) recapitulates these findings. Phylogenetic analysis showed that CFAP57 is highly conserved in organisms that assemble motile cilia. CFAP57 is allelic with the BOP2/IDA8/FAP57 gene identified previously in Chlamydomonas reinhardtii. Two independent, insertional fap57 Chlamydomonas mutant strains show reduced swimming velocity and altered waveforms. Tandem mass tag (TMT) mass spectroscopy shows that FAP57 is missing, and the "g" inner dyneins (DHC7 and DHC3) and the "d" inner dynein (DHC2) are reduced, but the FAP57 paralog FBB7 is increased. Together, our data identify a homozygous variant in CFAP57 that causes PCD that is likely due to a defect in the inner dynein arm assembly process.


Assuntos
Axonema/metabolismo , Transtornos da Motilidade Ciliar/genética , Códon sem Sentido , Dineínas/metabolismo , Proteínas/genética , Células 3T3 , Adulto , Animais , Axonema/fisiologia , Células Cultivadas , Chlamydomonas reinhardtii , Cílios/metabolismo , Cílios/fisiologia , Transtornos da Motilidade Ciliar/patologia , Sequência Conservada , Humanos , Masculino , Camundongos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas/química , Proteínas/metabolismo , Mucosa Respiratória/metabolismo
5.
PLoS Pathog ; 16(8): e1008736, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32745149

RESUMO

Human cytomegalovirus (HCMV) is one of the main causative agents of congenital viral infection in neonates. HCMV infection also causes serious morbidity and mortality among organ transplant patients. Glycoprotein B (gB) is a major target for HCMV neutralizing antibodies, yet the underlying neutralization mechanisms remain largely unknown. Here we report that 3-25, a gB-specific monoclonal antibody previously isolated from a healthy HCMV-positive donor, efficiently neutralized 14 HCMV strains in both ARPE-19 cells and MRC-5 cells. The core epitope of 3-25 was mapped to a highly conserved linear epitope on antigenic domain 2 (AD-2) of gB. A 1.8 Å crystal structure of 3-25 Fab in complex with the peptide epitope revealed the molecular determinants of 3-25 binding to gB at atomic resolution. Negative-staining electron microscopy (EM) 3D reconstruction of 3-25 Fab in complex with de-glycosylated postfusion gB showed that 3-25 Fab fully occupied the gB trimer at the N-terminus with flexible binding angles. Functionally, 3-25 efficiently inhibited HCMV infection at a post-attachment step by interfering with viral membrane fusion, and restricted post-infection viral spreading in ARPE-19 cells. Interestingly, bivalency was required for HCMV neutralization by AD-2 specific antibody 3-25 but not the AD-4 specific antibody LJP538. In contrast, bivalency was not required for HCMV binding by both antibodies. Taken together, our results reveal the structural basis of gB recognition by 3-25 and demonstrate that inhibition of viral membrane fusion and a requirement of bivalency may be common for gB AD-2 specific neutralizing antibody.


Assuntos
Anticorpos Antivirais/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Epitopos/imunologia , Proteínas do Envelope Viral/imunologia , Motivos de Aminoácidos , Anticorpos Neutralizantes/imunologia , Sequência Conservada , Citomegalovirus/química , Citomegalovirus/genética , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/virologia , Epitopos/química , Epitopos/genética , Humanos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Internalização do Vírus
6.
Sci Rep ; 10(1): 14214, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848162

