RESUMO
Snakebite envenomation is a serious health concern in tropical regions, resulting in high mortality. The World Health Organization (WHO) has declared it a neglected tropical disease and is working on strategies to reduce mortality. Russell's viper (Daboia russelii) is one of the most abundant venomous snakes found across Southeast Asia. Proteomic analysis of Russell's viper venom has demonstrated variation, with phospholipase A2 (PLA2) being the most abundant toxin across geographic boundaries. PLA2, a major constituent of the low-molecular-weight fraction of snake venom, hydrolyses phospholipids at the sn-2 position, releasing arachidonic acid and lysophospholipids. They are reported to cause various pharmacological effects, including hemolysis, anticoagulation, neurotoxicity, myotoxicity, and oedema. Though administration of antivenoms (ASV) is the primary treatment for envenomation, it has many drawbacks. Besides causing hypersensitivity reactions and life-threatening anaphylaxis, treatment with ASV is further complicated due to its inability to neutralize low-molecular-weight toxins. Thus, there is a greater need to produce next-generation antivenoms that can target specific toxins in the venom. In this review, we explored the classification of Russell's viper and the variation in its proteomic profile across Southeast Asia to date. In addition, we have also summarized the mechanism of action of PLA2 and discussed various isoforms of PLA2 found across different regions with their respective pharmacological effects. Finally, the drawbacks of commercially available antivenoms and the molecules investigated for inhibiting the low-molecular-weight toxin, PLA2 are discussed.
RESUMO
Autophagy is a key step involved in many unicellular eukaryotic diseases, including leishmaniasis, for cellular remodelling and differentiation during parasite's lifecycle. Lipids play a significant role in the infection process that begins with Leishmania major invading host cells. MicroRNAs (miRNAs), a family of small, 22-24 nucleotide noncoding regulatory RNAs, target mRNAs to modify gene expression and, subsequently, proteome output may have a regulatory role in altering the host cell processes. We observed miR-146a-3p expression increases in a time-dependent manner post Leishmania major infection. Transfecting miR-146a-3p mimic increases the expression of ATG7, an autophagy gene that encodes an E1-like enzyme in two ubiquitin-like conjugation systems required for autophagosome progression. HPGD (15-hydroxyprostaglandin dehydrogenase) operates as an enzyme, converting prostaglandin to its non-active form. Microarray data and western studies reveal that miR-146a-3p targets and inhibits HPGD, thereby increasing prostaglandin activity in lipid droplets. Herein, our research focuses on miR-146a-3p, which boosts ATG7 expression while reducing HPGD post Leishmania major infections helping us comprehend the intricate network of microRNA, autophagy, and lipid metabolism in leishmaniasis.
Assuntos
Autofagia , Leishmania major , Leishmaniose Cutânea , Metabolismo dos Lipídeos , MicroRNAs , MicroRNAs/metabolismo , MicroRNAs/genética , Leishmania major/genética , Leishmania major/fisiologia , Leishmania major/metabolismo , Leishmaniose Cutânea/parasitologia , Animais , Camundongos , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína 7 Relacionada à Autofagia/genética , Camundongos Endogâmicos BALB C , Macrófagos/parasitologia , Macrófagos/metabolismo , Humanos , Transfecção , Western BlottingRESUMO
IL-6 and IL-17 are paradoxical cytokines that progress inflammatory states in chronic diseases, including cancer. In lung cancer, their role has been elucidated to favor cancer development by modulating signaling mechanisms critical to cellular growth. The intrinsic ability of these cytokines to influence macroautophagy is yet another reason to facilitate lung cancer. Here, we employed a systems immunology approach to discover the mechanistic role of these cytokines in cancer development. In a biological system, at later stages, the activation of NFkB stimulates immunosuppressive phenotypes to achieve tolerating effects in a transformed cell. We found that the upregulation of cytokines signaled M2 macrophages to modulate tumor responses through the activation of autophagic intermediates and inflammasome mediators. This caused immune perturbations