RESUMO
Post-translational modification of G-protein coupled receptors (GPCRs) plays a central role in tissue hemostasis and cancer. The molecular mechanism of post-translational regulation of protease-activated receptors (PARs), a subgroup of GPCRs is yet understudied. Here we show that the cell-surface transmembrane E3 ubiquitin ligase ring finger 43 (RNF43) is a negative feedback regulator of PAR2 , impacting PAR2 -induced signaling and colon cancer growth. RNF43 co-associates with PAR2 , promoting its membrane elimination and degradation as shown by reduced cell surface biotinylated PAR2 levels and polyubiquitination. PAR2 degradation is rescued by R-spondin2 in the presence of leucine-rich repeat-containing G-protein-coupled receptor5 (LGR5). In fact, PAR2 acts jointly with LGR5, as recapitulated by increased ß-catenin levels, transcriptional activity, phospho-LRP6, and anchorage-independent colony growth in agar. Animal models of the chemically induced AOM/DSS colon cancer of wt versus Par2/f2rl1 KO mice as also the 'spleen-liver' colon cancer metastasis, allocated a central role for PAR2 in colon cancer growth and development. RNF43 is abundantly expressed in the Par2/f2rl1 KO-treated AOM/DSS colon tissues while its level is very low to nearly null in colon cancer adenocarcinomas of the wt mice. The same result is obtained in the 'spleen-liver' model of spleen-inoculated cells, metastasized to the liver. High RNF43 expression is observed in the liver upon shRNA -Par2 silencing. "Limited-dilution-assay" performed in mice in-vivo, assigned PAR2 as a member of the cancer stem cell niche compartment. Collectively, we elucidate an original regulation of PAR2 oncogene, a member of cancer stem cells, by RNF43 ubiquitin ligase. It impacts ß-catenin signaling and colon cancer growth.
Assuntos
Neoplasias do Colo , Receptor PAR-2 , Camundongos , Animais , Receptor PAR-2/genética , beta Catenina/genética , Células-Tronco NeoplásicasRESUMO
Cancer cells depend on specific oncogenic pathways or present a genetic alteration that leads to a particular disturbance. Still, personalized and targeted biological therapy remains challenging, with current efforts generally yielding disappointing results. Carefully assessing onco-target molecular pathways can, however, potently assist with such efforts for the selection of patient populations that would best respond to a given drug treatment. RNF43, an E3 ubiquitin ligase that negatively regulates Wnt/frizzled (FZD) receptors by their ubiquitination, internalization, and degradation, controls a key pathway in cancer. Recently, additional target proteins of RNF43 were described, including p85 of the PI3K/AKT/mTOR signaling pathway and protease-activated receptor 2 (PAR2), a G-protein-coupled receptor that potently induces ß-catenin stabilization, independent of Wnts. RNF43 mutations with impaired E3 ligase activity were found in several types of cancers (e.g., gastrointestinal system tumors and endometrial and ovarian cancer), pointing to a high dependency on FZD receptors and possibly PAR2 and the PI3K/AKT/mTOR signaling pathway. The development of drugs toward these targets is essential for improved treatment of cancer patients.
Assuntos
Neoplasias , Ubiquitina-Proteína Ligases , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Transdução de Sinais/efeitos dos fármacos , Terapia de Alvo Molecular , Animais , Serina-Treonina Quinases TOR/metabolismo , Ubiquitinação , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Frizzled/metabolismo , Receptores Frizzled/genética , Receptor PAR-2/metabolismo , Receptor PAR-2/genética , Mutação , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
G-protein-coupled receptors (GPCRs) are involved in a wide array of physiological and disease functions, yet knowledge of their role in colon cancer stem cell maintenance is still lacking. In addition, the molecular mechanisms underlying GPCR-induced post-translational signaling regulation are poorly understood. Here, we find that protease-activated receptor 4 (PAR4) unexpectedly acts as a potent oncogene, inducing ß-catenin stability and transcriptional activity. Both PAR4 and PAR2 are able to drive the association of methyltransferase EZH2 with ß-catenin, culminating in ß-catenin methylation. This methylation on a lysine residue at the N-terminal portion of ß-catenin suppresses the ubiquitination of ß-catenin, thereby promoting PAR-induced ß-catenin stability and transcriptional activity. Indeed, EZH2 is found to be directly correlated with high PAR4-driven tumors, and is abundantly expressed in large tumors, whereas very little to almost none is expressed in small tumors. A truncated form of ß-catenin, ∆N133ß-catenin, devoid of lysine, as well as serine/threonine residues, exhibits low levels of ß-catenin and a markedly reduced transcriptional activity following PAR4 activation, in contrast to wt ß-catenin. Our study demonstrates the importance of ß-catenin lysine methylation in terms of its sustained expression and function. Taken together, we reveal that PAR-induced post-transcriptional regulation of ß-catenin is centrally involved in colon cancer.
