Lecture 21 Autophagy 1 Flashcards
Define macroautophagy
A mechanism to digest IC material
Why do cells need degradation x5
homeostasis signalling removing damaged components recycling nutrients reprogramming cells (differentiation)
Name 2 mechanisms of degradation
- The ubiquitin/proteasome system (UPS)
* Autophagy
Name 3 flavours of autophagy
Macroautophagy
Microautophagy
Chaperone-mediated autophagy
Briefly explain macroautophagy
autophagosomes fuse to lysosome
Briefly explain microautophagy
Lysosome invaginates to capture the cargo material
Briefly explain chaperone-mediated autophagy
Specific proteins are unfolded by HSC70 and threaded 1 by 1 threaded via LAMP-2A for degradation by the lysosome
Which pathways are/are not lysosomal
Proteasome - not
Macro/chaperone mediated/micro autophagy - is
Which pathways do/do not degrade individual proteins
proteasome - individual
macroautophagy - bulk
chaperone-mediated/micro - individual
Which pathway is a major turnover route for short-lived proteins
proteasome
Which pathway can remove whole organelles
Macroautophagy
Which pathway turns over specific, long-lived proteins
Chaperone-mediated autophagy/microautophagy
Molecules released by … can support what
the macroautophagy pathway
metabolism
Which pathway has a relatively low capacity
Chaperone/micro
Name the 4 functions of macroautophagy
Nutrient recycling
Cellular remodelling
Removal of damaged components
Killing IC pathogens
Nutrient recycling:
When is autophagy rapidly unregulated?
This causes…
Under nutrient starvation
Causes non-selective bulk degradation of the cytosol
What occurs in cells lacking autophagy vs WT in starvation
Death vs the WT which get smaller due as they digest themselves
When do autophagy-deficient mice die
during neonatal starvation
Give an example where autophagy is needed for survival
Cancer cells in solid tumours
Autophagy is the only mechanism for…
the degradation of organelles
Autophagy is essential for forming which cell types?
Erythropoiesis (red blood cell differentiation)
Removal of sperm-derived mitochondria in C.Elegans fertilisation
Describe the process of erythropoiesis
reticulocyte denucleated
mitochondrial clearance
You only inherit mitochondria from…
mother
Why are components removed by autophagy
Cellular components accumulate damage over time
examples of when damaged components needed to be removed by autophagy
mechanical damage in muscles
damaged mitochondria selectively removed
How can mitochondria become damaged
CCCP decoupled the mitochondria respiratory train i.e. depolarises the mitochondria
What happens to the lysosomal capacity as we age
decreases
What disease is autophagy related to
Neurodegenerative and ageing
What causes age-related degeneration
Reduced autophagy
What cells are most susceptible to damage
Long lived neurons
metabolic muscle cells
Name the hypothesis associated to autophagy and what it states
The dietary restriction hypothesis:
Starvation/exercise leads to increased autophagy which in turn leads to increased damage repair
What could starvation prevent according to the hypothesis
prevent ageing
Eat2 mutant worms vs WT
Calorific restricted worms
Live significantly longer vs WT
How do we know eat2 mutant worms live longer via an autophagy dependent pathway
Removing autophagy in these worms has no affect on dietary restriction
What happens to the effect of dietary restriction on life span, the higher up through evolution you go
The less the effect is
How does dietary restriction increase autophagy
If starving, ATP levels drop. AMPK/SIRT1 detect to ATP/AMP levels and activate autophagy and inhibit TORC1 which inhibits autophagy
What does TORC1 recongise
Torc1 recognises amino acids. If cell is deficient in amino acids, you upregulate autophagy.
What pathogens can escape into the cytoplasm
TB
Salmonella
How are pathogens usually killed
Encased in a phagolysosome
Fuses to lysosome
Digestion
Why is the cytoplasm favourable for pathogens
They use nutrients here to grow
Macrophages cannot detect them
How does autophagy play a role in killing IC pathogens
Cells recognise the bacteria in the cytosol and targets them for autophagy
Effect of inhibiting autophagy in myocardium infection
TB bacteria can escape into the cytoplasm and grow unchecked leading to infection
Effect of inhibiting autophagy in MRSA
They grow less well as autophagy promotes their growth
Recycling nutrients causes
How manipulate?
- to survive starvation
- helps cancer to grow
So could inhibit autophagy to prevent cancer yet cells accumulate damage over time so will get mutations if not removed
Damaged protein/organelle removal causes
How manipulate?
- Ageing
- Muscular dystrophy
- Neurodegeneration
- Cancer
Increase autophagy in all these cases
Cellular remodelling causes
- Erythrocyte differentiation
- Removing sperm-derived mitochondrial
Intracellular pathogen removal causes
How manipulate?
- Tuberculosis
- MRSA
- viruses
Inhibiting autophagy removes MRSA as cant hide in autophagosomes but makes more susceptible to TB
When/what/who were autophagosomes first discovered by?
EM
1963
Christian De Duve
When/who/how shown autophagy occurred in yeast?
1992
Yoshimori Oshumi
- Nitrogen-starved protease deficient S.cerevisiae
- In cells slightly mutated, starving them leads to accumulation of vesicles inside the vacuole
How many autophagy genes were identified?
1993 Performed genetic screen to identify 15 autophagy genes
Identifying the Atg genes allowed?
- Disruption of autophagy to investigate its functions
- A start on dissecting how the machinery works
- Observation of autophagy in live cells
Describe the 4 steps in the formation of autophagosomes
- Initiation
- Expansion into cup shapes (phagophore) – formed from fats using lipid transport
- Cup shape closes (autophagosome)
- Fusion with lysosome (autolysosome) using Syntaxin 17
How is the organelle made compared to most organelles?
Only organelle in the cell to be made de novo – every other vesicle is generated by budding off from the organelles
When are autophagosomes upregulated?
Starvation
Why are autophagosomes easy to visualise with EM?
It has 2 membranes
Where are autophagosomes formed?
At the ER
How does selective autophagy occur?
Ubiquitination
Protein aggregates
How are things targeted to phagosomes
Ubiquitination
What aids in selective autophagy
Adaptor proteins
How do adaptor proteins work
- Adaptor proteins have a ubiquitin binding domain on one end and an Atg binding motif on the other end (LC3)
- This binds and targets specific proteins/organelles e.g. mitochondria to the phagosome
Are adaptor proteins vital?
No, some proteins directly bind to the Atg8, independent of adaptors
Why do ubiquitin protein aggregates accumulate?
Removing autophagy means they accumulate ubiquitin protein aggregates
