4. Autophagy in cancer Flashcards
Which cell organelles perform degradation?
Degradation performed by:
- proteosomes
- lysosomes
What needs to be degraded in cells? Why degradation mechanisms exist?
Need degradation for cellular homeostasis:
- toxic waste
- misfolded proteins
- regulate pathway activity (stop working when protein degraded)
- recycle molecules and cells for building blocks
Why are there two organelles for degradation?
They degrade different size proteins:
- proteosomes - barrel shaped - can’t fit large proteins in - such as protein aggregates and organelles - only degrade smaller
- lysosomes - can degrade large proteins
What is needed for degradation in proteosomes to notice proteins meant for degradation?
Proteins for degradation are ubiquitinated by ubiguitin ligases -> noticed by proteosomes -> degraded
What is autophagy?
Autophagy - self-degradation - cellular process in which a cell breaks down and recycles its own components, such as damaged organelles, misfolded proteins, or other intracellular debris
What are the types of autophagy?
Types of autophagy:
- Micro-autophagy
- Chaperone-mediated autophagy
- Macro-autophagy (=autophagy) - the mechanism discussed in this lecture
Explain the mechanism of autopahgy (=macro-autophagy)
Autophagy (=macro-autophagy):
1) Upstream signal for autophagy induction
2) Pre-autophagosome engulfs degradation target
3) ATG8 (ex LC3) activation by fusion with a lipid
4) LC3 integration into pre-autophagosome membrane
5) Autophagosome fusion with lysosome - contains degrading enzymes
6) Degradation + recycling of nutrients
Explain autophagosome structure
Pre- autophagosome vesicle:
- double membrane
- surface proteins
Autophagosome vesicle:
+ activated ATG8 proteins (ex LC3)
What is an autophagosome
Autophagosome - double-membraned vesicle that plays a crucial role in the process of autophagy - fuses with lysosome for functional degradation
Why is autophagosome fusion with lysosome needed for degradation?
Lysosomes contain digestive enzymes - inside low pH acidic - protection mechanism if aggressive degradation enzymes spilled - would not be functional in cytosol
What was the first experiment done to investigate autophagy genes?
Random mutagenesis induced in yeast cells -> mutants with active / inactive autophagy -> grown in starvation media (hypothesised that autophagy is needed to survive in starvation) -> some yeats survived, others not -> first autophagy genes defined - autophagy related genes (ATG) - the ones missing in dead mutants with inactive autophagy
Explain the ATG proteins
Autophagy related genes (ATG) - genes needed to perform autophagy - 2 subfamilies of ATG8 proteins:
- LC3: LC3A, LC3B, LC3C (the ones focuse din this lecture)
- GABARAP
-> these are ubiquitin like molecules
What are the functions of ATG proteins?
ATG protein functions:
- autophagy cargo recruitment
- role in autophagosome growth and lysosome fusion
Explain LC3 (ATG8) protein activation
LC3 (ATG8) protein activation - ubiquitin-like conjugation:
1) LC3 inactive - protease cleaves off an end -> exposes glycine
2) cleaved LC3 modified by ubiquitin-like enzymes
3) PE lipid conjugated to LC3 - lipidation => LC3 activated - can integrated into pre-autophagosome membrane
What is used to assess lipidation of LC3 (ATG8) as a readout for autophagy?
Fluorescence microscopy: change in fluorescence when LC3 tagged
SDS PAGE: aactive / inactive LC3 proteins show up differently by size - active run lower