Assuntos
Neoplasias do Colo , beta Catenina , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Humanos , Lisina , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The essential role of G-protein coupled receptors (GPCRs) in tumor growth is recognized, yet a GPCR based drug in cancer is rare. Understanding the molecular path of a tumor driver gene may lead to the design and development of an effective drug. For example, in members of protease-activated receptor (PAR) family (e.g., PAR1 and PAR2), a novel PH-binding motif is allocated as critical for tumor growth. Animal models have indicated the generation of large tumors in the presence of PAR1 or PAR2 oncogenes. These tumors showed effective inhibition when the PH-binding motif was either modified or were inhibited by a specific inhibitor targeted to the PH-binding motif. In the second part of the review we discuss several aspects of some cardinal GPCRs in tumor angiogenesis.
Assuntos
Neoplasias/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Ativados por Proteinase/metabolismo , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/fisiopatologia , Domínios de Homologia à Plecstrina/genética , Domínios de Homologia à Plecstrina/fisiologia , Domínios Proteicos/genética , Domínios Proteicos/fisiologia , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Receptores Ativados por Proteinase/genética , Transdução de Sinais/fisiologiaRESUMO
The original version of this article unfortunately contained a mistake. The family name of Beatrice Uziely was mistakenly spelled as Uzieky. The correct name is now presented above.
RESUMO
G protein-coupled receptors (GPCRs) comprise the main signal-transmitting components in the cell membrane. Over the past several years, biochemical and structural analyses have immensely enhanced our knowledge of GPCR involvement in health and disease states. The present review focuses on GPCRs that are cancer drivers, involved in tumor growth and development. Our aim is to highlight the involvement of stabilized ß-catenin molecular machinery with a specific array of GPCRs. We discuss recent advances in understanding the molecular path leading to ß-catenin nuclear localization and transcriptional activity and their implications for future cancer therapy research.
Assuntos
Neoplasias/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , beta Catenina/metabolismo , Animais , Endotelinas/metabolismo , Via de Sinalização Hippo , Humanos , Neoplasias/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade Proteica , Transporte Proteico , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Receptores de Prostaglandina/metabolismo , Transdução de Sinais , Transcrição Gênica , Via de Sinalização WntRESUMO
G protein-coupled receptors (GPCRs), the largest family of cell receptors, act as important regulators of diverse signaling pathways. Our understanding of the impact of GPCRs in tumors is emerging, yet there is no therapeutic platform based on GPCR driver genes. As cancer progresses, it disrupts normal epithelial organization and maintains the cells outside their normal niche. The dynamic and flexible microenvironment of a tumor contains both soluble and matrix-immobilized proteases that contribute to the process of cancer advancement. An example is the activation of cell surface protease-activated receptors (PARs). Mammalian PARs are a subgroup of GPCRs that form a family of four members, PAR1â»4, which are uniquely activated by proteases found in the microenvironment. PAR1 and PAR2 play central roles in tumor biology, and PAR3 acts as a coreceptor. The significance of PAR4 in neoplasia is just beginning to emerge. PAR1 has been shown to be overexpressed in malignant epithelia, in direct correlation with tumor aggressiveness, but there is no expression in normal epithelium. In this review, the involvement of key transcription factors such as Egr1, p53, Twist, AP2, and Sp1 that control PAR1 expression levels specifically, as well as hormone transcriptional regulation by both estrogen receptors (ER) and androgen receptors (AR) are discussed. The cloning of the human protease-activated receptor 2; Par2 (hPar2) promoter region and transcriptional regulation of estrogen (E2) via binding of the E2â»ER complex to estrogen response elements (ERE) are shown. In addition, evidence that TEA domain 4 (TEAD4) motifs are present within the hPar2 promoter is presented since the YAP oncogene, which plays a central part in tumor etiology, acts via the TEAD4 transcription factor. As of now, no information is available on regulation of the hPar3 promoter. With regard to hPar4, only data showing CpG methylation promoter regulation is available. Characterization of the PAR transcriptional landscape may identify powerful targets for cancer therapies.
