module 6 part 2

Question 1

Cargo

Answer 1

GPCRs, hormone receptors, adhesion molecules, synaptic vesicle proteins etc

Question 2

Cargo adaptors

Answer 2

AP2, Epsin, Eps15, arrestins, Da2, ARH

Question 3

Accessory proteins

Answer 3

SNX9, AP180, AAK, amphiphysin, synaptojanin, endophilin, auxilin, Hsc70, dynamin

Question 4

Scaffold

Answer 4

Clathrin

Question 5

Clathrin binding

Answer 5

beta propellor structure
can bind adaptors and accessory proteins
Clathrin box or W box

Question 6

PI lipids

Answer 6

specific intracellular membrane markes

PIP2 = critical marker of plasma membrane, can recruit adaptors

Question 7

AP2 adaptor protein complex

Answer 7

AP2 - heterotetrameric (four subunits)
binds cargo proteins with YxxO and DxxxLL sequences in cytoplasmic tails
binds to PIP2 via a and u2 subunits
hub for binding clathrin and other accessory proteins

Question 8

AP2 conformational change

Answer 8

induced by u2 subunit phosphorylation by AAK

Question 9

AP2 as a hub

Answer 9

appendage domains = platforms for recruiting accessory proteins and other adaptors
binds to short peptide sequences

Question 10

Affinity

Answer 10

how tight interaction is between two components

Question 11

Avidity

Answer 11

additive strength from binding multiple interactions

Question 12

Coat formation requires both

Answer 12

multiple low affinity interactions can have high avidity. ensures activation via several sequences at once. easy to disassemble. dynamic and very stable

Question 13

Epsin

Answer 13

amphipathic helix binds to PIP2, forms helix that punches into lipid bilayer due to hydrophobic surface. acts as a wedge. lots bind and causes membrane to buckle

Question 14

Amphiphysin and SNX9

Answer 14

banana-shaped BAR domain via arginines and lycines
Positively charged, attracts -ve membrane to curve to match shape of protein. proteins polymerise and curvature gets required force to form spherical shape. also associates with already bent surfaces.

SH3 region recruits proteins to recruit dynamin to help pinching

Question 15

Uncoating

Answer 15

ATPase called Hsc70 is recruited by auxilin, binds to vertices of clathrin lattices and hydrolyses ATP to ADP. Conformation change occurs and drives uncoating.

Question 16

Synaptojanin

Answer 16

removes PIP2 by hydrolysing it to PI4P. no longer bound to adaptors which fall off as they can’t bind by themselves. also recruited by endophilin.

Question 17

A

Answer 17

Adaptors: AP2 = phosphorylated by AAK and drives conformational change to bind lipid and cargo to clathrin terminal domain

Question 18

B

Answer 18

Building of the coat: network of interactions, stabilise assembly and cargo capture.

Question 19

C

Answer 19

Clathrin: recruited and polymerisation begins. by AP180 and AP2. lattice stabilised by lipid and protein interactions. SNX9 - drives membrane curvature via curved BAR domains. Epsin - inserts amphipathic helix

Question 20

D

Answer 20

Dynamin: recruit by amphiphysin (banana), drives pinching/popping. requires GTP hydrolysis for conformational change.

Question 21

E

Answer 21

Energy input for uncoating. Hsc70 uses ATP to disassemble clathrin coat.

Question 22

Amyloid hypothesis

Answer 22

Alzheimers: amyloid plaques and intracellular neurofibrillary tangles. formed by misfolded/aggregation of amyloid peptide (AB)&raquo_space; from APP protein
disruption of calcium homeostasis, apoptosis induced, mitochondrial function, glucose metal, inflamm. responses

Question 23

Amyloid precursor protein

Answer 23

APP
transmembrane protein
cleaved by secretase enzymes - alpha, beta, gamme

Question 24

Lipoprotein receptor family

Answer 24

Low density lipoprotein receptor (LDLR) family
10 in humans
brain
clearance proteins for lipoproteins, proteases, and vitamin carriers
can bind to ApoE
LRP1 protein and SorLA NB for AD, assoc w/ APP and trafficking

Question 25

ApoE

Answer 25

chaperon for AB uptake – clearance. three isoforms. ApoE4 is weakest AB binder - risk in impaired AB clearance

Question 26

LRP1A

Answer 26

isoform of LRP1. interacts with APP, increases rate of endocytosis in CCV, increases level of AB production
linked by FE65 via NPXY sequence. also links to AP2 therefore clathrin.

Question 27

LRP1B

Answer 27

interacts with APP, decreases endocytosis and level of AB production

Question 28

SNX17

Answer 28

Endosome to cell surface recycling - APP, LRP1, other LPP receptors
PIP3 binding PX domain, recruits to endosome, interacts with APP via FERM, regulates recycling - rescue protein!
N-terminal PX domain
C-terminal FERM domain
KO = increased APP breakdown, AB production

Question 29

Retromer

Answer 29

mediates APP endosome to GOLGI trafficking within MEMBRANE TUBULES
cargo loading complex
captures cargo associates with SNZ proteins with BAR domains that form membrane tubules
reduced in patients
depletion increases APP breakdown and AB production

Question 30

SorLA

Answer 30

cytoplasmic domain binds directly to retromer. lumenal domain binds to APP. APP trafficking to golgi.

Question 31

Therapy

Answer 31

can’t KO SNX17 or retromer. introduce chaperone proteins that increase retromer’s stability - enhance neuroprotective effect

Question 32

APP transport to endosomes

Answer 32

from golgi
via AP4 (closely related to AP2)
uses mu4 subunit, binds to YXFFE motif

Question 33

AP clathrin adaptor family

Answer 33

1: Golgi-endosome
2: endocytosis
3: lysosomal organelles
4: Golgi-endosome

Question 34

KO of AP4

Answer 34

APP trapped in golgi

increased AB production