Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process necessary for normal recycling of cellular constituents and for appropriate response to cellular stress. Although several genes belonging to the core molecular machinery involved in autophagosome formation have been discovered, relatively little is known about the nature of signaling networks controlling autophagy upon intracellular or extracellular stimuli. We discovered ATG8-like proteins (MAP1LC3B, GABARAP and GABARAPL1) as novel interactors of MAPK15/ERK8, a MAP kinase involved in cell proliferation and transformation. Based on the role of these proteins in the autophagic process, we demonstrated that MAPK15 is indeed localized to autophagic compartments and increased, in a kinase-dependent fashion, ATG8-like proteins lipidation, autophagosome formation and SQSTM1 degradation, while decreasing LC3B inhibitory phosphorylation. Interestingly, we also identified a conserved LC3-interacting region (LIR) in MAPK15 responsible for its interaction with ATG8-like proteins, for its localization to autophagic structures and, consequently, for stimulation of the formation of these compartments. Furthermore, we reveal that MAPK15 activity was induced in response to serum and amino-acid starvation and that this stimulus, in turn, required endogenous MAPK15 expression to induce the autophagic process. Altogether, these results suggested a new function for MAPK15 as a regulator of autophagy, acting through interaction with ATG8 family proteins. Also, based on the key role of this process in several human diseases, these results supported the use of this MAP kinase as a potential novel therapeutic target.
MAP kinases; signal transduction; autophagy; LC3B; GABARAP; SQSTM1
Autophagy is a major degradative process responsible for the disposal of cytoplasmic proteins and dysfunctional organelles via the lysosomal pathway. During the autophagic process, cells form double-membraned vesicles called autophagosomes that sequester disposable materials in the cytoplasm and finally fuse with lysosomes. In the present study, we investigated the inhibition of autophagy by a synthesized compound, MHY1485, in a culture system by using Ac2F rat hepatocytes. Autophagic flux was measured to evaluate the autophagic activity. Autophagosomes were visualized in Ac2F cells transfected with AdGFP-LC3 by live-cell confocal microscopy. In addition, activity of mTOR, a major regulatory protein of autophagy, was assessed by western blot and docking simulation using AutoDock 4.2. In the result, treatment with MHY1485 suppressed the basal autophagic flux, and this inhibitory effect was clearly confirmed in cells under starvation, a strong physiological inducer of autophagy. The levels of p62 and beclin-1 did not show significant change after treatment with MHY1485. Decreased co-localization of autophagosomes and lysosomes in confocal microscopic images revealed the inhibitory effect of MHY1485 on lysosomal fusion during starvation-induced autophagy. These effects of MHY1485 led to the accumulation of LC3II and enlargement of the autophagosomes in a dose- and time- dependent manner. Furthermore, MHY1485 induced mTOR activation and correspondingly showed a higher docking score than PP242, a well-known ATP-competitive mTOR inhibitor, in docking simulation. In conclusion, MHY1485 has an inhibitory effect on the autophagic process by inhibition of fusion between autophagosomes and lysosomes leading to the accumulation of LC3II protein and enlarged autophagosomes. MHY1485 also induces mTOR activity, providing a possibility for another regulatory mechanism of autophagy by the MHY compound. The significance of this study is the finding of a novel inhibitor of autophagy with an mTOR activating effect.
