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1.
Mol Divers ; 2024 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-39043911

RESUMO

The excessive activation of the monkeypox virus (MPXV-Congo_8-156) is linked to various skin and respiratory disorders such as rashes, fluid-filled blisters, swollen lymph nodes and encephalitis (inflammation of the brain), highlighting MPXV-Congo_8-156 as a promising target for drug intervention. Despite the effectiveness of Cidofovir, in inhibiting MPXV activity, its limited ability to penetrate the skin and its strong side effects restrict its application. To address this challenge, we screened 500 compounds capable of penetrating the skin and gastrointestinal tract to identify potent MPXV inhibitors. Various characterization schemes and structural models of MPXV-Congo_8-156 were explored with bioinformatics tools like PROTPARAM, SOPMA, SWISS-MODEL and PROCHECK. Using molecular docking in PyRx, we evaluated the binding affinities of these compounds with MPXV-Congo_8-156 and identified the top five candidates ranging from - 9.2 to - 8.8 kcal/mol. ADMET analysis indicated that all five compounds were safer alternatives, showing no AMES toxicity or carcinogenicity in toxicological assessments. Molecular dynamics (MD) simulations, conducted for 100 ns each, confirmed the docking interactions of the top five compounds alongside the control (Cidofovir), validating their potential as MPXV inhibitors. The compounds with PubChem CID numbers 4061636, 4422538, 3583576, 4856107 and 4800629 demonstrated strong support in terms of root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration (Rg), solvent-accessible surface area (SASA) value, hydrogen bond analysis, and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) analysis. Thus, our investigation identified these five compounds as promising inhibitors of MPXV, offering potential therapeutic avenues. However, further in vivo studies are necessary to validate our findings.

2.
World J Microbiol Biotechnol ; 40(5): 141, 2024 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-38519797

RESUMO

Metarhizium robertsii, a vital entomopathogenic fungus for pest management, relies on various virulence-related proteins for infection. Identifying these proteins, especially those with unknown functions, can illuminate the fungus's virulence mechanisms. Through RNA-seq, we discovered that the hypothetical protein MAA_07646 was significantly upregulated during appressorium formation in M. robertsii. In this study, we characterized MAA_07646, finding its presence in both the nucleus and cytoplasm. Surprisingly, it did not affect vegetative growth, conidiation, or chemical tolerance. However, it played a role in heat and UV radiation sensitivity. Notably, ΔMAA_07646 exhibited reduced virulence in Galleria mellonella larvae due to impaired appressorium formation and decreased expression of virulence-related genes. In conclusion, MAA_07646 contributes to thermotolerance, UV resistance, and virulence in M. robertsii. Understanding its function sheds light on the insecticidal potential of M. robertsii's hypothetical proteins.


Assuntos
Metarhizium , Mariposas , Animais , Virulência , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Mariposas/metabolismo , Esporos Fúngicos
3.
Proteins ; 91(10): 1427-1436, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37254593

RESUMO

Thermus thermophilus is an extremely thermophilic organism that thrives at a temperature of 65°C. T. thermophilus genome has ~2218 genes, out of which 66% (1482 genes) have been annotated, and the remaining 34% (736 genes) are assigned as hypothetical proteins. In this work, biochemical and biophysical experiments were performed to characterize the hypothetical protein TTHA1873 from T. thermophilus. The hypothetical protein TTHA1873 acts as a nuclease, which indiscreetly cuts methylated and non-methylated DNA in divalent metal ions and relaxes the plasmid DNA in the presence of ATP. The chelation of metal ions with EDTA inhibits its activity. These results suggest that the hypothetical protein TTHA1873 would be a CRISPR-associated protein with non-specific DNase activity and ATP-dependent DNA-relaxing activity.


