Block B Lecture 3 - Protein Lipidation Flashcards
What is protein lipidation?
It is a type of post-translational modification (PTM) where a lipid (such as a fatty acid, isoprenoid or cholesterol) is covalently attached to a protein
(Slide 4)
Why is protein lipidation important?
As it can affect the activity of the protein and it gives distinct membrane affinities to target them to organelles and the plasma membrane
(Slide 4)
What are 3 common intracellular protein lipid modifications?
Answers Include:
S-Palmitoylation
N-Myristoylation
Farnesylation
Geranylgeranylation
(Slide 4)
What is S-Palmitoylation?
Addition of a 16 carbon palmitoyl group from the fatty acid palmitoyl-CoA. It does not have a specific consensus motif
(Slide 5)
What is a consensus motif?
A short, recurring sequence pattern in DNA, RNA, or proteins that is recognized by specific biological molecules
(Slide 5)
Where is the palmitoyl group added in S-Palmitoylation?
To the sulphur molecule of the thiol side chain of cysteine residues (resulting in a thioester linkage)
S in S-Palmitoylation = Sulphur
(Slide 5)
What class of enzymes mediate S-Palmitoylation?
zDHHC enzymes, which are a subtype of palmitoyl transferases (PATs)
(Slide 5)
What is the use of S-Palmitoylation?
The addition of the long hydrophobic group can anchor the protein to the membrane. It is a reversible modification, so therefore it can be used as an “on/off switch” for protein localisation
(Slide 5)
What 2 classes of enzymes can catalyse the release palmitoyl group from palmitoylated proteins, reversing S-Palmitoylation?
Acyl protein thioesterases (APTs) and palmitoyl protein thioesterases (PPTs)
(Slide 6)
What is “Dynamic S-palmitoylation”?
Cycles of palmitoylation and de-palmitoylation
(Slide 6)
What are the 2 steps of S-Palmitoylation?
- His154 on the enzyme is polarised by Asp153, and then acts as a base in extracting a proton and activating Cys156 into a thiolate nucleophile. This then attacks the palmitoyl coenzyme A thioester carbonyl thioester carbonyl carbon which results in a palmitoylated DHHC
- The cysteine residue of the protein substrate then likely attacks the the thioester carbonyl carbon of the palmitoylated DHHC enzyme and therefore ends up regenerating the enzyme, and transferring the palmitoyl group to the cysteine of the protein
(Slide 7)
What are the 2 steps of de-palmitoylation?
- His154 deprotonates Serine to make it more nucleophilic. Serine then attacks the thioester bond, which forms a covalent intermediate. Asp153 then stabilises the histidine through hydrogen bonding.
- Presumably the histidine deprotonates
(Slide 8)
What is N-Myristoylation?
When a saturated 14 carbon myristoyl group is attached to a protein
(Slide 9)
What is the consensus motif for N-Myristoylation?
MGXXX S/T (where X = any amino acid)
(Slide 9)
Where is the myristoyl group attached in N-Myristoylation?
To the N-terminal glycine of proteins (resulting in an amide bond)
N in N-Myristoylation = N-terminal
(Slide 9)
What does N-Myristoylation require?
The removal of methionine from the protein by methionine aminopeptidase (MAP)
(Slide 9)
What enzyme is N-Myristoylation mediated by?
N-myristoyltransferase (NMT)
(Slide 9)
What can N-Myristoylation be used for and why is this the case?
It can be used for protein localisation purposes as results in sufficient hydrophobicity and affinity so it interacts with membranes but not enough to permanently anchor the protein in the membrane, which is useful as it is irreversible
(Slide 9)
What 2 ways can N-Myristoylation occur?
Either co-translationally (during translation) following the cleavage of the N-terminal methionine residue in the growing polypeptide, or post-translationally (after translation) following caspase cleave in apoptotic cells
(Slide 10)
What is isoprenylation?
Attachment of a farnesyl (C15) (farnesylation) or geranylgeranyl (C20) pyrophosphate group (geranylgeranylation) to cysteine residues within 5 amino acids from the C-terminus
(Slide 11)
What classes of enzymes mediate farnesyl and geranylgeranyl attachment respectively?
Farnesyl transferases (FTases)
Geranylgeranyl transferases (GGTases)
(Slide 11)
What kind of enzymes are farnesyl transferases (FTases) and geranylgeranyl transferases (GGTases)?
Cytosolic enzymes
(Slide 11)
What is the consensus sequence for isoprenylation?
