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1.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34206009

RESUMO

Toll-like receptor (TLR) signaling plays a critical role in the induction and progression of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematous, experimental autoimmune encephalitis, type 1 diabetes mellitus and neurodegenerative diseases. Deciphering antigen recognition by antibodies provides insights and defines the mechanism of action into the progression of immune responses. Multiple strategies, including phage display and hybridoma technologies, have been used to enhance the affinity of antibodies for their respective epitopes. Here, we investigate the TLR4 antibody-binding epitope by computational-driven approach. We demonstrate that three important residues, i.e., Y328, N329, and K349 of TLR4 antibody binding epitope identified upon in silico mutagenesis, affect not only the interaction and binding affinity of antibody but also influence the structural integrity of TLR4. Furthermore, we predict a novel epitope at the TLR4-MD2 interface which can be targeted and explored for therapeutic antibodies and small molecules. This technique provides an in-depth insight into antibody-antigen interactions at the resolution and will be beneficial for the development of new monoclonal antibodies. Computational techniques, if coupled with experimental methods, will shorten the duration of rational design and development of antibody therapeutics.


Assuntos
Anticorpos Monoclonais/imunologia , Artrite Reumatoide/imunologia , Encefalite/imunologia , Epitopos/genética , Doença de Hashimoto/imunologia , Doenças Neurodegenerativas/imunologia , Receptor 4 Toll-Like/genética , Sequência de Aminoácidos/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Técnicas de Visualização da Superfície Celular , Encefalite/genética , Encefalite/patologia , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Doença de Hashimoto/genética , Doença de Hashimoto/patologia , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/patologia , Ligação Proteica/genética , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/imunologia
2.
Front Immunol ; 12: 688758, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34220846

RESUMO

Coronaviruses (CoVs) are a known global threat, and most recently the ongoing COVID-19 pandemic has claimed more than 2 million human lives. Delays and interference with IFN responses are closely associated with the severity of disease caused by CoV infection. As the most abundant viral protein in infected cells just after the entry step, the CoV nucleocapsid (N) protein likely plays a key role in IFN interruption. We have conducted a comprehensive comparative analysis and report herein that the N proteins of representative human and animal CoVs from four different genera [swine acute diarrhea syndrome CoV (SADS-CoV), porcine epidemic diarrhea virus (PEDV), severe acute respiratory syndrome CoV (SARS-CoV), SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), infectious bronchitis virus (IBV) and porcine deltacoronavirus (PDCoV)] suppress IFN responses by multiple strategies. In particular, we found that the N protein of SADS-CoV interacted with RIG-I independent of its RNA binding activity, mediating K27-, K48- and K63-linked ubiquitination of RIG-I and its subsequent proteasome-dependent degradation, thus inhibiting the host IFN response. These data provide insight into the interaction between CoVs and host, and offer new clues for the development of therapies against these important viruses.


Assuntos
Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteína DEAD-box 58/metabolismo , Interferons/antagonistas & inibidores , Interferons/imunologia , Receptores Imunológicos/metabolismo , Sequência de Aminoácidos/genética , Animais , COVID-19/patologia , Proteína DEAD-box 58/imunologia , Deltacoronavirus/genética , Deltacoronavirus/imunologia , Humanos , Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/imunologia , Fator Regulador 3 de Interferon/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Fosforilação , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/imunologia , Receptores Imunológicos/imunologia , Vírus da SARS/genética , Vírus da SARS/imunologia , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Suínos , Ubiquitinação/fisiologia
3.
Int J Mol Sci ; 22(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069753

RESUMO

Hydrocarbon stapling is a useful tool for stabilizing the secondary structure of peptides. Among several methods, hydrocarbon stapling at i,i + 1 positions was not extensively studied, and their secondary structures are not clarified. In this study, we investigate i,i + 1 hydrocarbon stapling between cis-4-allyloxy-l-proline and various olefin-tethered amino acids. Depending on the ring size of the stapled side chains and structure of the olefin-tethered amino acids, E- or Z-selectivities were observed during the ring-closing metathesis reaction (E/Z was up to 8.5:1 for 17-14-membered rings and up to 1:20 for 13-membered rings). We performed X-ray crystallographic analysis of hydrocarbon stapled peptide at i,i + 1 positions. The X-ray crystallographic structure suggested that the i,i + 1 staple stabilizes the peptide secondary structure to the right-handed α-helix. These findings are especially important for short oligopeptides because the employed stapling method uses two minimal amino acid residues adjacent to each other.


