Midterm one Flashcards
what are the four major macromolecuels
- Lipids
- Protein
- Nucleic acids
- Carbohydrates
What are lipids?
- Fatty, waxy, or oily compounds
- soluble in organic solvent and insoluble in polar solvents
-either hydrophobic or amphipathic
What are some reasons for lipid diversity? (6)
MS BRCL
1. Control membrane fluidity
- Signaling pathways
- Lipid rafts
- Regulations of protein binding
- Bilayer asymmetry
- Multiple membrane systems in cell
What is membrane fluidity and what can it affect?
-The ability of ease of molecules to move in the membrane, in relation to the viscosity of the membrane
-Can affect the diffusion of biomolesules in the membrane, thus affecting function
-Can be affected by lipid structure, cholesterol content, temp etc
What are signalling pathway?
Can act as messengers or precursors as messengers in signaling cascades
Common secondary messenger after activation of membrane receptor
ex. PKC activation
What is a lipid raft?
-Functional plasms membrane highly ordered microdomains that can house proteins for functional and signaling purposes
-Enriched in GPI anchored proteins and other proteins involved in signaling
-Will vary cell to cell
How is protein activation influenced/ regulated by lipids?
- Physical state of lipid system
- Presence of lipid rafts
- Lipid modifications
- Lipid environment can alter structure
What is Lipid Asymmetry?
Lipid distribution of lipids between two leaflets of the bilayer is not symmetric
What lipids compose the bilayer leaflets? BE ABLE TO RECOGNZIE PICTUYRES
Outer: SM, PC, cholesterol, and glycolipids
Inner: PS, PI and PE
SM: sphingolipids
PC: phosphatidylcholine
PS: phosphatidylserine
PI: phosphatidylinositol; cell to cell recognition and signalling pathways
PE: phosphatidylethanolamine
PG= phosphatidylglycerol
How is the lipid bilayer related to blood coagluation? what lipid is heavily involved?
Activity of coagulation enzymes in the absence of proper lipid environment is too slow
Externalization of PS forms complex that helps convert prothrombin to thrombin
VERY BIG fold increase in thrombin formation
Thrombin: converts soluble fibrinogen to insoluble fibrin to clot wounds
Lipid asymmetry important here
PS: phosphatidylserine
What is Scott syndrome?
Bleeding disorder where procoagulant activity is affected due to membrane distribution
Ca dependent mechanism exist that can abolish asymmetry Maintenace leading to PS externalization, due to “scramblase” activity
Scramblases activity requires cytosolic Ca
Scott syndrome has been characterized by an impairment of scramblase activity
Decreasing externalized PS of activity blood platelets
Only roughly 1/5th the amount of binding sites for Factor Xa
Have normal translocase activity (flippases and floppases)
Evidenced by sequestered fluorescent PS lipid (NBD-PS) in the inner leaflet
Activation of platelets
What are characteristics of Fatty Acid Lipids?
-present in many metabolic pathways
- can be synthesized from successive reactions of acetyl-CoA
-used as building blocks of lipid and triacylglycerols
-can be released form triacylglycerols into the blood stream as fuel by beta oxidization
-Contain hydrocarbon chain and carboxylic acid
-Most fatty acids are an even number of carbons (between 12-24)
-Saturated vs unsaturated (bent at double bond) ; mono vs poly
How is saturated/unsatirated related to food?
-Melting point plays a contributor in packing both saturated and unsaturated FA
Coconut oil is 74% saturated and solid at room temp
Olive oil is 85% unsaturated and liquid at room temp
What are the Functions of Fatty acids?
- signalling pathways
- Energy storage and source
- Triacyglycerols are stored in adipose tissue and can hold more energy than glucose
- Composition of hormones and lipids
- protein modification
- lipids can act as protein modifiers by regulating their activity
What are prostaglandins and how are they related to the immune response?
