Leishmania donovani Induces Autophagy in Human Blood-Derived Neutrophils.

Neutrophils, the essential components of the innate immune system, are recruited in large numbers to the pathogen site of entry. Several pathogens induce neutrophil autophagy; however, function of autophagic events during Leishmania parasite infection remain unknown. In this article, we report a finding that is new, to our knowledge, of how Leishmania-induced human polymorphonuclear neutrophil (hPMN) autophagy regulates the silent mode of parasite transfer to macrophages by influencing the engulfment of infected cells. Leishmania infection induced a time-dependent autophagy increase responsive to block by 3-methyladenine but sensitive to ULK1/2 inhibition only after 3 h. This suggested the prevalence of canonical autophagy during later hours, ULK1/2 inhibition being able to block only canonical autophagy. Interaction of Rubicon and Beclin-1 at 1 h postinfection affirmed the prevalence of noncanonical autophagy during early infection. There was a reduction in macrophage uptake of parasite-exposed hPMNs treated with 3-methyladenine or ULK1/2 inhibitor, suggesting the involvement of both noncanonical and canonical autophagy in neutrophil engulfment. Autophagy inducer rapamycin augmented neutrophil engulfment by macrophages. Redistribution of hPMN surface CD47 encouraged neutrophil uptake. Activation of ERK, phosphoinositide 3-kinase, and NADPH oxidase-mediated reactive oxygen species generation were induced after parasite binding. The lpg1-knockout parasites expressing defective lipophosphoglycan did not induce autophagy, indicating that lipophosphoglycan is necessary for interaction with the neutrophils. Autophagy induction was TLR2/4 independent because the receptor blockade did not interfere with infection-induced autophagy. In summary, the engulfment of neutrophils by the macrophages was influenced by the escalation of hPMN autophagy, which is an important event during Leishmania infection.

Sestrins: Darkhorse in the regulation of mitochondrial health and metabolism.

Every disease is an outcome of one or more stress signals which get convened at the interface of the mitochondria. Mitochondria and metabolism are inextricably anchored to each other and a disruption in either can result in the generation of stressors, which can lead to detrimental health consequences. Stowing everything in one frame reflects that the proteins involved in the sensing of stressors are fundamental for the initiation of various pathologies and their detailed study is necessary for proper understanding of disease mechanisms. Sestrins, a class of evolutionarily conserved, stress inducible genes are activated by a wide range of stressors such as oxidative, genotoxic, and metabolic and play a role in cellular homeostasis. In addition, recent reports have highlighted their importance in governing the mitochondrial dynamics and metabolism. However, their spectrum of involvement in various pathologies has not been dissected out very well. This review will focus and discuss the role of Sestrins mainly Sestrin2 and associated nexus in the context of mitochondria, metabolism, and health.

Halictine-2 antimicrobial peptide shows promising anti-parasitic activity against Leishmania spp.

The protozoan parasite Leishmania spp. causes leishmaniases, a group of diseases creating serious health problems in many parts of the world with significant resistance to existing drugs. Insect derived antimicrobial peptides are promising alternatives to conventional drugs against several human disease-causing pathogens because they do not generate resistance. Halictine-2, a novel antimicrobial peptide from the venom of eusocial honeybee, Halictus sexcinctus showed significant anti-leishmanial activity in vitro, towards two life forms of the dimorphic parasite, the free-swimming infective metacyclic promastigotes and the intracellular amastigotes responsible for the systemic infection. The anti-leishmanial activity of the native peptide (P5S) was significantly enhanced by serine to threonine substitution at position 5 (P5T). The peptide showed a propensity to form α-helices after substitution at position-5, conferring amphipathicity. Distinct pores observed on the promastigote membrane after P5T exposure suggested a mechanism of disruption of cellular integrity. Biochemical alterations in the promastigotes after P5T exposure included generation of increased oxygen radicals with mitochondrial Ca2+ release, loss of mitochondrial membrane potential, reduction in total ATP content and increased mitochondrial mass, resulting in quick bioenergetic and chemiosmotic collapse leading to cell death characterized by DNA fragmentation. P5T was able to reduce intracellular amastigote burden in an in vitro model of Leishmania infection but did not alter the proinflammatory cytokines like TNF-α and IL-6. The ability of the P5T peptide to kill the Leishmania parasite with negligible haemolytic activity towards mouse macrophages and human erythrocytes respectively, demonstrates its potential to be considered as a future antileishmanial drug candidate.