Nrf2 regulates the expression of numerous cytoprotective genes in mammalian cells. The activity of Nrf2 is regulated by the Cul3 adaptor Keap1, yet little is known regarding mechanisms of regulation of Keap1 itself. Here, we have used immunopurification of Keap1 and mass spectrometry, in addition to immunoblotting, to identify sequestosome 1 (SQSTM1) as a cellular binding partner of Keap1. SQSTM1 serves as a scaffold in various signaling pathways and shuttles polyubiquitinated proteins to the proteasomal and lysosomal degradation machineries. Ectopic expression of SQSTM1 led to a decrease in the basal protein level of Keap1 in a panel of cells. Furthermore, RNA interference (RNAi) depletion of SQSTM1 resulted in an increase in the protein level of Keap1 and a concomitant decrease in the protein level of Nrf2 in the absence of changes in Keap1 or Nrf2 mRNA levels. The increased protein level of Keap1 in cells depleted of SQSTM1 by RNAi was linked to a decrease in its rate of degradation; the half-life of Keap1 was almost doubled by RNAi depletion of SQSTM1. The decreased level of Nrf2 in cells depleted of SQSTM1 by RNAi was associated with decreases in the mRNA levels, protein levels, and function of several Nrf2-regulated cell defense genes. SQSTM1 was dispensable for the induction of the Keap1-Nrf2 pathway, as Nrf2 activation by tert-butylhydroquinone or iodoacetamide was not affected by RNAi depletion of SQSTM1. These findings demonstrate a physical and functional interaction between Keap1 and SQSTM1 and reveal an additional layer of regulation in the Keap1-Nrf2 pathway.
Antioxidant; Gene Regulation; Oxidative Stress; Protein-Protein Interactions; Signal Transduction; Keap1; Nrf2; SQSTM1
Autophagy is an evolutionarily conserved lysosomal self-digestion process involved in degradation of long-lived proteins and damaged organelles. In recent years, increasing evidence indicates that autophagy is associated with a number of pathological processes, including cancer. In this review, we focus on the recent studies of the evolutionarily conserved autophagy-related genes (ATGs) that are implicated in autophagosome formation and the pathways involved. We discuss several key autophagic mediators (eg, Beclin-1, UVRAG, Bcl-2, Class III and I PI3K, mTOR, and p53) that play pivotal roles in autophagic signaling networks in cancer. We discuss the Janus roles of autophagy in cancer and highlighted their relationship to tumor suppression and tumor progression. We also present some examples of targeting ATGs and several protein kinases as anticancer strategy, and discuss some autophagy-modulating agents as antitumor agents. A better understanding of the relationship between autophagy and cancer would ultimately allow us to harness autophagic pathways as new targets for drug discovery in cancer therapeutics.
autophagy; cancer; autophagy-related gene (ATG); Beclin-1; Bcl-2; Class III and I PI3K; mTOR; p53
Autophagy is a self-degradation system of cellular components through an autophagosomal-lysosomal pathway. Over the last 15 yr, yeast genetic screens led to the identification of a number of genes involved in the autophagic pathway. Most of these autophagy genes are present in higher eukaryotes and regulate autophagy process for cell survival and homeostasis. Significant progress has recently been made to better understand the molecular mechanisms of the autophagy machinery. Especially, autophagy process, including the regulation of autophagy induction through mTOR and the nucleation and elongation in autophagosome formation through class III phosphatidylinositol 3-kinase complex and ubiquitin-like conjugation systems, became evident. While many unanswered questions remain to be answered, here, we summarize the recent process of autophagy with emphasis on molecules and their protein complexes along with advanced molecular mechanisms that regulate the autophagy machinery.
ATG2 protein, human; ATG5 protein, human; ATG9B protein, human; ATG12 protein, human; ATG16L1 protein, human; autophagy; class III phosphatidylinositol 3-kinases; MTOR protein, human; ULK1 protein, human
Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and coimmunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant huntingtin. We suggest that p62 may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagy machinery.
Autophagy plays an evolutionarily conserved role in host defense against pathogens. Autophagic protection mechanisms against microbes range from regulating immune signaling responses to directly targeting the pathogens for lysosomal degradation. Toll-like receptors (TLR s) that detect conserved molecular features shared by pathogens regulate several innate immune responses including autophagy. Our recent study demonstrates that autophagy reported in response to TLR4-stimulation in macrophages is selective autophagy of aggresome-like induced structures (ALIS), and p62 (also known as SQSTM1) plays an essential role in this process. Treatment of macrophages with either Escherichia coli or lipopolysaccharide (LPS) results in the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), leading to an increase in the levels of p62 mRNA and protein, assembly of ALIS and their autophagic degradation. This study revealed a signaling role for p62, distinct from its known function as a bacterial-targeting factor, which might be critical for cellular stress response during infection.
