Autophagy (Autofagia) Mohamed Elgendy MD, PhD
Autophagy (Autofagia) Mohamed Elgendy MD, PhD Autophagy (Autofagia) Mohamed Elgendy MD, PhD [email protected] “Auto+phagy” Greek for “Self Eating” Autophagy = Recycling Types of Autophagy • 1-Macro-autophagy • 2-Micro-autophagy • 3-Chaperon-mediated autophagy Macro-autophagy 1-Induction Lysosome Autophagosome Fusion LC 3 LC3 II PE LC 3 LC 3 Phagophore 2-Nucleation Autolysosome LC3 I 3-Maturation Other types of Autophagy • Macro-autophagy Delivery of cytoplasmic cargo to the lysosome through the intermediary of a double membranebound vesicle, referred to as an autophagosome, that fuses with the lysosome to form an autolysosome. • Micro-autophagy Cytosolic components are directly taken up by the lysosome itself through invagination of the lysosomal membrane • Chaperon-mediated autophagy Targeted proteins are translocated across the lysosomal membrane in a complex with chaperone proteins (such as Hsc-70) that are recognized by the lysosomal membrane receptor LAMP-2A, resulting in their unfolding and degradation Roles of autophagy • Housekeeping roles Removal of misfolded or aggregated proteins, clearing damaged organelles, such as mitochondria and ER. • Host-defense mechanism Degradation of intracellular pathogens • Role during embryonic development Balancing sources of energy at critical times • As a component of cellular integrated stress responses. Regulation of Autophagy 1-Induction Lysosome Autophagosome LC 3 LC 3 Phagophore Fusion Autolysosome 2-Nucleation 3-Maturation Regulation of Autophagy Starvation 1-Induction mTOR Rapamycin Promotes autophagy Inhibits autophagy AMPK Rheb TSC 2 Low Energy Akt PTEN Regulation of Autophagy Starvation 1-Induction mTOR Rapamycin Promotes autophagy Inhibits autophagy AMPK Rheb TSC 2 Low Energy Akt PTEN Regulation of Autophagy 1-Induction Lysosome Autophagosome LC 3 LC 3 Phagophore Fusion Autolysosome 2-Nucleation 3-Maturation Regulation of Autophagy 2-Nucleation Autophagosome LC 3 Phagophore LC3 II Atg 7 Atg 12 Atg 7 Atg 5 Atg 4 Atg 16 Atg 3 Atg 10 Conjugation Systems LC3 I Detection of Autophagy 1-Induction Lysosome Autophagosome Fusion LC 3 LC3 II PE LC 3 LC 3 Phagophore 2-Nucleation Autolysosome LC3 I 3-Maturation Regulation of Autophagy -Relatively understudied -Requires the small G protein Rab7 in its GTP-bound state Inactivation of LAMP-2 is the causative genetic lesion associated with Danon disease in humans, an X-linked condition that causes cardiomyocyte hypertrophy and accumulation of autophagosomes in heart muscle. Similar cardiac defects are observed in Lamp2-null mice, as well as skeletal abnormalities and periodontitis associated with inflammation arising from a failure to eliminate intracellular pathogens in the oral mucosa -Within the lysosome, cathepsin proteases B and D are required for turnover of autophagosomes LC 3 Fusion LC 3 -Requires Lamp-1 and Lamp-2 at the lysosome Lysosome Autolysosome 3-Maturation Detection of Autophagy Electron Microscopy Detection of Autophagy Fluorescence microscopy (increase in punctate LC3) Detection of Autophagy Fluorescence microscopy (Acidotropic dyes) Acridine Orange Lysotracker Red Detection of Autophagy Immunoblotting Detection of Autophagy Other methods -mTOR kinase activity (Kinase assays western blot or immunoprecipitation) -Transcriptional regulation of autophagy genes (qRT-PCR or Northern blot) Detection of Autophagy Flux measurement Klionsky et al., Autophagy 2008 Crosstalk between autophagy and other cellular processes Autophagy and Apoptosis - Autophagy as an alternative mode of programmed cell death : Apoptosis PCD type I Autophagic cell death PCD type II Autophagy : a matter of life or death? Life Death -Starvation -Role in development (e.g Atg5 