Lipid Signaling Flashcards
(53 cards)
evolution of lipid signaling
-lipids were initially thought to just support receptors involved in signaling
-thought that some lipids mediated inflammatory responses –> production of these lipids, like prostalglandins and leukotrienes, depends on activation and/or presence of specific lipid metabolizing enzymes
-when you get inflammation, you take Aspirins that inhibit specific enzymes to block production of inflammatory lipids
Ex. phospholipase A2 can release a very important lipid called arachidonic acid and this can be metabolized in 2 major pathways: cyclooxygenase and lipoxygenase and they can produce prostalglandins and leukotrienes
soluble vs interfacial kinetcs
-soluble- you have an enzyme and substrates and everything is in solution- they can bounce around and tumble and do everything in 3D –> enzyme finds substrate and generates products
-interfacial- you have soluble enzyme that can bind to the membrane- either by binding to another component or directly to its substrate and once it’s bound to the substrate, it can then affect its catalysis and generate products
–> products can be either soluble or stuck in the membrane
–> substrate is not roaming around free space- just moving along the planar surface and instead of thinking about millimolar [] or soluble []s, you have to think about the % of the substrate that’s in the membrane (mole %)
how do you determine whether or not the cPLA2 membrane localization is regulated in an agonist or Ca dependent way?
-CryIC- bacterial toxin that stimulates arachodonic acid and Ca increases in a receptor-DEpendent manner
-okadaic acid- stimulates arachodonic acid and Ca increases in a receptor-INdependent manner
-when they took these cells and had some PLA2 attached to GFP then added Ca ionophore A23187, you can see the enzyme going to a membrane (goes around nuclear envelope), add toxin, and okadaic acid then same thing happens
-nothing happens with GFP alone- enzyme is actually moving
-Ca increases was important
regulation of cPLA2
-when Ca levels rise, it binds to certain domains within the enzyme called C2 domain and helps bind the enzyme to the membrane
-other basic regions of the membrane bind other phospholipids (PIP2 and ceramide phosphate)- these help anchor enzyme into the membrane
-MAPK phosphorylate the enzymes- there’s activation of enzyme and PTM of it
how would you determine whether a PLA2 is regulated via interfacial activation?
-critical micellar [] of a lipid (CMC) = [] at which micelles form
-below the CMC, lipid exists in monomers and above that lipid micelles form (balls of lipids with aquaeous bubbles around
PI-PLC cyclic phosphodiesterase activity
-slow breakdown of the cyclic inositol phosphate to generate IP
-you can look at specific activity of added cyclic inositol phosphate to the mix
-if you don’t have detergent around, you have low activity but if you add 8 M triton, the activity shoots up
-if you add diC7PC with low CMC, the activity shoots up
-substrate interface causes enzyme activity to work very well and it hydrolyzes cAMP into inositol phosphate
the players: membrane lipids
-phosphatidic acid (PtdOh) is what starts them
-phosphatidylethanolamine (PtdEh)
-phosphatidylcholine (PtdCho)
-phosphatidylserine (PtdSer)
-phosphatidylinositol (PtdIns)
-sphingomyelin- sphingosine-liken backbone with choline atttached to it and helps with signaling –> forms major constituent of myelin in neurons but breakdown of lipid and generation of sphingosine that gets phosphorylated works as signaling molecules and involved in growth, senescence
PI3-K associated with polyma virus-mediated transformation
-immunoprecipitated T antigen and showed that it has tyrosine kinase activity
-it can phosphorylate enolase and used mutant that does not transform cells and when it’s immunoprecipitated will have tyrosine kinase activity associated with it
-while WT T antigen has PI3-K activity associated with it, the mutant form doesn’t have PI3-K activity associated with it –> PI3-K is involved in transforming cells
membrane lipids also show a preferential distribution in membranes
-if you look at the distribution of membranes within membrane, you see that the amino-containing phospholipids are enriched in the inner leaflet and the choline-containing phospholipids are enriched in the outer leaflet
-if you look at the total leaflet distribution in the membrane, it’s about 50/50 with 50% facing outside and 50% facing inside but if you look at the specific distribution, you see this difference and this has to do with the way the lipids pack because there’s inside of the membrane with curvature that goes in whereas on the outside it’s convex out –> different kinds of curvature help membranes pack
-there’s also neutral lipids like diacylglycerol (DAG) that flip between 2 leaflets of the membrane
signaling lipids
modulation of proteins, including enzyme activities, cell growth, cell aggregation, and apoptosis, inflammatory immune responses
what is the major difference between lipid-mediated signaling and other signaling systems?