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major public health concern. A handful of static structures now provide molecular insights into how SARS-CoV-2 and SARS-CoV interact with its host target, which is the angiotensin converting enzyme 2 (ACE2). Molecular recognition, binding and function are dynamic processes. To evaluate this, multiple 500 ns or 1 µs all-atom molecular dynamics simulations were performed to better understand the structural stability and interfacial interactions between the receptor binding domain of the spike (S) protein of SARS-CoV-2 and SARS-CoV bound to ACE2. Several contacts were observed to form, break and reform in the interface during the simulations. Our results indicate that SARS-CoV-2 and SARS-CoV utilizes unique strategies to achieve stable binding to ACE2. Several differences were observed between the residues of SARS-CoV-2 and SARS-CoV that consistently interacted with ACE2. Notably, a stable salt bridge between Lys417 of SARS-CoV-2 S protein and Asp30 of ACE2 as well as three stable hydrogen bonds between Tyr449, Gln493 and Gln498 of SARS-CoV-2 and Asp38, Glu35 and Lys353 of ACE2 were observed, which were absent in the ACE2-SARS-CoV interface. Some previously reported residues, which were suggested to enhance the binding affinity of SARS-CoV-2, were not observed to form stable interactions in these simulations. Molecular mechanics-generalized Born surface area based free energy of binding was observed to be higher for SARS-CoV-2 in all simulations. Stable binding to the host receptor is crucial for virus entry. Therefore, special consideration should be given to these stable interactions while designing potential drugs and treatment modalities to target or disrupt this interface.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Vírus da SARS/fisiologia , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Pandemias , Peptidil Dipeptidase A/química , Ligação Proteica , Conformação Proteica , Glicoproteína da Espícula de Coronavírus/química
7.
Nat Commun ; 11(1): 3969, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769976

RESUMO

Mevalonate diphosphate decarboxylases (MDDs) catalyze the ATP-dependent-Mg2+-decarboxylation of mevalonate-5-diphosphate (MVAPP) to produce isopentenyl diphosphate (IPP), which is essential in both eukaryotes and prokaryotes for polyisoprenoid synthesis. The substrates, MVAPP and ATP, have been shown to bind sequentially to MDD. Here we report crystals in which the enzyme remains active, allowing the visualization of conformational changes in Enterococcus faecalis MDD that describe sequential steps in an induced fit enzymatic reaction. Initial binding of MVAPP modulates the ATP binding pocket with a large loop movement. Upon ATP binding, a phosphate binding loop bends over the active site to recognize ATP and bring the molecules to their catalytically favored configuration. Positioned substrates then can chelate two Mg2+ ions for the two steps of the reaction. Closure of the active site entrance brings a conserved lysine to trigger dissociative phosphoryl transfer of γ-phosphate from ATP to MVAPP, followed by the production of IPP.


Assuntos
Carboxiliases/metabolismo , Enterococcus faecalis/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Carboxiliases/química , Sequência Conservada , Cristalografia por Raios X , Ligantes , Metais/metabolismo , Modelos Moleculares , Estrutura Secundária de Proteína , Especificidade por Substrato
8.
PLoS One ; 15(8): e0238032, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841304

RESUMO

Longan (Dimocarpus longan Lour.) is an important commercial fruit tree in southern China. The embryogenesis of longan affects the quality and yield of fruit. A large number of alternative splicing events occurs during somatic embryogenesis (SE), which is regulated by serine/arginine-rich (SR) proteins. However, the functions of SR proteins in longan are poorly understood. In this study, 21 Dlo-SR gene family members belonging to six subfamilies were identified, among which Dlo-RSZ20a, Dlo-SR30, Dlo-SR17, Dlo-SR53 and Dlo-SR32 were localized in the nucleus, Dlo-RSZ20b, Dlo-RSZ20c, Dlo-RSZ20d, Dlo-SC18, Dlo-RS2Z29, Dlo-SCL41, and Dlo-SR33 were localized in chloroplasts, and Dlo-RS43, Dlo-SC33, Dlo-SC37, Dlo-RS2Z33, Dlo-RS2Z16, Dlo-RS2Z24, Dlo-SCL43, Dlo-SR112, and Dlo-SR59 were localized in the nucleus and chloroplasts. The Dlo-SR genes exhibited differential expression patterns in different tissues of longan. The transcript levels of Dlo-RSZ20a, Dlo-SC18, Dlo-RS2Z29, DLo-SR59, Dlo-SR53, and Dlo-SR17 were low in all analyzed tissues, whereas Dlo-RS43, Dlo-RS2Z16, Dlo-RS2Z24, and Dlo-SR30 were highly expressed in all tissues. To clarify their function during SE, the transcript levels of Dlo-SR genes were analyzed at different four stages of SE, comprising non-embryonic callus (NEC), friable-embryogenic callus (EC), incomplete compact pro-embryogenic culture (ICpEC) and globular embryo (GE). Interestingly, the transcript levels of Dlo-RS2Z29 and Dlo-SR112 were increased in embryogenic cells compared with the NEC stage, whereas transcript levels of Dlo-RSZ20a, Dlo-RS43, Dlo-SC37, and Dlo-RS2Z16 were especially increased at the GE stage compared with the other stages. Alternative splicing events of Dlo-SR mRNA precursors (pre-mRNAs) was detected during SE, with totals of 41, 29, 35, and 44 events detected during NEC, EC, ICpEC, and GE respectively. Protein-protein interaction analysis showed that SR proteins were capable of interaction with each other. The results indicate that the alternative splicing of Dlo-SR pre-mRNAs occurs during SE and that Dlo-SR proteins may interact to regulate embryogenesis of longan.


