RESUMO
NLRP3 inflammasome is involved in several chronic inflammatory diseases. The inflammatory effect of the NLRP3 inflammasome is executed through IL-1ß and IL-18. Therefore, IL-1ß is one of the primary targets in chronic inflammatory conditions. However, current treatment regimens are dependent on anti- IL-1ß biologicals. The therapies targeting IL-1ß through inhibition of NLRP3 inflammasome are thus being actively explored. We identified safranal, a small molecule responsible for the essence of saffron as a potential inhibitor of the NLRP3 inflammasome. Safranal significantly suppressed the release of IL-1ß from ATP stimulated J774A.1 and bone marrow-derived macrophages (BMDMs) by regulating CASP1 and CASP8 dependent cleavage of pro-IL-1ß. Safranal markedly suppressed the expression of NLRP3 and its ATPase activity. Safranal treatment enhanced the expression of NRF2, whereas, si-RNA mediated silencing of Nrf2 abrogated the anti-NLRP3 effect of safranal. Furthermore, safranal inhibited ASC oligomerization and formation of ASC specks. Safranal also displayed anti-NLRP3 activity in multiple mice models. Treatment of animals with safranal reduced the production of IL-1ß in ATP elicited peritoneal inflammation, MSU induced air pouch inflammation, and MSU injected foot paw edema in mice. Thus, our data projects safranal as a potential preclinical drug candidate against NLRP3 inflammasome triggered chronic inflammation.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/antagonistas & inibidores , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Cicloexenos/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Terpenos/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Cicloexenos/uso terapêutico , Relação Dose-Resposta a Droga , Feminino , Humanos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Terpenos/uso terapêuticoRESUMO
The HIV transactivator protein, Tat, enhances HIV transcription by recruiting P-TEFb from the inactive 7SK snRNP complex and directing it to proviral elongation complexes. To test the hypothesis that T-cell receptor (TCR) signaling induces critical post-translational modifications leading to enhanced interactions between P-TEFb and Tat, we employed affinity purification-tandem mass spectrometry to analyze P-TEFb. TCR or phorbal ester (PMA) signaling strongly induced phosphorylation of the CDK9 kinase at Ser175. Molecular modeling studies based on the Tat/P-TEFb X-ray structure suggested that pSer175 strengthens the intermolecular interactions between CDK9 and Tat. Mutations in Ser175 confirm that this residue could mediate critical interactions with Tat and with the bromodomain protein BRD4. The S175A mutation reduced CDK9 interactions with Tat by an average of 1.7-fold, but also completely blocked CDK9 association with BRD4. The phosphomimetic S175D mutation modestly enhanced Tat association with CDK9 while causing a 2-fold disruption in BRD4 association with CDK9. Since BRD4 is unable to compete for binding to CDK9 carrying S175A, expression of CDK9 carrying the S175A mutation in latently infected cells resulted in a robust Tat-dependent reactivation of the provirus. Similarly, the stable knockdown of BRD4 led to a strong enhancement of proviral expression. Immunoprecipitation experiments show that CDK9 phosphorylated at Ser175 is excluded from the 7SK RNP complex. Immunofluorescence and flow cytometry studies carried out using a phospho-Ser175-specific antibody demonstrated that Ser175 phosphorylation occurs during TCR activation of primary resting memory CD4+ T cells together with upregulation of the Cyclin T1 regulatory subunit of P-TEFb, and Thr186 phosphorylation of CDK9. We conclude that the phosphorylation of CDK9 at Ser175 plays a critical role in altering the competitive binding of Tat and BRD4 to P-TEFb and provides an informative molecular marker for the identification of the transcriptionally active form of P-TEFb.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Infecções por HIV/metabolismo , HIV/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Provírus/metabolismo , Transcrição Gênica , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/virologia , Proteínas de Ciclo Celular , Ciclina T/genética , Ciclina T/metabolismo , Quinase 9 Dependente de Ciclina/genética , Feminino , HIV/genética , Infecções por HIV/genética , Humanos , Masculino , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação/genética , Fator B de Elongação Transcricional Positiva/genética , Ligação Proteica/genética , Provírus/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Protein synthesis requires accurate charging of tRNA with cognate amino acid as catalyzed by aminoacyl-tRNA synthetases. Crystal structures of tyrosyl-tRNA synthetase (YRSs) show remarkably diverse conformations for the KMSKS loop, hitherto classified as "open" and "closed". This traditional classification implied that the KMSKS loop adopts different conformations depending on occupancy of active site pocket. Our structural analyses of evolutionarily derived ensemble of differentially ligated YRSs using quantitative structural criterion demonstrate intrinsic conformational heterogeneity in KMSKS loop that is independent of occupancy of active site. Differential centroid distance analyses between KMSKS motif and Rossmann fold domain reveal an intriguing bimodal distribution. These insights have been used for a more consistent re-classification of YRS conformations as either compact or extended. Our data not only reflect inherent dynamics within the conformational landscape of KMSKS loops, but also have implications for structure-based drug design efforts.