-Prostaglandins (PG) are lipids that are derive from the metabolism of polyunsaturated fatty acids (the poly fats are the limiting step, can be modulated)
-prostaglandin synthase (COX2) is promoted by pro inflammatory markers (cytokines)
-PG therefore mediates inflammatory response and cancers
examples of hormones and fatty acid lipids
Prostaglandins: synthesized from arachidonic acid; involved in inflammation, vascular tone, pain etc
Lipoxins: synthesized from arachidonic acid; reduces inflammation
Thromboxane: synthesized from arachidonic acid; involved in blood clotting
Resolvins/protectins: derived from omega-3 fatty acids; reducing inflammation
Other hormones such as cortisol, estrogen, testosterone are also derived from cholesterol
What are Sphingolipids?
-Saturated fatty acids to a degree, most of the time associated with lipid wraps (described as compact lipid domain where signaling happens)
-Instead of glycerol backbone, has a sphingosine (18-C amino alcohol)
Fatty acid connects to the sphingosine through an amide linkage –> Results formation of a ceramide connects to the sphingosine through an amide linkage
how are gangliosides are degraded into ceramides? what happens
Degraded into ceramides by removal of sugar units of the oligosaccharide group
1.Be highly specific lysosomal enzyme
2.Mutation of these enzymes can lead to accumulation = gangliosidosis
3.Could lead to certain disease
what are the major lipids of the membrane?
1.Phosphoglycerides/glycerophospholipids
2.Spingolipids
3.Ganglioside
4.Cholesterol
- Many others; cardiolipin
(<5% mitochondria, 8% Golgi apparatus, 10% endoplasmic reticulum )
What are Gangliosides?
-Gangliosides contain varying amounts of sialic acids (typically 1-40
-Needs to be metabolically degraded, specific enzymes, if they are mutated then build up of ganglioside
-If taken up by lysosome then enzymes get to them and they accumulate in lysosome, start to affect the lysosome function
-Different structural makeup of ganglioside and each requires a specific enzyme
what is the glycerol backbone in Phosphoglycerides?
-Fatty acid esterified at C1 and C2
-Phosphate group at C3 through phosphate ester
What is cholesterol?
-Most common steroid in the human body
-Principal component of cell membrane (20-50%) ; Present in lower quantities in organelles (10%)
-Important for lipid wraps, forms their domains
-Behaves in ways where they aggregate together in common phases ex saturated lipids come together to form saturated domains, this causes them to vary in functionality
What is Tay Sachs disease?
-Genetic defect (autosomal recessive) leading to non-function production of hexosaminidase A; metabolic syndrome
-Accumulation of GM2 (notation to show number of gangliosides) in lysosome
-Leads to accumulation in ganglion cells (swelling)
-Typically seen in babies, resulting in loss of ability to move
What are the five families of hormones that derive from choelsterol?
1.Androgens
2.Estrogens
3.Progestins
4.Glucocorticoids
5.Mineralocorticoids
What is cardiolipin?
Has been shown to have a high affinity for curved membrane regions (ex. inner mitochondrial membrane)
Present in mitochondria, smaller more compact head group with more range on tail, helps with curvature
Four fatty acid chains, two PA lipids coming off glyceriol (diphosphatidylglycerol)
- 18C fatty acids with 2 unsaturated bonds
what is water?
Lipids have polar and non-polar components (overall polar though)
Can form up to four hydrogen bonds
Responsible for the formation of lipid related structure
In water you get H bond lattices of water, upon addition of hydrophobic molecules, some of the bonds will break
What is the hydeophobic affect
-Decreased entropy
-Will drive hydrophobic molecules together to diminish contact with water stability of H bonded water network make it difficult to dissolve hydrophobic compounds
Introduction of lipids leads to decreased stability and a decrease in entropy
OVERALL happens because: Large positive free energy is not favorable
Enthalpy change of taking a hydrocarbon and putting it into water is very small
Large negative change in entropy dominates the change in free energy
We overcome this by aggregating the non-polar molecules together
What is the water oil interface
Surfactant has a hydrophilic and hydrophobic mobility, partition of water oil interface
Depending on specific surfactant it can lead to different divisions OR at high concentrations you get c which gets you more of mixing of phases; comes back to concentration of components ex lipids of surfactant
what are Thermodynamics of lipid self
Critical micelle concentration: the concentration at which the lipid molecules aggregate and form micelles
Depends on the chemical structure of CHAIN LENGTH AND HEAD GROUP
How does soap work?
