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Introduction
Cell homeostasis is maintained by a precisely regulated
balance between synthesis and degradation of cellular
components. There are 2 powerful hydrolytic mechanisms
in eukaryotic cells: the proteasome and the
lysosome/vacuole. More than 90% of cellular proteins are long-lived.
These proteins and some cytoplasmic organelles are believed
to be degraded within a specific compartment, the
lysosome/vacuole[1]. There are at least 3 different pathways for
lysosomal protein degradation: Cvt (cytosol to vacuole
targeting pathway)[2], Vid (vacuolar import and degradation
path-way)[3] and
autophagy[4]. Autophagy plays important roles
in physiology and pathophysiology in all cell types. In
addition to its role in protein and organelle degradation,
autophagy may induce a type of programmed cell death that is
different from apoptosis, namely type II programmed cell
death[5]. Recently it has been found that autophagy may be
a transitory tactical response, and that it affects a range of
normal developmental processes, such as sporulation in
yeast and pupa formation in Drosophila
melanogaster[6,7]. Furthermore, autophagy may contribute to the extension of
lifespan induced by caloric
restriction[8]. Autophagy might also act as a means of defense against invasion by various
bacteria and viruses. There is a potential link between
autophagy and a number of diseases in humans. For example,
cancer, cardiomyopathy and neurodegenerative disorders
such as Alzheimer¡¯s, Parkinson¡¯s and Huntington¡¯s diseases,
amyotrophic lateral sclerosis and prion diseases are all
associated with increased autophagy
activity[9]. Consequently, study of the molecular basis of autophagy would result in a
better understanding of the role of autophagy in cell death
and ageing, and would also result in proposals for new
therapeutic approaches for neurodegenerative diseases.
The morphology of autophagy was first characterized in
studies of mammalian cells. With a few exceptions, however,
the molecular components of autophagy were initially
elucidated in yeast because of the convenience of gene analysis.
Recent studies in various eukaryotic systems have revealed
a conservation of the autophagic
mechanism[10]. In the past, the many terms used in the autophagy field have been highly
confusing: Aut (autophagocytosis), Apg (autophagy), Vps
(vacuolar protein-sorting) all have been used. Recently, the
autophagy-related genes and the products of these genes
were named ATG and Atg,
respectively[11].
Classification of autophagy
Autophagy is a ubiquitous physiological process that
occurs in all eukaryotic cells. There are three primary forms
of autophagy: macroautophagy, microautophagy and
chaperone-mediated autophagy (CMA). Macroautophagy is the
most prevalent form of autophagy. It comprises the
following processes: initially, a "C" shape double-membrane
structure appears in the cytoplasm, and then at both ends of this
membrane a structure grows, and finally closes to form a
vacuole. The bulk of the cytoplasm and some organelles are
wrapped into the vacuole (autophagosome). Then the
autophagosome targets the lysosome/vacuole, where its
outer membrane fuses with the lysosomal membrane and the
inter sac (autophagic body) enters the lysosome/vacuole.
The autophagic body is degraded in the lysosome/vacuole
so the carrying constituent components can be
recycled[12]. Microautophagy is a form with few features. In this pathway,
the membrane of the lysosome/vacuole invaginates, and then
finally pinches off to form an internal vacuolar vesicle that
contains material derived from the cytoplasm, akin to the
autophagic bodies formed in macroautophagy. The notable
difference between macroautophagy and microautophagy
is that in the latter the cytoplasm is directly up taken into the
lysosome/vacuole[13]. CMA differs from the other lysosomal
degradation pathways in that vesicular traffic is not involved.
Cytosolic proteins with particular peptide sequence motifs
are recognized by a complex of molecular chaperones, then
bind to a receptor in the lysosomal membrane, the
lysosome-associated membrane protein (Lamp) type 2a. Proteins are
delivered to lysosomes with the help of molecular
chaperones and Lamp 2a[14].
Autophagy is basically a non-selective process, in which
bulk cytoplasm is randomly sequestered into the cytosolic
autophagosome. However, in some cases it may select its
target. For example, autophagy can selectively eliminate some
organelles, such as injured or excrescent peroxisomes,
endoplasmic reticulum (ER) and
mitochondria[15]. A recent report shows that in yeast,
Saccharomyces cerevisiae, the cytosolic protein acetaldehyde dehydrogenase (Ald6) is
delivered to the vacuole and degraded by means of specific
autophagy[16].
Molecular mechanisms of macroautophagy
Macroautophagy, a major form of autophagy, is relatively
well characterized at present. Autophagy and the related
processes are dynamic, and many molecules involved in the
autophagic process have been identified. At least 25
specific yeast genes are exclusively involved in autophagy, and
there are more than 40 additional yeast genes required for
autophagy, but they also play roles in other
pathways[11]. However, the physiological functions of many of these genes
need to be further clarified.
Origin of the autophagosomal membrane There is still
debate on the origin of autophagosome membranes. Initially,
the ribosome-free region of the rough ER and Golgi were
proposed as the source of autophagosomal
membranes[17,18]. Now, it is generally accepted that the phagophore, a poorly
characterized organelle, may be the major source of the
autophagosomal membrane and related
structures[19].
Autophagosome formation
Two ubiquitin-like conjugation systems In yeast,
autophagy almost completely shuts off under growing
conditions, although every ATG gene is expressed.
Molecular biological and biochemical analyses of these gene
products uncovered the genetic and biophysical interactions
among the Atg proteins. One of the most remarkable
findings regarding the Atg proteins is the discovery of two
ubiquitin-like conjugation systems, Atg12-Atg5 and Atg8-
phosphatidylethanolamine (PE) (Figure 1). In fact, half of
the APG genes essential for autophagy are involved in these
conjugation systems, and these two conjugation systems
are well conserved among eukaryotes. Furthermore,
Atg12-Atg5 and Atg8 conjugation systems are somehow related to
each other[20-22]: if the former is defective, the latter can not
target to the pre-autophagosomal structure (PAS); the
levels of Atg8-PE also play an important role in the conjugation
of Atg12-Atg5.