KO, Atg7 KO) (Regression of salivary glands in Drosophila) -Role in tumor suppression Negative regulators :PI3K, Akt, Bcl-2 Positive regulators: Beclin-1, UVRAD, Bif1, PTEN. -Autophagy inducers as anticancer agents : e.g. Arsenic Trioxide, Temozolomide, SAHA, Ceramide, Obatoclax, Rapamycin • Brief and mild vs prolonged and excessive • Mechanistically and functionally different autophagy programs -Selective (specific) vs non-selective (non-specific) - Atg5-,Atg7-indepdent , Beclin-1-depedenet autophagy (nature 2009) -The magnitude of Beclin-1 induction: mild survival strong death • Intactness of apoptotic machinery Bcl-2 family links apoptosis to autophagy Stress/Damage DNA damage Oncogenic Growth Factor stress Decision Proapoptotic Noxa Puma Bmf Depreivation Bim Bad Bcl-2 family Mcl-1 Antiapoptotic Death/Survival A1 Bcl-2 Bcl-xL Bcl-B Regulation of Autophagy Starvation 1-Induction mTOR Rapamycin Promotes autophagy Inhibits autophagy AMPK Rheb TSC 2 Low Energy Akt PTEN Bcl-2 family as regulators of autophagy Mcl-1 Bcl-X Bcl-2 pLC3-GFP BH3 Beclin-1 Autophagy Beclin-1 Links apoptosis to autophagy • Interaction with the Bcl-2 family proteins • Cleavage by caspases Kroemer et al., Mol. Cell 2010 Autophagy and senescence -Autophagy mediates oncogene-induced senescence Young et al., Genes and Dev 2009 -Senescent cells may also die by autophagy Autophagy and ER stress Segredi et al., EMBO reports 2006 Autophagy and ER stress • The role of ATF4 in transcriptional regulation of autophagy genes LC3 and Beclin-1. • Crosstalk between autophagy and unfolded protein response (UPR). • Autophagy counterbalances ER expansion. • ER as a source of phagophore membrane biogenesis Autophgay and DNA damage response -Autophagy induced by HDACi counteracts DNA damage response -Autophagy may promote genomic stability by clearing damaged mitochondria Autophagy in health and disease • Cardiovascular and ischemic diseases Starvation during ischemia • Neurodegenrative diseases Intracellular aggregate accumulation plays a particularly significant role in the aetiology of neurodegenerative diseases, including dementia, Alzheimer’s, Huntington’s and Parkinson’s For example, polyglutamine-expansion repeats, as seen in mutant huntingtin (Huntington’s disease), mutant forms of α-synuclein (familial Parkinson’s disease) and different forms of tau (Alzheimer’s disease) are dependent on autophagy for their clearance from neurons. Neuronal-specific inactivation of the key autophagy genes Atg5 or Atg7 results in intracellular aggregate accumulation and neurodegeneration in mice • Cancer Autophagy and Cancer A friend or Enemy? • Autophagy regulators as tumor suppressors or oncogenes Morselli et al. / Biochimica et Biophysica Acta 2009 Morselli et al. / Biochimica et Biophysica Acta 2009 Targeting autophagy for therapeutic intervention Roy et al., Semin Immunopathol 2010 Autophagy and Cancer A friend or Enemy? • Brief specific versus prolonged nonspecific autophagy? • Different roles during different stages of tumorigenesis? Autophagy and immunity • Infection induces autophagy in many cases. • Autophagy induction during infection is regulated by cytokines (e.g IFN gamma) and pathogen recognition receptors (PRRs) that recognize conserved components of pathogens or products of their replication(PAMPs) and DAMPs (e.g. products of necrotic cells, abnormal reactive oxygen species, misfolded proteins) • The transcription factor NF-kB and some of its upstream regulators function to integrate diverse stress signals including immune signals with the autophagy pathway Autophagy and aging • Autophagy promotes longevity Rubinsztein et al., Cell 2011 Thank you (Grazie) Questions? [email protected]
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