Protein Aggregation

Question 1

What are NDD characterised by?

Answer 1

Clinical Symptoms (crossover between AD and PD with dementia)
Pathological features (crossover with Tau pathology in AD and PD)
Loss of specific neuronal populations

Question 2

What do all NDD have in common?

Answer 2

Specific protein aggregation and NDD - often linked to a genetic factor, even if rare.
These genetic mutations that directly link to protein aggregation suggestive of a role in the pathogenesis of the disease.

Question 3

What is the aggregate in AD?

Answer 3

APP and Tau - Genetic mutations in APP - rare

Question 4

What is the aggregate in PD?

Answer 4

LB and LN characterised by ub-asyn - mutations in SNCA - either polymorphisms (risk), amplifications (dosage effect) or missense (effecting folding) - rare

Question 5

What is the aggregate in CJD?

Answer 5

Prions - genetic mutations in Prp - rare

Question 6

What is the aggregate in HD?

Answer 6

Huntingtin - CAG repeats above 34 in Htt gene (95%)

Question 7

What are the characteristics of aggregated proteins?

Answer 7

Increased beta sheet structure, typically a hydrophobic core, which upon aggregation is typically exposed on the surface of the protein, this can encourage interactions with other proteins.
Prion - 3% in soluble form and 43% in aggregate

Question 8

What is the definition of an amyloid fibril?

Answer 8

A waxy tissue deposit that takes the form of starch, amylose and amylopetin

Question 9

Describe the process of protein folding

Answer 9

The folding funnel directs the folding of an unfolded amino acid sequence into a lower free energy conformation, this can take place through multiple pathways; nucleation, hydrophobic collapse. Cells have means of aiding protein folding, or ensuring the “correct” folding by preventing interactions with other proteins during the folding process.
This is through the use of chaperones - chaperones act either co-translationally or post-translationally and are never a part of the final structure. Chaperones are particularly useful in the context of the dynamic microenvironment which is the incredibly crowed

Question 10

Describe quality control systems in place for folding

Answer 10

The use of heat shock proteins, whose expression is upregulated in time of increased heat, a time at which proteins are more likely to misfold or unfold.

Question 11

Describe the role of Hsp70

Answer 11

Co-translational role: HSP 70 family bind to proteins as they are synthesized to prevent inappropriate association with other proteins and allow optimal protein configuration. Hsp90 acts downstream of this chaperone.

Question 12

Describe the role of BiP

Answer 12

Protein found in ER, binds to hydrophobic regions in the internal parts of folding proteins, preventing inappropriate folds and aggregation

Question 13

Describe the role of Hsp60

Answer 13

Creates a caged structure, the inside of which is hydrophilic, with the outer rims being hydrophobic - enticing the protein inwards, allowing it to perform hydrophobic collapse once inside

Question 14

What is the UPS?

Answer 14

Ubiquitin Proteasome System - it is a protein quality control system which targets proteins that are dsyfunctional, misfolded or are in a two high a concentration for degradation - it is a major component of cellular proteostasis.

Question 15

How does the UPS work?

Answer 15

The UPS works through ubiquitination of proteins, which are then targeted for degradation via the 26S proteosome.

1. E3 ubiquitin ligase recognises a protein and ligates a ubiquitin to it
2. This is done through a process of ubiquitin transfer from an E2 ubiquitin conjugating enzyme
3. The subsequent ubiquitins are attached to lysin 48 to generate a polyUb tail
4. The polyUb tail is recognised by the 26S proteosome and degraded

Question 16

Describe the three pathways leading to lysosomes

Answer 16

1. CMA
2. Macroautophagy (mitophagy is a subset)
3. Microautophagy

Question 17

Why is the CNS so dependent on protein degradation pathways and lysosomes in particular?

Answer 17

Unlike other cells, neurones are post-mitotic so the build up of misfolded proteins can not be diluted through a process of cell division.
Additionally the long, yet thin neuronal processes are paritcularly dependent on efficient trafficking of vesicles required for synpatic transmission. The build up of proteins in these processes could alter synaptic transmission.

Question 18

What are the 5 steps of CMA?

Answer 18

1. Recognition - Hsc70 recognises proteins targetted for proteolysis, this sequence is found in asyn (KFERQ)
2. Binding - Binding of Hsc70 and Protein to LAMP2A monomers, this triggers the assembly of a LAMP2A multimer
3. Unfolding - The protein is unfolded
4. Translocation - Hsc70 is thought to pull through
5. Disassociation

Question 19

Why are newly folded proteins at a great risk for misfolding?

Answer 19

Due to the highly hostile environment of the cytosol - the dense concentration of the cytosol, many proteins which could potential interact with it and disrupt contact formation - some particular contacts are required - for example nucleation and

Question 20

What are the models of protein folding?

Answer 20

Nucleation condensation model
Hydrophobic collapse
and formation of distal secodnary structures
–> these all fold along a folding funnel

Question 21

Do only specific proteins form amyloid fibrils?

Answer 21

The SH3 domain of bovine phosphatidyl 3’-kinase forms amyloid fibrils in a low pH - this can demonstrate how environment can act as a huge influence onf the folding

Question 22

Tell me something promiscouis

Answer 22

Well misfolded proteins have a large percentage of exposed hydrophobic side chains, thus they often have the propensity to be promiscious in terms of there interaction, this is to avoid being encapsulated by a sleeve of water, which presents unfavourable interactions to the protein

Question 23

Define a chaperone

Answer 23

A chaperone is a protein that can interact and aid the folding of a polypeptide chain into its native state without being present in it final conformation.

Question 24

Where do E3 ligases transfer their Ubs to and from.

Answer 24

Transferred from E2 Ub conjugating enzyme, to the C terminal glycine on ubiquitin to specific lysine residues on the protein being targetted for degradation

Question 25

Where do Ub’s attach on the polyUb tail?

Answer 25

Lysine 48

Question 26

What is E1?

Answer 26

Ubiquitin activating enzyme. which then transfers Ub to E2

Question 27

What is the CMA sequence?

Answer 27

KFERQ

Question 28

What is PAS?

Answer 28

Phagophore assembly site

Question 29

What triggers macroautophagy?

Answer 29

mTOR suppression (nutrient starvation). This phosphorylates ULK1, which triggers phagofore formation via translocation of the beclin-1-phosphoinositide 3-kinase complex

Question 30

What happens to PARK1 in normal mito?

Answer 30

It is localised to the IMM and cleaved by PARL

Question 31

What are the steps of mitophagy?

Answer 31

1. PARK1 translocates to OMM and phosphorylates Ub in cytosol
2. This recruits parkin which is phosphorylated
3. Various mitochondrial substrates are ubiquitinated by parkin including Mfn
4. P62 associates with ub mitochondria and fuses wiht LC3 PE on the phagophore

Question 32

What does PE stand for?

Answer 32

Phosphatidylethanolamine