Assuntos
Modelos Moleculares , Tirosina-tRNA Ligase/química , Sítios de Ligação , Cristalografia por Raios X , Bases de Dados de Proteínas , Conformação Proteica , Tirosina-tRNA Ligase/metabolismoRESUMO
BACKGROUND: Some species of fungi can cause serious human diseases, particularly to immuno-compromised individuals. Opportunistic fungal infections are a leading cause of mortality, and present an emerging challenge that requires development of new and effective therapeutics. Aminoacyl-tRNA synthetases (aaRSs) are indispensable components of cellular protein translation machinery and can be targeted for discovery of novel anti-fungal agents. RESULTS: Validation of aaRSs as potential drug targets in pathogenic microbes prompted us to investigate the genomic distribution of aaRSs within three fungi that infect humans - A. niger, C. albicans and C. neoformans. Hidden Markov Models were built for aaRSs and related proteins to search for homologues in these fungal genomes. Here, we provide a detailed and comprehensive annotation for 3 fungal genome aaRSs and their associated proteins. We delineate predicted localizations, subdomain architectures and prevalence of unusual motifs within these aaRSs. Several fungal aaRSs have unique domain appendages of unknown function e.g. A. niger AsxRS and C. neoformans TyrRS have additional domains that are absent from human homologs. CONCLUSIONS: Detailed comparisons of fungal aaRSs with human homologs suggest key differences that could be exploited for specific drug targeting. Our cataloging and structural analyses provide a comprehensive foundation for experimentally dissecting fungal aaRSs that may enable development of new anti-fungal agents.
Assuntos
Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Aspergillus niger/genética , Candida albicans/genética , Cryptococcus neoformans/genética , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/metabolismo , Aspergillus niger/metabolismo , Candida albicans/metabolismo , Biologia Computacional , Cryptococcus neoformans/metabolismo , Conjuntos de Dados como Assunto , Genoma Fúngico , Humanos , Cadeias de Markov , Modelos Moleculares , Dados de Sequência Molecular , Fases de Leitura Aberta , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Transporte Proteico , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Mutation(s) in proteins are a natural byproduct of evolution but can also cause serious diseases. Aminoacyl-tRNA synthetases (aaRSs) are indispensable components of all cellular protein translational machineries, and in humans they drive translation in both cytoplasm and mitochondria. Mutations in aaRSs have been implicated in a plethora of diseases including neurological conditions, metabolic disorders and cancer. RESULTS: We have developed an algorithmic approach for genome-wide analyses of sequence substitutions that combines evolutionary, structural and functional information. This pipeline enabled us to super-annotate human aaRS mutations and analyze their linkage to health disorders. Our data suggest that in some but not all cases, aaRS mutations occur in functional and structural sectors where they can manifest their pathological effects by altering enzyme activity or causing structural instability. Further, mutations appear in both solvent exposed and buried regions of aaRSs indicating that these alterations could lead to dysfunctional enzymes resulting in abnormal protein translation routines by affecting inter-molecular interactions or by disruption of non-bonded interactions. Overall, the prevalence of mutations is much higher in mitochondrial aaRSs, and the two most often mutated aaRSs are mitochondrial glutamyl-tRNA synthetase and dual localized glycyl-tRNA synthetase. Out of 63 mutations annotated in this work, only 12 (~20%) were observed in regions that could directly affect aminoacylation activity via either binding to ATP/amino-acid, tRNA or by involvement in dimerization. Mutations in structural cores or at potential biomolecular interfaces account for ~55% mutations while remaining mutations (~25%) remain structurally un-annotated. CONCLUSION: This work provides a comprehensive structural framework within which most defective human aaRSs have been structurally analyzed. The methodology described here could be employed to annotate mutations in other protein families in a high-throughput manner.
