RESUMO
BACKGROUND: The most fatal form of Visceral leishmaniasis or kala-azar is caused by the intracellular protozoan parasite Leishmania donovani. The life cycle and the infection pathway of the parasite are regulated by the small GTPase family of Rab proteins. The involvement of Rab proteins in neurodegenerative amyloidosis is implicated in protein misfolding, secretion abnormalities and dysregulation. The inter and intra-cellular shuttlings of Rab proteins are proposed to be aggregation-prone. However, the biophysical unfolding and aggregation of protozoan Rab proteins is limited. Understanding the aggregation mechanisms of Rab protein will determine their physical impact on the disease pathogenesis and individual health. OBJECTIVE: This work investigates the acidic pH-induced unfolding and aggregation of a recombinant Rab2 protein from L. donovani (rLdRab2) using multi-spectroscopic probes. METHODS: The acidic unfolding of rLdRab2 is characterised by intrinsic fluorescence and ANS assay, while aggregation is determined by Thioflavin-T and 90° light scattering assay. Circular dichroism determined the secondary structure of monomers and aggregates. The aggregate morphology was imaged by transmission electron microscopy. RESULTS: rLdRab2 was modelled to be a Rab2 isoform with loose globular packing. The acidinduced unfolding of the protein is a plausible non-two-state process. At pH 2.0, a partially folded intermediate (PFI) state characterised by ~ 30% structural loss and exposed hydrophobic core was found to accumulate. The PFI state slowly converted into well-developed protofibrils at high protein concentrations demonstrating its amyloidogenic nature. The native state of the protein was also observed to be aggregation-prone at high protein concentrations. However, it formed amorphous aggregation instead of fibrils. CONCLUSION: To our knowledge, this is the first study to report in vitro amyloid-like behaviour of Rab proteins in L donovani. This study provides a novel opportunity to understand the complete biophysical characteristics of Rab2 protein of the lower eukaryote, L. donovani.
Assuntos
Amiloide , Leishmania donovani , Proteínas Recombinantes , Proteína rab2 de Ligação ao GTP , Leishmania donovani/metabolismo , Leishmania donovani/química , Leishmania donovani/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Amiloide/química , Amiloide/metabolismo , Proteína rab2 de Ligação ao GTP/metabolismo , Proteína rab2 de Ligação ao GTP/química , Proteína rab2 de Ligação ao GTP/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Concentração de Íons de Hidrogênio , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Agregados Proteicos , Dicroísmo CircularRESUMO
INTRODUCTION: Visceral leishmaniasis (VL) is caused by Leishmania donovani. The purine and pyrimidine pathways are essential for L. donovani. Simultaneously inhibiting multiple targets could be an effective strategy to eliminate the pathogen and treat VL. OBJECTIVE: We aimed to target the essential enzymes of L. donovani and inhibit them using a multi-target approach. MATERIALS AND METHODS: A systematic analytical method was followed, in which first reported inhibitors of two essential enzymes (adenine phosphoribosyl-transferase [APRT] and dihydroorotate dehydrogenase [DHODH]) were collected and then ADMET and PASS analyses were conducted using the Lipinski rule and Veber's rule. Additionally, molecular docking between screened ligands and proteins were performed. The stability of complexes was analyzed using molecular dynamics (MD) simulations and MMPBSA analysis. RESULTS: Initially, 6,220 unique molecules were collected from the PubChem database, and then the Lipinski rule and Veber's rule were used for screening. In total, 203 compounds passed the ADMET test; their antileishmanial properties were tested by PASS analysis. As a result, 15 ligands were identified. Molecular docking simulations between APRT or DHODH and these 15 ligands were performed. Four molecules were found to be plant-derived compounds. Lig_2 and Lig_3 had good docking scores with both proteins. MD simulations were performed to determine the dynamic behavior and binding patterns of complexes. Both MD simulations and MMPBSA analysis showed Lig_3 is a promising antileishmanial inhibitor of both targets. CONCLUSION: Promising plant-derived compounds that might be used to combat VL were obtained through a multi-target approach.