in the tumor microenvironment, which were associated with cancer inflammation. To address these inflammatory states, we performed triggered event analysis to examine whether overexpressing immune effectors or downregulating immune suppressors may have an effect on cancer reversal. Interestingly, the inhibition of immune regulators opposed the model outcome to an increased immune response. Therefore, IL6-IL17-mediated regulation of lung cancer may address tumor malignancy and potentiate the development of newer therapeutics for NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Interleucina-6 , Interleucina-17 , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Citocinas/uso terapêutico , Microambiente TumoralRESUMO
Increasing research suggests that sphingolipid metabolism is essential for the progression and metastasis of cancer. The underlying mechanistic insight into the dysregulation of sphingolipid metabolism affecting pathways is poorly investigated. As a result, the goal of the current study was to glean knowledge from the systems biology approach to investigate how the sphingolipid metabolism affects the signal transduction network in non-small cell lung cancer (NSCLC), the most common type of cancer in terms of occurrence and death globally. Our paper includes system-level models representing the diseased and healthy states elucidating that sphingolipids and its enzymes mediate PI3K/AKT pathway. Notably, its activation of downstream signaling mediators has led to cancer growth. Considering the critical role of sphingolipids in NSCLC, our study advocates the target CERS6 which can be potentially inhibited using hsa-miR-520c-3p to combat NSCLC for future precision medicine.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , MicroRNAs , Humanos , Carcinoma Pulmonar de Células não Pequenas/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Pulmonares/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Regulação Neoplásica da Expressão Gênica , Proliferação de Células , Transdução de Sinais , Esfingolipídeos , Linhagem Celular TumoralRESUMO
The outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in December 2019 and caused coronavirus disease 2019 (COVID-19), which causes pneumonia and severe acute respiratory distress syndrome. It is a highly infectious pathogen that promptly spread. Like other beta coronaviruses, SARS-CoV-2 encodes some non-structural proteins (NSPs), playing crucial roles in viral transcription and replication. NSPs likely have essential roles in viral pathogenesis by manipulating many cellular processes. We performed a sequence-based analysis of NSPs to get insights into their intrinsic disorders, and their functions in viral replication were annotated and discussed in detail. Here, we provide newer insights into the structurally disordered regions of SARS-CoV-2 NSPs. Our analysis reveals that the SARS-CoV-2 proteome has a chunk of the disordered region that might be responsible for increasing its virulence. In addition, mutations in these regions are presumably responsible for drug and vaccine resistance. These findings suggested that the structurally disordered regions of SARS-CoV-2 NSPs might be invulnerable in COVID-19.
Assuntos
COVID-19 , Vacinas , Humanos , SARS-CoV-2RESUMO
In many regions of the world, Leishmaniasis is a cause of substantial mortality and ailment. Due to impediment in available treatment, development of novel and effective treatments is indispensable. Significance of autophagy has been accentuated in infectious disease as well as in Leishmaniasis, and it is having capability to be manifested as a therapeutic target. By evincing autophagy as a novel therapeutic regime, this study emphasized on the critical role of ATG4.1-ATG8 and ATG5-ATG12 complexes in Leishmania species. The objective here was to identify ATG8 as a potential therapeutic target in Leishmania. R71T, P56E, R18P are the significant mutations which shows detrimental effect on ATG8 while Arg276, Arg73, Cys75 of ATG4.1 and Val88, Pro89, Glu116, Asn117, and Gly120 are interacting residues of ATG8. Along with this, we also bring into spotlight an enticing role of Thiabendazole derivatives that interferes with the survival mechanisms by targeting ATG8. Further, the study claims that thiabendazole can be a potential drug candidate to target autophagy process in the infectious disease Leishmaniasis.
