24 - Regulated Protein Degradation in Neuronal Development Flashcards
For cell surface receptors / ligands to mediate repulsive signaling, what needs to happen?
The cells need to physically separate
What is the most important mediator of repulsive ephrin signaling?
ADAM-10
What does ADAM-10 do?
Causes cleavage of the ephrin ligand
Where is ADAM-10 expressed?
At the cell surface, near the Eph receptor
What is the significance of ADAM-10?
It allows the cell to physically separate, which is needed for repulsive signaling
How is ADAM-10 activated (in ephrin signaling)?
Upon binding of Eph to ephrin
True or false: overexpression of Eph and ephrin can rescue the deletion of ADAM-10
False: overexpression would not lead to cleavage, which is necessary for the proper phenotype
What is the phenotype of deleting ADAM-10?
Disorganized array of axons at the tectum (no repulsion)
What is an example of ripping?
Notch signaling
What processes utilize Notch signaling?
Embryonic development, especially neurogenesis and neuronal development
What is the structure of Notch?
A single pass transmembrane receptor with a large extracellular domain, and a small transmembrane and intracellular domain
What type of molecules are Notch ligands?
Single pass cell surface proteins
What are the names of some Notch ligands?
Delta and Serrate (flies), Delta-like and Jagged (mammals)
What happens when Notch binds to Delta?
It induces proteolytic cleavage of the intracellular domain of Notch
What does NICD stand for?
Notch intracellular domain
Where is Notch cleaved?
Once close to the extracellular leaflet, and once at the transmembrane region
What does the NICD do after cleavage?
It translocates to the nucleus to activate gene expression
Which cleavage of Notch does ADAM-10 mediate?
Cleavage at the extracellular domain
Which enzyme catalyzes the cleavage of Notch at the extracellular domain?
ADAM-10
Which cleavage of Notch does the presenilin / gamma-secretase complex mediate?
Cleavage at the transmembrane domain
Which enzyme catalyzes the cleavage of Notch at the transmembrane domain?
The presenilin / gamma-secretase complex
What is the order of cleavages for Notch?
First the extracellular cleavage, then the transmembrane cleavage
How is ADAM-10 activated (in Notch signaling)?
By binding of Notch to Delta
How is the presenilin / gamma-secretase complex actiavted?
Upon cleavage of Notch by ADAM-10
What is ripping?
Regulated intramembrane proteolysis
What are the specific characteristics (3) of the Notch signaling pathway?
- Release of NCID is irreversible
- Not a good pathway for signal amplification
- Directness
What types of decisions are not suited for Notch signaling?
Rapid, repeat changes in stimulus over time
What types of decisions are suited for Notch signaling?
Strong, decisive decisions (cell fate)
Why is Notch signaling not suited for amplification?
One activated receptor only generates one active transcription factor
True or false: NCID is a transcription factor for a particular gene
False: it can be a transcription factor for many genes
How is Notch signaling a direct signaling pathway?
There are no intermediates between cell-surface receptor engagement and nuclear activity
What proteins can MMPs degrade?
Collagen, fibronectin, etc.
What events (4) do MMPs participate in?
- Cell migration
- Wound healing / cell protrusion
- Ovulation
- Angiogenesis
What is the broad function of MMPs?
Remodeling of the extracellular environment
How are MMPs and ADAMs regulated?
Through transcription / translation, or proteolytic activation
How are MMPs and ADAMs regulated through transcription / translation?
A tight regulation that occurs only under specific conditions
How are MMPs and ADAMs regulated through proteolytic activation?
They are produced as inactive zymogens, that need to be activated by MMPs or other proteases
What does the ubiquitin pathway regulate?
Regulated degradation of cytosolic proteins
Why would a cell want to degrade cytosolic proteins (3 reasons)?
- When the protein is old/damaged/inactive
- When the protein is unable to fold correctly or is damaged
- Protein removal for attenuation of cell signaling
What is the nature of the proteasome pathway?
Rapid and irreversible regulation of cell signaling
What is the purpose of ubiquitin?
Tag proteins for degradation
What is the purpose of the proteasome?
Degrade cytosolic proteins into short peptides
Why must misfolded proteins be removed?
Their accumulation and aggregation can be toxic to the cell, so they must be removed
What considerations (2) need to be considered when degrading proteins?