Atg12-Atg5 conjugation system Atg12, a small
hydrophilic protein of 186 amino acids with no apparent
homology to ubiquitin, can covalently link to a unique target
protein, Atg5[23]. The mode of conjugation of Atg12 and
Atg5 is quite similar to that of ubiquitination. Atg12 is first
activated in an ATP-dependent manner by Atg7 (it functions
as an ubiquitin-activating enzyme, E1), leading to the
formation of a thioester bond between the C-terminal glycine in
Atg12 and a cysteine residue in
Atg7[24]. The C-terminal glycine in Atg12 is then transferred to the cysteine in Atg10
(it functions as a ubiquitin-conjugating enzyme, E2),
forming a new thioester bond, and Atg7 is
released[25]. Finally, the C-terminal glycine in Atg12 forms an isopeptide bond
with the e-amino group of lysine 149 in Atg5, and Atg10 is in
its free state again. The formation of the Atg12-Atg5
conjugate is indispensable to autophagosome formation. It seems
that this ubiquitin-like system is a constitutive process,
because the formation of the Atg12-Atg5 conjugate is not
dependent upon starvation or other autophagy-inducing
conditions[26]. Atg12 and Atg5 form a conjugate immediately
after their synthesis, and free forms of these are hardly
detectable. The conjugation reaction between Atg5 and
Atg12 is irreversible, and so far no protease has been found
to deconjugate this conjugate. Atg16 also binds
preferentially to the Atg12-Atg5 conjugate. Atg16 links with
Atg12-Atg5 through self-oligomerization, and its C-terminal
coiled-coil region may be responsible for this oligomerization.
Therefore, Atg16 forms a 350 kDa multimeric complex with
the Atg12-Atg5 conjugates. It should be pointed out that
Atg16 only binds to Atg5, and not to
Atg12[27,28]. This new complex is necessary for the elongation of the isolation
membranes (used for formation of the autophagosomal
membrane). A small fraction of the Atg12-Atg5·Atg16
complex initially associates with a small crescent-shaped vesicle
evenly. As the membrane elongates, Atg12-Atg5 shows
asymmetric localization and most of these proteins
associate with the convex surface of the isolation membrane. This
complex will dissociate from the membrane upon completion
of autophago-some formation; thus, it is not present in the
mature autophagosome[29]. The molecular basis of this
transient association of Atp12-Atg5 conjugates with the
autophago-some membrane is not yet known.
In mammalian cells, Atg5 and Atg12 are conjugated to
each other in the same way as they are in yeast, but the
complex interacts with Atg16L, forming an ~800 kDa
structure instead of a 350 kDa complex in
yeast[27]. Atg16L is a 63 to 74 kDa protein, which has a binding region and coiled-coil
region similar to that of Atg16. However, Atg16L has a long
C-terminal extension containing 7 WD repeats, but the role
of the WD repeats in autophagy has not yet been elucidated.
Atg8 conjugation system The second ubiquitin-like
protein essential for autophagy is Atg8 (Aut7/Apg8). Atg8
is a 117-amino acid protein and is present in the early
isolation membranes, autophagosomes and autophagic
bodies[30]. This feature makes Atg8 a good marker for studying
membrane dynamics during autophagy. Cell fractionation
studies have shown that Atg8 is mostly membrane-bound;
approximately half of it is peripherally bound to the membrane
and the other half behaves like an intrinsic membrane protein.
Atg4, a novel cysteine protease, is responsible for
processing Atg8 by cleaving a single Arg residue from it,
consequently exposing Gly in the C-terminus of
Atg8[31]. Atg8 can be activated by Atg7 (E1) in an ATP-dependent manner and
transferred to a conjugating E2 enzyme,
Atg3[32]. Atg7 is a unique enzyme that activates two different ubiquitin-like
proteins, Atg12 and Atg8, and assigns them to their proper
E2 enzymes, Atg10 and Atg3, respectively. Interestingly, in
the final step, Atg8 does not form a conjugate with other
proteins, but interacts with PE, an abundant membrane
phospholipid[32]. This lipidation reaction leads to a
conformational change of Atg8 that is necessary for the membrane
dynamics of autophagy[33]. In addition, Atg8-PE is
deconjugated by Atg4, which cleaves the lipid-protein
linkage and provides a new source of cytoplasmic Atg8. The
cycle of conjugation and deconjugation is important for the
normal progression of autophagy.
Microtubule-associated protein 1 light chain 3 (LC3), the
mammalian orthologue of Atg8, targets to the
autophago-somal membranes in an Atg5-dependent manner and remains
there even after Atg12-Atg5 dissociates. Thus LC3 is the
only credible marker of the autophagosome in mammalian
cells[12]. In wild-type cells, LC3 is detected in 2 forms: LC3-I
(18 kDa) and LC3-II (16 kDa)[34]. Twenty-two amino acids in
the C-terminus of the newly synthesized LC3 are cleaved
immediately by the mammalian orthologue of the yeast
cysteine proteinase Atg4, autophagin, to produce an active
cytosolic form, LC3-I[35]. Then with the catalysis of Atg7 and
Atg3, LC3-I undergoes a series of ubiquitination-like
reactions, and is modified to LC3-II. LC3-I is located in the
cytoplasm, while LC3-II is a tightly membrane bound protein
and is attached to PAS and autophagosomes. The relative
amount of membrane-bound LC3-II reflects the abundance
of autophagosomes, so the induction and inhibition of
autophagy can be monitored through measuring total and free
LC3-II levels by means of
immunoassay[34]. In addition, studies have shown that the Atg12 and LC3 systems have a
functional relationship. In ATG5-/-
cells, LC3-II is not generated at
all[21]. As a result, LC3 cannot target the autophagosomal
membranes. The recent generation of transgenic mice
expressing green fluorescent protein (GFP) fused to LC3
provides a useful tool to investigate autophagy in various
mammalian organs in vivo[36].
