Molecular Biology Of The Cell Flashcards
What are the 5 main classes of lipids?
1) Free fatty acids
2) Triacylglycerols
3) Phospholipids
4) Glycolipids
5) Steroids
Where and how are fatty acids stored?
The are often stored in cells in the form of triacylglycerols, molecules composed of 3 fatty acids attached to a glycerol molecule, via Ester linkages. Fatty acids are reduced and anhydrous, making them an ideal storage molecule.
What is the function of Ester linkages?
The linkages help to neutralise the carboxylic acid groups and hence keep the pH in cells within a normal range.
What does fatty acid metabolism produce and why?
Fatty acid metabolism ultimately ends,up in acetyl CoA production. Acetyl CoA is produced by both types of major food molecules (the other being sugars/polysaccharides) within the mitochondria of cells. Therefore, it is the location where most of the cellular oxidation reactions occur and where the majority of cellular ATP is produced.
Outline the 3 primary sources of fat
1) The diet
2) De novo biosynthesis (liver) : lipogenesis
3) Storage depots in adipose (in conditions of starvation)
Outline the origin of bile salts
Bile salts are generated from cholesterol, by the liver and stored in the gallbladder. Bile salts have a hydrophobic face, the planar organic molecules, and a hydrophilic face, with carboxylate groups and hydroxyl groups.
Explain the role of bile salts
During digestion, they pass from the bile duct into the intestine. They emulsify fats in the intestine, aiding their digestion and absorption fats and also that of fat-soluble vitamins (e.g. A, D, E and K).
What happens when there is a lack of bile salts?
This results in the majority of fat passing through the gut undigested and unabsorbed, resulting in steatorrhea (fatty stool).
What is Orlistat/ tetrahydrolipstatin?
It is a potent inhibitor of gastric and pancreatic lipases. It is a chemically synthesised derivative of lipstatin, a product of Streptomyces toxytricini. Orlistat reduces fat absorption by 30%, which is almost completely excreted by the faecal route. Large multi-centre randomised clinical trials have shown that Orlistat is effective in treating obesity for up to 2 years. It’s main side effects include abdominal pain, urgency to defecate, increased flatus (wind) and steatorrhea.
How are different lipids and cholesterol are transported around the body?
Lipids are transported in the plasma (essentially aqueous) by lipoproteins, as the break down of lipids produces very hydrophobic molecules. The lipoproteins can be categorised according to their density:
1) Chylomicrons (CM): produced in the intestines and play a role in dietary (exogenous) fat transportation.
2) Very low density lipoproteins (VLDL): produced in the liver and play a role in endogenous (originate from within the organism) fat transportation.
3) Intermediate density lipoproteins (IDL): produced by VLDLs, they serve as low density lipoprotein precursors.
4) Low density lipoproteins (LDL): produced by IDLs, they play a role in cholesterol transport (from the liver to peripheral tissue).
5) High density lipoproteins (HDL): produced by the liver, they play a role in reverse cholesterol transport (from peripheral tissue back to the liver).
Outline the transportation of dietary fats
Digested dietary products are absorbed by enterocytes that line the brush border of the small intestine. Triacylglycerols are resynthesised under the control of several enzymes prior to their incorporation into chylomicrons (CM). Chylomicrons then travel from the lacteals, the lymphatic vessels of the small intestine which absorb digested fats, to the thoracic duct and to the left subclavian vein where they enter the bloodstream. They acquire apoproteins from HDL following release into the blood stream.
Outline the function of lipoprotein lipase
Lipoprotein lipase is located inside the capillary endothelial cells lining a variety of tissues including adipose, heart and skeletal muscle. It emulsifies triacylglycerols, allowing them to be more easily digested. Fatty acids undergo beta-oxidation and glycerol is returned to the liver for use in gluconeogenesis.
Outline the anatomy of lipoproteins
Lipoproteins solve the problem of transporting hydrophobic molecules in an aqueous environment. A phospholipid monolayer contains cholesterol and apoproteins surround a core of cholesterol esters and triacylglycerols.
Outline cholesterol esterificatiom
When cholesterols are esterified, to make cholesterol esters, they are made even more hydrophobic. Cholesterol esters are synthesised in the plasma from cholesterol and the acyl chain of phosphatidylcholine (lecithin) via a reaction catalysed by lecithin-cholesterol acyltransferase (LCAT), forming lysophosphatidylcholine as a byproduct. This esterification of cholesterol makes it pack more tightly within the hydrophobic core of lipoproteins.