p62; Nrf2; TLR4; autophagy; ALIS
Farnesoid X Receptor (FXR) is a member of the nuclear receptor superfamily and is a ligand-activated transcription factor essential for maintaining liver and intestinal homeostasis. FXR is protective against carcinogenesis and inflammation in liver and intestine as demonstrated by the development of inflammation and tumors in the liver and intestine of FXR knock-out mice. However, mechanisms for the protective effects of FXR are not completely understood. This study reports a novel role of FXR in regulating expression of Sqstm1, which encodes for p62 protein. p62 plays an important role in maintaining cellular homeostasis through selective autophagy and activating signal transduction pathways, such as NF-κB to support cell survival and caspase-8 to initiate apoptosis. FXR regulation of Sqstm1 may serve as a protective mechanism.
Methods and Results
This study showed that FXR bound to the Sqstm1 gene in both mouse livers and ileums as determined by chromatin immunoprecipitation. In addition, FXR activation enhanced transcriptional activation of Sqstm1 in vitro. However, wild-type mice treated with GW4064, a synthetic FXR ligand, showed that FXR activation induced mRNA and protein expression of Sqstm1/p62 in ileum, but not in liver. Interestingly, FXR-transgenic mice showed induced mRNA expression of Sqstm1 in both liver and ileum compared to wild-type mice.
Our current study has identified a novel role of FXR in regulating the expression of p62, a key factor in protein degradation and cell signaling. Regulation of p62 by FXR indicates tissue-specific and gene-dosage effects. Furthermore, FXR-mediated induction of p62 may implicate a protective mechanism of FXR.
Induction of autophagy in response to starvation is a highly conserved ability of eukaryotic cells, indicating a critical and ancient role of this process in adapting to nutrient conditions. The target of rapamycin (TOR) pathway is major conduit for such signals, and in most cell types TOR activity is necessary and sufficient to suppress autophagy under favorable growth conditions. Recent studies have begun to reveal how TOR activity is regulated in response to nutritional cues, and are shedding new light on the mechanisms by which TOR controls the autophagic machinery. In addition, a variety of signals, stressors and pharmacological agents that induce autophagy independent of nutrient conditions have been identified. In some cases these signals appear to have been spliced into the core TOR pathway, whereas others are able to bypass the control mechanisms regulated by TOR. Increasing evidence is pointing to an important role for both positive and negative feedback loops in controlling this pathway, leading to an emerging view that TOR signaling not only regulates autophagy but is also highly sensitive to cellular rates of autophagy and other TOR-dependent processes.
Autophagy is a fundamental homeostatic process in which cytoplasmic targets are sequestered within double-membraned autophagosomes and subsequently delivered to lysosomes for degradation. Accumulating evidence supports the pivotal role of autophagy in host defense against intracellular pathogens implicating both innate and adaptive immunity. Many of these pathogens cause common zoonotic infections worldwide. The induction of the autophagic machinery by innate immune receptors signaling, such as TLRs, NOD1/2, and p62/SQSTM1 in antigen-presenting cells results in inhibition of survival and elimination of invading pathogens. Furthermore, Th1 cytokines induce the autophagic process, whereas autophagy also contributes to antigen processing and MHC class II presentation, linking innate to adaptive immunity. However, several pathogens have developed strategies to avoid autophagy or exploit autophagic machinery to their advantage. This paper focuses on the role of host cell autophagy in the regulation of immune response against intracellular pathogens, emphasizing on selected bacterial and protozoan zoonoses.
