4 - Protein Misfolding and Disease

Question 1

How can protein folding be described?

Answer 1

Protein folding is a stochastic search for the correct fold on a biased energy landscape.

Question 2

How is the equilibreum between folded and unfolded proteins maintained?

Answer 2

Molecular Chaperones

Other proteostatic control mechanisms such as quality control, proteosomes

Question 3

What can misfolding lead to?

Answer 3

Misfolding can lead to improper trafficking of the protein, such as occurs in emphysema, or produce toxic folds that lead to amyloid fibrils, or the loss of the population of the working protein because the misfolded ones are being degraded.

Question 4

What is UPR?

Answer 4

The unfolded protein response

Question 5

How is UPR activated

Answer 5

Cells recognise an imbalance in native state formation, the abundance of misfolded proteins. This leads to the activation of the UPR.

Question 6

Describe the four main actions of the UPR, often held in parallel

Answer 6

• Increased synthesis of chaperones
• Decreased protein translation
• Activation of the 26S proteosome pathway
• Induction of apoptosis through caspase activation

Question 7

What is the main aim of induction of apoptosis in UPR?

Answer 7

To prevent toxic productions of misfolded proteins from affecting other cells

Question 8

What is the main aim of decreased protein synthesis in UPR?

Answer 8

Two-fold:

1. Decrease number of misfolded proteins produced
2. Prevents wasting of energy, as stress causing misfolding clearly still present

Question 9

What is the aim of activating the 26S proteosome pathway in UPR?

Answer 9

To increase degradation of misfolded proteins, which are typically toxic and harmful to the cell.
Additionally misfolded proteins can often induce misfolding in other proteins, due to exposure of hydrophobic residues which will interact with nascent chain emerging from ribosome - prion response.

Question 10

What protein is associated with Cataracts?

Answer 10

Alpha B crystallin

Question 11

What protein is associated with Cystic Fibrosis?

Answer 11

CFTR - mutant ion channel

Question 12

What protein are associated with Emphysema and Liver Cirrhosis?

Answer 12

Antitrypsin

Question 13

What protein is associated with Cancer?

Answer 13

p53

Question 14

What protein is associated with Type II diabetes?

Answer 14

Amylin

Question 15

What protein is associated with coagulation and artherosclerosis?

Answer 15

Antithrombin

Question 16

What protein is associated with Parkinson’s, Alzheimer’s and ALS>

Answer 16

Alpha Synuclein
Beta Amyloid and Tau
SOD, TDP-43

Question 17

What protein is associated with Systemic Amyloidosis?

Answer 17

Transthyretin
Lysozyme
Β2M
Prion diseases (Kuru, BSE and Prp^c)

Question 18

What treatments have been proposed for disease caused by protein aggregation?

Answer 18

1. Augmentation Therapy
2. Gene Therapy
3. UPR targeting

Question 19

What would augmentation therapy entail?

Answer 19

Restoration of the active population of protein, via means of misfolded clearance such as treatment of antitrypsin

Question 20

What would Gene therapy as a treatment entail?

Answer 20

If mutant variant present which only produces misfolded protein can use virus encourage WT expression of normal variant, such as CF trials

Question 21

How can the probability of misfolding be predicted?

Answer 21

The probability of a protein to aggregate, or the aggregation rate of a protein can be predicted mathematically based on sequence dependent and environment dependent factors, including hydrophobicity, charge distribution, formation of various secondary structures or the pH, ionic strength and protein concentration of the solution.

Question 22

Describe Systemic Amyloidosis.

Answer 22

Family of disease characterised by the production of amyloid fibrils which can be made by any common protein that forms a soluble precursor which holds the capacity to aggregate.

Question 23

How many proteins are known to form a fibrous structure?

Answer 23

20, thought to aggregate following conformation change under acidic conditions, such as in the lysosome or endosome. Another theory proposes they form through failure in quality control mechanisms, such as proteosomes or macrophage function.

Question 24

What two categories describe the types of amyloid fibrils?

Answer 24

Proteins that form amyloid fibril can be sorted into two categories: normal proteins that have an inherent tendency to fold improperly and mutant proteins whose mutation makes them prone to misfolding. These mutations commonly just worsen the inherent misfolding ability, causing early onset.

