Autophagy and Immunity Qingqing Wang Institute of Immunology Zhejiang University School of Medicine Nov 2, 2012 Phagy = eating/lysosomal degradation Autophagy = self-eating Mechanism of breakdown of cytoplasm within the lysosome Autophagy: a basic cellular process in eukaryotes Autophagy Highly conserved and regulated process that maintains cellular homeostasis and protects cells against starvation and microbe invasion Autophagy Autophagosome: A double membrane bound compartment that engulfs cytosol and degrades the cytoplasmic contents. Large: nm May originate from ER or from fusion of lipid-containing vesicles that form sequestration crescent. Autophagic pathways Plants and animals Bacteria, viruses, parasites Highly regulated processes Stages: induction, execution, maturation Autophagy genes Based on S. cerevisiae yeast studies. ATG in yeast, homologs in other species. Involved in: Tumor suppression Starvation responses Preventing premature cell senescence 3 Stages Initiation: nutrient starvation, growth factor-mediated starvation Execution Maturation Induction TOR (Target Of Rapamycin) kinase Inhibits autophagy when phosphorylated. Also regulates protein and amino acid synthesis. Rapamycin and nutrient starvation dephosphorylate TOR, inducing autophagy. Tamoxifen induces autophagy too (TOR?) Induction Trimeric G proteins High amino acids inactivate G proteins, so aa depletion may induce autophagy via G proteins. PI3Ks (Phosphatidylinositol-3-kinases) Essential for starvation induced autophagy. 3-MA, wortmannin, LY target PI3Ks, and result in inhibition of autophagy. Execution Covalent linkage of Atg5 and Atg12 Covalent lipidation of Atg8 Enzymes Atg3, Atg7, and Atg10 are homologs of ubiquitylation enzymes but are used to modify pathway components instead of labeling them for degradation. Maturation GTPases (Rab24) mediate vesicle fusion. Intermediate autophagosomes Fuse with endosomal vesicles. Acquire LAMP, accumulate DAMP proteins. Mature autolysosomes Fuse with lysosomes. Acquire cathepsins and acid phosphatases. Detection methods Microscopy Electron microscopy ultrastructure, morphology, volumetrics, staining. Biochemistry Enzyme activity assays. Radioactive degradation studies. Marker studies Autophagosomal or organelle markers. Fluorescence or immunodetection. The physilogical significance of autophagy There are many ways to die Programmed cell death Type I: caspase mediated Type II: autophagy-mediated Apoptosis Programmed cell death Type I: caspase mediated Type II: autophagy-mediated Entire cell dismantled within membrane-enclosed vesicles Taken up by phagocytes, preventing release of intracellular components from dying cells Normal morphogenesis, removing genetically damaged cells, proper tissue homeostasis, invading microbes Mechanism Sequential activation of cysteine proteases (caspases) Caspase 1-related: 1,4,5,13,14 Cytokine processing and pro-inflammatory cell death Initiator caspases: 2, 8, 9, 10 Effector caspases: 3,6,7 Regulated process Extrinsic: stimulation of Fas or TNFR surface receptors Intrinsic: altered mitochondrial membrane integrity Necrosis Cell swelling and rupture Release in intracellular components Activation of inflammatory response Can be regulated Can occur in concert with or instead of apoptosis (e.g. if apoptosis is blocked) Pyroptosis Caspase-1-dependent cell death Convert IL-1B and IL-18 to active forms Induced by Shigella IpaB and Salmonella SipB Autophagy and diseases Oncology Liver Breast Colon Lung Beclin-1 Brain Alzheimer Multiple Sclerosis Parkinsons disease Hungtingdons disease Miscellaneous Myodegerative disease Cardiac stress a-antitrypsin deficiency Cellular Process Apoptosis Aging Growth Antigen presentation TLR signaling Infection Autophagy and immunity Role of autophagy Does autophagy benefit the host as a defense mechanism? Does autophagy benefit the microbe by facilitating survival and replication? Or both? Bacterial susceptibility Astragalus-Mesorhizobium: Bacteria differentiate within membrane compartments until they can fix nitrogen and establish symbiosis. Nutrient starvation: bacterial degradation observed. Autophagy? Bacterial susceptibility Rickettsiae conorii Sensitive to NO produced by IFN, TNF-. Correlations between autophagosome-like structures and bacterial degradation. Are autophagosomes destroying bacteria or just cleaning up after bacteria are killed? Bacterial susceptibility Listeria monocytogenes Enter host cells by phagocytosis, escape from phagosomes, multiply in cytoplasm. ActA (actin) mutants are engulfed in autophagosome-like compartments. Wortmannin reduces bacterial entry into autophagosomes. Nutrient depletion increases bacterial entry into autophagosomes. Bacterial subversion Porphyromonas gingivalis Infects human coronary artery endothelial cells. Localizes to autophagosome-like compartments. Wortmannin: compartments resembled lysosomes and acquired cathepsin earlier. Bacterial survival decreased. Bacterial subversion Brucella abortus Endosomal uptake, then autophagosomes. Wortmannin reduced survival, cell starvation increased survival. VirB mutants have Type IV secretion mutation that inhibits intracellular transport and growth. Mutants are localized to membrane compartments that acquire cathepsin earlier, resembling lysosomes. Bacterial subversion Legionella pneumophila Replicates in autophagosome-like compartments in macrophages. Dot/icm mutants are defective in Type IV secretion involved with organelle trafficking or intracellular multiplication. Mutants localized to lysosomal-like vesicles, not autophagosomes. Pregnant pause model says that autophagosome maturation is delayed to allow for pathogen development. Bacterial subversion Coxiella burnetti Replicates within autophagosome-like acidic vesicles. Rab7 mutants had altered size and numbers of vesicles containing bacteria. Dot/icm homologs in Coxiella were able to return Dot/icm deficient Legionella to a wild phenotype. Viral susceptibility Herpes-virus PKR kinase phosphorylates eukaryotic translation-initiation factor eIF2 to inhibit and deregulate cellular translation. PKR can also induce autophagy, apoptosis, and activate NF-KB. ICP34.5 produced by herpes simplex virus 1 to antagonize PKR function by dephosphorylating eIF2. Viral subversion Positive-strand RNA viruses Poliovirus, murine hepatitis virus (MHV), equine arterivirus (EAV), SARS human corona virus. Require membranes for replication. Autophagosomes are induced during infection, but are they part of the viral replication process or the host response to eliminate pathogens? TIP OF THE ICEBERG?