CAAX (where C= cysteine, A = alanine and X = any amino acid)
(Slide 11)
What is an example of a modification which can happen after isoprenylation?
Methylation can occur after Ras-converting CAAX endopeptidase (RCE1) cleaves the “AAX” part of the isoprenylation motif
(Slide 12)
Why is Rce1 cleaving the “AAX” of the isoprenylation motif required before other modifications, such as methylation, can occur on isoprenylated proteins?
As this is responsible for converting a hydrophilic C-terminus into a hydrophobic domain which has an affinity for membranes
(Slide 12)
What enzyme mediates the methylation of isoprenylated proteins?
Isoprenylcysteine carboxyl methyltransferase
(Slide 12)
What does farnesylation and geranylgeranylation result in?
A stable thioester bond
(Slides 13 and 14)
Are farnesylation and geranylgeranyl reversible or irreversible?
Irreversible
(Slides13 and 14)
What does methylation do when it is combined with farnesylation or geranylgeranylation?
The methylation itself is reversible and it increases the protein’s affinity for the membrane
(Slides 13 and 14)
What is a major lipid modification of extracellular proteins and what does it do?
A glycosylphosphatidylinositol (GPI) anchor, which anchors proteins to the outer leaflet (outer layer of the lipid bilayer) of the cell membrane, and GPI anchors also regulate sorting to the apical membrane in polarised epithelial cells
(Slides 17 and 21)
What do GPI-anchored proteins function as?
Adhesion molecules, protease inhibitors, enzymes and receptors
(Slide 17)
What makes up the core structure which all GPI anchors share?
A phospholipid, a glycan backbone, and ethanolamine phosphate (EtNP)
(Slide 18)
Where is GPI attached to?
The C-terminus
(Slide 18)
Is GPI a post-translational or co-translational modification?
It is a post-translational modification
(Slide 18)
Where is GPI synthesised and attached?
The endoplasmic reticulum (ER)
(Slide 18)
What do GPI-anchored proteins have which allow them to be targeted to the endoplasmic reticulum (ER)?
A cleavable signal sequence also known as a signal peptide sequence
(Slide 19)
What does GPI transamidase do?
It replaces a C-terminal hydrophobic region of a protein with a preformed GPI anchor on the luminal (membrane) side of the endoplasmic reticulum (ER)
(Slide 19)
What happens after a GPI anchor is placed onto a protein in the luminal side of the ER?
The GPI is further modified in the ER and golgi
(Slide 19)
What are GPI-anchored proteins believed to associate with (where are they usually found)?
Lipid rafts
(Slide 20)
What are lipid rafts?
Membrane microdomains which are enriched with glycosphingolipids, cholesterol, and certain types of lipidated proteins
(Slide 20)
What do lipid rafts do?
They organise the plasma membrane into domains which can serve as platforms for a variety of cellular functions, such as vesicular trafficking and signal transduction
(Slide 20)
How do GPI anchors regulate sorting to the apical membrane in polarised epithelial cells?
As the GPI-anchor is recognised, which results in GPI-AP (GPI-anchored proteins) being packed into distinct vesicles in the golgi and then delivered to the apical plasma membrane
(Slide 21)
What do GPI anchored proteins do once they are sorted to the apical membrane (in polarised epithelial cells)?
They are able to form high molecular weight complexes via oligomerisation, which then may drive budding of an apical vesicle
(Slide 21)
What are Ras proteins?
Soluble, monomeric GTP-binding proteins which regulate cell growth, proliferation and survival
(Slide 23)
What kind of genes are Ras genes?
Proto-oncogenes
(Slide 23)
What are proto-oncogenes?
Genes which control normal cell growth and division. If they become mutated they can become overactive and become oncogenes, which can cause cancer
(Slide 23)
What are the 3 major isoforms of Ras?
H-Ras, N-Ras and K-Ras
(Slide 23)
What 3 things happen to all Ras protein isoforms after synthesis?
They are farnesylated in the cytosol, the the “AAX” section of their farnesylation motif is cleaved by Rce1 before they are methylated by ICMT
(Slide 24)
What does a farnesyl group provide and what does this allow a protein to do?
It provides a weak membrane affinity, which is sufficient for transient (short lasting) membrane binding
(Slide 24)
Why do fatty acid / isoprene groups provide the ability for a protein to transiently interact with membranes?
As they increase the molecular hydrophobicity of proteins
(Slide 25)
Since fatty acid / isoprene groups only allow proteins to transiently interact with membranes, what does this mean?
An additional membrane-binding mechanism is required for full membrane-binding
(Slide 25)
What occurs to H-Ras and N-Ras after they are farnesylated in order to enable stable membrane binding?