In vesicle has aq on inside and outside (like a cell sued to trap hydrophilic particles in vesicles)
In micelles the stuff inside is hydrophobic tho
Soap will form micelle’s structure when you start to wash hands, agitate hydrophobic stuff ex dirt
Agitate micelles which start to trap them inside micelles
On outside of micelle is hydrophilic head groups which is easily washed away with water
How is vaping related to lung surfactants
Vitamin E is a common bimolecular marker found in bracheoli which is related to lung stress with vaping
They wash the respiratory system, and the unwanted debris come out
Vitamin E is one of the many dilutants of different chemicals, used in vape flavoring
When vitamin e you get a much softer membrane which means it cannot withstand certain pressure with constant expansion/exhale leading to the collapse which leads to alveoli death
Initially people think vitamin E is good for you but it’s bad here. In vaping industry, they base safety regulations of FDA which tests eating/consumption NOT investigating inhaled which is why it passed
Lipid shape parameters (P)
-Shape parameters: ratio of head size to tail size will give certain structure ex. More planar or curved bilayers
-Curves can come in the form of folds in bilayer (helps with rounding affect and some processes ex. Endo and exocytosis, membranes go in and out)
-Shape parameter influence shape and affects function.
What is the bilayer?
Form planar regions where you can incorporate lysolipids (phospholipids with a cleaved tail so head tail is bigger than tail region to form the curved part. There is also certain charges which will help with curvatures.
Gel like = solid, L alpha is more fluid
Previously thought of saturated vs unsaturated fatty acid BUT here is the melting point that determines what phase; at room temp they are almost always in liquid phase but if more saturated or below temp then rigid phase
membrane curvature
membrane curvature
Depending on size parameter you will get positive or negative curvature
There is a bar domain which helps stabilize curvature; proteins form dimers that is stabilized upon binding to lipid membrane,
-Binding via electrostatic interaction because membrane is negatively charged (strong binding = more curve) -Bind to more readily curved liposomes therefore some membranes have a higher affinitive for curvature since there is more electrostatic interaction opportunities
Certian residues stabilize curvature
There is also specific lipid domains that help, like PI lipids, which are used for signaling and bind to specific protein with the domains close to membrane which stabilize the curve process
What are the different states of cholesterol in the membrane?
Cholesterol has affinity the Ld phase (which changes La to Ld)
-Ld is more disorder -Lo is more ordered -Lb is more solid General effect is to inhibit Lbeta but result in a less fluid and more ordered structure than L alpha
-Degree of cholesterol will determine of Lo or Ld phase, once you bypass the lipid threshold for cholesterol you will enter the Lo phase
-Never just all one type, ex. If Ld phase there will be small amount of Lbeta still, it will just be negligible
What are the techniques to observe membrane?
- Fluorecence Microscopy
- differential scanning calorimetry (DSC)
- Scattering experiments (look at membrane and phase behavior through neutrons)
What is fluorecence microscopy and what are its pros and cons?
-Can use flourescently labelled lipids to look at lipid phases; visualize so most people can understand
-Pros
Easy to obtain
Quick to visualize, see different phases, people understand well
Nondestructive probe, doesn’t damage system
-Cons
Exogenous (bulky) probe –-> probes that you attach to lipids are bulky which can cause the lipids to deviate from their natural structure
Photobleaching –> fluorescent probe has the possibility of being exposed to light which will then degrade. This causes it to be less accurate
Auto fluorecent cells are cells that have innate florescent behavior so if you are using fluorescent to quantity a certain behavior in the cell it can throw off numbers
What is the process of differential scanning calorimetry (DSC)
Use two cells, one sample and one reference, can be done with solids but when using lipids normally the one of interest is in an aqueous solution and the reference will be in water
Add heat to cells slowly at the same rate
Once you get close to melting point you are breaking the interactions of gel phase
Need energy to break the bonds, there is difference in heat transfer because as system absorbs heat (to break the bonds) the cell must compensate by adding heat
Therefore, in water nothing will happen, no absorb or release of heat
However there needs to be energy to go from gel to disordered aq phase. On graph the area under peak is enthalpy related to that specific event
In some cases, there is a lipid ripple phase (intermediate/ pre transition peak)), the intermolecular forces start to breakdown which may cause a small hump before dissolving into full phase
Peak is full melting phase
How can scattering experiments be used to observe membrane and phase behaviour?