Assuntos
Leishmania donovani , Leishmaniose Visceral , Leishmania donovani/química , Simulação de Dinâmica Molecular , Simulação de Acoplamento Molecular , Di-Hidro-Orotato Desidrogenase , Leishmaniose Visceral/prevenção & controle , Compostos Fitoquímicos/farmacologiaRESUMO
Visceral leishmaniasis (VL, also known as kala-azar) is a vector borne disease caused by obligate intracellular protozoan parasite Leishmania donovani. To overcome the limitations of currently available drugs for VL, molecular target-based study is a promising tool to develop new drugs to treat this neglected tropical disease. One such target we recently identified from L. donovani (Ld) genome (WGS, clinical Indian isolate; BHU 1220, AVPQ01000001) is a small GTP-binding protein, Rab6 protein. We now report a specific inhibitor of the GTPase activity of Rab6 protein of L. donovani (LdRab6) without restricting host enzyme activity. First, to understand the nature of LdRab6 protein, we generated recombinant LdRab6 mutant proteins (rLdRab6) by systematically introducing deletion (two cysteine residues at C-terminal) and mutations [single amino acid substitutions in the conserved region of GTP (Q84L)/GDP(T38N) coding sequence]. The GTPase activity of rLdRab6:GTP and rLdRab6:GDP locked mutant proteins showed ~ 8-fold and ~ 1.5-fold decreases in enzyme activity, respectively, compared to the wild type enzyme activity. The mutant protein rLdRab6:ΔC inhibited the GTPase activity. Sequence alignment analysis of Rab6 protein of L. donovani with Homo sapiens showed identical amino acids in the G conserved region (GTP/GDP-binding sites) but it differed in the C-terminal region. We then evaluated the inhibitory activity of trans-dibenzalacetone (DBA, a synthetic analog of curcumin with strong antileishmanial activity reported earlier by us) in the GTPase activity of LdRab6 protein. Comparative molecular docking analysis of DBA and specific inhibitors of Rab proteins (Lovastatin, BFA, Zoledronate, and NE10790) indicated that DBA had optimum binding affinity with LdRab6 protein. This was further confirmed by the GTPase activity of DBA-treated LdRab6 which showed a basal GTP level significantly lower than that of the wild-type rLdRab6. The results confirm that DBA inhibits the GTPase activity of LdRab6 protein from L. donovani (LdRab6), a potential target for its antileishmanial effect.
Assuntos
Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/parasitologia , Pentanonas/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Sequência de Aminoácidos , Sítios de Ligação , Curcumina/farmacologia , Humanos , Leishmania donovani/química , Leishmania donovani/enzimologia , Leishmania donovani/genética , Leishmaniose Visceral/tratamento farmacológico , Simulação de Acoplamento Molecular , Pentanonas/química , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Tyrosine aminotransferase (TAT) catalyzes the transamination of amino acids in Leishmania sp.. TAT from Leishmania donovani has been found to be extremely stable at extreme temperatures and pH conditions. This study was conceived to map the functions of the non-conserved N-terminal and conserved C-terminal domain of TAT. N-terminal (NTAT) and C-terminal (CTAT) domain of TAT was truncated and cloned into the pET28a(+) vector. The truncated proteins were expressed, purified, and biochemically characterized. The Km of NTAT and CTAT for the tyrosine-pyruvate pair was determined to be 3.468 ± 0.796 mM and 4.581 ± 0.627 mM, repectively. Temperature and pH stability studies found NTAT to be stable like TAT but CTAT was extremely susceptible to temperature and pH changes. Upon docking and simulation for 100 ns, NTAT had lower SASA values. From UV spectroscopic study, PLP bound better to CTAT than NTAT because of the reduced SASA of NTAT. The sensitivity of CTAT was reasoned when the urea denaturation studies showed two-state denaturation which differed from NTAT's and TAT's biphasic folding mechanism. From this study, the authors hypothesize that the N-terminal is responsible for PLP stabilization and C-terminal protects the active site from extreme conditions.