Assuntos
Doenças Transmissíveis , Leishmania , Leishmaniose , Autofagia/genética , Humanos , Leishmaniose/tratamento farmacológico , TiabendazolRESUMO
The emergence of drug resistance is a major concern for combating against Cutaneous Leishmaniasis, a neglected tropical disease affecting 98 countries including India. Miltefosine is the only oral drug available for the disease and Miltefosine transporter proteins play a pivotal role in the emergence of drug-resistant Leishmania major. The cause of resistance is less accumulation of drug inside the parasite either by less uptake of the drug due to a decrease in the activity of P4ATPase-CDC50 complex or by increased efflux of the drug by P-glycoprotein (P-gp, an ABC transporter). In this paper, we are trying to allosterically modulate the behavior of resistant parasite (L. major) towards its sensitivity for the existing drug (Miltefosine, a phosphatidylcholine analog). We have used computational approaches to deal with the conservedness of the proteins and apparently its three-dimensional structure prediction through ab initio modeling. Long scale membrane-embedded molecular dynamics simulations were carried out to study the structural interaction and stability. Parasite-specific motifs of these proteins were identified based on the machine learning technique, against which a peptide library was designed. The protein-peptide docking shows good binding energy of peptides Pg5F, Pg8F and PC2 with specific binding to the motifs. These peptides were tested both in vitro and in vivo, where Pg5F in combination with PC2 showed 50-60% inhibition in resistant L. major's promastigote and amastigote forms and 80-90% decrease in parasite load in mice. We posit a model system wherein the data provide sufficient impetus for being novel therapeutics in order to counteract the drug resistance phenotype in Leishmania parasites.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/farmacologia , Regulação Alostérica/efeitos dos fármacos , Leishmania major/metabolismo , Fosforilcolina/análogos & derivados , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/toxicidade , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/farmacologia , Transportadores de Cassetes de Ligação de ATP/toxicidade , Animais , Antiprotozoários/metabolismo , Antiprotozoários/farmacologia , Linhagem Celular , Biologia Computacional/métodos , Resistência a Medicamentos/efeitos dos fármacos , Leishmania major/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Aprendizado de Máquina , Camundongos , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Biblioteca de Peptídeos , Peptídeos/síntese química , Fosforilcolina/metabolismo , Fosforilcolina/farmacologiaRESUMO
Macrophage phenotype plays a crucial role in the pathogenesis of Leishmanial infection. Pro-inflammatory cytokines signals through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway that functions in parasite killing. Suppression of cytokine signaling (SOCS) is a well-known negative feedback regulator of the JAK/STAT pathway. However, change in the expression levels of SOCSs in correlation with the establishment of infection is not well understood. IL6 is a pleotropic cytokine that induces SOCS1 and SOCS3 expression through JAK-STAT signaling. Mathematical modeling of the TLR2 and IL6 signaling pathway has established the immune axis of SOCS1 and SOCS3 functioning in macrophage polarization during the early stage of Leishmania major infection. The ratio has been quantified both in silico and in vitro as 3:2 which is required to establish infection during the early stage. Furthermore, phosphorylated STAT1 and STAT3 have been established as an immunological cross talk between TLR2 and IL6 signaling pathways. Using synthetic biology approaches, peptide based immuno-regulatory circuits have been designed to target the activity of SOCS1 which can restore pro-inflammatory cytokine expression during infection. In a nutshell, we explored the potential of synthetic biology to address and rewire the immune response from Th2 to Th1 type during the early stage of leishmanial infection governed by SOCS1/SOCS3 immune axis.