Functional Involvement of Leishmania donovani Tryparedoxin Peroxidases during Infection and Drug Treatment.

The parasite Leishmania donovani causes visceral leishmaniasis, a potentially fatal disease. The parasites survive within mammalian macrophages and express a unique set of enzymes, the tryparedoxin peroxidases, for their defense against oxidative stress generated by the host. In this study, we demonstrate different roles of two distinct enzymes, the mitochondrial tryparedoxin peroxidase (mTXNPx) and the cytosolic tryparedoxin peroxidase (cTXNPx), in defending the parasites against mitochondrial and exogenous oxidative stress during infection and drug treatment. Our findings indicate a greater increase in cTXNPx expression in response to exogenous oxidative stress and a higher elevation of mTXNPx expression in response to mitochondrial or endogenous stress created by respiratory chain complex inhibitors. Overexpression of cTXNPx in Leishmania showed improved protection against exogenous stress and enhanced protection against mitochondrial stress in parasites overexpressing mTXNPx. Further, parasites overexpressing cTXNPx infected host cells with increased efficiency at early times of infection compared to control parasites or parasites overexpressing mTXNPx. The mTXNPx-overexpressing parasites maintained higher infection at later times. Higher mTXNPx expression occurred in wild-type parasites on exposure to miltefosine, while treatment with antimony elevated cTXNPx expression. Parasites resistant to miltefosine or antimony demonstrated increased expression of mTXNPx, as well as cTXNPx. In summary, this study provides evidence of distinct roles of the two enzymes defined by virtue of their localization during infection and drug treatment.

RBX1-mediated ubiquitination of SESN2 promotes cell death upon prolonged mitochondrial damage in SH-SY5Y neuroblastoma cells.

Sestrins are evolutionary conserved stress-inducible genes which regulate the axis of cell survival and cell death. Suppression of Sestrin 2 (SESN2) has been linked with increase in oxidative stress and cell death but mechanistic details related to regulation of SESN2 during mitochondrial damage remain unknown. Our study shows that prolonged CCCP-induced mitochondrial damage decreases SESN2 levels and viability of SH-SY5Y cells while overexpression of SESN2 significantly rescues the viability of cells. Further, we demonstrate that Ring box protein 1 (RBX1) is a novel interactive partner and E3 ligase for SESN2 which mediates its K-48-linked ubiquitination upon extensive mitochondrial damage. Downregulation of RBX1 causes stabilization in levels of SESN2. Notably, silencing of RBX1 expression substantially declines cell death and generation of mitochondrial ROS in response to prolonged mitochondrial damage. Taken together, we suggest that SESN2 is critical to protect cells against detrimental effect of mitochondrial damage and RBX1 is a negative regulator of SESN2 which hampers its stabilization.

Elevated ergosterol protects Leishmania parasites against antimony-generated stress.