Assuntos
Células Epiteliais/patologia , Neoplasias/genética , Neoplasias/patologia , Animais , Sequência de Bases , Humanos , Regiões Promotoras Genéticas , Receptores Ativados por Proteinase/genética , Receptores Ativados por Proteinase/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Despite the fact that G protein-coupled receptors (GPCRs) are the largest signal-conveying receptor family and mediate many physiological processes, their role in tumor biology is underappreciated. Numerous lines of evidence now associate GPCRs and their downstream signaling targets in cancer growth and development. Indeed, GPCRs control many features of tumorigenesis, including immune cell-mediated functions, proliferation, invasion and survival at the secondary site. Technological advances have further substantiated GPCR modifications in human tumors. Among these are point mutations, gene overexpression, GPCR silencing by promoter methylation and the number of gene copies. At this point, it is imperative to elucidate specific signaling pathways of "cancer driver" GPCRs. Emerging data on GPCR biology point to functional selectivity and "biased agonism"; hence, there is a diminishing enthusiasm for the concept of "one drug per GPCR target" and increasing interest in the identification of several drug options. Therefore, determining the appropriate context-dependent conformation of a functional GPCR as well as the contribution of GPCR alterations to cancer development remain significant challenges for the discovery of dominant cancer genes and the development of targeted therapeutics.
Assuntos
Neoplasias/metabolismo , Animais , Humanos , Neoplasias/patologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Studying primordial events in cancer is pivotal for identifying predictive molecular indicators and for targeted intervention. While the involvement of G-protein-coupled receptors (GPCRs) in cancer is growing, GPCR-based therapies are yet rare. Here, we demonstrate the overexpression of protease-activated receptor 2 (PAR2), a GPCR member in the fallopian tubes (FTs) of high-risk BRCA carriers as compared to null in healthy tissues of FT. FTs, the origin of ovarian cancer, are known to express genes of serous tubal intraepithelial carcinoma (STICs), a precursor lesion of high-grade serous carcinoma (HGSC). PAR2 expression in FTs may serve as an early prediction sensor for ovarian cancer. We show now that knocking down Par2 inhibits ovarian cancer peritoneal dissemination in vivo, pointing to the central role of PAR2. Previously we identified pleckstrin homology (PH) binding domains within PAR1,2&4 as critical sites for cancer-growth. These motifs associate with PH-signal proteins via launching a discrete signaling network in cancer. Subsequently, we selected a compound from a library of backbone cyclic peptides generated toward the PAR PH binding motif, namely the lead compound, Pc(4-4). Pc(4-4) binds to the PAR PH binding domain and blocks the association of PH-signal proteins, such as Akt or Etk/Bmx with PAR2. It attenuates PAR2 oncogenic activity. The potent inhibitory function of Pc(4-4) is demonstrated via inhibition of ovarian cancer peritoneal spread in mice. While the detection of PAR2 may serve as a predictor for ovarian cancer, the novel Pc(4-4) compound may serve as a powerful medicament in STICs and ovarian cancer. This is the first demonstration of the involvement of PAR PH binding motif signaling in ovarian cancer and Pc(4-4) as a potential therapy treatment.
