Ravi Manjithaya - Academia.edu (original) (raw)

Papers by Ravi Manjithaya

Cell Biology International

Aggrephagy is a selective autophagic degradation intracellular mechanism that clears toxic misfol... more Aggrephagy is a selective autophagic degradation intracellular mechanism that clears toxic misfolded protein aggregates such as α‐synuclein. Here, we identify and demonstrate that the small molecule, XCT 790 alleviates α‐synuclein‐mediated adverse effects in a yeast model of proteotoxicity. XCT 790 induced general autophagy and also enhanced starvation‐induced autophagy. Mechanistically, we showed that XCT 790 clears toxic α‐synuclein aggregates in an autophagy‐dependent manner. Interestingly, XCT 790 did not demonstrate a synergistic effect on autophagy induction in the presence of another autophagy inducer such as 6‐Bio.

Macroautophagy/autophagy functions as a part of the innate immune system in clearing intracellula... more Macroautophagy/autophagy functions as a part of the innate immune system in clearing intracellular pathogens. Although this process is well known, the mechanisms that control antibacterial autophagy are not clear. In this study we show that during intracellular <i>Salmonella typhimurium</i> infection, the activity of TFEB (transcription factor EB), a master regulator of autophagy and lysosome biogenesis, is suppressed by maintaining it in a phosphorylated state on the lysosomes. Furthermore, we have identified a novel, antibacterial small molecule autophagy (xenophagy) modulator, acacetin. The xenophagy effect exerted by acacetin occurs in an MTOR (mechanistic target of rapamycin kinase)-independent, TFEB-dependent manner. Acacetin treatment results in persistently maintaining active TFEB in the nucleus and also in TFEB mediated induction of functional lysosomes that target <i>Salmonella</i>-containing vacuoles (SCVs). The enhanced proteolytic activity due to...

Yeast, 2021

Unicellular organisms, like yeast, have developed mechanisms to overcome environmental stress con... more Unicellular organisms, like yeast, have developed mechanisms to overcome environmental stress conditions like nutrient starvation. Autophagy and sporulation are two such mechanisms employed by yeast cells. Autophagy is a well‐conserved, catabolic process that degrades excess and unwanted cytoplasmic materials and provides building blocks during starvation conditions. Thus, autophagy maintains cellular homeostasis at basal conditions and acts as a survival mechanism during stress conditions. Sporulation is an essential process that, like autophagy, is triggered due to stress conditions in yeast. It involves the formation of ascospores that protect the yeast cells during extreme conditions and germinate when the conditions are favorable. Studies show that autophagy is required for the sporulation process in yeast. However, the exact mechanism of action is not clear. Furthermore, several of the core autophagy gene knockouts do not sporulate and at what stage of sporulation they are inv...

Frontiers in Aging Neuroscience, 2020

Aggregated tau is a hallmark neuropathological feature in numerous neurodegenerative disorders. P... more Aggregated tau is a hallmark neuropathological feature in numerous neurodegenerative disorders. Previous studies aiming to validate aggregated tau pathology as a pathogenic driver of neurodegeneration in correlation to characteristic behavioral phenotypes have had shortcomings. Although studies on soluble tau pathology have effectively addressed these shortcomings, the role of soluble tau in the molecular pathogenesis of neurodegeneration is not yet unequivocally established. In sporadic Alzheimer's disease (AD), the relevance of soluble tau pathology in endolysosomal dysfunction and autophagic stress, some of the earliest disease manifestations, is unclear. In this study, we report that soluble 4R0N tau overexpression affects the expression levels of certain markers associated with the endolysosomal system and autophagy. Moreover, through live-cell imaging, we found that the vesicular dynamics of early endosomes were affected with respect to spatiotemporal parameters and vesicle maturation. Additionally, we observed the localization of amyloid precursor protein (APP) along the endocytic pathway and found that upon overexpression of soluble 4R0N tau, APP was preferentially localized to the endocytic compartments implicated in the amyloidogenic pathway. Overall, our observations indicate that soluble 4R0N tau abrogates the dynamics of the endolysosomal system, autophagy, and affects the trafficking of APP. Since the amyloidogenic processing of APP occurs during its progression through the endocytic pathway, our results suggest that the generation of amyloid-β (Aβ) might also be modulated.

Journal of Cell Science, 2020

Foot-and-mouth disease virus (FMDV) is a picornavirus that causes contagious acute infection in c... more Foot-and-mouth disease virus (FMDV) is a picornavirus that causes contagious acute infection in cloven-hoofed animals. FMDV replication associated viral protein expression induces endoplasmic reticulum (ER) stress and unfolded protein response (UPR), in turn inducing autophagy to restore cellular homeostasis. We observed that inhibition of BiP, a master regulator of ER stress and UPR, decreased FMDV infection confirming their involvement. Further, we show that the FMDV infection induces UPR mainly through PKR-like ER kinase (PERK)-mediated pathway. Knockdown of PERK and chemical inhibition of PERK activation resulted in decreased expression of FMDV proteins along with the reduction of autophagy marker protein LC3B-II. There are conflicting reports on the role of autophagy in FMDV multiplication. Our study systematically demonstrates that during FMDV infection, PERK mediated UPR stimulated an increased level of endogenous LC3B-II and turnover of SQSTM1, thus confirming the activation...