Assuntos
Proteínas de Bactérias , Thermus thermophilus , Thermus thermophilus/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Plasmídeos/genética , Temperatura , Trifosfato de Adenosina/metabolismo
4.
Microb Pathog ; 185: 106403, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37879452

RESUMO

A common environmental bacteria called Stenotrophomonas maltophilia has become an organism responsible for significant nosocomial infection, mortality in immunocompromised patients, and significantly increasing morbidity and is challenging to treat due to the antibiotic resistance activity of the organism. and bacteriophage therapy is one of the promising treatments against the organism. In this research, we isolated, identified, and characterized Stenotrophomonas phage CM1 against S. maltophilia. Stenotrophomonas phage CM1 head was measured to have a diameter of around 224.25 nm and a tail length of about 159 nm. The phage was found to have noticeable elongated tail spikes around 125 nm in length, the Myoviridae family of viruses, which is categorized under the order Caudovirales. The ideal pH for growth was around 7, demonstrated good thermal stability when incubated at 37-60 °C for 30 min or 60 min, and phage infectivity decreased marginally after 30 min of incubation at 1-5% chloroform concentration. Phage was 3,19,518 base pairs long and had an averaged G + C composition of 43.9 %; 559 open-reading frames (ORFs) were found in the bacteriophage genome, in which 508 of them are hypothetical proteins, 22 of them are other known proteins, 29 of them are tRNAs, and one of them is restriction enzyme. A phylogenetic tree was reconstructed, demonstrating that CM1 shares a close evolutionary relationship with other Stenotrophomonas phages.


Assuntos
Bacteriófagos , Humanos , Bacteriófagos/genética , Stenotrophomonas/genética , Filogenia , Genoma Viral , Myoviridae/genética , Fases de Leitura Aberta
5.
Arch Virol ; 168(10): 241, 2023 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-37668772

RESUMO

A novel mycovirus, Ceratobasidium bipartite virus 1 (CBV1), was identified in Ceratobasidium sp. AG-A strain SHX-YJLC-1 isolated from diseased potato stems. The complete genome of CBV1 consists of two double-stranded RNA (dsRNA) segments: dsRNA1 (2311 bp) and dsRNA2 (1761 bp). dsRNA1 contains a single open reading frame (ORF1) encoding an RNA-dependent RNA polymerase (RdRp), while dsRNA2 contains a single ORF (ORF2) encoding a hypothetical protein (HP) with unknown function. BLASTp analysis revealed that RdRp (75.04%) and HP (61.86%) encoded by the two ORFs have the highest sequence similarity to their counterparts in Rhizoctonia solani dsRNA virus 11 (RsRV11). The genome organization and phylogenetic analysis indicated that the closest relatives to CBV1 are members of the proposed family "Bipartitiviridae". Based on the collective results, CBV1 is inferred to be a new member of the proposed family "Bipartitiviridae". This is the first report on the complete genome sequence of the novel bipartitivirus CBV1, which infects Ceratobasidium sp. AG-A strain SHX-YJLC-1.


Assuntos
Basidiomycota , Micovírus , Filogenia , Fungos , Micovírus/genética , RNA de Cadeia Dupla/genética , RNA Polimerase Dependente de RNA/genética
6.
Int J Mol Sci ; 24(4)2023 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-36835042

RESUMO

Verticillium dahliae is a soil-borne pathogenic fungus that causes Verticillium wilt in host plants, a particularly serious problem in potato cultivation. Several pathogenicity-related proteins play important roles in the host infection process, hence, identifying such proteins, especially those with unknown functions, will surely aid in understanding the mechanism responsible for the pathogenesis of the fungus. Here, tandem mass tag (TMT) was used to quantitatively analyze the differentially expressed proteins in V. dahliae during the infection of the susceptible potato cultivar "Favorita". Potato seedlings were infected with V. dahliae and incubated for 36 h, after which 181 proteins were found to be significantly upregulated. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that most of these proteins were involved in early growth and cell wall degradation. The hypothetical, secretory protein with an unknown function, VDAG_07742, was significantly upregulated during infection. The functional analysis with knockout and complementation mutants revealed that the associated gene was not involved in mycelial growth, conidial production, or germination; however, the penetration ability and pathogenicity of VDAG_07742 deletion mutants were significantly reduced. Therefore, our results strongly indicate that VDAG_07742 is essential in the early stage of potato infection by V. dahliae.