In addition to LC3, at least another two mammalian
orthologs of yeast Atg8 have been
identified[37,38]: g-aminobutyric acid type A receptor-associated protein
(GABARAP) and Golgi-associated ATPase enhancer of 16
kDa (GATE-16). The 2 proteins also covert to membrane
bound forms (form II), which are recovered in membrane
fractions[39]. These results suggest that all mammalian Atg8
homologues receive common modifications to associate with
autophagosomal membranes, but the functions of these
orthologs and their modified form II need to be further
studied.
Two kinase complexes
Atg 1 protein kinase Atg1 is a serine/threonine protein
kinase, which forms a protein complex with different
regulatory proteins such as Atg13, Vac8, Atg17 and Cvt9. The
complex controls the switch between Cvt and autophagy
pathways (Figure 2). The composition of this complex is
dynamic and it may vary depending on nutrient condi-tions[40]. Under nutrient-rich conditions, Atg13 is hyperphos-phorylated so that its association with Atg1 is blocked. On
the other hand, under nutrient starvation conditions or after
treatment with rapamycin, Atg13 becomes partially
dephos-phorylated. This dephosphorylation leads to an Atg1-Atg13
interaction and subsequent generation of autophagosomes
instead of Cvt vesicles and, thus, activates autophagy. Vac8,
the vacuolar inheritance protein that acts in the Cvt pathway
is not essential for starvation-induced
autophagy[41]. Vac8 is also a phosphoprotein and may help to facilitate the
phosphorylation of Atg13. Atg1 also interacts with 2 other
proteins. One of these is Cvt9, which is only required for the
Cvt pathway[42]. Cvt9 is a large coiled-coil protein, and it
interacts with itself, possibly through the coiled-coil domain.
This could potentially crosslink the Atg1 complex into a
higher-order structure required early in the sequestration
process of autophagy. The other protein, Atg17, is only
required for the autophagic import and has been proposed
to play a role in Atg1-Atg13
interactions[43]. Recently, Atg17 has been found to interact with 2 Cvt pathway-specific
components, Atg24/Cvt13/Snx4 and Atg20/Cvt20, proteins
that contain PI3P-binding PX
domains[44,45]. But it remains a challenge to evaluate the meaning of interactions between
the autophagic-specific Atg17 protein and Cvt
pathway-specific Atg24 and Atg20 proteins. Genetic analysis suggests
that the Atg1 complex functions at a rather late stage of
autophagosome formation[22]. The Atg1 complex may
control membrane dynamics, rather than act as a signal
transducer. The kinase activity of Atg1 is upregulated
during induction of autophagy, thus the levels of kinase
activity seem to be important for the regulation of
autophago-some formation[43]. A recent report has shown that Atg1 may
only have a non-kinase structural role in autophagy
induction, although the kinase activity of Atg1 is involved
in autophagy; however, it plays more important roles in the
Cvt pathway[46].
Two putative human homologs of Atg1 have been
identified[47]: the UNC-51-like kinases ULK1 and ULK2. ULK1
has been shown by yeast two-hybrid screening to interact
with GATE-16 and GABARAP, two homologs of the yeast
autophagy protein Atg8.
Vps34/PI3K Early in 1982, it was found that 3-MA
(3-methyladenine) inhibits the formation of autophago-somes[48]. 3-MA is a PI3K
inhibitor[49]. Several studies have demonstrated that PI3K inhibitors interfere with the
formation of autophagosomes in rat
hepatocytes[50]. Yeast is known to contain only one PI3K,
Vps34[51]. Vps34 function is regulated by the protein kinase activity of Vps15, with
which it forms a stable, membrane-associated complex under
normal conditions. This complex links the Vps34 kinase to
cytoplasmic membranes. Vps34-Vps15 is present in 2
complexes, which are involved in a variety of membrane
transport events (Figure 3)[52]: complex I, which is composed of
Vps34-Vps15, Atg6 and Atg14, controls autophagy, whereas
complex II, which is composed of Vps34-Vps15, Atg6 and
Vps38, is essential for sorting of carboxypeptidase Y (CPY)
into the vacuole. Atg6 is a possible coiled-coil protein and is
associated with the membrane through Vps15 and Vps34.
Atg14 is a specific factor in the autophagy-specific PI3K
complex. Complex I functions primarily, but not exclusively,
at the PAS, whereas complex II functions at the endosome.
The lipid kinase activity associated with Vps34 is thought to
create lipid patches of PI3-P, the reaction product of class III
PI3K, at specific trans-Golgi locations, and these patches
then function in protein sorting into vesicles that travel from
the Golgi to the endosome.
In mammalian cells, there are 3 classes of PI3K. Class I
PI3K is an inhibitor of autophagy[53]. Class II PI3K activity is
thought to have no relevance to autophagy control. Class
III PI3K, a functional orthologue of yeast Vps34, is an
activator of autophagy and plays a crucial role at an early step
of autophagosome formation in mammalian
cells[54], and it is required for increasing the size of the sequestering membrane,
presumably through fusion events. PI3-P interacts with
proteins containing FYVE or PX motifs, thus recruiting such
proteins from the cytosol for autophagosome
biogenesis[55]. The activation of a population of PI3K located in a
determined membrane domain may be responsible for autophagosome biogenesis. In addition, the presence of
PI3-P in a specific membrane location may generate
significant asymmetries and drive membrane curvature of
PAS[54]. Finally, PI3-P may be converted to higher-order
polyphos-phoinositides (PI), which are involved in diverse signaling
functions. The mammalian cell orthologue of Vps15 is p150.
It is associated with class III PI3K, and interacts with beclin-1,
a functional orthologue of yeast Atg6 in mammalian
cells[56]. Beclin1, which is the first autophagy-related tumor
suppressor gene reported so far, is required for both autophagy and
Cvt pathways. The characterization of the tumor
suppressor activity of beclin-1 could establish an important
relationship between cancer and the autophagic pathway. Beclin-1
was originally isolated as a bcl-2-interacting protein that can
downregulate bcl-2, but it is still unclear whether this
interaction is instrumental in
autophagy[57].