Parasite lipids can serve as signaling molecules, important membrane components, energy suppliers, and pathogenesis factors critical for survival. Functional roles of lipid changes in response to drug-generated stress in parasite survival remains unclear. To investigate this, Leishmania donovani parasites, the causative agents of kala-azar, were exposed to the antileishmanial agent potassium antimony tartrate (PAT) (half-maximal inhibitory concentration ∼ 284 µg/ml). Analysis of cell extracts using gas chromatography-mass spectrometry showed significant increases in very long-chain fatty acids (VLCFAs) prior to an increase in ergosterol in PAT-treated parasites as compared with vehicle-treated controls. Ergosterol biosynthesis inhibition during PAT treatment decreased cell viability. VLCFA inhibition with specific inhibitors completely abrogated ergosterol upsurge followed by a reduction in cell viability. Following PAT-induced VLCFA increase, an upsurge in reactive oxygen species (ROS) occurred and inhibition of this ROS with antioxidants abrogated ergosterol increase. Genetically engineered parasites expressing low constitutive ergosterol levels showed more susceptibility to PAT as compared with wild-type control cells but ergosterol supplementation during PAT treatment increased cell viability. In conclusion, we propose that during antimony treatment, the susceptibility of parasites is determined by the levels of cellular ergosterol that are regulated by oxidative stress generated by VLCFAs.

Proteomic-based approach to gain insight into reprogramming of THP-1 cells exposed to Leishmania donovani over an early temporal window.

Leishmania donovani, a protozoan parasite, is the causative agent of visceral leishmaniasis. It lives and multiplies within the harsh environment of macrophages. In order to investigate how intracellular parasite manipulate the host cell environment, we undertook a quantitative proteomic study of human monocyte-derived macrophages (THP-1) following infection with L. donovani. We used the isobaric tags for relative and absolute quantification (iTRAQ) method and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to compare expression profiles of noninfected and L. donovani-infected THP-1 cells. We detected modifications of protein expression in key metabolic pathways, including glycolysis and fatty acid oxidation, suggesting a global reprogramming of cell metabolism by the parasite. An increased abundance of proteins involved in gene transcription, RNA splicing (heterogeneous nuclear ribonucleoproteins [hnRNPs]), histones, and DNA repair and replication was observed at 24 h postinfection. Proteins involved in cell survival and signal transduction were more abundant at 24 h postinfection. Several of the differentially expressed proteins had not been previously implicated in response to the parasite, while the others support the previously identified proteins. Selected proteomics results were validated by real-time PCR and immunoblot analyses. Similar changes were observed in L. donovani-infected human monocyte-derived primary macrophages. The effect of RNA interference (RNAi)-mediated gene knockdown of proteins validated the relevance of the host quantitative proteomic screen. Our findings indicate that the host cell proteome is modulated after L. donovani infection, provide evidence for global reprogramming of cell metabolism, and demonstrate the complex relations between the host and parasite at the molecular level.

Beclin-1-p53 interaction is crucial for cell fate determination in embryonal carcinoma cells.

Emerging interest on the interrelationship between the apoptotic and autophagy pathways in the context of cancer chemotherapy is providing exciting discoveries. Complexes formed between molecules from both pathways present potential targets for chemotherapeutics design as disruption of such complexes could alter cell survival. This study demonstrates an important role of Beclin-1 and p53 interaction in cell fate decision of human embryonal carcinoma cells. The findings provide evidence for p53 interaction with Beclin-1 through the BH3 domain of the latter. This interaction facilitated Beclin-1 ubiquitination through lysine 48 linkage, resulting in proteasome-mediated degradation, consequently maintaining a certain constitutive level of Beclin-1. Disruption of Beclin-1-p53 interaction through shRNA-mediated down-regulation of p53 reduced Beclin-1 ubiquitination suggesting requirement of p53 for the process. Reduction of ubiquitination consequently resulted in an increase in Beclin-1 levels with cells showing high autophagic activity. Enforced overexpression of p53 in the p53 down-regulated cells restored ubiquitination of Beclin-1 reducing its level and lowering autophagic activity. The Beclin-1-p53 interaction was also disrupted by exposure to cisplatin-induced stress resulting in higher level of Beclin-1 because of lesser ubiquitination. This higher concentration of Beclin-1 increased autophagy and offered protection to the cells from cisplatin-induced death. Inhibition of autophagy by either pharmacological or genetic means during cisplatin exposure increased apoptotic death in vitro as well as in xenograft tumours grown in vivo confirming the protective nature of autophagy. Therefore, Beclin-1-p53 interaction defines one additional molecular subroutine crucial for cell fate decisions in embryonal carcinoma cells.