Assuntos
Aminoacil-tRNA Sintetases/genética , Doença/genética , Evolução Molecular , Anotação de Sequência Molecular , Mutação , Algoritmos , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/química , Citoplasma/enzimologia , Genômica , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação ProteicaRESUMO
Glutathione is a thiol-containing tripeptide that plays important roles in redox-related processes. The first step in glutathione biosynthesis is catalysed by γ-GCS (γ-glutamylcysteine synthetase). The crystal structure of Escherichia coli γ-GCS has revealed the presence of a disulfide bond. As the disulfide-bonding cysteine residues Cys372 and Cys395 are not well conserved among γ-GCS enzymes in this lineage, we have initiated a biochemical genetic strategy to investigate the functional importance of these and other cysteine residues. In a cysteine-free γ-GCS that was non-functional, suppressor analysis yielded combinations of cysteine and aromatic residues at the position of the disulfide bond, and one mutant that lacked any cysteine residues. Kinetic analysis of the wild-type and mutant enzymes revealed that the disulfide bond was not involved in determining the affinity of the enzyme towards its substrate, but had an important role in determining the stability of the protein, and its catalytic efficiency. We show that in vivo the γ-GCS enzyme can also exist in a reduced form and that the mutants lacking the disulfide bond show a decreased half-life. These results demonstrate a novel means of regulation of γ-GCS by the redox environment that works by an alteration in its stability.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Glutamato-Cisteína Ligase/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Domínio Catalítico/genética , Sequência Conservada , Cristalografia por Raios X , Cisteína/química , DNA Bacteriano/genética , Estabilidade Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Genes Bacterianos , Teste de Complementação Genética , Glutamato-Cisteína Ligase/química , Glutamato-Cisteína Ligase/genética , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxirredução , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
To examine the global function of the key surface-exposed loops of streptokinase, bearing substrate-specific exosites, namely, the 88-97 loop in the α domain, the 170 loop in the ß domain, and the coiled-coil region (Leu321-Asn338) in the γ domain, mutagenic as well as peptide inhibition studies were carried out. Peptides corresponded to the primary structure of an exosite, either individual or stoichiometric mixtures of various disulfide-constrained synthetic peptide(s) inhibited plasminogen activation by streptokinase. Remarkably, pronounced inhibition of substrate plasminogen activation by the preformed streptokinase-plasmin activator complex was observed when complementary mixtures of different peptides were used compared to the same overall concentrations of individual peptides, suggesting co-operative interactions between the exosites. This observation was confirmed with streptokinase variants mutated at one, two, or three sites simultaneously. The single/double/triple exosite mutants of streptokinase showed a nonadditive, synergistic decline in kcat for substrate plasminogen activation in the order single > double > triple exosite mutant. Under the same conditions, zymogen activation by the various mutants remained essentially native- like in terms of nonproteolytic activation of partner plasminogen. Multisite mutants also retain affinity to form 1:1 stoichiometric activator complexes with plasmin when probed through sensitive equilibrium fluorescence studies. Thus, the present results strongly support a model of streptokinase action, wherein catalysis by the streptokinase-plasmin complex operates through a distributed network of substrate-interacting exosites resident across all three domains of the cofactor protein.
Assuntos
Proteínas de Bactérias/química , Plasminogênio/química , Streptococcus/enzimologia , Estreptoquinase/química , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Biocatálise , Domínio Catalítico , Ativação Enzimática , Humanos , Cinética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estreptoquinase/genéticaRESUMO
Because of its natural ability to utilize both xylose and arabinose, the halotolerant and osmotolerant yeast Debaryomyces hansenii is considered as a potential microbial platform for exploiting lignocellulosic biomass. To gain better understanding of the xylose metabolism in D. hansenii, we have cloned and characterized a xylitol dehydrogenase gene (DhXDH). The cloned gene appeared to be essential for xylose metabolism in D. hansenii as the deletion of this gene abolished the growth of the cells on xylose. The expression of DhXDH was strongly upregulated in the presence of xylose. Recombinant DhXdhp was expressed and purified from Escherichia coli. DhXdhp was highly active against xylitol and sorbitol as substrate. Our results showed that DhXdhp was thermo-sensitive, and except this, its biochemical properties were quite comparable with XDH from other yeast species. Furthermore, to make this enzyme suitable for metabolic engineering of D. hansenii, we have improved its thermotolerance and modified cofactor requirement through modelling and mutagenesis approach.