Atg 9 complex Atg9 is an integral membrane protein,
containing several potential transmembrane
domains[58]. A fraction of Atg9 is located in the PAS together with other
autophagy proteins, but it is absent in the membranes of
mature autophagosomes[59,60]. If cells lack Atg9, neither the
Cvt pathway nor autophagy can take place. However, Cvt
and autophagy can still accumulate protease-sensitive
prApe1 (the precursor to aminopeptidase I), indicating that
Atg9 has an effect prior to closure of the double-bilayer
vesicle[61]. A soluble protein, Atg2 physically interacts with
Apg9, and this interaction is indispensable to
autophago-some formation[62]. The localization of Atg2 to the PAS
requires the activity of several proteins, such as Atg1 and
Atg9, but the kinase activity of Atg1 seems to be of
questionable importance to this function. Atg18 is also
indispensable to correct Atg2 localization, suggesting that there
is a potential link between this protein and the Atg9
system[63].
Mammalian orthologs of Atg2 and Atg9 are present in
the genome sequence databases, but they have not been
studied in detail.
PAS All the abovementioned Atg proteins have a
function before or during the formation of autophagosomes.
Thus, it is important to find out the distribution of these Atg
proteins in cytoplasm. By using fluorescent protein fused
with Atg proteins, it has been shown that Atg8 stains
autophagosomes, autophagic bodies in the vacuoles, and
also the intermediate isolation membrane. However, Atg5
shows a single bright dot structure next to the vacuole. This
structure has been named the PAS, and Atg8 also
co-localizes with it[64,22]. In addition, no Golgi, ER or late-endosomal
markers has been found in the PAS, suggesting that this is a
novel structure that has not been described so
far[59]. However, little is known about the existence of a PAS-like
structure in mammalian cells. Almost all Atg proteins are
co-localized in the PAS, suggesting that it is an organizing
center of the autophagosome. The autophagy-specific PI3K
complex, PI3-P, may recruit different proteins such as Atg18,
Atg20, Atg21, Atg24, and Atg27 to the
PAS[54,65,66]. The interaction between PI3-P and these proteins is a requisite for
the re-recruitment of Atg proteins to the PAS. None of these
proteins contains PX or FYVE motifs, so the domain of the
lipdation has not been identified. Atg9 also has a strong
effect on organization of the PAS, whereas defects in the
Atg1 kinase complex show little effect on the PAS structure.
Indeed, a recent study suggests that, in nutrient-rich
conditions, the PAS does not form in the absence of prApe1,
Atg19 or Atg11[67].
Autophagosome fusion with the vacuole/lysosome
In yeast, the fusion of autophagosomes with the vacuole
requires several factors that are involved in other types of
vesicular transport. Molecular genetic studies have
indicated that the machinery required for homotypic vacuole
fusion is also required for the fusion of autophagosomes
with the vacuole. This machinery includes: the SNARE pro
teins Vam3, Vam7, Vti1, and Ykt6; the NSF, SNAP, and GDI
homologs Sec17, Sec18, and Sec19; the Rab protein Ypt7;
members of the class C Vps/HOPS complex; and Ccz1 and
Mon1[68]. However, it remains to be determined whether
different SNARE and/or other fusion components operate
during autophagy. If the fusion process begins prior to
completion of the double-membrane vesicle, the cargo will remain in
the cytosol, so the timing of vesicle fusion with the
lysosome/vacuole must be precisely regulated. Atg8-PE, which
is located on the outer surface of the autophagosome, is
removed prior to fusion as a result of a second cleavage by
Atg4. Removal of Atg8-PE from autophagosomes can
prevent premature fusion with lysosomes.
Atg12-Atg5·Atg16 may have the same function. The expression of
Atg8ÄR is a mutated form of Atg8 lacking the ultimate arginine
residue, bypassing the need for the initial Atg4-dependent cleavage
step. However, the mutation causes a loss of ability of Atg
to carry out the second cleavage event that releases Atg8
from PE and results in a partial defect in
autophagy[31].
In mammalian cells, autophagosome fusion with
lysosomes is a more complex process in which the autophagosome
requires a series of maturation steps prior to its fusion with
the lysosome. Similar to yeast, the activity of monomeric
GTPases (Rab22, Rab24) and mammalian orthologs of SNARE protein family members and the NSF protein are
needed for correct autophagosome
maturation[69,70]. Prior to their fusion with lysosomes, autophagosomes have to fuse
with endosomes or endosome-derived vesicles.
Overexpres-sion of a mutant form of the regulator of endosome sorting,
SKD1 ATPase, which is unable to hydrolyze ATP, hampers
endosome function and causes a massive accumulation of
nascent autophagosomes[71]. Rab7, which is associated with
autophagic vacuoles, is involved in maturation of
autophago-somes. Overexpression of a Rab7 dominant negative mutant
hampers fusion between autophagosomes and the late
endosome/lysosomal compartment, leading to the
accumulation of autophagosomes with a concomitant decrease in the
degradation of long-lived proteins[72]. In addition, the
cyto-skeletal elements are also involved in either autophagosome
maturation or autophagosome-lysosome fusion. The
microtubule is another important factor for this event, because
treatment of cells with microtubule-destabilizing drugs
blocks autophagosome maturation. For example, cells treated
with cytochalasin D, an agent that disrupts actin filaments,
display a significant reduction in autophagosome
formation[73], whereas the microtubule stabilization mediated by a new
antitumor drug, taxol, increases the fusion of amphisomes
with lysosomes[74].
