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ATU2 > Protein Folding and Disease > Flashcards

Protein Folding and Disease Flashcards

1
Q

What does biological folding require?

A

Elaborate machinery and energy input

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2
Q

Give examples of bond formation/isomerisation that is too slow to support life unaided.

A

Spontaneous peptidyl-propyl amide bond isomerisation

Spontaneous disulphide bind formation

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3
Q

In the cell what is there a constant competition between?

A

Folding, misfolding and aggregation

- Always in equilibrium to the unfolded polypeptide

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4
Q

What does a folding funnel show?

A

The 3D energy landscape

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5
Q

Where does aggregation occur from?

A

A partially folded trapped state
OR
Intrinsically unfold proteins - aggregation occurs from the unfolded state

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6
Q

What are the to environments in which proteins commence folding?

A

Cytoplasm

Secretory pathway

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7
Q

What did Anfinsen teach us?

A

Refolding is spontaneous at low protein concentration and temperature in vivo

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8
Q

What do chaperones and folding enzymes allow?

A

Multiple attempts at proper folding

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9
Q

What are the three outcomes for misfiling?

A

Degradation - Gauchers disease
Improper trafficking - CF
Toxic conformer - FAP, Lysozyme amyloidosis

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10
Q

In the disease lysozyme amyloidosis what is the protein called and what is the precursor?

A

Lysozyme

mixed alpha beta fold

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11
Q

In FAP what is the protein called and what is the precursor?

A

Transthyretin, all beta

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12
Q

Give examples of functional amyloid

A

Melanin made on pMel17

Secretory hormones - small peptides

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13
Q

Define the characteristics of amyloid aggregates

A

Thread like amyloid fibrils about 10nm in diameter and rich in beta sheet structure

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14
Q

What are the common properties of amyloid fibrils?

A

Fibre diffraction - cross beta structure
Negative Stain EM
Congo red birefringence - the dye gets ordered
Nucleated growth

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15
Q

What are the examples that amyloid like fibrils can be made from?

A

Polylysine
Polyglutamate
Polythreonine
and myoglobin (when under the right conditions)

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16
Q

What did cyro-EM studies of amyloid fibrils show?

A

One form of SH3 fibrils is a hollow tube only about 10nm in diameter but several microns in length
Built of four protofilaments each with cross beta structure

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17
Q

What are amyloid fibrils composed of?

A

Different numbers of inter twined protofilaments

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18
Q

Define the characteristics of human lysozyme

A 
Small 130 amino acids 
4 disulphide bonds
Enzyme glycosidase
Soluble, globular protein
Mixed alpha beta fold 
X-Ray and NMR structure available
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19
Q

What are the mutations of human lysozyme that form amyloid in vivo?

A

I56T and D67H

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20
Q

What did the X-ray structures of human lysozyme tell us?

A

Amyloidogenic variants fold to a native like structure and are catalytically active - highlighting that this is not a folding disease - have the same X-ray structures

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21
Q

Which features imbue the amyloidogenic character of these lysozyme variants?

A

Lysozyme variants are less stable than wild type and more aggregation prone

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22
Q

What are the common properties of amyloidgoenic lysozyme variants?

A
  1. Correctly folded and catalytically active
  2. Less stable
  3. Aggregation prone
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23
Q

Does the reduction in stability explain their ability to form amyloid?

A

No, other variants are found that are equally destabilised yet are not involved in disease

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24
Q

How does the D67H variant of lysozyme exchange with solvent?

A

Deuterium/hydrogen exchange

- Exchanges cooperatively - transient unfolding of the entire beta domain

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25
Q

What does soft ionisation allow?

A

Proteins can be introduced into the gas phase whilst maintaining their integrity

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26
Q

How does the D67H exchange with the solvent?

A

Transient unfolding one the entire beta domain

Cooperatively and concurrently

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27
Q

Give brief details about Jeff Kelly

A

Professor of Chemistry
Molecule to Market
Published 353 papers with a h=73
Discovered tafamidis

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28
Q

What type of disease are the transthyretin amyloidosis?

A

In trans gain of proteotoxcity

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29
Q

What type of tissue does TTR aggregation destroy?

A

post-mitotic tissue

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30
Q

What are the two diseases associated with TTR?