Assuntos
Ascomicetos , Solanum tuberosum , Verticillium , Solanum tuberosum/microbiologia , Virulência/genética , Proteínas , Doenças das Plantas/microbiologia
7.
Proteins ; 89(4): 468-472, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33236809

RESUMO

We report the crystal structure of PYCH_01220, a hypothetical protein in Pyrococcus yayanosii CH1. This protein is composed of two domains, named Domain A and Domain B. While Domain B is not significantly homologous to known protein structures, Domain A is structurally analogous to the C-terminal ribonuclease domain of Escherichia coli colicin D. Domain A has a positively charged surface patch rendered by 13 basic residues, eight arginine or lysine residues of which are evolutionarily conserved. Electrophoretic mobility shift assays showed that PYCH_01220 binds to DNA, and charge-inversion mutations on this patch negatively affect the DNA binding, suggesting that the function of PYCH_01220 might involve nucleic acid-binding via the positively charged patch.


Assuntos
Proteínas Arqueais , DNA , Pyrococcus/química , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Proteínas de Escherichia coli/química , Modelos Moleculares , Ligação Proteica , Domínios Proteicos
8.
Cell Immunol ; 361: 104272, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33445051

RESUMO

Visceral leishmaniasis (VL) is a potentially fatal parasitic disease causing high morbidity and mortality in developing countries. Vaccination is considered the most effective and powerful tool for blocking transmission and control of diseases. However, no vaccine is available so far in the market for humans. In the present study, we characterized the hypothetical protein LDBPK_252400 of Leishmania donovani (LdHyP) and explored its prophylactic behavior as a potential vaccine candidate against VL. We found reduced hepato-splenomegaly along with more than 50% parasite reduction in spleen and liver after vaccination in mice. Protection in vaccinated mice after the antigen challenge correlated with the stimulation of antigen specific IFN-γ expressing CD4+T cell (~4.6 fold) and CD8+T cells (~2.1 fold) in vaccinated mice in compared to infected mice, even after 2-3 months of immunization. Importantly, antigen-mediated humoral immunity correlated with high antigen specific IgG2/IgG1 responses in vaccinated mice. In vitro re-stimulation of splenocytes with LdHyP enhances the expression of TNF-α, IFN-γ, IL-12 and IL-10 cytokines along with lower IL-4 cytokine and IL-10/IFN-γ ratio in vaccinated mice. Importantly, we observed ~3.5 fold high NO production through activated macrophages validates antigen mediated cellular immunity induction, which is critical in controlling infection progression. These findings suggest that immunization with LdHyP mount a very robust immunity (from IL-10 towards TFN-γ mediated responses) against L. donovani infection and could be explored further as a putative vaccine candidate against VL.


Assuntos
Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/tratamento farmacológico , Animais , Antígenos de Protozoários/imunologia , Citocinas/imunologia , Imunidade Celular/imunologia , Imunização/métodos , Leishmania donovani/imunologia , Leishmania donovani/patogenicidade , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/imunologia , Linfócitos T/imunologia , Vacinação/métodos
9.
Fish Shellfish Immunol ; 108: 73-79, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33285163

RESUMO

A unique strain of Vibrio harveyi is the causative agent of scale drop and muscle necrosis disease (SDMND) in Asian sea bass (Lates calcarifer). This study investigated the protein profiles of SDMND-causing Vibrio harveyi isolates compared to the reference V. harveyi ATCC 14126 strain. A distinct protein band of 33 kDa, namely HP33, found from only V. harveyi SDMND was subjected to analysis by LC-MS/MS and the identified peptide sequences matched to an unknown hypothetical protein. Detection of HP33 coding sequence was investigated at both genomic and transcriptional levels and the results consistently supported the protein analysis. Recombinant HP33 protein was then produced using Escherichia coli system. The rHP33 protein did not cause mortality or visible clinical signs to Asian sea bass. However, the rHP33 protein was able to stimulate antibody response in Asian sea bass as evidenced by Western blotting and agglutination tests. Here, we proposed that rHP33 might be a good protein target for development of subunit vaccine and/or immunostimulant to protect Asian sea bass from SDMND.