Degradation of autophagic body The main purpose of autophagy in yeast is to degrade cytoplasm and recycle the
resulting macromolecules for use in the synthesis of
essential components during nutrient stress. Degradation of
autophagy bodies requires a low pH, proteinase B (Prb1) and
Atg15/Cvt17[75,76]. Prb1 is a hydrolase that is involved in the
activation of many other vacuolar zymogens, which
indirectly affects the vesicle breakdown. Atg15/Cvt17 has
sequence similarity to a family of lipases, and seems to
function directly in vesicle breakdown. It is the only putative
lipase that has been identified that has a role in the
degradation of autophagic bodies[76]. So far, we know that the
expression of Atg15 is low in the vesicle, but the mechanism or
site of action of Atg15 is unknown. Another protein, Atg22,
has also been implicated in this last step. Atg22 is an
integral membrane protein, and is needed only for the
degradation of autophagic bodies[77].
Molecular mechanisms of CMA
CMA is one of several lysosomal pathways of
proteo-lysis. CMA is activated by physiological stressors such as
prolonged starvation. During starvation, macroautophagy
is activated first, but this process quickly declines and CMA
is then activated. The mechanisms by which these 2
lysosomal protein degradation pathways interact, if any, are
unknown. We now know that cytosolic proteins can be
degraded by CMA in rat liver, kidney, heart, and other tissues.
However, to date, there are no physiological phenomena
similar to CMA found in yeast. Only the cytosolic proteins with
exposed pentapeptide sequence motifs related to KFERQ
can act as substrate proteins, and be recognized by a
complex of molecular chaperones whose major constitutive form
is the heat shock 70 kDa protein
(hsc70)[78]. The substrate protein and molecular chaperone complex bind to the
lysosomal membrane and interact with lamp2a. The protein is
unfolded by the molecular chaperone complex prior to
importation into the lysosome. The protein is pulled into the
lysosomal lumen with the help of lysosomal hsc70 (ly-hsc70)[14,79]. The level of lamp2a can be a rate-limiting step in
CMA. A lamp2 knockout mouse without any of the lamp2
isoforms showed a reduction in rate of protein degradation
and an increase in accumulation of autophagic vacuoles in
heart, skeletal muscle, and other
tissues[80]. The amount of lamp2a in the lysosomal membrane is regulated, in part, by
changes in its degradation rate. For example, during
starvation lamp2a levels increase due to a decrease in lamp2a
degradation. The mechanisms of lamp2a degradation include
an initial cleavage by the lysosomal protease cathepsin A.
In fact, cathepsin A knockout mice had elevated levels of
lamp2a in the lysosomal membrane and showed higher ac
tivity of CMA[81]. A metalloprotease yet to be identified also
contributes to lamp2a degradation and works in cooperation
with cathepsin A. A more rapid adjustment of lamp2a in the
lysosomal membrane occurs through changing the
proportions of lamp2a sequestered in the lysosomal matrix.
Regulation of autophagy
Autophagy is probably the main mechanism for
degradation of long-lived proteins and cytoplasmic organelles. A
great number of extracellular stimuli (starvation, hormone or
therapeutic treatment) as well as intracellular stimuli
(accumulation of misfolded proteins, invasion of microorganisms) are able to modulate the autophagic
response. In yeast and mammalian cells, autophagy is a
fundamental biological event that occurs under normal
growth conditions. Thus, there must be an array of
mechanisms by which extracellular and/or intracellular signals can
be accepted and transmitted to the regulatory factors to
promote or inhibit autophagy when it is needed.
TOR/mTOR There are a number of signaling complexes
and pathways involved in the initiation and maturation of
autophagy. The central player in these signaling pathways
is TOR, the target of rapamycin. TOR is a serine/threonine
kinase involved in most regulatory pathways that control
the response to changes in nutrient conditions and energy
metabolism. TOR acts as a good gate-keeper in autophagy
and exerts an inhibitory effect on autophagy (Figure 4). TOR
kinase may inhibit autophagy through two general mechanisms. First, TOR acts in a signal transduction
cascade through various downstream effectors to control both
translation and transcription[82]. Second, TOR appears to
directly or indirectly affect the Atg proteins, resulting in
interference with the formation of
autophagosomes[83]. In mammalian cells, there is a TOR orthologue, mTOR, which
appears to modulate autophagy in a manner similar to that
observed in yeast.
Protein phosphatase 2A (PP2A) In yeast, TOR
phosphorylates the protein Tap42, causing its association with
PP2A[84]. This interaction significantly reduces the enzyme
activity of PP2A. Inhibition of TOR by nutrient stress or
rapamycin results in the dephosphorylation and
dissociation of Tap42 from PP2A. PP2A may then dephosphorylate
its targets, which eventually leads to a variety of
antiprolifera-tive responses and induction of autophagy. PP2A is a
phosphatase that acts on several TOR substrates, including
glutaminase (Gln3). Dephosphorylation of Gln3 by PP2A
promotes its dissociation from urease 2 (Ure2) and its
successive translocation into the nucleus, where it activates the
transcription of several genes[85,86]. Some of those genes are
parts of the autophagy machinery such as Atg8 and Atg14[87,88].
4E-BP1 mTOR has a serine/threonine kinase activity,
and eukaryotic initiation factor 4E binding protein-1 (4E-BP1)
is one of its substrates[89]. 4E-BP1 is an inhibitor of
translation and can be directly phosphorylated by mTOR. After
phosphorylation, 4E-BP1 will dissociate from eukaryotic
initiation factor 4E (eIF4E). Free eIF4E binds to the 5¡¯
terminal cap structure of RNAs and promotes the progress of
translation.
p70S6 kinase p70S6 kinase (p70S6) is also a candidate
of the substrates of mTOR, which can be called S6K for short.
S6K is a protein kinase of ribosomal 40S subunit S6.
Phosphorylation of S6 upregulates the translation of mRNAs
containing 5¡¯ terminal oligopyrimidine tract (5¡¯ Top). The 5¡¯ Top
mRNAs account for approximately 20% of all cell mRNAs
and are foundations of protein biological synthesis. The
major products of 5¡¯ Top mRNAs include ribosomal protein,
elongation factor (EFla, EF2), and polyA binding protein.