Autophagy, 2019

Macroautophagy/autophagy functions as a part of the innate immune system in clearing intracellula... more Macroautophagy/autophagy functions as a part of the innate immune system in clearing intracellular pathogens. Although this process is well known, the mechanisms that control antibacterial autophagy are not clear. In this study we show that during intracellular Salmonella typhimurium infection, the activity of TFEB (transcription factor EB), a master regulator of autophagy and lysosome biogenesis, is suppressed by maintaining it in a phosphorylated state on the lysosomes. Furthermore, we have identified a novel, antibacterial small molecule autophagy (xenophagy) modulator, acacetin. The xenophagy effect exerted by acacetin occurs in an MTOR (mechanistic target of rapamycin kinase)-independent, TFEB-dependent manner. Acacetin treatment results in persistently maintaining active TFEB in the nucleus and also in TFEB mediated induction of functional lysosomes that target Salmonella-containing vacuoles (SCVs). The enhanced proteolytic activity due to deployment of lysosomes results in clamping down Salmonella replication in SCVs. Acacetin is effective as a xenophagy compound in an in vivo mouse model of infection and reduces intracellular Salmonella burden.

Molecular Biology of the Cell, 2019

Autophagy is an evolutionarily conserved intracellular lysosomal degradation pathway. It is a mul... more Autophagy is an evolutionarily conserved intracellular lysosomal degradation pathway. It is a multistep process involving de novo formation of double membrane autophagosomes that capture cytosolic constituents (cargo) and eventually fuse with lysosomes wherein the cargo gets degraded and resulting simpler biomolecules get recycled. In addition to their autophagy function, several of the autophagy-related proteins work at the interface of other vesicular trafficking pathways. Hence, development of specific autophagy modulators that do not perturb general endo-lysosomal traffic possesses unique challenges. In this article, we report a novel small molecule EACC that inhibits autophagic flux by blocking autophagosome–lysosome fusion. Strikingly, unlike other late stage inhibitors, EACC does not have any effect on lysosomal properties or on endocytosis-mediated degradation of EGF receptor. EACC affects the translocation of SNAREs Stx17 and SNAP29 on autophagosomes without impeding the co...

Frontiers in Cell and Developmental Biology, 2018

Growing amount of evidence in the last two decades highlight that macroautophagy (generally refer... more Growing amount of evidence in the last two decades highlight that macroautophagy (generally referred to as autophagy) is not only indispensable for survival in yeast but also equally important to maintain cellular quality control in higher eukaryotes as well. Importantly, dysfunctional autophagy has been explicitly shown to be involved in various physiological and pathological conditions such as cell death, cancer, neurodegenerative, and other diseases. Therefore, modulation and regulation of the autophagy pathway has emerged as an alternative strategy for the treatment of various disease conditions in the recent years. Several studies have shown genetic or pharmacological modulation of autophagy to be effective in treating cancer, clearing intracellular aggregates and pathogens. Understanding and controlling the autophagic flux, either through a genetic or pharmacological approach is therefore a highly promising approach and of great scientific interest as spatiotemporal and cell-tissue-organ level autophagy regulation is not clearly understood. Indeed, chemical biology approaches that identify small molecule effectors of autophagy have thus a dual benefit: the modulators act as tools to study and understand the process of autophagy, and may also have therapeutic potential. In this review, we discuss different strategies that have appeared to screen and identify potent small molecule modulators of autophagy.

Cell death discovery, 2018

The precise role of autophagy in remains largely unknown. Although a limited number of autophagy ... more The precise role of autophagy in remains largely unknown. Although a limited number of autophagy genes have been identified in this apicomplexan, only Atg8 has been characterized to a certain extent. On the basis of the expression levels of Atg8 and the putative Atg5, we report that the basal autophagy in this parasite is quite robust and mediates not only the intraerythrocytic development but also fresh invasion of red blood cells (RBCs) in the subsequent cycles. We demonstrate that the basal autophagy responds to both inducers and inhibitors of autophagy. In addition, the parasite survival upon starvation is temporally governed by the autophagy status. Brief periods of starvation, which induces autophagy, help survival while prolonged starvation decreases autophagy leading to stalled parasite growth and reduced invasion. Thus, starvation-induced autophagy is context dependent. Importantly, we report characterization of another autophagy marker in this parasite, the putative Atg5 (...