- Farnesylation enables transient membrane interactions
- Transient membrane interactions bring the protein to membrane anchored zDHHC enzymes
- These enzymes palmitoylate cysteine residues which are adjacent to the farnesylated cysteine
- The combination of farnesylation and palmitoylation on nearby residues causes a much greater membrane affinity, resulting in stable membrane binding of H-Ras and N-Ras
(Slide 26)
How does the stable membrane binding of K-Ras occurs?
It requires farnesylation to enable transient membrane interaction and a polybasic domain.
The polybasic domain provides a second membrane anchor by interaction with negatively-charged phospholipids
(Slide 27)
What 2 things mediate the reversibility of the membrane binding of H-Ras and N-Ras?
Acyl protein thioesterase (APT) and dynamic palmitoylation
(Slide 28)
What do the different lipidations of the Ras isoforms allow?
It allows them to have distinct subcellular localisation and membrane distribution
(Slide 28)
How does de-palmitoylation / dynamic palmitoylation regulate Ras activity?
As de-palmitoylation causes dissociation from the membrane so that Ras cannot interact with effectors as efficiently
(Slide 28)
How can polybasic domain activity (such as in K-Ras) be regulated?
Via phosphorylation of nearby residues
(Slide 28)
What are the 4 main steps of the secretory pathway?
- Proteins enter the secretory pathway at the endoplasmic reticulum (ER)
- Protein transport occurs via vesicles from the ER to the Golgi
- Intra-Golgi protein processing and transportation occurs
- Vesicle transport of proteins occurs from the Golgi to either the plasma membrane or lysosomes
(Slide 30)
What proteins do N-Myristoylation, isoprenylation and S-Palmitoylation occur on?
N-Myristoylation and isoprenylation usually occur on soluble proteins whereas S-Palmitoylation occurs on both soluble and transmembrane proteins
(Slide 31)
What is LRP6 and how does S-Palmitoylation play a part in its function?
It is a single pass membrane protein which plays a crucial role in Wnt signaling and cellular processes like development, cell proliferation, and differentiation.
S-Palmitoylation of LRP6 is essential in ensuring its correct localisation to the plasma membrane
(Slide 31)
What diseases / conditions is the loss-of-function of LRP6 associated with?
Increased plasma lipids
Hypertension
Diabetes
Osteoporosis
(Slide 31)
What is osteoporosis?
A bone disease that develops when bone mineral density and bone mass decreases, or when the quality or structure of bone changes.
It results in bones weakening and becoming more likely to break
(Slide 31)
What does preventing the S-palmitoylation of LRP6 lead to?
It’s accumulation at the endoplasmic reticulum (ER)
(Slide 33)
Why is S-Palmitoylation needed in order for LRP6 to exit the plasma membrane?
Different membranes within cells have a different thickness, which results in them having different transmembrane domain (TMD) lengths. The length of the ER TMD (15) is shorter than that of the plasma membrane (20).
This difference is important in order to allow “matching” of a protein’s hydrophobic TMD with the hydrophobic interior of the membrane. If this hydrophobic matching is disrupted, a “hydrophobic mismatch” occurs, which leads to protein aggregation and trapping of LRP6 in the ER.
For reference, the TMD is 23 amino acids long
(Slides 34 and 36)
How does the TMD domain of LRP6 interact differently with the plasma membrane and the ER membrane?
The length of the hydrophobic TMD domain matches that of the hydrophobic region (TMD) of the plasma membrane.
However, the length of the hydrophobic TMD of LRP6 is too large to be accommodated within the ER membrane bi-layer, which results in a “mismatch”
(Slide 35)
How does S-Palmitoylation help with the hydrophobic mismatch between the TMD of LRP6 and the ER membrane bilayer?
S-palmitoylation causes the TMD of LRP6 to tilt with respect to the plane of the membrane, which stabilises the LRP6 and allows the TMD to be- accommodated within the membrane of the ER
(Slide 37)
What does prevention of S-Palmitoylation lead to in the context of protein stability?
It results in the protein undergoing ubiquitination and subsequent degradation
(Slide 38)
What is micro-localisation?
The precise spatial distribution or positioning of molecules, cells, or proteins at a sub-cellular level, typically within specialized regions of a cell membrane or organelles.
(Slide 38)
What role does S-Palmitoylation play in membrane micro-localisation?
It plays an important role in targeting proteins to cholesterol-rich microdomains in cell membranes such as lipid rafts and caveolae
(Slide 38)
