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Y2 Mol Bio > Autophagy and Rho GTPases > Flashcards

Autophagy and Rho GTPases Flashcards

1
Q

Describe autophagy

A

A mechanism to digest intracellular material. Creates a membrane which expands de novo to capture cytosol in a vesicle and digest it

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2
Q

Why is GFP not appropriate for tracking autophagy?

A

It is pH sensitive, so degrades in acidic environments, such as the lysosome

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3
Q

Why are organelles degraded?

A

Homeostasis
Removing damaged components
Signalling
Recycling nutrients
Reprogramming cells (differentiation)

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4
Q

Outline the basic ubiquitin/proteasome system (UPS)

A

Ubiquitin tags cargo proteins to be degraded in the proteasome

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5
Q

Describe chaperone-mediated autophagy

A

Lysosomal
Low capacity and degrades individual proteins
Typically turns over long-lived proteins
LAMP-2 receptor on the surface of the lysosome recognises specific target proteins by their amino acid tags

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6
Q

Describe the proteasome

A

2 outer α subunits, 2 inner β subunits
Non-lysosomal
Degrades individual, (typically) short-lived proteins

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7
Q

Describe microautophagy

A

less well understood than chaperone-mediated
lysosomes invaginate directly on the surface, allowing cargo to get sucking into the lysosome (similar to endocytosis)

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8
Q

What are the 4 functions macroautophagy serves?

A

Lysosomal
Can remove whole organisms
1. Recycling nutrients
2. Cellular remodelling
3. Removing damaged components
4. Killing intracellular pathogens

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9
Q

How does macroautophagy recycle nutrients?

A

Under starvation, it causes non-selective bulk degeneration of the cytosol to recycle nutrients for metabolism
Cells lacking autophagy die under starvation

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10
Q

How does macroautophagy promote cellular remodelling?

A

Autophagy degrades organelles which is essential for some cell differentiation, e.g removing mitochondria from sperm

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11
Q

How does macroautophagy removing damaged components relate to aging?

A

Lysosomal capacity decreases as we age
Long-lived/highly metabolic cells e.g neurons and muscle are most susceptible to age related degradation.
This pathway is boosted to reduce ageingm cancer, dystrophy and neurodegeneration

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12
Q

What is the dietary restriction hypothesis?

A

Starvation/exercise leads to autophagy and damage repair

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13
Q

What intracellular pathogens do macroautophagy kill?

A

Pathogens in the cytosol, e.g MRSA, tuberculosis and viruses

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14
Q

What steps are used to study autophagy?

A

Disrupting autophagy
Dissecting the machinery
Observing the machinery

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15
Q

What is the kinase complex in autophagy responsible for?

A

Turning the pathway on/off

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16
Q

How are proteins linked to autophagosome membranes?

A

Adaptor proteins with Atg8 interacting motif (AIM) and ubiquitin binding domain (UBD) link these.
Some proteins don’t need adaptors as they already have an AIM on them. Phosphorylation regulates this interaction

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17
Q

Outline the steps of autolysosome formation?

A
  1. Initiation and expansion of membrane
  2. Closure of membrane containing Atg8
  3. Fusion of lysosome
  4. Acidification and maturation of autolysosome
  5. Degradation of material
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18
Q

What does neuronal-specific autophagy disruption in mice cause?

A

Increased apoptosis
Ubiquitinated aggregates accumulating

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19
Q

What neurodegenerative diseases do proteinopathies have a role in?

A

Huntingin aggregates in Huntington’s
α-synuclein in parkinson’s
Amyloid β plaques in Alzheimers

20
Q

How does huntington’s disease lead to neurodegeneration?

A
  1. > 35 CAG repeats encode a disease forming polyglutamine tract
  2. This misfolds and aggregates
  3. The proteins are ubiquitinated, forming an aggresome
  4. This undergoes Autophagic degradation
21
Q

What possible mechanisms in Huntington’s make it toxic?

