RESUMEN
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
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
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.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa , Plasmodium falciparum , Plasmodium falciparum/genética , Unión Proteica , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN/metabolismo , Reparación del ADNRESUMEN
BACKGROUND AND PURPOSE: Mitochondrial oxidative stress, inflammation and apoptosis primarily underlie gastric mucosal injury caused by the widely used non-steroidal anti-inflammatory drugs (NSAIDs). Alternative gastroprotective strategies are therefore needed. Sirtuin-3 pivotally maintains mitochondrial structural integrity and metabolism while preventing oxidative stress; however, its relevance to gastric injury was never explored. Here, we have investigated whether and how sirtuin-3 stimulation by the phytochemical, honokiol, could rescue NSAID-induced gastric injury. EXPERIMENTAL APPROACH: Gastric injury in rats induced by indomethacin was used to assess the effects of honokiol. Next-generation sequencing-based transcriptomics followed by functional validation identified the gastroprotective function of sirtuin-3. Flow cytometry, immunoblotting, qRT-PCR and immunohistochemistry were used measure effects on oxidative stress, mitochondrial dynamics, electron transport chain function, and markers of inflammation and apoptosis. Sirtuin-3 deacetylase activity was also estimated and gastric luminal pH was measured. KEY RESULTS: Indomethacin down-regulated sirtuin-3 to induce oxidative stress, mitochondrial hyperacetylation, 8-oxoguanine DNA glycosylase 1 depletion, mitochondrial DNA damage, respiratory chain defect and mitochondrial fragmentation leading to severe mucosal injury. Indomethacin dose-dependently inhibited sirtuin-3 deacetylase activity. Honokiol prevented mitochondrial oxidative damage and inflammatory tissue injury by attenuating indomethacin-induced depletion of both sirtuin-3 and its transcriptional regulators PGC1α and ERRα. Honokiol also accelerated gastric wound healing but did not alter gastric acid secretion, unlike lansoprazole. CONCLUSIONS AND IMPLICATIONS: Sirtuin-3 stimulation by honokiol prevented and reversed NSAID-induced gastric injury through maintaining mitochondrial integrity. Honokiol did not affect gastric acid secretion. Sirtuin-3 stimulation by honokiol may be utilized as a mitochondria-based, acid-independent novel gastroprotective strategy against NSAIDs.
Asunto(s)
Sirtuina 3 , Ratas , Animales , Sirtuina 3/metabolismo , Ratas Sprague-Dawley , Antiinflamatorios no Esteroideos/farmacología , Indometacina/toxicidad , Mucosa Gástrica/metabolismo , Apoptosis , Inflamación/metabolismoRESUMEN
Gastroduodenal inflammation and ulcerative injuries are increasing due to expanding socio-economic stress, unhealthy food habits-lifestyle, smoking, alcoholism and usage of medicines like non-steroidal anti-inflammatory drugs. In fact, gastrointestinal (GI) complications, associated with the prevailing COVID-19 pandemic, further, poses a challenge to global healthcare towards safeguarding the GI tract. Emerging evidences have discretely identified mitochondrial dysfunctions as common etiological denominators in diseases. However, it is worth realizing that mitochondrial dysfunctions are not just consequences of diseases. Rather, damaged mitochondria severely aggravate the pathogenesis thereby qualifying as perpetrable factors worth of prophylactic and therapeutic targeting. Oxidative and nitrosative stress due to endogenous and exogenous stimuli triggers mitochondrial injury causing production of mitochondrial damage associated molecular patterns (mtDAMPs), which, in a feed-forward loop, inflicts inflammatory tissue damage. Mitochondrial structural dynamics and mitophagy are crucial quality control parameters determining the extent of mitopathology and disease outcomes. Interestingly, apart from endogenous factors, mitochondria also crosstalk and in turn get detrimentally affected by gut pathobionts colonized during luminal dysbiosis. Although mitopathology is documented in various pre-clinical/clinical studies, a comprehensive account appreciating the mitochondrial basis of GI mucosal pathogenesis is largely lacking. Here we critically discuss the molecular events impinging on mitochondria along with the interplay of mitochondria-derived factors in fueling mucosal damage. We specifically emphasize on the potential role of aberrant mitochondrial dynamics, anomalous mitophagy, mitochondrial lipoxidation and ferroptosis as emerging regulators of GI mucosal pathogenesis. We finally discuss about the prospect of mitochondrial targeting for next-generation drug discovery against GI disorders.
