Membrane Structure and Lipids.pdf

Membrane Structure and Lipids Presented by Dr. Kherie Rowe Reading: Lippincott Reviews in Biochemistry, Assistant Professor of 8th Ed., Chapter 17, pp. 223-233; Chapter 15, Biochemistry pp. 191; Chapter 18, pp. 244. [email protected]...

Membrane Structure and Lipids Presented by Dr. Kherie Rowe Reading: Lippincott Reviews in Biochemistry, Assistant Professor of 8th Ed., Chapter 17, pp. 223-233; Chapter 15, Biochemistry pp. 191; Chapter 18, pp. 244. [email protected] 1 1 Learning Objectives 1. Describe the defining physicochemical properties of lipids 2. Compare and contrast the following terms: lipid, fat, oil, detergent 3. Describe the properties of membrane lipids that are key for membrane formation and membrane properties 4. List the general types of lipids found in membranes 5. Describe the role of cholesterol in membranes 6. Explain the types of lipid movement in membranes and which require facilitation by proteins 7. Draw a schematic of membrane topology in a cell 2 2 Why Study Lipids? The structures of lipids drive the formation of important biological elements, membranes, lipid droplets, lipoproteins. They act as signaling molecules, inflammatory molecules and are known to be key in some disorders including metabolic disorders, cardiovascular disease, oncology and others. Many studies have positively correlated essential fatty acids with reduction of cardiovascular morbidity and mortality, infant development, cancer prevention, optimal brain and vision functioning, arthritis, hypertension, diabetes mellitus and neurological/neuropsychiatric disorders. 3 3  Lecture Outline 1. Lipid properties and structural features 2. Fatty acids and fat 3. Membrane lipids and membrane structure 4. Lipid components of membranes - Glycerophospholipids - Plasmalogens and cardiolipin - Sphingolipids and glycolipids - Cholesterol 5. Detergents 6. Membrane topology and lipid motion in membranes 4 4  What are Lipids? Fats, Oils, Detergents, Waxes Lipids Non-polar or amphipathic molecules Biological origin Constituents of membranes, fats, oils, waxes, detergents Biological Function Energy storage Compartmentation – membranes Signaling molecules (e.g., steroids) Vitamins Dietary Lipids Energy Heart disease 5 Lipids – Hydrophobic (non-polar) or amphipathic molecules of biological origin. 1. Not soluble in water except as aggregates in the form of micelles or vesicles or membrane sheets. Even so, they are more properly referred to as dispersions rather than having been dissolved. 2. All lipids are non-polar to some extent. 3. Many Lipids are amphipathic where the polar and non-polar parts are segregated within the molecule. Amphipathic lipids are required constituents of micelles, vesicles, and bilayer sheets. 5  What are Lipids? Fats, Oils, Detergents, Waxes Lipids – hydrophobic or amphiphilic (amphipathic) small molecules occurring in nature. Generally, insoluble or poorly soluble in water. Waxes – long chain or branched hydrocarbons, or esters. Hydrophobic solid or semisolid. Bees’ wax, candle wax. Oils – Hydrophobic liquid. Can be hydrocarbons, triglycerides, or fatty acids of varying length, but can be other chemical types as well. Most vegetable oils are triglycerides. Fat – In medicine and biology: triglycerides. In a broad context, a hydrophobic solid, or semisolid. Membrane lipids: Amphipathic. Detergents – natural or synthetic amphiphilic compounds that act as surfactants (form micelles). Phosphatidyl Choline: Saturated FA and unsaturated FA. 6 Verbiage: 1. Be familiar with the differences in the terms above 2. The key aspect of lipids is their hydrophobicity: All lipids are hydrophobic to some extent. Some may also have a polar part (amphipathic). Completely non-polar biological molecules (waxes, triglycerides, cholesterol esters) tend to form solids or fat (unless they are small, like the hydrocarbons in gasoline). 3. Amphipathic molecules are those with both polar and non-polar parts. i. These can form: Bilayers, vesicles, micelles, other exotic structures ii. Depending on the exact shape, they are classified as Detergents – tend to form micelles Membranous lipids – can form bilayers as vesicles, or membranes. Example of Membrane Lipids: Phosphatidylethanolamine molecules. 1. Polar head group 2. Glycerol backbone 3. Fatty acids, the principal hydrophobic component. 