Assuntos
Leishmania donovani/enzimologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Tirosina Transaminase/química , Tirosina Transaminase/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Simulação por Computador , Humanos , Cinética , Leishmania donovani/química , Leishmania donovani/genética , Leishmaniose Visceral/parasitologia , Domínios Proteicos , Proteínas de Protozoários/genética , Alinhamento de Sequência , Tirosina Transaminase/genéticaRESUMO
Leucyl-tRNA synthetases (LRS) catalyze the linkage of leucine with tRNALeu. A large insertion CP1 domain (Connective Polypeptide 1) in LRS is responsible for post-transfer editing of mis-charged aminoacyl-tRNAs. Here, we characterized the CP1 domain of Leishmania donovani, a protozoan parasite, and its role in editing activity and interaction with broad spectrum anti-fungal, AN2690. The deletion mutant of LRS, devoid of CP1 domain (LRS-CP1Δ) was constructed, followed by determination of its role in editing and aminoacylation. Binding of AN2690 and different amino acids with CP1 deletion mutant and full length LRS was evaluated using isothermal titration calorimetry (ITC) and molecular dynamics simulations. The recombinant LRS-CP1Δ protein did not catalyze the aminoacylation and the editing reaction when compared to full-length LRS. Thus, indicating that CP1 domain was imperative for both aminoacylation and editing activities of LRS. Binding studies with different amino acids indicated selectivity of isoleucine by CP1 domain over other amino acids. These studies also indicated high affinity of AN2690 with the editing domain. Molecular docking studies indicated that AN2690-CP1 domain complex was stabilized by hydrogen bonding and hydrophobic interactions resulting in high binding affinity between the two. Our data suggests CP1 is crucial for the function of L.donovani LRS.
Assuntos
Antiprotozoários/farmacologia , Compostos de Boro/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Leishmania donovani/química , Leucina-tRNA Ligase/antagonistas & inibidores , Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/antagonistas & inibidores , Sequência de Aminoácidos , Antifúngicos/química , Antifúngicos/farmacologia , Antiprotozoários/química , Sítios de Ligação , Compostos de Boro/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Reposicionamento de Medicamentos , Expressão Gênica , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Leishmania donovani/enzimologia , Leishmania donovani/genética , Leucina-tRNA Ligase/química , Leucina-tRNA Ligase/genética , Leucina-tRNA Ligase/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA de Transferência de Leucina/química , RNA de Transferência de Leucina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Aminoacilação de RNA de Transferência/genéticaRESUMO
The development of an accurate protein-based antigen detection assay for diagnosis of active visceral leishmaniasis (VL) would represent a major clinical advance. VL is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani. The gold standard confirmatory diagnostic test for VL is the demonstration of parasites or their DNA from aspirates from spleen, lymph node, and bone marrow or from blood buffy coats. Here we describe the production and use of monoclonal antibodies (mAbs) for the development of a sensitive and specific antigen detection capture ELISA for VL diagnosis. This test simultaneously detects six leishmania protein biomarkers that we have previously described (Li-isd1, Li-txn1, Li-ntf2, Ld-mao1, Ld-ppi1 and Ld-mad1). The initial clinical validation of this new mAb-based multiplexed capture ELISA showed a sensitivity of ≥93%. The test was negative with 35 urine samples from healthy control subjects as well as with 30 patients with confirmed non-VL tropical diseases (cutaneous leishmaniasis, n = 6; Chagas disease, n = 6; schistosomiasis, n = 6; and tuberculosis, n = 12). These results strongly support the possible utility of this mAb-based multiplexed capture ELISA as a promising diagnostic test for active VL as well as for monitoring the treatment efficacy of this disease. The test is ready for upscaling and validation for clinical use.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Protozoários/urina , Leishmania donovani/química , Leishmania infantum/química , Leishmaniose Visceral/diagnóstico , Urinálise/métodos , Urina/química , Adolescente , Adulto , Idoso , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Biomarcadores/urina , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto JovemRESUMO
Leishmania donovani tyrosine aminotransferase (LdTAT) is an essential enzyme that catalyzes the first step of amino acid catabolism. To understand LdTAT activity at different pH, molecular dynamics simulations were performed and trajectory and T-pad analysis pad were conducted. Fluorescence spectroscopy of LdTAT at various pH was measured to understand structural stability. UV studies on PLP were performed to determine the binding of the enzyme to cofactor PLP at different pH. The MD simulations showed that the structure of LdTAT was stable and no structural denaturation was observed at pHâ¯2, 7 and 12. LdTAT exhibited the highest activity at pHâ¯-8 and fluorescent spectroscopy also corroborated by exhibiting the highest intensity at pHâ¯-8. Moreover, no structural denaturation was observed during the pH gradient. UV studies concluded that the aldimine bond forms only around neutral pH and redshift was observed on enzyme binding. From our observation, we hypothesize that the activity of LdTAT is a close interplay between the structure and charges of K286 and PLP. This study may provide significant insight into understanding parasitic enzymes like LdTAT during the life-cycle of Leishmania parasite. Knowledge of such enzyme mechanisms can pave the way for the design and delivery of enzyme-specific inhibitors.
