RESUMO
DNA meiotic recombinase 1 (disrupted meiotic cDNA, Dmc1) protein is homologous to the Escherichia coli RecA protein, was first identified in Saccharomyces cerevisiae. This gene has been well studied as an essential role in meiosis in many species. However, studies on the dmc1 gene in reptiles are limited. In this study, a cDNA fragment of 1,111 bp was obtained from the gonadal tissues of the Chinese soft-shell turtle via RT-PCR, containing a 60 bp 3' UTR, a 22 bp 5' UTR, and an ORF of 1,029 bp encoding 342 amino acids, named Psdmc1. Multiple sequence alignments showed that the deduced protein has high similarity (>95 %) to tetrapod Dmc1 proteins, while being slightly lower (86-88 %) to fish species.Phylogenetic tree analysis showed that PsDmc1 was clustered with the other turtles' Dmc1 and close to the reptiles', but far away from the teleost's. RT-PCR and RT-qPCR analyses showed that the Psdmc1 gene was specifically expressed in the gonads, and much higher in testis than the ovary, especially highest in one year-old testis. In situ hybridization results showed that the Psdmc1 was mainly expressed in the perinuclear cytoplasm of primary and secondary spermatocytes, weakly in spermatogonia of the testes. These results indicated that dmc1 would be majorly involved in the developing testis, and play an essential role in the germ cells' meiosis. The findings of this study will provide a basis for further investigations on the mechanisms behind the germ cells' development and differentiation in Chinese soft-shell turtles, even in the reptiles.
Assuntos
Gametogênese , Filogenia , Tartarugas , Animais , Feminino , Masculino , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Clonagem Molecular , Gametogênese/genética , Meiose/genética , Ovário/metabolismo , Espermatócitos/metabolismo , Testículo/metabolismo , Tartarugas/genética , Tartarugas/metabolismoRESUMO
The PP2A-B55 phosphatase regulates a plethora of signaling pathways throughout eukaryotes. How PP2A-B55 selects its substrates presents a severe knowledge gap. By integrating AlphaFold modeling with comprehensive high-resolution mutational scanning, we show that α helices in substrates bind B55 through an evolutionary conserved mechanism. Despite a large diversity in sequence and composition, these α helices share key amino acid determinants that engage discrete hydrophobic and electrostatic patches. Using deep learning protein design, we generate a specific and potent competitive peptide inhibitor of PP2A-B55 substrate interactions. With this inhibitor, we uncover that PP2A-B55 regulates the nuclear exosome targeting (NEXT) complex by binding to an α-helical recruitment module in the RNA binding protein 7 (RBM7), a component of the NEXT complex. Collectively, our findings provide a framework for the understanding and interrogation of PP2A-B55 function in health and disease.
Assuntos
Ligação Proteica , Proteína Fosfatase 2 , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2/química , Especificidade por Substrato , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/química , Modelos Moleculares , Sequência de AminoácidosRESUMO
Phosphatidylethanolamine N-methyltransferase (PmtA) catalyzes the biosynthesis of phosphatidylcholine (PC) from phosphatidylethanolamine (PE). Although PC is one of the major phospholipids constituting bilayer membranes in eukaryotes, certain bacterial species encode PmtA, a membrane-associated methyltransferase, to produce PC, which is correlated with cellular stress responses, adaptability to environmental changes, and symbiosis or virulence with eukaryotic hosts. Depending on the organism, multiple PmtAs may be required for producing monomethyl- and dimethyl-PE derivatives along with PC, whereas in organisms such as Rubellimicrobium thermophilum, a single enzyme is sufficient to direct all three methylation steps. In this study, we present the x-ray crystal structures of PmtA from R. thermophilum in complex with dimethyl-PE and S-adenosyl-l-homocysteine, as well as in its lipid-free form. Moreover, we demonstrate that the enzyme associates with the cellular membrane via electrostatic interactions facilitated by a group of critical basic residues and can successively methylate PE and its methylated derivatives, culminating in the production of PC.