Assuntos
Proteínas de Bactérias/genética , Bass , Doenças dos Peixes/imunologia , Imunogenicidade da Vacina , Necrose/veterinária , Vibrioses/veterinária , Vibrio/imunologia , Escamas de Animais/patologia , Animais , Proteínas de Bactérias/imunologia , Doenças dos Peixes/microbiologia , Doenças Musculares/imunologia , Doenças Musculares/microbiologia , Doenças Musculares/veterinária , Necrose/imunologia , Necrose/microbiologia , Vibrio/genética , Vibrioses/imunologia , Vibrioses/microbiologia
10.
Exp Parasitol ; 221: 108048, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33307096

RESUMO

Leishmaniasis is a disease caused by trypanosomatid protozoa of the genus Leishmania. In the Americas, the species Leishmania amazonensis is predominantly associated with American cutaneous leishmaniasis (ACL) while L. infantum is an agent of visceral leishmaniasis (VL). The genome sequences of Leishmania spp. have shown that each genome can contain about 8000 genes encoding proteins, more than half of which have an unknown function (''hypotheticals") at the time of publication. To understand the biology and genome of the organisms, it is important to discover the function of these "hypothetical" proteins; however, few studies have focused on their characterizations. Previously, LinJ.30.3360 (a protein with unknown function) was identified as immunogenic to canine serum with VL and a good antigen to diagnose the visceral form in dogs. Here, we show that the LinJ.30.3360 protein is conserved in L. infantum, L. tarantolae, L. donovani, L. major, L. mexicana, L. braziliensis, L. panamensis, Leptomonas pyrrhocoris, and Leptomonas seymouri. It has been annotated as a MORN (Membrane Occupation and Recognition Nexus) domain protein. However, since the function of this motif is unknown, functional inferences based on the primary sequence are not possible. The protein has a folded ß-leaf secondary structure, and phosphorylation was the only post-translational modification (PTM) found using prediction approach. Experiments have shown that it is located close to the flagellar pocket and presents similar abundance in both L. amazonensis and L. infantum. Furthermore, because it is a conserved protein in trypanosomatids but not in mammals and also because of its antigenicity, LinJ.30.3360 may constitute a potential drug target and/or vaccine for leishmaniasis.


Assuntos
Leishmania infantum/química , Leishmania mexicana/química , Proteínas de Protozoários/química , Animais , Western Blotting , Sequência Conservada , Imuno-Histoquímica , Leishmania infantum/genética , Leishmania mexicana/genética , Masculino , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína
11.
Proteins ; 88(9): 1226-1232, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32337767

RESUMO

Cyanophages, widespread in aquatic systems, are a class of viruses that specifically infect cyanobacteria. Though they play important roles in modulating the homeostasis of cyanobacterial populations, little is known about the freshwater cyanophages, especially those hypothetical proteins of unknown function. Mic1 is a freshwater siphocyanophage isolated from the Lake Chaohu. It encodes three hypothetical proteins Gp65, Gp66, and Gp72, which share an identity of 61.6% to 83%. However, we find these three homologous proteins differ from each other in oligomeric state. Moreover, we solve the crystal structure of Gp72 at 2.3 Å, which represents a novel fold in the α + ß class. Structural analyses combined with redox assays enable us to propose a model of disulfide bond mediated oligomerization for Gp72. Altogether, these findings provide structural and biochemical basis for further investigations on the freshwater cyanophage Mic1.


Assuntos
Bacteriófagos/química , Cianobactérias/virologia , Dissulfetos/química , Proteínas Virais/química , Sequência de Aminoácidos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Dissulfetos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Água Doce/microbiologia , Água Doce/virologia , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Oxirredução , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Virais/genética , Proteínas Virais/metabolismo
12.
Funct Integr Genomics ; 17(1): 39-52, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27778111

RESUMO

In silico derived properties on experimental validation revealed that hypothetical protein Alr2954 of Anabaena sp. PCC7120 is ADP-ribose pyrophosphatase, which belongs to nudix hydrolase superfamily. Presence of ADP-ribose binding site was attested by ADP-ribose pyrophosphatase activity (K m 44.71 ± 8.043 mM, V max 7.128 ± 0.417 µmol min-1 mg protein-1, and K cat/K m 9.438 × 104 µM-1 min-1). Besides ADP-ribose, the enzyme efficiently hydrolyzed various nucleoside phosphatases such as 8-oxo-dGDP, 8-oxo-dADP, 8-oxo-dGTP, 8-oxo-dATP, GDP-mannose, ADP-glucose, and NADH. qRT-PCR analysis of alr2954 showed significant expression under different abiotic stresses reconfirming its role in stress tolerance. Thus, Alr2954 qualifies to be a member of nudix hydrolase superfamily, which serves as ADP-ribose pyrophosphatase and assists in multiple abiotic stress tolerance.