Assuntos
Interleucina-6/metabolismo , Janus Quinases/antagonistas & inibidores , Leishmania/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Macrófagos/metabolismo , Peptídeos/química , Inibidores de Proteínas Quinases/química , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Peptídeos/farmacologia , Fosforilação , Ligação Proteica , Conformação Proteica , Inibidores de Proteínas Quinases/farmacologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Receptor 2 Toll-Like/metabolismoRESUMO
The tumor suppressor F-box protein 31 (FBXO31) is indispensable for maintaining genomic stability. Its levels drastically increase following DNA damage, leading to cyclin D1 and MDM2 degradation and G1 and G2/M arrest. Prolonged arrest in these phases leads to cellular senescence. Accordingly, FBXO31 needs to be kept at low basal levels in unstressed conditions for normal cell cycle progression during growth and development. However, the molecular mechanism maintaining these basal FBXO31 levels has remained unclear. Here, we identified the F-box family SCF-E3 ubiquitin ligase FBXO46 (SCFFBXO46) as an important proteasomal regulator of FBXO31 and found that FBXO46 helps maintain basal FBXO31 levels under unstressed conditions and thereby prevents premature senescence. Using molecular docking and mutational studies, we showed that FBXO46 recognizes an RXXR motif located at the FBXO31 C terminus to direct its polyubiquitination and thereby proteasomal degradation. Furthermore, FBXO46 depletion enhanced the basal levels of FBXO31, resulting in senescence induction. In response to genotoxic stress, ATM (ataxia telangiectasia-mutated) Ser/Thr kinase-mediated phosphorylation of FBXO31 at Ser-278 maintained FBXO31 levels. In contrast, activated ATM phosphorylated FBXO46 at Ser-21/Ser-67, leading to its degradation via FBXO31. Thus, ATM-catalyzed phosphorylation after DNA damage governs FBXO31 levels and FBXO46 degradation via a negative feedback loop. Collectively, our findings reveal that FBXO46 is a crucial proteasomal regulator of FBXO31 and thereby prevents senescence in normal growth conditions. They further indicate that FBXO46-mediated regulation of FBXO31 is abrogated following genotoxic stress to promote increased FBXO31 levels for maintenance of genomic stability.
Assuntos
Senescência Celular , Proteínas F-Box/metabolismo , Proteínas Ligases SKP Culina F-Box/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Instabilidade Genômica , Humanos , Simulação de Acoplamento Molecular , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , UbiquitinaçãoRESUMO
IL-6 has been proposed to favor the development of Th2 responses and play an important role in the communication between cells of multicellular organisms. They are involved in the regulation of complex cellular processes such as proliferation, differentiation and act as key player during inflammation and immune response. Th2 cytokines play an immunoregulatory role in early infection. Literature says in mice infected with L. major, IL-6 may promote the development of both Th1 and Th2 responses. IL-4 is also considered to be the signature cytokine of Th-2 response. IL-10 was initially characterized as a Th2 cytokine but later on it was proved to be a pleiotropic cytokine, secreted from different cell types including the macrophages. A major challenge is to understand how these complex non-linear processes are connected and regulated. Systems biology approaches may be used to tackle this challenge in an iterative process of quantitative mathematical analysis. In this study, we created an in silico model of IL6 mediated macrophage activation which suffers from an excessive impact of the negative feedback loop involving SOCS3. The strategy adopted in this framework may help to reduce the complexity of the leishmanial IL6 model analysis and also laydown various physiological or pathological conditions of IL6 signaling in future.
Assuntos
Interleucina-6/metabolismo , Leishmaniose/metabolismo , Transdução de Sinais , Biologia de Sistemas , Animais , Simulação por Computador , Camundongos , Modelos Biológicos , Filogenia , Análise de Componente Principal , Receptores Toll-Like/metabolismoRESUMO
Systems and synthetic biology in the coming era has the ability to manipulate, stimulate and engineer cells to counteract the pathogenic immune response. The inherent biological complexities associated with the creation of a device allow capitalizing the biotechnological resources either by simply administering a recombinant cytokine or just reprogramming the immune cells. The strategy outlined, adopted and discussed may mark the beginning with promising therapeutics based on the principles of synthetic immunology.