Assuntos
Proteínas de Bactérias , Fosfatidilcolinas , Fosfatidiletanolamina N-Metiltransferase , Fosfatidilcolinas/biossíntese , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Modelos Moleculares , Fosfatidiletanolaminas/metabolismo , Fosfatidiletanolaminas/biossíntese , Cristalografia por Raios X , Metilação , Membrana Celular/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosil-Homocisteína/química , Conformação Proteica , Ligação Proteica , Metiltransferases/metabolismo , Metiltransferases/química , Sequência de AminoácidosRESUMO
Jasmonic acid (JA), an important plant hormone, plays a crucial role in defending against herbivorous insects. In this study, we have identified a new Bowman-Birk type protease inhibitor (BBTI) protein in maize that is regulated by the JA pathway and exhibits significant antifeedant activity, which is notably induced by exogenous Methyl Jasmonate and Ostrinia furnacalis feeding treatments. Bioinformatics analysis revealed significant differences in the BBTI protein among different maize inbred lines, except for the conserved domain. Prokaryotic and eukaryotic expression systems were constructed and expressed, and combined with bioassays, it was demonstrated that the antifeedant activity of BBTI is determined by protein modifications and conserved domains. Through RT-qPCR detection of BBTI and JA regulatory pathway-related genes' temporal expression in different maize inbred lines, we identified the regulatory mechanism of BBTI synthesis under the JA pathway. This study successfully cloned and identified the MeJA-induced anti-feedant activity gene BBTI and conducted functional validation in different maize inbred lines, providing valuable insights into the response mechanism of insect resistance induced by the plant JA pathway. The increased expression of the anti-feedant activity gene BBTI through exogenous MeJA induction may offer a potential new strategy for mediating plant defense against Lepidoptan insects.
Assuntos
Acetatos , Ciclopentanos , Regulação da Expressão Gênica de Plantas , Mariposas , Oxilipinas , Proteínas de Plantas , Zea mays , Zea mays/genética , Zea mays/efeitos dos fármacos , Zea mays/metabolismo , Zea mays/parasitologia , Oxilipinas/farmacologia , Oxilipinas/metabolismo , Ciclopentanos/farmacologia , Ciclopentanos/metabolismo , Animais , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Acetatos/farmacologia , Mariposas/efeitos dos fármacos , Mariposas/fisiologia , Reguladores de Crescimento de Plantas/farmacologia , Reguladores de Crescimento de Plantas/metabolismo , Inibidores de Proteases/farmacologia , Inibidores de Proteases/metabolismo , Herbivoria , Sequência de Aminoácidos , FilogeniaRESUMO
ß-Glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus (Bgl1) has been denoted as having an attractive catalytic profile for various industrial applications. Bgl1 catalyses the final step of in the decomposition of cellulose, an unbranched glucose polymer that has attracted the attention of researchers in recent years as it is the most abundant renewable source of reduced carbon in the biosphere. With the aim of enhancing the thermostability of Bgl1 for a broad spectrum of biotechnological processes, it has been subjected to structural studies. Crystal structures of Bgl1 and its complex with glucose were determined at 1.47 and 1.95â Å resolution, respectively. Bgl1 is a member of glycosyl hydrolase family 1 (GH1 superfamily, EC 3.2.1.21) and the results showed that the 3D structure of Bgl1 follows the overall architecture of the GH1 family, with a classical (ß/α)8 TIM-barrel fold. Comparisons of Bgl1 with sequence or structural homologues of ß-glucosidase reveal quite similar structures but also unique structural features in Bgl1 with plausible functional roles.