When nutrition is sufficient, TOR is turned on and the
activity of the enzyme S6K increases. Recently, it has been found
that S6K is needed for the entire process of autophagy
activation, and it must be activated first for maximal
activation of autophagy[90]. However, there is a contradictory
phenomenon, in that TOR, an activator of S6K, is an
inhibitor of autophagy. The possible explanation is that S6K needs
to be active under starvation conditions in which its
activator, TOR, is turned off. Perhaps, after TOR is switched
off, any active S6K remains active for some time, ensuring
maximal autophagy induction can be achieved, but other
mechanisms might then gradually deactivate the p70S6
kinase, thereby preventing excessive autophagy, which
could be harmful.
Hormones The hormones glucagon and ecdysone in
Drosophila larvae inhibit TOR by downregulating PI3K-I,
resulting in an increase in autophagy. Conversely, the
hormone insulin appears to have an inhibitory effect on the
autophagic pathway[91,92]. In response to food, insulin is
produced; insulin binds a receptor on the surface of cells
and triggers a signaling cascade. Insulin first activates its
tyrosine kinase receptors, causing these receptors to
phosphorylate themselves. P85 as a regulatory subunit of PI3K
activates PI3K by association with these phosphorylated
insulin receptors. The active PI3K transfers to the inter
surface of the cell membrane and phosphorylates the lipid
phosphatidylinositol, leading to the activation of protein
kinase B (AKT/PKB) and other enzymes. This is followed by
the activation of TOR. The latter then negatively regulates
Atg proteins to prevent autophagy activation.
Amino acids Amino acids, which are the final products
of autophagic protein degradation, act as negative feedback
regulators for the process. In almost all cell types, especially
in hepatocytes, a combination of leucine and a few other
amino acids is very effective in inhibiting
autophagy[93-95]. It was discovered that the addition of amino acids (leucine in
particular) in the absence of insulin or other growth factors
resulted in a strong and fairly rapid stimulation of the
phosphorylation of ribosomal protein
S6K[96,97]. Phosphorylation of 4E-BP1, similar to that of S6K, requires the presence of
amino acids, and amino acids alone, but not insulin alone,
stimulate phosphorylation of this protein in a
rapamycin-sensitive manner[98,99]. Furthermore, leucine activates
glutamate dehydrogenase, which contributes to the
ability of leucine to potentiate insulin production in
b-cells[100]. Amino acids and insulin act synergistically on both
processes to inhibit autophagy[96,97]. The amino acid/TOR
signaling pathway can be confronted by the AMP-dependent
protein kinase (AMPK). However, this effect can be blocked
by rapamycin and two inhibitors of PI3K, wortmannin and
LY294002[101]. Recently, it has been found that amino acids
can modulate activation of the kinase Raf-1. Raf-1 acts
upstream of the mitogen-activated protein kinase (MAPK) and
extracellular signal-regulated kinase (Erk1/2)
cascades[102]. However, the mechanism by which amino acids regulate the
activation of Raf-1 remains to be elucidated.
ATP Autophagic sequestration is ATP-dependent, and
the depletion of ATP inhibits autophagic
sequestration[103]. Because of adenylate kinase equilibrium in the cell, a fall in
ATP is often associated with an increase in AMP. AMPK,
which serves as a general integrator of metabolic responses
to changes in energy availability, is activated in response to
elevations of the AMP/ATP ratio. Thus, autophagy can be
suppressed by AMP through activation of AMPK. In addition, high levels of AMP can be reached under hypoxia
and other conditions of energy depletion, which also
suppress autophagy[104,105]. Different studies suggest that in
liver and muscle cells AMPK negatively modulates protein
synthesis by impairing the mTOR-dependent signals to
p70S6 kinase and 4E-BP1[106], interfering with the occurrence
of autophagy. Although AMPK is involved in the control of
mTOR signaling, its role in autophagy needs to be clarified.
However, Snf1, the yeast homologue of AMPK, has been
identified as a positive regulator of
autophagy[107].
Atg proteins TOR signaling negatively regulates the
association between Atg1 and Atg13. Under nutrient-rich
conditions, active TOR causes hyperphosphorylation of
Atg13, preventing or modulating its association with
Atg1[43].
It is not clear whether TOR directly phosphorylates Atg13.
TOR inactivation by starvation or rapamycin treatment
promotes the rapid dephosphorylation of Atg13, a process that
seems to be independent of PP2A. Dephosphorylated Atg13
binds to Atg1. This association promotes
autophosphoryla-tion and activation of Atg1, leading to the induction of
autophagy. The inhibition of TOR signaling also promotes
the assembling of other regulatory proteins in the membrane
of PAS due to increased Atg1 kinase activity. In other words,
inactivation of TOR is necessary for the prolongation of
pre-autophagosomal membranes and enhancement of the
expression of autophagic specific genes such as Atg8.
PI3K-I/PKB The PI3K-I/PKB pathway is involved in the
negative modulation of autophagy (Figure 4). If PI3K-I is
activated, it will phosphorylate PI4P and
PI(4,5)P2 to produce
PI(3,4)P2 and
PI(3,4,5)P3[108]. These lipids have a role in
the PAS structure to recruit proteins necessary for the
biogenesis of autophagosomes. These lipids bind to protein
kinase B (Akt/PKB) and its activator
phosphoinositide-dependent kinase-1 (PDK1) via its pleckstrin homology (PH)
domains. Upon lipid binding, Akt/PKB is then
activated[108]. Furthermore, PDK1 phosphorylates other kinases,
including p70S6 kinase, making them acquire kinase
activity[109]. Activation of this pathway by expression of a constitutive
active form of PDK1 and PKB has an inhibitory effect on
autophagy. The phosphatase PTEN, which selectively
hydrolyzes PI(3,4,5)P3, has a stimulatory effect on autophagy
by relieving class I PI3K/PKB
inhibition[55]. Rapamycin can reverse most of the inhibition of autophagy because of
activation of the class I PI3K pathway, which suggests that
mTOR is a downstream target. The activation of PI3K/PKB
has been shown to relieve the inhibitory effects of the
tuberous sclerosis complex (TSC1/TSC2, hamartin/tuberin) on
mTOR/p70S6 kinase signaling[110]. TSC2 has a
GTPase-activating activity towards monomeric Rheb, which controls
mTOR/p70S6 kinase signaling[111], so TSC2 can promote the
conversion of Rheb from the GTP-bound state (inhibitor of
TOR) to the GDP-bound state (activator of TOR).