A

SSA - senile systemic amyloidosis

FAP - familial amyloid polyneuropathy and cardiomyopathy

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31
Q

What protein is implicated in SSA?

A

Wild type

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32
Q

What [rotein is implicated in FAP?

A

mutant and wt TTR

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33
Q

What is the age of onset for SSA?

A

Greater than 60

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34
Q

What is the age of onset for FAP?

A

15-60

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35
Q

Describe TTR

A

127 amino acids
Beta sheet rich
55kDa homotetramer
Present in serum and cerebral spinal fluid

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36
Q

What does TTR stand for?

A

Transport Thyroxine Retinol binding protein

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37
Q

What does TTR not do in humans?

A

Thyroxine carrier function is not used - negative cooperativity whereas there is thyroxine binding protein that has much higher affinity 6x10^9

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38
Q

How many mutations are known to give rise to transthyretin amyloidosis?

A

Over 50

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39
Q

What is believed to be the cause of disease?

A

Tetramer dissociates - monomers misfiled and form oligomers then protofibrils then fibrils
Native monomer is not amyloidogenic

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40
Q

Where is the weak point in TTR?

A

Along the 2 fold axis - dissociates into two dimers here

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41
Q

When was the proof of principle that kinetic stabilisation of transthyretin prevents fibril formation?

A

1996

- 3 equivalents of small molecule inhibits TTR aggregation

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42
Q

What is the most conservative approach?

A

Do not presuppose what the toxic species is

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43
Q

What is the highly penetrant version of FAP in Portugal?

A

V30M FAP

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44
Q

What did the family who had V30M also have that meant they did not develop FAP?

A

T119M

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45
Q

What does the T119M mutation seem to do?

A

Protect against V30M amyloidogenesis in trans through mixed tetramer formation

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46
Q

What does the T119M subunit incorporation do?

A

Increases the dissociative kinetic barrier preventing amyloidogenesis and amyloidosis

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47
Q

What does the addition of small molecules do?

A

Activation barrier tuning with small molecules mediated by native state kinetic stabilisation
- Prevent amyloidogenesis by kinetic stabilisation of the native state

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48
Q

At what pH and time did two equivalents of small molecule inhibit TTR aggregation?

A

Titrated in different concentrations of thyroxine relative to TTR
pH 5
72 hours

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49
Q

What are the properties required for a TTR amyloid drug?

A

Bind tightly to TTR with negative cooperativity
Bind one or more binding sites
Bind with high selectivity to TTR in plasma
Does not interfere with thyroid hormone receptor

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50
Q

What is the efficacy score for TTR amyloid drug?

A

A combination of binding affinity for TTR and binding selectivity

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51
Q

What small molecule can be used with TTR?

A

A small analogue of thyroxine - TTR can bind thyroxine but this function is not used in vivo

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52
Q

What did the attachment of the cysteine to TTR do?

A

Made a heterotetramer and a place to attach an extra moiety

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53
Q

What was discovered about the linker?

A

The longer the linker the more inhibition - needs to be able to bind round to the AS
Otherwise the biaromatic moiety can’t bind round and fit in
Don’t need to work against negative cooperativity

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54
Q

What type of kinetic stabilisers are excellent for TTR amyloidogenesis inhibitors?

A

Benzoxazole based TTR kinetic stabilisers

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55
Q

What drug was designed with air of X-Ray crystallography?

A

Tafamidis

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56
Q

With tafamidis what neurological examination changes?

A

Sensation, muscle strength and lower limn reflexes change from baseline

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57
Q

What else does tafamidis improve?

A

Large fibre function, five nerve conduction measurements, vibratory threshold, heart rate and response to deep breathing

Improved cachexia and autonomic neuropathy

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58
Q

What statistical significance was found in all endpoints?

A

Improved small nerve fibre function
Improved large nerve fibre function
Reversal in slope of modified body mass index
Improvement in lower extremity neurological exam

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59
Q

What is the proteostasis network?

A

Compilation of integrated biological pathways that influence the proteome and its function from birth to death - protein synthesis, folding, trafficking and degradation
Maintenance of the proteome

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60
Q

Are the proteins involved in maintaining the proteasome conserved?

A

Yes from yeast to fungi to man

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61
Q

Where are chaperone regulators increased greatly?