Assuntos
beta-Glucosidase , beta-Glucosidase/química , beta-Glucosidase/metabolismo , Cristalografia por Raios X , Caldicellulosiruptor/enzimologia , Modelos Moleculares , Conformação Proteica , Proteínas de Bactérias/química , Domínio Catalítico , Glucose/metabolismo , Sequência de AminoácidosRESUMO
Overgrowth of Gardnerella vaginalis causes an imbalance in vaginal microecology. The pathogenicity of G. vaginalis is directly regulated by the cAMP receptor protein (CRP). In this study, we resolve the crystal structure of CRPGv at a resolution of 2.22 Å and find some significant differences from homologous proteins. The first 23 amino acids of CRPGv are inserted into the ligand binding pocket, creating a strong steric barrier to ligand entry that has not been seen previously in its homologues. In the absence of ligands, the two α helices used by CRPGv to bind oligonucleotide chains are exposed and can specifically bind TGTGA-N6-TCACA sequences. cAMP and other ligands of CRP homologs are not cofactors of CRPGv. There is no coding gene of the adenylate cyclase, and cAMP could not be identified in G. vaginalis by liquid chromatography tandem mass spectrometry. We speculate that CRPGv may achieve fine regulation through a conformational transformation different from that of its homologous proteins, and this conformational transformation is no longer dependent on small molecules, but may be aided by accessory proteins. CRPGv is the first discovered CRP that is not ligand-regulated, and its active conformation provides a structural basis for drug screening.
Assuntos
Gardnerella vaginalis , Ligantes , Gardnerella vaginalis/genética , Gardnerella vaginalis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/química , Cristalografia por Raios X , Sítios de Ligação , Modelos Moleculares , Sequência de Aminoácidos , Ligação Proteica , Conformação ProteicaRESUMO
5-Methyluridine (m5U) rRNA modifications frequently occur at U747 and U1939 (Escherichia coli numbering) in domains II and IV of the 23S rRNA in Gram-negative bacteria, with the help of S-adenosyl-l-methionine (SAM)-dependent rRNA methyltransferases (MTases), RlmC and RlmD, respectively. In contrast, Gram-positive bacteria utilize a single SAM-dependent rRNA MTase, RlmCD, to modify both corresponding sites. Notably, certain archaea, specifically within the Thermococcales group, have been found to possess two genes encoding SAM-dependent archaeal (tRNA and rRNA) m5U (Arm5U) MTases. Among these, a tRNA-specific Arm5U MTase (PabTrmU54) has already been characterized. This study focused on the structural and functional characterization of the rRNA-specific Arm5U MTase from the hyperthermophilic archaeon Pyrococcus horikoshii (PhRlmCD). An in-depth structural examination revealed a dynamic hinge movement induced by the replacement of the iron-sulfur cluster with disulfide bonds, obstructing the substrate-binding site. It revealed distinctive characteristics of PhRlmCD, including elongated positively charged loops in the central domain and rotational variations in the TRAM domain, which influence substrate selectivity. Additionally, the results suggested that two potential mini-rRNA fragments interact in a similar manner with PhRlmCD at a positively charged cleft at the interface of domains and facilitate dual MTase activities akin to the protein RlmCD. Altogether, these observations showed that Arm5U MTases originated from horizontal gene transfer events, most likely from Gram-positive bacteria.
Assuntos
Proteínas Arqueais , Metiltransferases , Especificidade por Substrato , Proteínas Arqueais/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/química , Metiltransferases/metabolismo , Metiltransferases/química , Metiltransferases/genética , Pyrococcus horikoshii/enzimologia , Pyrococcus horikoshii/genética , Modelos Moleculares , Cristalografia por Raios X , S-Adenosilmetionina/metabolismo , Sequência de AminoácidosRESUMO
Extracellular proteases from haloarchaea, also referred to as halolysins, are in increasing demand and are studied for their various applications in condiments and leather industries. In this study, an extracellular protease encoding gene from the haloarchaeon Halorubellus sp. PRR65, hly65, was cloned and heterologously expressed in E. coli. The novel halolysin Hly65 from the genus Halorubellus was characterized by complete inhibition of phenylmethanesulfonyl fluoride (PMSF) on its enzyme activity. Experimental determination revealed a triad catalytic active center consisting of Asp154-His193-Ser348. Deletion of the C-terminal extension (CTE) resulted in loss of enzyme activity, while dithiothreitol (DTT) did not inhibit the enzyme activity, suggesting that Hly65 may function as a monomer. The Km, Vmax and Kcat for the Hly65 were determined to be 2.91 mM, 1230.47 U·mg-1 and 1538.09 S-1, respectively, under 60 °C, pH 8.0 and 4.0 M NaCl using azocasecin as a substrate. Furthermore, a three-dimensional structure prediction based on functional domains was obtained in this study which will facilitate modification and reorganization of halolysins to generate mutants with new physiological activities.