Assuntos
Anabaena/enzimologia , Escherichia coli/genética , Pirofosfatases/genética , Estresse Fisiológico/genética , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/metabolismo , Sequência de Aminoácidos/genética , Sítios de Ligação , Clonagem Molecular , Simulação por Computador , Nucleotídeos de Desoxiadenina/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Escherichia coli/enzimologia , Hidrólise , Simulação de Acoplamento Molecular , Pirofosfatases/química , Pirofosfatases/isolamento & purificação , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
13.
Biochem Biophys Res Commun ; 482(4): 1176-1182, 2017 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-27919686

RESUMO

The uropathogenic Escherichia coli strain CFT073 contains multiple iron and heme transport systems, which facilitate infection of the host urinary tract. To elucidate the molecular and cellular function of ChuY, a hypothetical gene in the heme degradation/utilization pathway, we solved the crystal structure of ChuY at 2.4 Å resolution. ChuY has high structural homology with human biliverdin and flavin reductase. We confirmed that ChuY has flavin mononucleotide (FMN) reductase activity, using NAD(P)H as a cofactor, and shows porphyrin ring binding affinity. A chuY deletion-insertion strain showed reduced survival potential compared to wild-type and complemented strains in mammalian cells. Current results suggest ChuY acts as a reductase in heme homeostasis to maintain the virulence potential of E. coli CFT073.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Animais , Biliverdina/química , Cristalografia por Raios X , Escherichia coli/patogenicidade , Proteínas de Escherichia coli/química , FMN Redutase/química , Deleção de Genes , Genômica , Células HEK293 , Heme/química , Hemina/química , Homeostase , Humanos , Ferro/química , Camundongos , NADP/química , Porfirinas/química , Conformação Proteica , Estrutura Secundária de Proteína , Células RAW 264.7 , Virulência
14.
Biochem Biophys Res Commun ; 482(2): 264-269, 2017 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-27845039

RESUMO

The crystal structure of a hypothetical protein MJ0366, derived from Methanocaldococcus jannaschii was solved at 1.9 Å resolution using synchrotron radiation. MJ0366 was crystallized as a monomer and has knot structural arrangement. Intriguingly, the solved structure consists of novel 'KNOT' fold conformation. The 31 trefoil knot was observed in the structure. The N-terminal and C-terminal ends did not participate in knot formation.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Methanocaldococcus/metabolismo , Modelos Químicos , Modelos Moleculares , Simulação por Computador , Cristalografia , Conformação Proteica , Dobramento de Proteína
15.
Parasitol Res ; 116(5): 1533-1544, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28389892

RESUMO

Computational approaches to predict structure/function and other biological characteristics of proteins are becoming more common in comparison to the traditional methods in drug discovery. Cryptosporidiosis is a major zoonotic diarrheal disease particularly in children, which is caused primarily by Cryptosporidium hominis and Cryptosporidium parvum. Currently, there are no vaccines for cryptosporidiosis and recommended drugs are ineffective. With the availability of complete genome sequence of C. hominis, new targets have been recognized for the development of effective and better drugs and/or vaccines. We identified a unique hypothetical protein (TU502HP) in the C. hominis genome from the CryptoDB database. A three-dimensional model of the protein was generated using the Iterative Threading ASSEmbly Refinement server through an iterative threading method. Functional annotation and phylogenetic study of TU502HP protein revealed similarity with human transportin 3. The model is further subjected to a virtual screening study form the ZINC database compound library using the Dock Blaster server. A docking study through AutoDock software reported N-(3-chlorobenzyl)ethane-1,2-diamine as the best inhibitor in terms of docking score and binding energy. The reliability of the binding mode of the inhibitor is confirmed by a complex molecular dynamics simulation study using GROMACS software for 10 ns in the water environment. Furthermore, antigenic determinants of the protein were determined with the help of DNASTAR software. Our findings report a great potential in order to provide insights in the development of new drug(s) or vaccine(s) for treatment and prophylaxis of cryptosporidiosis among humans and animals.