Beclin1/PI3K- III The beclin 1/PI3K-III complex is
involved in the formation of autophagosomes and initiation of
autophagy. 3-MA, wortmannin, and LY294002, three
PI3K-III inhibitors, interfere with this
pathway[50]. Recently, it has been shown that in muscle cells, amino acids can negatively
regulate autophagy by interference with the activity of class
III PI3K[55]. Further studies showed that all beclin forms a
complex with PI3K, whereas ~50% of PI3K remains free from
beclin. Indirect immunofluorescence microscopy
demonstrated that the majority of beclin and PI3K localized to the
trans-Golgi network (TGN). Some PI3K also distributed in
the late endosome[112]. This suggests that beclin and PI3K
control autophagy by functioning in PI3-P sorting into
vesicles that travel from the Golgi to the endosome as a
complex at the TGN. In addition, an increase in the class III
PI3K product, PI3-P, can also stimulate
autophagy[49]. Beclin-1, as an important element of mammalian autophagy, is
consistently mono-allellically deleted in 40-75% of human
sporadic breast, prostate and ovarian cancers. In
in vitro cultured MCF-7 cells, overexpression of beclin-1 induces
autophagy and is associated with inhibition of MCF-7 cell
proliferation[56]. The protein beclin-1 is able to shuttle between
the nucleus and the cytoplasm[113]. Its role in the nucleus is
unknown, but nuclear beclin-1 does not control autophagy
and does not inhibit tumorigenicity of breast carcinoma cells.
Beclin-1 contains a leucine-rich nuclear export signal that is
required for its autophagy and tumor suppressor
functions[113]. This nuclear export traffic is modulated by nuclear export
protein chromosome region maintenance
1(CRM1)[113]. The CRM1-dependent nuclear export traffic plays an important
role in the regulation of autophagy. However, when the
traffic is inhibited by daunomycin B or a mutation of the
nuclear export signal, beclin-1 exists almost exclusively in
the nucleus. Thus, autophagy induction by starvation
cannot be initiated and its anti-tumor effect is also blunted.
GTPases
Heterotrimeric G proteins and partners In the human
colon cancer cell line HT-29, it was found that the trimeric
Gi3 (ai3bg) control the autophagic pathway at the sequestration
step[114]. The activity of autophagy was low when the
Gai3 protein was in the GTP-bound form, and it was stimulated
when GDP was bound to the Gai3
protein[115]. The localization of the
Gai3 protein is also essential in controlling
autophagy. It must first associate with the ER or Golgi in
order to control autophagy. The guanine nucleotide cycle
of the Gai3 protein is dependent upon the activity of the G
alpha interacting protein (GAIP). GAIP belongs to the
regulators of the G-protein signaling (RGS) family and is a
GTPase-activating protein towards the
Gai3 protein. The phosphorylation of a conserved serine residue in the RGS domain of
GAIP stimulates its GAP activity, and consequently the
autophagic pathway[116]. In addition, GAIP is a cytoplasmic
substrate for the MAP kinase Erk1/2 and its
phosphorylation is reduced in the presence of amino acids. The different
domains of the activator of G-protein signaling 3 (AGS3) are
all involved in the regulation of
autophagy[117]: its N-terminal part containing 7 tetratricopeptide regulatory (TPR)
repeats can decrease the occurrence of autophagy; its
C-terminal part containing G-protein regulatory (GPR or GoLoco)
motifs can interact and stabilize the
Gai3 protein in its GDP-bound conformation. Recently, investigators showed that
AGS3 had a stimulatory effect on autophagy in human colon
cancer cells[118], but this stimulatory effect could be
counteracted by GoLoco motifs. The
Gai3 protein and its partners (GAIP and AGS3) act prior to the formation of the
autophagosome. They may be involved in the control of
membrane flux to the autophagic pathway. What is
noticeable is that the Gai3 protein and its partner proteins can
control the balance between the flow of membranes in the
exocytic pathway and the delivery of membrane
constituents to the macroautophagic pathway.
Monomeric G proteins Rab proteins are monomeric
GTPases necessary for vesicular transport in the
exo/endocytic pathway, in which Rab22a is associated with early
and late endosomes. However, Rab22aQ64L, a mutant with
low GTPase activity of Rab22a co-localizes with the
autophagic vacuoles[69]. In addition, another monomeric GTPase,
Rab24, existing preferentially in a GTP-bound state when
expressed in cultured cells, is redistributed and co-localized
with MAP1-LC3 during starvation. It has been shown that
Rab24 has a role in autophagy. The GTP membrane-bound
forms of Rab proteins are able to recruit cytosolic proteins,
which target vesicles to appropriate sites on the acceptor
membrane[119]. Whether or not this function of Rab proteins
is essential for membrane fusion, transport, and the
maturation of autophagic vesicles is still to be investigated.
Calcium Autophagy is dependent on the presence of
sequestered Ca2+ in some intracellular storage compartments
that are sensitive to interference by a number of
Ca2+-perturbing agents[120]. ER is the major reservoir for intracellular
calcium, thus thapsigargin, which inhibits the ER
calcium/ATPase promotes the release of intracellular calcium from
ER and thereby lowers ER calcium levels leading to notable
inhibition of autophagy. This implies that depletion of
sequestered, rather than of cytosolic, intracellular
Ca2+ should be responsible for the common mechanism of autophagy
inhibition. Inhibitors of Ca2+-activated protein kinases
(KN-62, H-7, W-7) had little or no effect on autophagy, indicating
that the Ca2+ requirement of autophagy was not mediated by
these kinases. Recent studies have shown that elements
that modify the lysosomal calcium levels, such as phorbol
myristate acetate, ionophore A23187, and phentolamine, may
modulate the total volume of autophagic
vacuoles[121].