1. Note that the middle glycerol position (2 position) contains an 6 unsaturated fatty acid. This is typical for many lipids. The fatty acid in this position can be released in response to hormones and act as a signaling molecule or precursor (e. g. Arachidonic acid forming prostaglandins). 6  Lecture Outline 1. Lipid properties and structural features 2. Fatty acids and fat 3. Membrane lipids and membrane structure 4. Lipid components of membranes - Glycerophospholipids - Plasmalogens and cardiolipin - Sphingolipids and glycolipids - Cholesterol 5. Detergents 6. Membrane topology and lipid motion in membranes 7 7  Fatty Acids Saturated (palmitate) Monounsaturated Trans fatty acids: Unnatural, dietarily important Essential fatty acids: – Linoleic – Arachidonic acid precursor – Linolenic 8 Saturated versus unsaturated fatty acids (FA) Fatty acids with only one double bond are called mono-unsaturated Fatty acids with two or more double bonds are called poly-unsaturated The degree of unsaturation affects fluidity of both membrane lipids where fatty acids are constituents, as well as of fat. - Saturated fatty acids are the least fluid. Fat with high percentage of saturated fat is more solid; it is also less healthy in the diet - Mono-unsaturated fatty acids are more fluid than saturated FAs, but less so than poly-unsaturated FAs. - Poly-unsaturated FAs are more fluid than the others, depending on the degree (how many double bonds) and their placement in the hydrocarbon chain. Linoleic acid and linolenic acid are both essential fatty acids; they are required in the diet because they cannot be made by the body and are required for a number of functions. Trans-fatty acids: These are produced mostly from chemical hydrogenation of poly-unsaturated oil to alter their physical properties. They can also be produced 8 with prolonged heating of unsaturated or polyunsaturated oils, such as in deep fat fryers (think French fries). Most double bonds made in nature are cis-double bonds, so trans-fatty acids in high amounts are processed into triglycerides and membrane lipids along with normal fatty acids. Although they are unsaturated their structure makes them pack more like saturated hydrocarbons, and they have similar deleterious health effects. Size of fatty acids: Fatty acids are designated by the number of carbons. Only even numbers of carbons occur normally when synthesized in the human body, but odd ones can be found in the diet. You can have 4 up to about 24 carbons. The most common are in the range of 16-22 carbons, but the others occur as well See the following website for a list of some common fatty acids: https://en.wikipedia.org/wiki/Fatty_acid 8  Glycerides & Fat Glycerol Fats: Glycerol plus Fatty Acids (FA) Triglycerides (TG, TAG) Major energy storage form Fatty Acid Connected by ester bonds (detergent) (15 Kg in a typical person) Oils: most vegetable oils are principally triglycerides Mono-acyl Do not form bilayers Glycerol (detergent) Ester bonds: Labile: easy to make and break and transfer to other molecules Much more hydrophobic than the Triglyceride Fat carboxylic acid of fatty acids 9 Constitution of Fat Fat, in humans, is constituted of triglycerides (abbreviated TG or TAG). Triglycerides can also be oils. Whether triglycerides are oils (liquid) or fat (semisolid or solid), depends on the length of the fatty acid chains and their degree of unsaturation (the number of double bonds in the hydrocarbon chain). Triglycerides are constituted of 1 glycerol and three fatty acyl chains (FA for fatty acid) The fatty acids are attached to a glycerol backbone via ester bonds: ester bonds are relatively readily reversible. Glycerols can have one two or three FA attached: Glycerol plus 1 FAs is MonoAcylGlycerol (MAG) Glycerol plus 2 FAs is DiAcylGlycerol (DAG) Glycerol plus 3 FAs is TriAcylGlycerol (TAG or TG). Fatty acids and MAGs can act as detergents, and this is an important part of dispersing fat during digestion. 9 The carboxylic acid group of fatty acids is polar while the rest of the hydrocarbon chain is non-polar (hydrophobic). When the carboxylic acid reacts with the glycerol hydroxyl group to form an ester, the ester is more hydrophobic than the original carboxylic acid and hydroxyl groups. So, triglycerides are generally hydrophobic because of the ester bond formation. 