What is the function of apoproteins
They are what are recognised by the various receptors on tissues. When recognised by receptors on skeletal muscle, and adipose, the free fatty acids and cholesterol, within a chylomicron, can be taken up by the tissue.
What are LDLs?
Often referred to as “bad cholesterol” as prolonged elevation of LDL levels leads to atherosclerosis (hardening of the arteries), leading to things like myocardial infarctions and strokes. They transport cholesterol synthesised in the liver to peripheral tissues with more than 40% of their weight made up of cholesterol esters.
What are HDLs?
Often referred to as “good cholesterol” as they function to take cholesterol from peripheral tissues back to the liver for use or disposal (reverse cholesterol transport). They help to lower the total serum cholesterol.
Outline the mechanism for beta-oxidation of fatty acids
1) Firstly, fatty acids are converted into an acyl CoA species, using the enzyme acyl-CoA synthetase (ACS) on the outer mitochondrial membrane. This requires energy, so ATP is hydrolysed twice to give a adenosine monophosphate (AMP).
2) To translate the species into the matrix, it is coupled to the molecule carnitine to form acyl carnitine. Carnitine and acyl carnitine are moved to and from the matrix by the enzyme translocase.
3) The acyl CoA species then undergoes a sequence of oxidation, hydration and thiolysis (cleavage) reactions (collectively called beta-oxidation).
5) This results in the production of 1 molecule of acetyl CoA and an acyl CoA species which is 2 carbons shorter than the original.
What is primary carnitine disorder
This is an autosomal recessive disorder, which occurs in 1 in 100,000 live births in the USA per year (1 in 40,000 live births in Japan and 1 in 500 live births in the Faroe Islands). Symptoms appear during infancy or early childhood and include encephalopathies (brain diseases), cardiomyopathies, muscle weakness and hypoglycaemia. Mutations in a gene known as SLC22A5, which encodes a carnitine transporter, result in reduced ability of cells to take up carnitine, needed for the beta-oxidation of fatty acids. Carnitor/Levocarnitine can be used as a supplement to treat this.
Outline the beta-oxidation of palmitic acid
The beta-oxidation reactions continue to consecutively remove 2-carbon units from the acyl CoA, thereby producing acetyl CoA. On the final cycle (4-carbon fatty acyl CoA intermediate), two acetyl CoA molecules are formed. From 7 beta-oxidation reactions, the 16-carbon palmitoyl CoA molecule (“activated” palmitic acid) produces 8 molecules of acetyl CoA. During each cycle, 1 molecule of FADH2 and NADH are each produced. This produces a net 129 ATP molecules in oxidative phosphorylation. (35 from beta-oxidation as NADH produces 3 ATP and FADH2 produces 2 ATP; and 96 from acetyl CoA as each produces 12 ATP).
When can fatty acid produced acetyl CoA enter the TCA cycle?
Acetyl CoA generated by beta-oxidation enters the TCA cycle only if beta-oxidation and carbohydrate metabolism are balanced since oxaloacetate is needed for entry. Hence the adage “fat burns in the flame of carbohydrate”.
Outline ketone body formation
When endogenous fat break down predominates (e.g. during fasting), acetyl CoA forms acetoacetate, D-3-hydroxybutyrate and acetone - known collectively as ketone bodies.
Outline fatty acid biosynthesis/lipogenesis
Fatty acids are formed sequentially by decarboxylative condensation reactions involving the molecules acetyl CoA and malonyl-CoA.
1) Elongation of the acyl group to make fatty acids longer than 16 carbons occurs separately from palmitate synthesis in the mitochondria and endoplasmic recticulum.
2) The fatty acid undergoes reduction and dehydration by the sequential action of ketoreductase (KR), dehydratase (DH), and enol reductase (ER) activity. The growing fatty acyl group is linked to an acyl carrier protein (ACP).
3) Desaturation of fatty acids requires the action of fatty acyl-CoA desaturates, which generate double bonds.
Outline the differences between synthesis and beta-oxidation
1) In beta-oxidation, Coenzyme-A (CoA) is used as a carrier protein, whereas in lipogenesis, an acyl carrier protein (ACP) is used.