Assuntos
Hidrocarbonetos/química , Peptídeos/química , Estabilidade Proteica/efeitos dos fármacos , Alcenos/química , Sequência de Aminoácidos/genética , Aminoácidos/química , Dicroísmo Circular/métodos , Cristalografia por Raios X/métodos , Oligopeptídeos/química , Prolina/química , Conformação Proteica em alfa-Hélice/fisiologia , Estrutura Secundária de Proteína/fisiologia , Raios X
4.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072847

RESUMO

Many proteins have a multimeric structure and are composed of two or more identical subunits. While this can be advantageous for the host organism, it can be a challenge when targeting specific residues in biochemical analyses. In vitro splitting and re-dimerization to circumvent this problem is a tedious process that requires stable proteins. We present an in vivo approach to transform homodimeric proteins into apparent heterodimers, which then can be purified using two-step affinity-tag purification. This opens the door to both practical applications such as smFRET to probe the conformational dynamics of homooligomeric proteins and fundamental research into the mechanism of protein multimerization, which is largely unexplored for membrane proteins. We show that expression conditions are key for the formation of heterodimers and that the order of the differential purification and reconstitution of the protein into nanodiscs is important for a functional ABC-transporter complex.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Lipoproteínas/genética , Complexos Multiproteicos/genética , Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Adenosina Trifosfatases/genética , Sequência de Aminoácidos/genética , Proteínas de Bactérias/ultraestrutura , Dimerização , Transferência Ressonante de Energia de Fluorescência , Lipoproteínas/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Multimerização Proteica/genética , Subunidades Proteicas/genética
5.
Molecules ; 26(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072901

RESUMO

The areca (Areca catechu L.) nut kernel (ANK) is a good potential protein source for its high protein content of 9.89-14.62 g/100 g and a high yield of around 300,000 tons per year in China. However, utilization of the areca nut kernel is limited. To expand the usage of ANK in pharmaceutical or foods industries, areca nut kernel globulin was extracted and angiotensin-I converting enzyme (ACE) inhibition peptides were prepared and identified using gel chromatography, reversed phase HPLC separation, UPLC-ESI-MS/MS analysis and in silico screening. Finally, a novel ACE-inhibitory heptapeptide (Ala-Pro-Lys-Ile-Glu-Glu-Val) was identified and chemically synthesized. The combination pattern between APKIEEV and ACE, and the inhibition kinetics, antihypertensive effect and endothlein-1 inhibition activity of APKIEEV were studied. The results of the molecular docking demonstrated that APKIEEV could bind to four active sites (not the key active sites) of ACE via short hydrogen bonds and demonstrated high ACE-inhibitory activity (IC50: 550.41 µmol/L). Moreover, APKIEEV exhibited a significantly lowering effect on both the systolic blood pressure and diastolic blood pressure of spontaneously hypertensive rats, and had considerable suppression ability on intracellular endothelin-1. These results highlight the potential usage of APKIEEV as ingredients of antihypertensive drugs or functional foods.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Areca/metabolismo , Globulinas/farmacologia , Sequência de Aminoácidos/genética , Animais , Anti-Hipertensivos/química , Pressão Sanguínea/efeitos dos fármacos , Globulinas/metabolismo , Hipertensão/metabolismo , Masculino , Simulação de Acoplamento Molecular/métodos , Nozes/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptídeos/química , Peptidil Dipeptidase A/metabolismo , Hidrolisados de Proteína/química , Hidrolisados de Proteína/metabolismo , Ratos , Ratos Endogâmicos SHR , Espectrometria de Massas em Tandem/métodos
6.
Viruses ; 13(5)2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070055