Assuntos
Clonagem Molecular , D-Xilulose Redutase/química , D-Xilulose Redutase/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Saccharomycetales/enzimologia , Sequência de Aminoácidos , D-Xilulose Redutase/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Saccharomycetales/química , Saccharomycetales/genética , Alinhamento de Sequência , Xilose/metabolismoRESUMO
Mutations in the insulin gene can impair proinsulin folding and cause diabetes mellitus. Although crystal structures of insulin dimers and hexamers are well established, proinsulin is refractory to crystallization. Although an NMR structure of an engineered proinsulin monomer has been reported, structures of the wild-type monomer and hexamer remain undetermined. We have utilized hydroxyl radical footprinting and molecular modeling to characterize these structures. Differences between the footprints of insulin and proinsulin, defining a "shadow" of the connecting (C) domain, were employed to refine the model. Our results demonstrate that in its monomeric form, (i) proinsulin contains a native-like insulin moiety and (ii) the C-domain footprint resides within an adjoining segment (residues B23-B29) that is accessible to modification in insulin but not proinsulin. Corresponding oxidation rates were observed within core insulin moieties of insulin and proinsulin hexamers, suggesting that the proinsulin hexamer retains an A/B structure similar to that of insulin. Further similarities in rates of oxidation between the respective C-domains of proinsulin monomers and hexamers suggest that this loop in each case flexibly projects from an outer surface. Although dimerization or hexamer assembly would not be impaired, an ensemble of predicted C-domain positions would block hexamer-hexamer stacking as visualized in classical crystal lattices. We anticipate that protein footprinting in combination with modeling, as illustrated here, will enable comparative studies of diabetes-associated mutant proinsulins and their aberrant modes of aggregation.
Assuntos
Mutação , Proinsulina/química , Proinsulina/genética , Biologia Computacional/métodos , Cristalografia por Raios X/métodos , Diabetes Mellitus/metabolismo , Dimerização , Radicais Livres , Humanos , Insulina/química , Espectrometria de Massas/métodos , Modelos Moleculares , Peptídeos/química , Conformação Proteica , Estrutura Terciária de Proteína , Software , Solventes/química , SíncrotronsRESUMO
To identify new structure-function correlations in the γ domain of streptokinase, mutants were generated by error-prone random mutagenesis of the γ domain and its adjoining region in the ß domain followed by functional screening specifically for substrate plasminogen activation. Single-site mutants derived from various multipoint mutation clusters identified the importance of discrete residues in the γ domain that are important for substrate processing. Among the various residues, aspartate at position 328 was identified as critical for substrate human plasminogen activation through extensive mutagenesis of its side chain, namely D328R, D328H, D328N, and D328A. Other mutants found to be important in substrate plasminogen activation were, namely, R319H, N339S, K334A, K334E, and L335Q. When examined for their 1:1 interaction with human plasmin, these mutants were found to retain the native-like high affinity for plasmin and also to generate amidolytic activity with partner plasminogen in a manner similar to wild type streptokinase. Moreover, cofactor activities of the mutants precomplexed with plasmin against microplasminogen as the substrate as well as in silico modeling studies suggested that the region 315-340 of the γ domain interacts with the serine protease domain of the macromolecular substrate. Overall, our results identify the presence of a substrate specific exosite in the γ domain of streptokinase.
