Lipid signaling

Question 1

most basic phospholipid

Answer 1

phosphatidic acid - phosphate, glycerol, two nonpolar tails

Question 2

why do lipid molecules aggregate in solution

Answer 2

hydrophobic effect, nonpolar tail of each lipid molecule surrounded by ordered molecules which are released upon aggregation. So driven by entropy.

Question 3

three types of aggregating lipid structures formed in aqueous solution

Answer 3

micelle, bilayer, liposome (resembles cellular architecture)

Question 4

another name for liposomes

Answer 4

nanoparticles

Question 5

functions of membrane lipids

Answer 5

1. principle barrier for cell
2. Mediate cell-cell interactions
3. Serve as second messengers
4. Affect membrane protein activities

Question 6

how is lipid signaling regulated?

Answer 6

1. Substrate availability
2. Lipid kinases (alteration of their intrinsic activity)
3. Lipid phosphatases
4. Membrane localization (ex “interfacial activation”)

Question 7

T/F. Composition of cell membrane lipids is similar for both outer and inner monolayer

Answer 7

False, asymmetry both prevalent and important

Question 8

Which lipid can famously move freely between leaflets?

Answer 8

Diacylglycerol (DAG)

Question 9

what is the major diff between lipid-mediated signaling and other signaling systems?

Answer 9

Lipids are hydrophobic, therefore their generation, effect and metabolism must involve hydrophobic environment or at least be amphipathic

Question 10

Name some examples of lipid signaling functions

Answer 10

DAG (activates protein signaling mediators), IP3 (elevates intracellular Ca2+), Ptdins(2,4,5)P3 (activates PDK/AKT), Sphingosine-P (stimulates growth - receptor mediated), ceramide (apoptosis)

Question 11

three major types of lipid metabolizing signaling enzymes

Answer 11

phospholipases (PLA2), lipid kinases (DAG kinase), and lipid phosphatases (PTEN)

Question 12

soluble kinetics vs interfacial kinetics

Answer 12

interfacial works through mediator, more difficult to study than soluble

Question 13

two conceptual modes for interfacial enzyme catalysis

Answer 13

scooting mode, hopping mode

Question 14

cleavage products of different phospholipases

Answer 14

1. PLA2 : phospholipid —-> LysoPL and free fatty acid
2. PLC : removes head group and PO43-, makes DAG
3. PLD: just removes head group, makes phosphatidic acid

Question 15

4 major types of PLA2

Answer 15

1. Small secreted
2. Cytosolic – regulated by Ca and phosphorylation (can be activated therefore by PLCs, see below).
   Ca-independent
3. PAF acetylhydrolases (act on platelet activating factor (PAF)).
4. This specific PLA2 helps degrade PAF; hydrolyzes acetyl group.
5. PAF is very potent => this phospholipase is important in regulating levels

Question 16

T/F. After PLA2 cleavage, both products are released from the membrane

Answer 16

False, they remain embedded in the membrane

Question 17

eiconoids are derived from

Answer 17

arachidonic acids, proken down by PLA2

Question 18

phosphatidylinositol cycle (PI cycle)

Answer 18

PI to PIP to PIP2, generates IP3 and DAG, IP3 increases calcium, PIP2 stimulates DAG which is endogenous activator of PKC and leads to growth and proliferation

Question 19

What is the min number (and identity) of signaling molecules generated by the PI cycle after activation of the PI-PLC only?

Answer 19

Two - IP3 (water soluble) and DAG (hydrophobic)

Question 20

What do PLCs do?

Answer 20

Hydrolyze PIP2 to generate IP3 and DAG, three major classes that are activated by different mechanisms

Question 21

What is one way you could prove interfacial activation was occuring in your enzyme?

Answer 21

Add weak and strong detergent vs. no detergent. In presence of strong detergent (think about micelle structure), you have an increase in the lipid environment => more enzyme activity.

Question 22

What kinase can phosphorylate PIP2, and what is the product of this phosphorylation?

Answer 22

PI3 kinase, makes PIP3

Question 23

Why is PIP3 an important signaling molecule?

Answer 23

It activates PDK, which phosphorylates and activates AKB (also known as PKB). PDK and AKB are brought by PIP3 to the membrane surface, and then AKB goes forth and activates other junk like mTOR, this activity very important for growth

Question 24

What is PTENs activity?

Answer 24

PTEN is a phosphatase, catalyzes PIP3 —-> PIP2, considered a tumor suppressor

Question 25

What is a ratiometric approach to measuring intracellular calcium levels?

Answer 25

Fura-2 fluorophore binding to Ca2+, bound fluoresces at 340 nM and unbound at 380 nM

Question 26

phosphatidylcholine (PC) cycle

Answer 26

PLD phosphatase hydrolyzes PC into free choline and phosphatidic acid (PA) - now PA can go and be made into more DAG. PLD activation is receptor mediated

Question 27

give an example of lipid kinase action/importance

Answer 27

sphingosine signals growth arrest, but sphingosine-PO4 leads to survival and proliferation. sphingosine kinase (SK1) activated by phosphorylation by ERK2

Question 28

give an example of lipid phosphatase action/importance

Answer 28

Important for converting PIP3 -> PIP2 because phosphorylation at the 3rd position prevents cleavage by PLC. PIP3 is a potent signaling molecule