- Data collection: investigation of samples with X-ray or neutron scattering technique
- Data reduction: raw data corrected by background or instrumental effects, normalized etc.
- Modeling: modeling structure (ex. Bilayer) can look at electron or neutron density distribution associated with a structure
- Fitting data adjusts parameters chosen by model to best match data
- Parameter extraction: fitted model will be based on preset parameters (can lock value)
- Probability profiles: information of electron or neutron density
- Analysis and interpretation: parameters and profiles can be used to gain insight on structure or function
- Software tools: large number of tools for analyzing data available
What is the process of using scattering techniques to observe membrane behaviour?
Make sample based on area of interest
Background reduction you want a baseline reading to subtract from all the data
Model the reduced data. Structural model to help depict data
Match data to theories
Based on scattering pattern to pick theory and then get parameters based on it
You can also get profiles which helps with depicting what structural component you are looking at, differ between x rays and neutrons because they highlight different aspects of membrane (x rays interact with electrons in sample so more e rich areas lit up, so in a phospholipid the phosphate (head) is more lit up; look at the neutrons of sample)
What are some techniques for looking at lipid structure?
- X ray scattering
- Neutron scattering
- Mass spectrometry: has been used for lateral organization but not transverse, and characterization –> separate all the lipids of the bilayer and tun it through process –> just look at single area/ specific structure to get a lipid profile then compare against literature
- Solid state NMR: for info related hydrocarbons, used to confirm lipid asymmetry
- CryoTEM; one of more accessible approaches, quick measurements, get a picture that is easy to interpret; electrical microscopy angel, visualizing technique, flash freeze sample at specific time, cannot look at dynamic process
- Cross section of bilayer
What is small angle X-ray scattering (SAS)? what are the two types?
Beak of neutron/xray –> hits sample and angle is detected –> machine has different techniques based on how machine was built
Can give insight on structural parameters of vesicle systems
- SAX: nanoscale resolution (1-100nm); structural properties
Some structural parameters measured with the visualization of SAX
-Bilayer thickness, head-to-head thickness, area per lipid, lipid volume, water thickness, and hydrophobic thickness
- WAXS: atomic resolution 0.1-1nm); atomic details
WAX (wide resolution) the farther your sample detector is the more scattering angle you can get so you need bigger detector to capture all the incoming signals
If you use wide angle than refraction can give more detail
Data gives intensity curve based on where data hits; means scattering event occurs more than others
What are the pros and cons of SAX small angle x ray scattering (SAS)?
-Pros
1 Nanometer scale resolution
2 Quantitative information
3 Dynamics studies
4 Low sample requirements
Cons
1 Average structural description
2 Radiation damage
3 Instrument access
4 Data interpretation
what is small angle neutron scattering (SANS)
Neutron will interact with the sample and scatter at an angle at the detector ; looks at scattering intensity
-Can look at :
- Size and structure
- Domain formation
3.Monitoring asymmetry
-Process
1 Neutron source
2 Hit the scanner
3 Depending on how it scatters you have a circular map giving intensity (how many points hit that specific q value) vs q value (where it refracts)
what are the pros and cons of small angle neutron scattering (SANS)
Pros
-Sensitive to H
-Contrast variation methods
-Dynamic studies
-Less damaging than x rays
Cons
-Lower flux compared to x rays
-Expensive and hard to get access
-More complex analysis
-Large amount of samples required
specific technqiues/tools in lipid research (structure and membrane)
- small angle X-ray scattering (SAS)
- small angle neutron scattering (SANS)
3.cyro electron microscopy
(make note of name, pro/con, and what it says)
What is contrast variation?
