RESUMEN
Mitochondria are double membrane-bound cellular work-horses constantly functioning to regulate vital aspects of cellular metabolism, bioenergetics, proliferation and death. Biogenesis, homeostasis and regulated turnover of mitochondria are stringently regulated to meet the bioenergetic requirements. Diverse external and internal stimuli including oxidative stress, diseases, xenobiotics and even age profoundly affect mitochondrial integrity. Damaged mitochondria need immediate segregation and selective culling to maintain physiological homeostasis. Mitophagy is a specialised form of macroautophagy that constantly checks mitochondrial quality followed by elimination of rogue mitochondria by lysosomal targeting through multiple pathways tightly regulated and activated in context-specific manners. Mitophagy is implicated in diverse oxidative stress-associated metabolic, proliferating and degenerative disorders owing to the centrality of mitopathology in diseases as well as the common mandate to eliminate damaged mitochondria for restoring physiological homeostasis. With improved health care and growing demand for precision medicine, specifically targeting the keystone factors in pathogenesis, more exploratory studies are focused on mitochondrial quality control as underlying guardian of cellular pathophysiology. In this context, mitophagy emerged as a promising area to focus biomedical research for identifying novel therapeutic targets against diseases linked with physiological redox perturbation. The present review provides a comprehensive account of the recent developments on mitophagy along with precise discussion on its impact on major diseases and possibilities of therapeutic modulation.
Asunto(s)
Metabolismo Energético/genética , Mitocondrias/genética , Mitofagia/genética , Estrés Oxidativo/genética , Animales , Autofagia/genética , Homeostasis/genética , Humanos , Lisosomas/genética , Lisosomas/metabolismo , Mitocondrias/metabolismo , Control de Calidad , Estrés Fisiológico/genéticaRESUMEN
The subcellular mechanism by which nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in gastric cancer and normal mucosal cells is elusive because of the diverse cyclooxygenase-independent effects of these drugs. Using human gastric carcinoma cells (AGSs) and a rat gastric injury model, here we report that the NSAID indomethacin activates the protein kinase Cζ (PKCζ)-p38 MAPK (p38)-dynamin-related protein 1 (DRP1) pathway and thereby disrupts the physiological balance of mitochondrial dynamics by promoting mitochondrial hyper-fission and dysfunction leading to apoptosis. Notably, DRP1 knockdown or SB203580-induced p38 inhibition reduced indomethacin-induced damage to AGSs. Indomethacin impaired mitochondrial dynamics by promoting fissogenic activation and mitochondrial recruitment of DRP1 and down-regulating fusogenic optic atrophy 1 (OPA1) and mitofusins in rat gastric mucosa. Consistent with OPA1 maintaining cristae architecture, its down-regulation resulted in EM-detectable cristae deformity. Deregulated mitochondrial dynamics resulting in defective mitochondria were evident from enhanced Parkin expression and mitochondrial proteome ubiquitination. Indomethacin ultimately induced mitochondrial metabolic and bioenergetic crises in the rat stomach, indicated by compromised fatty acid oxidation, reduced complex I- associated electron transport chain activity, and ATP depletion. Interestingly, Mdivi-1, a fission-preventing mito-protective drug, reversed indomethacin-induced DRP1 phosphorylation on Ser-616, mitochondrial proteome ubiquitination, and mitochondrial metabolic crisis. Mdivi-1 also prevented indomethacin-induced mitochondrial macromolecular damage, caspase activation, mucosal inflammation, and gastric mucosal injury. Our results identify mitochondrial hyper-fission as a critical and common subcellular event triggered by indomethacin that promotes apoptosis in both gastric cancer and normal mucosal cells, thereby contributing to mucosal injury.
