module 6 part 2 Flashcards
Cargo
GPCRs, hormone receptors, adhesion molecules, synaptic vesicle proteins etc
Cargo adaptors
AP2, Epsin, Eps15, arrestins, Da2, ARH
Accessory proteins
SNX9, AP180, AAK, amphiphysin, synaptojanin, endophilin, auxilin, Hsc70, dynamin
Scaffold
Clathrin
Clathrin binding
beta propellor structure
can bind adaptors and accessory proteins
Clathrin box or W box
PI lipids
specific intracellular membrane markes
PIP2 = critical marker of plasma membrane, can recruit adaptors
AP2 adaptor protein complex
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
AP2 conformational change
induced by u2 subunit phosphorylation by AAK
AP2 as a hub
appendage domains = platforms for recruiting accessory proteins and other adaptors
binds to short peptide sequences
Affinity
how tight interaction is between two components
Avidity
additive strength from binding multiple interactions
Coat formation requires both
multiple low affinity interactions can have high avidity. ensures activation via several sequences at once. easy to disassemble. dynamic and very stable
Epsin
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
Amphiphysin and SNX9
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
Uncoating
ATPase called Hsc70 is recruited by auxilin, binds to vertices of clathrin lattices and hydrolyses ATP to ADP. Conformation change occurs and drives uncoating.
Synaptojanin
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.
A
Adaptors: AP2 = phosphorylated by AAK and drives conformational change to bind lipid and cargo to clathrin terminal domain
B
Building of the coat: network of interactions, stabilise assembly and cargo capture.
C
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
D
Dynamin: recruit by amphiphysin (banana), drives pinching/popping. requires GTP hydrolysis for conformational change.
E
Energy input for uncoating. Hsc70 uses ATP to disassemble clathrin coat.
Amyloid hypothesis
Alzheimers: amyloid plaques and intracellular neurofibrillary tangles. formed by misfolded/aggregation of amyloid peptide (AB)»_space; from APP protein
disruption of calcium homeostasis, apoptosis induced, mitochondrial function, glucose metal, inflamm. responses
Amyloid precursor protein
APP
transmembrane protein
cleaved by secretase enzymes - alpha, beta, gamme
Lipoprotein receptor family
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
ApoE
chaperon for AB uptake – clearance. three isoforms. ApoE4 is weakest AB binder - risk in impaired AB clearance
LRP1A
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.
LRP1B
interacts with APP, decreases endocytosis and level of AB production
SNX17
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
Retromer
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
SorLA
cytoplasmic domain binds directly to retromer. lumenal domain binds to APP. APP trafficking to golgi.
Therapy
can’t KO SNX17 or retromer. introduce chaperone proteins that increase retromer’s stability - enhance neuroprotective effect
APP transport to endosomes
from golgi
via AP4 (closely related to AP2)
uses mu4 subunit, binds to YXFFE motif
AP clathrin adaptor family
1: Golgi-endosome
2: endocytosis
3: lysosomal organelles
4: Golgi-endosome
KO of AP4
APP trapped in golgi
increased AB production