Frontiers in molecular neuroscience, 2018

Mechanistic insights into aggrephagy, a selective basal autophagy process to clear misfolded prot... more Mechanistic insights into aggrephagy, a selective basal autophagy process to clear misfolded protein aggregates, are lacking. Here, we report and describe the role of Estrogen Related Receptor α (ERRα, HUGO Gene Nomenclature ESRRA), new molecular player of aggrephagy, in keeping autophagy flux in check by inhibiting autophagosome formation. A screen for small molecule modulators for aggrephagy identified ERRα inverse agonist XCT 790, that cleared α-synuclein aggregates in an autophagy dependent, but mammalian target of rapamycin (MTOR) independent manner. XCT 790 modulates autophagosome formation in an ERRα dependent manner as validated by siRNA mediated knockdown and over expression approaches. We show that, in a basal state, ERRα is localized on to the autophagosomes and upon autophagy induction by XCT 790, this localization is lost and is accompanied with an increase in autophagosome biogenesis. In a preclinical mouse model of Parkinson's disease (PD), XCT 790 exerted neuropr...

Autophagy, 2017

Due to the involvement of macroautophagy/autophagy in different pathophysiological conditions suc... more Due to the involvement of macroautophagy/autophagy in different pathophysiological conditions such as infections, neurodegeneration and cancer, identification of novel small molecules that modulate the process is of current research and clinical interest. In this work, we developed a luciferase-based sensitive and robust kinetic high-throughput screen (HTS) of small molecules that modulate autophagic degradation of peroxisomes in the budding yeast Saccharomyces cerevisiae. Being a pathway-specific rather than a target-driven assay, we identified small molecule modulators that acted at key steps of autophagic flux. Two of the inhibitors, Bay11 and ZPCK, obtained from the screen were further characterized using secondary assays in yeast. Bay11 inhibited autophagy at a step before fusion with the vacuole whereas ZPCK inhibited the cargo degradation inside the vacuole. Furthermore, we demonstrated that these molecules altered the process of autophagy in mammalian cells as well. Strikingly, these molecules also modulated autophagic flux in a novel model plant, Aponogeton madagascariensis. Thus, using small molecule modulators identified by using a newly developed HTS autophagy assay, our results support that macroautophagy is a conserved process across fungal, animal and plant kingdoms.

Journal of the Indian Institute of Science, 2017

Introduction Autophagy, an intracellular evolutionarily conserved process, involves engulfment of... more Introduction Autophagy, an intracellular evolutionarily conserved process, involves engulfment of unwanted proteins and organelles by double-membrane vesicles, called autophagosomes, which then fuse with the lysosomes/vacuole, and the engulfed cargo is subsequently degraded. It is a cell survival mechanism under stress conditions and it also play important roles in many other intra-cellular processes like protein and organelle turnover and transport of some of the vacuolar enzymes. This process can be divided into various steps, including autophagy induction, nucleation, autophagosome formation, maturation, fusion with the lysosomes/vacuole, degradation of the cargo, and recycling of the precursor molecules, such as amino acids, lipids, and nucleotides, back to the cytoplasm. Autophagy is a tightly regulated cellular mechanism and its flux varies depending on the cell type(s) of an organism. Autophagy is involved in various physiological roles, such as cellular homeostasis, embryonic development, antigen presentation, protein quality control, and maintenance of the amino-acid pool during starvation conditions. It is also implicated in various pathophysiological diseases, such as infection, cancer, diabetes, and neurodegeneration. Autophagosomes: The "Pac-Man" like double membrane vesicles involved in macroautophagy.

Autophagy is a conserved cellular degradation pathway wherein a double membrane vesicle, called a... more Autophagy is a conserved cellular degradation pathway wherein a double membrane vesicle, called as an autophagosome captures longlived proteins, damaged or superfluous organelles and delivers to the lysosome for degradation1. We have identified a novel role for septins in autophagy. Septins are GTP-binding proteins that localize at the bud-neck and are involved in cytokinesis in budding yeast2. We show that septins under autophagy prevalent conditions are majorly localized to the cytoplasm in the form of punctate structures. Further, we report that septins not only localize to pre-autophagosomal structure (PAS) but also to autophagosomes in the form of punctate structures. Interestingly, septins also form small non-canonical rings around PAS during autophagy. Furthermore, we observed that in one of the septin Ts" mutant,cdc10-5, the anterograde trafficking of Atg9 was affected at the non-permissive temperature (NPT). All these results suggest a role of septins in early stages o...