Studies on the antileishmanial mechanism of action of the arylimidamide DB766: azole interactions and role of CYP5122A1.

Arylimidamides (AIAs) are inspired by diamidine antimicrobials but show superior activity against intracellular parasites. The AIA DB766 {2,5-bis[2-(2-i-propoxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride} displays outstanding potency against intracellular Leishmania parasites and is effective in murine and hamster models of visceral leishmaniasis when given orally, but its mechanism of action is unknown. In this study, through the use of continuous DB766 pressure, we raised Leishmania donovani axenic amastigotes that displayed 12-fold resistance to this compound. These DB766-resistant (DB766R) parasites were 2-fold more sensitive to miltefosine than wild-type organisms and were hypersensitive to the sterol 14α-demethylase (CYP51) inhibitors ketoconazole and posaconazole (2,000-fold more sensitive and over 12,000-fold more sensitive than the wild type, respectively). Western blot analysis of DB766R parasites indicated that while expression of CYP51 is slightly increased in these organisms, expression of CYP5122A1, a recently identified cytochrome P450 associated with ergosterol metabolism in Leishmania, is dramatically reduced in DB766R parasites. In vitro susceptibility assays demonstrated that CYP5122A1 half-knockout L. donovani promastigotes were significantly less susceptible to DB766 and more susceptible to ketoconazole than their wild-type counterparts, consistent with observations in DB766R parasites. Further, DB766-posaconazole combinations displayed synergistic activity in both axenic and intracellular L. donovani amastigotes. Taken together, these studies implicate CYP5122A1 in the antileishmanial action of the AIAs and suggest that DB766-azole combinations are potential candidates for the development of synergistic antileishmanial therapy.

A Novel Role of Secretory Cytosolic Tryparedoxin Peroxidase in Delaying Apoptosis of Leishmania-Infected Macrophages.

The cytosolic tryparedoxin peroxidase (cTXNPx) of Leishmania donovani is a defensive enzyme. Apart from the nonsecretory form, the cTXNPx is released in the spent media of Leishmania cultures and also in the host cell cytosol. The secretory form of the enzyme from the parasite interacts with multiple proteins in the host cell cytosol, the apoptosis-inducing factor (AIF) being one of them. Immunoprecipitation with anti-cTXNPx and anti-AIF antibodies suggests a strong interaction between AIF and cTXNPx. Consequent to parasite invasion, the migration of AIF to the nucleus to precipitate apoptosis is inhibited in the presence of recombinant cTXNPx expressed in the host cell. This inhibition of AIF movement results in lesser host cell death, giving an advantage to the parasite for continued survival. Staurosporine-induced AIF migration to the nucleus was also inhibited in the presence of recombinant cTXNPx in the host cell. Therefore, this study demonstrates the ability of a Leishmania parasite enzyme, cTXNPx, to interfere with the migration of the host AIF protein, providing a survival advantage to the Leishmania parasite.

Leishmania cell surface prohibitin: role in host-parasite interaction.

Proteins selectively upregulated in infective parasitic forms could be critical for disease pathogenesis. A mammalian prohibitin orthologue is upregulated in infective metacyclic promastigotes of Leishmania donovani, a parasite that causes visceral leishmaniasis. Leishmania donovani prohibitin shares 41% similarity with mammalian prohibitin and 95-100% within the genus. Prohibitin is concentrated at the surface of the flagellar and the aflagellar pole, the aflagellar pole being a region through which host-parasite interactions occur. Prohibitin is attached to the membrane through a GPI anchor. Overexpression of wild-type prohibitin increases protein surface density resulting in parasites with higher infectivity. However, parasites overexpressing a mutant prohibitin with an amino acid substitution at the GPI anchor site to prevent surface expression through GPI-link show lesser surface expression and lower infective abilities. Furthermore, the presence of anti-prohibitin antibodies during macrophage-Leishmania interaction in vitro reduces infection. The cognate binding partner for Leishmania prohibitin on the host cell appears to be macrophage surface HSP70, siRNA mediated downregulation of which abrogates the capability of the macrophage to bind to parasites. Leishmania prohibitin is able to generate a strong humoral response in visceral leishmaniasis patients. The above observations suggest that prohibitin plays an important role in events leading to Leishmania-host interaction.