Assuntos
Clonagem Molecular , Escherichia coli , Concentração de Íons de Hidrogênio , Escherichia coli/genética , Cinética , Domínio Catalítico , Halobacteriaceae/genética , Halobacteriaceae/enzimologia , Halobacteriaceae/metabolismo , Sequência de Aminoácidos , Estabilidade Enzimática , Especificidade por Substrato , Temperatura , Temperatura Alta , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas Arqueais/química , Modelos Moleculares , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Cloreto de Sódio/metabolismo , Fluoreto de Fenilmetilsulfonil/farmacologia , CaseínasRESUMO
Histones are important chromatin-organizing proteins in eukaryotes and archaea. They form superhelical structures around which DNA is wrapped. Recent studies have shown that some archaea and bacteria contain alternative histones that exhibit different DNA binding properties, in addition to highly divergent sequences. However, the vast majority of these histones are identified in metagenomes and thus are difficult to study in vivo. The recent revolutionary breakthroughs in computational protein structure prediction by AlphaFold2 and RoseTTAfold allow for unprecedented insights into the potential function and structure of previously uncharacterized proteins. Here, we categorize the prokaryotic histone space into 17 distinct groups based on AlphaFold2 predictions. We identify a superfamily of histones, termed α3 histones, which are common in archaea and present in several bacteria. Importantly, we establish the existence of a large family of histones throughout archaea and in some bacteriophages that, instead of wrapping DNA, bridge DNA, thereby diverging from conventional nucleosomal histones.
Assuntos
Archaea , Bactérias , Histonas , Histonas/metabolismo , Histonas/química , Histonas/genética , Archaea/metabolismo , Archaea/genética , Bactérias/metabolismo , Bactérias/genética , Células Procarióticas/metabolismo , Filogenia , Nucleossomos/metabolismo , Modelos Moleculares , Proteínas Arqueais/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Sequência de AminoácidosRESUMO
Cilia are antenna-like organelles protruding from the surface of many cell types in the human body. Defects in ciliary structure or function often lead to diseases that are collectively called ciliopathies. Cilia and flagella-associated protein 410 (CFAP410) localizes at the basal body of cilia/flagella and plays essential roles in ciliogenesis, neuronal development and DNA damage repair. It remains unknown how its specific basal body location is achieved. Multiple single amino acid mutations in CFAP410 have been identified in patients with various ciliopathies. One of the mutations, L224P, is located in the C-terminal domain (CTD) of human CFAP410 and causes severe spondylometaphyseal dysplasia, axial (SMDAX). However, the molecular mechanism for how the mutation causes the disorder remains unclear. Here, we report our structural studies on the CTD of CFAP410 from three distantly related organisms, Homo sapiens, Trypanosoma brucei and Chlamydomonas reinhardtii. The crystal structures reveal that the three proteins all adopt the same conformation as a tetrameric helical bundle. Our work further demonstrates that the tetrameric assembly of the CTD is essential for the correct localization of CFAP410 in T. brucei, as the L224P mutation that disassembles the tetramer disrupts its basal body localization. Taken together, our studies reveal that the basal body localization of CFAP410 is controlled by the CTD and provide a mechanistic explanation for how the mutation L224P in CFAP410 causes ciliopathies in humans.
Assuntos
Corpos Basais , Trypanosoma brucei brucei , Corpos Basais/metabolismo , Humanos , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/genética , Modelos Moleculares , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cílios/metabolismo , Cristalografia por Raios X , Mutação , Sequência de Aminoácidos , Multimerização Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/químicaRESUMO
Lepidopteran silk is a complex mixture of proteins, consisting mainly of fibroins and sericins. Sericins are a small family of highly divergent proteins that serve as adhesives and coatings for silk fibers. So far, five genes encoding sericin proteins have been identified in Bombyx mori. Having previously identified sericin protein 150 (SP150) as a major sericin-like protein in the cocoons of the pyralid moths Galleria mellonella and Ephestia kuehniella, we describe the identification of its homolog in B. mori. Our refined gene model shows that it consists of four exons and a long open reading frame with a conserved motif, CXCXCX, at the C-terminus, reminiscent of the structure observed in a class of mucin proteins. Notably, despite a similar expression pattern, both mRNA and protein levels of B. mori SP150 were significantly lower than those of its pyralid counterpart. We also discuss the synteny of homologous genes on corresponding chromosomes in different moth species and the possible phylogenetic relationships between SP150 and certain mucin-like proteins. Our results improve our understanding of silk structure and the evolutionary relationships between adhesion proteins in the silk of different lepidopteran species.
