RESUMEN
Visceral leishmaniasis (VL) or Kala-azar, primarily caused by Leishmania donovani, is a major health concern in many countries including India. Growing unresponsiveness among the parasites toward the available drugs is alarming, and so, it is necessary to decipher the underlying mechanism of such development for designing new therapeutics. Moreover, even after successful treatment, some VL patients develop apparently harmless skin lesions known as post-kala-azar dermal leishmaniasis (PKDL) which may serve as a reservoir of the parasite in the transmission cycle. Furthermore, recent reports of para-kala-azar dermal leishmaniasis (para-KDL) cases having PKDL manifestation with concomitant VL, emphasize the necessity of more attention to address complex nature of the parasite for eradicating the disease effectively. In the present study, whole genome sequencing is performed with sodium stibogluconate (SSG) sensitive and resistant L. donovani strains along with SSG sensitive para-KDL strains, derived from the clinical isolates of Indian patients to identify the genomic variations among them. Notably, the analyses of chromosome somy values and genome wide mutation profile in the coding regions reveal distinct clustering of the para-KDL strains with 24 genes being mutated uniquely in this group. Such distinguishing genomic changes among the para-KDL strains could be significant for the parasites to become dermatotropic. Overall, the study reveals a possible correlation of the development of SSG resistance and the transition towards the manifestation of PKDL with chromosome aneuploidy and non-synonymous genetic variations in the coding sequences of the L. donovani strains from Indian patients.
Asunto(s)
Genoma de Protozoos , Leishmania donovani , Leishmaniasis Cutánea , Leishmaniasis Visceral , Gluconato de Sodio Antimonio , Humanos , India/epidemiología , Leishmania donovani/genética , Leishmaniasis Cutánea/epidemiología , Leishmaniasis Cutánea/parasitología , Leishmaniasis Visceral/epidemiología , Leishmaniasis Visceral/parasitologíaRESUMEN
The emergence of resistance to available antileishmanial drugs advocates identification of new drug targets and their inhibitors for visceral leishmaniasis. Here, we identified Leishmania donovani heat shock protein 78 (LdHSP78), a putative caseinolytic protease, as important for parasite infection of host macrophages and a potential therapeutic target. Enrichment of LdHSP78 in infected humans, hamsters, and parasite amastigotes suggested its importance for disease persistence. Heterozygous knockouts of L. donovani HSP78 (LdHSP78+/-) and Leishmania mexicana HSP78 (LmxHSP78+/-) were generated using a flanking UTR-based multifragment ligation strategy and the CRISPR-Cas9 technique, respectively to investigate the significance of HSP78 for disease manifestation. The LdHSP78+/- parasite burden was dramatically reduced in both murine bone marrow-derived macrophages and hamsters, in association with enrichment of proinflammatory cytokines and NO. This finding implies that LdHSP78+/- parasites cannot suppress immune activation and escape NO-mediated toxicity in macrophages. Furthermore, phosphorylation of the mitogen-activated protein kinase p38 was enhanced and phosphorylation of extracellular signal-regulated kinase 1/2 was decreased in cells infected with LdHSP78+/- parasites, compared with WT parasites. Virulence of the LdHSP78+/- strain was restored by episomal addition of the LdHSP78 gene. Finally, using high-throughput virtual screening, we identified P1,P5-di(adenosine-5')-pentaphosphate (Ap5A) ammonium salt as an LdHSP78 inhibitor. It selectively induced amastigote death at doses similar to amphotericin B doses, while exhibiting much less cytotoxicity to healthy macrophages than amphotericin B. In summary, using both a genetic knockout approach and pharmacological inhibition, we establish LdHSP78 as a drug target and Ap5A as a potential lead for improved antileishmanial agents.