2) In beta-oxidation, the reducing power comes from FAD/NAD+, whereas in lipogenesis, the reducing power comes from NADPH.
3) Beta-oxidation occurs in the mitochondrial matrix, whereas lipogenesis occurs in the cytoplasm of cells.
Name the 2 enzymes involved in fatty acid biosynthesis
1) Acetyl CoA Carboxylase
2) Fatty acid synthase
Outline the role of fatty acid synthesis in cancer
In adults, de novo biosynthesis is restricted mainly to the liver, adipose tissue and lactating breast. Evidence suggests that reactivation of fatty acid synthesis also occurs in certain cancer cells. The inhibition of FASN by cerulenin (an antifungal antibiotic) has shown to reduce Timor growth of obstinate cancer cells.
Which family of enzymes catalyse they initial step in each cycle of beta-oxidation?
A family of different acyl-CoA-dehydrogenase catalyse the initial step in each cycle of beta-oxidation within the mitochondrial matrix. Each acyl-CoA-dehydrogenase can bind a fatty acid chain of varying lengths:
>Short-chain acyl-CoA-dehydrogenase (<6C)
>Medium-chain acyl-CoA-dehydrogenase (6C-12C)
>Long-chain acyl-CoA-dehydrogenase (13C-21C)
>Very long-chain acyl-CoA-dehydrogenase (>22C)
Outline medium-chain acyl-CoA-dehydrogenase deficiency (MCADD)
This is an autosomal recessive disorder mainly occurring in Caucasians. It occurs in 1 in 10,000 births in the UK per year. If undiagnosed, it can be fatal. It is thought to account for 1% of deaths from Sudden Infant Death Syndrome (SIDS). If diagnosed, using a MCADD screening (heel prick test on newborns), patients should never go without food for longer than 10-12 hours and adhere to a high carbohydrate diet. Patients with an illness resulting in appetite loss or severe vomiting may need i.v. glucose to make sure that the body is not dependent on fatty acids for energy.
Outline the metabolic features of the brain
The brain and nervous system accounts 2% of the body’s total mass, but uses 20% of resting metabolic rate. It requires a continuous supply of glucose, as it cannot metabolise fatty acids. Ketone bodies (e.g. beta-hydroxybutyrate) can partially substitute for glucose. Too little glucose (hypoglycaemia) causes faintness and coma. Too much glucose (hyperglycaemia) can cause irreversible damage. Even in the fasting state, glucose remains the main metabolic fuel of the brain.
Outline the metabolic features of skeletal muscle
Skeletal muscle account for about 40% of the body’s total mass. It’s ATP requirements vary depending on exercise. The requirements of light contraction of skeletal muscle, are met by oxidative phosphorylation, oxygen, together with glucose and fatty acids in the blood being used as fuel for the muscle. During vigorous contraction, oxygen becomes a limiting factor as ATP consumption is faster than the ATP supply from oxidative phosphorylation. This leads to the muscle stores of glycogen being broken down to produce ATP. Under anaerobic conditions, pyruvate is converted to lactate, which leaves the muscle and reaches the liver via the blood.
Outline the metabolic features of the heart
The heart account for 1% of total body mass but uses 10% of resting metabolic rate. As it must beat constantly, it is therefore designed for completely aerobic respiration, making it rich in mitochondria, utilising the TCA cycle substrates (e.g, free fatty acids, ketone bodies). Loss of oxygen supply to the heart can therefore be devastating, as it leads to cell death and myocardial infarction when the energy demand becomes much greater than the energy supply. The main metabolic fuel of the heart in the fasting state is fatty acids.
Outline the metabolic features of the liver
The liver undertake a wide repertoire of metabolic processes:
1) It is highly metabolically active
2) It can convert nutrient types.
3) It plays a central role in maintains blood glucose at 4.0-5.5 mM.
4) It is a storage organ, storing glucose as glycogen.
5) It plays a key role in lipoprotein metabolism (the transport of triglycerides and cholesterols).
Outline carbohydrate metabolism
1) Carbohydrates are broken down into simple sugars and enter the glycolytic pathway leading to the production of pyruvate.
