RESUMO
The EccC enzyme of Mycobacterium tuberculosis ESX-1 secretion system is involved in EsxAB virulence factor secretion and offers an attractive target for antivirulence inhibitors development against M. tuberculosis. The EccCb1 polypeptide of the EccC enzyme contains two Ftsk/SpoIIIE type ATPase domains (D2 and D3) and binds to the EsxAB factor at the C-terminal region of the D3 domain. In the current study, we have determined a low-resolution structure of EccCb1, and its mechanism involved in ATPase activity and EsxAB factor binding. Small-angle X-ray scattering data yielded a double hexameric ring structure of EccCb1 in solution and was further confirmed by SEC-MALS and dynamic light scattering. ATPase activity of wild-type, D2, and D3 mutants showed that D2-K90A and D3-K382A mutations led to a complete loss of enzyme activity. The full-length EccCb1 showed â¼3.7-fold lower catalytic efficiency than D2 domain and â¼1.7 fold lower than D3 domain. The EsxAB factor binds EccCb1 with Kd â¼ 11.3 ± 0.6â nM and its affinity is enhanced â¼2 fold in presence of ATP + Mg2+. These data indicate the involvement of ATPase activity in EsxAB factor translocation. Molecular dynamics simulation on wild-type, ATP + Mg2+, and EsxAB + ATP + Mg2+ bound EccCb1 double-ring structure showed enhanced stability of enzyme upon ATP + Mg2+ and EsxAB binding. Overall, our study showed a low-resolution structure of EccCb1, and the mechanism involved in ATPase activity and EsxAB factor recognition, which can be targeted for the development of antivirulence drugs against M. tuberculosis.
Assuntos
Mycobacterium tuberculosis , Sistemas de Secreção Tipo VII , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Humanos , Magnésio/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculose/microbiologia , Sistemas de Secreção Tipo VII/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Falcipain-2 (FP2) and falcipain-3 (FP3) constitute the major hemoglobinases of Plasmodium falciparum. Previous biochemical and structural studies have explained the mechanism of inhibition of these enzymes by small molecules. However, a residue-level protein-protein interaction (PPI) with its natural macromolecular substrate, hemoglobin is not fully characterized. Earlier studies have identified a short motif in the C-terminal of FP2, an exosite protruding away from the active site, essential for hemoglobin degradation. Our structural and mutagenesis studies suggest that hemoglobin interacts with FP2 via specific interactions mediated by Glu185 and Val187 within the C-terminal motif, which are essential for hemoglobin binding. Since FP3 is also a major hemoglobinase and essential for parasite survival, we further demonstrate its interactions with hemoglobin. Our results suggest that Asp194 of FP3 is required for hemoglobin hydrolysis and residue-swap experiments confirmed that this position is functionally conserved between the two hemoglobinases. Residues involved in protein-protein interactions constitute important targets for drug-mediated inhibition. Targeting protein-protein interactions at exosites may likely be less susceptible to emergence of drug resistance and thus is a new field to explore in malaria.
Assuntos
Cisteína Endopeptidases/metabolismo , Hemoglobinas/metabolismo , Plasmodium falciparum/enzimologia , Ácido Aspártico/química , Clonagem Molecular , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Ácido Glutâmico/química , Hemoglobinas/química , Hidrólise , Estrutura Molecular , Mutagênese , Plasmodium falciparum/genéticaRESUMO
Proteolytic activity constitutes a fundamental process essential for the survival of the malaria parasite and is thus highly regulated. Falstatin, a protease inhibitor of Plasmodium falciparum, tightly regulates the activity of cysteine hemoglobinases, falcipain-2 and 3 (FP2, FP3), by inhibiting FP2 through a single surface exposed loop. However, the multimeric nature of falstatin and its interaction with FP2 remained unexplored. Here we report that the N-terminal falstatin region is highly disordered, and needs chaperone activity (heat-shock protein 70, HSP70) for its folding. Protein-protein interaction assays showed a significant interaction between falstatin and HSP70. Further, characterization of the falstatin multimer through a series of biophysical techniques identified the formation of a falstatin decamer, which was extremely thermostable. Computational analysis of the falstatin decamer showed the presence of five falstatin dimers, with each dimer aligned in a head-to-tail orientation. Further, the falstatin C-terminal region was revealed to be primarily involved in the oligomerization process. Stoichiometric analysis of the FP2-falstatin multimer showed the formation of a heterooligomeric complex in a 1:1 ratio, with the participation of ten subunits of each protein. Taken together, our results report a novel protease-inhibitor complex and strengthens our understanding of the regulatory mechanisms of major plasmodium hemoglobinases.
