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Printable Version
The Translation Post Vol.2 Issue 2
Featured Article - Autophagy, a Survival Guide
By John Mountzouris, Ph.D., Director Product Support |
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Introduction
Achieving a spatial and temporal balance between formation and degradation of
cellular components is essential to cellular homeostasis, and significant
resources of the cell are devoted regulating this balance, given that
disruption can present dire consequences to normal cell growth and
development. Two cellular instruction sets for regulating survival/extinction
at the individual level of shortlived proteins (the ubiquitin pathway) and at
the grand level of the cell (apoptosis) have received great attention over the
last fifteen years, and the emerging details have advanced both fundamental
cellular research and pharmaceutical initiatives to exploit this knowledge for
therapeutic purposes. Autophagy, the third leg on this stool, is a catabolic
trafficking pathway for bulk destruction/ turnover of long-lived proteins and
organelles (the only means for the latter) via regulated lysosomal degradaton.
It was first identified in differentiating kidney cells (Clark, 1957), and it
role in cell survival glimpsed early on, as Ashford and Porter noted in 1967
that hydrolysis may be "providing the protoplast with breakdown products for
use in a reoriented physiology," with the membrane "shield[ing] the rest of
the cell from the general spread of the degradative process." Although the
gross phenomenon of autophagy had been identified for over forty years via
electron microscopy, 1) the advent of newer techniques permitting the
identification within the last decade of the full set of genes in yeast, 2)
the discovery of human homologues tied to specific disease states, and 3) the
definition of signaling pathways regulating autophagy have accelerated
interest in elucidating the full molecular details of this important process.
General Mechanisms
Autophagy can be broken down into a few basic steps: signaling, sequestration
of cytoplasm, completion of vesicle formation, targeting of the completed
vesicle to the lysosome/vacuole followed by docking and fusion, and breakdown.
In higher eukaryotes, the lysosomal pathway of intracellular degradation is
further partitioned into three distinct pathways: macroautophagy, chaperone-medited
autophagy, and microautophagy. The most detail and significance is understood
regarding macrophagy, which will be the core of this review, but it is worth
reviewing briefly the general aspects of each.
Macroautophagy begins with the formation in the cytoplasm of the autophagosome,
a multilamellar or double membrane vescular structure. The autophagosome
engulfs cytoplasmic constituents such as proteins, lipids, and damaged
organelles such as mitochondria, endoplasmic reticulum, and ribosomes. The
outer membrane of the autophagosome fuses with the lysosome in mammalian cells
(vacuole in yeast or plants) to deliver the sequestered cargo. The lysosome
holds a variety of hydrolytic enzymes. The inner membrane of the fused
structure (autophagolysosome), then dissolves, and the digestion of the
interior contents generates nucleotides, amino acids, and free fatty acids
that can be recycled to provide raw materials and energy to the cell.
Microautophagy circumvents the autophagosomic step of macrophagy, and begins
with the direct uptake of cytosolic material via invaginations and pinching
off of the lysosomal membrane. The internalized cytosolic components are
digested by lysosomal enzymes released when the vacuolar membrane
disintegrates, as in macroautophagy.
In chaperone-mediated autophagy, specific chaperone proteins bind to target
proteins containing a KFERQ sequence and channel these proteins to the surface
of the lysosome. These proteins bind to Lamp2a and are then transported across
the lysosomal membrane with the assistance of lysosomal chaperones, after
which they are degraded by vacuolar proteases.
Morphology
The classical
phemonological signs of apoptosis include cell shrinkage, membrane blebbing,
phagocytic engulfment of the fragmented cell, and DNA fragmentation. In
contrast, upon induction of autophagy, a membrane cisterna (fold of membrane)
known as the isolation membrane appears and curves around part of the
cytoplasm. Sealing of the edges of the isolation membrane results in a unique
double membrane vesicle, the autophagosome, clearly visible by electron
microscopy. Soon after forming, autophagosomes fuse with a lysosome, where
degradation of the delivered material for recycling takes place. The membranes
of isolation membranes and autophagosomes differ visibly from other membranes
in the cell in having few intramembrane proteins.