A

A toxic oligomer which may damage the proteasome
Aggresomes
Adaptor sequestration

22
Q

Describe parkinson’s disease

A

Affects 1-2 per 1000
Loss of dopinergic neurons
Main neuropathology is α-synuclein aggregates (Lewy bodies) which are rarely mutated
5-10% familial cases

23
Q

How do α-synuclein mutations affect autophagy?

A

α-synuclein is normally degraded by chaperone-mediated autophagy.
An A53T mutation in α-syn blocks LAMP2 on the lysosome, preventing autolysosome formation

24
Q

What mutations cause mitochondria to accumulate in parkinson’s

A

PINK1- loss of function in mitochondrial kinase
PARKIN- E3 ubiquitin ligase

25
Q

Why is mitochondria accumulating damaging to cells?

A

Mitochondria is the main soure of reactive oxygen species (ROS) which damage cellular components and cause mutations

26
Q

What happens if autophagy is downregulated?

A

Organelles are damaged
Protein toxicity
ROS- oxidative stress
DNA damage

27
Q

What importance does Atg6 (Beclin1) have in ovarian, breast and prostate cancers?

A

Monoallelically deleted in 40-75% of these cancers
This is because it is involved in autophagy

28
Q

How does autophagy drive tumour survival?

A

It inhibits apoptosis
Survival during oxygen/nutrient shortage and chemotherapy

29
Q

How does a cell initiate apoptosis?

A

Bcl-2 binds to mitochondria
Membrane is permeablised
Caspase is activated

30
Q

Describe small GTPases

A

21kDa proteins
One of the largest groups of signalling protein
Change conformation upon activation
Bind and activate downstream effectors

31
Q

Why is it difficult to differentate active/inactive GTPases?

A

Subtle changes happen, so antibodies cannot differentiate

32
Q

What does GTPase signalling depend on?

A

Bound nucleotide

33
Q

Describe nucleotide binding sites in GTPases

A

P-loop which coordinates phosphate
Mg2+ is essential for nucleotide binding
Switches 1 and 2 bind effectors
Glu61 positions water
Counteracting negative charge and phosphates with p-loop (12GxxGKT17), hydrogen bonds and lysine.

34
Q

What are some active GTPase mutants?

A

Q61L is a catalytic mutant
G12V pushes out Q61, disturbing the p-loop

35
Q

What does Rac1-GTP lead to production of?

A

PAK kinase family
wave2- induces lamellipodia protrusions

36
Q

What role does Arg85 have in GAPs?

A

Strong positive charge which counteracts phosphate, weakening the phosphate bond.
Flexible which helps it insert into the active site to stabilise Q61 and transition state

37
Q

What stabilises the attacking water and Mg2+ in GAPs?

A

Thr35

38
Q

What are GEFs

A

Exchange GDP for GTP
Stabilise nucleotide-free, Mg2+ free GTPases
Dbl-homology domain
DOCK-family
Sec7 domain

39
Q

Describe the RacW56F mutant

A

RacW56F is insensitive to Tiam1 (it’s GEF), but sensitive to ITSN (Intersectin-1)
A subtle chage can turn Rac to Cdc42

40
Q

Give an example of a universal GEF?

A

Vav1/2/3

41
Q

What organelles assist in cell motility and what GTPases drive them?

A

Rac1- lamellipodium which protrudes towards the signal
Cdc42- Filipodium drives protrusion
RhoA- actin stress fibres which contract actomyosin apparatus, moving the whole cell forward

42
Q

What are the antagonistic signals in cell motility?

A

Cdc42/Rac are protrusion signals
RhoA is a contractile signal

43
Q

How does 2D and 3D cell motility differ?

A

2D is random and fast
3D is directional, often slower but translocates more efficiently

44
Q

What does increasing Rac1 activity do?

A

Activates protrusion in the right place

45
Q

What concequence does lack of SDC4-1 have on mice?

A

Healing defect due to cells having no direction

Y2 Mol Bio (10 decks)

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