Asunto(s)
Apoptosis/efectos de los fármacos , GTP Fosfohidrolasas/metabolismo , Mucosa Gástrica/enzimología , Indometacina/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/enzimología , Dinámicas Mitocondriales/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína Quinasa C/metabolismo , Neoplasias Gástricas/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Apoptosis/genética , Línea Celular Tumoral , Dinaminas , GTP Fosfohidrolasas/genética , Mucosa Gástrica/patología , Humanos , Sistema de Señalización de MAP Quinasas/genética , Proteínas Asociadas a Microtúbulos/genética , Mitocondrias/genética , Dinámicas Mitocondriales/genética , Proteínas Mitocondriales/genética , Proteínas de Neoplasias/genética , Proteína Quinasa C/genética , Ratas , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
The indispensable role of macrophage migration inhibitory factor (MIF) in cancer cell proliferation is unambiguous, although which specific roles the cytokine plays to block apoptosis by preserving cell growth is still obscure. Using different cancer cell lines (AGS, HepG2, HCT116, and HeLa), here we report that the silencing of MIF severely deregulated mitochondrial structural dynamics by shifting the balance toward excess fission, besides inducing apoptosis with increasing sub-G0 cells. Furthermore, enhanced mitochondrial Bax translocation along with cytochrome c release, down-regulation of Bcl-xL, and Bcl-2 as well as up-regulation of Bad, Bax, and p53 indicated the activation of a mitochondrial pathway of apoptosis upon MIF silencing. The data also indicate a concerted down-regulation of Opa1 and Mfn1 along with a significant elevation of Drp1, cumulatively causing mitochondrial fragmentation upon MIF silencing. Up-regulation of Drp1 was found to be further coupled with fissogenic serine 616 phosphorylation and serine 637 dephosphorylation, thus ensuring enhanced mitochondrial translocation. Interestingly, MIF silencing was found to be associated with decreased NF-κB activation. In fact, NF-κB knockdown in turn increased mitochondrial fission and cell death. In addition, the silencing of CD74, the cognate receptor of MIF, remarkably increased mitochondrial fragmentation in addition to preventing cell proliferation, inducing mitochondrial depolarization, and increasing apoptotic cell death. This indicates the active operation of a MIF-regulated CD74-NF-κB signaling axis for maintaining mitochondrial stability and cell growth. Thus, we propose that MIF, through CD74, constitutively activates NF-κB to control mitochondrial dynamics and stability for promoting carcinogenesis via averting apoptosis.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Dinámicas Mitocondriales , FN-kappa B/metabolismo , Transducción de Señal , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Regulación hacia Abajo , Dinaminas , GTP Fosfohidrolasas/metabolismo , Silenciador del Gen , Humanos , Factores Inhibidores de la Migración de Macrófagos/deficiencia , Factores Inhibidores de la Migración de Macrófagos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Mitocondriales/metabolismo , Transporte de Proteínas , Regulación hacia ArribaRESUMEN
Retromer complex plays a crucial role in intracellular protein trafficking and is conserved throughout the eukaryotes including malaria parasite, Plasmodium falciparum, where it is partially conserved. The assembly of retromer complex in RBC stages of malarial parasite is extremely difficult to explore because of its complicated physiology, small size, and intra-erythrocytic location. Nonetheless, understanding of retromer assembly may pave new ways for the development of novel antimalarials targeting parasite-specific protein trafficking pathways. Here, we investigated the assembly of retromer complex in P. falciparum, by an immunosensing method through highly sensitive Surface Plasmon Resonance (SPR) technique. After taking leads from the bioinformatics search and literature, different interacting proteins were identified and specific antibodies were raised against them. The sensor chip was prepared by covalently linking antibody specific to one component and the whole cell lysate was passed through it in order to trap the interacting complex. Antibodies raised against other interacting components were used to detect them in the trapped complex on the SPR chip. We were able to detect three different components in the retromer complex trapped by the immobilized antibody specific against a different component on a sensor chip. The assay was reproduced and validated in a different two-component CD74-MIF system in mammalian cells. We, thus, illustrate the assembly of retromer complex in P. falciparum through a bio-sensing approach that combines SPR with immunosensing requiring a very small amount of sample from the native source.