The methods by which the neutron scattering intensity from a multicomponent complex can be separated into that from the components through the use of hydrogen- deuterium substitution in the complex or solvent
The difference in scattering length density between materials is the “contrast”
What is cyro electron microscopy
Allows for the visualization at the near atomic level
Vesicles need to be frozen and embeded specialized grid
Programs can be used to analyze image to obtain structural data
what are pros/cons of cyro electron microscopy
Pros
-Preservation of sample
-Versatile (size range)
-Minimal radiation
Cons
-Cost/specialization
-Computationally intensive
-Non moving system
brief history of lipid rafts; 1988
-Single and Nicolson;
the fluid mosaic model of the structure of cell membranes –> systems/bilayers that have variety of different lipids in systems which give memebrane some function which might lead to downstream signaling
-Kai Simons
Determination of lipid rafts
Hypothesized a specific interaction between glycosphingolipids and apical proteins
Luminal leaflet has to be concentrated by sphingolipids for vesicle transport
Built on the idea of lipids domains forming on cellular membranes
Saw that different protiens were packaged in different areas of the cell; differenjt lipids sent to different areas of the membrane
what are lipid rafts?
Can get bigger and smaller in size; transient; smaller and always dynamic; appearing and disapearing depending on functional needs
What are the two types of lipid raft proteins?
- GPI anchored protiens
GPI- GlycosylPhosphatidylInositol
Attached to the outer leaflet of the cell membrane
Can function in cell adhesion, as enzyme, receptors, proteases, or structural purposes
- Src Kinase (ex. C-Src)
Typically found on inner leaflet
Dynamic association
Dysregulation has been linked to cancer progression (hyperactivated)
Can function in signalling, adhesion, migration, survival and angiogenesis
Lipid raft targeting has been a focus of cancer therapeutics
What is one problem with models? how do you get a system assymetric?
-One problem with models is that you lack behavior of an actual biological sample
-Distribution of phospholipids in bilayers
Has to do with the composition of membranes
Overall lipid composition on inner and outter leaflets but there is also asymmetry in inner and outter
How does asymmetric distribution occur?
1.Phosphoglycerides: synthesized on cytosolic face of ER (major site of lipid syntesis
-Can freely exchange leaflet in ER
-Not the case in the PM
2.Golgi: can be involved in lipid modification and processing
3.ER does not have sig asymmetry but plasma membrane and golgi have the P4 translocate enzyme in turns of lipases and flopases
- Protiens
- Vertical synthesis
-SM: synthesized on luminal face of golgi –> outside of PM
-Ceramide made in ER is major building block of sphingolipids
-studying occurs with some sort of probe; involves ability to go from one leaflet to another –> hydrophobic/philic proeprties may become an issue
-Ideally probe free system; neutrons system is less invasvie
what are the three ways proteins keep the lipid bilayer asymmetric?
Flippases: movement of lipids from outer to inner leaflet (require energy)
Floppases: movement of lipids from inner to outer leaflet (require energy)
Scramblases: bidirectional movement causing randomness
What is lipid flip flop and the methods to look at it?
Membrane phase influences flip flop
Membrane geometry influences flip flop
Methods to look at flipflop
1.NMR
2.ESR (bulky probe, may affect transfers ability of lipid)
3.Neutron scattering
4.MD (molecular dynamics, want to see in real life)
5.SFVS (some frequency vibration spectroscopy) (lots of bilayers fixed to a certain plate, free floating vesicle in a certian solution)
6. mediated flip flop
What is TR SANS???
Looking at h contrast variation
Idea of protien or dna match to solvent
Monitor change in neutron scattering intensity over the function of time
Use protiated and deuterated lipids will give good contrast
Symmetric vs asymmetric will have different SLD and therefore different scattering intensity curve
Do as a function of time
Start with asymmetric system but then symmetric will be your average mix scrammled LEB?
Roughly 50/50 eq state (maybe like 60/40 depedning on curvature, maybe more lipids on outside)
Average SLD of two lipids and match sysetm to it, solvent system will be matched to the symmetric theroretical scrammbling whoich is why asymetric graph line levels
what are issues with mediated flip flop?
