Browsing by Subject "Autophagy"
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Item Autophagy in Antiviral Immunity(2012-08-15) Orvedahl, Anthony Walter; Levine, BethAutophagy is an evolutionarily conserved pathway in which cytoplasmic material is sequestered in a double-membrane vesicle and delivered to the lysosome for degradation. During times of stress, autophagy functions to generate essential nutrients through the degradation of non-essential cytoplasmic contents. It is also the only known mechanism for removal of damaged or superfluous organelles and cytoplasmic contents that are too large to be degraded by the proteasome. Given the critical role for autophagy in stress response and in maintaining cell cytoplasmic quality control, it is not surprising that autophagy plays an essential role in the host response to infection, and that microbes have evolved mechanisms to counteract or evade autophagy. In this work, we studied the role of autophagy inhibition in a mouse model of herpes simplex virus type I (HSV-1) encephalitis, investigated the role of autophagy in protection against Sindbis virus infection of the central nervous system, and identified novel host genes involved in targeting viral proteins to the autophagy pathway. We found that the HSV-1 encoded neurovirulence protein ICP34.5 interacted with the host autophagy protein Beclin 1, and that this interaction was essential for HSV-1 neurovirulence. This was the first example of a viral virulence protein that targets host autophagy, and provided evidence that autophagy functions in innate immunity to viruses. In the second study, we found that the host autophagy gene Atg5 was required to protect against lethal Sindbis virus CNS diseases, and that autophagy targeted viral proteins for degradation in brains of infected mice and cells in vitro. We found that the autophagy adaptor protein p62 was involved in targeting viral proteins for autophagic degradation and this promoted survival of infected cells. This study demonstrated that clearance of viral proteins by autophagy was an important mechanism for cellular and organismal survival during viral infection. Lastly, we performed a genome-wide siRNA screen to identify novel host factors required for autophagic targeting of viral proteins. We identified previously unappreciated cellular networks and genes that were involved in targeting viral proteins for autophagy. One of these factors, SMURF1, is an E3 ubiquitin ligase that not only functions to target viral proteins, but is also involved in targeting damaged mitochondria for autophagic clearance. [Keywords: autophagy; innate immunity; virus; infection; Herpes Simplex Virus Tupe I (HSV-1); Sindbis virus; mitophagy; central nervous system (CNS); pb2/SQSTM1; SMURF1]Item Cardiomyocyte Autophagy is Induced by Protein Aggregation in Heart Disease(2009-06-19) Tannous, Paul; Hill, Joseph A.Autophagy is associated with diverse forms of myocardial stress. When I initiated my studies activators of this pathway had not been identified in the heart, nor was it clear weather autophagy is an adaptive or maladaptive response in the stressed myocardium. My initial research focused on autophagy in hypertension-induced heart failure, the most common cardiovascular disease in Western nations. Early evidence demonstrated generation of reactive oxygen species, protein damage, and protein aggregation in the acute period of pressure overload. Given the simultaneous presence of autophagosomes and aggregates, and autophagy's role in bulk degradation, I postulated these events were mechanistically linked. I designed experiments to test the hypotheses that protein aggregates are activators of autophagy in the heart, and that autophagy functions in aggregate clearance. Here I report novel findings that link pressure overload-induced protein aggregation to increased cardiomyocyte autophagy. Specifically, in the pressure-stressed ventricle 1) generation of reactive oxygen species is an early pathological event, 2) there is extensive protein aggregation with higher-order processing into aggresomes, 3) protein aggregation induces cardiomyocyte autophagy, and 4) in this setting autophagy functions in its role as a mechanism of bulk protein degradation. These findings are the first to demonstrate proteinopathy of non-genetic etiology contributes to hypertension-induced heart failure and that protein aggregates are robust activators of cardiomyocyte autophagy. To directly address the role of autophagy in cardiomyocyte clearance of toxic protein species, I turned my attention to CryABR120G-induced desmin-related cardiomyopathy (DRCM), an aggregate-associated disease with autosomal dominant inheritance. My studies demonstrated that 1) autophagy is activated by CryABR120G-induced