Scientific reports, Jan 30, 2015

Alzheimer's disease is one of the devastating illnesses mankind is facing in the 21(st) centu... more Alzheimer's disease is one of the devastating illnesses mankind is facing in the 21(st) century. The main pathogenic event in Alzheimer's disease is believed to be the aggregation of the β-amyloid (Aβ) peptides into toxic aggregates. Molecules that interfere with this process may act as therapeutic agents for the treatment of the disease. Use of recognition unit based peptidomimetics as inhibitors are a promising approach, as they exhibit greater protease stability compared to natural peptides. Here, we present peptidomimetic inhibitors of Aβ aggregation designed based on the KLVFF (P1) sequence that is known to bind Aβ aggregates. We improved inhibition efficiency of P1 by introducing multiple hydrogen bond donor-acceptor moieties (thymine/barbiturate) at the N-terminal (P2 and P3), and blood serum stability by modifying the backbone by incorporating sarcosine (N-methylglycine) units at alternate positions (P4 and P5). The peptidomimetics showed moderate to good activity in...

Journal of Cell Biology, 2010

Autophagy is important for many cellular processes such as innate immunity, neurodegeneration, ag... more Autophagy is important for many cellular processes such as innate immunity, neurodegeneration, aging, and cancer. Although the signaling events triggering autophagy have been studied, little is known regarding the signaling mechanisms by which autophagy is redirected to achieve selective removal of cellular components. We have used the degradation of a peroxisomal marker to investigate the role of protein kinases in selective autophagy of peroxisomes (pexophagy) in Saccharomyces cerevisiae. We show that the Slt2p mitogen-activated protein kinase (MAPK) and several upstream components of its signal transduction pathway are necessary for pexophagy but not for pexophagosome formation or other nonselective and selective forms of autophagy. Other extracellular signals that activate this pathway do not trigger pexophagy on their own, suggesting that this MAPK cascade is necessary but not sufficient to trigger pexophagy. We propose that pexophagy requires the simultaneous activation of thi...

Molecular Biology of the Cell, 2006

Sterol glucosyltransferase, Ugt51/Atg26, is essential for both micropexophagy and macropexophagy ... more Sterol glucosyltransferase, Ugt51/Atg26, is essential for both micropexophagy and macropexophagy of methanol-induced peroxisomes in Pichia pastoris. However, the role of this protein in pexophagy in other yeast remained unclear. We show that oleate- and amine-induced peroxisomes in Yarrowia lipolytica are degraded by Atg26-independent macropexophagy. Surprisingly, Atg26 was also not essential for macropexophagy of oleate- and amine-induced peroxisomes in P. pastoris, suggesting that the function of sterol glucoside (SG) in pexophagy is both species and peroxisome inducer specific. However, the rates of degradation of oleate- and amine-induced peroxisomes in P. pastoris were reduced in the absence of SG, indicating that P. pastoris specifically uses sterol conversion by Atg26 to enhance selective degradation of peroxisomes. However, methanol-induced peroxisomes apparently have lost the redundant ability to be degraded without SG. We also show that the P. pastoris Vac8 armadillo repea...

Developmental Cell, 2008

Autophagy, an intrinsically nonselective process, can also target selective cargo for degradation... more Autophagy, an intrinsically nonselective process, can also target selective cargo for degradation. The mechanism of selective peroxisome turnover by autophagy-related processes (pexophagy), termed micropexophagy and macropexophagy, is unknown. We show how a Pichia pastoris protein, PpAtg30, mediates peroxisome selection during pexophagy. It is necessary for pexophagy, but not for other selective and nonselective autophagy-related processes. It localizes at the peroxisome membrane via interaction with peroxins, and during pexophagy it colocalizes transiently at the preautophagosomal structure (PAS) and interacts with the autophagy machinery. PpAtg30 is required for formation of pexophagy intermediates, such as the micropexophagy apparatus (MIPA) and the pexophagosome (Ppg). During pexophagy, PpAtg30 undergoes multiple phosphorylations, at least one of which is required for pexophagy. PpAtg30 overexpression stimulates pexophagy even under peroxisome-induction conditions, impairing peroxisome biogenesis. Therefore, PpAtg30 is a key player in the selection of peroxisomes as cargo and in their delivery to the autophagy machinery for pexophagy.

Frontiers in Cell and Developmental Biology

Macroautophagy (henceforth autophagy) an evolutionary conserved intracellular pathway, involves l... more Macroautophagy (henceforth autophagy) an evolutionary conserved intracellular pathway, involves lysosomal degradation of damaged and superfluous cytosolic contents to maintain cellular homeostasis. While autophagy was initially perceived as a bulk degradation process, a surfeit of studies in the last 2 decades has revealed that it can also be selective in choosing intracellular constituents for degradation. In addition to the core autophagy machinery, these selective autophagy pathways comprise of distinct molecular players that are involved in the capture of specific cargoes. The diverse organelles that are degraded by selective autophagy pathways are endoplasmic reticulum (ERphagy), lysosomes (lysophagy), mitochondria (mitophagy), Golgi apparatus (Golgiphagy), peroxisomes (pexophagy) and nucleus (nucleophagy). Among these, the main focus of this review is on the selective autophagic pathway involved in mitochondrial turnover called mitophagy. The mitophagy pathway encompasses dive...