Idea of using probes
Bigger probes may cause deformaties in bilayer which will affect the halflife
Protiens may have harder time going through transverse membrane (due to hydrophobic/philic) and will want to go through probe because easier –> probe leads acess from inner to outter leaflet
Can get rid of asymetry; laterally diffusing over transverse
what is the inside out rule?
Goes back to lipid asymetry and protien
Cytosol and exocytosis side
Negative charges can attract positive amino acids which helps with structures certian transmembrane proteins
So asymmtric distribution of lipids causes different distribution of charges which can affect the function
what are the three types of membrane proteins
1.Peripheral
Can be removed or added as needed
2.Lipid anchored
Strong lipid modification
3.Integral membrane proteins
Also known as “intrinsic” –> spans membrane
what are characteristics of integral membrane proteins?
-Associate through strong hydrophobic interactions and the interactions exposed to the cytosol is hydrophilic
Can be isolated by:
1.Detergents – dissolve sections of the transmembrane with some phospholipid attached (maintain function hopefully)
2.Chaotropic agents (don’t care much about preserving function, fully denature, but you can gain insight on a.a seq)
3.Denaturing conditions
Are amphiphiles
1.Fatty acid interacts with r group
2.Hydrophobic surface residues in core
3.Polar residues on extended portions
Demonstrated by surface labelling
Labelled protein domain with agents that cannot penetrate the membrane
What are some transmembrane proteins?
** they will cross 0 line on the hydropathy plot
1.glycophorin A
2.Bacteriorhodopsin
3.OmpX and PapC usher
4.Ion channels
5.Resting K+ channels
6. Voltage gates channels
What is glycophorin A and its functions?
-transmembrane protein
-Located on red blood cells
Functions:
1.Membrane integrity
2.Cell to cell interactions
3.Lipid raft association
4.Blood type (MNS) –> carries blood group antigens
what is a hydropathy plot?
Online tool that can be used to predict transmembrane regions based on residue seq
Uses a hydrophobicity sale as a predictive tool
Get sequence. Above 0 is hydrophobic and below is hydrophilic
This will tell you if protein spans membrane. Once you gain region that passes threshold/0 then you have confidence it spans membrane. Whever ABOVE 0 is a transmembrane region
what is Bacteriorhodopsin?
Bacteriorhodopsin; a helices, varies in function
-transmembrane protein
Residues must organize exposing hydrophobic regions to fatty acids hiding polar backbone; alpha helices, beta sheets
Bacteriorhodopsin (alpha helices); 7 transmembrane helices, has a retinal chromophore, changes from cis to trans upon light absorption to activate light driven proton pump = ATP production
Cis to trans allows protons to go across membrane
What are OmpX and PapC usher
-transmembrane protein
Beta barrels occur in porins = channel- forming proteins
Initially seen in bacteria –> organelles
Seen in cell membrane of bacteria or organelles. Forms pore on inside to transport ions or small molecules –> typically in an outer membrane to shutter small molecules through the protective outer layer –> typically just for movement
what is Resting K+ channels
-transmembrane protein
Relevant during resting state of a cell
Allow movement of K out of cell through confirmational changes
Activity regulated by voltage across the membrane
Vestibule: cavity or chamber-like structure that is part of a transmembrane proteins
what is an ion channel
Certain molecules will have easier time than others, different proteins are required
what are voltage gated channels ?
Once they are in the membrane, most of function is from helices rather than extracellular components
Tetramers and monomers
what is a topogenic sequence?
Topogenic sequence: a peptide seqence essential for a protien to properly insert and orientate in the membrane
Single pass vs multipass
what are topogical class type one proteins?
N terminus signal sequence enter translocon until reaching a stop transfer anchor sequence (STA)
Typically will be hydrophobic residues to make a helix
Stops translocation through the translocon and will release into the bilayer
Synthesizes remaining C-terminal portion until stop codon
Can move laterally between protien sub units
what are topogical class type two proteins?
Instead of STA they will have a signaling seq that functions in bringing it to the locon and
The postive charges of concentrated residues on protien affect whether the initial part will go through the translocon or if it will sequeeze around? IN TYPE TWO THEY WILL NOT GO THROUGH
Orientated in the translocon with N terminal towards cytosol
Believed to be mediated by positively charged residues
Alongated with an internal signal anchor (SA) sequence; also binds SRP
Laterally moves out and anchors into membrane
Completed with C terminus being released into lumen side
what are topogical class type three proteins?
