Lipid  membrane    

Learning outcomes •  Describe the composition of the plasma membrane •  Functions of the plasma membrane with reference to specific molecules •  Fatty acid composition and functions •  Describe phospholipid structure •  Phospholipid functions

Membrane

Thickness: 7.5-10 nm

Membrane Functions 1. Compartmentalization (Intracellular compartments) 2. Scaffold for biochemical activity 3. Selectively-permeable barrier 4. Transporting solutes 5. Responding to external signals 6. Intercellular interactions 7. Energy transduction

The membrane is a lipid bilayer

Lipid bilayer comprises Phospholipid head groups (hydrophilic) And Associated fatty acids (hydrophobic)

e

Saturated C16 or C18 FA

Phosphodiester linkage

Derived from polar alcohol •  smallest = H (from H-OH) •  least common in membranes •  phosphatidic acid

Unsaturated C16 – C22 FA

Saturated fatty acids - No double bonds - Space-saving - More rigid membranes Alpha linolenic acid

COOH CH3

Arachidonic acid

DHA

EPA

Unsaturated fatty acids - More than 1 double bond - Each double bond confers ‘kink’ - Occupies more space - Promotes fluidity ofmembranes

ω-3 fatty acids

ω-6 fatty acids

α-linolenic (18:3)

linoleic (18:2) Δ-6-desaturase Δ-6-desaturase

octadecatetraenoic (18:4)

γ-linolenic (18:3)

elongase eicosatetraenoic (20:4)

dihomo-γ-linolenic (20:3)

Δ-5-desaturase eicosapentanoic (20:5)

arachidonic (20:4)

PG3 and LT5

PG2 and LT4

The Importance of the fatty acids •  Determine the fluidity of the membrane length of chain (usually 18-22) Level of unsaturation (# of C=C bonds)

•  Lipids rarely move from one layer to another •  Lipids exchange places with their neighbors •  Lipids rotate around their axis

Membranes need to be fluid to: Enable the membrane proteins to diffuse rapidly Allow distribution of lipids and proteins properly in the lipid bilayer Ensure membranes can fuse with one another when necessary

Cholesterol in the Membrane

•  Cholesterol is present where unsaturated lipids predominate •  It ‘fills the gaps’ and stabilizes the bilayer •  It stiffens the bilayer ∴ decreases fluidity & permeability

Extracellular face

75%

Outer layer stiffened by cholesterol Inner layer (fluidity determined by PUFA)

5%

20%

Rigid membrane

Fluidity determined by fatty acid composition of phospholipid Saturated: Palmitic (C16:0), Stearic (C18:1) Common fatty acids

Fluid membrane

Unsaturated: Arachidonic, Docosahexaenoic AA (20:4) DHA (22:6)

Membranes are Asymmetrical Glycolipids are found only on the extracellular surface (Sugar added in the Golgi)

•  Inner & outer surfaces have different lipids •  Proteins in the bilayer have a specific orientation due to its function

Phospholipids Phospholipids are Amphipathic (Amphipathic molecules are mostly lipid-like in structure, but have a hydrophilic region at one end

Choline Ethanolamine Serine Inositol

BASE

Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine Phosphatidylinositol

Phosphatidylinositol bisphosphate (PIP2)

Glycerol backbone

Inositol trisphosphate

Phospholipases PLA1 PLA2 PLC PLD

PLA1 CH2O..Stearic Acid

CH2O..Stearic Acid

CHO..AA

CHOH

CH2O..P..Base

CH2O..P..Base

Phospholipid

SA

Lysophospholipid

CH2O..Stearic Acid

CH2O..Stearic Acid

CHO..AA

CHOH

PLA2

CH2O..P..Base

Phospholipid

AA

CH2O..P..Base

Lysophospholipid

CH2O..Stearic Acid

CH2O..Stearic Acid

CHO..AA

CHO..AA

CH2O..P..Base PIP2

CH2OH

PLC IP3

Diacylglycerol

Action of phospholipase D

Phosphatidylinositol bisphosphate (PIP2) Membrane

3 phosphate groups ie PIP2

PLC hydrolyses PIP2

2 second messengers are produced

PIP2 hydrolysis

- stimulated by ligand-receptor Interactions - G protein-mediated - Leads to increased intracellular calcium concentration

PKC isozymes and some roles of PKC 1.  Conventional (require DAG, Ca2+ and Phospholipid) PKCα, PKCβ (I and II), PKCγ

2. Novel (require DAG but not Ca2+) PKCδ, PKCε (I and II), PKCη and PKCθ

3. Atypical (require neither DAG nor Ca2+) PKCι, PKCζ (I and II) and PK-N1, N2

PKC is a serine-threonine kinase Roles (tissue- and cell-specific) include Receptor desensitization Modulation of membrane structure events Regulation of transcription Modulation of immune responses Regulation of cell growth Learning and memory.

Some lipid molecules act as signalling molecules Diacylglycerol AA Ceramide

Production of arachidonic acid

ACTIONS OF AA Activates ion channels directly Activates PKC Precursor of many signalling lipids

Phospholipases A2 PLA2 – categorized in terms of localization and association with calcium. (20 types identified; 9 in humans)

Secretory

Cytosolic Ca2+-independent (cytoplasmic) PAF-acetyl hydrolyses Roles: Inflammation, cell death, cell signaling, maintenance of membrane phospholipids.