Assuntos
Cisteína Endopeptidases , Plasmodium falciparum , Dobramento de ProteínaRESUMO
The human transporter associated with antigen processing (TAP) protein belongs to the ATP-binding cassette (ABC) transporter superfamily and is formed by the heterodimerization of TAP1 and TAP2 subunits. TAP selectively pumps cytosolic peptides into the lumen of the endoplasmic reticulum in an ATP-dependent manner. The catalytic cycle of the ATPase domain of TAP is not understood at the molecular level. The structures of catalytic intermediates of the ATPase domain of TAP will contribute to the understanding of the chemical mechanism of ATP hydrolysis. In order to understand this mechanism, the ATPase domain of human TAP1 (NBD1) was expressed and purified, crystallized in nucleotide-free and transition-state complex forms and X-ray crystallographic studies were performed. The NBD1 protein was crystallized (i) in the nucleotide-free apo form; (ii) in complex with ADP-Mg(2+), mimicking the product-bound state; (iii) in complex with vanadate-ADP-Mg(2+), mimicking the ATP-bound state; and (iv) in complex with azide-ADP-Mg(2+), also mimicking the ATP-bound state. X-ray diffraction data sets were collected for apo and complexed NBD1 using an in-house X-ray diffraction facility at a wavelength of 1.5418 Å. The apo and complexed NBD1 crystals belonged to the primitive hexagonal space group P6(2), with one monomer in the asymmetric unit. Here, the crystallization, data collection and preliminary crystallographic analysis of apo and complexed NBD1 are reported.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Difosfato de Adenosina/química , Adenosina Trifosfatases/química , Azidas/química , Vanadatos/química , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Transportadores de Cassetes de Ligação de ATP/metabolismo , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/metabolismo , Azidas/metabolismo , Cristalização , Cristalografia por Raios X , Humanos , Ligação Proteica , Vanadatos/metabolismoRESUMO
The Ergp55 protein belongs to the Ets family of transciption factors. The Ets transcription factors are involved in various developmental processes and the regulation of cancer metabolism. They contain a highly similar DNA-binding domain known as the ETS domain and have diverse functions in oncogenesis and physiology. The Ets transcription factors differ in their DNA-binding preference at the ETS site and the mechanisms by which they target genes are not clearly understood. To understand its DNA-binding mechanism, the ETS domain of Ergp55 was expressed and purified. The ETS domain was crystallized in the native form and in complex forms with DNA sequences from the E74 and cfos promoters. An X-ray diffraction data set was collected from an ETS-cfos DNA complex crystal at a wavelength of 0.9725â Å on the BM14 synchrotron beamline at the ESRF, France. The ETS-cfos DNA complex crystal belonged to space group C222(1), with four molecules in the asymmetric unit. For structure analysis, initial phases for the ETS-cfos DNA complex were obtained by the molecular-replacement technique with Phaser in the CCP4 suite using the coordinates of Fli-1 protein (PDB entry 1fli) and cfos DNA (PDB entry 1bc7) as search models. Structure analysis of the ETS-cfos DNA complex may possibly explain the DNA-binding specificity and its mechanism of interaction with the ETS domain of Ergp55.
Assuntos
Oligonucleotídeos/química , Proteína Proto-Oncogênica c-ets-1/química , Proteínas Proto-Oncogênicas c-fos/genética , Sequência de Bases , Cristalização , Cristalografia por Raios X , Escherichia coli , Humanos , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteína Proto-Oncogênica c-ets-1/biossíntese , Proteína Proto-Oncogênica c-ets-1/isolamento & purificaçãoRESUMO
P25 and P28 proteins are essential for Plasmodium parasites to infect mosquitoes and are leading candidates for a transmission-blocking malaria vaccine. The Plasmodium vivax P25 is a triangular prism that could tile the parasite surface. The residues forming the triangle are conserved in P25 and P28 from all Plasmodium species. A cocrystal structure shows that a transmission-blocking antibody uses only its heavy chain to bind Pvs25 at a vertex of the triangle.
