Lecture 18: Protein Lipidation Flashcards
protein lipidation (def.)
a covalent modification of proteins by lipids
protein lipidation can play a key role in _____ and _____ of modified protein
localization and function
most modifications are ___ but some can be ____
stable (permanent)
dynamic (regulated addition and removal)
protein lipidation promotes localization in membrane by ____
acting as an anchor in membrane
protein lipidation can be ___ or ____
co-translational
post-translational
Five main categories of Protein Lipidation
1) glypiation
2) cholesteroylation
3) prenylation
4) Acylation
5) Other
glypiation is the addition of _____
GPI anchor
cholesteroylation is the addition of _____
cholesterol
prenylation is the addition of _____
farnesyl and geranylgeranyl
acylation is the addition of _____
fatty acids (myristate and palmitate)
other types of protein lipidation is the addition of _____
phospholipids
several proteins are subject to _____ because it promotes _____
multiple lipidation
strong association with membrane
nature of lipidation affects ___ and ____
localization and properties
5 main lipids
1) myristate
2) palmitate
3) palmitoleic
4) farnesyl
5) geranylgeranyl
protein lipidation with myristate linkage? to associated residue? stability?
amide/H2N-Gly –> stable
protein lipidation with palmitate linkage? to associated residue? stability?
thioester/cysteine –> dynamic
amide/ H2N-Cys –> stable
protein lipidation with palmitoleic linkage? to associated residue? stability?
oxyester/serine –> stable
protein lipidation with farnesyl linkage? to associated residue? stability?
thioether/cysteine –> stable
protein lipidation with geranylgeranyl linkage? to associated residue? stability?
thioether/cysteine –> stable
2 saturated lipids
1) myristate
2) palmitate
3 unsaturated lipids
1) palmitoleic
2) farnesyl
3) geranylgeranyl
cholesterol can be ___
inserted into membrane
protein cholesteroylation helps with ____
protein insertion into membrane
main targets for protein cholesteroylation are ___
protein of the Hedgehog family (Hh)
Hedgehog protein family (def.)
family of secreted signaling proteins needed for growth and patterning
Hedgehog protein family are morphogens that can ____
elicit developmental fate over significant
distances
Hedgehogs are produced as ______ and large extension at ______
larger precursors with signal sequence
the C-terminus
Hedgehogs require ____
extensive processing
Cholesteroylation of Hh proteins method
1) cleavage in conserved Gly-Cys-Phe (GCF) on C-terminus after translocation
2) auto-processing mediated by C-terminal extension
3) Transfer of cholesterol to C-terminal glycine
4) Stable palmitoylation at N-terminus on Cys
Both the _____ and _____ (protein lipidation) are essential for function of Hh proteins
cholesteroylation
stable palmitoylation
the protein modifications of Hh proteins allows the ____
secreted signaling domain to remain tethered to PM
the protein modifications of Hh proteins help the protein to form____
micelles
Protein isoprenylation occurs on _____
cysteine residue part of the “-CAAX” or “-CXC” motifs at the C-terminus
(A-aliphatic residues)
Protein isoprenylation forms ___ bond
Irreversible thioether
Isoprene has how many carbons?
5
Farnesyl residue has how many carbons?
15
geranylgeranyl residue has how many carbons?
20
General rule: when to add geranylgeranyl vs farnesyl residue?
-geranylgeranyl: CAAX, CXC –> X = leucine (L)
- farnesyl: anything else
the synthesis pathway of cholesterol contains ____
many lipids used in protein lipidation as intermediates
Protein isoprenylation of small GTPases is needed for ___
attachment to membrane
Geranylgeranylated proteins display ____affinity for membranes (especially ___)
high
di-geranylgeranylated proteins
geranylgeranyl group is _____ than farnesyl group
10-40X more hydrophobic
Some prenylated proteins (____) must be extracted from membranes by ____
Rabs
specialized chaperones (GDIs, Guanine nucleotides dissociation inhibitors)
GDI keeps protein bound to ___
GDP
fatty acylation with ___ and ____ acts to mediate ____
myristate and palmitate
special types of membrane targeting
fatty acylation uses how many types of fatty acids?
3; all saturated but of different lengths
3 types of fatty acids for fatty acylation
- C8:0, octanoate (ghrelin etc…)
- C14:0, myristate
- C16:0, palmitate
fatty acylation increases protein ___ and promotes _____
hydrophobicity
weak membrane interaction
myristate (# carbons?)
14
palmitate (# carbons?)
16
____% of human proteome is myristoylated
~0.5%
N-myristoylation adds ____
myristate co-translationally to N-terminal
glycine
enzyme needed for N-myristoylation?