Assuntos
Bombyx , Filogenia , Sericinas , Bombyx/genética , Bombyx/metabolismo , Animais , Sericinas/metabolismo , Sericinas/genética , Sericinas/química , Sequência de Aminoácidos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/química , Seda/metabolismo , Seda/genética , Seda/químicaRESUMO
Human noroviruses (HuNoVs) are a leading cause of acute viral gastroenteritis worldwide. Infectious outbreaks due to recombinant NoV genotype called GII.P16-GII.2 have been frequently reported since 2016. In this study, we expressed the major capsid protein VP1 from three GII.2 NoV strains using the recombinant baculovirus expression system. The assembly, histo-blood group antigen (HBGA)-binding patterns, and cross-blocking abilities of VP1 proteins were investigated. All the three NoV VP1 proteins successfully assembled into virus-like particles (VLPs). The HBGA-binding assay demonstrated a temporal binding pattern. The latest isolate bound to saliva samples of all blood types. Sequence alignment suggested that the observed gain in HBGA-binding ability was attributed to a limited number of amino acid mutations. Using chimeric VP1 proteins, we demonstrated that synergistic effects resulted in enhanced binding ability. Bile salts increased GII.2 VLP avidity for HBGAs except GII.2-2011/M1. In vitro blockade assay of salivary HBGA-VLP binding demonstrated the presence of cross-blocking effects among different strains. This study provides insight into the evolutionary binding characteristics and cross-blocking effects of GII.2 NoVs to facilitate the development of measures to control this type of viruses.
Assuntos
Antígenos de Grupos Sanguíneos , Proteínas do Capsídeo , Norovirus , Norovirus/genética , Norovirus/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/imunologia , Humanos , Antígenos de Grupos Sanguíneos/metabolismo , Infecções por Caliciviridae/virologia , Infecções por Caliciviridae/imunologia , Ligação Proteica , Genótipo , Saliva/virologia , Gastroenterite/virologia , Sequência de AminoácidosRESUMO
BACKGROUND: HIV-1 has well-established mechanisms to disrupt essential pathways in people with HIV, such as inflammation and metabolism. Moreover, diversity of the amino acid sequences in fundamental HIV-1 proteins including Tat and Vif, have been linked to dysregulating these pathways, and subsequently influencing clinical outcomes in people with HIV. However, the relationship between Tat and Vif amino acid sequence variation and specific immune markers and metabolites of the tryptophan-kynurenine (Trp-Kyn) pathway remains unclear. Therefore, this study aimed to investigate the relationship between Tat/Vif amino acid sequence diversity and Trp-Kyn metabolites (quinolinic acid (QUIN), Trp, kynurenic acid (KA), Kyn and Trp/Kyn ratio), as well as specific immune markers (sCD163, suPAR, IL-6, NGAL and hsCRP) in n = 67 South African cART-naïve people with HIV. METHODS: Sanger sequencing was used to determine blood-derived Tat/Vif amino acid sequence diversity. To measure Trp-Kyn metabolites, a LC-MS/MS metabolomics platform was employed using a targeted approach. To measure immune markers, Enzyme-linked immunosorbent assays and the Particle-enhanced turbidimetric assay was used. RESULTS: After adjusting for covariates, sCD163 (p = 0.042) and KA (p = 0.031) were higher in participants with Tat signatures N24 and R57, respectively, and amino acid variation at position 24 (adj R2 = 0.048, ß = -0.416, p = 0.042) and 57 (adj R2 = 0.166, ß = 0.535, p = 0.031) of Tat were associated with sCD163 and KA, respectively. CONCLUSIONS: These preliminary findings suggest that amino acid variation in Tat may have an influence on underlying pathogenic HIV-1 mechanisms and therefore, this line of work merits further investigation.