Assuntos
Alergia e Imunologia , Biologia Sintética/métodos , Big Data , Citocinas/genética , Citocinas/uso terapêutico , Humanos , Modelagem Computacional Específica para o Paciente , Biologia Sintética/tendênciasRESUMO
Miltefosine [hexadecylphosphocholine (HPC)] is the only orally bioavailable drug for the disease visceral leishmaniasis, which is caused by the protozoan parasite Leishmania donovani. Although miltefosine has direct leishmanicidal effects, evidence is mounting for its immune system-dependent effects. The mechanism of such indirect antileishmanial effects of miltefosine remains to be discovered. As platelet-activating factor and HPC share structural semblances and both induce killing of intracellular Leishmania, we surmised that platelet-activating factor (PAF) receptor had a significant role in the antileishmanial function of miltefosine. The proposition was supported by molecular dynamic simulation of HPC docking into PAF receptor and by comparison of its leishmanicidal function on PAF receptor-deficient macrophages and mice under HPC treatment. We observed that compared with wild-type macrophages, the PAF receptor-deficient macrophages showed 1) reduced binding of a fluorescent analog of HPC, 2) decreased TNF-α production, and 3) lower miltefosine-induced killing of L. donovani. Miltefosine exhibited significantly compromised leishmanicidal function in PAF receptor-deficient mice. An anti-PAF receptor Ab led to a significant decrease in miltefosine-induced intracellular Leishmania killing and IFN-γ production in a macrophage-T cell coculture system. These results indicate significant roles for PAF receptor in the leishmanicidal activity of HPC. The findings open new avenues for a more rational understanding of the mechanism of action of this drug as well as for improved therapeutic strategies.
Assuntos
Antiprotozoários/farmacologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/metabolismo , Fosforilcolina/análogos & derivados , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antígenos de Protozoários/imunologia , Antiprotozoários/administração & dosagem , Antiprotozoários/química , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Técnicas de Inativação de Genes , Interferon gama/biossíntese , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/genética , Ligantes , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Modelos Moleculares , Conformação Molecular , Fosforilcolina/administração & dosagem , Fosforilcolina/química , Fosforilcolina/farmacologia , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/química , Glicoproteínas da Membrana de Plaquetas/deficiência , Ligação Proteica , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/deficiênciaRESUMO
Leishmania major is a parasite that resides and replicates in macrophages. We previously showed that the parasite enhanced CD40-induced Raf-MEK-ERK signaling but inhibited PI3K-MKK-p38MAPK signaling to proleishmanial effects. As Raf and PI3K have a Ras-binding domain but exert opposite effects on Leishmania infection, we examined whether Ras isoforms had differential roles in Leishmania infection. We observed that L. major enhanced N-Ras and H-Ras expression but inhibited K-Ras expression in macrophages. L. major infection enhanced N-Ras activity but inhibited H-Ras and K-Ras activity. TLR2 short hairpin RNA or anti-TLR2 or anti-lipophosphoglycan Abs reversed the L. major-altered N-Ras and K-Ras expressions. Pam3CSK4, a TLR2 ligand, enhanced N-Ras expression but reduced K-Ras expression, indicating TLR2-regulated Ras expression in L. major infection. Whereas N-Ras silencing reduced L. major infection, K-Ras and H-Ras silencing enhanced the infection both in macrophages in vitro and in C57BL/6 mice. BALB/c-derived macrophages transduced with lentivirally expressed N-Ras short hairpin RNA and pulsed with L. major-expressed MAPK10 enhanced MAPK10-specific Th1-type response. CD40-deficient mice primed with these macrophages had reduced L. major infection, accompanied by higher IFN-γ but less IL-4 production. As N-Ras is activated by Sos, a guanine nucleotide exchange factor, we modeled the N-Ras-Sos interaction and designed two peptides from their interface. Both the cell-permeable peptides reduced L. major infection in BALB/c mice but not in CD40-deficient mice. These data reveal the L. major-enhanced CD40-induced N-Ras activation as a novel immune evasion strategy and the potential for Ras isoform-targeted antileishmanial immunotherapy and immunoprophylaxis.