Tissue/Biofluid Specific Molecular Cartography of Leishmania donovani Infected BALB/c Mice: Deciphering Systemic Reprogramming.

Pathophysiology of visceral leishmaniasis (VL) is not fully understood and it has been widely accepted that the parasitic components and host immune response both contribute to the perpetuation of the disease. Host alterations during leishmaniasis is a feebly touched area that needs to be explored more to better understand the VL prognosis and diagnosis, which are vital to reduce mortality and post-infection sequelae. To address this, we performed untargeted metabolomics of Leishmania donovani (Ld) infected, uninfected and treated BALB/c mice's tissues and biofluids to elucidate the host metabolome changes using gas chromatography-mass spectrometry. Univariate and multivariate data treatments provided numerous significant differential hits in several tissues like the brain, liver, spleen and bone marrow. Differential modulations were also observed in serum, urine and fecal samples of Ld-infected mice, which could be further targeted for biomarker and diagnostic validations. Several metabolic pathways were found to be upregulated/downregulated in infected (TCA, glycolysis, fatty acids, purine and pyrimidine, etcetera) and treated (arginine, fumaric acid, orotic acid, choline succinate, etcetera) samples. Results also illustrated several metabolites with different pattern of modulations in control, infected and treated samples as well as in different tissues/biofluids; for e.g. glutamic acid identified in the serum samples of infected mice. Identified metabolites include a range of amino acids, saccharides, energy-related molecules, etcetera. Furthermore, potential biomarkers have been identified in various tissues-arginine and fumaric acid in brain, choline in liver, 9-(10) EpOME in spleen and bone marrow, N-acetyl putrescine in bone marrow, etcetera. Among biofluids, glutamic acid in serum, hydrazine and deoxyribose in urine and 3-Methyl-2-oxo pentanoic acid in feces are some of the potential biomarkers identified. These metabolites could be further looked into for their role in disease complexity or as a prognostic marker. The presented profiling approach allowed us to attain a metabolic portrait of the individual tissue/biofluid modulations during VL in the host and represent a valuable system readout for further studies. Our outcomes provide an improved understanding of perturbations of the host metabolome interface during VL, including identification of many possible potential diagnostic and therapeutic targets.

Oestrogen modulates human macrophage apoptosis via differential signalling through oestrogen receptor-alpha and beta.

Human macrophages express oestrogen receptors and are therefore competent to respond to the hormone present in their microenvironment, which is implicated in sexual dimorphism observed in several immune and autoimmune phenomena. An earlier study from this laboratory demonstrated 17beta-oestradiol (E2) induced apoptosis in macrophages derived from human peripheral blood monocytes and THP-1 acute monocytic leukaemia cell line when Bcl-2 was down-regulated; however, the involvement of E2 receptor subtypes in the modulation of death pathways in these cells remain unknown. Using macrophages derived from THP-1 human acute monocytic leukaemia cells as a model, we demonstrate that plasma membrane associated oestrogen receptor (ER) -alpha participate in E2 induced Bcl-2 increase, through activation of the mitogen activated protein kinase (MAPK) pathway whereas cytosolic ER-beta transmits signals for the pro-apoptotic event of Bax translocation. The mechanistic basis of Bax translocation comprised of ER-beta mediated increase in intracellular pH, facilitated by activation of the Na(+)-H(+) exchanger. Intracellular alkalinization accompanied by concomitant Bcl-2 increase and Bax migration does not cause cellular apoptosis; however, siRNA mediated down-regulation of ER-alpha during E2 exposure leads to inhibition of Bcl-2 increase and consequently apoptosis due to the unopposed action of mitochondrial Bax. In summary, this study underscores the importance of integrative signalling modality from multiple oestrogen receptor pools in modulating oestrogen effects on human monocyte-derived macrophage apoptotic signalling pathway, which opens new vistas to explore the use of selective oestrogen receptor modulators in apoptosis-based therapies.

