lf206 lecture 13 - proteostatsis and autophagy

proteostasis is the dynamic regulation of the ______ _________ within the cell

balance is maintained by biosynthetic pathways and degradation pathways i.e. proteasomal _______ and _______

protein compliment

degradation

autophagy

what is autophagy?

process of self-digestion within the cell involving delivery to lysosome for degradation

cytoplasmic material is recycled

why do cells use autophagy? [3]

1. provides amino acids, nucleotides, lipids and sugars under low nutrient conditions

2. removes aggregates, damaged organelles and invading pathogens

3. cell differentiation and developmental remodelling (e.g. removing nucleus from an erythrocyte)

types of autophagy?

1. chaperone-mediated autophagy (proteins that display a particular motif, transported to lysosome)

2. microautophagy (direct interaction between lysosome and what needs to be degraded, i.e vesicles, small aggregates)

3. macroautophagy (isolation membrane encapsulates cell contents to be degraded)

during macroautophagy, a __________ forms to encapsulate cell contents

types of macroautophagy [2]

autophagosome

1. canonical autophagy - non-specific

2. xenophagy - of proteins

lysosome is central for intracellular _________

proteins enter vesicles called early _______ which will mature into late endosome

as pH ________, enzymes within late endosome _______

late ______ fuses with lysosome to form an _________

degradation

endosome

decreases

activates

endosome

endolysosome

how is lysosome pH maintained?

• __-type ATPases hydrolyse ___ to pump protons ___ the lysosome

• this creates a _____ gradient

• this is maintained by losing _____ or gaining _____

V-type

ATP

into

voltage

cations (positive)

anions

CHAPERONE MEDIATED AUTOPHAGY RECOGNITION

• Hsc70 identifies ______ motifs of LAMP (_______ ______ ______ _______) proteins on _____ surface

• describe the motif

  • has up to 2 ______ charged residues - _ & _

  • up to 2 _______ resides - I, F, _ or _

  • a single _______ charged residue- _ or _

  • a single _ on either the N or C terminus

• the motif can be in any _____

• KFERQ

• lysosome associated membrane proteins

• lysosome

• positively - K, R

• hydrophobic - L, V

• negatively - E, D

• Q

• order

CHAPERONE MEDIATED AUTOPHAGY

1. inactive _____-2A

2. Hsc70 recognises ______ motif and delivery of _____ to LAMP-2A

3. _______ of LAMP-2A by Hsp__

4. _________ of protein mediated by Lys-Hsc70

5. unused LAMP-2A is captured by ___ and transferred to lipid ______ to break them down by ______ A and a metalloproteinase

1. LAMP

2. KFERQ, protein

3. multimerisation, 90

4. translocation

5. Hsc70, microdomain, cathepsin

DOWN-REGULATION OF CHAPERONE MEDIATED AUTOPHAGY

1. mTORC2 complex ______ Akt1 by __________

2. Akt1 phosphorylates ____

3. phosphorylated ___ keeps LAMP-2A _____

4. this occurs in ____ nutrient conditions as the conditions _____ mTORC2

1. activates, phosphorylation

2. GFAP

3. GFAP, inactive

4. high, stimulate

Tau is a ______ in _____-mediated autophagy

in neurodegenerative diseases we see CMA dysfunction meaning Tau ______ → Alzheimer’s

too much up-regulation of CMA, degradation of tumour _____ and pro-_______ proteins favour/promote _______

substrate

chaperone

accumulates

suppressors

apoptotic

cancers

in microautophagy, autophagic ______ are taken up directly by late _____ and lysosomes

some microautophagy substrates have ______ motifs and are delivered by _____ - it binds to ________ in late endosome membrane

cargo is degraded in _______ or lysosomal lumen

cargoes

endosomes

KFERQ

Hsc70

phosphatidylserine

endolysosomal

MACROAUTOPHAGY

1. a ______ grows and captures cargo

2. it fuses with itself forming an __________

3. autophagosome fuses with a late ______ to form an _______

4. the autophagosome/_______ fuse with a lysosome to form an _______

5. _______ occurs

1. phagophore

2. autophagosome

3. endosome, amphisome

4. amphisome, autolysosome

5. degradation

steps of forming autophagosomes [4]

autophagosomes are used only in which type of autophagy?

1. initiation

2. nucleation

3. elongation

4. closure

macroautophagy

INITIATION

• mTORC2 phosphorylates ______ which activates mTORC_

• mTORC1 phosphorylates ____ in ___ places

• phosphorylation of ____ inhibits _________

• ________ is stimulated during starvation

1. ULK1 complex associate at __ membrane and capture _____ vesicles

   1. these vesicles are cycled between ____ membrane and trans-_____ network as they maintain membranes

2. ____ is associated with these vesicles

3. ULK1 ____ phosphorylates ____ (BECN) which activates PI3K

4. PI3K is a _____ kinase,

   1. adding ______ to lipids

5. turning phosphatidylinositol to phosphatidylinositol (3)-phosphate (PI3P)

• Akt1

• mTORC1

• ULK1, 2

• ULK1, macroautophagy

• macroautophagy

1. ER, ATG9

   1. plasma, Golgi

2. PI3K

3. kinase, Beclin

4. lipid

   1. phosphates

NUCLEATION

1. ____ proteins bind to PI3P on the ATG9 vesicles

2. _______ is recruited by WIPI proteins

3. LC3-1 is recruited by ATG16L1

4. binding of WIPI and PI3P lipids also recruit ___ which binds the ______some

1. WIPI

2. ATG16

4. ATG2, omegasome