A

In higher order eukaryotes

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62
Q

How many genes do eukaryotes have to monitor the proteome?

A

Around 600

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63
Q

What are the various types of molecular chaperones and folding enzymes?

A 
Hsp70 and Hsp40
Hsp90
sHSPs
Chp
Pfd
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64
Q

What is Gauchers disease?

A

A genetic disease in which a fatty substance accumulates in cells and certain organs

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65
Q

What type of disease is Gauchers the most common?

A

Lysosomal storage diseases

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66
Q

What is Gauchers disease characterised by?

A

Bruising, low platelets, enlargement of the liver and spleen, fatigue and anaemia

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67
Q

What enzyme is involved in Gauchers disease?

A

Hereditary deficiency in the enzyme glucosylceramidase

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68
Q

What does the enzyme do?

A

Acts on the fatty acid glucysoylceramide - when the enzyme is absent glucosylceramide accumulates particularly in white blood cells, most often macrophages

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69
Q

What is the threshold value for lysosomal GC enzymatic activity?

A

10%

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70
Q

At what percentage do you have Gauchers disease?

A

8%
Hence why this is a good disease to try and treat - not trying to get 100% of the enzyme folded, just need to increase by 2%

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71
Q

What does GC convert glucosylceramide into?

A

Glucose and ceramide

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72
Q

What happens when the mutation N370S is incorporated into GC?

A

Would be stable in the lysosome, however the large pH difference between the lysosome (5) and ER (7.4) means that the protein is not stable in the ER hence degraded ERAD

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73
Q

What did immunofluorescence show?

A

Wildtype co-localises to the lysosome (LAMP4)

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74
Q

What mutation in GC showed trafficking at a permissive temperature?

A

L444P

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75
Q

What happened to this mutation?

A

At 30 degrees the protein can fold and traffic to the lysosome
At 37 degrees the protein can not make it to the lysosome

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76
Q

What are proteostasis regulators?

A

Small molecules or biologicals that control the concentration, conformation, quaternary structure and location of proteins comprising the proteome by manipulating the proteostasis network, often by influencing the signaling pathways that control the proteostasis network

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77
Q

What were the two proteostasis regulators that increased L444P GC activity in patient-derived fibroblasts?

A

Celastrol and MG-132

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78
Q

What concentration of MG-132 increased L44P activity in patient derived fibroblasts?

A

Around 0.8 microM

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79
Q

What sugar side chain is sensitive to endo H?

A

Mannose rich glycans in the ER

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80
Q

What sugar side chain is resistant to EndoH?

A

Complex glycan in the Golgi

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81
Q

What did EndoH analysis of L444P show?

A

MG-132 allows its trafficking to the Golgi

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82
Q

What did immunofluorescence with proteostasis regulators show?

A

They enhance proper GC trafficking to the lysosome as discerned by immunofluorescence

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83
Q

What is the mechanism of protein homeostasis regulators NOT revealed by?

A

Known pharmacology
Celasrtrol - heat shock response activation of the proteostasis network do not explain
MG-132 - proteasome inhibition does not explain

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84
Q

What does the Unfolded Protein Response do?

A

Remodels the ER proteostasis network through three branches

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85
Q

What are the three branches of the UPR?

A

ATF6
PERK
IRE1
- BiP binds to these proteins and switches them off - when they are activated BiP dissociates off to help the protein folding chaperone activity

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86
Q

Where does IRE1 sit?

A

ER membrane

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87
Q

What happens when BiP dissociates from IRE1?

A

Dimerise and trans-phosphorylate - become active

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88
Q

What does IRE1 then do?

A

Alter a type of mRNA splicing within the cytoplasm - XBP1 - normally an intron however splicing by IRE1 switches XBP1 on

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89
Q

What is XBP1 when active?

A

A TF - turns on transcription and translation of chaperone, lipid synthesis components and ERAD proteins

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90
Q

Does MG-132 and Celastrol affect IRE1?

A

Yes - look at XBP1 splicing

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91
Q

What does the AFT6 arm of the UPR do?

A

The N-terminal section of AFT6 becomes a TF - leads to the induction of ER resident proteins

92
Q

DO MG-132 and celastrol affect the AFT6 arm of UPR?

A

Yes - induce the cleavage of ATF6 indicating the activation

93
Q

What does the PERK arm of the UPR do?