RESUMO

The understanding of the molecular mechanisms driving the fitness of the SARS-CoV-2 virus and its mutational evolution is still a critical issue. We built a simplified computational model, called SpikePro, to predict the SARS-CoV-2 fitness from the amino acid sequence and structure of the spike protein. It contains three contributions: the inter-human transmissibility of the virus predicted from the stability of the spike protein, the infectivity computed in terms of the affinity of the spike protein for the ACE2 receptor, and the ability of the virus to escape from the human immune response based on the binding affinity of the spike protein for a set of neutralizing antibodies. Our model reproduces well the available experimental, epidemiological and clinical data on the impact of variants on the biophysical characteristics of the virus. For example, it is able to identify circulating viral strains that, by increasing their fitness, recently became dominant at the population level. SpikePro is a useful, freely available instrument which predicts rapidly and with good accuracy the dangerousness of new viral strains. It can be integrated and play a fundamental role in the genomic surveillance programs of the SARS-CoV-2 virus that, despite all the efforts, remain time-consuming and expensive.


Assuntos
Biologia Computacional/métodos , Aptidão Genética/genética , SARS-CoV-2/genética , Sequência de Aminoácidos/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/genética , COVID-19/metabolismo , Humanos , Modelos Teóricos , Mutação/genética , Ligação Proteica/genética , Receptores Virais/metabolismo , SARS-CoV-2/patogenicidade , Software , Glicoproteína da Espícula de Coronavírus/genética
7.
Int J Mol Sci ; 22(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068603

RESUMO

Genomic and phylogenetic analyses of various invertebrate phyla revealed the existence of genes that are evolutionarily related to the vertebrate's decapeptide gonadotropin-releasing hormone (GnRH) and the GnRH receptor genes. Upon the characterization of these gene products, encoding peptides and putative receptors, GnRH-related peptides and their G-protein coupled receptors have been identified. These include the adipokinetic hormone (AKH) and corazonin (CRZ) in insects and their cognate receptors that pair to form bioactive signaling systems, which network with additional neurotransmitters/hormones (e.g., octopamine and ecdysone). Multiple studies in the past 30 years have identified many aspects of the biology of these peptides that are similar in size to GnRH and function as neurohormones. This review briefly describes the main activities of these two neurohormones and their receptors in the fruit fly Drosophila melanogaster. The similarities and differences between Drosophila AKH/CRZ and mammalian GnRH signaling systems are discussed. Of note, while GnRH has a key role in reproduction, AKH and CRZ show pleiotropic activities in the adult fly, primarily in metabolism and stress responses. From a protein evolution standpoint, the GnRH/AKH/CRZ family nicely demonstrates the developmental process of neuropeptide signaling systems emerging from a putative common ancestor and leading to divergent activities in distal phyla.


Assuntos
Evolução Molecular , Hormônios de Inseto/genética , Proteínas de Insetos/genética , Neuropeptídeos/genética , Neurotransmissores/genética , Oligopeptídeos/genética , Ácido Pirrolidonocarboxílico/análogos & derivados , Sequência de Aminoácidos/genética , Animais , Drosophila melanogaster/genética , Hormônio Liberador de Gonadotropina , Humanos , Filogenia , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genética
8.
Nat Commun ; 12(1): 3225, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050176

RESUMO

Non-Ribosomal Peptides (NRPs) represent a biomedically important class of natural products that include a multitude of antibiotics and other clinically used drugs. NRPs are not directly encoded in the genome but are instead produced by metabolic pathways encoded by biosynthetic gene clusters (BGCs). Since the existing genome mining tools predict many putative NRPs synthesized by a given BGC, it remains unclear which of these putative NRPs are correct and how to identify post-assembly modifications of amino acids in these NRPs in a blind mode, without knowing which modifications exist in the sample. To address this challenge, here we report NRPminer, a modification-tolerant tool for NRP discovery from large (meta)genomic and mass spectrometry datasets. We show that NRPminer is able to identify many NRPs from different environments, including four previously unreported NRP families from soil-associated microbes and NRPs from human microbiota. Furthermore, in this work we demonstrate the anti-parasitic activities and the structure of two of these NRP families using direct bioactivity screening and nuclear magnetic resonance spectrometry, illustrating the power of NRPminer for discovering bioactive NRPs.