- Need to avoid potential dangers of unrestrained proteolytic activity
- Sites of proteolysis must be confined/sequestered from other components of the cytosol
What do lysosomes do?
Mediate the degradation of proteins derived from endocytosis
How does a lysosome shield proteolysis from the cytosol?
Through a lipid membrane
What is the structure of the proteasome?
A large multi-subunit protein structure, cylindrical in shape
What manmade object is similar to the proteasome?
A paper shredder
How is a paper shredder similar to the proteasome (2 ways)?
- It has a clear direction
2. Closed box (well protected)
What is meant by the proteasome having a “clear direction”?
Proteins go in one way, and amino acids go out the other way (specified direction)
What is meant by the proteasome being a “closed box”?
The proteolytic environment is well protected from the outside environment
What does the cap protein of the proteasome do?
Recognize the protein for degradation
What does the core protein of the proteasome do?
Performs proteolysis
What does the regulatory protein of the proteasome do?
Releases the amino acids
What is the sedimentation of the entire proteasome?
26S
What is the sedimentation of the cap and regulatory proteins in the proteasome?
19S
What is the sedimentation of the core protein in the proteasome?
20S
How does the core protein of the proteasome mediate proteolysis?
It is lined by many proteases
How do proteins enter the proteasome?
By binding to the lid structure
In what state does the protein enter the proteasome?
As a linear chain
How are proteins misfolded when entering the proteasome?
They are threaded as a linear chain into the proteasome
What is needed for the protein to be threaded into the proteasome?
ATP
What is the purpose of ATP in the proteasome?
Misfold the protein and thread it into the proteasome
How does a cell mark a protein for degradation?
By addition of unbiquitin
What is ubiquitin?
A small molecule used as a tag for protein degradation
What mediates the binding of ubiquitin to a protein / other ubiquitins?
Lysine linked connections
What does the cap protein recognize to allow a protein to go into the proteasome?
A polyubiquitin tag
What class of proteins add ubiquitin to proteins?
Cellular ubiquitin ligases
Which proteins conjugate ubiquitin to a molecule?
E1, E2, and E3
What is E1?
A ubiquitin activating enzyme
What does E1 do?
Activates ubiquitin to create a high energy intermediate (E1-S-ubiquitin)
What type of protein is the high energy intermediate after E1?
A thiol ester (E1-S-ubiquitin)
What is needed for E1 to create the thiol ester?
ATP
What is E2?
Ubiquitin-carrier proteins, or UBCs
What does UBCs stand for?
Ubiquitin-Conjugating enzymes
What is another name for UBC?
E2
What does E2 do?
Transfers the ubiquitin from E1 to the target protein
What is E3?
A ubiquitin-protein ligase
What does E3 do?
Binds to E2 and the target protein to facilitate transfer of ubiquitin
Which parts of the ubiquitin conjugation pathway are general to many proteins?
E1 and E2
Which parts of the ubiquitin conjugation pathway are specific to one particular protein?
E3
For each turn of the ubiquitin conjugation pathway, how many ubiquitins are added to the protein?
One
What processes does E3 catalyze?
Transfer of ubiquitin from E2 to the target protein, and covalent attachment of ubiquitin to the target protein
What does DUB stand for?
Deubiquitinating enzymes
What do DUBs do?
Remove ubiquitin from polyubiquitin chains
What does Smurf1 stand for?
Smad ubiquitin regulatory factor 1
What is Smurf1?
An E3 ligase in neuronal polarization
What protein phosphorylated Smurf1?
PKA
What does phosphorylation of Smurf1 do (generally)?
Changes its affinity for its substrates
What do the two substrates of Smurf1 do?
They have opposing effects on axon formation
What is the effect on its substrates when Smurf1 is phosphorylated?
Reduced degradation of PAR-6, and increased degradation of RhoA
What does PAR-6 do?
It is a scaffold protein that helps mediate axon formation
What does RhoA do?
It is a growth-inhibiting small GTPase that inhibits axon formation
What are the downstream targets of Smurf1?
RhoA and PAR-6
Which substrate of Smurf1 promotes axon formation?
PAR-6
Which substrate of Smurf1 inhibits axon formation?
RhoA
When Smurf1 is phosphorylated, what is the net result?
Promotion of axon formation