Asunto(s)
Técnicas Biosensibles , Complejos Multiproteicos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Resonancia por Plasmón de Superficie , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Western Blotting , Biología Computacional , Células Hep G2 , Humanos , Inmunoprecipitación , Cinética , Ratones , Complejos Multiproteicos/genética , Complejos Multiproteicos/inmunología , Células 3T3 NIH , Plasmodium falciparum/genética , Plasmodium falciparum/inmunología , Unión Proteica , Transporte de Proteínas , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/inmunologíaRESUMEN
Alba family proteins are small, basic, dimeric nucleic acid-binding proteins, which are widely distributed in archaea and a number of eukaryotes. This family of proteins bears the distinct features of regulation through acetylation/deacetylation, hence named as acetylation lowers binding affinity (Alba). Alba family proteins bind DNA cooperatively with no apparent sequence specificity. Besides DNA, Alba proteins also interact with diverse RNA species and associate with ribonucleo-protein complexes. Initially, Alba proteins were recognized as chromosomal proteins and supposed to be involved in the maintenance of chromatin architecture and transcription repression. However, recent studies have shown increasing evidence of functional plasticity among Alba family of proteins that widely range from genome packaging and organization, transcriptional and translational regulation, RNA metabolism, and development and differentiation processes. In recent years, Alba family proteins have attracted growing interest due to their widespread occurrence in large number of organisms. Presence in multiple copies, functional crosstalk, differential binding affinity, and posttranslational modifications are some of the key factors that might regulate the biological functions of Alba family proteins. In this review article, we present an overview of the Alba family proteins, their salient features and emphasize their functional role in different organisms reported so far.
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Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Cromatina/genética , Cromatina/metabolismo , Cristalografía por Rayos X , ADN/química , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Familia de Multigenes/genética , Unión Proteica/genética , Conformación Proteica , ARN/genética , ARN/metabolismo , Relación Estructura-ActividadRESUMEN
We synthesized a new series of conjugated hydrazones that were found to be active against malaria parasite in vitro, as well as in vivo in a murine model. These hydrazones concentration-dependently chelated free iron and offered antimalarial activity. Upon screening of the synthesized hydrazones, compound 5f was found to be the most active iron chelator, as well as antiplasmodial. Compound 5f also interacted with free heme (KD [equilibrium dissociation constant] = 1.17 ± 0.8 µM), an iron-containing tetrapyrrole released after hemoglobin digestion by the parasite, and inhibited heme polymerization by parasite lysate. Structure-activity relationship studies indicated that a nitrogen- and sulfur-substituted five-membered aromatic ring present within the benzothiazole hydrazones might be responsible for their antimalarial activity. The dose-dependent antimalarial and heme polymerization inhibitory activities of the lead compound 5f were further validated by following [(3)H]hypoxanthine incorporation and hemozoin formation in parasite, respectively. It is worth mentioning that compound 5f exhibited antiplasmodial activity in vitro against a chloroquine/pyrimethamine-resistant strain of Plasmodium falciparum (K1). We also evaluated in vivo antimalarial activity of compound 5f in a murine model where a lethal multiple-drug-resistant strain of Plasmodium yoelii was used to infect Swiss albino mice. Compound 5f significantly suppressed the growth of parasite, and the infected mice experienced longer life spans upon treatment with this compound. During in vitro and in vivo toxicity assays, compound 5f showed minimal alteration in biochemical and hematological parameters compared to control. In conclusion, we identified a new class of hydrazone with therapeutic potential against malaria.
Asunto(s)
Antimaláricos/farmacología , Benzotiazoles/farmacología , Hidrazonas/farmacología , Animales , Antimaláricos/síntesis química , Antimaláricos/química , Benzotiazoles/síntesis química , Benzotiazoles/química , Cloroquina/química , Cloroquina/farmacología , Resistencia a Múltiples Medicamentos , Hidrazonas/síntesis química , Hidrazonas/química , Hierro/química , Masculino , Ratones , Plasmodium falciparum/efectos de los fármacos , Plasmodium yoelii/efectos de los fármacos , Pirimetamina/química , Pirimetamina/farmacologíaRESUMEN
Translocation of various proteins to the subcellular organelles is an essential mechanism to regulate the metabolic pathways and often vacuolar protein sorting (VPS) proteins are involved in this transportation. Plasmodium falciparum VPS29 (PfVPS29) is predicted to be a functional component in the assembly of the retromer complex; however, so far detailed characterization of PfVPS29 in its native form is not yet done. We report the successful expression and purification of tag-free recombinant PfVPS29 with a yield of 5.6 mg from 1 L of Escherichia coli culture. PfVPS29 was purified by combined anion-exchange and size exclusion chromatography. The protein showed a single band in SDS-PAGE and it exhibited molecular mass of 21.7 kDa as measured by MALDI-TOF mass spectrometry. Secondary structure was elucidated by circular dichroism spectroscopy. It was found to be a monomeric protein in solution as evident from dynamic light scattering studies, chemical cross-linking experiments and size exclusion chromatography. Subsequently, polyclonal anti-PfVPS29 antibody was generated and used for evaluating protein expression by western blot and following subcellular localization in P. falciparum by confocal immunofluoroscence microscopy. PfVPS29 was found to be located in cytoplasm and expressed from early trophozoite to schizont stages with maximum expression in trophozoite stage. This study provides purification, biophysical characterization and subcellular localization of PfVPS29 in different asexual stages of P. falciparum.