Assuntos
Antibacterianos/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Biologia Computacional/métodos , Descoberta de Drogas/métodos , Peptídeos/isolamento & purificação , Algoritmos , Sequência de Aminoácidos/genética , Antibacterianos/biossíntese , Produtos Biológicos/metabolismo , Conjuntos de Dados como Assunto , Humanos , Espectrometria de Massas , Redes e Vias Metabólicas/genética , Metabolômica/métodos , Metagenômica/métodos , Microbiota/genética , Família Multigênica , Biossíntese Peptídica , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Microbiologia do Solo
9.
Gene ; 791: 145711, 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-33984445

RESUMO

Clubroot disease, caused by Plasmodiophora brassicae infection, occurs in cruciferous vegetable crops in many areas of the world, sometimes leading to yield loss. In this study, a differentially expressed protein (0305), was found between control and clubroot-diseased Chinese cabbage (Brassica rapa L.) roots through two-dimensional electrophoresis. Mass spectrometry analysis showed that Bra003466 was highly matched to protein 0305. Because the sequence of Bra003466 had 89% percent identity with ATG6 of Arabidopsis thaliana and other Brassica, the gene was named as BrATG6. However, 790 bp sequences were mismatched with the cDNA sequence of the Bra003466 gene from the Brassica database. In this study, we cloned the cDNA of Bra003466 and found the BrATG6 was highly expressed in roots among all organs. When plants were inoculated with P. brassicae Woronin, the expression of BrATG6 was significantly increased in infected roots of Chinese cabbage. This result was verified by reverse transcription-qPCR and in situ hybridization. Examination of disease resistance showed that, compared with wild type plants, A. thaliana ATG6 deletion mutants were more easily infected by P. brassicae than WT. This shows that BrATG6 may play a potential role in the resistance of B. rapa to P. brassicae infection.


Assuntos
Brassica rapa/genética , Resistência à Doença/genética , Infecções por Protozoários/genética , Sequência de Aminoácidos/genética , Arabidopsis/genética , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Brassica/genética , Brassica rapa/metabolismo , Genes de Plantas/genética , Doenças das Plantas/genética , Raízes de Plantas/genética , Plasmodioforídeos/genética , Plasmodioforídeos/patogenicidade
10.
Methods Mol Biol ; 2295: 219-247, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34047980

RESUMO

The fatty acid biosynthetic cycle is predicated on an acyl carrier protein (ACP) scaffold where two carbon acetyl groups are added in a chain elongation process through a series of repeated enzymatic steps. The chain extension is terminated by hydrolysis with a thioesterase or direct transfer of the acyl group to a glycerophospholipid by an acyltransferase. Methods for analysis of the concentrations of acyl chains attached to ACPs are lacking but would be informative for studies in lipid metabolism. We describe a method to profile and quantify the levels of acyl-ACPs in plants, bacteria and mitochondria of animals and fungi that represent Type II fatty acid biosynthetic systems. ACPs of Type II systems have a highly conserved Asp-Ser-Leu-Asp (DSLD) amino acid sequence at the attachment site for 4'-phosphopantetheinyl arm carrying the acyl chain. Three amino acids of the conserved sequence can be cleaved away from the remainder of the protein using an aspartyl protease. Thus, partially purified protein can be enzymatically hydrolyzed to produce an acyl chain linked to a tripeptide via the 4'-phosphopantetheinyl group. After ionization and fragmentation, the corresponding fragment ion is detected by a triple quadrupole mass spectrometer using a multiple reaction monitoring method. 15N isotopically labeled acyl-ACPs generated in high amounts are used with an isotope dilution strategy to quantify the absolute levels of each acyl group attached to the acyl carrier protein scaffold.


Assuntos
Proteína de Transporte de Acila/análise , Proteína de Transporte de Acila/isolamento & purificação , Cromatografia Líquida/métodos , Proteína de Transporte de Acila/metabolismo , Acil Coenzima A/metabolismo , Sequência de Aminoácidos/genética , Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Sequência Conservada/genética , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos/genética , Lipídeos/química , Lipogênese/genética , Mitocôndrias/metabolismo , Plantas/metabolismo , Espectrometria de Massas em Tandem/métodos
11.
Nat Commun ; 12(1): 2987, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016967