Autophagic Proteins/Signaling Pathways
In eucayrotic cells
autophagy occur constitutively at low levels in all cells to perform
housekeeping functions such as destruction of dysfunctional organelles.
Dramatic upregulation occurs (e.g., cytoplasmic and organelle turnover) in the
presence of external stressors (starvation, hormonal imbalance, oxidation,
extreme temperature, and infection), and internal needs (generation of source
materials for architectural remodeling, removal of protein aggregates).
Autophagy is highly regulated through the coordinated action of various
kinases, phosphatases, and guanosine triphosphatases (GTPases). For example,
mediators of phosphoinositide-3 (PI3) kinase signaling pathways and trimeric G
proteins play major roles in regulating the formation of autophagosomes.
Target of rapamycin (TOR) kinase, a highly conserved environmental sensor
protein, is the predominant inhibitor of autophagy. Interestingly, this
negative regulator of autophagy is a significant target for cancer
therapeutics. Rapamcyin, which inhibits TOR and is used in model studies to
induce autophagy, is an FDA approved drug with indications for
immunosuppression and cardiovascular therapy. The eukaryotic initiation factor
2 (eIF2 ) kinase Gcn2 and its downstream target Gcn4, a transcriptional
transactivator of autophagy genes, induce autophagy under conditions of
cellular starvation. Additionally, the PI3K/Akt signaling pathway inhibits
autophagy in response to insulin-like and other growth factor signals.
Downstream of TOR kinase, an assortment of proteins essential for autophagy
have been defined in yeast, with higher eucaryotic orthologs in the majority
of cases. The proteins participate functionally as follows:
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Protein serine/threonine kinase complex that relays upstream signals from
TOR kinase: Atg1, Atg13, Atg17
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Lipid kinase signaling complex that engages vesicle nucleation: Atg6, Atg14,
Vps34, and Vps15
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Ubiquitin-like conjugation pathways that facilitate vesicle expansion: Atg8
and Atg12 networks
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Recycling pathway for removal of autophagy proteins from autophagosomes: Atg2,
Atg9, Atg18
Two mammalian homologues receiving significant recent attention are LC3 and
beclin. LC3 (rat microtubule-associated protein light chain 3), a homolog of
yeast Apg8p, is localized in autophagosome membranes after processing and is a
widely applied marker for autophagy. Following synthesis, the C-terminus of rat
LC3 is cleaved by Atg4 to produce LC3-I, which resides in the cytosol. LC3-I is
converted to LC3-II by Atg7 and Atg3. LC3-II is modified by
phosphatidylethanolamine (PE) at the C-terminus and binds tightly to
autophagosomal membrane. The amount of LC3-II was correlated with the extent of
the autophagosome formation. LC3-II is the first mammalian protein identified
that specifically associates with the autophagosome membranes. Two novel
variants of rat LC3, named LC3A and LC3B, respectively, show different
expression patterns in rat tissues.
Beclin 1, a Bcl-2 interacting protein, is the mammalian orthologue of the yeast
Apg6/Vps30 gene. It can complement the defect in autophagy present in apg6 yeast
strains and stimulate autophagy when overexpressed in mammalian cells (6).
Beclin 1 is monoallelically deleted in human breast and ovarian cancers and is
expressed at reduced levels in those tumors. Overexpression of Beclin can
promote autophagy and inhibit tumorigenesis in cultured breast carcinoma cells,
and conversely, that heterozygous disruption of the Beclin gene can promote
tumorigenesis in mice. Beclin associates with the human class III
phosphatidylinositol 3- kinase (PI3K), hVps34. The lipid product of Vps34,
PI(3)P, is required not only for autophagy, but also for assembly of proteins
involved in endocytosis and trafficking of enzymes from the trans-Golgi network
to the lysosomes. Beclin is required for hVps34 to function in autophagy.