Assuntos
Antígenos de Protozoários/química , Antígenos de Protozoários/metabolismo , Antígenos de Superfície/química , Antígenos de Superfície/metabolismo , Culicidae/parasitologia , Vacinas Antimaláricas/química , Vacinas Antimaláricas/metabolismo , Plasmodium vivax/química , Plasmodium vivax/crescimento & desenvolvimento , Animais , Modelos Moleculares , Plasmodium vivax/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
ESX-1-secreted protein regulator (EspR; Rv3849) is a key regulator in Mycobacterium tuberculosis that delivers bacterial proteins into the host cell during infection. EspR binds directly to the Rv3616c-Rv3614c promoter and activates transcription and secretes itself from the bacterial cell by the ESX-1 system. The three-dimensional structure of EspR will aid in understanding the mechanisms by which it binds to the Rv3616c-Rv3614c promoter and is involved in transcriptional activation. This study will significantly aid in the development of EspR-based therapeutics against M. tuberculosis. The full-length EspR gene from M. tuberculosis (H37Rv strain) was cloned and overexpressed as a soluble protein in Escherichia coli. The protein was purified by affinity chromatography using His-tagged protein followed by size-exclusion chromatography. EspR was crystallized using polyethylene glycol 3350 as precipitant. The crystals diffracted to 3.2â Å resolution using synchrotron radiation of wavelength 0.97625â Å. The crystal belonged to space group P3(1)21 and contained three monomers in the asymmetric unit. Native and heavy-atom-derivatized data sets were collected from EspR crystals for use in ab initio structure-solution techniques.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Homeodomínio/metabolismo , Mycobacterium tuberculosis/química , Proteínas de Bactérias/genética , Clonagem Molecular , Cristalização , Dados de Sequência Molecular , Difração de Raios XRESUMO
The EccC enzyme of ESX-1 system contains (i) a membrane bound Rv3870 with single ATPase domain and (ii) a cytoplasmic Rv3871 with two ATPase domains and involved in secretion of ESAT6/CFP10 factor out of the cell. In current study, we have structurally and biochemically characterized the ATPase domain (442-747 residues) of Rv3870 enzyme. The ΔRv3870 eluted as oligomer (~813 kDa) from Superdex 200 (16/60) column, as identified based on molecular mass standard and dynamics light scattering. The SAXS analysis yielded a tetrameric ring envelope of ΔRv3870, quite consistent to dynamic light scattering data. The ΔRv3870 exhibited ATPase activity having kinetic parameters, Km ~ 100 ± 40 µM, kcat ~ 1.81 ± 0.27 min-1 and Vmax ~ 54.41 µM/min/mg. ATPase activity using nine ΔRv3870 mutants showed 70-91% decrease in catalytic efficiency of the enzyme. ΔRv3870 binds Rv3871 with KD ~ 484.0 ± 10.3 nM and its catalytic efficiency is enhanced ~6.7-fold in presence of Rv3871. CD data revealed the high TM ~ 82.2 ± 0.5 °C for ΔRv3870 and enhanced in presence of ATP + Mg2+, as observed in dynamics simulation on ΔRv3870 hexameric models. Overall, our structural and biochemical studies on ΔRv3870 have explained the mechanism, which will contribute in development of antivirulence inhibitors against M. tuberculosis.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/enzimologia , Nucleotídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Estabilidade Enzimática , Cinética , Magnésio/metabolismo , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Soluções , Homologia Estrutural de Proteína , Temperatura , Difração de Raios XRESUMO
Mycobacterium tuberculosis HddA enzyme phosphorylates the M7P substrate and converts it to M7PP product in GDP-D-α-D-heptose biosynthetic pathway. For structural and functional studies on MtbHddA, we have purified the enzyme, which eluted as a monomer from size exclusion column. Purified MtbHddA had ATPase activity. The SAXS analysis supported globular monomeric scattering profile of MtbHddA in solution. The CD analysis showed that MtbHddA contains 45% α-helix, 18% ß-stands, and 32% random coil structures and showed unfolding temperature (TM) ~ 47.5 °C. The unfolding temperature of MtbHddA is enhanced by 1.78±0.41 °C in ATP+Mg2+ bound state, 2.12±0.41 °C in Mannose bound state and 3.07±0.41 °C in Mannose+ ATP+Mg2+ bound state. The apo and M7P +ATP + Mg2+ complexed models of MtbHddA showed that enzyme adopts a classical GHMP sugar kinase fold with conserved ATP+Mg2+ and M7P binding sites. The dynamics simulation analysis on four MtbHddA models showed that ATP+Mg2+ and M7P binding enhanced the stability of active site conformation of MtbHddA. Our study provides important insights into MtbHddA structure and activity, which can be targeted for therapeutic development against M. tuberculosis.
Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Magnésio/química , Mycobacterium tuberculosis/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfatos Açúcares/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cátions Bivalentes , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Magnésio/metabolismo , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , Fosfatos Açúcares/metabolismo , TermodinâmicaRESUMO
Head and neck cancer (HNC) is among the ten leading malignancies worldwide, with India solely contributing one-third of global oral cancer cases. The current focus of all cutting-edge strategies against this global malignancy are directed towards the heterogeneous tumor microenvironment that obstructs most treatment blueprints. Subsequent to the portrayal of established information, the review details the application of single cell technology, organoids and spheroid technology in relevance to head and neck cancer and the tumor microenvironment acknowledging the resistance pattern of the heterogeneous cell population in HNC. Bioinformatic tools are used for study of differentially expressed genes and further omics data analysis. However, these tools have several challenges and limitations when analyzing single-cell gene expression data that are discussed briefly. The review further examines the omics of HNC, through comprehensive analyses of genomics, transcriptomics, proteomics, metabolomics, and epigenomics profiles. Patterns of alterations vary between patients, thus heterogeneity and molecular alterations between patients have driven the clinical significance of molecular targeted therapies. The analyses of potential molecular targets in HNC are discussed with connotation to the alteration of key pathways in HNC followed by a comprehensive study of protein kinases as novel drug targets including its ATPase and additional binding pockets, non-catalytic domains and single residues. We herein review, the therapeutic agents targeting the potential biomarkers in light of new molecular targeted therapies. In the final analysis, this review suggests that the development of improved target-specific personalized therapies can combat HNC's global plight.
RESUMO
M. tuberculosis GmhA enzyme catalyzes the isomerization of D-sedoheptulose 7-phosphate into D-glycero-D-α-manno-heptose-7-phosphate in GDP-D-glycero-α-D-manno-heptose biosynthetic pathway. The D-glycero-α-D-manno-heptose is a major constituent of lipopolysaccharide and contributes to virulence and antibiotic resistance to mycobacteria. In current study, we have performed the structural and biochemical analysis of M. tuberculosis GmhA, the first enzyme involved in D-sedoheptulose 7-phosphate isomerization in GDP-D-α-D-heptose biosynthetic pathway. The MtbGmhA enzyme exits as tetramer and small angle X-ray scattering analysis also yielded tetrameric envelope in solution. The MtbGmhA enzyme binds to D-sedoheptulose 7-phosphate with Km ~ 0.31 ± 0.06 mM-1 and coverts it to D-glycero-D-α-manno-heptose-7-phosphate with catalytic efficiency (kcat/Km) ~ 1.45 mM-1 s-1. The residues involved in D-sedoheptulose 7-phosphate and Zn2+ binding were identified using modeled MtbGmhA + D-sedoheptulose 7-phosphate + Zn2+ structure. To understand the role in catalysis, six site directed mutants of MtbGmhA were generated, which showed significant decrease in catalytic activity. The circular dichroism analysis showed ~ 46% α-helix, ~ 19% ß-sheet and ~ 35% random coil structures of MtbGmhA enzyme and melting temperature ~ 53.5 °C. Small angle X-ray scattering analysis showed the tetrameric envelope, which fitted well with modeled MtbGmhA tetramer in closed conformation. The MtbGmhA dynamics involved in D-sedoheptulose 7-phosphate and Zn2+ binding was identified using dynamics simulation and showed enhanced stability in presence of these ligands. Our biochemical data and structural knowledge have provided insight into mechanism of action of MtbGmhA enzyme, which can be targeted for novel antibiotics development against M. tuberculosis.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Proteínas de Bactérias/metabolismo , Lipopolissacarídeos/biossíntese , Mycobacterium tuberculosis/enzimologia , Fosfatos Açúcares/metabolismo , Aldose-Cetose Isomerases/química , Dicroísmo Circular , Modelos MolecularesRESUMO
The article by Sharma et al. [(2018), Acta Cryst. F74, 656-663] is corrected.