Asunto(s)
Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Secuencia de Aminoácidos , Western Blotting , Dicroismo Circular , Clonación Molecular , Citoplasma/metabolismo , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Estadios del Ciclo de Vida , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Plasmodium falciparum/fisiología , Estructura Secundaria de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/aislamiento & purificaciónRESUMEN
The liver efficiently restores function after damage induced during malarial infection once the parasites are cleared from the blood. However, the molecular events leading to the restoration of liver function after malaria are still obscure. To study this, we developed a suitable model wherein mice infected with Plasmodium yoelii (45% parasitemia) were treated with the antimalarial α/ß-arteether to clear parasites from the blood and, subsequently, restoration of liver function was monitored. Liver function tests clearly indicated that complete recovery of liver function occurred after 25 days of parasite clearance. Analyses of proinflammatory gene expression and neutrophil infiltration further indicated that hepatic inflammation, which was induced immediately after parasite clearance from the blood, was gradually reduced. Moreover, the inflammation in the liver after parasite clearance was found to be correlated positively with oxidative stress and hepatocyte apoptosis. We investigated the role of heme oxygenase 1 (HO-1) in the restoration of liver function after malaria because HO-1 normally renders protection against inflammation, oxidative stress, and apoptosis under various pathological conditions. The expression and activity of HO-1 were found to be increased significantly after parasite clearance. We even found that chemical silencing of HO-1 by use of zinc protoporphyrin enhanced inflammation, oxidative stress, hepatocyte apoptosis, and liver injury. In contrast, stimulation of HO-1 by cobalt protoporphyrin alleviated liver inflammation and reduced oxidative stress, hepatocyte apoptosis, and associated tissue injury. Therefore, we propose that selective induction of HO-1 in the liver would be beneficial for the restoration of liver function after parasite clearance.
Asunto(s)
Antimaláricos/uso terapéutico , Hemo-Oxigenasa 1/metabolismo , Hígado/patología , Malaria/tratamiento farmacológico , Malaria/patología , Plasmodium yoelii/crecimiento & desarrollo , Animales , Pruebas de Función Hepática , Malaria/parasitología , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Plasmodium falciparum , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Inmunoprecipitación de Cromatina , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/análisis , Lisina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/metabolismo , Unión Proteica , Proteínas Protozoarias/análisis , Proteínas de Unión al ARN/química , Sirtuina 2/metabolismo , Transcripción GenéticaRESUMEN
Non-steroidal anti-inflammatory drugs (NSAIDs) are most extensively used over-the-counter FDA-approved analgesic medicines for treating inflammation, musculoskeletal pain, arthritis, pyrexia and menstrual cramps. Moreover, aspirin is widely used against cardiovascular complications. Owing to their non-addictive nature, NSAIDs are also commissioned as safer opioid-sparing alternatives in acute trauma and post-surgical treatments. In fact, therapeutic spectrum of NSAIDs is expanding. These "wonder-drugs" are now repurposed against lung diseases, diabetes, neurodegenerative disorders, fungal infections and most notably cancer, due to their efficacy against chemoresistance, radio-resistance and cancer stem cells. However, prolonged NSAID treatment accompany several adverse effects. Mechanistically, apart from cyclooxygenase inhibition, NSAIDs directly target mitochondria to induce cell death. Interestingly, there are also incidences of dose-dependent effects where NSAIDs are found to improve mitochondrial health thereby suggesting plausible mitohormesis. While mitochondria-targeted effects of NSAIDs are discretely studied, a comprehensive account emphasizing the multiple dimensions in which NSAIDs affect mitochondrial structure-function integrity, leading to cell death, is lacking. This review discusses the current understanding of NSAID-mitochondria interactions in the pathophysiological background. This is essential for assessing the risk-benefit trade-offs of NSAIDs for judiciously strategizing NSAID-based approaches to manage pain and inflammation as well as formulating effective anti-cancer strategies. We also discuss recent developments constituting selective mitochondria-targeted NSAIDs including theranostics, mitocans, chimeric small molecules, prodrugs and nanomedicines that rationally optimize safer application of NSAIDs. Thus, we present a comprehensive understanding of therapeutic merits and demerits of NSAIDs with mitochondria at its cross roads. This would help in NSAID-based disease management research and drug development.