Cell Biology International

Aggrephagy is a selective autophagic degradation intracellular mechanism that clears toxic misfol... more Aggrephagy is a selective autophagic degradation intracellular mechanism that clears toxic misfolded protein aggregates such as α‐synuclein. Here, we identify and demonstrate that the small molecule, XCT 790 alleviates α‐synuclein‐mediated adverse effects in a yeast model of proteotoxicity. XCT 790 induced general autophagy and also enhanced starvation‐induced autophagy. Mechanistically, we showed that XCT 790 clears toxic α‐synuclein aggregates in an autophagy‐dependent manner. Interestingly, XCT 790 did not demonstrate a synergistic effect on autophagy induction in the presence of another autophagy inducer such as 6‐Bio.

Macroautophagy/autophagy functions as a part of the innate immune system in clearing intracellula... more Macroautophagy/autophagy functions as a part of the innate immune system in clearing intracellular pathogens. Although this process is well known, the mechanisms that control antibacterial autophagy are not clear. In this study we show that during intracellular <i>Salmonella typhimurium</i> infection, the activity of TFEB (transcription factor EB), a master regulator of autophagy and lysosome biogenesis, is suppressed by maintaining it in a phosphorylated state on the lysosomes. Furthermore, we have identified a novel, antibacterial small molecule autophagy (xenophagy) modulator, acacetin. The xenophagy effect exerted by acacetin occurs in an MTOR (mechanistic target of rapamycin kinase)-independent, TFEB-dependent manner. Acacetin treatment results in persistently maintaining active TFEB in the nucleus and also in TFEB mediated induction of functional lysosomes that target <i>Salmonella</i>-containing vacuoles (SCVs). The enhanced proteolytic activity due to...

Yeast, 2021

Unicellular organisms, like yeast, have developed mechanisms to overcome environmental stress con... more Unicellular organisms, like yeast, have developed mechanisms to overcome environmental stress conditions like nutrient starvation. Autophagy and sporulation are two such mechanisms employed by yeast cells. Autophagy is a well‐conserved, catabolic process that degrades excess and unwanted cytoplasmic materials and provides building blocks during starvation conditions. Thus, autophagy maintains cellular homeostasis at basal conditions and acts as a survival mechanism during stress conditions. Sporulation is an essential process that, like autophagy, is triggered due to stress conditions in yeast. It involves the formation of ascospores that protect the yeast cells during extreme conditions and germinate when the conditions are favorable. Studies show that autophagy is required for the sporulation process in yeast. However, the exact mechanism of action is not clear. Furthermore, several of the core autophagy gene knockouts do not sporulate and at what stage of sporulation they are inv...

Frontiers in Aging Neuroscience, 2020

Aggregated tau is a hallmark neuropathological feature in numerous neurodegenerative disorders. P... more Aggregated tau is a hallmark neuropathological feature in numerous neurodegenerative disorders. Previous studies aiming to validate aggregated tau pathology as a pathogenic driver of neurodegeneration in correlation to characteristic behavioral phenotypes have had shortcomings. Although studies on soluble tau pathology have effectively addressed these shortcomings, the role of soluble tau in the molecular pathogenesis of neurodegeneration is not yet unequivocally established. In sporadic Alzheimer's disease (AD), the relevance of soluble tau pathology in endolysosomal dysfunction and autophagic stress, some of the earliest disease manifestations, is unclear. In this study, we report that soluble 4R0N tau overexpression affects the expression levels of certain markers associated with the endolysosomal system and autophagy. Moreover, through live-cell imaging, we found that the vesicular dynamics of early endosomes were affected with respect to spatiotemporal parameters and vesicle maturation. Additionally, we observed the localization of amyloid precursor protein (APP) along the endocytic pathway and found that upon overexpression of soluble 4R0N tau, APP was preferentially localized to the endocytic compartments implicated in the amyloidogenic pathway. Overall, our observations indicate that soluble 4R0N tau abrogates the dynamics of the endolysosomal system, autophagy, and affects the trafficking of APP. Since the amyloidogenic processing of APP occurs during its progression through the endocytic pathway, our results suggest that the generation of amyloid-β (Aβ) might also be modulated.

Journal of Cell Science, 2020

Foot-and-mouth disease virus (FMDV) is a picornavirus that causes contagious acute infection in c... more Foot-and-mouth disease virus (FMDV) is a picornavirus that causes contagious acute infection in cloven-hoofed animals. FMDV replication associated viral protein expression induces endoplasmic reticulum (ER) stress and unfolded protein response (UPR), in turn inducing autophagy to restore cellular homeostasis. We observed that inhibition of BiP, a master regulator of ER stress and UPR, decreased FMDV infection confirming their involvement. Further, we show that the FMDV infection induces UPR mainly through PKR-like ER kinase (PERK)-mediated pathway. Knockdown of PERK and chemical inhibition of PERK activation resulted in decreased expression of FMDV proteins along with the reduction of autophagy marker protein LC3B-II. There are conflicting reports on the role of autophagy in FMDV multiplication. Our study systematically demonstrates that during FMDV infection, PERK mediated UPR stimulated an increased level of endogenous LC3B-II and turnover of SQSTM1, thus confirming the activation...