Crucial role of cytosolic tryparedoxin peroxidase in Leishmania donovani survival, drug response and virulence.

Leishmania donovani, the causative agent of visceral leishmaniasis, uses a cascade of enzymes that include cytosolic tryparedoxin peroxidase (cTXNPx) for detoxification of peroxides, an event pivotal for survival of digenic parasites living in two disparate biological environments. In this study, we observed an increase in promastigote cTXNPx levels after exposure to H(2)O(2) and this group did not show any cell death; however, exposure to a combination of H(2)O(2) and nitric oxide resulted in significant reduction of cTXNPx levels accompanied by high cell death. The protective relationship between higher levels of cTXNPx and survival was further substantiated by the improved ability of L. donovani promastigotes overexpressing cTXNPx to withstand exposure to H(2)O(2) and nitric oxide combination as compared with vector transfectants. In addition, cTXNPx transfectants demonstrated increased virulence, causing higher parasite burden in macrophages as compared with vector transfectants. Interestingly, the cTXNPx transfectants as promastigotes or amastigotes were resistant to clearance by the anti-leishmanial drug antimony, suggesting a cTXNPx link to drug response. Mechanistically, cTXNPx overexpression was protective against changes in Ca(2+) homeostasis but not against mitochondrial hyperpolarization brought about by exposure to H(2)O(2) and nitric oxide. Therefore, this study provides a link between cTXNPx expression to survival, virulence and drug response in L. donovani.

Leishmania donovani parasite requires Atg8 protein for infectivity and survival under stress.

The importance of autophagy in parasites with a digenetic life cycle like Leishmania spp. is significant. The parasite survives as promastigotes in the insect gut and as immotile amastigotes in mammals. This study demonstrates increased autophagy in Leishmania parasite during progression of in vitro life cycle and upon exposure to stress stimuli like starvation, oxidative stress, and drugs. Autophagy inhibition during stress exposure increased cell death, indicating the importance of autophagy in cellular defense against adverse conditions. Atg8 protein, a homolog of mammalian autophagy protein LC3 is expressed in Leishmania parasite but its function remains unknown. Overexpression of Atg8 (Atg8-OE) rendered the parasites resistant to stress and capable of infecting macrophages in substantial numbers; however, disruption of the Atg8 gene (ΔAtg8) resulting in suppression of Atg8 protein expression, increased susceptibility to stress and reduced the capability to cause infection. A critical event in the Leishmania parasite lifecycle is the differentiation of promastigote forms to the disease causing amastigote forms. The failure of ΔAtg8 parasites lacking Atg8 protein to differentiate into amastigotes, unlike the Atg8-OE and vector-transfected parasites, clearly indicated Atg8 involvement in a crucial event. The inability of ΔAtg8 parasites to infect macrophages in vitro was verified in an in vivo mouse model of leishmaniases where infection could not be induced by the ΔAtg8 parasites. Autophagy is known to be involved in the remodeling of damaged organelles. The accumulation of Atg8 around damaged mitochondria suggested increase of autophagy in the vicinity of the organelle. This buildup was prevented when mitochondria generated reactive oxygen species that were quenched, suggesting them as possible signaling molecules for sensing mitochondrial instability. In summary, our study provides new evidences for a crucial role of Atg8 protein in sustaining Leishmania parasite survival during life cycle and stress exposure, differentiation to amastigotes, and their infective abilities.

SESN2 facilitates mitophagy by helping Parkin translocation through ULK1 mediated Beclin1 phosphorylation.