Assuntos
Proteínas de Bactérias/química , Fibrinolíticos/química , Modelos Moleculares , Streptococcus/enzimologia , Estreptoquinase/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fibrinolíticos/metabolismo , Humanos , Mutagênese , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Streptococcus/genética , Estreptoquinase/genética , Estreptoquinase/metabolismo , Relação Estrutura-AtividadeRESUMO
Aminoacyl-tRNA synthetases are an indispensable component of ribosomal protein translational machinery and Plasmodium Tyrosyl-tRNA synthetase (PfTyrRS) is a validated drug target. This manuscript illustrates the dynamic conformational landscape of PfTyrRS in the context of substrate binding. Molecular dynamics simulations of PfTyrRS in the presence and absence of ligand show conformational heterogeneity for both the protein and the bound ligand. Diverse conformations for the evolutionarily conserved ATP binding motif (KMSKS) have been observed in both apo- and holo PfTyrRS. Further, the presented attributes of the tyrosyl-adenylate conformational sub-states in situ along with their implications on the strength of intermolecular interactions would be a pertinent benchmark for molecular design studies. In addition, an analysis of the ligand hydration pattern foregrounds the structurally conserved water-mediated inter-molecular interactions. The quantitative assessment of the conformational landscape, based on the fluctuations of the distance between the ligand binding pockets, of apo-PfTyrRS and holo-PfTyrRS highlights the nature of diversity in conformational sampling for the two cases. Evidently, the holo-PfTyrRS adopts a rather compact conformation compared to the apo-PfTyrRS. An intriguing asymmetry in the dynamics of the two monomers is contextualized with the functional asymmetry of the symmetrically dimeric PfTyrRS. Importantly, the network of non-bonded contacts in the apo- and holo- simulated ensembles has been analyzed. The graph-theoretic analysis-based novel insights concerning the nature of information flow as a function of ligation state would prove valuable in understanding PfTyrRS functions. The results presented here contend that understanding allostery in PfTyrRS is essential to astutely design structure-based inhibitors.
Assuntos
Plasmodium/enzimologia , Polimorfismo Genético/genética , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/genética , Biologia Computacional , Conformação Proteica , Especificidade por Substrato , Tirosina-tRNA Ligase/metabolismoRESUMO
With the goal of identifying hitherto unknown surface exosites of streptokinase involved in substrate human plasminogen recognition and catalytic turnover, synthetic peptides encompassing the 170 loop (CQFTPLNPDDDFRPGLKDTKLLC) in the beta-domain were tested for selective inhibition of substrate human plasminogen activation by the streptokinase-plasmin activator complex. Although a disulfide-constrained peptide exhibited strong inhibition, a linear peptide with the same sequence, or a disulfide-constrained variant with a single lysine to alanine mutation showed significantly reduced capabilities of inhibition. Alanine-scanning mutagenesis of the 170 loop of the beta-domain of streptokinase was then performed to elucidate its importance in streptokinase-mediated plasminogen activation. Some of the 170 loop mutants showed a remarkable decline in k(cat) without any alteration in apparent substrate affinity (K(m)) as compared with wild-type streptokinase and identified the importance of Lys(180) as well as Pro(177) in the functioning of this loop. Remarkably, these mutants were able to generate amidolytic activity and non-proteolytic activation in "partner" plasminogen as wild-type streptokinase. Moreover, cofactor activities of the 170 loop mutants, pre-complexed with plasmin, against microplasminogen as the substrate showed a similar pattern of decline in k(cat) as that observed in the case of full-length plasminogen, with no concomitant change in K(m). These results strongly suggest that the 170 loop of the beta-domain of streptokinase is important for catalysis by the streptokinase-plasmin(ogen) activator complex, particularly in catalytic processing/turnover of substrate, although it does not seem to contribute significantly toward enzyme-substrate affinity per se.
Assuntos
Plasminogênio/metabolismo , Estreptoquinase/metabolismo , Alanina/química , Sequência de Aminoácidos , Domínio Catalítico , Relação Dose-Resposta a Droga , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese , Mutação , Peptídeos/química , Plasminogênio/química , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Pichia angusta (syn. Hansenula polymorpha) represents one of the rare yeast that can grow and ferment both xylose and glucose at higher temperature (50°C). However, little is known about the enzymes involved in xylose utilization from this species. Previous studies indicated the presence of one xylose reductase and two xylitol dehydrogenase genes in P. angusta. In this study, we have expressed both xylitol dehydrogenases (PaXdh1p and PaXdh2p) in Escherichia coli and purified them as 6X-Histidine-tagged proteins. Biochemical characterization of the recombinant proteins reveals that both PaXdh1p and PaXdh2p are thermotolerant enzymes. PaXdh2p contains a catalytic and a structural Zn atom. However, the structural Zn atom is not present in PaXdh1p. Both enzymes also differ in their affinity for the substrate as well as in the catalytic efficiency. Through mutagenesis and modeling approaches, we have also identified residues important for catalysis and substrate binding.
