1
Q
What are the functions of biomembranes?
A
- Barrier between cell and its environmement
- Protein constituents allow selective permeability and uptake
- Communication between cell and its environment
2
Q
What are the characteristics of the phospholipid bi-layer?
A
- Charged hyrophilic head groups form H bonds with water
- neutral hydrocarbon chains are hydrophobic associate via van der Waals forces (AMPHIPATHIC)
- Energetically favorable arrangement
- Impermeable to macromolecules and small charged ions
3
Q
What are the two physical states in which the phospholipid bi-layer exists?
A
- Gel-like state at low temperatures
- Liquid-crystalline state at higher temperatures (membranes MUST exist like this)
4
Q
What is the temperature at which a membrane changes from the gel-like state to the liquid crystalline state?
A
Transition temperature
5
Q
What are the six classes of membrane lipids?
A
- Fatty acids
- Triacyglycerols
- Glycerophospholipids
- Sphingolipids
- Waxes
- Isoprene-based lipids
6
Q
What are fatty acids?
A
Alkyl chains terminated by carboxylic acid groups
7
Q
What are the characteristics of fatty acids which affect the transition temperature?
A
- Degree of saturation (double bonds decrease van der waals force)
- Chain length (longer chains mean more interactions)
8
Q
What are the two main types of phospholipids?
A
Phosphoglycerides and phosphoceramides
9
Q
What are the characteristics of phosphoglycerides?
A
Based on glycerol backbone
- Glycerol substituted for phosphodiester group attached to an alcohol headgroup and two ester linked fatty acids
10
Q
What is the common precursor of phospholipids?
A
Phosphatic acid
11
Q
How are triacylglycerols used for energy?
A
- Hydrolysed by lipase to fatty acids and glycerol and released from adipose tissue to be released to energy requiring tissues
- Fatty acids broken down step by step to Acetyl CoA which is processed in the citric acid cycle
12
Q
What are the six most common head groups of phospholipids?
A
- Phosphatic acid (PA)
- Phosphatiydyserine (PS)
- Phosphatidylenthanolamine (PE)
- Phosphatidylcholine (PC)
- Phosphtidiylinositol (PI)
- Phosphadidylglycerol (PG)
13
Q
What can phosphoinositol further synthesize?
A
Can be phsophorylated by photolipase C to form DAG and IP3 which are both important second messengers
14
Q
What is the structure of cardioplin/PG?
A
- dimeric structure with four acyl groups and two negative charges
- Found in membranes of bacteria and mitochondria, mainly on the inner layer where it interacts with a large number of mitochondrial proteins especially those related to oxidative phosphorylation
- Cone shape allows curvatures
15
Q
What are the characteristics of sphingolipids?
A
- Sphingosine with acyl chain and head chain that is either ethanolaminephosphoceramide (EPC), shingomyelin (SM) or inositolphosphoceramide (IPC)
- More stable and less prone to hydrolysis, hence found on outer thicker membranes
16
Q
What are the a) the simplest and b) most common gylcolipids?
A
a) glucosyceramide
b) GM2 ganglioside
17
Q
What are waxes?
A
- esters of long chain alcohols with long chain fatty acids
- Insoluble in water and so are used to keep moisture in/out
18
Q
What are terpenes and what is included in this classs?
A
Lipids formed by isoprene units
- steroids are terpene derived
- odors are often monoterpenes due to their volatility (e.g menthol)
- retinol is a diterpene
19
Q
What are the functions of cholesterol?
A
- Stabilise lipid membranes by immobilising first few hydrocarbon groups of phospholipid molecules, acting as a buffer for sudden changes in temperature of the environment by preventing phase shifts/ crystallisation of hydrocarbons
- In cell membranes regulate cell signalling
- Precursor to vitamin D, sex hormones and corticosteroids
20
Q
What is the structure of the lysosome membrane?
A
- Single membrane
- Hydrolytic enzymes degrade proteins and lipids
- low pH maintained by membrane enzyme which pumps H+ into the lumen
21
Q
What is the structure of the mitochondrial membrane?
A
- Inner membrane is 76% protein
- high proportion of cardioplin
- outer membrane is porous
22
Q
Describe fatty acid synthesis
A
- Takes place in the cytoplasm
- Intermediates liked to acyl carrier protein (ACP) and enzymes joined by single polypeptide of fatty acid synthase
- Fatty acid chain grown through seuqential addition of 2xC units from acetyl CoA (donated by malonyl ACP) driven by the release of CO2
23
Q
How are proteins targeted to the membrane?
A
Through covalent lipid modifications: fatty acylation, prenylation, addition of GPI anchors (present on all eukaryote outer membranes) or cholesterol groups
24
Q
What are the 3 types of fatty acylation?