Mitophagy, the selective degradation of mitochondria by autophagy, is crucial for the maintenance of healthy mitochondrial pool in cells. The critical event in mitophagy is the translocation of cytosolic Parkin, a ubiquitin ligase, to the surface of defective mitochondria. This study elucidates a novel role of SESN2/Sestrin2, a stress inducible protein, in mitochondrial translocation of PARK2/Parkin during mitophagy. The data demonstrates that SESN2 downregulation inhibits BECN1/Beclin1 and Parkin interaction, thereby preventing optimum mitochondrial accumulation of Parkin. SESN2 interacts with ULK1 (unc-51 like kinase 1) and assists ULK1 mediated phosphorylation of Beclin1 at serine-14 position required for binding with Parkin prior to mitochondrial translocation. The trigger for SESN2 activation and regulation of Parkin translocation is the generation of mitochondrial superoxide. Scavenging of mitochondrial superoxide lower the levels of SESN2, resulting in retardation of Parkin translocation. Importantly, we observe that SESN2 mediated cytosolic interaction of Parkin and Beclin1 is PINK1 independent but mitochondrial translocation of Parkin is PINK1 dependent. Together, these findings suggest the role of SESN2 as a positive regulator of Parkin mediated mitophagy.

Sestrin2 facilitates glutamine-dependent transcription of PGC-1α and survival of liver cancer cells under glucose limitation.

Differential utilization of metabolites and metabolic plasticity can confer adaptation to cancer cells under metabolic stress. Glutamine is one of the vital and versatile nutrients that cancer cells consume avidly for their proliferation, and therefore mechanisms related to glutamine metabolism have been identified as targets. Recently, sestrin2 (SESN2), a stress-inducible protein, has been reported to regulate survival in glutamine-depleted cancer cells; based on this, we explored if SESN2 could regulate glutamine metabolism during glucose starvation. This report highlights the role of SESN2 in the regulation of glutamine-dependent activation of the mitochondrial biogenesis marker peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) under glucose scarcity in liver cancer cells (HepG2). We demonstrate that down-regulation of SESN2 induces a decrease in the levels of intracellular glutamine and PGC-1α under glucose deprivation, concomitant with a decline in cell survival, but no effect was observed on the invasive or migration potential of the cells. Under similar metabolic conditions, SESN2 forms a complex with c-Jun N-terminal kinase (JNK) and forkhead box protein O1 (FOXO1). Absence of SESN2 or inhibition of JNK reduces nuclear translocation of FOXO1, consequently causing transcriptional inhibition of PGC-1α. Notably, our observations demonstrate a reduction in cell viability under high glutamine and low glucose conditions during SESN2 down-regulation that could be rescued on JNK inhibition. To recover from acetaminophen-induced mitochondrial damage, SESN2 was crucial for glutamine-mediated activation of PGC-1α in HepG2 cells. Collectively, we demonstrate a novel role of SESN2 in mediating activation of PGC-1α by modulating glutamine metabolism that facilitates cancer cell survival under glucose-limited metabolic conditions.

Zinc depletion promotes apoptosis-like death in drug-sensitive and antimony-resistance Leishmania donovani.

Micronutrients are essential for survival and growth for all the organisms including pathogens. In this manuscript, we report that zinc (Zn) chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethylenediamine (TPEN) affects growth and viability of intracellular pathogen Leishmania donovani (LD) by a concentration and time dependent manner. Simultaneous addition of zinc salt reverses the effect of TPEN. Further experiments provide evidence of apoptosis-like death of the parasite due to Zn-depletion. TPEN treatment enhances caspase-like activity suggesting increase in apoptosis-like events in LD. Specific inhibitors of cathepsin B and Endoclease G block TPEN-induced leishmanial death. Evidences show involvement of reactive oxygen species (ROS) potentially of extra-mitochondrial origin in TPEN-induced LD death. Pentavalent antimonials remained the prime source of treatment against leishmaniasis for several decades; however, antimony-resistant Leishmania is now common source of the disease. We also reveal that Zn-depletion can promote apoptosis-like death in antimony-resistant parasites. In summary, we present a new finding about the role of zinc in the survival of drug sensitive and antimony-resistant LD.