Assuntos
D-Xilulose Redutase/genética , D-Xilulose Redutase/metabolismo , Pichia/enzimologia , Sequência de Aminoácidos , Cromatografia de Afinidade , Clonagem Molecular , Coenzimas/metabolismo , D-Xilulose Redutase/química , Análise Mutacional de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Estabilidade Enzimática , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Pichia/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade por Substrato , Temperatura , Zinco/metabolismoRESUMO
Malaria remains the leading cause of deaths globally, despite significant advancement towards understanding its epidemiology and availability of multiple therapeutic interventions. Poor efficacy of the approved vaccine, and the rapid emergence of antimalarial drug resistance, warrants an urgent need to expedite the process of development of new lead molecules targeting malaria. Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes crucial for ribosomal protein synthesis and are valid antimalarial targets. This study explores the prospects of (re-)positioning the repertoire of approved drugs and natural products as potential malarial aaRS inhibitors. Molecular docking of these two sets of small-molecules to lysyl-, prolyl-, and tyrosyl- synthetases from Plasmodium followed by a comparison of the top-ranking docked compounds against human homologs facilitated identification of promising molecular scaffolds. Raltitrexed and Cefprozil, an anticancer drug and an antibiotic, respectively, showed stronger binding to Plasmodium aaRSs compared to human homologs with > 4 kcal/mol difference in the docking scores. Similarly, a difference of ~ 3 kcal/mol in Glide scores was observed for docked Calcipotriol, a drug used for psoriasis treatment, against the two lysyl-tRNA synthetases. Natural products such as Dihydroxanthohumol and Betmidin, having aromatic rings as a substructure, showed preferential docking to the purine binding pocket in Plasmodium tyrosyl-tRNA synthetase as evident from the calculated change in binding free energies. We present detailed analyses of the calculated intermolecular interaction for all top-scoring docked poses. Overall, this study provides a compelling foundation to design and develop specific antimalarials.
RESUMO
The role of a prominent surface-exposed loop (residues 88-97) in the alpha domain of streptokinase (SK), in human plasminogen (HPG) activation was explored through its selective mutagenesis and deletion studies. We first made a conformationally constrained derivative of the loop by the substitution of sequences known to possess a strong propensity for beta-turn formation. The mutant so formed (termed SK88-97-Beta Turn), when tested for co-factor activity against substrate HPG, after first forming a 1:1 molar complex with human plasmin (HPN), showed a nearly 6-fold decreased co-factor activity compared to the wild-type, native SK. The major catalytic change was observed to be at the k(cat) level, with relatively minor changes in Km values against HPG. Real-time binary interaction (i.e. the 1:1 complexation between SK, or its mutant/s, with HPG), and ternary complexation studies (i.e. the docking of a substrate HPG molecule into the preformed SK-HPG complex) using Surface Plasmon Resonance were done. These studies revealed minor alterations in binary complex formation but the ternary interactions of the substitution and/or deletion mutants were found to be decreased for full-length HPG compared to that for native SK.HPG. In contrast, their ternary interactions with the isolated five-kringle domain unit of plasminogen (K1-5) showed Kd values comparable to that seen with the native SK.HPG complex. Taking into consideration the overall alterations observed in catalytic levels after site-specific mutagenesis and complete loop deletion of the 88-97 loop, on the one hand, and its known position at the SK-HPG interface in the binary complex, suggests the importance of this loop. The present results suggest that the 88-97 loop of the alpha domain of SK contributes towards catalytic turn-over, even though its individual contribution towards enzyme-substrate affinity per se is minimal.
Assuntos
Plasminogênio/química , Plasminogênio/metabolismo , Estreptoquinase/química , Estreptoquinase/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Domínio Catalítico/genética , Primers do DNA/genética , Ativação Enzimática , Humanos , Técnicas In Vitro , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Estreptoquinase/genética , TermodinâmicaRESUMO
Using several tens of rationally-selected substitutions, insertions and deletions of predominantly non-contiguous residues, we have remodeled the solvent-exposed face of a beta sheet functioning as the substrate-binding and catalytically-active groove of a thermophile cellulase (Rhodothermus marinus Cel12A) to cause it to resemble, both in its structure and function, the equivalent groove of a mesophile homolog (Trichoderma reesei Cel12A). The engineered protein, a mesoactive-thermostable cellulase (MT Cel12A) displays the temperature of optimal function of its mesophile ancestor and the temperature of melting of its thermophile ancestor, suggesting that such 'grafting' of a mesophile-derived surface onto a thermophile-derived structural scaffold can potentially help generate novel enzymes that recombine structural and functional features of homologous proteins sourced from different domains of life.