Assuntos
Leishmania donovani/enzimologia , Tirosina Transaminase/metabolismo , Domínio Catalítico , Humanos , Concentração de Íons de Hidrogênio , Leishmania donovani/química , Leishmania donovani/metabolismo , Leishmaniose Visceral/parasitologia , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Fosfato de Piridoxal/metabolismo , Tirosina Transaminase/químicaRESUMO
Nucleoside hydrolases are a strategic target for the development of drugs to treat leishmaniasis, a neglected disease that affects 700 thousand to one million people annually. The present study aimed to identify Leishmania donovani nucleoside hydrolase (LdNH) inhibitors from the leaves of Ormosia arborea, a tree endemic to Brazilian ecosystems, through a strategy based on 1H NMR analyses and chemometrics. The aqueous EtOH extract of O. arborea leaves inhibited LdNH activity by 95%. The extract was fractionated in triplicate (13 in each step, making a total of 39 fractions). Partial least squares discriminant analysis (PLS-DA) was used to correlate the 1H NMR spectra of the fractions with their LdNH inhibitory activity and thus to identify the spectral regions associated with the bioactivity. The strategy aimed at isolating the probable bioactive substances and led to two new A-type proanthocyanidins, linked to a p-coumaroyl unit (1 and 2), which appeared as noncompetitive inhibitors of LdNH (IC50: 28.2 ± 3.0 µM and 25.6 ± 4.1 µM, respectively). This study confirms the usefulness of the NMR-based chemometric methods to accelerate the discovery of drugs from natural products.
Assuntos
Fabaceae/química , Leishmania donovani/química , N-Glicosil Hidrolases/antagonistas & inibidores , Brasil , Ecossistema , Fabaceae/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/metabolismoRESUMO
Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the ß5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the ß4 and ß5 proteasome subunits. This induced pocket exploits ß4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.
Assuntos
Antiprotozoários/administração & dosagem , Leishmania donovani/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/diagnóstico por imagem , Inibidores de Proteassoma/administração & dosagem , Proteínas de Protozoários/antagonistas & inibidores , Animais , Antiprotozoários/química , Sítios de Ligação , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Leishmania donovani/química , Leishmania donovani/enzimologia , Leishmania infantum/química , Leishmania infantum/enzimologia , Leishmaniose Visceral/parasitologia , Masculino , Camundongos , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismoRESUMO
Noncanonical base pairs play important roles in assembling the three-dimensional structures critical to the diverse functions of RNA. These associations contribute to the looped segments that intersperse the canonical double-helical elements within folded, globular RNA molecules. They stitch together various structural elements, serve as recognition elements for other molecules, and act as sites of intrinsic stiffness or deformability. This work takes advantage of new software (DSSR) designed to streamline the analysis and annotation of RNA three-dimensional structures. The multiscale structural information gathered for individual molecules, combined with the growing number of unique, well-resolved RNA structures, makes it possible to examine the collective features deeply and to uncover previously unrecognized patterns of chain organization. Here we focus on a subset of noncanonical base pairs involving guanine and adenine and the links between their modes of association, secondary structural context, and contributions to tertiary folding. The rigorous descriptions of base-pair geometry that we employ facilitate characterization of recurrent geometric motifs and the structural settings in which these arrangements occur. Moreover, the numerical parameters hint at the natural motions of the interacting bases and the pathways likely to connect different spatial forms. We draw attention to higher-order multiplexes involving two or more G·A pairs and the roles these associations appear to play in bridging different secondary structural units. The collective data reveal pairing propensities in base organization, secondary structural context, and deformability and serve as a starting point for further multiscale investigations and/or simulations of RNA folding.