RESUMO

The elongasome, or Rod system, is a protein complex that controls cell wall formation in rod-shaped bacteria. MreC is a membrane-associated elongasome component that co-localizes with the cytoskeletal element MreB and regulates the activity of cell wall biosynthesis enzymes, in a process that may be dependent on MreC self-association. Here, we use electron cryo-microscopy and X-ray crystallography to determine the structure of a self-associated form of MreC from Pseudomonas aeruginosa in atomic detail. MreC monomers interact in head-to-tail fashion. Longitudinal and lateral interfaces are essential for oligomerization in vitro, and a phylogenetic analysis of proteobacterial MreC sequences indicates the prevalence of the identified interfaces. Our results are consistent with a model where MreC's ability to alternate between self-association and interaction with the cell wall biosynthesis machinery plays a key role in the regulation of elongasome activity.


Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Parede Celular/ultraestrutura , Sequência Conservada/genética , Microscopia Crioeletrônica , Cristalografia por Raios X , Mutagênese , Filogenia , Conformação Proteica em alfa-Hélice/genética , Conformação Proteica em Folha beta/genética , Domínios Proteicos/genética , Multimerização Proteica , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
12.
Molecules ; 26(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925288

RESUMO

The consequences of manipulations in structure and amino acid composition of native cyclolinopeptide A (CLA) from linen seeds, and its linear precursor on their biological activities and mechanisms of action, are reviewed. The modifications included truncation of the peptide chain, replacement of amino acid residues with proteinogenic or non-proteinogenic ones, modifications of peptide bond, and others. The studies revealed changes in the immunosuppressive potency of these analogs investigated in a number of in vitro and in vivo experimental models, predominantly in rodents, as well as differences in their postulated mechanism of action. The modified peptides were compared with cyclosporine A and parent CLA. Some of the synthesized and investigated peptides show potential therapeutic usefulness.


Assuntos
Sequência de Aminoácidos/genética , Peptídeos Cíclicos/genética , Peptídeos/genética , Relação Estrutura-Atividade , Animais , Roupas de Cama, Mesa e Banho , Imunossupressores/química , Imunossupressores/farmacologia , Modelos Animais , Peptídeos/química , Peptídeos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Sementes/química
13.
Int J Biol Macromol ; 181: 1104-1123, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-33895178

RESUMO

Vicieae tribe, Leguminosae family (Fabaceae), has been extensively studied. In particular, the study of lectins. The purification, physicochemical and structural characterizations of the various purified lectins and the analysis of their relevant biological activities are ongoing. In this review, several works already published about Vicieae lectins are addressed. Initially, we presented the purification protocols and the physicochemical aspects, such as specificity for carbohydrates, optimal activity in the face of variations in temperature and pH, as well metals-dependence. Following, structural characterization studies are highlighted and, finally, various biological activities already reported are summarized. Studies on lectins in almost all genera (Lathyrus, Lens, Pisum and Vicia) are considered, with the exception of Vavilovia which studies of lectins have not yet been reported. Like other leguminous lectins, Vicieae lectins present heterogeneous profiles of agglutination profiles for erythrocytes and other cells of the immune system, and glycoproteins. Most Vicieae lectins consist of two subunits, α and ß, products of a single precursor protein derived from a single gene. The differences between the isoforms result from varying degrees of proteolytic processing. Along with the identification of these molecules and their characteristics, biological activities become very relevant and robust for both basic and applied research.


Assuntos
Carboidratos/química , Lectinas/química , Lectinas/isolamento & purificação , Vicia/química , Sequência de Aminoácidos/genética , Carboidratos/genética , Lectinas/genética , Lectinas/ultraestrutura
14.
Int J Mol Sci ; 22(7)2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807484

RESUMO

Transcription factors play a crucial role in regulating biological processes such as cell growth, differentiation, organ development and cellular signaling. Within this group, proteins equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown to be critically important for efficient regulation of the BCL11B target genes and could therefore represent a promising target for novel drug therapies. Here, we report the structural basis for BCL11B-BCL11B interaction mediated by the N-terminal ZF domain. By combining structure prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of the single ZF domain and directly involved in forming the dimer interface. These findings not only provide deep insight into how BCL11B acquires its active structure but also represent an important step towards rational design or selection of potential inhibitors.