Protein synthesis pathway In rat hepatocytes, the
phosphorylation of the ribosomal S6 protein, a p70S6 kinase
substrate, is also related to the inhibition of autophagy. The
degree of S6 phosphorylation determines the degree of
occupancy of the endoplasmic reticulum by ribosomes, and
thus determines the rate of autophagic sequestration and
the rate of ER-linked protein
synthesis[97]. So it is possible that there is a relationship between the control of protein
synthesis and of autophagy. Furthermore, the finding of the
involvement of eukaryotic initiation factor-2 alpha
(eIF2a) kinases in autophagy strengthens this view. The eIF2a
kinases are members of a family of evolutionarily conserved
serine/threonine kinases that regulate stress-induced
translational arrest. Recently, it was demonstrated that two
eIF2a kinases (GCN2 and PKR) positively controled autophagic
sequestration in yeast and mammalian cells in response to
nutrient deprivation by phosphorylating the translation
factor eIF2a[122]. However, a general inhibition of protein
synthesis does not trigger autophagy. For example, when
translation is inhibited by cycloheximide, autophagy is not
induced, but the formation of smaller autophagosomes is
observed. These results indicate that protein synthesis is
required for the expansion of the preautophagosome vesicle[123]. In addition, the de
novo synthesis of proteins is also required after the sequestration step during the
maturation of autophagosomes.
Other regulatory factors The regulation of autophagy
is very complex. In addition to the above mentioned
molecular mechanisms, microtubule-associated protein, integrin, and
some other kinases such as tyrosine protein kinase II,
naringin-sensitive protein kinase, death-associated related
protein kinase-1 (DRP-1), dea, th-associated protein kinase
(DAPk) all have a role[124]. However, the molecular
mechanisms by which these regulatory factors contribute to the
control of autophagy are still largely unknown and their
downstream targets also remain to be identified.
Autophagy and diseases
Autophagy is an important gate-keeping mechanism for
the stabilization of cell homeostasis, which is required for
eliminating discarded or damaged organelles and/or
cytoplasmic components and remodeling cytoplasm.
Autophagy has been studied for more than 40 years, but it is only in
the last 10 years that the molecular basis of autophagy has
been gradually understood through the utilization of yeast
genes. The discovery of the ATG genes and the dissection
of the signaling pathways involved in the regulation of
autophagy have greatly increased our knowledge of the
occurrence and development of this lysosomal degradation
pathway. In yeast, many questions about the molecular
mechanisms of autophagy have already been
investigated, but there are a great number of tasks ahead, such as
clarification of the putative links between the different signaling
complexes and elucidation of the specific mechanisms
mediating autophagosome biogenesis, transport, fusion and autophagic degradation. Although the molecular machinery of
the autophagic pathway is well conserved during evolution
in multicellular organisms, especially in mammals, this
process is much more complex in mammals than it is in yeast. At
present, many autophagic genes in mammals still remain
unknown and their functions in autophagy also remain
undiscovered. The creation and analysis of transgenic and
knockout animal models will help to understand the
evolutionarily-acquired complexity of autophagy-mediated
processes in mammals. Some gene (beclin-1,
ATG5, and ATG7) knockout mice have already been used in experiments to
explore the functions of these genes and the relationships
between autophagy and some
diseases[21,125,126]. For example, with a targeted
beclin-1 mutant mouse model, it has been shown that heterozygous disruption of
beclin-1 increases the frequency of spontaneous malignancies and accelerates
the development of hepatitis B virus-induced premalignant
lesions. In addition, it has also been demonstrated that
beclin-1 is a haplo-insufficient tumor-suppressor gene, and
therefore it is possible that the genetic disruption of
autophagy, either by mutations of downstream
autophagy-execution genes or by mutations in upstream
autophagy-regulatory signaling pathways, may be an important
mechanism of oncogenesis[126]. Thus, mutation of
beclin-1 or other autophagy genes might contribute to the pathogenesis of
human cancers. A growing number of pathological conditions, including cancer and neurodegenerative
disorders, are associated with autophagy, so it is very
important to reveal the molecular relationships between
autophagy and diverse diseases. In our laboratory, we found
that autophagy may have dual functions in cultured tumor
cells. On the one hand, it may delay the apoptotic process in
one cell type; but on the other hand, it may promote cell
death in other cell types (Yan et al, unpublished
observa-tions). Autophagy plays important roles in the degradation
of misfolded or aggregated proteins and, therefore, may play
a role in certain neurodegenerative diseases that feature the
misfolding and aggregation of disease proteins, such as
Huntington¡¯s disease and Parkinson¡¯s disease. We have
studied the degradation of mutant huntingtin by autophagy.
We found that autophagy upregulated cathepsins and
enhanced the clearance of huntingtin fragments, but mutant
huntingtin was relatively resistant to degradation by
cathepsin D[127]. Over-stimulation of autophagy by mutant
huntingtin resulted in mislocalization and dysfunction of
mitochondria (Qin et al, unpublished observations). In our
recent studies, we also found that an autophagic mechanism
was involved in excitotoxicity. Activation of the NMDA
(N-methyl-D-aspartate)- and KA (kainic acid)-type glutamate
receptors stimulated autophagy and lysosomal enzymes. The
apoptotic death of striatal neurons was blocked by 3-MA
and a cathepsin B inhibitor, suggesting that the activation
of autophagy probably contributes to
excitotoxicity[128]. These studies have opened a new field for investigating the
pathogenic mechanisms in neurodegenerative diseases
related to protein misfolding, aggregation and excitotoxicity.
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