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Cell Biology > Lecture 10 - Protein degradation including Autophagy > Flashcards

Lecture 10 - Protein degradation including Autophagy Flashcards

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1
Q

What three kinds of proteins are degraded by cells?

A

Incomplete or mis-sense proteins (ER or cytosol)
Post-synthetic damage proteins (30% of newly made protein)
Unwanted proteins

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2
Q

Give examples of where incomplete or mis-sense proteins might originate from

A
  • cellular errors (incorperation of innaccurate amino acids or analogues)
  • proteins with disruptive mutations
  • products of premature termination
  • products of proteolytic cleavage
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3
Q

Give examples of where post-synthetic damaged proteins might originate from

A
  • mis-folded proteins
  • protein aging e.g. oxygen free radical damage
  • Denaturation e.g heat shock
  • UV damage
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4
Q

Give examples of where unwanted proteins might originate from

A
  • inactive or used proteins (acute regulation of signalling)
  • free subunits of multimeric complexes
  • excess proteins
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5
Q

What are the three intracellular protein degradation routes?

A 
Soluble proteins (from cytosol or ER lumen) - targeted to the proteosome
Soluble proteins/organelles/pathogens - target to the lysosome (and autophagy)
Membrane proteins - targeted to the Lysosome (e.g. via receptor mediated endocytosis)
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6
Q

How could autphagy be exploited for medicience?

A
  • if activated could get rid of protein plaques

- alzheimers/parkinsons

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7
Q

What proteins give the degradation ability to lysosomes?

A

hydrolases

  • 63
  • degrade DNA, RNA, lipid, protein, carbohydrate
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8
Q

What is ubiquitin tagging for?

A

to target proteins/organelles etc to

  • proteosome
  • endosome
  • lysosome
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9
Q

What chemicals inhibit Endocytosis?

A
  • Wortmannin

- Bafilomycin

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10
Q

What chemical inhibit Autophagy?

A
  • Wortmannin

- Bafilomycin

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11
Q

What chemical inhibits the proteosome?

A
  • Epoxomicin

- Lactacystin

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12
Q

What are the features of ubiquitin?

A

76 aa polypeptide
labels proteins
hydrophobic globular core
attaches to lysine side chains of proteins (which terminate in an amino group) via C-terminal end
Added on with ubiquitin ligase E3 working in conjunction with 2 ubiquitin conjugating enzymes (E2, E1)

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13
Q

What is the process of ubiquitination?

A
  1. Target protein emits a degradation signal, (the exposing of an E- amino group on the lysine side chain
  2. Ubiquitin-activating enzyme E1 uses ATP (to AMP) to activate ubiquitin for conjugation then transfers it to an E2 enzyme
  3. The E2 enzyme interacts with a specific E3 partner = ubiquitin ligase primed with ubiquitin, and tranfers ubiquitin to the taret protein
  4. process can be repeated many times as ubiquitin it self can be ubiquitinated, creating a polyubuitquitin side chain which targets the protein for degradation
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14
Q

How does ubuiquitination determine the fate of the target protein for degradation?

A

the shape/structure of the ubiquitin side chain determines the fate of the protein

  • depending on which of ubiquitins 8 lysine residues are ubiquitinated
  • ‘molecular tag’
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15
Q

How many lysine residues does ubiquitin have?

A

8

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16
Q

What happens if one of the 63 hydrolases in a lysosome are missing?>

A

Lysosomal storage disorder

  • the substrate required for the missing hydrolase accumulates in the lysosomes because of ineffective digestion
  • swollen lysosomes
  • e.g. Tasachs
  • often leads to neuronal defects
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17
Q

What are the different types of ubiquitination/ubiquitin structures?

A
  • Homotypic (same lysine residue ubiquitinated)
  • Mixed chain (different lysine residues ubiquitinated then homotypic chains branching)
  • Heterologous (mixtures of ubiquitin and other ubiquitinlike proteins)
  • Multiple monoubiquitination
  • Di-ubiquitin
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18
Q

Where can proteins be targeted to through ubiquitination?

A

1) Proteosome
2) Lysosome
- autophagy
- endocytosis

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19
Q

What proteins bind to ubquitin to target it for degradation and how do they bind?

A

Ubiquitin binding proteins

  • bind to the conformation of a poly ubiquitinated protein ‘molecular tag’ and target it for degradation via proteosome/lysosome
  • specific depending on where they’re targeted to
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20
Q

What types of ubiquitin binding proteins target proteins to the proteosome?

A 
Intrinsic receptors (RPN10, PRN13)
'Shuttling' receptors (RAD23A, UBQLN1, scDdi1)
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21
Q

What are the features of Intrinsic proteasome uqbiquitin binding proteins?