Assuntos
Proteínas Repressoras/metabolismo , Proteínas Repressoras/ultraestrutura , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/ultraestrutura , Sequência de Aminoácidos/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Transferência Ressonante de Energia de Fluorescência/métodos , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Proteínas Repressoras/genética , Fatores de Transcrição/metabolismo , Dedos de Zinco/genética
16.
Int J Mol Sci ; 22(9)2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33926148

RESUMO

Autoimmunity may have its origins of early repertoire selection in developmental B cells. Such a primary repertoire is probably shaped by selecting B cells that can efficiently perform productive signaling, stimulated by self-antigens in the bone marrow, such as DNA. In support of that idea, we previously found a V segment from VH10 family that can form antibodies that bind to DNA independent of CDR3 usage. In this paper we designed four antibody fragments in a novel single-chain pre-BCR (scpre-BCR) format containing germinal V gene segments from families known to bind DNA (VH10) or not (VH4) connected to a murine surrogate light chain (SLC), lacking the highly charged unique region (UR), by a hydrophilic peptide linker. We also tested the influence of CDR2 on DNA reactivity by shuffling the CDR2 loop. The scpre-BCRs were expressed in bacteria. VH10 bearing scpre-BCR could bind DNA, while scpre-BCR carrying the VH4 segment did not. The CDR2 loop shuffling hampered VH10 reactivity while displaying a gain-of-function in the nonbinding VH4 germline. We modeled the binding sites demonstrating the conservation of a positivity charged pocket in the VH10 CDR2 as the possible cross-reactive structural element. We presented evidence of DNA reactivity hardwired in a V gene, suggesting a structural mechanism for innate autoreactivity. Therefore, while autoreactivity to DNA can lead to autoimmunity, efficiently signaling for B cell development is likely a trade-off mechanism leading to the selection of potentially autoreactive repertoires.


Assuntos
Região Variável de Imunoglobulina/genética , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Sequência de Aminoácidos/genética , Animais , Anticorpos Antinucleares/genética , Arginina/genética , Arginina/metabolismo , Autoantígenos/genética , Autoimunidade/imunologia , Sequência de Bases/genética , DNA/imunologia , Células Germinativas/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina/ultraestrutura , Camundongos , Anticorpos de Domínio Único/ultraestrutura , Relação Estrutura-Atividade
17.
Molecules ; 26(9)2021 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-33923273

RESUMO

Many gram-negative bacteria use type IV secretion systems to deliver effector molecules to a wide range of target cells. These substrate proteins, which are called type IV secreted effectors (T4SE), manipulate host cell processes during infection, often resulting in severe diseases or even death of the host. Therefore, identification of putative T4SEs has become a very active research topic in bioinformatics due to its vital roles in understanding host-pathogen interactions. PSI-BLAST profiles have been experimentally validated to provide important and discriminatory evolutionary information for various protein classification tasks. In the present study, an accurate computational predictor termed iT4SE-EP was developed for identifying T4SEs by extracting evolutionary features from the position-specific scoring matrix and the position-specific frequency matrix profiles. First, four types of encoding strategies were designed to transform protein sequences into fixed-length feature vectors based on the two profiles. Then, the feature selection technique based on the random forest algorithm was utilized to reduce redundant or irrelevant features without much loss of information. Finally, the optimal features were input into a support vector machine classifier to carry out the prediction of T4SEs. Our experimental results demonstrated that iT4SE-EP outperformed most of existing methods based on the independent dataset test.


Assuntos
Evolução Molecular , Bactérias Gram-Negativas/genética , Interações Hospedeiro-Patógeno/genética , Sistemas de Secreção Tipo IV/genética , Sequência de Aminoácidos/genética , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/genética , Infecções Bacterianas/microbiologia , Biologia Computacional , Bactérias Gram-Negativas/patogenicidade , Humanos , Sistemas de Secreção Tipo IV/química
18.
Int J Mol Sci ; 22(9)2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33925935