2) Decarboxylation and reduction of pyruvate produces acetyl CoA which can enter the TCA cycle. This cycle produces reduced co-factors which are reoxidised by the electron transport chain which in turn is coupled to ATP production (Oxidative phosphorylation).
3) During extreme exercise, the ATP demands of the muscle outstrip the oxygen supply needed for aerobic respiration and lactate is produced.
4) During fasting, rather than enter the TCA, much of the acetyl CoA produced results in ketone body production.
What happens to excess substrates?
Excess glucose-6-phosphate can be used to generate glycogen in liver and muscle. Similarly, excess Acetyl CoA can be used to generate fatty acids, which are stored as triglycerides in adipose tissue.
Outline the other uses of pyruvate and glucose-6-phosphate outside of carbohydrate metabolism
1) Pyruvate and other TCA cycle intermediates can also be a source of some amino acids. The backbone of these molecules can be used to used to make nucleotides.
2) Glucose-6-phosphate via the pentose phosphate pathway can also be used as a source for nucleotide production in a pathway that generates the bulk of the NADPH needed for anabolic pathways e.g. cholesterol synthesis.
How does the body avoid hypoglycaemia?
During fasting, if plasma glucose concentrations fall below 3mM then the body will enter a hypoglycaemic coma. In the short term, to avoid hypoglycaemia the body can:
1) Breakdown of liver glycogen stores occurs to maintain plasma glucose levels.
2) Releases free fatty acids from adipose tissue.
3) Convert Acetyl CoA into ketone bodies via the liver.
What is gluconeogenesis necessary?
Both fatty acids and ketone bodies can be used by muscle, making more of the plasma glucose available for the brain. However, within 12-18 hr all glycogen stores are typically exhausted, hence the need for another pathway to generate glucose – gluconeogenesis.
Outline the origin of the substrates needed for gluconeogenesis
1) Lactate is generated by skeletal muscle during strenuous exercise, when the rate of glycolysis exceeds the rate of the TCA cycle and the electron transport chain. Lactate can be taken up by the liver and utilised to regenerate pyruvate by lactate dehydrogenase (LDH), also known as the Cori cycle.
2) Amino acids can be derived from the diet or during times of starvation (e.g. from the breakdown of skeletal muscle).
3) Triglyceride hydrolysis yields fatty acids and glycerol, the glycerol backbone being used to generate dihydroxyyacetone phosphate (DHAP) a molecule you may recall from step 5 of glycolysis.
Why is gluconeogenesis not simply a reversal of glycolysis?
As the three essentially irreversible reactions, catalysed by the kinases hexokinase, phosphofructokinase and pyruvate kinase in glycolysis, have to be bypassed with four additional enzymes in gluconeogenesis. ΔG for the straight reversal of glycolysis would be +90 kJ/mol which is energetically unfavourable. ΔG for gluconeogenesis is -38 kJ/mol.
Deaminiation of all 20 amino acids gives rise to which 7 molecules?
1) Pyruvate
2) Acetyl CoA,
3) Acetoacetyl CoA,
4) α-Ketoglutarate,
5) Succinyl CoA,
6) Fumerate and
7) Oxaloacetate.
Urea is lost as a waste product.
What are glutogenic amino acids?
Their skeletons can give rise to glucose via gluconeogenesis.
What are ketogenic amino acids?
Give rise to skeletons which cannot enter gluconeogenesis but can be used to synthesise fatty acids and ketone bodies. Fatty acids can also be converted into ketone bodies and used by tissues such as muscle and brain.
What role do triglycerides play in gluconeogenesis?
Triglycerides are broken down into fatty acids and glycerol. Glycerol can be converted to DHAP and enter the gluconeogenic pathway upstream.
Can fatty acids be converted into glucose by gluconeogenisis?
Fatty acids cannot be converted into glucose by gluconeogenesis. 2C atoms enter the TCA cycle as acetyl CoA by combining with oxaloacetate to form citrate. As the cycle progresses, two carbon atoms are sequentially lost as CO2 before oxaloacetate is eventually regenerated. Hence, no net synthesis of oxaloacetate or pyruvate is possible from acetyl CoA .
Outline aerobic respiration during moderate exercise
During moderate levels of exercise, where oxygen supply is adequate, the ATP demands of muscle can be met by oxidative phosphorylation using glucose and other substrates as fuels. Glucose is transported from the blood into muscle cells where it can undergo metabolism by glycolysis and the TCA cycle to ultimately generate ATP by the re-oxidation of cofactors.