Chondrocytes in the growth plate and articular cartilage, and osteocytes subsumed in Haversian bone exist in environmental niches that are characterized by a limited oxygen supply. In these tissues, cells display a hitherto unrecognized state in which there is evidence of autophagy. The autophagic condition serves to promote cell survival. When the response is triggered, the cell cannibalizes itself to generate energy; if extended, then it can activate Type II apoptosis. We opine that survival is dependent on niche conditions and regulated by crosstalk between mTOR, AMPK and HIF-1 and HIF-2. Recent studies suggest that HIF-2 is a potent regulator of chondrocyte autophagy and that this protein acts as a brake to the stimulatory function of HIF-1. Accordingly, the oxemic state of the tissue, its nutrient supply as well as the energetic state of the cells regulates autophagic flux. From a clinical viewpoint, it may be possible to enhance skeletal cell survival through drugs that modulate the autophagic state and prevent the induction of apoptosis.
autophagy; HIF-1; HIF-2; AMPK; mTOR; chondrocyte; osteocyte
Recent publications showed that the kinase MTOR localizes to lysosomes and its activation depends on amino acids inside the lysosomal lumen, implying that autophagic protein degradation is a positive regulator of MTOR in this setting. Since decreased MTOR activity results in autophagy induction, drug treatments that block autolysosomal degradation (a commonly used technique to estimate autophagic flux) may actually interfere not only with lysosomal breakdown, but also increase autophagosome generation through impaired MTOR signaling.
Atg8; autophagy; bafilomycin A1; chloroquine; LC3; leupeptin; lysosome; MTOR; TOR; v-ATPase
Mammalian target of rapamycin (mTOR) is a protein kinase involved in translation control and long-lasting synaptic plasticity. mTOR functions as the central component of two multi-protein signaling complexes, mTORC1 and mTORC2, which can be distinguished from each other based on their unique compositions and substrates. Although majority of evidence linking mTOR function to synaptic plasticity comes from studies utilizing rapamycin, studies in genetically-modified mice also suggest that mTOR couples receptors to the translation machinery for establishing long-lasting synaptic changes that are the basis for higher order brain function, including long-term memory. Finally, perturbation of the mTOR signaling cascade appears to be a common pathophysiological feature of human neurological disorders, including mental retardation syndromes and autism spectrum disorders.
Neurons have highly dynamic cellular processes for their proper functions such as cell growth, synaptic formation, or synaptic plasticity by regulating protein synthesis and degradation. Therefore, the quality control of proteins in neurons is essential for their physiology and pathology. Autophagy is a cellular degradation pathway by which cytosolic components are sequestered in autophagosomes and degraded upon their fusion with lysosomal components. Thus, the autophagic pathway may play important roles in neuronal cell survival and neuronal function under physiological condition and pathological conditions. Recent several findings suggest that the loss of basal autophagy or imbalance of autophagic flux leads to neurodegeneration. Autophagosomes accumulate abnormally in affected neurons of several neurodegenerative diseases such as Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), or Frontotemporal dementia (FTD). Thus, the understanding how autophagy is associated with several neurological diseases would be the first step for new therapeutic intervention in neurological disorders. In this review, I will discuss the molecular mechanism of autophagy in neurons and autophagy-associated neurodegenerative diseases.
neuron; autophagy; neurodegenerative disease; homeostasis
Autophagy is an evolutionarily conserved process of cellular self-eating and is a major pathway for degradation of cytoplasmic material by the lysosomal machinery. Autophagy functions as a cellular response in nutrient starvation, but it is also associated with the removal of protein aggregates and damaged organelles and therefore plays an important role in the quality control of proteins and organelles. Although it was initially believed that autophagy occurs randomly in the cell, during the last years, there is growing evidence that sequestration and degradation of cytoplasmic material by autophagy can be selective. Given the important role of autophagy and selective autophagy in several disease-related processes such as neurodegeneration, infections, and tumorigenesis, it is important to understand the molecular mechanisms of selective autophagy, especially at the organismal level. Drosophila is an excellent genetically modifiable model organism exhibiting high conservation in the autophagic machinery. However, the regulation and mechanisms of selective autophagy in Drosophila have been largely unexplored. In this paper, I will present an overview of the current knowledge about selective autophagy in Drosophila.