RESUMO
While the role of G-protein-coupled receptors (GPCR) in cancer is acknowledged, their underlying signaling pathways are understudied. Protease-activated receptors (PAR), a subgroup of GPCRs, form a family of four members (PAR1-4) centrally involved in epithelial malignancies. PAR4 emerges as a potent oncogene, capable of inducing tumor generation. Here, we demonstrate identification of a pleckstrin-homology (PH)-binding motif within PAR4, critical for colon cancer growth. In addition to PH-Akt/PKB association, other PH-containing signal proteins such as Gab1 and Sos1 also associate with PAR4. Point mutations are in the C-tail of PAR4 PH-binding domain; F347 L and D349A, but not E346A, abrogate these associations. Pc(4-4), a lead backbone cyclic peptide, was selected out of a mini-library, directed toward PAR2&4 PH-binding motifs. It effectively attenuates PAR2&4-Akt/PKB associations; PAR4 instigated Matrigel invasion and migration in vitro and tumor development in vivo. EGFR/erbB is among the most prominent cancer targets. AYPGKF peptide ligand activation of PAR4 induces EGF receptor (EGFR) Tyr-phosphorylation, effectively inhibited by Pc(4-4). The presence of PAR2 and PAR4 in biopsies of aggressive breast and colon cancer tissue specimens is demonstrated. We propose that Pc(4-4) may serve as a powerful drug not only toward PAR-expressing tumors but also for treating EGFR/erbB-expressing tumors in cases of resistance to traditional therapies. Overall, our studies are expected to allocate new targets for cancer therapy. Pc(4-4) may become a promising candidate for future therapeutic cancer treatment.
Assuntos
Neoplasias do Colo , Receptores de Trombina , Proteínas Sanguíneas , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Desenho de Fármacos , Receptores ErbB/genética , Humanos , Oncogenes , Fosfoproteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Trombina/genética , Receptores de Trombina/metabolismoRESUMO
Wolbachia is an intracellular endosymbiont of Brugia malayi parasite whose presence is essential for the survival of the parasite. Treatment of B. malayi-infected jirds with tetracycline eliminates Wolbachia, which affects parasite survival and fitness. In the present study we have tried to identify parasite proteins that are affected when Wolbachia is targeted by tetracycline. For this Wolbachia depleted parasites (B. malayi) were obtained by tetracycline treatment of infected Mongolian jirds (Meriones unguiculatus) and their protein profile after 2-DE separation was compared with that of untreated parasites harboring Wolbachia. Approximately 100 protein spots could be visualized followed by CBB staining of 2-D gel and included for comparative analysis. Of these, 54 showed differential expressions, while two new protein spots emerged (of 90.3 and 64.4 kDa). These proteins were subjected to further analysis by MALDI-TOF for their identification using Brugia coding sequence database composed of both genomic and EST sequences. Our study unravels two crucial findings: (i) the parasite or Wolbachia proteins, which disappeared/down-regulated appear be essential for parasite survival and may be used as drug targets and (ii) tetracycline treatment interferes with the regulatory machinery vital for parasites cellular integrity and defense and thus could possibly be a molecular mechanism for the killing of filarial parasite. This is the first proteomic study substantiating the wolbachial genome integrity with its nematode host and providing functional genomic data of human lymphatic filarial parasite B. malayi.