Assuntos
Infecções por HIV , HIV-1 , Inflamação , Cinurenina , Triptofano , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Humanos , Triptofano/metabolismo , Infecções por HIV/virologia , Infecções por HIV/genética , Masculino , HIV-1/genética , Adulto , Feminino , Cinurenina/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Pessoa de Meia-Idade , Biomarcadores/sangue , Receptores de Superfície Celular , Antígenos de Diferenciação Mielomonocítica , Antígenos CDRESUMO
MOTIVATION: For the alignment of large numbers of protein sequences, tools are predominant that decide to align two residues using only simple prior knowledge, e.g. amino acid substitution matrices, and using only part of the available data. The accuracy of state-of-the-art programs declines with decreasing sequence identity and when increasingly large numbers of sequences are aligned. Recently, transformer-based deep-learning models started to harness the vast amount of protein sequence data, resulting in powerful pretrained language models with the main purpose of generating high-dimensional numerical representations, embeddings, for individual sites that agglomerate evolutionary, structural, and biophysical information. RESULTS: We extend the traditional profile hidden Markov model so that it takes as inputs unaligned protein sequences and the corresponding embeddings. We fit the model with gradient descent using our existing differentiable hidden Markov layer. All sequences and their embeddings are jointly aligned to a model of the protein family. We report that our upgraded HMM-based aligner, learnMSA2, combined with the ProtT5-XL protein language model aligns on average almost 6% points more columns correctly than the best amino acid-based competitor and scales well with sequence number. The relative advantage of learnMSA2 over other programs tends to be greater when the sequence identity is lower and when the number of sequences is larger. Our results strengthen the evidence on the rich information contained in protein language models' embeddings and their potential downstream impact on the field of bioinformatics. Availability and implementation: https://github.com/Gaius-Augustus/learnMSA, PyPI and Bioconda, evaluation: https://github.com/felbecker/snakeMSA.
Assuntos
Cadeias de Markov , Proteínas , Alinhamento de Sequência , Análise de Sequência de Proteína , Alinhamento de Sequência/métodos , Proteínas/química , Análise de Sequência de Proteína/métodos , Software , Aprendizado Profundo , Algoritmos , Biologia Computacional/métodos , Sequência de AminoácidosRESUMO
Peptides are valuable for therapeutic development, with multicyclic peptides showing promise in mimicking antigen-binding potency of antibodies. However, our capability to engineer multicyclic peptide scaffolds, particularly for the construction of large combinatorial libraries, is still limited. Here, we study the interplay of disulfide pairing between three biscysteine motifs, and designed a range of triscysteine motifs with unique disulfide-directing capability for regulating the oxidative folding of multicyclic peptides. We demonstrate that incorporating these motifs into random sequences allows the design of disulfide-directed multicyclic peptide (DDMP) libraries with up to four disulfide bonds, which have been applied for the successful discovery of peptide binders with nanomolar affinity to several challenging targets. This study encourages the use of more diverse disulfide-directing motifs for creating multicyclic peptide libraries and opens an avenue for discovering functional peptides in sequence and structural space beyond existing peptide scaffolds, potentially advancing the field of peptide drug discovery.