Asunto(s)
Antiprotozoarios/farmacología , Fosfatos de Dinucleósidos/farmacología , Proteínas de Choque Térmico/antagonistas & inhibidores , Leishmania donovani/metabolismo , Leishmaniasis Visceral/tratamiento farmacológico , Macrófagos/parasitología , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Sistemas CRISPR-Cas , Cricetinae , Técnicas de Inactivación de Genes , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Leishmania donovani/genética , Leishmaniasis Visceral/genética , Leishmaniasis Visceral/metabolismo , Macrófagos/metabolismo , Ratones , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Host- as well as parasite-specific factors are equally crucial in allowing either the Leishmania parasites to dominate, or host macrophages to resist infection. To identify such factors, we infected murine peritoneal macrophages with either the virulent (vAG83) or the non-virulent (nvAG83) parasites of L. donovani. Then, through dual RNA-seq, we simultaneously elucidated the transcriptomic changes occurring both in the host and the parasites. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed (DE) genes, we showed that the vAG83-infected macrophages exhibit biased anti-inflammatory responses compared to the macrophages infected with the nvAG83. Moreover, the vAG83-infected macrophages displayed suppression of many important cellular processes, including protein synthesis. Further, through protein-protein interaction study, we showed significant downregulation in the expression of many hubs and hub-bottleneck genes in macrophages infected with vAG83 as compared to nvAG83. Cell signaling study showed that these two parasites activated the MAPK and PI3K-AKT signaling pathways differentially in the host cells. Through gene ontology analyses of the parasite-specific genes, we discovered that the genes for virulent factors and parasite survival were significantly upregulated in the intracellular amastigotes of vAG83. In contrast, genes involved in the immune stimulations, and those involved in negative regulation of the cell cycle and transcriptional regulation, were upregulated in the nvAG83. Collectively, these results depicted a differential regulation in the host and the parasite-specific molecules during in vitro persistence and clearance of the parasites.
Asunto(s)
Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Leishmania donovani/crecimiento & desarrollo , Leishmania donovani/inmunología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/parasitología , Análisis de Secuencia de ARN , Animales , Células Cultivadas , Biología Computacional , Ratones , Anotación de Secuencia MolecularRESUMEN
A novel library of C2-substituted tryptamines (based on diverse C2-aroyl/arylimino indoles and indole-diketopiperazine hybrids) possessing antimitotic properties were designed, synthesized and screened for their inhibitory activity against tubulin polymerization, and against proliferation of A549 lung cancer, HeLa cervical cancer, MCF7 breast cancer and HePG2 liver cancer cell lines. The design of molecules were inspired from known antimitotic compounds and natural products. The molecular docking of the designed compounds indicated that they bind to the colchicin binding site of tubulin. They were synthesized by a unique iodine catalysed oxidative ring opening reaction of 1-aryltetrahydro-ß-carbolines. Among the compounds synthesized quite a few compounds induced cytotoxicity on the cancer cells by disrupting the tubulin polymerization. They were found to be non-toxic for healthy cells. Immuno Fluorescence study for the most active molecules (between ~6⯵M concentration) against A549 and HeLa cells demonstrated complete disruption and shrinkage of the microtubule structures. These compounds also inhibited indoleamine-2, 3-dioxygenase with low micromolar IC50.
Asunto(s)
Antimitóticos , Dioxigenasas/antagonistas & inhibidores , Triptaminas , Moduladores de Tubulina , Antimitóticos/química , Antimitóticos/farmacología , Línea Celular , Dioxigenasas/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Triptaminas/química , Triptaminas/farmacología , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologíaRESUMEN
Nonalcoholic fatty liver disease (NAFLD), which is characterized by excess accumulation of triglyceride in hepatocytes, is the major cause of chronic liver disease worldwide and no approved drug is available. The mechanistic target of rapamycin (mTOR) complexes has been implicated in promoting lipogenesis and fat accumulation in the liver, and thus, serve as attractive drug targets. The generation of non- or low cytotoxic mTOR inhibitors is required because existing cytotoxic mTOR inhibitors are not useful for NAFLD therapy. New compounds based on the privileged adenosine triphosphate (ATP) site binder quinoline scaffold conjugated to glucose and galactosamine derivatives, which have significantly low cytotoxicity, but strong mTORC1 inhibitory activity at low micromolar concentrations, have been synthesized. These compounds also effectively inhibit the rate of lipogenesis and lipid accumulation in cultured hepatocytes. This is the first report of glycomimetic-quinoline derivatives that reduce lipid load in hepatocytes.