- N-myristoyl transferase I (Nmt I)
Nmt I is associated with ___
ribosome
Nmt I transfers ___ following removal of ____
myristate
initiating methionine
N-myristoylation bond formed?
amide bond
N-myristoylation stability?
Stable (not reversible)
N-myristoylation often works with ____ to regulate membrane association
second signal
S-Palmitoylation is typically added _____ to _____
post-translationally to thiol group of specific cysteine residue
S-Palmitoylation stability?
Reversible, dynamic
Most common lipid modification
Protein Palmitoylation
S-Palmitoylation is easily detectable because ____ which is cleavable with ____
it involves a thioester bond
hydroxylamine
S-Palmitoylation is typically used to ____
regulate protein function
S-Palmitoylation happens what type of protein?
many different types of proteins
S-Palmitoylation happens on what residue? near what?
usually Cys residue close to membrane surface, sometimes near myristoyl or prenyl group
proteins with this domain acts as palmitoyl acyltransferases (PATs) in many organisms?
DHHC-domain (zDHHC containing proteins)
aspartate-histidine-histidine-cysteine
Glypiation occurs when? stability? targets? function?
-post-translational
-stable
- extracellular proteins
- targets proteins to rafts
Cholesteroylation occurs when? stability? targets?
-post-translational on newly exposed C-terminal glycine
-stable
- found on members of hedgehog family
Isoprenylation occurs when? stability? targets? function?
- post-translational on cysteine within -CAAX, -CCAAX or -CXC
-stable
-promotes membrane recruitment and further processing
-N-palmitoylation occurs when? stability? targets? function?
- post-translational on N-terminal cysteine
- found on members of hedgehog family
- stable (amide bond)
-N-myristoylation occurs when? stability? targets? function?
- co-translational on N-terminal glycine of cytosolic protein
- stable (amide bond)
*S-palmitoylation occurs when? stability? targets? function?
-post-translational on cysteine, various positions
- thioester bond cleavable with hydroxylamine
- reversible/dynamic (thioester bond)
Cancer cells often increase the expression of _____
N-myristoyltransferase
PCLX-001 treatment results in _____
the degradation of N-myristoylated proteins
PCLX-001 selectively ____
kills blood cancer cells in vitro
PCLX-001 induces cells death by _____
triggering apoptosis
composition of lipids in organelles depends on ___
what organelle it is?
Synthesis of lipid is ____ in the cells.
location-specific
Final steps in the synthesis of phospholipids (eg. PC) are catalyzed by _____
membrane-bound enzymes on the cytosolic surface of the endoplasmic reticulum
Cholesterol synthesis is completed _____
on the ER membrane
Addition of headgroups to ceramide to produce glycosphingolipids (sugar) or sphingomyelin (choline) occurs ______
on the lumenal face of cis and trans-Golgi membranes
Trans-bilayer movement (flip-flop) of charged lipids is _____ in artificial liposomes
extremely slow
Generation of Membrane Asymmetry at ER membrane
Phospholipids synthesized on cytosolic side of ER membrane are transferred to lumenal leaflet by a scramblase (maintains
equilibrium across bilayer)
Membrane Asymmetry at ER membrane? ATP?
ATP independent
Scramblase at PM action? stimulated by? ATP?
-moves lipids in both directions
-simulated by calcium
-ATP independent
movement of amino-phospholipids at PM action? stimulated by? ATP?
- PS and PE moved from outer to inner leaflet by specific flippase
-creates asymmetry
-ATP dependent
floppase works at ___ and is responsible for ____
PM
export of lipids from the cell
floppase action? ATP?
moves phospholipids from inner to outer leaflet at PM
ATP hydrolysis promotes activity
floppase best characterized as a member of ______ protein family
ABC (ATP-binding cassette)
Membrane asymmetry critically controls _____
transmembrane protein function
Movement of ____ from the ______ to other
compartments relies on ______
sphingolipids
Golgi complex
vesicle-mediated transport
Phospholipids and cholesterol can move between organelles by ______
different mechanisms that do not involve vesicles
Phospholipids and cholesterol movement to cell surface? speed? ATP?
extremely rapid movement to cell surface
- ATP-independent
drug that block COPI function
BFA
transport of phospholipids and cholesterol is not eliminated by ____
BFA (drug that blocks COPI - vesicle transport)
mechanism for transport of phospholipids and cholesterol involves____
Lipid Transfer Proteins (LTPs)
proteins CERT and ORP5 do what?
create unique interorganelle contact sites to facilitate exchange of specific lipids
phospholipids, ceramide and cholesterol transport involves ___ and/or ______
specific transfer proteins
inter-organelle membrane contact sites (MCS)
