Lecture 6 - Autophagy Flashcards
What is autophagy?
Auto-phagy self-eating, at the subcellular level
Autophagy, or cellular self-digestion, is a cellular pathway involved in
protein and organelle degradation
Autophagy relies on
Dynamic rearrangement of intracellular membranes to allow an organized breakdown and recycling of cytoplasmic portions
Protein classification according to turnover
Intracellular proteins can be classified into: short-lived proteins (half-life, 10–20 min) and long-lived proteins
most short-lived proteins are
degraded by the ubiquitin-proteasome system
most long-lived proteins are
degraded in lysosomes via the autophagic pathway
More than 99% of intracellular proteins are
long-lived
Autophagy- 1 of 2 degradative systems
- the ubiquitin-proteasome system (short-lived proteins)
- the vacuolar degradative pathway/lysosomal system, (long-lived proteins)
non-selective vacuolar degradation process is a
highly conserved pathway within eukaryotes
Three types of autophagy are known:
(i) chaperone-mediated autophagy (CMA)
(ii) microautophagy
(iii) macroautophagy
Macroautophagy = Autophagy
CMA:
selective motif tagged protein translocation directly through the lysosomal membrane
Microautophagy:
trapping and engulfing of cytosolic regions by lysosomes
Macroautophagy:
formation and accumulation of double membrane intermediate vesicles
primary mechanism for cytoplasm-to-lysosome delivery
macroautophagy
Macroautophagy (from here onwards referred to as Autophagy)
Definition
or cellular self-digestion, is a cellular pathway involved in protein and organelle degradation
Three basic steps can describe the autophagic pathway:
(1). Formation of isolation membrane and preautophagosome
(2). Formation of Autophagosome
(3). Formation of Autophagolysosome
How is autophagy induced
Nutrient depletion
ER stress
mTOR kinase senses nutrient conditioninduction via Regulatory complex
ROS stress
Hypoxia
Toxic compounds
Radiation
High temperature
mTOR-upstream sensor
mTOR is a sensor for amino acids and ATP (ADP/AMP) and can integrate hormone stimuli via the class I PI3-kinase/PKB pathway
it directly or indirectly causes hyperphosphorylation of the autophagy protein Atg13.
This protein modulates Atg1
Induction
Increased mTOR activity inhibits autophagy
mTOR affects downstream
protein/protein affinities
When mTOR is inhibited
autophagosome formation is favored and kinase activity of Atg1 increases
when mTOR is stimulated by nutrients
Atg13 becomes hyperphosphorylated which reduces its affinity to- and association with Atg1, autophagy not being favored
LC-3, a key protein and structural component
Atg8/PE or its mammalian homologue, microtubule associated protein 1-light chain 3 (MAP1-LC3) is required as a structural component of the autophagosome and is therefore recruited to the autophagosome membrane
Where in the cell localizes mTOR?
association of mTOR with the outer mitochondrial membrane, where it is ideally situated to sense changes in the ATP/AMP ratio
it also localizes with LC3-positive membranes, suggesting an involvement in the autophagosome maturation step
2 conjugation machineries of autophagy proteins (Atg) are involved in the vesicle formation
How does it work?
Macroautophagy: cargoes are sequestered within a unique doublemembrane cytosolic vesicle, an autophagosome.
Sequestration can be either nonspecific, involving the engulfment of bulk cytoplasm, or selective
targeting specific cargoes such as organelles or invasive microbes
origin of the membrane is unknown (ER-membrane, de-novo synthesis or mitochondrial membrane are current models)
Fusion with a lysosome provides hydrolases. Lysis of the autophagosome inner membrane and breakdown of the contents occurs in the autolysosome
and the resulting macromolecules are released back into the cytosol
Levels of autophagy
Almost all tissues have a basal level of autophagy, but to varying degrees
Not all tissues respond equally with starvation induced autophagy
How is autophagy measured?
Electron microscopy, the gold standard
Fluorescence Microscopy
LC3, Beclin, other Atg proteins
Quantification of cells with numerous LC3 punctae
Quantification of autophagosomes per cell
Already fused? Or not yet fused? What is the fused proportion?
Co-localization analysis between autophagosomal and lysosomal signal.
How is it measured?
Western Blotting for key proteins
LC3 I/II
Beclin-1
p62
LC3-II is specifically targeted to the
Atg12-Atg5-Atg16 complex and remains associated with the limiting membrane, the autophagosomes and the autophagolysosomes
LC3-II is found both on the
the lumenal and cytosolic surfaces of autophagosomes
The lumenal pool is degraded after fusion with lysosomes (loss of signal with increased degradation activity), while the LC3-II on the cytosolic side can be delipidated and recycled
In higher eukaryotes, LC3 is the only known protein that
specifically associates with autophagosomes and autolysosomes
LC3-II levels correlate with
autophagosome number
HOWEVER: cellular LC3-II levels correlate with the numbers of autophagosomes in the cells at a snapshot in time
Normal flux (A) vs block in degradation (B) and additional fusion with endosome prior to fusion (C)
How is flux measured
Autophagy is crucial
Autophagy knock-out mice die within the first day of life
An impaired autophagic machinery is associated with many pathologies
Autophagy can lead to cell death
with autophagy = type II cell death
Cell death with Autophagy is a physiologically
controlled mechanism
Autophagy can be utilized as an avenue for
cellular demise, especially during development (programmed cell death)
Thinking points
Why are cells different in their basal protein degradation activity?