A

ER membrane kinase - dimerises and phosphorylates when BiP dissociates - phosphorylates eIF2 that then causes selective translation of GCN4/ATF4 and increase in CHOP

94
Q

Does MG-132 and celastrol effect the PERK arm of the UPR?

A

Yes - CHOP expression levels increase

95
Q

What are UPR activators?

A

Proteostasis regulators that re-sculpt the folding free energy landscape through up regulation of the proteostasis network components including chaperones that bind to folding intermediates and enzymes that lower transition states

96
Q

What does the addition of a small molecule do?

A

Upregulates UPR - through PERK, ATF6 and IRE1

97
Q

What role does MG-132 have in Tay-Sachs disease?

A

Also facilities this enzyme

98
Q

What do proteostasis regulators do?

A

Expand the minimal export threshold

99
Q

What do small molecule do when they bind to enzymes?

A

Stabilise the folded state of the mutant enzyme in the ER - increasing its concentration and enabling its trafficking from the ER

100
Q

What does NN-DNJ do?

A

At sub-inhibitory concentrations enhance the trafficking of GC to lysosomes

  • 2-fold increase in activity of N370S (most common GD mutation)
  • Increased activity lasts for 6 days
  • Ligand binding stabilises the native state
101
Q

How can NN-DNJ stabilise but inhibits?

A

The affinity for the substrate much outweighs the affinity for the inhibitor - once in the lysosome will bind the substrate - just gets it out of the ER

102
Q

What fraction of the proteome is synthesised by ribosomes docked on the ER?

A

⅓

103
Q

What type of proteins fold in the ER before being trafficked?

A 
Secreted proteins
PM proteins
ER
Golgi
Eadosomal and lysosomal proteins
104
Q

What happens to proteins that fail to fold in the ER?

A

ERAD

105
Q

Describe the order of the secretory pathway

A

ER - cis-Golgi - medial-Golgi - trans-Golgi - trans-Golgi network

106
Q

What does the signal recognition particle do?

A

Recognises signal sequences (typically N-terminal)
pauses translation effectively preventing the protein from folding in the cytosol
Delivers the ribosome and nascent chain to the ER membrane by interaction with SRPR

107
Q

What is the SRP?

A

Cytosolic ribonucleoportein complex signal recognition particle

108
Q

When is the SRP displaced?

A

Once the ribosome binds to the SEC61 translocon - translation resumes

109
Q

What is the SEC61 translocon?

A

An aqueous pore across the ER membrane - opened when proteins translocate across the membrane
Translocated whilst translated

110
Q

Describe BiP

A

Binding Immunoglobulin protein

  • ER luminal hsp70 chaperone
  • Roles in protein translocation, folding and degradation
111
Q

What does BiP do?

A

Binds to proteins as they are inserted into the ER and retains the proteins in the ER

Also has a major role in the assembly of proteins in the ER, particularly non-glycosylated proteins

112
Q

How can BiP bind to unfolded regions of proteins?

A

ATP hydrolysis

Exchange of ATP for ADP causes BiP to dissociate and provides an opportunity

113
Q

What kind of environment is the ER?

A

Oxidising - favours the formation of disulphide bonds

114
Q

What is the protein that promotes the correct pairing of disulphide bonds between cysteine?

A

Protein Disulphide isomerase

115
Q

What in effect does PDI do?

A

Make (oxidise), break (reduce) and re-arrange (isomerise) disulphide bonds

116
Q

What chain is used in N-linked glycosylation?

A

Pre-formed oligosaccharide chain

117
Q

What is the pre-formed oligosaccharide chain transferred from and to?

A

From the dolichol lipid precursor to asparagine by Oligo-saccharyltransferase

118
Q

What is the pre-formed oligosaccharide chain composed of?

A

9 mannose
3 terminal glucose
2 N-acetylglucosamines

119
Q

What is the consensus sequence for N-linked glycosylation?

A

Asn-X-Thr/Ser

X = anything but proline

120
Q

What roles does glycosylation have?

A
  • Improves protein solubility
  • Provides binding sites for calnexin and calreticulin facilitating integration with PDI
  • Is used to monitor protein folding
121
Q

How is protein folding monitored with glycosylation?