A
- N-meristoylation
- S-Palmitoylation
- N-Palmitoylation
25
What are the 2 types of prenylation?
- Farnesylation
| - Geranylgeranylation
26
What is the strucutre of a GPI anchor?
- Phosphoinositol is a phosphlipid which extends the membrane with two fatty acyl chains which extend into the bi-layer
- Glycosylated to a variable number of sugar units and a phoethanolamine unit which creates an amide link to the COOH terminal of the amino acid
27
How does a protein become anchored by GPI?
- Protein undergoes transamilation reaction within the ER
- Cleaved with part remaining in the ER and part being attached to the GPI anchor
- This will then leave the ER through the golgi destined for the membrane and is also usually heavily glycosylated
28
Which types of proteins are anchored by GPI?
- No apparent pattern but more common in protozoa
- Include enzymes, protozoan coats, adhesion molecules and receptors
- Some can exist both GPI and transmembrane
29
What is protein N-myristoylation?
- Addition of myristic acid to glycine through an amide linkage
- Protein made in the ER has initiator methionine cleaved off by methonine peptidase so that glycine may form a bond with meristate
- This process is non-reversible but allows proteins to associate with membranes reversibly
- Involved in HIV signal transduction pathway
30
Give an example of how N-meristoylation allows a protein to be reversibly membrane bound?
Recoverin protein with modification
- When Ca2+ free it is soluble as meristoyl is water soluble and small
- When Ca2+ meristoyl group is hidden in the hydrophobic pocket and so allowed to be hydrophobic
31
Describe protein palmitoylation?
- Reversible post-translational modificarion
- Addition of palmitic acid to cyteine through thioster linkage
- Used for peripheral and integral proteins
- Used to anchor to a specific membrane
32
Which signal transduction pathways are palmitoylated?
- Protein tyrosine kinases
| - G-proteins involved in vesicle movement
33
What is prenylation?
- Addition of farnesyl (C15|) through thioster linkage to cysteine residue at C terminal
- Irreversible
- Associated with Ras and rab motifs (vesicle movement)
34
What does the localisation of Ras isoforms in the cell depend on?
- Farnesylation and palmitoylation
- Reversible palmitoylation allows ras to cycle between cellular compartments and perform different tasks in different areas
35
What are phosphoinositides?
- Based on phosphoinositol: glyerophospholipid with glycerol backbone and two fatty acid chains
- Phosphorylation at 3,4, and 5 positions alone or in combination give 7 different species with different subceullular distributions
- Provide reversible recruitment of proteins to specific cell membranes
- 3-phsophatase linked to cancer
36
What are the functions of phosphoinositides?
- Intracellular trafficking
- Signalling
- Cytoskeletal remodelling
37
What is the distribution of the different phosphoinositides?
- PI(4)P - Golgi
- PI(3)P - Early endosomes
- PI(4,5)P2 - plasma membranes
- PI (3,5)P2 - multi-vesicular endosomes
38
What is the basic strucutre of G-coupled protein receptors?
- 7 transmembrane domains (in hydrophobic core)
- 3 intraculular loops
- 3 extracellular loops
- Intracellular C terminal and extracellular N terminal
39
What is the function of G-coupled receptors?
- Allow extracellular ligand which cannot cross plasma membrane to provoke a response
- activates heterodimeric G protein which switches between active and inactive forms activating membrane bound proteins which activate a signalling cascade in the cell
40
What does the diversity of GCPRs show?
- Number of different target tissues using different hormones to get diffferent major response with similar strucuture
- Not sequentially related suggesting converging evolution
41
What is the structure of the heteordimeric G protein?
Has alpha, beta and gamma subunits
- Alpha and gamma are membrane bound, beta bound to gamma
- Alpha binds GDP (inactive) or GTP (active) depending on state
- When inactive all tightly associated with eachother
42
How is the exchange of GTP and GDP mediated at GCPRs?
- Receptor activates GEF (guanine exchange factor) which swaps GDP for GTP allowing trimer to dissociate into alpha and beta/gamma which both interact with effectors
- RGS regulators activate GAP for hydrolysis of GTP and reassembly of the trimer
43
What effects can GPCR activation have?
- opening of ion channels (inhibition of cAMP or AC)
- Increases in intracellular Ca2+
- Differences in gene trancription for differences in differentiation, cell survival and development
44
Describe GPCR activation of AC
- Alpha subunit binds to AC activating it to convert ATP into cAMP
- This activates PKA which is usually a heteromer can become a monomer
- May then enter nucleus and bind to CREB which can change protein transcription e.g diesterases which can cleave chaperone protein of PKA resulting in further activity
45
How can GPCR act through phosphodiesterase (PE)?