-lipids are hydrophobic or at least amphipathic
-therefore the generation effects and metabolism must involve hydrophobic environment and this presents some challenges
major types of lipid metabolizing signaling enzymes
- phospholipases
- lipid kinases
- lipid phosphatases
phospholipases
- phospholipase A2 (PLA2)
- phospholipase C (PLC)
- phospholipase D (PLD)
lipid kinases
- phosphoinositide kinases
- diacylglycerol kinases (DAGK)
- sphingosine kinases
lipid phosphatases
- phosphoinositide phosphatases (PTEN-phosphatase and tensin homolog)
- phosphatidic acid phosphatases
soluble vs interfacial kinetic
-soluble- you have an enzyme and substrates and everything is in solution- they can bounce around and tumble and do everything in 3D
-enzyme finds substrate and generates products
-interfacial- you have soluble enzyme that can bind to the membrane- either by binding to another component or directly to the substrate and once it’s bound to the substrate, it can then affect its catalysis and generate a product
-products can either be soluble or stuck in membrane as well
–> substrate is not roaming around free space- just moving along planar surface –> have to think about the percentage of the substrate that is in the membrane
general principle
- substrate availability- enzyme is present, on/off the membrane, and nothing is happening until substrate gets generated in a vicinity that enzyme can get to then you see enzymatic activity- nothing happening to the intrinsic activity of the membrane, just whether substrate becomes available
- alteration of intrinsic activity- enzyme is inactive –> something happens –> binds to allosteric effector and activity goes up and enzyme is activated
- interfacial activation- there could be an enzyme that is either active or inactive while it’s soluble but once it binds to membrane, it becomes activated –> interfacial activation
two major conceptual modes
- scooting- enzyme binds to the surface of a membrane and scoots around until it finds its substrate
- hopping- enzyme might bind to a membraen and scoot around to find its substrate and then come off and go to another membrane
PLA2s mediate the generation fo a lysophospholipid and free fatty acid
-you can see the glycerol backbone- SN1, 2 and 3
-SN2 has fatty acid backbone and does SN1
-PLA2s will hydrolyze the fatty acid off the 2nd position and it’ll be free fatty acid and lysophospholipid is left –> called this because when they were IDed they could add them to cells and they would lyse
-free fatty acid can go and be signaling molecule
Ex. the group IA phospholipase 2A with phospholipid substrate modeled in the activate site
-tryptophans help bring enzyme to the surface and anchor it there and when it does, it takes the phospholipid in membrane surface and lifts it out of the membrane then cleaves 2nd position off
-once 2nd position is off and fatty acid is free, the substrate drops back down and the enzyme is free to go and find another substrate
what are the five major types of PLA2s?
- cytosolic PLA2s- very specific arachondonic acid- inflammatory responses
- small secreted PLA2s
- Ca-independent PLA2s
- lipoprotein-associated PLA2s
- PAF acetylhydrolases
how would you determine whether or not the cPLA2 membrane localization is regulated in an agonist or Ca-dependent way?
-CryIC- bacterial toxin that stimulates arachodonic acid and Ca increases in a receptor-dependent manner
-okadaic acid- stimulates arachodonic acid and Ca increases in a receptor-INdependent manner
-when they took these cells and had some PLA2 attached to the GFP then added Ca ionophore A23187, you can see the enzyme going to a membrane (goes around the nuclear envelope)
-add toxin and okadaic acid and the same thing happens
-nothing happens with GFP alone- enzyme is actually moving
regulation of cPLA2
-when Ca levels rise, it binds to certain domains within the enzyme called C2 domain and helps bind the enzyme to the membrane
-other basic regions of the membrane bind other phospholipids (PIP2 and ceramide phosphate)- help anchor enzyme into the membrane
-MAPK phosphorylate the enzymes- there’s activation of enzyme and PTM of it
-phosphorylation is most likely important at low agonist and Ca []s
how would you determine whether a PLA2 is regulated via interfacial activation?
-look at the activity of PLA2 against different []s of substrate and you can see that the enzyme is active just when the substrate reaches the critical micelle concentration (CMC)
-enzyme has slow activity then when CMC hits and you form micelle with substrates, the activity shoots up