Assuntos
Brugia Malayi/metabolismo , Brugia Malayi/microbiologia , Proteínas de Helminto/metabolismo , Parasitos/metabolismo , Parasitos/microbiologia , Tetraciclina/farmacologia , Wolbachia/efeitos dos fármacos , Animais , Anticorpos Anti-Helmínticos , Brugia Malayi/efeitos dos fármacos , Brugia Malayi/imunologia , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Parasitos/efeitos dos fármacos , Reação em Cadeia da Polimerase , Proteoma/análise , Reprodutibilidade dos TestesRESUMO
Protease-activated receptor-2 (PAR2) plays a central role in cancer; however, the molecular machinery of PAR2-instigated tumors remains to be elucidated. We show that PAR2 is a potent inducer of ß-catenin stabilization, a core process in cancer biology, leading to its transcriptional activity. Novel association of low-density lipoprotein-related protein 6 (LRP6), a known coreceptor of Frizzleds (Fz), with PAR2 takes place following PAR2 activation. The association between PAR2 and LRP6 was demonstrated employing co-immunoprecipitation, bioluminescence resonance energy transfer (BRET), and confocal microscopy analysis. The association was further supported by ZDOCK protein-protein server. PAR2-LRP6 interaction promotes rapid phosphorylation of LRP6, which results in the recruitment of Axin. Confocal microscopy of PAR2-driven mammary gland tumors in vivo, as well as in vitro confirms the association between PAR2 and LRP6. Indeed, shRNA silencing of LRP6 potently inhibits PAR2-induced ß-catenin stabilization, demonstrating its critical role in the induced path. We have previously shown a novel link between protease-activated receptor-1 (PAR1) and ß-catenin stabilization, both in a transgenic (tg) mouse model with overexpression of human PAR1 (hPar1) in the mammary glands, and in cancer epithelial cell lines. Unlike in PAR1-Gα13 axis, both Gα12 and Gα13 are equally involved in PAR2-induced ß-catenin stabilization. Disheveled (DVL) is translocated to the cell nucleus through the DVL-PDZ domain. Collectively, our data demonstrate a novel PAR2-LRP6-Axin interaction as a key axis of PAR2-induced ß-catenin stabilization in cancer. This newly described axis enhances our understanding of cancer biology, and opens new avenues for future development of anti-cancer therapies.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Receptores Acoplados a Proteínas G/metabolismo , beta Catenina/química , Sequência de Aminoácidos , Apoptose , Proteína Axina/genética , Proteína Axina/metabolismo , Biomarcadores Tumorais/genética , Proliferação de Células , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Neoplasias/genética , Fosforilação , Conformação Proteica , RNA Interferente Pequeno/genética , Receptor PAR-2 , Receptores Acoplados a Proteínas G/genética , Homologia de Sequência , Transdução de Sinais , Células Tumorais Cultivadas , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
AIM: Wolbachia is a promising antifilarial chemotherapeutic target. Translation initiation factor-1 (Tl IF-1) is an essential factor in prokaryotes. Functional characterization of Wolbachia's novel proteins/enzymes is necessary for the development of adulticidal drugs. MATERIALS & METHODS: Mutant, Wol Tl IF-1 R45D was constructed by site directed mutagenesis. Fluorimetry and size exclusion chromatography were used to determine the biophysical characteristics. Mobility shift assay and fluorescence resonance energy transfer were used to investigate the functional aspect of Wol Tl IF-1 with its mutant. RESULTS: Both wild and mutant were in monomeric native conformations. Wild exhibits nonspecific binding with ssRNA/ssDNA fragments under electrostatic conditions and showed annealing and displacement of RNA strands in comparison to mutant. CONCLUSION: Point mutation impaired RNA chaperone activity of the mutant and its interaction with nucleotides.
Assuntos
Arginina , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fator de Iniciação 1 em Procariotos/genética , Fator de Iniciação 1 em Procariotos/metabolismo , Wolbachia/genética , Wolbachia/metabolismo , Animais , Proteínas de Bactérias/química , Evolução Biológica , Brugia Malayi/microbiologia , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Mutagênese Sítio-Dirigida , Filogenia , Mutação Puntual , Fator de Iniciação 1 em Procariotos/química , Ligação Proteica , RNA/metabolismo , Alinhamento de SequênciaRESUMO