Assuntos
Perfilação da Expressão Gênica , Genômica , Proteínas de Plantas/genética , Sapindaceae/genética , Motivos de Aminoácidos , Sequência Conservada , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Técnicas de Embriogênese Somática de Plantas , Regiões Promotoras Genéticas/genética , Mapeamento de Interação de Proteínas , Sapindaceae/metabolismo
9.
PLoS One ; 15(8): e0237177, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760115

RESUMO

LEA3 proteins, a family of abiotic stress proteins, are defined by the presence of a tryptophan-containing motif, which we name the W-motif. We use Pfam LEA3 sequences to search the Phytozome database to create a W-motif definition and a LEA3 sequence dataset. A comprehensive analysis of these sequences revealed four N-terminal motifs, as well as two previously undiscovered C-terminal motifs that contain conserved acidic and hydrophobic residues. The general architecture of the LEA3 sequences consisted of an N-terminal motif with a potential mitochondrial transport signal and the twin-arginine motif cut-site, followed by a W-motif and often a C-terminal motif. Analysis of species distribution of the motifs showed that one architecture was found exclusively in Commelinids, while two were distributed fairly evenly over all species. The physiochemical properties of the different architectures showed clustering in a relatively narrow range compared to the previously studied dehydrins. The evolutionary analysis revealed that the different sequences grouped into clades based on architecture, and that there appear to be at least two distinct groups of LEA3 proteins based on their architectures and physiochemical properties. The presence of LEA3 proteins in non-vascular plants but their absence in algae suggests that LEA3 may have arisen in the evolution of land plants.


Assuntos
Sequência Conservada , Proteínas de Plantas/genética , Motivos de Aminoácidos , Evolução Molecular , Proteínas de Plantas/química , Plantas/genética , Domínios Proteicos
10.
Nat Commun ; 11(1): 3695, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728065

RESUMO

Pseudogenes are ideal markers of genome remodelling. In turn, the mouse is an ideal platform for studying them, particularly with the recent availability of strain-sequencing and transcriptional data. Here, combining both manual curation and automatic pipelines, we present a genome-wide annotation of the pseudogenes in the mouse reference genome and 18 inbred mouse strains (available via the mouse.pseudogene.org resource). We also annotate 165 unitary pseudogenes in mouse, and 303, in human. The overall pseudogene repertoire in mouse is similar to that in human in terms of size, biotype distribution, and family composition (e.g. with GAPDH and ribosomal proteins being the largest families). Notable differences arise in the pseudogene age distribution, with multiple retro-transpositional bursts in mouse evolutionary history and only one in human. Furthermore, in each strain about a fifth of all pseudogenes are unique, reflecting strain-specific evolution. Finally, we find that ~15% of the mouse pseudogenes are transcribed, and that highly transcribed parent genes tend to give rise to many processed pseudogenes.