RESUMEN
Alba domain proteins, owing to their functional plasticity, play a significant role in organisms. Here, we report an intrinsic DNase activity of PfAlba6 from Plasmodium falciparum, an etiological agent responsible for human malignant malaria. We identified that tyrosine28 plays a critical role in the Mg2+ driven 5'-3' DNase activity of PfAlba6. PfAlba6 cleaves both dsDNA as well as ssDNA. We also characterized PfAlba6-DNA interaction and observed concentration-dependent oligomerization in the presence of DNA, which is evident from size exclusion chromatography and single molecule AFM-imaging. PfAlba6 mRNA expression level is up-regulated several folds following heat stress and treatment with artemisinin, indicating a possible role in stress response. PfAlba6 has no human orthologs and is expressed in all intra-erythrocytic stages; thus, this protein can potentially be a new anti-malarial drug target.
RESUMEN
Non-canonical nucleotides, generated as oxidative metabolic by-products, significantly threaten the genome integrity of Plasmodium falciparum and thereby, their survival, owing to their mutagenic effects. PfHAM1, an evolutionarily conserved inosine/xanthosine triphosphate pyrophosphohydrolase, maintains nucleotide homeostasis in the malaria parasite by removing non-canonical nucleotides, although structure-function intricacies are hitherto poorly reported. Here, we report the X-ray crystal structure of PfHAM1, which revealed a homodimeric structure, additionally validated by size-exclusion chromatography-multi-angle light scattering analysis. The two monomeric units in the dimer were aligned in a parallel fashion, and critical residues associated with substrate and metal binding were identified, wherein a notable structural difference was observed in the ß-sheet main frame compared to human inosine triphosphate pyrophosphatase. PfHAM1 exhibited Mg++-dependent pyrophosphohydrolase activity and the highest binding affinity to dITP compared to other non-canonical nucleotides as measured by isothermal titration calorimetry. Modifying the pfham1 genomic locus followed by live-cell imaging of expressed mNeonGreen-tagged PfHAM1 demonstrated its ubiquitous presence in the cytoplasm across erythrocytic stages with greater expression in trophozoites and schizonts. Interestingly, CRISPR-Cas9/DiCre recombinase-guided pfham1-null P. falciparum survived in culture under standard growth conditions, indicating its assistive role in non-canonical nucleotide clearance during intra-erythrocytic stages. This is the first comprehensive structural and functional report of PfHAM1, an atypical nucleotide-cleansing enzyme in P. falciparum.
RESUMEN
Gastric cancer (GC) is a deadly malignancy that demands effective therapeutic intervention capitalizing unique drug target/s. Here, we report that indomethacin, a cyclooxygenase non-selective non-steroidal anti-inflammatory drug, arrests GC cell growth by targeting mitochondrial deacetylase Sirtuin 3 (SIRT3). Interaction study revealed that indomethacin competitively inhibited SIRT3 by binding to nicotinamide adenine dinucleotide (NAD)-binding site. The Cancer Genome Atlas data meta-analysis indicated poor prognosis associated with high SIRT3 expression in GC. Further, transcriptome sequencing data of human gastric adenocarcinoma cells revealed that indomethacin treatment severely downregulated SIRT3. Indomethacin-induced SIRT3 downregulation augmented SOD2 and OGG1 acetylation, leading to mitochondrial redox dyshomeostasis, mtDNA damage, respiratory chain failure, bioenergetic crisis, mitochondrial fragmentation, and apoptosis via blocking the AMPK/PGC1α/SIRT3 axis. Indomethacin also downregulated SIRT3 regulators ERRα and PGC1α. Further, SIRT3 knockdown aggravated indomethacin-induced mitochondrial dysfunction as well as blocked cell-cycle progression to increase cell death. Thus, we reveal how indomethacin induces GC cell death by disrupting SIRT3 signaling.