protein aggregation, 2) aggregate formation is inversely proportional to the degree of autophagic activity and 3) blunting autophagy accelerates pathological myocardial remodeling and the onset of heart failure. Extending this work to clinical medicine, we observed increased autophagy in the skeletal muscle from patients with desmin-related skeletal myopathy. Cumulatively these data are the first to demonstrate autophagy is induced in DRCM and functions as a protective cellular response. These findings suggest autophagy is a pathway amenable to therapeutic intervention in patients suffering from myofibrillar myopathy, a disease class for which there are limited therapeutic options.Item Characterization of Vibrio VopS, an AMPylator of Rho GTPases(2009-06-19) Yarbrough, Melanie Leann; Orth, KimVibrio parahaemolyticus is a gram-negative marine bacterium that causes gastroenteritis associated with the consumption of contaminated shellfish. The emergence of pandemic strains of V. parahaemolyticus has increased the need for characterization of the virulence factors of this pathogen. Sequencing of the genome of a clinical isolate revealed the presence of two type III secretion systems (T3SSs), one on each chromosome. The T3SS on chromosome one (T3SS1) has been shown to be responsible for cytotoxicity in HeLa cells, and it shares a high degree of homology to the T3SS of the Yersinia spp. Our studies have shown that infection of HeLa cells with a strain of V. parahaemolyticus capable of secreting only from T3SS1 indicated that T3SS1 mediates several events during infection including the rapid induction of autophagy, cell rounding, and finally lysis of the cell. Defining the T3SS1-mediated events of infection gives insight into virulence mechanisms of V. parahaemolyticus that have not been well characterized and provide a basis for the elucidation of the functions associated with T3SS1 effectors. One of the T3SS effectors, VopS, contains a Filamentation induced by cAMP (Fic) domain that we have shown is critical for the function of this effector. Our studies have found that VopS inhibits Rho GTPase signaling during infection by directly modifying Rho, Rac, and Cdc42, preventing their interaction with downstream effectors. These observations reveal a unique activity for VopS, which targets a pathway that is critical in the cellular response to V. parahaemolyticus infection. In addition, they provide insight into a novel post-translational modification that may expand our knowledge of eukaryotic cell signaling. Fic domains are found in proteins from several bacterial and eukaryotic species and are recognized by their conserved motif, HPFX(D/E)GNGR. The presence of Fic domains in higher eukaryotes suggested that this modification could be utilized in cell signaling. Our preliminary studies indicated that AMPylation is utilized by eukaryotes. We have shown that a Fic protein from humans, HYPE, possesses auto-AMPylation activity, confirming our hypothesis that these domains are involved in AMPylation. Ongoing and future studies seek to identify the substrates of HYPE activity and identify other components involved in this new layer of eukaryotic cell signaling.Item Characterization of VOPQ, A Type III Secreted Effector Protein from Vibrio Parahaemolyticus(2009-06-15) Burdette, Dara Lesley; Orth, KimVibrio parahaemolyticus is a Gram-negative bacterium responsible for gastroenteritis associated with the consumption of raw or undercooked shellfish. Its most well-characterized virulence factors are hemolysins that cause b-hemolysis on a special blood agar. Mutants lacking these hemolysins are still virulent in animal and tissue culture models of infection. These phenomena can be attributed in part to one of two type III secretion systems; one on chromosome 1 and the other on chromosome 2. We demonstrate that Vibrio parahaemolyticus utilizes the type III secretion system on chromosome 1 to induce a temporally regulated series of events that initiates with the induction of autophagy, followed by cellular rounding and finally cellular lysis and death. To the best of our knowledge, no other Gram-negative extracellular bacterium has been shown to induce autophagy during infection. To understand the mechanism of Vibrio parahaemolyticus induced cell death, we focused our analysis on VopQ, a type III secreted effector encoded by the type III locus on chromosome 1. We demonstrate that VopQ contributes to cytotoxicity as DvopQ strains induce cell lysis less efficiently. In addition, VopQ is necessary and sufficient for the induction of autophagy during infection. VopQ-mediated autophagy occurs independently of phosphatidylinositol 3-kinases and prevents phagocytosis. Additional experiments using Saccharomyces cerevisiae demonstrate