Assuntos
Adenina/química , Guanina/química , Dobramento de RNA , RNA/metabolismo , Pareamento de Bases , Escherichia coli/química , Ligação de Hidrogênio , Leishmania donovani/química , Modelos Moleculares , Conformação de Ácido Nucleico , RNA/química , Saccharomyces cerevisiae/química , Software , Thermus thermophilus/químicaRESUMO
Anthroponotic visceral leishmaniasis is a life-threatening disease caused by Leishmania donovani (Kinetoplastida: Trypanosomatidae) in East Africa and the Indian subcontinent. Unlike promastigote growth and differentiation in the sand fly gut or in axenic culture, L. donovani promastigote-into-amastigote development has been studied by high-throughput gene expression profiling. In this study, we have identified abundant constitutive proteins in axenically cultured promastigotes by two-dimension electrophoresis and matrix-assisted laser desorption-ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry. Most proteins involved in the trypanothione-based redox antioxidant system are expressed constitutively throughout axenic L. donovani promastigote growth and differentiation (tryparedoxin, trypanothione peroxidase, generic peroxidoxin, iron-superoxide dismutase, and elongation factor 1ß). These findings are in agreement with previous data on other Old World species (i.e., L. major and L. infantum), whereas New World species (i.e., L. amazonensis and L. pifanoi) and Crithidia fasciculata show different expression patterns.
Assuntos
Leishmania donovani/química , Leishmania donovani/crescimento & desenvolvimento , Proteoma/análise , Proteínas de Protozoários/análise , Eletroforese em Gel Bidimensional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
New series of quinoline-based thiadiazole analogs (1-20) were synthesized, characterized by EI-MS, 1H NMR and 13C NMR. All synthesized compounds were subjected to their antileishmanial potential. Sixteen analogs 1-10, 12, 13, 16, 17, 18 and 19 with IC50 values in the range of 0.04⯱â¯0.01 to 5.60⯱â¯0.21⯵M showed tremendously potent inhibition as compared to the standard pentamidine with IC50 value 7.02⯱â¯0.09⯵M. Analogs 11, 14, 15 and 20 with IC50 8.20⯱â¯0.35, 9.20⯱â¯0.40, 7.20⯱â¯0.20 and 9.60⯱â¯0.40⯵M respectively showed good inhibition when compared with the standard. Structure-activity relationships have been also established for all compounds. Molecular docking studies were performed to determine the binding interaction of the compounds with the active site target.
Assuntos
Quinolinas/farmacologia , Tiadiazóis/farmacologia , Tripanossomicidas/farmacologia , Domínio Catalítico , Leishmania donovani/química , Leishmania major/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Oxirredutases/química , Oxirredutases/metabolismo , Ligação Proteica , Quinolinas/síntese química , Quinolinas/metabolismo , Relação Estrutura-Atividade , Tiadiazóis/síntese química , Tiadiazóis/metabolismo , Tripanossomicidas/síntese química , Tripanossomicidas/metabolismoRESUMO
Current diagnostic tests for visceral leishmaniasis (VL) are either not adapted for use in resource-poor settings or are insufficiently accurate in Eastern Africa. Only the direct agglutination test (DAT), based on whole Leishmania promastigotes, is highly reliable in all endemic regions, but its implementation is hampered by the need for a cold chain, minimal laboratory conditions, and long incubation times. Integrating the DAT antigen(s) in an immunochromatographic rapid diagnostic test (RDT) would overcome these disadvantages. Unfortunately, the identity of the DAT antigen(s) involved in the agglutination reaction is unknown. For this study, we reviewed all publications that might shed some light on this issue. We conclude that the DAT antigen is a mixture of Leishmania-specific epitopes of protein, carbohydrate, and lipid nature. To develop an accurate RDT for VL diagnosis in Eastern Africa, we suggest to complement the classical protein antigen discovery with approaches to identify carbohydrate and lipid epitopes.
