Autophagy: a brief description
Autophagy, literally ‘self-eating’, is a process by which cells break down intracellular contents, often for recycling or turnover of nutrients and material, regulation of organelle function, or removal of misfolded proteins and inclusion bodies (Dikic & Elazar, 2018). In general, autophagy is a homeostatic process that protects against cellular stress, and accordingly, can be upregulated when cells face certain pressures. Three main pathways exist; microautophagy, chaperone-mediated autophagy, and macroautophagy. Microautophagy describes lysosomal membrane invagination and vesicle scission into the lumen, in a process similar to the formation of multi-vesicular bodies, which leads to the digestion of the vesicles and their contents. While this is a constitutive process of membrane homeostasis, it becomes upregulated during nitrogen restriction (Li, Li, & Bao, 2012). Chaperone-mediated autophagy describes direct translocation of soluble cytosolic proteins across the lysosomal membranes, due to their recognition and selection by cytosolic chaperones such as heat shock cognate protein 70 (HSC70) (Chiang, Terlecky, Plant, & Dice, 1989), which are then translocated across the lysosomal membrane via interaction with lysosome-associated membrane protein type 2A (LAMP2A) (Kaushik & Cuervo, 2018). Substrates of chaperone-mediated autophagy may consist of proteins from other sub-cellular compartments, which are transported into the cytosol for degradation as part of a quality control mechanism, for example in the removal of misfolded proteins. This can occur via HSC70-mediated recognition of a KFERQ-like sequence present in some 40% of the mammalian proteome, highlighting the importance of chaperone-mediated autophagy in protein turnover (Dice, 1990).
The term “autophagy” is however often used as shorthand for macroautophagy, which describes the process of degradation of larger portions of the cytosol, which may include targeted organelles such as mitochondria, by the formation of a double-membraned vesicle known as the autophagosome. This is also a homeostatic process required for removal and recycling of damaged organelles and aggregated and misfolded proteins, but is also upregulated by hypoxia, oxidative or edoplasmatic reticulum (ER) stress, protein aggregation, cell damage, or nutrient starvation in many cell types (Dikic & Elazar, 2018). Autophagosome formation begins with nucleation of a pre-autophagosomal structure, called the omegasome, from the ER. Cellular stress causes activation of Unc-51-like kinase (ULK1) complex, which then phosphorylates the Class III phosphatidylinositol 3-kinase (PI3K) complex, made up of PI3K, Beclin 1, VPS34, and ATG14L (Dikic & Elazar, 2018). Autophagy genes (ATG ) were first discovered and numbered in yeast, and their mammalian homologs are often named ATGL- for ”ATG-like”. Local production of phosphatidylinositol-3-phosphate (PI3P) by the PI3K complex recruits PI3P-binding proteins such as WD repeat domain phosphoinositide-interacting protein 2 (WIPI2) to the growing phagophore membrane. This is then extended, enclosing and partitioning a portion of the cytosol containing autophagy substrates. An important step in this process is the recruitment of ATG16L1, that occurs via lipid interactions and binding to WIPI2. ATG16L1 itself binds to a conjugate of ATG12 and ATG5 (Otomo, Metlagel, Takaesu, & Otomo, 2013). The ATG12-5 complex has E3 ligase-like activity, and is involved in lipidation of the ubiquitin-like cytoplasmic protein LC3 (a homolog to yeast ATG8), by coupling it to phosphoethanolamine, thus relocalising LC3 from the cytosol and into the growing phagophore membrane (Kabeya et al., 2000). The observation of these LC3-positive structures by fluorescent microscopy, as well as the gel motility shift observed on lipidation of LC3 (from soluble LC3-I to membrane-associated lipidated LC3-II), is often used to assess cellular autophagy induction (Klionsky et al., 2016). Membrane-associated LC3-II is involved in sequestering targeted material to the growing phagophore, via interaction with receptor proteins such as the stress-inducible autophagy substrate p62/Sequestosome 1 (SQSTM1), which itself binds to intracellular ubiquitinylated material and protein aggregates targeted for destruction (Bjorkoy et al., 2005; Ichimura et al., 2008). Other ubiquitin-independent LC3-binding receptors can be stabilized on the surface of organelles such as damaged mitochondria, marking them as cargo for inclusion within the growing phagophore, and subsequent recycling by autophagy (Koentjoro, Park, & Sue, 2017). LC3-II is required for optimal growth of the forming autophagosome, but LC3 and related ATG8-family GABARAP proteins are also essential for autophagosome completion, an incompletely understood process by which the phagophore membrane fuses to entirely surround and enclose the sequestered cytosol and cargo (T. N. Nguyen et al., 2016; Weidberg et al., 2011). This is an important step, without which the autophagosome does not subsequently fuse with lysosomes; premature fusion of a lysosome with an unsealed autophagosome would result in leakage of lysosomal contents into the cytosol.
Once the autophagosome is sealed, ATG proteins must be removed from the cytosol-facing surface of the autophagosome membrane for recycling, to avoid being degraded within the lysosome after fusion. It is possible that presence of some of these proteins on the autophagosome inhibit lysosome fusion. In yeast, phospholipid phosphatases and the protease ATG4 are involved in removal of PI3P and ATG8 from the autophagosome surface, respectively, without which, lyosomal fusion does not occur (Reggiori & Ungermann, 2017). As ATG8/LC3-II and PI3P presence in the membrane recruits many other ATG proteins to the autophagosome, their removal likely induces dissociation of the remaining autophagosome-forming machinery, and therefore allows subsequent lysosomal fusion. Completed autophagosomes are transported along the cytoskeleton and fuse with lysosomes via the action of tethering proteins and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) (Nakamura & Yoshimori, 2017), which help to physically fuse the lipid membranes together, allowing lysosomal enzymes to mix with the enclosed cytosolic compartment, degrading its contents. Liberated amino acids and nutrients are then exported from the completed autolysosome back into the cytosol for recycling.
Although the function of autophagy was first identified as a mechanism required for both homeostatic and responsive turnover of cellular contents, it has also been identified as an intracellular defence mechanism against pathogens. Whereas viral particles can be small enough for destruction by the proteasome, intracellular bacteria need to be identified and targeted by autophagy machinery, leading to their inclusion within autophagosomes and subsequent degradation, not only within phagocytes but also within non-immune cells (Nakagawa et al., 2004). Ubiquitinylation of intracellular pathogens can target them for inclusion within growing autophagosomes, via recruitment of receptor proteins such as P62 described above. Alternatively, mechanisms exist which cause direct recruitment of autophagy machinery such as ATG16L1 to pathogens or pathogen-containing membrane compartments. For example, the C-terminal WD-repeat domain of ATG16L1 interacts via a conserved motif identified on the cytosolic tail of late endosome membrane protein TMEM59 (Boada-Romero et al., 2013), allowing TMEM59 to direct autophagic destruction of internalised bacteria. The same conserved motif binding the ATG16L1 WD-repeat domain was also identified on the cytosolic tail of TLR2 as well as on the cytosolic protein NOD2 (Boada-Romero et al., 2013), explaining the mechanism by which these proteins can recruit ATG16L1 complexes to bacteria-containing phagosomes (Sanjuan et al., 2007) and to the site of bacterial entry into the cell (Travassos et al., 2010), respectively, to induce a non-canonical autophagic pathway. The destruction of intracellular pathogens by autophagy has a special term, xenophagy, and is of particular relevance to this review.