Assuntos
Criptosporidiose/tratamento farmacológico , Cryptosporidium/metabolismo , Descoberta de Drogas/métodos , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Animais , Sequência de Bases , Criança , Criptosporidiose/diagnóstico , Cryptosporidium/classificação , Cryptosporidium/genética , Genoma de Protozoário/genética , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Filogenia , Estrutura Secundária de Proteína , Proteínas de Protozoários/genética , Reprodutibilidade dos Testes , Zoonoses/parasitologia
16.
Arch Biochem Biophys ; 596: 10-21, 2016 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-26926257

RESUMO

The genome of Leishmania donovani, the causative agent of visceral leishmaniasis, codes for approximately 65% of both conserved and non-conserved hypothetical proteins. Studies on 'conserved hypothetical' proteins are expected to reveal not only new and crucial aspects of Leishmania biochemistry, but it could also lead to discovery of novel drug candidates. Conserved hypothetical protein, LdBPK_070020, is a 31.14 kDa protein, encoded by an 810 bp gene. BLAST analysis of LdBPK_070020, performed against NCBI non-redundant database, showed 80-99% similarity with conserved hypothetical proteins of Leishmania belonging to other species. Using homologues recombination method, we have performed gene knockout of LdBPK_070020 and effects of the same were investigated on the parasite. The gene knocked out strain shows significant retardation in growth with respect to wild type. Detailed biochemical studies indicated towards important role of LdBPK_070020 in the parasite survival and growth.


Assuntos
Leishmania donovani , Bases de Dados de Proteínas , Técnicas de Silenciamento de Genes , Leishmania donovani/genética , Leishmania donovani/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Análise de Sequência de Proteína
17.
Extremophiles ; 20(3): 275-82, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26936147

RESUMO

TTHA0829 from Thermus thermophilus HB8 has a molecular mass of 22,754 Da and is composed of 210 amino acid residues. The expression of TTHA0829 is remarkably elevated in the latter half of logarithmic growth phase. TTHA0829 can form either a tetrameric or dimeric structure, and main-chain folding provides an N-terminal cystathionine-ß-synthase (CBS) domain and a C-terminal aspartate-kinase chorismate-mutase tyrA (ACT) domain. Both CBS and ACT are regulatory domains to which a small ligand molecule can bind. The CBS domain is found in proteins from organisms belonging to all kingdoms and is observed frequently as two or four tandem copies. This domain is considered as a small intracellular module with a regulatory function and is typically found adjacent to the active (or functional) site of several enzymes and integral membrane proteins. The ACT domain comprises four ß-strands and two α-helices in a ßαßßαß motif typical of intracellular small molecule binding domains that help control metabolism, solute transport and signal transduction. We discuss the possible role of TTHA0829 based on its structure and expression pattern. The results imply that TTHA0829 acts as a cell-stress sensor or a metabolite acceptor.


Assuntos
Aspartato Quinase/química , Proteínas de Bactérias/química , Corismato Mutase/química , Cistationina beta-Sintase/química , Thermus thermophilus/genética , Aspartato Quinase/genética , Aspartato Quinase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Corismato Mutase/genética , Corismato Mutase/metabolismo , Cistationina beta-Sintase/genética , Cistationina beta-Sintase/metabolismo , Domínios Proteicos , Thermus thermophilus/enzimologia
18.
Exp Parasitol ; 171: 71-76, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27765655

RESUMO

The protein encoding zone of Mitochondrial DNA region (inherited from single lineage) seems most suitable and effective for taxonomic, systematic, ecological, evolutionary, DNA barcoding, cryptic species and population studies, exploiting nucleotide/amino acid datasets (1D/2D/3D conformational level). Nowadays, expeditious computerized methods are in trend for analyzing genetic material to demonstrate variations at various levels of protein structures. Structural proteomics have implemented here for genetic identification, differentiation and relationship of species from information rich data of mt COI gene of the family Diplostomidae with inclusion of molecular tools. Various aspects have been utilized herein for re-validation and infallible discrimination of Trematode diplostomoid metacercariae (Tetracotyle lucknowensis Pandey, 1971; T. xenentodoni Chakrabarti, 1970; T. fausti Rai and Pande, 1969; T. muscularius Chakrabarti, 1970 and Diplostomulum minutum Pandey, 1968), the infective stage in the life cycle, causing severe damage to fish host, whose adults are found mainly in fish eating birds and mammals.