RESUMEN
We have investigated the impact of persistent intravascular hemolysis on liver dysfunction using the mouse malaria model. Intravascular hemolysis showed a positive correlation with liver damage along with the increased accumulation of free heme and reactive oxidants in liver. Hepatocytes overinduced heme oxygenase-1 (HO-1) to catabolize free heme in building up defense against this pro-oxidant milieu. However, in a condition of persistent free heme overload in malaria, the overactivity of HO-1 resulted in continuous transient generation of free iron to favor production of reactive oxidants as evident from 2',7'-dichlorofluorescein fluorescence studies. Electrophoretic mobility shift assay documented the activation of NF-κB, which in turn up-regulated intercellular adhesion molecule 1 as evident from chromatin immunoprecipitation studies. NF-κB activation also induced vascular cell adhesion molecule 1, keratinocyte chemoattractant, and macrophage inflammatory protein 2, which favored neutrophil extravasation and adhesion in liver. The infiltration of neutrophils correlated positively with the severity of hemolysis, and neutrophil depletion significantly prevented liver damage. The data further documented the elevation of serum TNFα in infected mice, and the treatment of anti-TNFα antibodies also significantly prevented neutrophil infiltration and liver injury. Deferoxamine, which chelates iron, interacts with free heme and bears antioxidant properties that prevented oxidative stress, NF-κB activation, neutrophil infiltration, hepatocyte apoptosis, and liver damage. Furthermore, the administration of N-acetylcysteine also prevented NF-κB activation, neutrophil infiltration, hepatocyte apoptosis, and liver damage. Thus, hepatic free heme accumulation, TNFα release, oxidative stress, and NF-κB activation established a link to favor neutrophil infiltration in inducing liver damage during hemolytic conditions in malaria.
Asunto(s)
Hemo/metabolismo , Hemólisis , Hígado/fisiopatología , Malaria/fisiopatología , FN-kappa B/metabolismo , Infiltración Neutrófila , Animales , Secuencia de Bases , Western Blotting , Cartilla de ADN , Modelos Animales de Enfermedad , Ensayo de Cambio de Movilidad Electroforética , Hemo Oxigenasa (Desciclizante)/metabolismo , Etiquetado Corte-Fin in Situ , Hígado/metabolismo , Malaria/parasitología , Masculino , Ratones , Ratones Endogámicos BALB C , Estrés Oxidativo , Plasmodium yoelii/aislamiento & purificación , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We have investigated the gastroprotective effect of SEGA (3a), a newly synthesized tryptamine-gallic acid hybrid molecule against non-steroidal anti-inflammatory drug (NSAID)-induced gastropathy with mechanistic details. SEGA (3a) prevents indomethacin (NSAID)-induced mitochondrial oxidative stress (MOS) and dysfunctions in gastric mucosal cells, which play a pathogenic role in inducing gastropathy. SEGA (3a) offers this mitoprotective effect by scavenging of mitochondrial superoxide anion (O(2)(·-)) and intramitochondrial free iron released as a result of MOS. SEGA (3a) in vivo blocks indomethacin-mediated MOS, as is evident from the inhibition of indomethacin-induced mitochondrial protein carbonyl formation, lipid peroxidation, and thiol depletion. SEGA (3a) corrects indomethacin-mediated mitochondrial dysfunction in vivo by restoring defective electron transport chain function, collapse of transmembrane potential, and loss of dehydrogenase activity. SEGA (3a) not only corrects mitochondrial dysfunction but also inhibits the activation of the mitochondrial pathway of apoptosis by indomethacin. SEGA (3a) inhibits indomethacin-induced down-regulation of bcl-2 and up-regulation of bax genes in gastric mucosa. SEGA (3a) also inhibits indometacin-induced activation of caspase-9 and caspase-3 in gastric mucosa. Besides the gastroprotective effect against NSAID, SEGA (3a) also expedites the healing of already damaged gastric mucosa. Radiolabeled ((99m)Tc-labeled SEGA (3a)) tracer studies confirm that SEGA (3a) enters into mitochondria of gastric mucosal cell in vivo, and it is quite stable in serum. Thus, SEGA (3a) bears an immense potential to be a novel gastroprotective agent against NSAID-induced gastropathy.