Assuntos
Antígenos CD40/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Proteínas Monoméricas de Ligação ao GTP/imunologia , Animais , Antígenos CD40/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Evasão da Resposta Imune/efeitos dos fármacos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , Imunoterapia , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/patologia , Leishmaniose Cutânea/prevenção & controle , Lipopeptídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Proteína Quinase 10 Ativada por Mitógeno/genética , Proteína Quinase 10 Ativada por Mitógeno/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Monoméricas de Ligação ao GTP/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Proteína Son Of Sevenless de Drosófila/genética , Proteína Son Of Sevenless de Drosófila/imunologia , Células Th1/imunologia , Células Th1/patologia , Receptor 2 Toll-Like , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
BACKGROUND: Modulated immune signal (CD14-TLR and TNF) in leishmaniasis can be linked to EGFR pathway involved in wound healing, through crosstalk points. This signaling network can be further linked to a synthetic gene circuit acting as a positive feedback loop to elicit a synchronized intercellular communication among the immune cells which may contribute to a better understanding of signaling dynamics in leishmaniasis. METHODS: Network reconstruction with positive feedback loop, simulation (ODE 15s solver) and sensitivity analysis of CD14-TLR, TNF and EGFR was done in SimBiology (MATLAB 7.11.1). Cytoscape and adjacency matrix were used to calculate network topology. PCA was extracted by using sensitivity coefficient in MATLAB. Model reduction was done using time, flux and sensitivity score. RESULTS: Network has five crosstalk points: NIK, IκB-NFκB and MKK (4/7, 3/6, 1/2) which show high flux and sensitivity. PI3K in EGFR pathway shows high flux and sensitivity. PCA score was high for cytoplasmic ERK1/2, PI3K, Atk, STAT1/3 and nuclear JNK. Of the 125 parameters, 20% are crucial as deduced by model reduction. CONCLUSIONS: EGFR can be linked to CD14-TLR and TNF through the MAPK crosstalk points. These pathways may be controlled through Ras and Raf that lie upstream of signaling components ERK ½ (c) and JNK (n) that have a high PCA score via a synthetic gene circuit for activating cell-cell communication to elicit an inflammatory response. Also a disease resolving effect may be achieved through PI3K in the EGFR pathway. GENERAL SIGNIFICANCE: The reconstructed signaling network can be linked to a gene circuit with a positive feedback loop, for cell-cell communication resulting in synchronized response in the immune cell population, for disease resolving effect in leishmaniasis.
Assuntos
Retroalimentação Fisiológica , Redes Reguladoras de Genes , Leishmania/imunologia , Leishmaniose/imunologia , Leishmaniose/metabolismo , Transdução de Sinais , Genes erbB-1 , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Análise de Componente Principal , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Selection pressure governs the relative mutability and the conservedness of a protein across the protein family. Biomolecules (DNA, RNA and proteins) continuously evolve under the effect of evolutionary pressure that arises as a consequence of the host parasite interaction. IPCS (Inositol phosphorylceramide synthase), SPL (Sphingosine-1-P lyase) and SPT (Serine palmitoyl transferase) represent three important enzymes involved in the sphingolipid metabolism of Leishmania. These enzymes are responsible for maintaining the viability and infectivity of the parasite and have been classified as druggable targets in the parasite metabolome. RESULTS: The present work relates to the role of selection pressure deciding functional conservedness and divergence of the drug targets. IPCS and SPL protein families appear to diverge from the SPT family. The three protein families were largely under the influence of purifying selection and were moderately conserved baring two residues in the IPCS protein which were under the influence of positive selection. To further explore the selection pressure at the codon level, codon usage bias indices were calculated to analyze genes for their synonymous codon usage pattern. IPCS gene exhibited slightly lower codon bias as compared to SPL and SPT protein families. CONCLUSION: Evolutionary tracing of the proposed drug targets has been done with a viewpoint that the amino-acids lining the drug binding pocket should have a lower evolvability. Sites under positive selection (HIS20 and CYS30 of IPCS) should be avoided during devising strategies for inhibitor design.