VopQ induces autophagy and cell death through an evolutionarily conserved mechanism. Results presented herein delineate a novel virulence mechanism used by Vibrio parahaemolyticus to cause disease. This study also highlights the effector VopQ as a novel inducer of autophagy and a key mediator of cytotoxicity during infection.Item Mechanistic Studies of Autophagy Initiation in Mammalian Cells(2011-08-26T17:35:06Z) Shang, Libin; Wang, XiadongMacroautophagy (herein referred to as autophagy) is an evolutionarily conserved self-digestive process cells use to adapt to starvation and other stresses. During autophagy, portions of cytoplasmic materials are engulfed into specialized double-membrane structures to form autophagosomes, which then fuse with lysosomes to degrade their cargos and regenerate nutrients. Initiation of autophagy has been extensively studied in budding yeast Saccharomyces cerevisiae. However, various significant differences exist between yeast and mammals. To pinpoint how mammalian autophagy is initiated, I first adopted proteomic approaches to identify associating partners of Unc-51-like kinase 1 (Ulk1), key initiator for mammalian autophagy. Two novel proteins, mAtg13 and Atg101, were found to interact with Ulk1 stoichiometrically. Knockdown of either mAtg13 or Atg101 led to decreased autophagy, and autophagy could be rescued with exogenous expression, suggesting the two proteins were critical for mammalian autophagy initiation. I then observed Ulk1 undergoes dramatic dephosphorylation upon starvation, particularly at serine 638 and serine 758. I found phosphorylations of Ulk1 are mediated by mammalian target-of-rapamycin (mTOR) kinase and AMP-activated protein kinase (AMPK). AMPK interacts with Ulk1 in a nutrient-dependent manner, and proper phosphorylations on Ulk1 are crucial for Ulk1/AMPK association, as a single serine-to-alanine mutation (S758A) at Ulk1 impairs this interaction. Compared to its wild-type counterpart, this Ulk1-S758A mutant initiates starvation-induced autophagy faster at early time points, but does not alter the maximum capacity of autophagy when starvation prolongs. With this layer of regulation, mammalian autophagy is capable of responding to environmental changes more promptly than previously considered.Item Mitophagy in Heart Failure-A Selective Autophagic Degradation of Mitochondria(2009-06-05) Yang, Kai-Chun (Daniel); Hill, Joseph A.INTRODUCTION: Cardiovascular disease is associated with declines in mitochondrial function. Autophagy is a lysosomal-dependent process through which cytoplasmic proteins and organelles can be degraded and has recently been shown to participate in remodeling of the myocardium in a variety of cardiac pathologies. Autophagy can be either non-selective or selective for damaged protein aggregates or organelles. Reactive oxygen species (ROS) generated in mitochondria causes mitochondrial permeability transition (MPT) and induces selective degradation of mitochondria (mitophagy). We hypothesized that mitophagy contributes to remodeling of the heart under severe oxidative stress. METHODS: Mice were subjected to hemodynamic overload by severe thoracic aortic constriction (sTAC). qPCR was used to measure the abundance of mtDNA relative to nuclear DNA. Changes in proteins and cardiac function were also assessed. RESULTS: Decreases in mtDNA abundance were time dependent after sTAC (-47%at day 2, p<0.1; -73%at day 4, p<0.05) (n=2 each) and correlated with increased mortality (37% at day 2; 75% at day 4). The decline in mtDNA was greater in the basal septum (-88%p<0.01) than in the left ventricular free wall (- 42% p<0.15) (n=4) day 8 post-sTAC. The basal septum is where we have observed the largest increases in autophagic activity and protein carbonylation, a ROS-mediated protein modification. Daily injections with cyclosporine (CsA), an inhibitor of both MPT and calcineurin, blunted load-induced mtDNA loss (-28%ith CsA vs -83%with vehicle treatment, p<0.01) (n=3) at 4 days post-sTAC. Furthermore, CsA improved survival at 4 days-post sTAC (40% mortality with CsA vs 75% with vehicle) (n=5-8). Mice with increased ROS generation due to a disruption of the cardiac isoform of the cytochrome-c oxidase subunit COXVIaH were more sensitive to pressure overload-induced loss of mtDNA and mitochondrial proteins. CONCLUSION: MtDNA abundance declines in this model of load-induced heart failure and is associated with increased autophagic activity and ROS generation. Short-term application of CsA can blunt mtDNA loss and improve survival.Item Novel Insights into the Regulation of Autophagy in Saccharomyces Cerevisiae(2011-12-15) Wu, Xi; Tu, BenjaminAutophagy is an evolutionarily conserved pathway for the degradation of intracellular contents. How autophagy is