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.