Assuntos
Pseudogenes/genética , Transcrição Genética , Animais , Sequência Conservada/genética , Evolução Molecular , Ontologia Genética , Genoma , Humanos , Camundongos Endogâmicos C57BL , Anotação de Sequência Molecular , Especificidade da Espécie
11.
BMC Evol Biol ; 20(1): 85, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32664916

RESUMO

BACKGROUND: ATP-binding cassette (ABC) transporters are involved in the active transportation of various endogenous or exogenous substances. Two ABCG2 gene subfamily members have been identified in birds. A detailed comparative study of the ABCG2 and ABCG2-like genes aid our understanding of their evolutionary history at the molecular level and provide a theoretical reference for studying the specific functions of ABCG2 and ABCG2-like genes in birds. RESULTS: We first identified 77 ABCG2/ABCG2-like gene sequences in the genomes of 41 birds. Further analysis showed that both the nucleic acid and amino acid sequences of ABCG2 and ABCG2-like genes were highly conserved and exhibited high homology in birds. However, significant differences in the N-terminal structure were found between the ABCG2 and ABCG2-like amino acid sequences. A selective pressure analysis showed that the ABCG2 and ABCG2-like genes were affected by purifying selection during the process of bird evolution. CONCLUSIONS: We believe that multiple members of the ABCG2 gene subfamily exist on chromosome 4 in the ancestors of birds. Over the long course of evolution, only the ABCG2 gene was retained on chromosome 4 in birds. The ABCG2-like gene on chromosome 6 might have originated from chromosome replication or fusion. The structural differences between the N terminus of ABCG2 protein and those of ABCG2-like proteins might lead to functional differences between the corresponding genes.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Aves/genética , Evolução Molecular , Homologia de Sequência de Aminoácidos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Animais , Cromossomos/genética , Sequência Conservada/genética , Éxons/genética , Regulação da Expressão Gênica , Genoma , Íntrons/genética , Família Multigênica , Fases de Leitura Aberta/genética , Fosforilação , Filogenia , Domínios Proteicos , Sítios de Splice de RNA/genética , Seleção Genética , Sintenia/genética
12.
BMC Evol Biol ; 20(1): 91, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32727363

RESUMO

BACKGROUND: The SIAMESE (SIM) locus is a cell-cycle kinase inhibitor (CKI) gene that has to date been identified only in plants; it encodes a protein that promotes transformation from mitosis to endoreplication. Members of the SIAMESE-RELATED (SMR) family have similar functions, and some are related to cell-cycle responses and abiotic stresses. However, the functions of SMRs are poorly understood in maize (Zea mays L.). RESULTS: In the present study, 12 putative SMRs were identified throughout the entire genome of maize, and these were clustered into six groups together with the SMRs from seven other plant species. Members of the ZmSMR family were divided into four groups according to their protein sequences. Various cis-acting elements in the upstream sequences of ZmSMRs responded to abiotic stresses. Expression analyses revealed that all ZmSMRs were upregulated at 5, 20, 25, and 35 days after pollination. In addition, we found that ZmSMR9/11/12 may have regulated the initiation of endoreplication in endosperm central cells. Additionally, ZmSMR2/10 may have been primarily responsible for the endoreplication regulation of outer endosperm or aleurone cells. The relatively high expression levels of almost all ZmSMRs in the ears and tassels also implied that these genes may function in seed development. The effects of treatments with ABA, heat, cold, salt, and drought on maize seedlings and expression of ZmSMR genes suggested that ZmSMRs were strongly associated with response to abiotic stresses. CONCLUSION: The present study is the first to conduct a genome-wide analysis of members of the ZmSMR family by investigating their locations in chromosomes, identifying regulatory elements in their promoter regions, and examining motifs in their protein sequences. Expression analysis of different endosperm developmental periods, tissues, abiotic stresses, and hormonal treatments suggests that ZmSMR genes may function in endoreplication and regulate the development of reproductive organs. These results may provide valuable information for future studies of the functions of the SMR family in maize.


Assuntos
Evolução Molecular , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Família Multigênica , Zea mays/genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos de Plantas/genética , Sequência Conservada/genética , Endosperma/genética , Duplicação Gênica , Genes de Plantas , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Análise de Regressão , Especificidade da Espécie , Estresse Fisiológico/efeitos dos fármacos , Sintenia/genética
13.
J Transl Med ; 18(1): 281, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32650788