Calcium can play an important role in the regulation of autophagy. We previously reported that exogenously introduced calcium in the form of calcium phosphate precipitates (CPP) induces autophagy. Here we showed that CPP-induced autophagy required the classical autophagic machinery, including the autophagosome initiating molecules FIP200 and Beclin 1, as well as molecules involved in the autophagosome membrane extension, Atg4, Atg5 and Atg3. On the other hand, Atg9 seemed to place a restriction on CPP-induced autophagy. Loss of Atg9 led to enhanced LC3 punctation and enhanced p62 degradation. CPP-induced autophagy was independent of mTOR and reactive oxygen species. It also did not affect MAP kinase activation and ER stress. DFCP1 is an ER-resident molecule that binds to phosphatidylinositol 3-phosphate. CPP activated DFCP1 punctation in a class III phosphatidylinositol-3-kinase and calcium dependent manner, and caused the association of DFCP1 puncta with the autophagosomes. Consistently, ER membranes, but not Golgi or mitochondrial membranes, colocalized with CPP-induced LC3 positive autophagosomes. These data suggest that CPP-induced autophagosome formation involves the interaction with the ER membrane.
Protein synthesis and autophagic degradation are regulated in an opposite manner by mammalian target of rapamycin (mTOR), whereas under certain conditions it would be beneficial if they occured in unison to handle rapid protein turnover. We observed a distinct cellular compartment at the trans-side of the Golgi apparatus, the ‘TOR-autophagy spatial coupling compartment’ (TASCC), where (auto)lysosomes and mTOR accumulated during Ras-induced senescence. mTOR recruitment to the TASCC was amino acid- and Rag guanosine triphosphatase (GTPase)-dependent, and disruption of mTOR localization to the TASCC suppressed interleukin-6/8 synthesis. TASCC-formation was observed during macrophage differentiation and in glomerular podocytes; both displayed increased protein secretion. The spatial coupling of cells’ catabolic and anabolic machinery could augment their respective functions and facilitate the mass synthesis of secretory proteins.
Lysosomal regulation is a poorly understood mechanism that is central to degradation and recycling processes. Here we report that LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and mTOR activator 1) downregulation affects lysosomal activation, through mechanisms that are not solely due to mTORC1 inhibition. LAMTOR1 depletion strongly increases lysosomal structures that display a scattered intracellular positioning. Despite their altered positioning, those dispersed structures remain overall functional: (i) the trafficking and maturation of the lysosomal enzyme cathepsin B is not altered; (ii) the autophagic flux, ending up in the degradation of autophagic substrate inside lysosomes, is stimulated. Consequently, LAMTOR1-depleted cells face an aberrant lysosomal catabolism that produces excessive reactive oxygen species (ROS). ROS accumulation in turn triggers p53-dependent cell cycle arrest and apoptosis. Both mTORC1 activity and the stimulated autophagy are not necessary to this lysosomal cell death pathway. Thus, LAMTOR1 expression affects the tuning of lysosomal activation that can lead to p53-dependent apoptosis through excessive catabolism.
LAMTOR1; apoptosis; p53; ROS; lysosomes
Autophagy is the regulated process by which cytoplasmic organelles and long-lived proteins are delivered for lysosomal degradation. Increased numbers of autophagosomes and autolysosomes often represent prominent ultrastructural features of degenerating or dying neurons. This morphology is characteristic not only of neurons undergoing pathologic degeneration, but also during developmental programmed cell death of some neuronal populations. In recent years, a growing number of reports highlight potentially important roles for autophagy-related processes in relation to protein aggregation, regulated cell death pathways, and neurodegeneration. While starvation-induced autophagy involves nonselective bulk degradation of cytoplasm, mechanisms that regulate selective targeting of damaged organelles form an emerging area. As the study of autophagy evolves from physiologic homeostasis to pathologic situations, consideration of terminology and definitions becomes important. Increased autophagic vacuoles do not necessarily correlate with increased autophagic activity or flux. Instead, the striking accumulation of autophagic vacuoles in dying or degenerating neurons likely reflects an imbalance between the rates of autophagic sequestration and completion of the degradative process. In other words, these cells can be thought of as undergoing “autophagic stress.” The concept of autophagic stress may reconcile apparently conflicting roles of autophagy-related processes in adaptive, homeostatic responses and in pathways of neurodegeneration and cell death.