OBJECTIVES: Wolbachia, an endosymbiont of filarial nematode, is considered a promising target for therapy against lymphatic filariasis. Transcription elongation factor GreA is an essential factor that mediates transcriptional transition from abortive initiation to productive elongation by stimulating the escape of RNA polymerase (RNAP) from native prokaryotic promoters. Upon screening of 6257 essential bacterial genes, 57 were suggested as potential future drug targets, and GreA is among these. The current study emphasized the characterization of Wol GreA with its domains. METHODOLOGY/PRINCIPAL FINDINGS: Biophysical characterization of Wol GreA with its N-terminal domain (NTD) and C-terminal domain (CTD) was performed with fluorimetry, size exclusion chromatography, and chemical cross-linking. Filter trap and far western blotting were used to determine the domain responsible for the interaction with α2ßß'σ subunits of RNAP. Protein-protein docking studies were done to explore residual interaction of RNAP with Wol GreA. The factor and its domains were found to be biochemically active. Size exclusion and chemical cross-linking studies revealed that Wol GreA and CTD exist in a dimeric conformation while NTD subsists in monomeric conformation. Asp120, Val121, Ser122, Lys123, and Ser134 are the residues of CTD through which monomers of Wol GreA interact and shape into a dimeric conformation. Filter trap, far western blotting, and protein-protein docking studies revealed that dimeric CTD of Wol GreA through Lys82, Ser98, Asp104, Ser105, Glu106, Tyr109, Glu116, Asp120, Val121, Ser122, Ser127, Ser129, Lys140, Glu143, Val147, Ser151, Glu153, and Phe163 residues exclusively participates in binding with α2ßß'σ subunits of polymerase. CONCLUSIONS/SIGNIFICANCE: To the best of our knowledge, this research is the first documentation of the residual mode of action in wolbachial mutualist. Therefore, findings may be crucial to understanding the transcription mechanism of this α-proteobacteria and in deciphering the role of Wol GreA in filarial development.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Fatores de Transcrição/metabolismo , Wolbachia/enzimologia , Sequência de Aminoácidos , Cromatografia em Gel , Reagentes de Ligações Cruzadas/metabolismo , Fluorometria , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Conformação Proteica , Elongação da Transcrição Genética , Fatores de Transcrição/químicaRESUMO
The intracellular alphaproteobacteria, Wolbachia, is considered to be a future antimacrofilarial drug target as it is obligatory for filarial endurance. Characterizing wolbachial proteins is necessary to understand wolbachial mechanisms and also for discovering new drug entities. Translation initiation factor-1 (Tl IF-1) is an indispensable prokaryotic factor concerned with bacterial viability. This factor is prioritized as one of the most potent antibacterial drug target. To investigate its role in filarial biology, recombinant Wol Tl IF-1 was purified on metal ion column. The factor was found folded in its monomeric native conformation, and contained a buried fluorophore. Molecular modeling revealed that the factor belonged to the Oligomer Binding family, and consisted of the highly conserved S1 domain with 81.6% of the amino acids occupying the allowed regions in Ramachandran plot. In addition, Wol Tl IF-1 exhibited selective binding to the 30S ribosomal subunit, which declined progressively with tetracycline addition. Tetracycline perturbs interaction of Thr18 and Asn32 of the factor with ribosomal protein S4. The factor was immune-localized in adult, microfilariae (Mf) and infective larvae (L3) of Brugia malayi by immunoblotting. High expression was also observed in Wolbachia within B. malayi Mf, L3 and female adult parasite along the gravid uteri by the confocal microscopy. Therefore, Wol Tl IF-1 appears to be an essential Wolbachia factor whose inhibition leads to extensive cell apoptosis and premature killing of adult worms, validating the antifilarial potential of the factor.
Assuntos
Antibacterianos/farmacologia , Brugia Malayi/microbiologia , Fator de Iniciação 1 em Procariotos/biossíntese , Biossíntese de Proteínas/efeitos dos fármacos , Tetraciclina/farmacologia , Wolbachia/efeitos dos fármacos , Wolbachia/genética , Animais , Feminino , Perfilação da Expressão Gênica , Immunoblotting , Masculino , Camundongos Endogâmicos BALB C , Microscopia Confocal , Modelos Moleculares , Fator de Iniciação 1 em Procariotos/química , Fator de Iniciação 1 em Procariotos/isolamento & purificação , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Dobramento de Proteína , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismoRESUMO
Wolbachia, the intracellular alpha-proteobacteria are required for the development, fertility and survival of filarial parasites. Wolbachia Translation initiation factor-1 (Wol Tl IF-1) is one of the factors required for Wolbachia growth and viability. In the present study, we cloned, over expressed and purified Wol Tl IF-1 that exhibited strong immuno-reactivity with various categories of bancroftian sera. Immunization with the recombinant protein resulted into significant reduction in microfilarial density (70-72%) and adult worm establishment (61-63%) in susceptible Mastomys coucha. Protection offered by Wol Tl IF-1 was found associated with humoral immune arm as observed by an increased antibody level with preponderance of IgE, IgM, IgG1 and IgG2a isotypes. The anti-Wol Tl IF-1 antibodies promoted profound adherence of peritoneal exudates cells to the surface of microfilariae and infective larvae causing cytotoxicity and their death. The present study indicates potential of recombinant Wol Tl IF-1 as a promising vaccine candidate against human lymphatic filarial infection.