regulated, especially upon changes in metabolic and nutritional state, remains poorly understood. In Saccharomyces cerevisiae, autophagy is normally triggered by nutrient starvation. However, by using a prototrophic strain, I discovered that autophagy can be strongly induced upon switch from a rich medium (YPL) to a minimal medium (SL) without nutrient starvation. This new autophagy-inducing condition was termed SL-induced autophagy. Growth measurement confirmed that SL-induced autophagy was important for cellular homeostasis and growth following medium switch. A genetic screen uncovered IML1, NPR2, NPR3 and PBP1, which are all required for SL-induced autophagy, but not for nitrogen-starvation-induced autophagy. Iml1p, Npr2p and Npr3p function in the same complex and regulate autophagosome formation. During SL-induced autophagy, Iml1p can localize to the pre-autophagosomal structures, consistent with the role of the Iml1p complex in autophagosome formation. Moreover, a conserved domain in Iml1p was identified to be required for SL-induced autophagy as well as complex formation. I discovered that sulfur containing amino acids, but not non-sulfur containing amino acids, can specifically inhibit SL-induced autophagy. I further demonstrated that cysteine is a key metabolite that inhibits SL-induced autophagy by regulating cellular processes related to cysteine metabolism. Cysteine does not suppress SL-induced autophagy by regulating oxidative stress, protein urmylation and thiolation of cytosolic tRNAs. Future studies will be required to reveal the exact mechanism through which cysteine inhibits SL-induced autophagy. I also discovered that autophagy can be significantly induced upon depletion of a Fe-S cluster containing protein, Rli1p, and other factors that are also involved in rRNA processing and translation initiation. Interestingly, IML1, NPR2, NPR3 and PBP1 are also important for Rli1p-depletion-induced mitophagy. These results strongly suggest the mechanistic link between SL-induced autophagy and ribosome biogenesis or translation regulation. Collectively, my studies have demonstrated the existence of additional mechanisms that regulate autophagy in response to relatively more subtle changes in metabolic and nutritional state.Item Starvation Response in Caenorhabditis elegans(2009-01-14) Kang, Chanhee; Avery, LeonWhen the supply of environmental nutrients is limited, multicellular animas can make physiological and behavioral changes so as to cope with nutrient starvation. Although starvation response is essential for the survival of animals during nutrient deprivation, uncontrolled or uncoordinated starvation responses could be harmful. Autophagy, a lysosomal degradation pathway for long-lived proteins and cytoplasmic organelles, is known to be an important starvation response, which promotes both cell and organism survival by providing fundamental building blocks to maintain energy homeostasis during starvation. Under different conditions, however, autophagy may instead act to promote cell death through an autophagic cell death pathway. Why autophagy acts in some instances to promote survival but in others to promote death is poorly understood. Here I show that physiological levels of autophagy act to promote survival in Caenorhabditis elegans during starvation, whereas insufficient or excessive levels of autophagy contribute to death. I find that inhibition of autophagy decreases survival of wild-type worms during starvation. Furthermore, I find that in gpb-2 starvation-hypersensitive mutants, starvation induces excessive autophagy in pharyngeal muscles, which in turn, causes damage that may contribute to death. These results demonstrate that, depending on level of its activation, autophagy can have either prosurvival or prodeath functions, providing in vivo evidence that an uncontrolled starvation response could be harmful to animals. Thus, it is important that animals ensure that their starvation response is coordinated between individual cells. However, the mechanisms by which animals sense starvation systemically remain elusive. Here I use gpb-2 mutants to identify molecules and mechanisms that modulate starvation signaling. I found that specific amino acids could suppress the starvation-induced death of gpb-2 mutants, and that MGL-1 and MGL-2, C. elegans homologs of metabotropic glutamate receptors, were involved. MGL-1 and MGL-2 acted in AIY and AIB neurons respectively. Treatment with leucine suppressed starvation-induced stress resistance and life span extension in wild-type worms, and mutation of mgl-1 and mgl-2 abolished these effects of leucine. Theses results suggest that metabotropic glutamate receptor homologs in AIY and AIB neuron may modulate a systemic starvation response in C. elegans.