Assuntos
Celulases/química , Engenharia de Proteínas/métodos , Rhodothermus/enzimologia , Homologia Estrutural de Proteína , Temperatura , Trichoderma/enzimologia , Sequência de Aminoácidos , Domínio Catalítico/genética , Celulases/genética , Celulases/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Desnaturação Proteica/genética , Dobramento de Proteína , Estrutura Secundária de Proteína , Rhodothermus/genética , Rhodothermus/metabolismo , Propriedades de Superfície , Termodinâmica , Temperatura de Transição , Trichoderma/genética , Trichoderma/metabolismoRESUMO
Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.
Assuntos
Antineoplásicos/farmacologia , Ativadores de Enzimas/farmacologia , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática , Ativadores de Enzimas/química , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos Transgênicos , Ligação Proteica , Proteína Fosfatase 2/química , Transdução de Sinais , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0162956.].
RESUMO
Methylation of DNA and histone proteins are mutually involved in the epigenetic regulation of gene expression mediated by DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs). DNMTs methylate cytosine residues within gene promoters, whereas HMTs catalyze the transfer of methyl groups to lysine and arginine residues of histone proteins, thus causing chromatin condensation and transcriptional repression, which play an important role in the pathogenesis of cancer. The potential reversibility of epigenetic alterations has encouraged the development of dual pharmacologic inhibitors of DNA and histone methylation as anticancer therapeutics. Dietary flavones can affect epigenetic modifications that accumulate over time and have shown anticancer properties, which are undefined. Through DNA binding and in silico protein-ligand docking studies with plant flavones viz. Apigenin, Chrysin and Luteolin, the effect of flavones on DNA and histone methylation was assessed. Spectroscopic analysis of flavones with calf-thymus DNA revealed intercalation as the dominant binding mode, with specific binding to a GC-rich sequence in the DNA duplex. A virtual screening approach using a model of the catalytic site of DNMT and EZH2 demonstrated that plant flavones are tethered at both ends inside the catalytic pocket of DNMT and EZH2 by means of hydrogen bonding. Epigenetic studies performed with flavones exhibited a decrease in DNMT enzyme activity and a reversal of the hypermethylation of cytosine bases in the DNA and prevented cytosine methylation in the GC-rich promoter sequence incubated with the M.SssI enzyme. Furthermore, a marked decrease in HMT activity and a decrease in EZH2 protein expression and trimethylation of H3K27 were noted in histones isolated from cancer cells treated with plant flavones. Our results suggest that dietary flavones can alter DNMT and HMT activities and the methylation of DNA and histone proteins that regulate epigenetic modifications, thus providing a significant anticancer effect by altering epigenetic processes involved in the development of cancer.
RESUMO
Despite a significant drop in malaria deaths during the past decade, malaria continues to be one of the biggest health problems around the globe. WD40 repeats (WDRs) containing proteins comprise one of the largest and functionally diverse protein superfamily in eukaryotes, acting as scaffolds for assembling large protein complexes. In the present study, we report an extensive in silico analysis of the WDR gene family in human malaria parasite Plasmodium falciparum. Our genome-wide identification has revealed 80 putative WDR genes in P. falciparum (PfWDRs). Five distinct domain compositions were discovered in Plasmodium as compared to the human host. Notably, 31 PfWDRs were annotated/re-annotated on the basis of their orthologs in other species. Interestingly, most PfWDRs were larger as compared to their human homologs highlighting the presence of parasite-specific insertions. Fifteen PfWDRs appeared specific to the Plasmodium with no assigned orthologs. Expression profiling of PfWDRs revealed a mixture of linear and nonlinear relationships between transcriptome and proteome, and only nine PfWDRs were found to be stage-specific. Homology modeling identified conservation of major binding sites in PfCAF-1 and PfRACK. Protein-protein interaction network analyses suggested that PfWDRs are highly connected proteins with ~1928 potential interactions, supporting their role as hubs in cellular networks. The present study highlights the roles and relevance of the WDR family in P. falciparum, and identifies unique features that lay a foundation for further experimental dissection of PfWDRs.