RESUMO

BACKGROUND: The recent outbreak by SARS-CoV-2 has generated a chaos in global health and economy and claimed/infected a large number of lives. Closely resembling with SARS CoV, the present strain has manifested exceptionally higher degree of spreadability, virulence and stability possibly due to some unidentified mutations. The viral spike glycoprotein is very likely to interact with host Angiotensin-Converting Enzyme 2 (ACE2) and transmits its genetic materials and hijacks host machinery with extreme fidelity for self propagation. Few attempts have been made to develop a suitable vaccine or ACE2 blocker or virus-receptor inhibitor within this short period of time. METHODS: Here, attempt was taken to develop some therapeutic and vaccination strategies with a comparison of spike glycoproteins among SARS-CoV, MERS-CoV and the SARS-CoV-2. We verified their structure quality (SWISS-MODEL, Phyre2, and Pymol) topology (ProFunc), motifs (MEME Suite, GLAM2Scan), gene ontology based conserved domain (InterPro database) and screened several epitopes (SVMTrip) of SARS CoV-2 based on their energetics, IC50 and antigenicity with regard to their possible glycosylation and MHC/paratope binding (Vaxigen v2.0, HawkDock, ZDOCK Server) effects. RESULTS: We screened here few pairs of spike protein epitopic regions and selected their energetic, Inhibitory Concentration50 (IC50), MHC II reactivity and found some of those to be very good target for vaccination. A possible role of glycosylation on epitopic region showed profound effects on epitopic recognition. CONCLUSION: The present work might be helpful for the urgent development of a suitable vaccination regimen against SARS CoV-2.


Assuntos
Betacoronavirus/imunologia , Biologia Computacional/métodos , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Epitopos/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , Infecções por Coronavirus/prevenção & controle , Glicosilação , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Concentração Inibidora 50 , Anotação de Sequência Molecular , Pandemias , Estrutura Secundária de Proteína , Glicoproteína da Espícula de Coronavírus/química
14.
PLoS One ; 15(6): e0230652, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32603331

RESUMO

Toxin-antitoxin systems (TAS) are commonly found on bacterial plasmids and are generally involved in plasmid maintenance. In addition to plasmid maintenance, several plasmid-mediated TAS are also involved in bacterial stress response and virulence. Even though the same TAS are present in a variety of plasmid types and bacterial species, differences in their sequences, expression and functions are not well defined. Here, we aimed to identify commonly occurring plasmid TAS in Escherichia coli and Klebsiella pneumoniae and compare the sequence, expression and plasmid stability function of their variants. 27 putative type II TAS were identified from 1063 plasmids of Klebsiella pneumoniae in GenBank. Among these, ccdAB and pemIK were found to be most common, also occurring in plasmids of E. coli. Comparisons of ccdAB variants, taken from E. coli and K. pneumoniae, revealed sequence differences, while pemIK variants from IncF and IncL/M plasmids were almost identical. Similarly, the expression and plasmid stability functions of ccdAB variants varied according to the host strain and species, whereas the expression and functions of pemIK variants were consistent among host strains. The specialised functions of some TAS may determine the host specificity and epidemiology of major antibiotic resistance plasmids.


Assuntos
Enterobacteriaceae/genética , Plasmídeos/genética , Sistemas Toxina-Antitoxina/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência Conservada , Farmacorresistência Bacteriana/genética , Enterobacteriaceae/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica
15.
Gene ; 757: 144929, 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32622990

RESUMO

Phaeodactylum tricornutum is a model microalgae that is widely used to study diatom physiology and ecology. Since the meiotic process and sexual cycle have never been observed directly, P. tricornutum has been considered to be an asexual species. However, phylogenetic analysis of the P. tricornutum genome has revealed a series of meiosis-specific gene homologues in this species. We identified two copies of differently transcribed SPO11 homologs that contain the conserved motifs of Winged-helix and Toprim domains. The homolog PtSPO11-3 interacts with TopoVIB in yeast two-hybrid analysis, whereas the homolog PtSPO11-2 could rescue the sporulation defect of a Spo11 yeast mutant strain. PtSPO11-2 was also found to be significantly up-regulated at low temperatures in P. tricornutum and its key catalytic residue was important to the homolog's function in sporulation. The results herein provide positive clue that meiosis and sexual reproduction could exist in this diatom.