Similar orientation to type I however does not have signal sequence
Similar to type II, however terminals are opposite
Signal anchor sequence is located near N terminus
Have positive residues on C terminal side, help orient SA sequence
what are topogical class type four proteins?
Idea that they are multi spanning
How many spanning regions you have will say if coo or nh group will be on same or opposite sides
Ex if even number then terminals will orientate on same side; alternatively there is one on outside and one on inside if odd number
what does lipid modifications for proteins act for?
Allows for an otherwise water soluble protein to interact strongly with membranes
Allows sorting of proteins into particular membrane domains (eg rafts)
Acts as a functional switch (on/off = with or without)
Types of modifications
- Prenylation
- Myristylation
- Thioacylation
- Cholesterol modification
- GPI anchoring
What is micro domain targetting?
S-acylated proteins co-purify with cholesterol-rich detergent-
insoluble membranes domains
– Prenylation = branched = bulky
– Acylation = saturated = not
bulky
Looked at detergent resistant
membranes (DRM) from Madin- Darby canine kidey cells
– Isolated and proteins were
characterized
* Ras – Was it or was it not found?
* Src family kinases – Was it or was it not found?
Therapeutic use of Isoprenoids//FTase inhibitors; rational drug design
Example of rational drug design
Aim was to inhibit farnesylation of Ras, which is often mutated in overactive cancers
Several canditdates showed promise in cell culture and mice but crashed in Phase III trials
Reason: likely GG-ation of K-Ras and N-Ras
Combination of Ftase and GGTase inhibitor is toxic
-For therapetuc use; lets say ypou dont want a spoecific protien to reach the membrabn; form an inhibior that targets gernaylgeranyl –> in cases of no gg the cell can still adapt so there still might be some loalization on membrane –. If you inhibit one enzyme, other protiens may go and restore function as theyve evolved to have that behaviour
myristoylated- classic example of N myristoylated protein: SRC (kinase)
Localized to inner leaflet of PM
* Myristoyl moiety, as well as polybasic
region nearby, accounts for membrane
affinity
* Membrane association necessary for
biological activity as substrates are
membrane-associated
* Phosphorylation of serines in polybasic
region results in membrane dissociation
* Ex) c-Src had higher kinase activity with
mod
Prenylation; what are Isoprenoids ?
Prenylation- covalently attached lipid built of isoprene subunits
-Typically, farnesyl (C15) and geranylgeranyl (C20)
Isoprene; base unit
FPP; helps it traverse the membrane;
Proteins will have residue req that is prone to these modifications. For exame
Farnesyltransferase and geranylgernyltransferases responsible for modification
Post addition mods can be followed to help with insertion and stability
Typically done at C terminal of protein
palmitoylation- what is thioaceylation ?
Linkage of FA to cysteine residue by reversible labile thioester linkage [palmitate (C16) most common] -75%
- Exclusively post-translational
– Enzymes are membrane associated (S-acyl
transferases) - Initially hard to study due to lack of consensus sequence
- Reversible
Occurs in peripheral or transmembrane domains
* Often can co-exists as dual lipid modification – such as with myristoyl
Cholesterol modification
-Occurs in Hedgehog (Hh) family of signaling proteins involved in development
- Conserved GCF tripeptide gets cleaved by auto-processing domain
- Cholesterol can act as trafficking signal
example; ZDHHC2 in Cancer
Member of the ZDHHC family,
originally named as reduced
expression associated with
metastasis protein (REAM)
– Tumor-suppressor protein
– Expression has implication in
cancers
- Acts as a palmitoyltransferase
– Substrate?
* CKAP4 – cell surface receptor for antiproliferative factor (APF)
* APF leads to
depalmitoylation nuclear
translocation gene
regulation
What is the french flag model of Hh signalling
Morphogen will form a gradient with individual cells
in terms of concentration. In response to the
concentration a specific differentiation event will
occur in relation to the concentration.