Autophagy, 2019

Macroautophagy/autophagy functions as a part of the innate immune system in clearing intracellula... more Macroautophagy/autophagy functions as a part of the innate immune system in clearing intracellular pathogens. Although this process is well known, the mechanisms that control antibacterial autophagy are not clear. In this study we show that during intracellular Salmonella typhimurium infection, the activity of TFEB (transcription factor EB), a master regulator of autophagy and lysosome biogenesis, is suppressed by maintaining it in a phosphorylated state on the lysosomes. Furthermore, we have identified a novel, antibacterial small molecule autophagy (xenophagy) modulator, acacetin. The xenophagy effect exerted by acacetin occurs in an MTOR (mechanistic target of rapamycin kinase)-independent, TFEB-dependent manner. Acacetin treatment results in persistently maintaining active TFEB in the nucleus and also in TFEB mediated induction of functional lysosomes that target Salmonella-containing vacuoles (SCVs). The enhanced proteolytic activity due to deployment of lysosomes results in clamping down Salmonella replication in SCVs. Acacetin is effective as a xenophagy compound in an in vivo mouse model of infection and reduces intracellular Salmonella burden.

Molecular Biology of the Cell, 2019

Autophagy is an evolutionarily conserved intracellular lysosomal degradation pathway. It is a mul... more Autophagy is an evolutionarily conserved intracellular lysosomal degradation pathway. It is a multistep process involving de novo formation of double membrane autophagosomes that capture cytosolic constituents (cargo) and eventually fuse with lysosomes wherein the cargo gets degraded and resulting simpler biomolecules get recycled. In addition to their autophagy function, several of the autophagy-related proteins work at the interface of other vesicular trafficking pathways. Hence, development of specific autophagy modulators that do not perturb general endo-lysosomal traffic possesses unique challenges. In this article, we report a novel small molecule EACC that inhibits autophagic flux by blocking autophagosome–lysosome fusion. Strikingly, unlike other late stage inhibitors, EACC does not have any effect on lysosomal properties or on endocytosis-mediated degradation of EGF receptor. EACC affects the translocation of SNAREs Stx17 and SNAP29 on autophagosomes without impeding the co...

Frontiers in Cell and Developmental Biology, 2018

Growing amount of evidence in the last two decades highlight that macroautophagy (generally refer... more Growing amount of evidence in the last two decades highlight that macroautophagy (generally referred to as autophagy) is not only indispensable for survival in yeast but also equally important to maintain cellular quality control in higher eukaryotes as well. Importantly, dysfunctional autophagy has been explicitly shown to be involved in various physiological and pathological conditions such as cell death, cancer, neurodegenerative, and other diseases. Therefore, modulation and regulation of the autophagy pathway has emerged as an alternative strategy for the treatment of various disease conditions in the recent years. Several studies have shown genetic or pharmacological modulation of autophagy to be effective in treating cancer, clearing intracellular aggregates and pathogens. Understanding and controlling the autophagic flux, either through a genetic or pharmacological approach is therefore a highly promising approach and of great scientific interest as spatiotemporal and cell-tissue-organ level autophagy regulation is not clearly understood. Indeed, chemical biology approaches that identify small molecule effectors of autophagy have thus a dual benefit: the modulators act as tools to study and understand the process of autophagy, and may also have therapeutic potential. In this review, we discuss different strategies that have appeared to screen and identify potent small molecule modulators of autophagy.

Cell death discovery, 2018

The precise role of autophagy in remains largely unknown. Although a limited number of autophagy ... more The precise role of autophagy in remains largely unknown. Although a limited number of autophagy genes have been identified in this apicomplexan, only Atg8 has been characterized to a certain extent. On the basis of the expression levels of Atg8 and the putative Atg5, we report that the basal autophagy in this parasite is quite robust and mediates not only the intraerythrocytic development but also fresh invasion of red blood cells (RBCs) in the subsequent cycles. We demonstrate that the basal autophagy responds to both inducers and inhibitors of autophagy. In addition, the parasite survival upon starvation is temporally governed by the autophagy status. Brief periods of starvation, which induces autophagy, help survival while prolonged starvation decreases autophagy leading to stalled parasite growth and reduced invasion. Thus, starvation-induced autophagy is context dependent. Importantly, we report characterization of another autophagy marker in this parasite, the putative Atg5 (...