Assuntos
Testes de Aglutinação/normas , Antígenos de Protozoários/química , Epitopos/química , Leishmania donovani/química , Leishmania infantum/química , Leishmaniose Visceral/diagnóstico , África Oriental/epidemiologia , Antígenos de Protozoários/imunologia , Carboidratos/química , Carboidratos/imunologia , Epitopos/imunologia , Humanos , Soros Imunes/química , Leishmania donovani/imunologia , Leishmania infantum/imunologia , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Lipídeos/química , Lipídeos/imunologia , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Previous metabolic studies have demonstrated that leishmania parasites are able to synthesise proline from glutamic acid and threonine from aspartic acid. The first committed step in both biosynthetic pathways involves an amino acid kinase, either a glutamate 5-kinase (G5K; EC2.7.2.11) or an aspartokinase (EC2.7.2.4). Bioinformatic analysis of multiple leishmania genomes identifies a single amino acid-kinase gene (LdBPK 262740.1) variously annotated as either a putative glutamate or aspartate kinase. To establish the catalytic function of this Leishmania donovani gene product, we have determined the physical and kinetic properties of the recombinant enzyme purified from Escherichia coli. The findings indicate that the enzyme is a bona fide G5K with no activity as an aspartokinase. Tetrameric G5K displays kinetic behaviour similar to its bacterial orthologues and is allosterically regulated by proline, the end product of the pathway. The structure-activity relationships of proline analogues as inhibitors are broadly similar to the bacterial enzyme. However, unlike G5K from E. coli, leishmania G5K lacks a C-terminal PUA (pseudouridine synthase and archaeosine transglycosylase) domain and does not undergo higher oligomerisation in the presence of proline. Gene replacement studies are suggestive, but not conclusive that G5K is essential. ENZYMES: Glutamate 5-kinase (EC2.7.2.11); aspartokinase (EC2.7.2.4).
Assuntos
Ácido Glutâmico/metabolismo , Leishmania donovani/química , Fosfotransferases (Aceptor do Grupo Carboxila)/metabolismo , Prolina/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Ácido Aspártico/metabolismo , Biocatálise , Clonagem Molecular , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Teste de Complementação Genética , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Leishmania donovani/enzimologia , Fosfotransferases (Aceptor do Grupo Carboxila)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Filogenia , Prolina/análogos & derivados , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , TermodinâmicaRESUMO
The pathogenicity of protozoan parasites is frequently attributed to their ability to circumvent the deleterious effects of ROS and Fe-S clusters are among their susceptible targets with paramount importance for parasite survival. The biogenesis of Fe-S clusters is orchestrated by ISC system; the sulfur donor IscS and scaffold protein IscU being its core components. However, among protozoan parasites including Leishmania, no information is available regarding biochemical aspect of IscU, its interaction partners and regulation. Here, we show that Leishmania donovani IscU homolog, LdIscU, readily assembles [2Fe-2S] clusters and, interestingly, follows Michaelis-Menten enzyme kinetics. It is localized in the mitochondria of the parasite and interacts with LdIscS to form a stable complex. Additionally, LdIscU and Fe-S proteins activity is significantly upregulated in resistant isolates and during stationary growth stage indicating an association between them. The differential expression of LdIscU modulated by Fe-S proteins demand suggests its potential role in parasite survival and drug resistance. Thus, our study provides novel insight into the Fe-S scaffold protein of a protozoan parasite.
Assuntos
Resistência a Medicamentos , Regulação da Expressão Gênica , Proteínas Ferro-Enxofre/biossíntese , Leishmania donovani/metabolismo , Proteínas de Protozoários/biossíntese , Animais , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Leishmania donovani/química , Leishmania donovani/genética , Masculino , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , CoelhosRESUMO
To facilitate a greater understanding of the biological processes in the medically important Leishmania donovani parasite, a combination of differential and density-gradient ultracentrifugation techniques were used to achieve a comprehensive subcellular fractionation of the promastigote stage. An in-depth label-free proteomic LC-MS/MS analysis of the density gradients resulted in the identification of â¼50% of the Leishmania proteome (3883 proteins detected), which included â¼645 integral membrane proteins and 1737 uncharacterized proteins. Clustering and subcellular localization of proteins was based on a subset of training Leishmania proteins with known subcellular localizations that had been determined using biochemical, confocal microscopy, or immunoelectron microscopy approaches. This subcellular map will be a valuable resource that will help dissect the cell biology and metabolic processes associated with specific organelles of Leishmania and related kinetoplastids.