Asunto(s)
COVID-19 , Mitofagia , Alarminas , Humanos , Mitocondrias/patología , Dinámicas Mitocondriales , PandemiasRESUMEN
In recent years, the seasonal patterns of Tropical Cyclones (TC) in the Bay of Bengal have been shifting. While tropical depressions have been common in March-May (spring), they typically have been relatively weaker than the TCs during October-December. Here we show that the spatial pattern of recent warming trends during the last two decades in the southwestern Bay has allowed for stronger springtime pre-monsoon cyclones such as Amphan (May 2020, Super Cyclone) and Fani (April-May 2019, Extremely Severe Cyclone). The tracks of the pre-monsoon cyclones shifted westward, concurrent with an increasing rate of warming. This shift allowed both Fani and Amphan tracks to cross the northeastward warm Western Boundary Current (WBC) and associated warm anti-cyclonic eddies, while the weaker Viyaru (April 2013, Cyclonic Storm) did not interact with the WBC. A quantitative model linking the available along-track heat potential to cyclone's intensity is developed to understand the impact of the WBC on cyclone intensification. The influence of the warming WBC and associated anti-cyclonic eddies will likely result in much stronger springtime TCs becoming relatively common in the future.
RESUMEN
BACKGROUND: Intracellular protein trafficking is crucial for survival of cell and proper functioning of the organelles; however, these pathways are not well studied in the malaria parasite. Its unique cellular architecture and organellar composition raise an interesting question to investigate. METHODS: The interaction of Plasmodium falciparum Rab7 (PfRab7) with vacuolar protein sorting-associated protein 26 (PfVPS26) of retromer complex was shown by coimmunoprecipitation (co-IP). Confocal microscopy was used to show the localization of the complex in the parasite with respect to different organelles. Further chemical tools were employed to explore the role of digestive vacuole (DV) in retromer trafficking in parasite and GTPase activity of PfRab7 was examined. RESULTS: PfRab7 was found to be interacting with retromer complex that assembled mostly near DV and the Golgi in trophozoites. Chemical disruption of DV by chloroquine (CQ) led to its disassembly that was further validated by using compound 5f, a heme polymerization inhibitor in the DV. PfRab7 exhibited Mg2+ dependent weak GTPase activity that was inhibited by a specific Rab7 GTPase inhibitor, CID 1067700, which prevented the assembly of retromer complex in P. falciparum and inhibited its growth suggesting the role of GTPase activity of PfRab7 in retromer assembly. CONCLUSION: Retromer complex was found to be interacting with PfRab7 and the functional integrity of the DV was found to be important for retromer assembly in P. falciparum. GENERAL SIGNIFICANCE: This study explores the retromer trafficking in P. falciparum and describes amechanism to validate DV targeting antiplasmodial molecules.
Asunto(s)
Plasmodium falciparum/metabolismo , Vacuolas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Antimaláricos/farmacología , Cloroquina/farmacología , Humanos , Magnesio/metabolismo , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/metabolismo , Malaria Falciparum/parasitología , Modelos Moleculares , Plasmodium falciparum/efectos de los fármacos , Mapas de Interacción de Proteínas/efectos de los fármacos , Vacuolas/efectos de los fármacos , Proteínas de Unión a GTP rab7RESUMEN
The rapid emergence of resistance against frontline antimalarial drugs essentially warrants the identification of new-generation antimalarials. Here, we describe the synthesis of ( E)-2-isopropyl-5-methyl-4-((2-(pyridin-4-yl)hydrazono)methyl)phenol (18), which binds ferriprotoporphyrin-IX (FeIII-PPIX) ( Kd = 33 nM) and offers antimalarial activity against chloroquine-resistant and sensitive strains of Plasmodium falciparum in vitro. Structure-function analysis reveals that compound 18 binds FeIII-PPIX through the -CâN-NH- moiety and 2-pyridyl substitution at the hydrazine counterpart plays a critical role in antimalarial efficacy. Live cell confocal imaging using a fluorophore-tagged compound confirms its accumulation inside the acidic food vacuole (FV) of P. falciparum. Furthermore, this compound concentration-dependently elevates the pH in FV, implicating a plausible interference with FeIII-PPIX crystallization (hemozoin formation) by a dual function: increasing the pH and binding free FeIII-PPIX. Different off-target bioassays reduce the possibility of the promiscuous nature of compound 18. Compound 18 also exhibits potent in vivo antimalarial activity against chloroquine-resistant P. yoelii and P. berghei ANKA (causing cerebral malaria) in mice with negligible toxicity.