A
  • Ubiquitin binding domains
  • In the proteasome
  • bind the proteasome directly
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22
Q

What are the features of ‘shuttling’ proteasome uqbiquitin binding proteins?

A
  • Ubiquitin binding domain , proteasome binding domains

- target protein to the proteosome then bind to their receptors

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23
Q

What are some examples of Autophagosome ubiquitin binding proteins?

A

p62
NBR1
NDP52

24
Q

Where do Autophagosome uqbiquitin binding proteins bind?

A

-bind to the autosomal membrane

25
Q

What are some types and examples of Endosome ubiquitin binding proteins?

A 
ESCRT-0
-HRS 
-STAM
-GGA3
-TOM1
ESCRT-I
-TSG101
ESCRT-II
-VPS36
26
Q

What molecular tag’s are used to target proteins to the proteosome?

A
  • majority = poly-ubiquitinated (Lys48)
  • traditionally 4XLys48 binding motif = minimal targeting motif
  • potential substrates = Lys11, Lys 63 and monounbiquitin epitopes
  • occasionally proteins can be targeted to the proteosome without degradation
27
Q

What is the structure of the proteosome?

A

-around 50 protein subunits
-over 2000kDa
Eukaryotes - 26S proteasome
-20S α, β (β has proteolytic sites) proteolytic core complex
-1/2 19S regulatory complexes attached to one or both ends of the cylinder shaped core complex
-hexomeric ATPase domain

28
Q

Where are the Proteasome intrinsic receptors found?

A

‘cap’ of the proteosome (19S regulatory particle)

29
Q

What happens when ubiquitinated proteins bind to cap proteins (either intrinsically or via shuttling receptors)?

A

the AAA-ATPase cap unfolds proteins to allow access to ten proteolytic core and hexameric ATPase domain pulls them through the cylinder (requires ATP) and fragments the protein

30
Q

What is the purpose of the AAA-ATPase cap in the proteosome?

A

Unfolds proteins to allow access to the proteolytic core

31
Q

What is the mechanism of the AAA-ATPase cap?

A

Because the 20S particle’s central channel is narrow/gated by the N-terminal tails of the α ring subunits, the substrates must be unfolded

  1. Substrate binds via ubiquitin binding proteins
  2. Cap deubiquitinises the protein and unfolds it. A high amount of ATP causes a conformational change in the AAA-ATPase causing a strained ring structure that pulls on the substrate to direct through the proteolytic core
  3. This is either followed by translocation (rare) and denaturation or (frequent) substrate release
32
Q

Outline an example of proteasome mediated degradation

A

Regulation of p53
p53
-tumour supressor, p53 upregulates the expression of p53 responsive genes
-lots of lysine residues so is readily ubiquitinated
MDMX regulates the transcriptional activity of p53, con sequently levels of p53 can be regulated by regulating the levels of the regulator
MDM2 = ubiquitin ligase
if activated -> ubiquitination of p53 which is transported to the proteasome and degraded
This could be bad as if p53 is removed it can no longer act as a tumour supressor
BUT MDM2 can self ubiquitinated to to self regulate the levels of MDM2
-MDMX inhibits the transcriptional acivity of p53 and can stimulate the ubiquitination of MDM2

33
Q

How can MDMX/MDM2 be targets for cancer therapy drugs?

A

MDMX = inhibits p53 transcription
if ubiquitinated p5 levels may be allowed to rise, more tumour supression
MDM2 = ubiquitin ligase ubiqitinates p53
if ubiquitinated p5 levels may be allowed to rise, more tumour supression

34
Q

How does the human papillona virus cause cellular division and consequently cancer?

A

encodes a E3 ligase that ubiquitinates p53

35
Q

What are some characteristics of lysosomes?

A
  • membrane bound organelle containing lots of hydrolytic enzymes optimally active at an acidic pH
  • around 0.5μm in diameter often with an electron dense core (proteinatious but not a lot of fluid, fusing with a late endosome provides fluid and around 63 hydrolases)
36
Q

What are some functions of lysosomes?

A
  • degradation
  • autophagy
  • phagocytosis
  • disease
37
Q

How do hydrolases end up in the lysosome?

A
  1. As passage through golgi lysosome enzymes are tagged with a mannose-6-phosphate in the golgi lumen
  2. This allows enzymes to bind to the mannose-6-phosphate receptor
  3. Receptor and hydrolases get trafficked out by vesicular transport to early/late endosome
  4. Acidity causes receptor/hydrolase dissociation, the receptor is recyled to the golgi, hydrolase is trafficked to the lysosome by default fluid traffic
38
Q

How can a cell regulate the levels of a protein?

A

Regulate the levels of:

  • transcription
  • degradation
39
Q

What do ESCRT proteins do?

A

Promote membrane invagination, creating vesicles inside endosomes

40
Q

Why is the function of ESCRT proteins necessary?