RESUMEN
Vinblastine and its related compound vincristine are important mono terpenoid indole alkaloids accumulated in the leaves of Catharanthus roseus (Madagascar periwinkle). They serve as major anticancer drugs. Vinblastine is formed by the condensation of vindoline and catharanthine. The vindoline moiety is derived from tabersonine via vindoline biosynthesis pathway. The reaction sequence from tabersonine to vindoline is now well established and the enzymes involved in this pathway are identified. However, to date, the structures of the enzymes involved in the vindoline biosynthesis pathway are not known, leading to limited mechanistic understanding of the substrate binding and catalysis. The purpose of this work is to provide structural insight regarding all the steps of the vindoline pathway via rigorous homology modeling, molecular docking, and molecular dynamics analyses. Substrate and cofactors required for each step were docked onto the computationally built and validated three-dimensional (3D) model of the corresponding enzyme, and the catalytic reaction was analyzed from the structural point of view. Possible binding modes of the substrates and cofactors were generated and corresponding binding residues were identified. Enzyme-substrate models were verified based on structure evaluation methods and molecular dynamics based approaches. Findings of our analysis would be useful in rational designing of these important enzymes aimed toward bio-production of vindoline.
Asunto(s)
Catharanthus/enzimología , Proteínas de Plantas/química , Vinblastina/análogos & derivados , Acetiltransferasas/química , Acetiltransferasas/metabolismo , Apoenzimas/química , Apoenzimas/metabolismo , Vías Biosintéticas , Dominio Catalítico , Metiltransferasas/química , Metiltransferasas/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , Oxigenasas/química , Oxigenasas/metabolismo , Vinblastina/biosíntesis , Vinblastina/químicaRESUMEN
Podophyllotoxin (ptox) is a therapeutically important lignan derived from Podophyllum hexandrum and is used as a precursor for the synthesis of anticancer drugs etoposide, teniposide and etopophose. In spite of its enormous economic significance, genomic information on this endangered medicinal herb is scarce. We have performed de novo transcriptome analysis of methyl jasmonate (MeJA)-treated P. hexandrum cell cultures exhibiting enhanced ptox accumulation. The results revealed the maximum up-regulation of several isoforms of cinnamyl alcohol dehydrogenase (CAD). CAD catalyzes the synthesis of coniferyl alcohol and sinapyl alcohol from coniferaldehyde (CAld) and sinapaldehyde respectively. Coniferyl alcohol can produce both lignin and lignan while sinapyl alcohol produces only lignin. To isolate the CAD isoforms favoring ptox, we deduced full length cDNA sequences of four CAD isoforms: PhCAD1, PhCAD2, PhCAD3 and PhCAD4 from the contigs of the transcriptome data. In vitro enzyme assays indicated a higher affinity for CAld over sinapaldehyde for each isoform. In silico molecular docking analyses also suggested that PhCAD3 has a higher binding preference with CAld over sinapaldehyde, followed by PhCAD4, PhCAD2, and PhCAD1, respectively. The transgenic cell cultures overexpressing these isoforms independently revealed that PhCAD3 favored the maximum accumulation of ptox as compared to lignin followed by PhCAD4 and PhCAD2, whereas, PhCAD1 favored both equally. Together, our study reveals transcriptome-wide identification and characterization of ptox specific CAD isoforms from P. hexandrum. It provides a useful resource for future research not only on the ptox biosynthetic pathway but on overall P. hexandrum, an endangered medicinal herb with immense therapeutic importance.