How is increased demand for ATP met in aerobic respiration?
As muscle contracts, the demand for ATP increases (e.g. requirements of muscle actomyosin ATPase and cation balance). Increased demand for glucose is met by an increase in the number of glucose transporters on the membranes of muscle cells.
What role does Adrenalin play in aerobic respiration?
Adrenalin plays a key role in meeting the demand for ATP by increasing the rate of glycolysis in muscle, increasing the rate of gluconeogensis by the liver and increasing the release of fatty acids from adipocytes.
Outline anaerobic respiration
Under anaerobic conditions, the demands of the contracting muscle for ATP cannot be met by oxidative phosphorylation and similarly, the transport of glucose from the blood cannot keep up with the demands of glycolysis. Glycogen within the muscle is therefore broken down to meet these demands. To replenish NAD+ levels and maintain glycolysis, pyruvate is taken up by the liver and converted into lactate by lactate dehydrogenase. Lactate can then be used by the liver to generate glucose by gluconeogenesis.
Outline the control of metabolic pathways
Blood glucose concentrations are typically maintained at around 4mM. Control of metabolic pathways is typically centred around reactions that are irreversible steps. At these points, increases in the rate of enzyme activity greatly increases the rate of the downstream steps. For the greatest levels of control it is desirable that these control steps are reasonably early in the pathway.
Control can be at several levels including:
1) product inhibition
2) under the influence of signalling molecules such as hormones
Where are the suitable isoforms of Hexokinase found?
Hexokinase catalyses the first irreversible step in the glycolysis pathway. Muscles and the liver contain suitably different forms (isoforms) of this enzyme. Both isoforms catalyse the same reaction. However they are maximally active at different concentrations of glucose.
What is the Michaelis constant?
Parameters such as the Michaelis constant (KM) allows comparison of relative activities of enzymes, by indicating the concentration of substrate at which an enzyme functions at a half-maximal rate (Vmax).
What is the KM of Hexokinase I and where is it found?
The KM of Hexokinase I found in muscle is 0.1mM, which means it is active at low concentrations of glucose and is essentially operating at maximal velocity at all times.
What product inhibits Hexokinase I?
Hexokinase I is also highly sensitive to inhibition by the product glucose-6-phosphate. This means that under anaerobic conditions when the rate of the TCA cycle drops, and glycolysis therefore slows, Hexokinase I is inhibited by accumulating levels of glucose-6-phosphate.
What is the KM of Hexokinase VI and where is it found?
Hexokinase IV found in liver, has a high KM of around 4mM and is much less sensitive to blood glucose concentrations than Hexokinase I. It is also less sensitive to the inhibitory effects of glucose-6-phosphate.
What is Glucose-6-Phosphatase and where is it found?
Glucose 6-phosphatase, found in the liver but not in muscle, can catalyse the reverse reaction to hexokinase, generating glucose from glucose-6-phosphate.
Outline the 4 hormones used to control blood glucose levels
1) Insulin: secreted when glucose levels rise: it stimulates uptake and use of glucose and storage as glycogen and fat. It also suppresses the production of glucagon.
2) Glucagon: secreted when glucose levels fall: it stimulates production of glucose by gluconeogenesis and breakdown of glycogen and fat.
(both are secreted by islets of the pancreas).
3) Adrenalin (or epinephrine): has strong and fast metabolic effects to mobilise glucose for “flight or fight”.
4) Glucocorticoids: steroid hormones which increase synthesis of metabolic enzymes concerned with glucose availability.
What are the 5 effects of insulin secretion, due to glucose levels rising after a meal?
On having a meal, blood glucose levels initially rise which is controlled by increased secretion of insulin (and reduced glucagon) from islets.
This has several effects including:
1) Increased glucose uptake by liver – used for glycogen synthesis and 2) Glycolysis (acetyl-CoA produced is used for fatty acid synthesis).
3) Increased glucose uptake and glycogen synthesis in muscle.
4) Increased triglyceride synthesis in adipose tissue.
5) Increased usage of metabolic intermediates due to a general stimulatory effect on the body’s synthesis and growth.
What controls the fall of blood glucose levels after a meal?