Frontiers in molecular neuroscience, 2018

Mechanistic insights into aggrephagy, a selective basal autophagy process to clear misfolded prot... more Mechanistic insights into aggrephagy, a selective basal autophagy process to clear misfolded protein aggregates, are lacking. Here, we report and describe the role of Estrogen Related Receptor α (ERRα, HUGO Gene Nomenclature ESRRA), new molecular player of aggrephagy, in keeping autophagy flux in check by inhibiting autophagosome formation. A screen for small molecule modulators for aggrephagy identified ERRα inverse agonist XCT 790, that cleared α-synuclein aggregates in an autophagy dependent, but mammalian target of rapamycin (MTOR) independent manner. XCT 790 modulates autophagosome formation in an ERRα dependent manner as validated by siRNA mediated knockdown and over expression approaches. We show that, in a basal state, ERRα is localized on to the autophagosomes and upon autophagy induction by XCT 790, this localization is lost and is accompanied with an increase in autophagosome biogenesis. In a preclinical mouse model of Parkinson's disease (PD), XCT 790 exerted neuropr...

Autophagy, 2017

Due to the involvement of macroautophagy/autophagy in different pathophysiological conditions suc... more Due to the involvement of macroautophagy/autophagy in different pathophysiological conditions such as infections, neurodegeneration and cancer, identification of novel small molecules that modulate the process is of current research and clinical interest. In this work, we developed a luciferase-based sensitive and robust kinetic high-throughput screen (HTS) of small molecules that modulate autophagic degradation of peroxisomes in the budding yeast Saccharomyces cerevisiae. Being a pathway-specific rather than a target-driven assay, we identified small molecule modulators that acted at key steps of autophagic flux. Two of the inhibitors, Bay11 and ZPCK, obtained from the screen were further characterized using secondary assays in yeast. Bay11 inhibited autophagy at a step before fusion with the vacuole whereas ZPCK inhibited the cargo degradation inside the vacuole. Furthermore, we demonstrated that these molecules altered the process of autophagy in mammalian cells as well. Strikingly, these molecules also modulated autophagic flux in a novel model plant, Aponogeton madagascariensis. Thus, using small molecule modulators identified by using a newly developed HTS autophagy assay, our results support that macroautophagy is a conserved process across fungal, animal and plant kingdoms.

Journal of the Indian Institute of Science, 2017

Introduction Autophagy, an intracellular evolutionarily conserved process, involves engulfment of... more Introduction Autophagy, an intracellular evolutionarily conserved process, involves engulfment of unwanted proteins and organelles by double-membrane vesicles, called autophagosomes, which then fuse with the lysosomes/vacuole, and the engulfed cargo is subsequently degraded. It is a cell survival mechanism under stress conditions and it also play important roles in many other intra-cellular processes like protein and organelle turnover and transport of some of the vacuolar enzymes. This process can be divided into various steps, including autophagy induction, nucleation, autophagosome formation, maturation, fusion with the lysosomes/vacuole, degradation of the cargo, and recycling of the precursor molecules, such as amino acids, lipids, and nucleotides, back to the cytoplasm. Autophagy is a tightly regulated cellular mechanism and its flux varies depending on the cell type(s) of an organism. Autophagy is involved in various physiological roles, such as cellular homeostasis, embryonic development, antigen presentation, protein quality control, and maintenance of the amino-acid pool during starvation conditions. It is also implicated in various pathophysiological diseases, such as infection, cancer, diabetes, and neurodegeneration. Autophagosomes: The "Pac-Man" like double membrane vesicles involved in macroautophagy.

Autophagy is a conserved cellular degradation pathway wherein a double membrane vesicle, called a... more Autophagy is a conserved cellular degradation pathway wherein a double membrane vesicle, called as an autophagosome captures longlived proteins, damaged or superfluous organelles and delivers to the lysosome for degradation1. We have identified a novel role for septins in autophagy. Septins are GTP-binding proteins that localize at the bud-neck and are involved in cytokinesis in budding yeast2. We show that septins under autophagy prevalent conditions are majorly localized to the cytoplasm in the form of punctate structures. Further, we report that septins not only localize to pre-autophagosomal structure (PAS) but also to autophagosomes in the form of punctate structures. Interestingly, septins also form small non-canonical rings around PAS during autophagy. Furthermore, we observed that in one of the septin Ts" mutant,cdc10-5, the anterograde trafficking of Atg9 was affected at the non-permissive temperature (NPT). All these results suggest a role of septins in early stages o...