- Phosphodiesterase will convert cGMP to GMP
| - Causing closing of cGMP dependent channels
46
How does the cholera subunit act through AC and GPCR?
- In the presence of G protein ARF, A subunit of chloera toxin ADP-ribosylates Galpha subunit, inhibits endogenous GTPase activity so G protein remains on and inhibits endogenous GTPase activity
47
What is the function of the phosphatidyl Inositol-3 Kinase pathway?
- Major growth pathway
- Phosphorylates membrane bound inositol phospholipids
- Acts as socking sites for intracellular proteins which form signalling complexes
48
What does the activation of PI-PLC on PIP2 result in?
The formation of 2 second messengers;
- Diacylglycerol (DAG) which is hydrophobic and remains in plasma membrane and activates PKC
- IP3 which is hydrophilic and brings about an increase in Ca2+
49
How does IP3 increase cytosolic Ca?
- Binds to IP3 receptor on ER allosterically, allowing Ca release (which can also activate PKC)
- Depletion of Ca stores in ER is detected by specific Ca channels in the plasma membrane, allowing Ca ion entry
50
By which method (other than cleavage from PIP2) can DAG be made?
- Action of phospholipase D
- This is activated by G-protein coupled receptors or PKC. This reaction produces phosphatidylcholind hydrolysis leading to phosphatic acid
51
What determines the nature of response to DAG?
- Levels to which activated by PLC or PLD
| - Subtypes of DAG activated
52
What is the structure of PKC?
- Has catalytic C terminus which is active when cleaved from N terminus which is highly conserved within subtypes (regulatory domain)
- Receptors for activated C-kinase (RACKs) only bind PKC when appropriate cofactors are present
53
What are the 3 subtypes of PKC?
```
Conventional:
- Ca dependent
- DAG dependent
- Phosphatidylserine dependent
Novel
- Ca independent
- DAG dependent
- phosphatidylserine independent
Atypical
- Ca independent
- DAG independent
- Phosphatidylserine dependent
```
54
What effects can elevated Ca levels have?
- Can bind to various proteins at different sites to activate them (e.g PKC)
- Many effects mediated by calmodulin (CaM) which is a non-enzyme protein allosterically activated when binding 4 Ca ions and can act as allosteric activator
55
What effect does the binding of Ca to CaM have?
- Induces conformational change
- Can then bind to inhibitory domain of protein to activate
- Protein auto-phosphorylates to become completely active
56
How can lipids be extracted to be analysed?
Application of solvent mixture of ethanol, chloroform and water results in the formation of two layers: the lower lipid layer and the upper aqueous layer which contains the other biomolecules (proteins)
57
Describe gas chromatography of lipids
- Volatalile lipids can undergo chromatography without modifaction
- Non-volatile lipids must be converted to FAMEs: base hydrolysis, acidify and extraction, conerting to methyl esters before dissolving in DCM
- Level of retardation shows length of chains and number of double bonds, as well as ammount in sample, but does not show head groups
58
How can lipids be extracted?
Beligh-dyer method
- Addition of chlorophorm and aquaeous layer
- Lipid becomes enriched in bottom cholorophorm layer
- Avoids hydrolysis and oxidation of lipids, preparation for tlc
59
How can lipids be visualised in thin-layer chromatography?
- Spraying with iodine solution (will appear as brown spots)
- radiolabelling with H3
- Visualises head groups
60
What is a survey scan?
Mass spectrometry without fractionation, shows different ions that are present
61
What are the different scanning techniques of a tandem mass spectrometer?
- Product ion = selection of species in Q1 for detection of daughter ion in Q3
- Precursor ion = records species from Q1 that gives pecific fragment in Q3
62
What is the significance of PC and PE?
Compose over 50% of all membrane phospholipids
63
Describe the CDP-ethanolamie pathway (Kennedy pathway)
3 enzymatic steps
1) Ethanolamine kinase catalyses the ATP-dependent phosphorylation of ethanolamine (to ethanolamine-P)
2) RATE LIMITING STEP, ECT (ethanolamine cytidyltransferase) uses CTP and Ethanolamine-P to form high energy donor CDP-ethanolamine with release of pyrophosphate
3) Ethanolamine phosphotransferase (EPT) catalyses transfer of Ethanolamine on to dicyglycerol to form PE and CMP as biproducts
BL3303
flashcards
Decks in class (15)
# Cards
-
Lipids63
-
Membrane Trafficking30
-
Principles of Cell Signalling71
-
Ion channels13
-
Ras signalling13
-
Glut4 translocation18
-
Ubiquitination19
-
Hippo pathway and cancer16
-
Ca signalling19
-
Plant transport51
-
SUMO15
-
Cell fractionation10
-
Visualising gene expression and protein localisation13
-
Western blotting11
-
Willin21
