Box 1 | Autophagy and its inhibitors

From the following article:

Self-eating and self-killing: crosstalk between autophagy and apoptosis

M. Chiara Maiuri, Einat Zalckvar, Adi Kimchi & Guido Kroemer

Nature Reviews Molecular Cell Biology 8, 741-752 (September 2007)

doi:10.1038/nrm2239

Autophagy starts with the stepwise engulfment of cytoplasmic material (cytosol and/or organelles) by the phagophore (also called isolation membrane), which sequesters material in double-membraned vesicles named autophagosomes (also called autophagic vacuoles). In many cellular settings, the first regulatory process (see figure, step 1) involves the de-repression of the mTOR Ser/Thr kinase, which inhibits autophagy by phosphorylating autophagy protein-13 (Atg13). This phosphorylation leads to the dissociation of Atg13 from a protein complex that contains Atg1 kinase and Atg17, and thus attenuates the Atg1 kinase activity. When mTOR is inhibited, re-association of dephosphorylated Atg13 with Atg1 stimulates its catalytic activity and induces autophagy. Notably, the mammalian orthologue of the yeast Atg13 has not been identified to date. Among the initial steps of vesicle nucleation is the activation of mammalian Vps34, a class III phosphatidylinositol 3-kinase (PI3K), to generate phosphatidylinositol-3-phosphate (PtdIns3P) (step 2). Vps34 activation depends on the formation of a multiprotein complex in which beclin-1 (Becn1; the mammalian orthologue of Atg6), UVRAG (UV irradiation resistance-associated tumour suppressor gene) and a myristylated kinase (Vps15, or p150 in humans) participate.

Self-eating and self-killing: crosstalk between autophagy and apoptosis 

Two ubiquitin-like conjugation systems are part of the vesicle elongation process (step 3). One pathway involves the covalent conjugation of Atg12 to Atg5, with the help of the E1-like enzyme Atg7 and the E2-like enzyme Atg10. The second pathway involves the conjugation of phosphatidylethanolamine (PE) to LC3/Atg8 (LC3 is one of the mammalian homologues of Atg8) by the sequential action of the protease Atg4, the E1-like enzyme Atg7 and the E2-like enzyme Atg3. Lipid conjugation leads to the conversion of the soluble form of LC3 (named LC3-I) to the autophagic-vesicle-associated form (LC3-II). LC3-II is used as a marker of autophagy because its lipidation and specific recruitment to autophagosomes provides a shift from diffuse to punctate staining of the protein and increases its electrophoretic mobility on gels compared with LC3-I. Moreover, green fluorescent protein–LC3 fusion proteins can be used to visualize autophagosomes by fluorescence videomicroscopy91. The mechanism of retrieval in which the Atg9 complex participates is poorly studied (step 4).

Autophagosomes undergo maturation by fusion with lysosomes to create autolysosomes (steps 5 and 6). In the autolysosomes, the inner membrane as well as the luminal content of the autophagic vacuoles is degraded by lysosomal enzymes that act optimally within this acidic compartment4, 92. Pharmacological inhibitors and small interfering RNAs that are capable of inhibiting distinct steps of this process are shown (red blocking arrows). Bcl2 and Bcl-XL are regulators of beclin-1. Lamp2, lysosome-associated membrane glycoprotein-2.