Assuntos
Leishmania donovani/química , Proteínas de Membrana/isolamento & purificação , Redes e Vias Metabólicas/genética , Proteoma/isolamento & purificação , Proteômica/métodos , Proteínas de Protozoários/isolamento & purificação , Fracionamento Celular/instrumentação , Fracionamento Celular/métodos , Núcleo Celular/química , Núcleo Celular/metabolismo , Centrifugação com Gradiente de Concentração , Cromatografia Líquida , Expressão Gênica , Ontologia Genética , Leishmania donovani/genética , Leishmania donovani/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microcorpos/química , Microcorpos/metabolismo , Microssomos/química , Microssomos/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Anotação de Sequência Molecular , Proteoma/genética , Proteoma/metabolismo , Proteômica/instrumentação , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Frações Subcelulares , Espectrometria de Massas em Tandem , UltracentrifugaçãoRESUMO
Visceral leishmaniasis caused by the protozoan Leishmania donovani is the most severe form of leishmaniasis and it is potentially lethal if untreated. Despite the availability of drugs for treating the disease, the current drug regime suffers from drawbacks like antibiotic resistance and toxicity. New drugs have to be discovered in order to overcome these limitations. Our aim is to identify natural compounds from plant sources as putative inhibitors considering the occurrence of structural diversity in plant sources. Spermidine Synthase (SpdS) was chosen as the target enzyme as it plays a vital role in growth, survival, and due to its contribution in virulence. Our initial investigation started with a literature survey in identifying natural compounds that showed antileishmanial activity. Subsequently, we identified two monoterpenoid compounds, namely Geraniol and Linalool, that were structurally analogous to one of the substrates (putrescine) of SpdS. In the present study, homology model of L. donovani SpdS was generated and the binding affinity of the identified compounds was analyzed and also compared with the putrescine through molecular docking and dynamic studies. The pharmacokinetic properties of the identified compounds were validated and the binding efficiency of these ligands over the original substrate has been demonstrated. Based on these studies, Geraniol and Linalool can be considered as lead molecules for future investigations targeting SpdS. This study further emphasizes the choice of natural compounds as a good source of therapeutic agents.
Assuntos
Produtos Biológicos/farmacologia , Inibidores Enzimáticos/farmacologia , Leishmania donovani/enzimologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Espermidina Sintase/antagonistas & inibidores , Sequência de Aminoácidos , Sítios de Ligação , Produtos Biológicos/química , Inibidores Enzimáticos/química , Leishmania donovani/química , Ligantes , Reprodutibilidade dos Testes , Espermidina Sintase/química , Espermidina Sintase/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
Proliferating cell nuclear antigen (PCNA) acts as a sliding clamp to support DNA replication and repair. The structure of PCNA from Leishmania donovani (LdPCNA) has been determined at 2.73Å resolution. Structure consists of six crystallographically independent molecules which form two trimeric rings. The pore diameter of the individual trimeric ring is of the order of 37Å. The two rings are stacked through their front to front faces. In order to gain a stable packing, the rings are rotated by 42° about the pore axis and shifted by 7Å and tilted by 16° along the perpendicular direction to pore axis. This form of stacking reduced the effective diameter of the pore to 32Å. The sequence of LdPCNA consists of a long segment of 41 amino acid residues (186-Gly-Val-Ser-Asp-Arg-Ser-Thr-Lys-Ser-Glu-Val-Lys-Ala-Glu-Val-Lys-Ala-Glu-Ala-Arg-Asp-Asp-Asp-Glu-Glu-Pro-Leu-Ser-Arg-Lys-Tyr-Gly-Lys-Ala-Asp-Ser-Ser-Ala-Asn-Ala-Ile-226) whereas the corresponding segments in other PCNAs contain only eight residues corresponding to 186-Gly-Val-Ser-Asp-Arg------224-Asn-Ala-Ile-226. The enhanced length of this segment in LdPCNA may influence its mode of interaction with DNA and other proteins. The dissociation constants obtained using real time binding studies with surface plasmon resonance (SPR) for two peptides, Lys-Arg-Arg-Gln-Thr-Ser-Met-Thr-Asp-Phe-Tyr-His (P1) from human cyclin-dependent kinase inhibitor-1(CKI-1) and Lys-Thr-Gln-Gly-Arg-Leu-Asp-Ser-Phe-Phe-Thr-Val (P2) from flap endonuclease 1 (Fen-1) as well as with two small molecule inhibitors, (S)-4-(4-(2-amino-3-hydroxypropyl)-2, 6-diiodophenoxy) phenol hydrochloride (ADPH) and N-(3-methylthiophene-2-carboxylicacid)-N'-((3-hydroxy-2-naphthalenyl) methylene) hydrazide (MCMH) are 0.29±0.09µM, 0.37±0.08µM, 0.35±0.09µM and 1.20±0.08µM respectively. The corresponding values obtained using fluorescence spectroscopic methods were 0.22±0.06µM, 0.68±0.07µM, 0.44±0.07µM and 0.75±0.05µM respectively.