Assuntos
Evolução Molecular , Leishmania/enzimologia , Leishmania/genética , Esfingolipídeos/metabolismo , Aldeído Liases/química , Aldeído Liases/genética , Aldeído Liases/metabolismo , Aminoácidos/genética , Códon , Hexosiltransferases/química , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Leishmania/metabolismo , Filogenia , Serina C-Palmitoiltransferase/química , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismoRESUMO
Leishmania major causes cutaneous form of Leishmaniasis affecting 21 million people in developing countries. Overuse of the chemotherapeutics against leishmaniasis has resulted in the development of drug resistance in the parasite. To surmount this emerging threat we have attempted to target the surface molecules. Glycosylphosphatidylinositol is one such molecule that is present abundantly and thus our work revolves around the enzyme mannosyltransferase (GPI 14), an enzyme essential to add mannose on the glycosylphosphatidyl. It has been targeted for drug discovery on account of growing resistance to miltefosine in L. major. This paper serves as the first attempt to detect GPI 14 gene in L. major supported with modeling and molecular dynamic analysis of complete three dimensional structure of GPI 14. The functional analysis revealed multiple transmembrane regions in GPI 14 and a close phylogenetic relation with Trypanosoma species and Schistosoma mansoni with highest bootstrap values. The protein model obtained was subjected to minimization for 14ns simulation. Eight derivatives of N-4-(-5(trifluromethyl)-1-methyl-1H benzo[d]imidazole-2 yl) phenyl) were docked onto GPI 14. The contact frequency of GPI 14 with the docked compounds suggested the inhibition of mannosylation proposing the druggability for leishmaniasis therapy.
Assuntos
Desenho de Fármacos , Glicosilfosfatidilinositóis/biossíntese , Leishmania major/metabolismo , Leishmaniose Cutânea/metabolismo , Modelos Moleculares , Sequência de Aminoácidos , Animais , Antiprotozoários/farmacologia , Biocatálise , Simulação por Computador , Sequência Conservada , Leishmania major/efeitos dos fármacos , Simulação de Acoplamento Molecular/métodos , Filogenia , Ligação Proteica , Especificidade da Espécie , Eletricidade EstáticaRESUMO
The hallmark characteristic of macrophages lies in their inherent plasticity, allowing them to adapt to dynamic microenvironments. Leishmania strategically modulates the phenotypic plasticity of macrophages, creating a favorable environment for intracellular survival and persistent infection through regulatory cytokine such as interleukin (IL)-10. Nevertheless, these effector cells can counteract infection by modulating crucial cytokines like IL-12 and key components involved in its production. Using sophisticated tool of single-cell assay for transposase accessible chromatin (ATAC) sequencing, we systematically examined the regulatory axis of IL-10 and IL-12 in a time-dependent manner during Leishmania major infection in macrophages Our analysis revealed the cellular heterogeneity post-infection with the regulators of IL-10 and IL-12, unveiling a reciprocal relationship between these cytokines. Notably, our significant findings highlighted the presence of sleepy macrophages and their pivotal role in mediating reciprocity between IL-10 and IL-12. To summarize, the roles of cytokine expression, transcription factors, cell cycle, and epigenetics of host cell machinery were vital in identification of sleepy macrophages, which is a transient state where transcription factors controlled the epigenetic remodeling and expression of genes involved in pro-inflammatory cytokine expression and recruitment of immune cells.IMPORTANCELeishmaniasis is an endemic affecting 99 countries and territories globally, as outlined in the 2022 World Health Organization report. The disease's severity is compounded by compromised host immune systems, emphasizing the pivotal role of the interplay between parasite and host immune factors in disease regulation. In instances of cutaneous leishmaniasis induced by L. major, macrophages function as sentinel cells. Our findings indicate that the plasticity and phenotype of macrophages can be modulated to express a cytokine profile involving IL-10 and IL-12, mediated by the regulation of transcription factors and their target genes post-L. major infection in macrophages. Employing sophisticated methodologies such as single-cell ATAC sequencing and computational genomics, we have identified a distinctive subset of macrophages termed "sleepy macrophages." These macrophages exhibit downregulated housekeeping genes while expressing a unique set of variable features. This data set constitutes a valuable resource for comprehending the intricate host-parasite interplay during L. major infection.