Assuntos
Brugia Malayi/imunologia , Filariose Linfática/prevenção & controle , Fator de Iniciação 1 em Procariotos/imunologia , Células Th2/imunologia , Wolbachia/imunologia , Animais , Linfócitos B/imunologia , Adesão Celular/imunologia , Clonagem Molecular , Reações Cruzadas/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Citotoxicidade Imunológica , Feminino , Expressão Gênica , Antígenos de Histocompatibilidade Classe II/imunologia , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Ativação Linfocitária , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Murinae , Fator de Iniciação 1 em Procariotos/genética , Fator de Iniciação 1 em Procariotos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Wolbachia/genéticaRESUMO
Although recombinant vaccines have several advantages over conventional vaccines, protection induced by single antigen vaccines is often inadequate for a multicellular helminth parasite. Therefore, immunoprophylactic efficacy of cocktail antigen vaccines comprised of several combinations of three Brugia malayi recombinant proteins BmAF-Myo, Bm-iPGM and Bm-TPP were evaluated. Myosin+TPP and iPGM+TPP provided the best protection upon B. malayi infective larval challenge with â¼70% reduction in adult worm establishment over non-vaccinated animals that was significantly higher than the protection achieved by any single antigen vaccine. Myosin+iPGM, in contrast did not provide any enhance protection over the single recombinant protein vaccines. Specific IgG, IgM level, IgG antibody subclasses levels (IgG1, IgG2a, IgG2b, IgG3), lymphocyte proliferation, reactive oxygen species level and cytokines level were also determined to elucidate the characteristics of the protective immune responses. Thus the study undertaken provided more insight into the cocktail vaccination approach to combat LF.
Assuntos
Brugia Malayi/imunologia , Filariose Linfática/imunologia , Vacinas Protozoárias/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/imunologia , Animais , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Citocinas/biossíntese , Filariose Linfática/parasitologia , Filariose Linfática/prevenção & controle , Imunização , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Larva/imunologia , Ativação Linfocitária/imunologia , Murinae/parasitologia , Proteínas de Protozoários/administração & dosagem , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas Sintéticas/administração & dosagemRESUMO
The lymphatic filarial parasite, Brugia malayi contains Wolbachia endobacteria that are essential for development, viability and fertility of the parasite. Therefore, wolbachial proteins have been currently seen as the potential antifilarial drug targets. NAD(+)-dependent DNA ligase is characterized as a promising drug target in several organisms due to its crucial, indispensable role in DNA replication, recombination and DNA repair. We report here the cloning, expression and purification of NAD(+)-dependent DNA ligase of Wolbachia endosymbiont of B. malayi (wBm-LigA) for its molecular characterization. wBm-LigA has all the domains that are present in nearly all the eubacterial NAD(+)-dependent DNA ligases such as N-terminal adenylation domain, OB fold, helix-hairpin-helix (HhH) and BRCT domain except zinc-binding tetracysteine domain. The purified recombinant protein (683-amino acid) was found to be biochemically active and was present in its native form as revealed by the circular dichroism and fluorescence spectra. The purified recombinant enzyme was able to catalyze intramolecular strand joining on a nicked DNA as well as intermolecular joining of the cohesive ends of BstEII restricted lamda DNA in an in vitro assay. The enzyme was localized in the various life-stages of B. malayi parasites by immunoblotting and high enzyme expression was observed in Wolbachia within B. malayi microfilariae and female adult parasites along the hypodermal chords and in the gravid portion as evident by the confocal microscopy. Ours is the first report on this enzyme of Wolbachia and these findings would assist in validating the antifilarial drug target potential of wBm-LigA in future studies.