Question 25

Describe the fibril structure.

Answer 25

These are usually made of several wound protofilaments that can bunch together into varying degrees of twisting structure. The protofilaments have common structure comprising short 10nm wide beta-sheets called the cross-β-structure. The antiparallel strands run perpendicular to the axis of the fibril.
SH3, acylphosphatase and myoglobin can all form fibrils like this.

Question 26

How do amyloid fibres produce toxic characteristics?

Answer 26

Intracellularly - disrupt intracellular function, cytotoxicity may be caused by formation or breakdown of products that lyse the cell.
Extracellularly - once cell death has occurred the aggregates can accumulate in extracellular space, could rupture membranes/bind and stimulate apoptosis

Question 27

How can amyloidosis be diagnosed?

Answer 27

Through imaging using immunohistochemical staining is far more common, as is use of stains such as congo red, which stains the amyloid fibrils pink. Observation of this under a polarised microscope allows visualisation of the fibrils through their green birefringence.

Question 28

How does alpha-1 antitrypsin misfolding lead to disease?

Answer 28

The loss of the function of the serpin means that protease it inhibits (elastase) causes damage to the lungs leading to emphysema. The aggregates themselves also can become trapped within liver cells causing liver disease. This is thought to be caused by cotranslational misfolding.

Question 29

Why does the serpin alpha-1 antitrypsin misfold?

Answer 29

Because of the formation of an alternative structure that flips out the reactive centre loop by 80° at one end of the protein, which can interact with the other end of another antitrypsin by inserting the loop into a beta sheet structure leading to polymerisation. This prevents proper activity of the antitrypsin. Insertion of the reaction loop into an active antitrypsin can cause it to favour the inactive state as well.

Question 30

What is CFTR?

Answer 30

CFTR - Cystic Fibrosis Transmembrane Conductance Receptors, which are an ABC ion transporter protein responsible for pumping chloride ions across the epithelial membranes found in lung tissue, gut, vagina - any mucousal secreting surface.

Question 31

Describe CFTR protein

Answer 31

It is a 1480 amino acid proteins consisting of five domains, two cytoplasmic ATP binding domains (NBD1 and 2), two transmembrane domains (MSD1 and 2) each with 6 membrane-spanning helices and an R domain bound to the cytoplasmic side of one of the transmembrane domains.

Question 32

What stimulates the CFTR?

Answer 32

Phosphorylation of the R domain by PKA (due to the cAMP response) allows ATP binding and hence stimulates the channel.

Question 33

What is the genetic cause of Cystic Fibrosis?

Answer 33

Point mutation of Phenylalanine-508. 1 in 30 carriers, most abundant genetic disease in Europe in terms of carriers, originally thought to hold evolutionary selectivity over cholera, mucus build up prevents water loss.

Question 34

What genes have been found to be involved in AD?

Answer 34

Those involved in the metabolism of transmembrane β-amyloid precursor protein called APP, due to position on chromosome 21 those with Down’s syndrome often develop early onset. Other causes can be mutations in the presenilins, two transmembrane proteins involved in the secretory pathway, and disturbances to the allele for apolipoprotein E, a lipid transporter.

Question 35

If I say CAG you say…

Answer 35

36 and above polyglutamine repeats in the huntingtin gene exome 1 leads to huntington’s disease (N terminal)

Question 36

What is a possible treatment for HTT?

Answer 36

Over expression of Hsp70 or removal of the pro-apoptic gene

Question 37

What neurodegenerative disease is considered infectious?

Answer 37

Prion disease; upon binding they causes other host proteins of the same type, spreading the misfold in an exponential manner.

Question 38

What is the genetic cause of lysozyme amyloidosis?

Answer 38

A result of one of two point mutations in the gene, either in Ile56 to Thr or Asp67 to His, which causes only a slight perturbation but makes a partially unfolded state that can aggregate readily accessible leading to amyloid fibril formation.

Question 39

How are IDP structures determined?

Answer 39

Characterised by high net charge and low hydrophobicity, however the lack of fully folded secondary structure does not preclude a global architecture, and IDPs are often found to gain structure upon association with a ligand or binding partner. However, their lack of folding does mean that their structures cannot be determines by conventional X-ray crystallography or NMR methods.