9  Lecture Outline 1. Lipid properties and structural features 2. Fatty acids and fat 3. Membrane lipids and membrane structure 4. Lipid components of membranes - Glycerophospholipids - Plasmalogens and cardiolipin - Sphingolipids and glycolipids - Cholesterol 5. Detergents 6. Membrane topology and lipid motion in membranes 10 10  Major Membrane Lipid Types Glycerophospholipids – Phosphatidylcholine (PC) – Phosphatidylserine (PS) – Phosphatidylethanolamine (PE) Galacto – Phosphatidylinositol (PI) cerebroside Sphingolipids Phosphatidyl Choline (PC) Sphingomyelin – Phosphosphingolipids: sphingomyelin – Glycosphingolipids Cholesterol and Cholesterol esters Other Lipids Cholesterol Cardiolipin – CardioLipins, PAF, Plasmalogen, etc. 11 Membrane Lipids There are thousands of distinct lipid molecules. This arises mainly from the variation in the length and desaturation of fatty acid side chains. They can be mixed or matched in the various positions giving rise to a large variety of structures. This is one of the reasons lipid membranes are amorphous (don’t have a highly regular repeating pattern). There are also a variety of distinct head-groups, though fewer, and a variety of glycol-lipids. PAF: platelet activating factor (phospholipid mediator involved in initiating platelet aggregation, dilation of blood vessels, inflammation, allergic responses and shock). 11  Amphipathic & Membrane Lipids Formation of membranes, micelles, vesicles. Structures can form spontaneously, in vitro, or under regulated conditions in vivo. Whether a lipid forms vesicles, membranes or micelles depends on the individual lipid structure and the bulk composition. 12 Amphipathic lipids, depending on their structure, can form: 1. Monolayers (not shown) 2. Bilayers 3. Micelles Vesicles, or liposomes, are bilayers enclosing a limited aqueous compartment. They are typically spherically shaped, the bilayer forming the outer layer of the sphere. Bilayers occur in vesicles, but may also form larger, flatter sheets that constitute plasma membranes, the endoplasmic reticulum, etc. Bilayers have two monolayers juxtaposed with their non-polar (hydrophobic) parts together, and the polar parts interaction with water. To form vesicles or bilayer sheets, the lipids that constitute them must be amphipathic. Micelles are also formed from amphipathic lipid molecules called detergents. Detergents differ from membrane lipids in that they tend to not form bilayers, but rather form micelles. Detergents have the ability to carry hydrophobic molecules in their cores, thus effectively dissolving oils and fats. 12  Cells are Compartmentalized by Membranes Lipid bilayers act to compartmentalize many organelles and define the outer boundary of the cell. 13 13  Singer-Nicholson Model of Lipid Bilayers The Singer-Nicholson Model was the first to reconcile the apparent protein and lipid composition of membranes and illustrated the bilayer nature of cellular membranes. 14 14  EM of a Lipid Bilayer The characteristic ‘railroad track’ appearance of membranes in early electron micrographs were a clue to the structure of membranes as lipid bilayers. 15 15  Lecture Outline 1. Lipid properties and structural features 2. Fatty acids and fat 3. Membrane lipids and membrane structure 4. Lipid components of membranes - Glycerophospholipids - Plasmalogens and cardiolipin - Sphingolipids and glycolipids - Cholesterol 5. Detergents 6. Membrane topology and lipid motion in membranes 16 16  Lipid Components of Membranes One major class of lipids is the glycerophospholipids They have a glycerol backbone They have two fatty acids The middle position (2-position) fatty acid is typically unsaturated The end (1) position fatty acid may be saturated They have a phosphate at the other end of the glycerol They can have a headgroup attached to the phosphate 17 17  The Main Glycerol-Phospholipids Phosphatidylcholine (PC) Choline is a trimethylated ethanolamine PC is the most common phospholipid Choline can be synthesized in the body, but not in sufficient quantity. So, it is also required in the diet. PC can serve other function: Dipalmitoyl (C16)-PC is A Space-filling Model of PC part of lung surfactant and is important to reduce surface tension in the alveoli. Phosphatidylethanolamine (PE) Principal constituent of bacterial membranes Common constituent of eukaryotic/mammalian membranes 18 The structure of some phospholipids, the glycerol-phospholipids One major class of lipids is the glycerol-phospholipids. 