Alzheimer disease; Autophagy; Huntington disease; Neurodegeneration; Mitochondria; Lysosomal storage disease; Parkinson disease; Programmed cell death
Autophagy is a cellular lysosomal degradation pathway essential for regulation of cell survival and death to maintain homeostasis. This process is negatively regulated by mammalian target of rapamycin (mTOR) signaling and often counteracts efficacy of certain cancer therapeutic agents. NVP-BEZ235 (BEZ235) is a novel, orally bioavailable dual PI3K/mTOR inhibitor that has exhibited promising activity against non-small cell lung cancer (NSCLC) in preclinical models. The current study focuses on evaluating the role of BEZ235 in regulating autophagy. BEZ235 was effective in inhibiting the growth of NSCLC cells including induction of apoptosis. It also potently induced the expression of type-II LC3, indicating induction of autophagy. When BEZ235 was used in combination with the lysosomal or autophagic inhibitor chloroquine (CQ), enhanced inhibitory effects on monolayer growth and colony formation of NSCLC cells was observed. In addition, enhanced induction of apoptosis was also detected in cells exposed to the combination of BEZ235 and CQ. Moreover, the combination of BEZ235 and CQ was more effective than each single agent alone in inhibiting the growth of NSCLC xenografts in nude mice. Thus, induction of autophagy by BEZ235 appears to be a survival mechanism that may counteract its anticancer effects. Based on these, we suggest a strategy to enhance BEZ235's anticancer efficacy by blockade of autophagy.
BEZ235; PI3K; mTOR; autophagy; apoptosis; lung cancer
Short-chain fatty acids are the major by-products of bacterial fermentation of undigested dietary fibers in human large intestine. SCFAs, mostly propionate and butyrate, inhibit proliferation and induce apoptosis in colon cancer cells, but clinical trials had mixed results regarding the anti-tumor activities of SCFAs. Herein we demonstrate that propionate and butyrate induced autophagy in human colon cancer cells to dampen apoptosis whereas inhibition of autophagy potentiated SCFA induced apoptosis. Colon cancer cells, after propionate treatment, exhibited extensive characteristics of autophagic proteolysis: increased LC3-I to LC3-II conversion, acidic vesicular organelle development and reduced p62/SQSTM1 expression. Propionate-induced autophagy was associated with decreased mTOR activity and enhanced AMP kinase activity. The elevated AMPKα phosphorylation was associated with cellular ATP depletion and overproduction of reactive oxygen species due to mitochondrial dysfunction involving the induction of MPT and loss of Δψ. In this context, mitochondria biogenesis was initiated to recover cellular energy homeostasis. Importantly, when autophagy was prevented either pharmacologically (3-MA or chloroquine) or genetically (knockdown of ATG5 or ATG7), the colon cancer cells became sensitized toward propionate induced apoptosis through activation of caspase 7 and its downstream effector caspase-3. The observations indicate that propionate-triggered autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death, whereas application of an autophagy inhibitor (Chloroquine) is expected to enhance the therapeutic efficacy of SCFAs in inducing colon tumor cell apoptosis.