Assuntos
DNA de Protozoário/química , Leishmania donovani/química , Antígeno Nuclear de Célula em Proliferação/química , Proteínas de Protozoários/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Inibidor de Quinase Dependente de Ciclina p21/química , DNA de Protozoário/genética , DNA de Protozoário/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Endonucleases Flap/química , Expressão Gênica , Leishmania donovani/metabolismo , Modelos Moleculares , Fenóis/química , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Hydrophilic acylated surface proteins (HASPs) are acidic surface proteins which get localized on the surface of Leishmania parasite during infective stages through a "non-classical" pathway. In this study, we report the heterologous expression and purification of Leishmania donovani HASPA (r-LdHASPA) in E. coli system and its partial characterization. The structural aspects of the purified protein were analyzed using CD spectroscopy and modeling studies which indicate that r-LdHASPA consists of random coils. Studies in mouse macrophage RAW264.7 cell lines indicate that r-LdHASPA enhances reactive oxygen species (ROS) production. Co-immunoprecipitation (IP) studies indicate that r-LdHASPA interacts with certain macrophage proteins which however could not be identified unambiguously. The present study provides key insights into the structural and functional aspects of an important Leishmania protein, HASPA, which we believe could be useful for further research on vaccine/drug development.
Assuntos
Antígenos de Protozoários/genética , Leishmania donovani/química , Macrófagos/efeitos dos fármacos , Proteínas de Protozoários/genética , Espécies Reativas de Oxigênio/agonistas , Animais , Antígenos de Protozoários/isolamento & purificação , Antígenos de Protozoários/metabolismo , Antígenos de Protozoários/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Leishmania donovani/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Conformação Proteica em alfa-Hélice , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
Visceral leishmaniasis is a dreadful infectious disease and caused by the intracellular protozoan parasites, Leishmania donovani and Leishmania infantum. Despite extensive efforts for developing effective prophylactic vaccine, still no vaccine is available against leishmaniasis. However, advancement in immunoinformatics methods generated new dimension in peptide based vaccine development. The present study was aimed to identify T-cell epitopes from the vaccine candidate antigens like Lipophosphogylcan-3(LPG-3) and Nucleoside hydrolase (NH) from the L. donovani using in silico methods. Available best tools were used for the identification of promiscuous peptides for MHC class-II alleles. A total of 34 promiscuous peptides from LPG-3, 3 from NH were identified on the basis of their 100% binding affinity towards all six HLA alleles, taken in this study. These peptides were further checked computationally to know their IFN-γ and IL4 inducing potential and nine peptides were identified. Peptide binding interactions with predominant HLA alleles were done by docking. Out of nine docked promiscuous peptides, only two peptides (QESRILRVIKKKLVR, RILRVIKKKLVRKTL), from LPG-3 and one peptide (FDKFWCLVIDALKRI) from NH showed lowest binding energy with all six alleles. These promiscuous T-cell epitopes were predicted on the basis of their antigenicity, hydrophobicity, potential immune response and docking scores. The immunogenicity of predicted promiscuous peptides might be used for subunit vaccine development with immune-modulating adjuvants.