1. They have a glycerol backbone 2. They have two fatty acids 1. The middle position (2-position) fatty acid is typically unsaturated. 2. The end (1) position fatty acid may be saturated. 3. They have a phosphate at the other end of the glycerol 4. They can have a headgroup attached to the phosphate Headgroups: 1. Choline 2. Ethanolamine 3. Serine 4. Inositol In addition to constituting a major structural component of membranes, many lipids participate in specific cell signaling events. The Main Glycerol-Phospholipids 18 1. Phosphatidylcholine (PC) 1. Choline is a trimethylated ethanolamine 2. PC is the most common phospholipid 3. Choline can be synthesized in the body, but not in sufficient quantity. So, it is also required in the diet. 4. PC can serve other function: Dipalmitoyl (C16)-PC is part of lung surfactant and is important to reduce surface tension in the alveoli. 2. Phosphatidyl-ethanolamine (PE) 1. Principal constituent of bacterial membranes 2. Common constituent of eukaryotic/mammalian membranes 3. The right-hand figure shows a space-filling model of PC 18  The Main Glycerol-Phospholipids Phosphatidylserine (PS) Found on the inner leaflet of the plasma membrane Has a negatively charged headgroup. In contrast, PE and PC are zwitterionic Serves as a signal in apoptosis Less common than PC and PE Phosphatidyl-inositol (PI) Phosphatidyl-Inositol A relatively minor constituent in mammalian membranes The inositol headgroup can be phosphorylated, and is involved in intracellular signaling (e.g., PIP2, PIP3) IP3 acts to release calcium PIP3 acts as a signaling molecule 19 3. Phosphatidyl serine (PS) 1. Found on the inner leaflet of the plasma membrane 2. Has a negatively charged headgroup. PE and PC are zwitterionic 3. Serves as a signal in apoptosis. 4. Less common than PC and PE 4. Phosphatidyl-inositol (PI) 1. A relatively minor constituent in mammalian membranes. 2. The inositol headgroup can be phosphorylated, and is involved in intracellular signaling (e.g. PIP2, PIP3). 1. IP3 acts to release calcium 2. PIP3 acts as a signaling molecule. PLC: phospholipase C 19 Plasmalogens and Cardiolipin Some variations on lipid structure: Cardiolipin is a specialized lipid found principally in the mitochondrial inner membrane. The role of this lipid is to make the membrane more impermeable to ions. Plasmalogens can constitute a substantial percentage of lipids in some membranes (e.g., plasma membranes) and in myelin. They have a similar structure to phospholipids except that the end fatty acyl group has an ether linkage and a double bond rather than the ester linkage found in fat and glycerophospholipids. The synthesis of plasmalogens is initiated in peroxisomes. Peroxisomal biogenesis disorders, such as Zellweger syndrome, severely affect plasmalogen synthesis and causes plasmalogen deficiency with severe developmental problems. 20 20 Plasmalogens vs Diacylglycero- phospholipids Basic plasmalogen structure. (A) Plasmalogens are glycerophospholipids characterized by the presence of a vinyl-ether linkage at the sn-1 position and an ester-linkage at the sn-2 position. R1 and R2 represent straight-chain carbon groups. At the sn-1 position, the chemical moiety highlighted in red is an alkenyl group, which are used to measure plasmalogen abundance and molecular composition. These alkenyl groups are most commonly derived from C16:0, C18:0, or C18:1 fatty alcohols. The sn-2 position of plasmalogens is occupied typically by polyunsaturated fatty acids. X represents the head group, typically ethanolamine or choline for plasmalogens. In contrast, (B) diacylglycerophospholipids have ester-linkages at their sn-1 and sn-2 positions. As above, R1 and R2 represent straight-chain carbon groups and X represents the head group. 