3D-Structure of MAP-LC3 (microtubule-associated protein light chain 3, Protein Data Bank 1UGM).
Autophagy, Apoptosis, and Cell Death
The interplay between
autophagy and apoptosis is complex, with the accumulating data revealing cases
in which autophagy is either an antagonistic, agonist, or independent of
canonical programmed cell death via the apoptotic pathway. An inkling of the
connection at the genetic level is clear in a Drosophila system where
apoptosis and autophagy genes appear to jointly upregulate.
Examples where cellular damage imposed by autophagy precedes and may even
trigger apoptosis include 1) a dramatic increase in autophagy followed by
induction of apoptosis in primary sympathetic neurons deprived of neural
growth factor, and 2) a similar effect in TNF-a-induced apoptosis of T-lymphoblastic
leukemia cell lines, although in this case additional pro-death factors must
be present in concert with autophagy to promote apoptosis. The fact that
autophagy inhibitors delay apoptosis while caspase inhibitors do not impair
autophagy tend to support the notion that autophagy is in fact a precedent to
caspase-dependent cell death.
On the other hand, the enhanced induction of apoptosis in autophagic deficient
HT-29 colon carcinoma cells by sulindac sulfide highlights the role that
autophagy under certain cases may play in blocking the apoptotic pathway. In
this case, it has been proposed that destruction of damaged mitochondria by
autophagy may serve to delay signaling for the apoptotic cascade.
Yet other cases exist where cells reserve the capacity to switch between cell
death via autophagy or apoptosis, an intriguing talent that may offer cells
committed to cell death a backup method of self-execution if the pathway of
first choice is corrupted. Given that in addition physical interactions
between certain autophagic and apoptotic proteins (Beclin 1, BNIP3) have been
observed, it seems clear that an intricate system of cell death regulation not
previously appreciated is at play, with much remaining to be elucidated.
Autophagy and Disease
The nexus between
autophagy and disease, and whether this process can be regulated, is of
growing interest to basic researchers and pharmaceutical companies alike. The
wealth of evidence establishing a link between autophagy and pathological
conditions is too extensive to be exhaustively presented in this review, but a
general sketch of known relationships is offered.
Autophagy and Cancer
An abundance of evidence
indicates that tumor cells exhibit lower levels of autophagic activity. This
is in line with the literature describing circumvention of or resistance to
apoptosis by tumor cells that survive cytotoxic therapy. A number of
well-known oncogenes and tumor suppressor genes recalibrate autophagic
pathways, and thereby alter prospects for cell survival and proliferation. The
PTEN tumor suppressor gene, class I PI 3-kinase and Akt oncogenes, Ras and Myc
oncogenes are among the proteins that appear to act in this way.
Autophagy and Neurodegenerative Disease
The gradual depletion of
neuronal cell populations in certain brain disease, and the suspected failure
of cellular housekeeping (protein aggregation clearance, etc.) have implicated
a role for autophagy in certain neurological disorders. A number of studies
have demonstrated the presence of autophagy in both brain tissue and animal
models of Huntington's Hallmarks of autophagy have also been observed in
Alzheimer™s disease in both human in animal models. Autophagic degeneration is
also widespread in neurons under insult by Parkinson's disease.
Conclusion
All in all, it seems both
plausible and likely that dysfunction of a major pathway such as autophagy
will lead to cellular disruption with profound effects for the health of the
organism. The spectrum of cellular functions impacted by autophagy is wide,
offering significant opportunities for scientific discovery and application.
It is no accident that the journal Nature recently identified autophagy as one
of the 10 hottest fields over the next decade of research.
Abgent and Autophagy
Abgent offers a
comprehensive collection of antibodies against the autophagy pathway,
including: APG8a (MAP-LC3) and APG8b, APG3L, APG4, APG5, APG7, APG9, APG12,
APG16, Beclin, Rab24 and RGS19. Additionally, Abgent has set up collaborations
with experts in autophagy for antibody evaluation in mouse models of
neurodegenerative diseases.
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