Scientific reports, Jan 30, 2015

Alzheimer's disease is one of the devastating illnesses mankind is facing in the 21(st) centu... more Alzheimer's disease is one of the devastating illnesses mankind is facing in the 21(st) century. The main pathogenic event in Alzheimer's disease is believed to be the aggregation of the β-amyloid (Aβ) peptides into toxic aggregates. Molecules that interfere with this process may act as therapeutic agents for the treatment of the disease. Use of recognition unit based peptidomimetics as inhibitors are a promising approach, as they exhibit greater protease stability compared to natural peptides. Here, we present peptidomimetic inhibitors of Aβ aggregation designed based on the KLVFF (P1) sequence that is known to bind Aβ aggregates. We improved inhibition efficiency of P1 by introducing multiple hydrogen bond donor-acceptor moieties (thymine/barbiturate) at the N-terminal (P2 and P3), and blood serum stability by modifying the backbone by incorporating sarcosine (N-methylglycine) units at alternate positions (P4 and P5). The peptidomimetics showed moderate to good activity in...

Journal of Cell Biology, 2010

Autophagy is important for many cellular processes such as innate immunity, neurodegeneration, ag... more Autophagy is important for many cellular processes such as innate immunity, neurodegeneration, aging, and cancer. Although the signaling events triggering autophagy have been studied, little is known regarding the signaling mechanisms by which autophagy is redirected to achieve selective removal of cellular components. We have used the degradation of a peroxisomal marker to investigate the role of protein kinases in selective autophagy of peroxisomes (pexophagy) in Saccharomyces cerevisiae. We show that the Slt2p mitogen-activated protein kinase (MAPK) and several upstream components of its signal transduction pathway are necessary for pexophagy but not for pexophagosome formation or other nonselective and selective forms of autophagy. Other extracellular signals that activate this pathway do not trigger pexophagy on their own, suggesting that this MAPK cascade is necessary but not sufficient to trigger pexophagy. We propose that pexophagy requires the simultaneous activation of thi...

Molecular Biology of the Cell, 2006

Sterol glucosyltransferase, Ugt51/Atg26, is essential for both micropexophagy and macropexophagy ... more Sterol glucosyltransferase, Ugt51/Atg26, is essential for both micropexophagy and macropexophagy of methanol-induced peroxisomes in Pichia pastoris. However, the role of this protein in pexophagy in other yeast remained unclear. We show that oleate- and amine-induced peroxisomes in Yarrowia lipolytica are degraded by Atg26-independent macropexophagy. Surprisingly, Atg26 was also not essential for macropexophagy of oleate- and amine-induced peroxisomes in P. pastoris, suggesting that the function of sterol glucoside (SG) in pexophagy is both species and peroxisome inducer specific. However, the rates of degradation of oleate- and amine-induced peroxisomes in P. pastoris were reduced in the absence of SG, indicating that P. pastoris specifically uses sterol conversion by Atg26 to enhance selective degradation of peroxisomes. However, methanol-induced peroxisomes apparently have lost the redundant ability to be degraded without SG. We also show that the P. pastoris Vac8 armadillo repea...

Developmental Cell, 2008

Autophagy, an intrinsically nonselective process, can also target selective cargo for degradation... more Autophagy, an intrinsically nonselective process, can also target selective cargo for degradation. The mechanism of selective peroxisome turnover by autophagy-related processes (pexophagy), termed micropexophagy and macropexophagy, is unknown. We show how a Pichia pastoris protein, PpAtg30, mediates peroxisome selection during pexophagy. It is necessary for pexophagy, but not for other selective and nonselective autophagy-related processes. It localizes at the peroxisome membrane via interaction with peroxins, and during pexophagy it colocalizes transiently at the preautophagosomal structure (PAS) and interacts with the autophagy machinery. PpAtg30 is required for formation of pexophagy intermediates, such as the micropexophagy apparatus (MIPA) and the pexophagosome (Ppg). During pexophagy, PpAtg30 undergoes multiple phosphorylations, at least one of which is required for pexophagy. PpAtg30 overexpression stimulates pexophagy even under peroxisome-induction conditions, impairing peroxisome biogenesis. Therefore, PpAtg30 is a key player in the selection of peroxisomes as cargo and in their delivery to the autophagy machinery for pexophagy.

Frontiers in Cell and Developmental Biology

Macroautophagy (henceforth autophagy) an evolutionary conserved intracellular pathway, involves l... more Macroautophagy (henceforth autophagy) an evolutionary conserved intracellular pathway, involves lysosomal degradation of damaged and superfluous cytosolic contents to maintain cellular homeostasis. While autophagy was initially perceived as a bulk degradation process, a surfeit of studies in the last 2 decades has revealed that it can also be selective in choosing intracellular constituents for degradation. In addition to the core autophagy machinery, these selective autophagy pathways comprise of distinct molecular players that are involved in the capture of specific cargoes. The diverse organelles that are degraded by selective autophagy pathways are endoplasmic reticulum (ERphagy), lysosomes (lysophagy), mitochondria (mitophagy), Golgi apparatus (Golgiphagy), peroxisomes (pexophagy) and nucleus (nucleophagy). Among these, the main focus of this review is on the selective autophagic pathway involved in mitochondrial turnover called mitophagy. The mitophagy pathway encompasses dive...