21 21  Degradation of Phospholipids 22 Phospholipases Type A: Cleaves fatty acids from the glycerol backbone. Type B: Not shown, can cleave fatty acids at either the 1 or 2 positions, usually uses lyso-phosphatidylcholine as a substrate. Type C: Cleaves the head-group between the glycerol and the phosphate. Type D: Cleaves the head-group after the phosphate. Phospholipase A2 is important because it is activated through hormone signaling mechanisms. This can release arachidonic acid from PI or PC. The arachidonic acid is subsequently converted to prostaglandins which act as further downstream signals on nearby cells (paracrine signaling). Phospholipase C can release IP3 from PIP2 and generates DAG as well. This is part of hormone second messenger signaling systems (see next slide). There are many (~13) subtypes of phospholipase C, and they are tightly regulated. 22 Phosphatidyl Inositol Cleavage PI cleavage is highly regulated to produce IP3 IP3 mediates Ca2+ release from intracellular stores PI-specific phospholipase C is regulated through transmembrane signaling 23 23  Lecture Outline 1. Lipid properties and structural features 2. Fatty acids and fat 3. Membrane lipids and membrane structure 4. Lipid components of membranes - Glycerophospholipids - Plasmalogens and cardiolipin - Sphingolipids and glycolipids - Cholesterol 5. Detergents 6. Membrane topology and lipid motion in membranes 24 24  Sphingolipids and Glycolipids Sphingomyelin Galactocerebroside Ganglioside GM2 25 Sphingolipids and Glycolipids 1. Sphingomyelin – A constituent of many membranes, but more so in neuronal membranes and myelin. 1. Has a phosphocholine head-group, similar to phosphatidylcholine’s head-group. 2. Structure is based on serine rather than glycerol 3. Hydrocarbon chains are connected via aliphatic bonds or amide bonds. This differentiates the sphingolipids from glycerophospholipids, which use ester bonds. 4. Basic structure of ceramide – outlined by red dashed line. 5. Sphingosine – an important breakdown intermediate. 2. Galactocerebroside – a simple glycolipid 1. Note the ceramide basis structure 2. Galactose is attached in a glycosidic bond 3. Galactose can be further modified by addition of sulfate or other sugars 3. Ganglioside GM2 1. An example of a glycolipid 2. Note the complex sugars – these are modified from glucose or 25  galactose or other sugars by addition of N-acetyl groups or carboxylic acids, etc. Glycosphingolipids serve a role in intercellular communication. They are found on the outer leaflet of the plasma membrane where they interact with the extracellular environment. They play a role in regulation of the cellular interactions, growth and development. They act as antigenic determinants of the ABO blood groups and are also a source of various embryonic antigens important in particular stages of fetal development. 25  The Glycocalyx Glycolipids on epithelial cells 26 Glycolipids Glycolipids can from an extensive carbohydrate layer on the outer leaflet of the bilayer. The glycolipids also provide mechanical integrity by protecting the bilayer from breakage. 26  Cholesterol and Cholesterol Esters Cholesterol Up to 30% of membranes Modulates fluidity: Stiffens fluid membranes (rich in unsaturated FA) Prevents stiffening of membranes rich in saturated FA 27 Cholesterol 1) Cholesterol is a major constituent of lipid bilayer membranes. 2) Cholesterol can be obtained from the diet or synthesized from AcCoA in the liver. 3) Cholesterol is highly regulated. 4) Dysfunction of cholesterol regulation is implicated in cardiovascular disease. 5) Cholesterol is carried through the body in lipoprotein particles, such as Chylomicrons, low-density lipoprotein, and high-density lipoprotein particles. Structural role of cholesterol: 1) Cholesterol is an amphipathic molecule, like other membrane lipids, because of the presence of the polar hydroxyl group. 2) Orientation is oriented with the polar group to the outside. 3) A major role of cholesterol is modifying bilayer fluidity. 4) Cholesterol modulates membrane fluidity by broadening lipid phase transitions. Thus, gel-like, or stiff membranes become more fluid. Highly fluid membranes become stiffer, by the addition of cholesterol. 27 The synthesis and regulation of cholesterol will be covered in greater depth in later lectures. 27 Key Observations: 1. Membranes have a unique lipid composition suited to their role 2. Composition can vary greatly depending on the organelle or species 3. Cholesterol is a major, normal component of membrane composition Note, that because of the variation in fatty acid length and unsaturation, there are dozens of possible fatty acids that can be attached in these lipids leading to hundreds of chemically distinct lipids. However, some occur more commonly than others. 