L1 - Intro to Proteostasis and Autophagy Pathway Flashcards
Why is studying proteins so important? (2)
1) Make up most of dry mass of cell
2) Proteins mediate pretty much all essential process of the cell (DNA replication, protein synthesis, mediate processes, communications)
What is a proteome?
the entire set of all proteins that are expressed by a genome, cell, or organism at a certain time
Why is the proteome described as dynamic?
levels and states of the proteins in the cell changes depending on the external and internal environments
How does protein levels change within the cell? (4)
1) transcription regulation
2) protein synthesis
3) protein breakdown
4) protein folding
What could happen when the cell fails to achieve proteostasis?
can lead to lots of complications in the cellular environment and lead to diseases
1) Parkinson’s disease
2) Alzheimer’s disease
3) cystic fibrosis
How does the cell achieve proteostasis?
cell has a network of machineries to control protein folding, breakdown and recycling
What are the 3 major arms for maintaining proteostasis?
1) translation initial factors
2) molecular chaperones
3) recycling/breakdown systems
How does translation initiations factors help with proteostasis?
ensures that there is efficient translation to make more polypeptides
How do molecular chaperones help with proteostasis?
ensures that proteins can adopt their native structure to perform its physiological function
How do recycling and breakdown systems help with proteostasis?
dispose misfolded proteins, protein aggregates and aged proteins
Why do we need a recycling /junk removal system for proteins? (3)
1) get rid of misfolded proteins- protein folding is complex and mistakes are bound to happen
2) get rid of aged proteins - proteins half-lives and don’t last forever
3) metabolic control - AA control affects cells metabolic rate and whether or not it divides
How might the protein
degradation and recycling system be viewed as a double-edged sword?
- can breakdown proteins that are still needed
- might result in waste of energy
What are the two major protein degradation systems in the cell?
1) ubiquitin-proteasome system
2) autophagy-lysosome system
What is ubiquitin?
small protein involved in the ubiquitin-proteasome system for protein degradation
How does ubiquitin work?
modifies protein post-translation
What kind of bond links ubiquitin to another protein?
covalent attachment via a system involving E1, E2, and E3
How big is ubiquitin?
8.6 kDa
What kind of system is ubiquitin linked to a protein?
ubiquitin-proteasome system
What are the steps of ubiquitination?
1) E1 activates ubiquitin (formation of thioester linkage between C-term of ubiquitin and E1 active site Cys)
2) ubiquitin transferred to E2 (formation of thioester linkage with E2 active site Cys)
3) ubiquitin transferred from E2 enzyme to Lys of substrate via E3
How many ubiquitin molecules are added onto a protein?
depends, can be just one or multiple
What is the fate of ubiquitinated proteins?
Many different fates (ex: autophagy, proteolysis, ribosomal activity, DNA repair, etc)
Where are polyubiquitinated proteins targeted to and why?
targeted to proteasome for degradation
What is a proteasome and what is its general function?
proteins that recognizes and degrades polyubiquitinated proteins
How big are proteasomes?
2.0 MDa
What are the general steps that proteasome takes to degrade its target?
1) proteasome recognizes and binds to poly-ubiquitin+substrate complex
2) substrate protein unfolds
3) substrate complex de-ubiquitinated
4) protein folds into the degradation chamber of proteasome
5)
Is proteasome degradation energy dependent?
Yes! ATP-dependant process
What are the limitations of the UPS system
1) proteasome can only degrade proteins
2) diameter of the entrance of the proteasome degradation chamber is small (20A) so protein has to be unfolded and cannot handle protein aggregates/large molecules
What are the advantages of the autophagy/lysosome pathway?
1) no upper size limit
2) can degrade things other than proteins (bacteria, organelles, viruses)
What are the three different types of autophagy?
1) macroautophagy
2) microautophagy
3) chaperone-mediated autohpagy
What is macrophagy?
type of autophagy
- autophagosome encapsulates cargo to be degraded
- autophagosome fuses with lysosome
What is microautophagy?
type of autophagy
- does not need an autophagosome
- cargo directly enters lysosome via inward folding with lysosomal membrane
What is chaperone-mediated autophagy?
type of autophagy
- Hsc70 chaperone complex recognizes the target protein+pentapeptide
- chaperone-substrate complex binds to membrane protein on lysosome
- substrate unfolds and translocates into lysosome
What are the four main functions of macro autophagy?
1) remove cellular waste
2) recycle cellular material
3) manage energy during environmental changes
4) defend against foreign material
How come we care so much about autophagy?
linked to many diseases (neurodegenerative diseases, cancer, diabetes etc)
What were the main experimental methods used by Christian de Duve to discover lysosomes?
1) subcellular fractionation
2) transmission electron microscopy
What is subcellular fractionation?
experimental method
- use high speed ultracentrifugation to separate different objects inside cell lysate based on size/shape/density
What are the two main approaches to subcellular fractionation?
1) differential sedimentation
2) density gradient centrifugation
How does differential sedimentation work?
separate objects by doing a hard spin on a cell lysate
How does density gradient centrifugation work?
separate objects based on density using a tube with a gradient of increasing concentration of a dense material (sucrose)
How is TEM different from light microscopy?
uses electrons instead of light as an emission source to view smaller objects
Are there lysosomes in plant cells and budding yeast cells?
no, but there are equivalent organelles called lytic vacuoles
What is lysosome? (structure+function too)
a organelle made of a lipid bilayer containing digestive enzymes for the breakdown of proteins, nucleic acids, saccharides and lipids
How does lysosome maintain a low, acidic pH in its interior?
use of V-ATPase
Why is it beneficial to make the interior of the lysosome acidic?
safety measure
- lysosome contains digestive enzymes that are only functional in acidic environment
- in case these enzymes are released into the cytoplasm, they won’t digest cellular components because the cytoplasm is neutral
What does autophagy mean?
self+eating
What is the autophagosome?
vesicle structure that forms around cargo to be degraded in the autophagy/lysosome digestion pathway
How is autophagy regulated by insulin/glucagon?
1) insulin: low autophagy
2) glucagon: high autophagy
When does non-selective autophagy occur?
- response to cellular stresses such as starvation
When does selective autophagy occur?
- disease related inclusions/aggregates, invasive bacteria, damaged organelles
What are the general steps of autophagy?
1) formation of phagophore
2) elongation + expansion of phagophore
3) recognize + engulf cargo
4) enclosure + formation of autophagosome]
5) fusion of autophagosome with lysosome
6) breakdown + recycling of autophagosome IM and contents
What is the rate determining step of autophagy?
autophagosome formation
What are the 4 main technical challenges in studying autophagy?
1) lysosomes are heterogenous in shape/content
2) no good method for quantify extent of autophagy
3) autophagosome only exists for short time before fusing with lysosome
4) lack of specific marker for tracking autophagosome
Can the UPS system degrade things other than proteins?
No, the enzymes in the proteasome’s degradation chamber can only hydrolyze peptide bonds
what is V-ATPase and how does it work?
a vacuolar-type protein-ATPase that couples the energy of ATP hydrolysis to proton transport across lysosomal membrane to make the interior acidic