Secretory PLA2 (sPLA2):

   Originally from snake and bee venom Found extracellular especially in damaged tissue 10 groups (at least) Histidine residue at active site Require Ca2+ for activity characterized by a serine residue Roles: Linked with rheumatoid arthritis*, atherosclerosis, CNS inflammation, inflammatory bowel disease, skin inflammation, cancer, asthma Causes lysis of gram-positive bacteria *sPLA2-IIA primarily implicated in RA Released by macrophages Functions as a bacteriocidal  

Cytosolic PLA2 (cPLA2):

  4 groups (IVA, IVB, IVC and IVD named α, β, γ, δ). Active site is characterized by a serine residue. Ca2+-dependent Method of action: Ca2+-bound phosphorylated PLA2 translocates to intracellular membrane, cleaves phospholipid and releases arachidonic acid Pathological action (of head group/AA) can result in atherosclerosis, neuronal damage, multiple sclerosis, alziehmers, epilepsy. Groups IVA-C - high levels associated with colorectal, small bowel, and lung cancers

Ca2+-independent PLA2 (iPLA2):

   Group members: VIA-1, VIA-2, VIB Active site characterized by a serine residue MW: 63 - 90 kDa (comparable in size to cPLA2). Cytoplasmic Roles: Maintenance of homeostasis through remodeling of membrane phospholipids Involvement in apoptosis, muscle contraction, and obesity. No direct links to inflammation and role in cancer not clear  

PLA2 and inflammation

Arachidonic acid and metabolites

NOTES Arachidonic acid (C20:4) – 4 double bonds and therefore highly reactive Activates PKC Affects ions channels particularly K+ channels Affects transmitter release Double bonds mean it is a target for oxygenases Cyclooxygenases Lipoxygenases Epoxygenase

1) Cyclooxygenase 1 and 2:   • Converts arachadonic acid into cycloperoxides • Cycloperoxides can then form thromboxanes, prostaglandins, and prostacyclins. • Ultimately roles of molecules include cell death, inflammation, vasoconstriction, and vasodilation in platelets, the endothelium, and in smooth muscle. • COX inhibitors eg VIOXX may be associated with increased risk of stroke. • Abnormal COX activity linked with cancer   (2) Lipoxygenase (LOX):   Converts arachidonic acid into hydroperoxyeicosatetraenoic acid (HPETE) and subsequently leukotrienes. (Leukotrienes have roles in vascular function and inflammation) Abnormal LOX expression has been linked with cancers, including colon, pancreatic, lung, prostate, bladder, skin, and liver cancer   (3) Cytochrome P450 monooxygenase (CYP450):   Converts arachidonic acid into 2 products, hydroxyeicosatetraenoic acid (HETE) and epoxyeicosatrienoic acid (EET). These molecules are peroxisomal proliferation activated receptor agonists (PPAR) Roles in angiogenic and mitogenic signaling. PPAR PPAR receptor is located at the nuclear membrane and dimerizes with 9-cis retiniose acid receptor following ligand binding; this causes binding to DNA at PPAR response elements. PPAR response elements are located near genes involved in lipid metabolism 3 types PPAR receptors: alpha, beta, and gamma. PPAR gamma is a modulator of the inflammatory response in peripheral macrophages, and monocytes (8).  

AA gives rise to prostaglandins 1. NA, thrombin and bradykinin activate PLA2 leading to AA 2. 

AA metabolites include prostaglandins thromboxanes prostacyclin

3. PGH2 is parent prostaglandin 4. Subsequent metabolism is enzymedriven

Some enzymes

PGE2 is synthesized from PGH2 by PGE synthase (Several types; PGES1 is key enzyme). PGD2 is synthesized from PGH2 by PGD2 synthases (2 forms known). PGI2 is synthesized from PGH2 via prostacyclin synthase PGF2a is synthesized from PGH2 by PGF synthase

PGs interact with specific receptors (pre- or postsynaptic) in brain PGD2 interacts with DP1, 2 PGE2 interacts with EP1-4 PGF2 interacts with FPα, β

PGI2 interacts with IP Thromboxanes interact with TPα, β

PG Receptors

Modulate physiological and pathological cellular functions

AA gives rise to leukotrienes

NOTES AA is omega-6 fatty acid DHA is omega-3 fatty acid

Omega-3 fatty acids

α-Linolenic acid (ALA) is parent fatty acid EPA and DHA are precursors for lipid modulators ALA .. plant sources (flaxseed, walnuts, pecans, hazelnuts, and kiwifruit) EPA and DHA … salmon, tuna, and herring

Omega-3 fatty acids

Linoleic acid (LA) is parent fatty acid Most from vegetable oils (soybean oil, corn oil, borage oil, and acai berry) LA converted to γ-linolenic acid (GLA) GLA converted to arachidonic acid

Lipid Analysis

Technologies: Analytical techniques for fatty acids, phospholipids,  sphingolipids, triglycerides and steroids. Originally Thin layer chromatography (TLC) was used. Now replaced by faster technologies with better resolution. Gas chromatography-Mass Spectrometry (GC-MS) Used for low molecular weight lipids of all different classes (