28 28  Lecture Outline 1. Lipid properties and structural features 2. Fatty acids and fat 3. Membrane lipids and membrane structure 4. Lipid components of membranes - Glycerophospholipids - Plasmalogens and cardiolipin - Sphingolipids and glycolipids - Cholesterol 5. Detergents 6. Membrane topology and lipid motion in membranes 29 29  Amphipathic Lipids: Membrane Lipids vs Detergents Detergents versus other lipids Detergents have a higher headgroup size relative to the tail Common detergents: Lysophosphatidylcholine Monoacylglycerol Sodium dodecyl sulfate Cholate, chenodeoxycholate (cholesterol derivatives) Fatty acids Membrane Lipids: good size match of headgroup and fatty-acyl chains 30 Amphipathic lipids, depending on their structure, can form: 1. Monolayers (not shown) 2. Bilayers 3. Micelles Vesicles, or liposomes, are bilayers enclosing a limited aqueous compartment. They are typically spherically shaped, the bilayer forming the outer layer of the sphere. Bilayers occur in vesicles, but may also form larger, flatter sheets that constitute plasma membranes, the endoplasmic reticulum, etc. Bilayers have two monolayers juxtaposed with their non-polar (hydrophobic) parts together, and the polar parts interaction with water. To form vesicles or bilayer sheets, the lipids that constitute them must be amphipathic. Micelles are also formed from amphipathic lipid molecules called detergents. Detergents differ from membrane lipids in that they tend to not form bilayers, but rather form micelles. In most cases, this is because the polar headgroup is bigger than the hydrophobic acyl chain. However, the bile acid detergents, have 30 somewhat different micellar structures. In all cases, detergents carry hydrophobic molecules in their cores, thus effectively dissolving oils and fats. 30  Lecture Outline 1. Lipid properties and structural features 2. Fatty acids and fat 3. Membrane lipids and membrane structure 4. Lipid components of membranes - Glycerophospholipids - Plasmalogens and cardiolipin - Sphingolipids and glycolipids - Cholesterol 5. Detergents 6. Membrane topology and lipid motion in membranes 31 31  Membrane Topology Lipid Bilayer Asymmetry Some lipids are on both the inner and outer membrane leaflets, while others are confined principally to one or the other resulting in lipid bilayer asymmetry 1. Plasma membrane outer leaflet is in contact with extracellular space 2. Plasma membrane inner leaflet is in contact with the cytoplasmic space Phosphatidylserine and phosphatidyl- inositol are on the inner leaflet Glycolipids on the outside of the cell or in the lumen of organelles Specific proteins maintain membrane topology (scramblases, flippases) 32 32  Lipid Motion Membrane lipids have several types of intrinsic motion they can undergo: Lateral movement Rotation Fatty acyl chain flexing (fluidity) Flip-flop. This is very slow and requires protein assistance (flippases, floppases, scramblases) 33 Intrinsic lipid Motion 1. Most motions of lipids are fast and occur on a picosecond to microsecond scale. 1. Bond vibration and isomerization 2. Protrusion (individual lipids moving up and down in the bilayer) 3. Lateral diffusion 4. Rotational diffusion 5. Undulations (large scale ‘waves’ in the bilayer) 2. Flip-flop is a slow movement. Lipid flip-flop is the switching of the headgroup from one leaflet to the other. This requires moving the headgroup that is hydrophilic through the hydrophobic part of the bilayer. That is very energetically expensive. Therefore, it occurs only very slowly. Spontaneously, this will occur on the scale of hours. This is too slow to be of biological use, and therefore enzymes are required to facilitate this type of lipid movement. 33  Summary Lipids constitute fat, an energy source, and membrane lipids and detergents Fats are triglycerides Membranes comprise glycerophospholipids, cholesterol, proteins, sphingolipids, glycolipids, plasmalogens and other lipids Cholesterol serves to moderate membrane fluidity Lipids are distributed asymmetrically among the two leaflets. Movement from one leaflet to the other is slow and requires protein action Membranes effectively divide the cell into compartments. The leaflet in contact with the cytosol always remains in contact with the cytosol 34 34