Assuntos
Domínio Catalítico , Diatomáceas/genética , Endodesoxirribonucleases/metabolismo , Meiose , Microalgas/genética , Sequência Conservada , Diatomáceas/fisiologia , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Microalgas/fisiologia , Ligação Proteica , Multimerização Proteica
16.
Gene ; 757: 144948, 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32652106

RESUMO

Pseudogenes are duplicated or retrotransposed DNA sequences of native functional genes. Amplification of pseudogenes along with gene of interest often produces false positive results, which is an innate problem in Reverse Transcription-quantitative PCR (RT-qPCR). Selecting a reference gene without any interference from pseudogene amplification is therefore a challenge to overcome. Among the common reference genes used for normalization (ACTB, GAPDH, HPRT1, TUBB, RNA18SN1 and B2M), B2M was found to have no pseudogenes in silico, which has also been confirmed by PCR and RT-qPCR. We also assessed the effect of pseudogenes on the determination of the stability of reference genes through data mining. The phylogenetic analysis of pseudogenes and functional genes revealed high sequence similarity among mammals. In addition, we demonstrated the deduction of pseudogene amplification signal using ValidPrime Assay (VPA) under conditions where genomic DNA contamination could not be avoided. Hence, we recommend the use of pseudo-free reference gene with consistent expression in the samples of interest or use VPA normalization method, where genomic DNA or pseudogene amplification is inevitable.


Assuntos
Perfilação da Expressão Gênica/normas , Pseudogenes , Reação em Cadeia da Polimerase em Tempo Real/normas , Microglobulina beta-2/genética , Animais , Sequência Conservada , Evolução Molecular , Humanos , Padrões de Referência
17.
Nat Commun ; 11(1): 3424, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647180

RESUMO

We have determined the cryo-electron microscopic (cryo-EM) structures of two archaeal type IV pili (T4P), from Pyrobaculum arsenaticum and Saccharolobus solfataricus, at 3.8 Å and 3.4 Å resolution, respectively. This triples the number of high resolution archaeal T4P structures, and allows us to pinpoint the evolutionary divergence of bacterial T4P, archaeal T4P and archaeal flagellar filaments. We suggest that extensive glycosylation previously observed in T4P of Sulfolobus islandicus is a response to an acidic environment, as at even higher temperatures in a neutral environment much less glycosylation is present for Pyrobaculum than for Sulfolobus and Saccharolobus pili. Consequently, the Pyrobaculum filaments do not display the remarkable stability of the Sulfolobus filaments in vitro. We identify the Saccharolobus and Pyrobaculum T4P as host receptors recognized by rudivirus SSRV1 and tristromavirus PFV2, respectively. Our results illuminate the evolutionary relationships among bacterial and archaeal T4P filaments and provide insights into archaeal virus-host interactions.


Assuntos
Archaea/metabolismo , Proteínas Arqueais/química , Evolução Biológica , Sequência de Aminoácidos , Archaea/virologia , Proteínas Arqueais/ultraestrutura , Sequência Conservada , Glicosilação , Domínios Proteicos , Estrutura Secundária de Proteína
18.
BMC Bioinformatics ; 21(Suppl 12): 305, 2020 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-32703190

RESUMO

BACKGROUND: Horizontal gene transfer, i.e. the acquisition of genetic material from nonparent organism, is considered an important force driving species evolution. Many cases of horizontal gene transfer from prokaryotes to eukaryotes have been registered, but no transfer mechanism has been deciphered so far, although viruses were proposed as possible vectors in several studies. In agreement with this idea, in our previous study we discovered that in two eukaryotic proteins bacteriophage recombination site (AttP) was adjacent to the regions originating via horizontal gene transfer. In one of those cases AttP site was present inside the introns of cysteine-rich repeats. In the present study we aimed to apply computational tools for finding multiple horizontal gene transfer events in large genome databases. For that purpose we used a sequence of cysteine-rich repeats to identify genes potentially acquired through horizontal transfer. RESULTS: HMMER remote similarity search significantly detected 382 proteins containing cysteine-rich repeats. All of them, except 8 sequences, belong to eukaryotes. In 124 proteins the presence of conserved structural domains was predicted. In spite of the fact that cysteine-rich repeats are found almost exclusively in eukaryotic proteins, many predicted domains are most common for prokaryotes or bacteriophages. Ninety-eight proteins out of 124 contain typical prokaryotic domains. In those cases proteins were considered as potentially originating via horizontal transfer. In addition, HHblits search revealed that two domains of the same fungal protein, Glycoside hydrolase and Peptidase M15, have high similarity with proteins of two different prokaryotic species, hinting at independent horizontal gene transfer events. CONCLUSIONS: Cysteine-rich repeats in eukaryotic proteins are usually accompanied by conserved domains typical for prokaryotes or bacteriophages. These proteins, containing both cysteine-rich repeats, and characteristic prokaryotic domains, might represent multiple independent horizontal gene transfer events from prokaryotes to eukaryotes. We believe that the presence of bacteriophage recombination site inside cysteine-rich repeat coding sequence may facilitate horizontal genes transfer. Thus computational approach, described in the present study, can help finding multiple sequences originated from horizontal transfer in eukaryotic genomes.