Assuntos
Cisteína , Dissulfetos , Biblioteca de Peptídeos , Dissulfetos/química , Cisteína/química , Motivos de Aminoácidos , Descoberta de Drogas/métodos , Sequência de Aminoácidos , Peptídeos/química , Peptídeos/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Ligação Proteica , Humanos , Oxirredução , Dobramento de ProteínaRESUMO
Prediction of the intrinsic disorder in protein sequences is an active research area, with well over 100 predictors that were released to date. These efforts are motivated by the functional importance and high levels of abundance of intrinsic disorder, combined with relatively low amounts of experimental annotations. The disorder predictors are periodically evaluated by independent assessors in the Critical Assessment of protein Intrinsic Disorder prediction (CAID) experiments. The recently completed CAID2 experiment assessed close to 40 state-of-the-art methods demonstrating that some of them produce accurate results. In particular, flDPnn2 method, which is the successor of flDPnn that performed well in the CAID1 experiment, secured the overall most accurate results on the Disorder-NOX dataset in CAID2. flDPnn2 implements a number of improvements when compared to its predecessor including changes to the inputs, increased size of the deep network model that we retrained on a larger training set, and addition of an alignment module. Using results from CAID2, we show that flDPnn2 produces accurate predictions very quickly, modestly improving over the accuracy of flDPnn and reducing the runtime by half, to about 27 s per protein. flDPnn2 is freely available as a convenient web server at http://biomine.cs.vcu.edu/servers/flDPnn2/.
Assuntos
Biologia Computacional , Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Biologia Computacional/métodos , Bases de Dados de Proteínas , Conformação Proteica , Software , Sequência de Aminoácidos , Proteínas/química , Proteínas/metabolismo , Análise de Sequência de Proteína/métodosRESUMO
Background: Atopic dermatitis (AD) is a chronic inflammatory skin condition that has a significant impact on quality of life. The immune response and allergy symptoms in AD are triggered by the recognition of specific allergens by IgE antibodies. Cross-reactivity can lead to auto-IgE responses, potentially worsening AD symptoms. Our research aimed to enhance our understanding of allergenic sources, including A. fumigatus, and their role in AD. We focused on molecular mimicry between human AQP3 and A. fumigatus aquaporin. Methods: In our in-silico analysis, we compared the amino acid sequences of human aquaporin 3 (AQP3) and A. fumigatus aquaporin with 25 aquaporins from various allergenic sources, sourced from the UniProt and NCBI databases. Phylogenetic relationship analysis and homology-based modeling were conducted. We identified conserved antigenic regions located within the 3D structures. Results: The global identity levels among the studied aquaporins averaged 32.6%. One antigenic site exhibited a remarkable local region, with a conserved identity of 71.4%. We categorized the aquaporins into five monophyletic clades (A-E), with group B showing the highest identity (95%), including six mammalian aquaporins, including AQP3. When comparing A. fumigatus aquaporins, the highest identity was observed with Malassezia sympodialis at 35%. Both human and A. fumigatus aquaporins have three linear and three discontinuous epitopes. Conclusions: We identified potential linear and conformational epitopes of AQP3, indicating a possible molecular mimicry between humans and A. fumigatus aquaporins. This suggests autoreactivity and potential cross-reactivity, although further validation using in vitro and in vivo experiments is required.
Assuntos
Aquaporina 3 , Aquaporinas , Aspergillus fumigatus , Simulação por Computador , Mimetismo Molecular , Filogenia , Humanos , Aspergillus fumigatus/imunologia , Aspergillus fumigatus/metabolismo , Aquaporina 3/metabolismo , Aquaporinas/metabolismo , Aquaporinas/química , Aquaporinas/genética , Sequência de Aminoácidos , Alérgenos/imunologia , Alérgenos/metabolismo , Hipersensibilidade/imunologia , Hipersensibilidade/microbiologia , Modelos Moleculares , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/imunologiaRESUMO
Efforts towards an effective HIV-1 vaccine have remained mainly unsuccessful. There is increasing evidence for a potential role of HLA-C-restricted CD8+ T cell responses in HIV-1 control, including our recent report of HLA-C*03:02 among African children. However, there are no documented optimal HIV-1 CD8+ T cell epitopes restricted by HLA-C*03:02; additionally, the structural influence of HLA-C*03:02 on epitope binding is undetermined. Immunoinformatics approaches provide a fast and inexpensive method to discover HLA-restricted epitopes. Here, we employed immunopeptidomics to identify HLA-C*03:02 CD8+ T cell epitopes. We identified a clade-specific Gag-derived GY9 (GTEELRSLY) HIV-1 p17 matrix epitope potentially restricted to HLA-C*03:02. Residues E62, T142, and E151 in the HLA-C*03:02 binding groove and positions p3, p6, and p9 on the GY9 epitope are crucial in shaping and stabilizing the epitope binding. Our findings support the growing evidence of the contribution of HLA-C molecules to HIV-1 control and provide a prospect for vaccine strategies.