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Podofilotoxina/biosíntesis , Podophyllum/enzimología , Podophyllum/metabolismo , Isoformas de Proteínas/metabolismo , Acetatos/farmacología , Oxidorreductasas de Alcohol/genética , Ciclopentanos/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/genética , Oxilipinas/farmacología , Podophyllum/efectos de los fármacos , Isoformas de Proteínas/genética , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Pseudomonas aeruginosa, a Gram-negative pathogen uses a specialized set of Type III secretion system (T3SS) translocator proteins to establish virulence in the host cell. An understanding of the factors that govern translocation by the translocator protein-chaperone complex is thus of immense importance. In this work, experimental and computational techniques were used to probe into the structure of the major translocator protein PopB from P. aeruginosa and to identify the important regions involved in functioning of the translocator protein. This study reveals that the binding sites of the common chaperone PcrH, needed for maintenance of the translocator PopB within the bacterial cytoplasm, which are primarily localized within the N-terminal domain. However, disordered and flexible residues located both at the N- and C-terminal domains are also observed to be involved in association with the chaperone. This intrinsic disorderliness of the terminal domains is conserved for all the major T3SS translocator proteins and is functionally important to maintain the intrinsically disordered state of the translocators. Our experimental and computational analyses suggest that a "disorder-to-order" transition of PopB protein might take place upon PcrH binding. The long helical coiled-coil part of PopB protein perhaps helps in pore formation while the flexible apical region is involved in chaperone interaction. Thus, our computational model of translocator protein PopB and its binding analyses provide crucial functional insights into the T3SS translocation mechanism.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Pseudomonas aeruginosa/metabolismo , Secuencia de Aminoácidos , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos , Sitios de Unión , Biología Computacional , Secuencia Conservada , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Replegamiento Proteico , Pseudomonas aeruginosa/patogenicidad , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Propiedades de SuperficieRESUMEN
Plant receptor-like kinases (RLKs) are distinguished by having a tyrosine in the 'gatekeeper' position. Previously we reported Symbiosis Receptor Kinase from Arachis hypogaea (AhSYMRK) to autophosphorylate on the gatekeeper tyrosine (Y670), though this phosphorylation was not necessary for the kinase activity. Here we report that recombinant catalytic domain of AhSYMRK with a phosphomimic substitution in the gatekeeper position (Y670E) is catalytically almost inactive and is conformationally quite distinct from the corresponding native enzyme. Additionally, we show that gatekeeper-phosphorylated AhSYMRK polypeptides are inactive and depletion of this inactive form leads to activation of intramolecular autophosphorylation of AhSYMRK. Together, our results suggest gatekeeper tyrosine autophosphorylation to be autoinhibitory for AhSYMRK.
Asunto(s)
Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Tirosina/metabolismo , Sustitución de Aminoácidos , Arachis/enzimología , Dominio Catalítico , Modelos Moleculares , Mutación , Fosforilación , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genéticaRESUMEN
We have investigated the DNA-binding nature as well as the function of a putative Alba (Acetylation lowers binding affinity) family protein (PfAlba3) from Plasmodium falciparum. PfAlba3 possesses DNA-binding property like Alba family proteins. PfAlba3 binds to DNA sequence non-specifically at the minor groove and acetylation lowers its DNA-binding affinity. The protein is ubiquitously expressed in all the erythrocytic stages of P. falciparum and it exists predominantly in the acetylated form. PfAlba3 inhibits transcription in vitro by binding to DNA. Plasmodium falciparum Sir2 (PfSir2A), a nuclear localized deacetylase interacts with PfAlba3 and deacetylates the lysine residue of N-terminal peptide of PfAlba3 specific for DNA binding. PfAlba3 is localized with PfSir2A in the periphery of the nucleus. Fluorescence in situ hybridization studies revealed the presence of PfAlba3 in the telomeric and subtelomeric regions. ChIP and ChIP ReChIP analyses further confirmed that PfAlba3 binds to the telomeric and subtelomeric regions as well as to var gene promoter.