RESUMO
The M. tuberculosis GmhB protein converts the d-glycero-α-d-manno-heptose 1,7-bisphosphate (GMB) intermediate into d-glycero-α-d-manno-heptose 1-phosphate by removing the phosphate group at the C-7 position. To understand the structure and substrate binding mechanism, the MtbGmhB was purified which elutes as monomer on gel filtration column. The small angle x-ray scattering analysis shows that MtbGmhB forms fully folded monomer with shape profile similar to its modeled structure. The circular dichroism analysis shows 38% α-helix, 15% ß-sheets and 47% random coil structures in MtbGmhB, similar to haloalkanoic acid dehalogenase (HAD) phosphohydrolase enzymes. The modeled MtbGmhB structure shows the catalytic site, which forms a concave, semicircular surface using the three loops around GMB substrate binding site. Dynamic simulation analysis on (i) Apo (ii) GMB bound (iii) GMBâ¯+â¯Mg2+ bound (iv) Zn2+ +GMBâ¯+â¯Mg2+ bound MtbGmhB structures show that Zn2+ as well as Mg2+ ions stabilize the loop conformation and trigger the changes in GMB substrate binding to active site of MtbGmhB. Upon demetallization, the large conformational changes occurred in ions binding loops, and leads to difference in GMB substrate binding to MtbGmhB. Our study provides information about structure and substrate binding of MtbGmhB, which may contribute in therapeutic development against M. tuberculosis.
Assuntos
Guanosina Difosfato/biossíntese , Heptoses/biossíntese , Mycobacterium tuberculosis/enzimologia , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X , Sequência de Aminoácidos , Apoenzimas/química , Apoenzimas/metabolismo , Domínio Catalítico , Magnésio/metabolismo , Simulação de Acoplamento Molecular , Zinco/metabolismoRESUMO
Metacaspases are clan CD cysteine peptidases found in plants, fungi and protozoa that possess a conserved Peptidase_C14 domain, homologous to the human caspases and a catalytic His/Cys dyad. Earlier reports have indicated the role of metacaspases in cell death; however, metacaspases of human malaria parasite remains poorly understood. In this study, we aimed to functionally characterize a novel malarial protease, P. falciparum metacaspase-3 (PfMCA3). Unlike other clan CD peptidases, PfMCA3 has an atypical active site serine (Ser1865) residue in place of canonical cysteine and it phylogenetically forms a distinct branch across the species. To investigate whether this domain retains catalytic activity, we expressed, purified and refolded the Peptidase_C14 domain of PfMCA3 which was found to express in all asexual stages. PfMCA3 exhibited trypsin-like serine protease activity with ser1865 acting as catalytic residue to cleave trypsin oligopeptide substrate. PfMCA3 is inhibited by trypsin-like serine protease inhibitors. Our study found that PfMCA3 enzymatic activity was abrogated when catalytic serine1865 (S1865A) was mutated. Moreover, PfMCA3 was found to be inactive against caspase substrate. Overall, our study characterizes a novel metacaspase of P. falciparum, different from human caspases and not responsible for the caspase-like activity, therefore, could be considered as a potential chemotherapeutic target.
Assuntos
Caspases/metabolismo , Plasmodium falciparum/enzimologia , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Biocatálise , Inibidores de Caspase/farmacologia , Caspases/química , Caspases/genética , Domínio Catalítico , Concentração de Íons de Hidrogênio , Cinética , Plasmodium falciparum/genética , TemperaturaRESUMO
ERG3 (ETS-related gene) is a member of the ETS (erythroblast transformation-specific) family of transcription factors, which contain a highly conserved DNA-binding domain. The ETS family of transcription factors differ in their binding to promoter DNA sequences, and the mechanism of their DNA-sequence discrimination is little known. In the current study, crystals of the ETSi domain (the ETS domain of ERG3 containing a CID motif) in space group P41212 and of its complex with the E74 DNA sequence (DNA9) in space group C2221 were obtained and their structures were determined. Comparative structure analysis of the ETSi domain and its complex with DNA9 with previously determined structures of the ERGi domain (the ETS domain of ERG containing inhibitory motifs) in space group P65212 and of the ERGi-DNA12 complex in space group P41212 were performed. The ETSi domain is observed as a homodimer in solution as well as in the crystallographic asymmetric unit. Superposition of the structure of the ETSi domain on that of the ERGi domain showed a major conformational change at the C-terminal DNA-binding autoinhibitory (CID) motif, while minor changes are observed in the loop regions of the ETSi-domain structure. The ETSi-DNA9 complex in space group C2221 forms a structure that is quite similar to that of the ERG-DNA12 complex in space group P41212. Upon superposition of the complexes, major conformational changes are observed at the 5' and 3' ends of DNA9, while the conformation of the core GGA nucleotides was quite conserved. Comparison of the ETSi-DNA9 structure with known structures of ETS class 1 protein-DNA complexes shows the similarities and differences in the promoter DNA binding and specificity of the class 1 ETS proteins.
Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Proteína Proto-Oncogênica c-fli-1/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Middle East respiratory syndrome (MERS) is caused by MERS coronavirus (MERS-CoV). Thus far, MERS outbreaks have been reported from Saudi Arabia (2013 and 2014) and South Korea (2015). No specific vaccine has yet been reported against MERS. PURPOSE: To address the urgent need for an MERS vaccine, in the present study, we have designed two multi-epitope vaccines (MEVs) against MERS utilizing several in silico methods and tools. METHODS: The design of both the multi-epitope vaccines (MEVs) are composed of cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) epitopes, screened form thirteen different proteins of MERS-CoV. Both the MEVs also carry potential B-cell linear epitope regions, B-cell discontinuous epitopes as well as interferon-γ-inducing epitopes. Human ß-defensin-2 and ß-defensin-3 were used as adjuvants to enhance the immune response of MEVs. To design the MEVs, short peptide molecular linkers were utilized to link screened most potential CTL epitopes, HTL epitopes and the adjuvants. Tertiary models for both the MEVs were generated, refined, and further studied for their molecular interaction with toll-like receptor 3. The cDNAs of both MEVs were generated and analyzed in silico for their expression in a mammalian host cell line (human). RESULTS: Screened CTL and HTL epitopes were found to have high propensity for stable molecular interaction with HLA alleles molecules. CTL epitopes were also found to have favorable molecular interaction within the cavity of transporter associated with antigen processing. The selected CTL and HTL epitopes jointly cover upto 94.0% of worldwide human population. Both the CTL and HTL MEVs molecular models have shown to have stable binding and complex formation propensity with toll-like receptor 3. The cDNA analysis of both the MEVs have shown high expression tendency in mammalian host cell line (human). CONCLUSION: After multistage in silico analysis, both the MEVs are predicted to elicit humoral as well as cell mediated immune response. Epitopes of the designed MEVs are predicted to cover large human population worldwide. Hence both the designed MEVs could be tried in vivo as potential vaccine candidates against MERS.
RESUMO
Earlier studies on Plasmodium apoptosis revealed the presence of proteases with caspases like- activity, which are known as "metacaspases". Although this family of cysteine proteases is structurally similar to caspases with Cys-His dyad but their evolutionary significance and functional relevance remains largely unknown. These proteases are considered to be an important target against malaria due to their absence in humans. In this report, we have biochemically characterized metacaspase-2 (PfMCA-2) of P.falciparum. Enzymatic assay showed that PfMCA-2 efficiently cleaved arginine/lysine specific peptide, but not caspase-specific substrate. Consistently, PfMCA-2 activity was sensitive to effector caspases inhibitor, Z-FA-FMK, and mildly inhibited by aprotinin and E-64. However, general caspase inhibitors such as Z-VAD-FMK and Z-DEVD-FMK had no effect on PfMCA-2 activity. Z-FA-FMK inhibits parasite growth with an IC50 value of 2.7⯵M along with the notable morphological changes. PfMCA-2 specifically expressed in schizonts and gametocyte stages and there was a notable depletion of PfMCA-2 expression in Z-FA-FMK treated schizonts and gametocytes stages of parasite. Notably, PfMCA-2 cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease) and the proteolysis of PfTSN did not occur after treatment with the Z-FA-FMK. The production of large amount of reactive oxygen species in presence of Z-FA-FMK caused oxidative stress which in turn leads to loss of cell viability. The oxidative stress further generates positive feedback for the occurrence of cell death in term of phosphatidylserine externalization and DNA fragmentation in vitro.