Assuntos
Proteínas de Helminto/química , Trematódeos/genética , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Ciclo-Oxigenase 1/genética , DNA Mitocondrial/química , Doenças dos Peixes/parasitologia , Peixes , Proteínas de Helminto/genética , Imageamento Tridimensional , Metacercárias/química , Metacercárias/classificação , Metacercárias/genética , Mitocôndrias/enzimologia , Modelos Biológicos , Modelos Estruturais , Conformação Molecular , Filogenia , Conformação Proteica , Proteômica , Trematódeos/química , Trematódeos/classificação , Infecções por Trematódeos/parasitologia , Infecções por Trematódeos/veterinária
19.
Parasite Immunol ; 37(12): 646-56, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26457798

RESUMO

In this work, the effect of vaccination of a newly described Leishmania infantum antigenic protein has been studied in BALB/c mice infected with this parasite species. The LiHyD protein was characterized after a proteomic screening performed with the sera from dogs suffering visceral leishmaniasis (VL). Its recombinant version was expressed, purified and administered to BALB/c mice in combination with saponin. As a result of vaccination and 10 weeks after challenge using an infective dose of L. infantum stationary promastigotes, vaccinated mice showed lower parasite burdens in different organs (liver, spleen, bone marrow and footpads' draining lymph nodes) than mice inoculated with the adjuvant alone or the vaccine diluent. Protected mice showed anti-Leishmania IgG2a antibodies and a predominant IL-12-driven IFN-γ production (mainly produced by CD4(+) T cells) against parasite proteins, whereas unprotected controls showed anti-Leishmania IgG1 antibodies and parasite-mediated IL-4 and IL-10 responses. Vaccinated mice showed an anti-LiHyD IgG2a humoral response, and their spleen cells were able to secrete LiHyD-specific IFN-γ, IL-12 and GM-CSF cytokines before and after infection. The protection was correlated with the Leishmania-specific production on nitric oxide. Altogether, the results indicate that the new LiHyD protein could be considered in vaccine formulations against VL.


Assuntos
Citocinas/metabolismo , Leishmania infantum/imunologia , Leishmaniose Visceral/veterinária , Proteínas de Protozoários/imunologia , Vacinação/veterinária , Adjuvantes Imunológicos , Animais , Modelos Animais de Doenças , Cães , Feminino , Leishmaniose Visceral/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes
20.
Exp Parasitol ; 151-152: 56-63, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25655405

RESUMO

Members of the haloacid dehalogenase (HAD) superfamily are emerging as an important group of enzymes by virtue of their role in diverse chemical reactions. In different Plasmodium species their number varies from 16 to 21. One of the HAD superfamily members, PVX_123945, a hypothetical protein from Plasmodium vivax, was selected for examining its substrate specificity. Based on distant homology searches and structure comparisons, it was predicted to be a phosphatase. Thirty-eight metabolites were screened to identify potential substrates. Further, to validate the prediction, biochemical and kinetic studies were carried out that showed that the protein was a monomer with high catalytic efficiency for ß-glycerophosphate followed by pyridoxal 5'-phosphate. The enzyme also exhibited moderate catalytic efficiencies for α-glycerophosphate, xanthosine 5'-monophosphate and adenosine 5'-monophosphate. It also hydrolyzed the artificial substrate p-nitrophenyl phosphate (pNPP). Mg(2+) was the most preferred divalent cation and phosphate inhibited the enzyme activity. The study is the first attempt at understanding the substrate specificity of a hypothetical protein belonging to HAD superfamily from the malarial parasite P. vivax.


Assuntos
Glicerofosfatos/metabolismo , Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Plasmodium vivax/enzimologia , Fosfato de Piridoxal/metabolismo , Monofosfato de Adenosina/metabolismo , Biologia Computacional , Hidrolases/antagonistas & inibidores , Hidrolases/química , Cinética , Magnésio/metabolismo , Estrutura Molecular , Peso Molecular , Nitrofenóis/metabolismo , Compostos Organofosforados/metabolismo , Fosfatos/farmacologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/química , Dobramento de Proteína , Ribonucleotídeos/metabolismo , Especificidade por Substrato , Xantina
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