Short-chain fatty acids; autophagy; apoptosis; mitochondrial; colon cancer
Short-chain fatty acids (SCFAs) are the major by-products of bacterial fermentation of undigested dietary fibers in the large intestine. SCFAs, mostly propionate and butyrate, inhibit proliferation and induce apoptosis in colon cancer cells, but clinical trials had mixed results regarding the anti-tumor activities of SCFAs. Herein we demonstrate that propionate and butyrate induced autophagy in human colon cancer cells to dampen apoptosis whereas inhibition of autophagy potentiated SCFA induced apoptosis. Colon cancer cells, after propionate treatment, exhibited extensive characteristics of autophagic proteolysis: increased LC3-I to LC3-II conversion, acidic vesicular organelle development, and reduced p62/SQSTM1 expression. Propionate-induced autophagy was associated with decreased mTOR activity and enhanced AMP kinase activity. The elevated AMPKα phosphorylation was associated with cellular ATP depletion and overproduction of reactive oxygen species due to mitochondrial dysfunction involving the induction of MPT and loss of Δψ. In this context, mitochondria biogenesis was initiated to recover cellular energy homeostasis. Importantly, when autophagy was prevented either pharmacologically (3-MA or chloroquine) or genetically (knockdown of ATG5 or ATG7), the colon cancer cells became sensitized toward propionate-induced apoptosis through activation of caspase-7 and caspase-3. The observations indicate that propionate-triggered autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death, whereas application of an autophagy inhibitor (Chloroquine) is expected to enhance the therapeutic efficacy of SCFAs in inducing colon tumor cell apoptosis.
short-chain fatty acids; autophagy; apoptosis; mitochondrial; colon cancer
The process of macroautophagy (referred to hereafter as autophagy), is generally characterized by the prominent formation of autophagic vesicles in the cytoplasm. In the past decades, studies of autophagy have been vastly expanded. As an essential process to maintain cellular homeostasis and functions, autophagy is responsible for the lysosome-mediated degradation of damaged proteins and organelles, and thus misregulation of autophagy can result in a variety of pathological conditions in human beings. Although our understanding of regulatory pathways that control autophagy is still limited, an increasing number of studies have shed light on the importance of autophagy in a wide range of physiological processes and human diseases. The goal of the reviews in the current issue is to provide a general overview of current knowledge on autophagy. The machinery and regulation of autophagy were outlined with special attention to its role in diabetes, neurodegenerative disorders, infectious diseases and cancer.
autophagy; disease; physiology
Autophagy is a compensatory pathway involving isolation and subsequent degradation of cytosolic material and organelles in eukaryotic cells.1 The autophagic process can provide a “housekeeping” function by removing damaged proteins and organelles in a selective or nonselective fashion in order to exert a protective effect following stress.2 Remarkably, after being discovered to be much more of a targeted process than a random one, the role of autophagy became implicated in many normal cellular and disease processes.3 Several methodologies are routinely employed to monitor the entire autophagic process.4 Microtubule-associated protein light chain 3, a mammalian homolog of yeast Atg8, has been widely used as a specific marker to monitor autophagy in numerous cell types.5 While monitoring autophagic flux is extremely important, it is also beneficial to perform a detailed analysis by electron microscopy (EM) to evaluate changes in various autophagic structures, quantify the areas involved, and determine if any particular organelle(s) or area of the cell cytoplasm is being targeted for degradation.6 The following article describes methods to localize and quantify subcellular areas of autophagy using transmission EM. Also discussed are methods for subcellular localization of specific proteins by employing immunogold EM; this method becomes particularly useful in detecting early changes in cellular homeostasis that may occur before later signs of cellular insult can be observed morphologically.
autophagy; electron microscopy; immunogold; morphometry; ultra-structure
Nutrient starvation induces autophagy in eukaryotic cells through inhibition of TOR (target of rapamycin), an evolutionarily-conserved protein kinase. TOR, as a central regulator of cell growth, plays a key role at the interface of the pathways that coordinately regulate the balance between cell growth and autophagy in response to nutritional status, growth factor and stress signals. Although TOR has been known as a key regulator of autophagy for more than a decade, the underlying regulatory mechanisms have not been clearly understood. This review discusses the recent advances in understanding of the mechanism by which TOR regulates autophagy with focus on mammalian TOR (mTOR) and its regulation of the autophagy machinery.
mTOR; Atg1; ULK1; ULK2; Atg13