Asunto(s)
Antiinflamatorios no Esteroideos/efectos adversos , Apoptosis/efectos de los fármacos , Ácido Gálico/farmacología , Indometacina/efectos adversos , Mitocondrias/metabolismo , Gastropatías/inducido químicamente , Gastropatías/tratamiento farmacológico , Triptaminas/farmacología , Animales , Antiinflamatorios no Esteroideos/farmacología , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Mucosa Gástrica/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Indometacina/farmacología , Peroxidación de Lípido/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Superóxidos/metabolismo , Proteína X Asociada a bcl-2/biosíntesisRESUMEN
Macrophage migration inhibitory factor (MIF) is responsible for proinflammatory reactions in various infectious and non-infectious diseases. We have investigated the mechanism of anti-inflammatory activity of epoxyazadiradione, a limonoid purified from neem (Azadirachta indica) fruits, against MIF. Epoxyazadiradione inhibited the tautomerase activity of MIF of both human (huMIF) and malaria parasites (Plasmodium falciparum (PfMIF) and Plasmodium yoelii (PyMIF)) non-competitively in a reversible fashion (K(i), 2.11-5.23 µm). Epoxyazadiradione also significantly inhibited MIF (huMIF, PyMIF, and PfMIF)-mediated proinflammatory activities in RAW 264.7 cells. It prevented MIF-induced macrophage chemotactic migration, NF-κB translocation to the nucleus, up-regulation of inducible nitric-oxide synthase, and nitric oxide production in RAW 264.7 cells. Epoxyazadiradione not only exhibited anti-inflammatory activity in vitro but also in vivo. We tested the anti-inflammatory activity of epoxyazadiradione in vivo after co-administering LPS and MIF in mice to mimic the disease state of sepsis or bacterial infection. Epoxyazadiradione prevented the release of proinflammatory cytokines such as IL-1α, IL-1ß, IL-6, and TNF-α when LPS and PyMIF were co-administered to BALB/c mice. The molecular basis of interaction of epoxyazadiradione with MIFs was explored with the help of computational chemistry tools and a biological knowledgebase. Docking simulation indicated that the binding was highly specific and allosteric in nature. The well known MIF inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) inhibited huMIF but not MIF of parasitic origin. In contrast, epoxyazadiradione inhibited both huMIF and plasmodial MIF, thus bearing an immense therapeutic potential against proinflammatory reactions induced by MIF of both malaria parasites and human.
Asunto(s)
Antiinflamatorios/farmacología , Limoninas/farmacología , Factores Inhibidores de la Migración de Macrófagos/farmacología , Macrófagos/efectos de los fármacos , Animales , Azadirachta/química , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium yoelii/metabolismoRESUMEN
Plasmodium falciparum Alba domain-containing protein Alba3 (PfAlba3) is ubiquitously expressed in intra-erythrocytic stages of Plasmodium falciparum, but the function of this protein is not yet established. Here, we report an apurinic/apyrimidinic site-driven intrinsic nuclease activity of PfAlba3 assisted by divalent metal ions. Surface plasmon resonance and atomic force microscopy confirm sequence non-specific DNA binding by PfAlba3. Upon binding, PfAlba3 cleaves double-stranded DNA (dsDNA) hydrolytically. Mutational studies coupled with mass spectrometric analysis indicate that K23 is the essential residue in modulating the binding to DNA through acetylation-deacetylation. We further demonstrate that PfSir2a interacts and deacetylates K23-acetylated PfAlba3 in favoring DNA binding. Hence, K23 serves as a putative molecular switch regulating the nuclease activity of PfAlba3. Thus, the nuclease activity of PfAlba3, along with its apurinic/apyrimidinic (AP) endonuclease feature identified in this study, indicates a role of PfAlba3 in DNA-damage response that may have a far-reaching consequence in Plasmodium pathogenicity.