After a meal blood glucose levels start to fall and are controlled by:
1) Increased glucagon secretion (and reduced insulin) from islets.
2) Glucose production in liver resulting from glycogen breakdown and gluconeogenesis.
3) Utilisation of fatty acid breakdown as alternative substrate for ATP production (important for preserving glucose for brain).
How does Adrenalin help decrease glucose levels after a meal?
Adrenalin also stimulates glucose production in the liver, but also stimulates skeletal muscle towards glycogen breakdown and glycolysis, and adipose tissue towards fat lipolysis to provide other tissues with alternative substrate to glucose.
Outline the 5 effects of prolonged fasting
After prolonged fasting (i.e. longer than can be covered by glycogen reserves):
1) The glucagon/insulin ratio increases further
2) Adipose tissue begins to hydrolyse triglyceride to provide fatty acids for metabolism
3) TCA cycle intermediates are reduced in amount to provide substrate for gluconeogenesis
4) Protein breakdown provides amino acid substrates for gluconeogenesis
5) Ketone bodies are produced from fatty acids and amino acids in liver to substitute partially the brain’s requirement for glucose
What is diabetes mellitus?
Diabetes mellitus is a disorder of insulin release and signalling, resulting in an impaired ability to regulate blood glucose concentrations. The overall effect is that metabolism is controlled as if the person is undergoing starvation, regardless of dietary glucose uptake.
What are the two main types of diabetes mellitus?
1) Type I diabetes in which individuals fail to secrete enough insulin (β-cell dysfunction).
2) Type II diabetes in which individuals fail to respond appropriately to insulin levels (insulin resistance).
Outline the 4 main complications of diabetes mellitus
1) Hyperglycaemia with progressive tissue damage (e.g. retina, kidney, peripheral nerves)
2) Increase in plasma fatty acids and lipoprotein levels with possible cardiovascular complications
3) Increase in ketone bodies with the risk of acidosis
4) Hypoglycaemia with consequent coma if insulin dosage is imperfectly controlled
Outline the 3 key reasons as to why Glucagon is important
1) Glucagon is important in protection against hypoglycaemia.
2) A major site of action is the liver where glucagon stimulates gluconeogenesis and glycogenolysis.
3) Insulin deficiency and relative excess of glucagon leads to increased hepatic output of glucose and, thus, hyperglycaemia.
What is the Extracellular matrix?
It is a complex network of macromolecules (proteins and carbohydrates) filling spaces between cells and compromises of both fibrillar and non-fibrillar components. It is formed from the material deposited by cells which forms the “insoluble” part of the extracellular environment. It is generally composed of fibrillar (or reticular) proteins (e.g. collagens, elastin) embedded in a hydrated gel (proteoglycans or “ground substance”). It may be poorly organised (e.g. loose connective tissue) or highly organised (e.g. tendon, bone, basal lamina)
What are the key functions of the Extracellular matrix?
1) Provides physical support
2) Determines the mechanical and physicochemical properties of the tissue
3) Influences the growth, adhesion and differentiation status of the cells and tissues with which it interacts
4) Essential for development, tissue function and organogenesis
What are collective tissues
These are particularly rich in Extracellular matrix and component cells. All connective tissues have a distinct spectrum of collagens (types I, II, III and IV - basement membrane), multi-adhesive glycoproteins (fribronectin, fibrinogen and laminin - basement membrane) and proteoglycans (aggrecan, versivan, decorin and perlecan - basement membrane), that make them unique. Matrix components interact with specific cell surface receptors.
Outline the varied properties of connective tissues
The different types and arrangement of collagen coupled with the presence or absence of different components of the Extracellular matrix, gives a wide variety of connective tissues:
1) Vitreous humour (jelly that fills the interior of the eye): relatively soft and transparent.
2) Tendon and skin: tough and flexible
3) Bone: hard and dense
4) Cartilage: resilient and shock absorbing
Outline the 4 ways in which ECM abnormalities cause disorders
1) Gene mutations affecting matrix proteins
2) Gene mutations affecting ECM catabolism
3) Fibrotic disorders due to excessive ECM deposition
4) Excessive loss of ECM
What are collagens?
They are a family of fibrous proteins found in all multicellular organisms. They are major proteins in bone, tendon and skin, and are the most abundant proteins in mammals (25% of total protein mass).