A
  • After addition, two terminal glucose are trimmed by glucosidase I and II
  • This provides a binding site for calreticulin and calnexin that retain the proteins in the ER and prevent aggregation, promote folding by binding to PDI (ERp57)
    The final glucose is trimmed by glucosidase II
    If folding is complete the protein can exit the ER - if incomplete a glucose residue can be added by UDP-Glucose glycoprotein glycosyl-transferase and the cycle is repeated
122
Q

What does removal of a mannose mean?

A

Acts as a molecular clock that monitors protein folding

123
Q

What catalyses the removal of mannose? What else does this do?

A

Mannosidases - slow removal and reduce the addition of glucose by UGT

124
Q

What happens is sufficient mannose residues are removed?

A

The protein is diverted from the folding pathway to a degradation pathway

125
Q

What percentage of nascent proteins fold correctly in the ER?

A

> 95%

126
Q

What recognises misfiled proteins?

A

Adaptors in the ER lumen

127
Q

What happens to misfiled proteins?

A

Retro-translocated into the cytosol

128
Q

What is ERAD a branch of?

A

UPS

129
Q

Where do the adaptors deliver the misfolded proteins to?

A

E3 ubiquitin ligases - HRD1 etc

130
Q

What is SEL1L?

A

A ER membrane protein that recognised misfolded proteins

131
Q

What can SEL1L recruit?

A

Additional adaptors, the lectins XTP3-B and OS9 which recognise mannose trimmed substrates

132
Q

Where does ubiquination occur?

A

Cytosolic face of the ER membrane

133
Q

For integral membrane proteins where can ubiquitination take place?

A

Cytoplasmic regions prior to translocation

134
Q

What do soluble proteins require?

A

At least partial translocation prior to ubiquitination

135
Q

What are derlins?

A

Membrane proteins which promote luminal protein transport across the membrane that resemble membrane rhomboid proteases

136
Q

What is the ubiquinated substrate recognised by?

A

Cytosolic AAA ATPase p97/Cdc48

137
Q

What does p97/Cdc48 do?

A

Uses ATP hydrolysis to energise the extraction of the protein from the ER

138
Q

How might the proteasome extract some protein substrates from the ER membrane?

A

Via the AAA ATPase in the 19S cap

139
Q

What do the compensatory mechanisms induced by UPR signals do?

A
  • Reduce bulk protein synthesis
  • Promote the production of chaperones
  • Increase ERAD
  • Increase the amount of the ER
140
Q

What does the N terminus of AFT6 induce?

A

Expression of proteins involved in ER folding:

  • BiP
  • GRP94
  • PDI
141
Q

What does PERK do?

A

Phosphorylates the elongation factor eIF2 inhibiting mRNA translation

142
Q

What is IRE1?

A

An ER transmembrane protein with endoribonuclease activities

143
Q

How does IRE1 directly associate with misfolded proteins?

A

Via its ER luminal domain promoting oligomerisation

144
Q

What does XBP1 promote?

A

Expression of genes that regulate lipid biosynthetic enzymes and ERAD components

145
Q

What is XBP1 also required for?

A

Plasma cell differentiation

146
Q

What type of mRNAs does IRE1 degrade?

A

Those of secretory pathways

147
Q

What is CFTR?

A

An ATP gated plasma membrane channel

Transports chloride ions across the plasma membrane

148
Q

Where is CFTR expressed?

A

Epithelial cells

149
Q

What fraction of caucasians carry a gene encoding a mutant form of CFTR?

A

1/27

150
Q

What happens in the absence of CFTR function?

A

Build up of viscous mucous in the lungs - resulting in inflammation and infection - causes lung damage

151
Q

What type of mutation is F508?

A

Autosomal recessive

- 3 nucleotide deletion

152
Q

How is GRASP activated?

A

IRE1-mediated signalling arm activates GRASP dependent secretion via phosphorylation

153
Q

What does GRASP do?

A

Binds to CFTR F508 and target it to the cell membrane allowing it to function as a Cl channel

154
Q

What does transgenic expression of GRASP in mice do?

A

Rescue the phenotype

155
Q

What does human cytomegalovirus do to the ERAD pathway?

A

Hijack it to degrade MHC class I molecules

156
Q

What does HCMV encode?