Assuntos
Epitopos de Linfócito T , HIV-1 , Antígenos HLA-C , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Humanos , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/química , Antígenos HLA-C/imunologia , Antígenos HLA-C/metabolismo , Antígenos HLA-C/genética , HIV-1/imunologia , HIV-1/genética , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Ligação Proteica , Infecções por HIV/imunologia , Infecções por HIV/virologia , Antígenos HIVRESUMO
The MYB(v-myb avian myeloblastosis viral oncogene homolog) family of transcription factors is the largest class of genes among higher plant transcription factors, which can be divided into four subfamilies, with the R2R3-MYB being the most common subfamily type. R2R3-MYB transcription factors are widely involved in the regulation of organ development and secondary metabolite biosynthesis in plants. To investigate the role of R2R3-MYB family transcription factors in the synthesis of flavonoids and glandular trichome development in Artemisia argyi, this study screened and identified 92 R2R3-MYB transcription factors based on the whole genome data of A. argyi, and predicted their potential functions based on bioinformatics. The results showed that the amino acid lengths of the 92 transcription factors ranged from 168 to 547 aa, with relative molecular weights ranging from 19. 6 to 60. 5 kDa, all of which were hydrophilic proteins. Subcellular localization analysis showed that 89 AaMYB proteins were located in the nucleus, while three proteins were simultaneously located in the nucleus and cytoplasm. According to the classification of Arabidopsis R2R3-MYB family, the 92 A. argyi R2R3-MYB proteins were divided into 26 subfamilies, with similar gene structures within the same subfamily.Cis-acting element prediction results showed that light-responsive elements, methyl jasmonate elements, and abscisic acid elements were widely distributed in the promoter regions of R2R3-MYB genes. Transcriptome expression analysis results showed that the expression of AaMYB60, AaMYB63, and AaMYB86 in leaves was higher than that in stems and roots, indicating that these three transcription factors mainly function in leaves. Additionally, five candidate R2R3-MYB transcription factors involved in A. argyi flavonoid biosynthesis or glandular trichome development were selected through phylogenetic analysis. This study provides important genetic resources for the breeding of superior varieties and germplasm innovation of A. argyi in the future.
Assuntos
Artemisia , Regulação da Expressão Gênica de Plantas , Filogenia , Proteínas de Plantas , Fatores de Transcrição , Artemisia/genética , Artemisia/metabolismo , Artemisia/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Perfilação da Expressão Gênica , Sequência de AminoácidosRESUMO
We computationally predicted all phosphorylation sites in the sequence of the human laminin γ1-chain (LAMC1), and computationally identified, for the first time, all kinases for experimentally observed phosphorylated residues of the LAMC1 and all missense deleterious LAMC1 mutations found in different cancer types that interfere with LAMC1 phosphorylation. Also, we mapped the above data to all the biologically functional interaction sequences of the LAMC1. Five kinases (CKII, GPCRK1, PKA, PKC, and CKI) are most enriched for LAMC1 phosphorylation, and the significance of ecto-kinases in this process was emphasized. PKA and PKC targeted more residues inside and close to functional interaction sequences compared with other kinases and in the functional interaction sequence RPESFAIYKRTR. Most phosphorylation-interfering mutations were found in cutaneous melanoma and uterine endometrioid carcinoma. The mutation R255H interfered with the experimentally observed phosphorylation of LAMC1 inside the functional interaction sequence TDIRVTLNRLNTF, while the mutations S181Y and S213Y interfered with the experimentally observed phosphorylation of LAMC1 outside the functional interaction sequences. Mutations R359C,H, R589H, R657C,H, R663I,G, and T1207 interfered with the predicted phosphorylation inside or close to the functional interaction sequences, whereas other mutations interfered outside. PKA- and PKC-predicted phosphorylation was mostly interfered with by mutations inside functional interaction sequences. Phosphorylation- interfering mutations and functional interaction sequences were suggested to promote specific cancer types or cancer progression in general.