How are collagen fibrils aligned?
1) Skin: successive layers nearly at right angles to each other
2) Mature bone and cornea: same arrangement
These tissues resist tensile force in all directions
Outline the molecular arrangements of collagen fibres
28 collagen types exist in humans, designated by roman numerals. There are 42 genes encoding collagens in humans. Each collagen molecule comprises three alpha chains, forming a triple helix. Collagen molecules can be composed of one or more different alpha chains.
Outline the structure of type I collagen
Type I collagen is a heterotrimer, has chains from two different genes - its composition is [alpha1(I)]2 [alpha2(I)].
Outline the structure of types II and III collagen
Types II and III collagen are homotrimers, having only one chain type each – their compositions are, therefore, [alpha1(II)]3 and [alpha1(III)]3, respectively.
Outline the structure of the collagen triple helix
It has a characteristic gly-x-y repeat, where x is often proline and y is often hydroxyproline. In fibrillar collagens, each alpha chain is approximately 1000 amino acids, forming a left-handed helix. Three alpha chains form a stiff triple helical structure – every third position must be occupied by glycine, which is small enough to occupy the interior (H side chain).
Outline the assembly of collagen fibres
Three alpha chains form a triple-stranded collagen molecule, which can associate to form fibrils. Fibrils can then come together to form collagen fibre.
Outline collagen biosynthesis
All newly synthesised collagen chains have non-collagenous domains at N- and C-termini. These domains are removed after secretion in the case of fibrillar collagens but remain part of the collagen in most other types.
Outline the function of covalent cross-links in collagen
Crosslinking provides tensile strength and stability. Both lysine and hydroxy-lysine residues are involved. The type and extent of cross-links is tissue specific and changes with age.
Outline lysine and proline hydroxylation
Prolyl and lysyl hydroxylation, in protein produces hydrolysine and hydroxyproline. Their hydroxylases require Fe2+ and vitamin C and contributes to interchain hydrogen bond formation. Lysine and hydroxylysine are also modified in the formation of covalent crosslinkages. This takes place only after the collagen has been secreted. Vitamin C-deficiency results in underhydroxylated collagens, with dramatic consequences for tissue stability (scurvy).
What are Ehlers-Danlos syndromes (EDS)?
Ehlers–Danlos syndromes (EDS) are a group of inherited connective tissue disorders whose symptoms include stretchy skin and loose joints.Several disoders arise due to mutations in collagen, which negatively affect:
•collagen production
•collagen structure
•collagen processing
Do all collagens form fibrils?
No, some collagens do not form fibrils. These include:
1) Fibril-associated collagens (e.g types IX and XII): associate with fibrillar collagens and regulate the organisation of collagen fibrils.
2) Type IV collagen: a network-forming collagen and is present in all basement membranes, though its molecular constitution varies from tissue to tissue.
What are basement membranes?
Basement membranes (BMs, also called basal laminae) are flexible, thin mats of extracellular matrix underlying epithelial sheets and tubes. They surround muscle, peripheral nerve and fat cells and underlie most epithelia. They are highly specialised extracellular matrices containing a distinct repertoire of collagens, glycoproteins and proteoglycans. In the kidney, they form a key part of the filtration unit as the Glomerular basement membrane (GBM).
What are elastic fibres?
Whereas collagens are important for the tensile strength of tissues, elastic fibres are important for the elasticity of tissues, such as skin, blood vessels and lungs. Often, collagen and elastic fibres are interwoven to limit the extent of stretching. Elastic fibres consist of a core made up of the protein elastin, and microfibrils, which are rich in the protein fibrillin.
Outline the function of fibrillin
The integrity of elastic fibers depends upon microfibrils, containing the protein fibrillin. Mutations in the protein fibrillin-1 are associated with Marfan’s syndrome which has some diverse manifestations, involving primarily the skeletal, ocular, and cardiovascular systems.
What is elastin?
Elastin is an unusual protein consisting of two types of segments that alternate along the polypeptide chain: hydrophobic regions, and α-helical regions rich in alanine and lysine. Many lysine side chains are covalently cross-linked.
What are laminins?