RESUMO

Temporin is an antimicrobial peptide (AMP) family discovered in the skin secretion of ranid frog that has become a promising alternative for conventional antibiotic therapy. Herein, a novel temporin peptide, Temporin-PF (TPF), was successfully identified from Pelophylax fukienensis. It exhibited potent activity against Gram-positive bacteria, but no effect on Gram-negative bacteria. Additionally, TPF exhibited aggregation effects in different solutions. Three analogs were further designed to study the relationship between the aggregation patterns and bioactivities, and the MD simulation was performed for revealing the pattern of the peptide assembly. As the results showed, all peptides were able to aggregate in the standard culture media and salt solutions, especially CaCl2 and MgCl2 buffers, where the aggregation was affected by the concentration of the salts. MD simulation reported that all peptides were able to form oligomers. The parent peptide assembly depended on the hydrophobic interaction via the residues in the middle domain of the sequence. However, the substitution of Trp/D-Trp resulted in an enhanced inter-peptide interaction in the zipper-like domain and eliminated overall biological activities. Our study suggested that introducing aromaticity at the zipper-like domain for temporin may not improve the bioactivities, which might be related to the formation of aggregates via the inter-peptide contacts at the zipper-like motif domain, and it could reduce the binding affinity to the lipid membrane of microorganisms.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Proteínas Citotóxicas Formadoras de Poros/química , Agregados Proteicos/fisiologia , Sequência de Aminoácidos/genética , Proteínas de Anfíbios/química , Animais , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Secreções Corporais/metabolismo , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Ranidae/metabolismo , Estresse Salino , Pele/metabolismo
19.
Molecules ; 26(8)2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33924269

RESUMO

Human glutathione transferase A1-1 (hGSTA1-1) contributes to developing resistance to anticancer drugs and, therefore, is promising in terms of drug-design targets for coping with this phenomenon. In the present study, the interaction of anthraquinone and diazo dichlorotriazine dyes (DCTD) with hGSTA1-1 was investigated. The anthraquinone dye Procion blue MX-R (PBMX-R) appeared to interact with higher affinity and was selected for further study. The enzyme was specifically and irreversibly inactivated by PBMX-R, following a biphasic pseudo-first-order saturation kinetics, with approximately 1 mol of inhibitor per mol of the dimeric enzyme being incorporated. Molecular modeling and protein chemistry data suggested that the modified residue is the Cys112, which is located at the entrance of the solvent channel at the subunits interface. The results suggest that negative cooperativity exists upon PBMX-R binding, indicating a structural communication between the two subunits. Kinetic inhibition analysis showed that the dye is a competitive inhibitor towards glutathione (GSH) and mixed-type inhibitor towards 1-chloro-2,4-dinitrobenzene (CDNB). The present study results suggest that PBMX-R is a useful probe suitable for assessing by kinetic means the drugability of the enzyme in future drug-design efforts.


Assuntos
Anticarcinógenos/química , Corantes/química , Glutationa Transferase/genética , Neoplasias/tratamento farmacológico , Triazinas/química , Sequência de Aminoácidos/genética , Anticarcinógenos/uso terapêutico , Sítios de Ligação/efeitos dos fármacos , Dinitroclorobenzeno/química , Glutationa/antagonistas & inibidores , Glutationa/genética , Glutationa Transferase/antagonistas & inibidores , Humanos , Cinética , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica/efeitos dos fármacos
20.
Nat Commun ; 12(1): 1605, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33707415

RESUMO

Deep learning algorithms have been utilized to achieve enhanced performance in pattern-recognition tasks. The ability to learn complex patterns in data has tremendous implications in immunogenomics. T-cell receptor (TCR) sequencing assesses the diversity of the adaptive immune system and allows for modeling its sequence determinants of antigenicity. We present DeepTCR, a suite of unsupervised and supervised deep learning methods able to model highly complex TCR sequencing data by learning a joint representation of a TCR by its CDR3 sequences and V/D/J gene usage. We demonstrate the utility of deep learning to provide an improved 'featurization' of the TCR across multiple human and murine datasets, including improved classification of antigen-specific TCRs and extraction of antigen-specific TCRs from noisy single-cell RNA-Seq and T-cell culture-based assays. Our results highlight the flexibility and capacity for deep neural networks to extract meaningful information from complex immunogenomic data for both descriptive and predictive purposes.


Assuntos
Algoritmos , Aprendizado Profundo , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Sequência de Aminoácidos/genética , Animais , Bases de Dados Genéticas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos , Redes Neurais de Computação , RNA-Seq/métodos
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