A
  • ESCRT proteins promote invagination within the lysosome
  • hydrolases can only degrade the proteins in the lumen and so membrane proteins will only be partially degraded when the lysosome fuses with the limiting membrane of the late endosome e.g. LDL receptor
  • receptors can continue to signal as ther signal can be released from the endo some
  • however if it is internalised then the hydolases can degrade all of the protein
41
Q

What is the structure of ESCRT proteins?

A

-a complex of proteins

42
Q

What do ESCRT proteins recognise?

A

membrane bound ubiquitinated cargo

43
Q

What is the process of membrane invagination and formation of endosomes within the lysosome by ESCRT proteins?

A

-recognise membrane bound ubiquitinated cargo and a series of proteins bind via the ubiquitin binding domains (firstly ESCRT0)
then a series of proteins and complexes bind the cargo (ESCRTI, ESCRTII, ESCRTII(pinches off the membrane))
this results in the cargo becoming internalised in the endosome creating a MVB (multivesicular body)
-complexes eventually dissaciate with the AAA-ATPase protein VPS4

44
Q

What is autophagy?

A

a process by which cells capture their own cytoplasm and organelles and deliver them to the lysosome for nutrient release
-regulates energy homeostasis

45
Q

What are the 3 types of autophagy?>

A
  • Macroautophagy
  • Microautophagy
  • Chaperone mediated autophagy
46
Q

What are the features/process of Macroautophagy?

A

when a membrane forms inside the cell (isolation membrane, a double membrane) and eventually circularises and contents are delivered to lysosome and degraded and the nutrients released
-may just contain part of the cytosol

47
Q

what are the features/process of Microautophagy?

A
  • invaginations of the lysosomal membrane

- pinch off small amounts of cytosol and form a vesicle

48
Q

What is mitoautophagy?

A

Autophagy of a mitochondrium

49
Q

What are the features/process of chaperone mediated autophagy?

A

-proteins bind to heat shock protein HSC70 with co chaperones in the presence of lamp2A which directly targets the proteins to the lysosome for degradation

50
Q

How do virus’ exploit ESCRT proteins?

A

-hijack the ESCRT machinery to invaginate away from the cytosol (as ESCRT proteins normally do in the endosome) but on the plasma membrane to creat a ‘coat’

51
Q

What is an example of receptor mediated down regulation via ubiquitin and the lysosome

A

EGF-R
-mono-uquitinations sufficient for internalisation
Must be internalised via escrt proteins within the endosomes so it can be degraded fully and it’s signalling stopped

52
Q

How did autophagy originate?

A

in periods of nutrient depravation autophagy is activated

  • membranes and proteins are degrades
  • and fed into the TCA cycle and through glycolysis to produce energy to regulate energy homeostasis
53
Q

What are the process of mTOR signalling

A

Mammalian target of rapomycin
-Rapomycin inhibits mTOR
-mTOR complex is one of the major regulators of autophagy
-serine/threonine kinase
When there are lots of nutrients
-phosphorylates autophagy targets, inactivating them
When there are not many nutirents
-mTOR inhibited, don’t get phosphorylation of proteins/autophagy targets/substrates and autophagy is switched on

54
Q

What is the (detailed) process of autophagy?

A
  1. Autophagy is initiated when Rapomycin inhibits mTOR, and when ULKI-Atg13-FIP200-Atg200 complex not phosphorylated by mTOR it moves to the ER with the p13k complex and forms the isolation membrane
  2. LC3/ATG8 gets lipidated (addition of phosphotidylethianine) and with the Atg5 + 12 complex can bind to p62 which binds to ubiquitated proteins, recruiting them into the membrane before circularisation
  3. Completetion and circularisation of the autophagosome
    [ULK1 complex (targeted by mTOR kinase) + VPS34 (Pi3 kinase) enables circularisation]
  4. Maturation occurs when contents are delivered to the lysosome = autolysosome, delivering fluid and hydrolases
  5. cargo is degraded
55
Q

What is the (basic) process of autophagy?

A
  1. Initation of the isolation membrane
  2. Expansion and cargo recognition
  3. completion and circularisation of the autophagosome
  4. Maturation occurs when contents are delivered to the lysosome = autolysosome, delivering fluid and hydrolases
  5. cargo is degraded
56
Q

What does selective autophagy occur on

A
  • ubiquitinated proteins
  • damaged mitochondrium
  • bacteria
57
Q

What is the mode of action of the Kaposi’s sarcoma herpes virus (KSHV)?

A

Kaposi’s sarcoma herpes virus (KSHV) produces K3, an E3 ubiquitin-ligase which aids in ubiquitinating and down-regulating MHC-I molecules from the plasma membrane (a poly-ubiquitin K63-linked process)

Cell Biology (15 decks)

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