Assuntos
Bacteriófagos/genética , Transferência Genética Horizontal/genética , Genes Virais , Recombinação Genética/genética , Proteínas Virais/química , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , Domínios Proteicos , Proteínas Virais/classificação
19.
PLoS Biol ; 18(7): e3000780, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32687489

RESUMO

Cells adjust to nutrient deprivation by reversible translational shutdown. This is accompanied by maintaining inactive ribosomes in a hibernation state, in which they are bound by proteins with inhibitory and protective functions. In eukaryotes, such a function was attributed to suppressor of target of Myb protein 1 (Stm1; SERPINE1 mRNA-binding protein 1 [SERBP1] in mammals), and recently, late-annotated short open reading frame 2 (Lso2; coiled-coil domain containing short open reading frame 124 [CCDC124] in mammals) was found to be involved in translational recovery after starvation from stationary phase. Here, we present cryo-electron microscopy (cryo-EM) structures of translationally inactive yeast and human ribosomes. We found Lso2/CCDC124 accumulating on idle ribosomes in the nonrotated state, in contrast to Stm1/SERBP1-bound ribosomes, which display a rotated state. Lso2/CCDC124 bridges the decoding sites of the small with the GTPase activating center (GAC) of the large subunit. This position allows accommodation of the duplication of multilocus region 34 protein (Dom34)-dependent ribosome recycling system, which splits Lso2-containing, but not Stm1-containing, ribosomes. We propose a model in which Lso2 facilitates rapid translation reactivation by stabilizing the recycling-competent state of inactive ribosomes.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Evolução Molecular , Células HEK293 , Humanos , Modelos Moleculares , Peptídeos/química , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/ultraestrutura , Relação Estrutura-Atividade
20.
Arch Virol ; 165(10): 2389-2392, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32699979

RESUMO

A novel tobamovirus, brugmansia latent virus (BrLV), was discovered during a study of brugmansia (Brugmansia spp.) in the living collections held at the Royal Botanic Gardens, Kew. Here, we report the complete genome sequence of BrLV, which is 6,397 nucleotides long and contains the four open reading frames (RNA-dependent RNA polymerase, methyltransferase/helicase, movement, and coat proteins) typical of tobamoviruses. The complete genome sequence of BrLV shares 69.7% nucleotide sequence identity with brugmansia mild mottle virus (BrMMV) and 66.7 to 68.7% identity with other tobamoviruses naturally infecting members of the Solanaceae plant family. Phylogenetic analysis of the complete genome nucleotide sequence and the deduced amino acid sequences of the four tobamovirus proteins place BrLV in a subcluster with BrMMV within the Solanaceae-infecting tobamovirus subgroup as a new species.


Assuntos
Brugmansia/virologia , Proteínas do Capsídeo/genética , Genoma Viral , RNA Viral/genética , Tobamovirus/genética , Sequência de Bases , Sequência Conservada , Metiltransferases/genética , Fases de Leitura Aberta , Filogenia , Doenças das Plantas/virologia , RNA Replicase/genética , Alinhamento de Sequência , Tobamovirus/classificação , Tobamovirus/isolamento & purificação , Reino Unido , Sequenciamento Completo do Genoma
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