A

US2 and US11 - ER localised integral membrane proteins that target MHC class I molecules for degradation

157
Q

What do US2 and US11 act as?

A

Virus encoded adaptors for the ERAD pathway - cause MHC molecules to be delivered to E3 ligases

158
Q

What is US2 dependent on?

A

Signal peptide peptidase for MHC class I degradation

159
Q

What is US11 dependent on?

A

Derlin-1 and SEL1L for MHC degradation

160
Q

What secretes cholera toxin?

A

Vibrio Cholerae

161
Q

What does cholera toxin promote?

A

Electrolyte and water movement into the intestinal lumen resulting in severe diarrhoea and further spread of the bacterium

162
Q

What happens in the ER to cholera?

A

The CTA1 subunit dissociates - involves PDI
CTA1 is unfolded - retrotranslocated across the ER membrane
Escapes proteasomal degradation (few lysine)
Binds adenylate cyclase increasing cAMP - in turn activates PKA
PKA phosphorylates CFTR - promoting chloride ion secretion

163
Q

What are the cellular roles for protein degradation?

A
  • Constitutive turnover of proteins
  • Response to starvation
  • Removal of misfolded proteins
  • Regulation of cellular pathways
  • Antigen presentation
164
Q

Where are the two principal sites for degradation?

A

Cytosolic proteasome and the lysosome

165
Q

How are cytosolic proteins degraded?

A

Either the UPS or by autophagy

166
Q

What percentage do lysosomes and proteasome account for degradation?

A

80-90% of cellular protein degradation

167
Q

What percentage of the human genome is dedicated to the UPS system?

A

5%

- Many genes correspond to E3 ubiquitin ligases

168
Q

Describe ubiquitin

A

76 amino acids

Highly conserved in eukaryotes

169
Q

How is ubiquitin linked to proteins?

A

via linkage between glycine 76 of ubiquitin and the amino groups of lysine on the substrate protein

170
Q

What is the typical linkage for polyubiquitinated chains?

A

Lysine48-Glycine76

171
Q

What other lysine can be used in ubiquitination?

A

Lysine63

172
Q

What does E1 do?

A

Forms a thiol ester with the carboxyl group of glycine 76 of ubiquitin

173
Q

What does E2 do?

A

Transiently carriers ubiquitin as a thiol ester

174
Q

What does E3 do?

A

Transfers the ubiquitin to the substrate protein

175
Q

How many E1, E2 and E3 enzymes are there in the body?

A

E1 - 2
E2 - 30
E3 - 600 - confer specificity

176
Q

What is the proteasome formed of?

A

26S complex composed on the 20S core and 19S cap/lid

177
Q

How is entry into the 20S core restricted?

A

Narrow channel - meaning proteins must be linearised and unfolded to enter

178
Q

What are the three principal proteolytic activities of the 20S core?

A

Chemotryptic - hydrophobic
Tryptic - basic
Peptidylglutamylpeptidase - acidic

179
Q

How long are the peptides produced by the proteasome?

A

3-22 amino acids

- Further cleaved by cytosolic amino-peptidases

180
Q

What does the 19S cap recognised?

A

Poly-ubiquitinated proteins

Cleaves ubiquitin from the substrate (recycling)

181
Q

How is unfolding of the substrate protein mediated?

A

By AAA ATPase activity of the 19S cap

182
Q

What are defective ribosomal initiation products?

A

⅓ of newly synthesised proteins that aredegraded and defective
- Inaccurate transcription/translation errors

183
Q

What is one specific class of DRiPs?

A

Produced from mRNAs that lack stop codons

184
Q

What ubiquinates these DRIPS?

A

E3 ubiquitin ligase Ltn1

185
Q

How is Ltn1 stimulated?

A

Stalling of the ribosome at the mRNA poly(A) tail

186
Q

What is Ltn1 part of?

A

SThe ribosome quality control complex - associates with stalled ribosomes via the 60S subunit

187
Q

Where do chaperones bind?

A

Surface exposed hydrophobic patches - present on misfolded proteins but not native proteins - therefore recognise proteins that need degrading

188
Q

What is a key link between chaperones and the UPS?

A

The cytosolic E3 ubiquitin ligase constitutive HSC70 interacting protein (CHIP)

189
Q

What does CHIP do?