Assuntos
Cisteína Proteases/metabolismo , Plasmodium falciparum/enzimologia , Inibidores de Cisteína Proteinase/metabolismo , Dipeptídeos/metabolismo , Perfilação da Expressão Gênica , Cetonas/metabolismo , Leucina/análogos & derivados , Leucina/metabolismo , Plasmodium falciparum/crescimento & desenvolvimento , Especificidade por SubstratoRESUMO
Mycobacterium tuberculosis decaprenylphosphoryl-ß-D-ribose oxidase (MtbDprE1) acts in concert with decaprenylphosphoryl-ß-D-ribose 2-epimerase (MtbDprE2) and catalyzes the epimerization of DPR into DPA. DPA is the sole precursor for synthesis of arabinogalactan and lipoarabinomannan in the mycobacterial cell wall. MtbDprE1 is a unique antimalarial drug target and many covalent and non-covalent inhibitors against MtbDprE1 have been studied for their antituberculosis activities. In the current study, we have purified MtbDprE1 enzyme and synthesized six sulfur-rich 2-mercaptobenzothiazole and 1, 2, 3-triazole conjugated ligands and performed binding analysis with MtbDprE1. All ligands have shown competitive binding, as observed for other covalently and noncovalently bound MtbDprE1 inhibitors. Molecular docking analysis of six ligands with MtbDprE1 shows that they occupy the substrate binding pocket of MtbDprE1 and are stabilized by hydrogen bonds and van der Waals interactions. Our study shows that sulfur-rich 2-mercaptobenzothiazole ligands act as specific inhibitors against MtbDprE1 and could be used as antituberculosis agents.
Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Benzotiazóis/farmacologia , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/enzimologia , Triazóis/farmacologia , Oxirredutases do Álcool/metabolismo , Antituberculosos/metabolismo , Proteínas de Bactérias/metabolismo , Benzotiazóis/metabolismo , Ligação Competitiva , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Mycobacterium tuberculosis/efeitos dos fármacos , Triazóis/metabolismoRESUMO
The CarD protein is highly expressed in mycobacterial strains under basal conditions and is transcriptionally induced during multiple types of genotoxic stress and starvation. The CarD protein binds the ß subunit of RNA polymerase and influences gene expression. The disruption of interactions between CarD and the ß subunit of RNA polymerase has a significant effect on mycobacterial survival, resistance to stress and pathogenesis. To understand the structure of CarD and its interaction with the ß subunit of RNA polymerase, Mycobacterium tuberculosis CarD (MtbCarD) and the Thermus aquaticus RNA polymerase ß subunit were recombinantly expressed and purified. Secondary-structure analysis using circular-dichroism spectroscopy indicated that MtbCarD contains â¼ 60% α-helix, â¼ 7% ß-sheet and â¼ 33% random-coil structure. The C-terminal domain of MtbCarD (CarD(83-161)) was crystallized and its X-ray structure was determined at 2.1 Å resolution. CarD(83-161) forms a distorted Y-shaped structure containing bundles of three helices connected by a loop. The residues forming the distorted Y-shaped structure are highly conserved in CarD sequences from other mycobacterial species. Comparison of the CarD(83-161) structure with the recently determined full-length M. tuberculosis and T. thermophilus CarD crystal structures revealed structural differences in residues 141-161 of the C-terminal domain of the CarD(83-161) structure. The structural changes in the CarD(83-161) structure occurred owing to proteolysis and crystallization artifacts.
Assuntos
Proteínas de Bactérias/química , Mycobacterium tuberculosis/química , RNA Ribossômico/genética , Transcrição Gênica/fisiologia , Proteínas de Bactérias/fisiologia , Dicroísmo Circular , Cristalização , Cristalografia por Raios X , Primers do DNA , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Espectrofotometria Ultravioleta , Thermus thermophilus/químicaRESUMO
The Mycobacterium tuberculosis ESX-1 secreted protein regulator (EspR, Rv3849) is the key protein that delivers bacterial proteins into the host cell during mycobacterial infection. EspR binds directly to the espACD operon and is involved in transcriptional activation. In the current study, M. tuberculosis EspR has been crystallized and its X-ray structure has been determined at 3.3â Å resolution in a P3221 crystal form. EspR forms a physiological dimer in the crystal. Each EspR monomer contains an N-terminal helix-turn-helix DNA-binding domain and a C-terminal dimerization domain. The EspR structure in the P3221 crystal form was compared with previously determined EspR structures in P32, P21 and P212121 crystal forms. Structural comparison analysis indicated that the N-terminal helix-turn-helix domain of EspR acquires a rigid structure in the four crystal forms. However, significant structural differences were observed in the C-terminal domain of EspR in the P21 crystal form when compared with the P3221 and P32 crystal forms. The interaction, stabilization energy and buried surface area analysis of EspR in the four different crystal forms have provided information about the physiological dimer interface of EspR.