Assuntos
Leishmania major , Leishmaniose Cutânea , Humanos , Citocinas/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Macrófagos , Leishmaniose Cutânea/parasitologia , Interleucina-12/genética , Interleucina-12/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Cutaneous leishmaniasis caused by the intracellular parasite Leishmania major, exhibits significant public health challenge worldwide. With limited treatment options available, the identification of novel therapeutic targets is of paramount importance. Present study manifested the crucial role of ATG8 protein as a potential target in combating L. major infection. Using machine learning algorithms, we identified non-conserved motifs within the ATG8 in L. major. Subsequently, a peptide library was generated based on these motifs, and three peptides were selected for further investigation through molecular docking and molecular dynamics simulations. Surface Plasmon Resonance (SPR) experiments confirmed the direct interaction between ATG8 and the identified peptides. Remarkably, these peptides demonstrated the ability to cross the parasite membrane and exert profound effects on L. major. Peptide treatment significantly impacted parasite survival, inducing alterations in the cell cycle and morphology. Furthermore, the peptides were found to modulate autophagosome formation, particularly under starved conditions, indicating their involvement in autophagy regulation within L. major. In vitro studies revealed that the selected peptides effectively decreased the parasite load within the infected host cells. Encouragingly, inâ vivo experiments corroborated these findings, demonstrating a reduction in parasite burden upon peptide administration. Additionally, the peptides were observed to affect the levels of LC3II, a known autophagy marker within the host cells. Collectively, our findings highlight the efficacy of these novel peptides in targeting L. major ATG8 and disrupting parasite survival, wherein P2 is showing prominent effect on L. major as compared to P1. These results provide valuable insights into the development of innovative therapeutic strategies against leishmaniasis.
Assuntos
Interações Hospedeiro-Parasita , Leishmaniose Cutânea , Humanos , Simulação de Acoplamento Molecular , Peptídeos/farmacologia , Leishmaniose Cutânea/parasitologia , AutofagiaRESUMO
Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the Leishmania genus. Despite the efforts to control and treat the disease, it still remains a major public health problem in many countries. Synthetic biology is a rapidly evolving interdisciplinary field that combines biology, engineering, and computer science to design and construct novel biological systems. In recent years, synthetic biology approaches have shown great promise for developing new and effective strategies to combat leishmaniasis. In this perspective, we summarize the recent advances in the use of synthetic biology for the development of vaccines, diagnostic tools, and novel therapeutics for leishmaniasis.
RESUMO
Cutaneous Leishmaniasis, an infectious disease is globally the most prevalent form of leishmaniasis accounting for approximately 1 million cases every year as per world health organization. Infected individuals develop skin lesion which has been reported to be infiltrated by immune cells and parasite with high sodium accumulation creating hypertonic environment. In our work, we tried to mimic the hypertonic environment in virtual environment to study dynamicity of SHP-1 and NFAT5 along with their interactions through molecular dynamics simulation. We validated the SHP-1 and NFAT5 dynamics in infection and HSD conditions to study the impact of hypertonicity derived NFAT5 mediated response to L.major infection. We also evaluated our therapeutic peptides for their binding to SHP-1 and to form stable complex. Membrane stability with the peptides was analyzed to understand their ability to sustain mammalian membrane. We identified PepA to be a potential candidate to interact with SHP-1. Inhibition of SHP-1 through PepA to discern IL-10 and IL-12 reciprocity may be assessed in future and furnish us with a potential therapeutic molecule. HSD mice exhibited high pro-inflammatory response to L.major infection which resulted in reduced lesion size. Contrary to observations in HSD mice, infection model exhibited low pro-inflammatory response and increased lesion size with high parasite load. Thus, increase in NFAT5 expression and reduced SHP-1 expression may result in disease resolving effect which can be further studied through incorporation of synthetic circuit using PepA to modulate IL-10 and IL-12 reciprocity.