A

Bind to chaperones and hence associated with misfolded proteins

190
Q

What does CHIP mediated ubiquitination promote?

A

Interaction of CHIP with the 26S proteasome via the co-chaperone BAG-1

191
Q

Define autophagy

A

A process by which cytoplasmic components are delivered to the lysosome for degradation

192
Q

What are the three classes of autophagy?

A

Macroautophagy
Chaperone mediated autophagy
Microautophagy

193
Q

What is the typical diameter of a lysosome?

A

200-400nm

194
Q

What proteins do lysosomes contain?

A

An array of hydrolases - acidic pH optima

195
Q

What are lysosomal proteases called?

A

Cathespins

196
Q

What is the specificity of cathespins?

A

Broad

Includes endopeptidases and exopeptidase

197
Q

Name three major lysosomal enzymes:

A

Cathespin D - aspartate endopeptidase
Cathespin L - cysteine endopeptidase
Cathespin B - cysteine protease (endo and carboxypeptidase activity)

198
Q

What is macroautophagy?

A

Removal of cytoplasmic components for degradation in lysosomes

199
Q

What can be degraded by macroautophagy?

A

Long lived cytosolic proteins and protein aggregates

200
Q

What is macroautophagy enhanced by?

A

Starvation of cells - hence recycling amino acids for use by the starved cell, and other cellular stresses

201
Q

What are cytoplasmic components surrounded by in macroautophagy?

A

Double membranes

202
Q

What donates this membrane to form the autophagosome?

A

ER, golgi, mitochondria and PM

203
Q

How are autophagosomes transported to lysosomes?

A

Along microtubules to the MTOC

204
Q

What is autophagosomes fusion with lysosomes dependent on?

A

SNARE-dependent

205
Q

What does CMA involve?

A

The direct delivery of proteins to the lysosome and do not involve the formation of membranes bound autophagosomes

206
Q

What does CMA recognise?

A

KFERQ motifs - present in ⅓ of cytosolic proteins

Exposed if misfolded or complex disassembles

207
Q

Why does aggregation preclude degradation by the CMA?

A

Only degrades proteins as single subunits

208
Q

What enhances CMA?

A

Cellular stress including nutrient depravation

209
Q

What exactly recognises KFERQ motifs?

A

Hsc70

210
Q

How are substrate proteins bound?

A

By the cytosolic portion of the lysosome membrane protein LAMP-2A via the KFERQ motif

211
Q

What does substrate binding promote in LAMP2A?

A

Multimerisation

212
Q

What reasons are there to suggest that aggregation of alpha syncline into amyloid is a failure in proteostasis?

A
  • Proteostasis machinery fails to prevent the misfiling of alpha synuclian
  • Aggregates of alpha synuclein are not removed by the protein degradation machinery
  • Mutant forms of alpha synuclein and alpha synuclein aggregates can disrupt protein degradation pathways
213
Q

How is alpha synuclein degraded?

A

Both the UPS and autophagy - CMA may be dominant for monomeric alpha syn

214
Q

What does alpha synuclein possess making key degradation via the CMA?

A

KFERQ like motif

215
Q

Why are A53T and A30P not degraded?

A

Bound by Hsc70 and although deliver to the membrane they do not get to the lumen and impair degradation of CMA

216
Q

What can increased amounts of the wild type alpha san do?

A

Impair CMA

217
Q

What do many cells accumulate in PD?

A

Autophagosomes - failure in clearance

218
Q

What may LBs not be cleared by and inhibit?

A

Not cleared by macroautophagy and inhibit macroautophagy

219
Q

What does rapamycin target?

A

mTOR - hence promotes macroautophagy

220
Q

What does rapamycin regulate?

A

Macroautophagy

221
Q

What does active mTOR suppress?

A

Autophagy

222
Q

What does starvation do?

A

Remove mTOR inhibition promoting autophagy and recycling of amino acids

223
Q

What does rapamycin do to mTOR?

A

Inhibits - promotes macroautophagy

224
Q

What has rapamycin been reported to do?

A

Promote clearance of alpha synuclein and reduce neuronal death in animal models

225
Q

What is TFEB and what does it do?

A

TF

regulates macroautophagy and lysosome biogenesis

226
Q

What does over expression of feb DO?

A

Promotes clearance of alpha syncline in cells

ATU2 (1 decks)

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