Laminins are heterortrimeric proteins made up of an α chain, a β chain and a γ (gamma) chain, which form a cross shaped molecule. Laminins are very large proteins with each chain having a molecular weight of between 160 and 400 kDa. Laminins are multi-adhesive proteins which can interact with a variety of cell surface receptors including integrins and dystroglycan. They can self-associate as part of the basement membrane matrix, but can also interact with other matrix components such as type IV collagen, nidogen and proteoglycans.
What are fibronectins?
Fibronectins are a family of closely related glycoproteins of the extracellular matrix which are also found in body fluids. They can exist either as an insoluble fibrillar matrix or as a soluble plasma protein. They are derived from a single gene, with alternate splicing of mRNAs giving rise to the different types. Like laminin, fibronectins are multi-adhesive proteins, made up of a large multidomain molecule linked together by disulphide bonds.
Outline the function of fibronectins
Again, like laminin, fibronectins are able to interact with cell surface receptors and other matrix molecules. They play important roles in regulating cell adhesion and migration in a variety of processes, notably embryogenesis and tissue repair. They are also important for wound healing, helping to promote blood clotting. Fibronectins can also bind multiple ligands and cell receptors.
What are proteoglycans?
Proteoglycans are core proteins to which are covalently attached to one or more glycosaminoglycan (GAG) chains. Small proteoglycans can have a single GAG chain attached, whereas some large proteoglycans carry up to 100 GAG chains.
What are GAG chains?
GAG chains are made up of repeating disaccharide units with one of the two sugars being an amino sugar (a sugar in which a hydroxyl group is replaced with an amine group). Many GAGs are sulfated or carboxylated, and as a result carry a high negative charge. This charge attracts a cloud of cations including Na+, resulting in large amounts of water being sucked into the extracellular matrix.
List the 4 proteoglycan families grouped upon their structural and functional characteristics
1) Basement membrane proteoglycans : e.g. perlecan
2) Aggregating proteoglycans (interact with hyaluronan): e.g. aggrecan
3) Small leucine-rich proteoglycans: e.g.decorin
4) Cell surface proteoglycans: e.g. syndecans 1-4
What is the function of cartilage?
Cartilage has a matrix rich in collagen with large quantities of GAGs trapped within the meshwork. The balance of swelling pressure is negated by the tension in the collagen fibres, generating great tensile strength. For example the cartilage lining the knee joint (synovial cartilage) can support pressures in excess of hundreds of Kg/cm2.
List the 4 main groups of GAG chains according to the repeating disaccharide unit
1) Hyaluronan
2) Chondroitin sulfate and dermatan sulfate
3) Heparan sulfate
4) Keratan sulfate
What is Hyaluronan?
Hyaluronan (also called hyaluronic acid) is found in the extracellular matrix of soft connective tissues. It is distinct form the other GAGs as it is simply a carbohydrate chain without a core protein. It is unsulfated and made up of repeating disaccharides which can number up to 25,000 sugars. It can undergo a very high degree of polymerization, typically in the range of 10,000 disaccharides creating molecules of enormous sizes. This means that hyularonan chains can occupy a relatively large volume. It is typically of high viscosity e.g. in the vitreous humour of the eye and in synovial fluid of joints. In the latter location, hyaluronan plays a key role in protecting the cartilaginous surface from damage.
What is Aggrecan?
Aggrecan is a major constituent of the cartilage extracellular matrix. In aggrecan, the GAGs are highly sulfated, increasing their negative charge. Also present are large numbers of negatively carboxyl groups. These multiple negative charges attract cations such as Na+ that are osmotically active. This in turn leads to large quantities of water being retained by the highly negatively charged environment. Under compressive load, water is given up, but regained once the load is reduced. Therefore, aggrecan in the cartilage matrix is perfectly suited to resist compressive forces.
What are tissues?
These are a group or groups of cells whose type, organisation and architecture are integral to its function. Tissues are made up of cells, extracellular matrix and fluid.
What are the 5 main cell types?
1) Connective tissue cells: fibroblasts (many tissues), chondrocytes (cartilage), osteocytes (bone). Mesenchymal (connective tissue and muscle) cancers are sarcomas.
2) Contractile tissues: skeletal muscle, cardiac muscle, smooth muscle.
3) Haematopoietic cells: blood cells, tissue-resident immune cells, and the cells of the bone marrow from which they are derived. Haemopoietic cancers are leukaemias (from bone marrow cells) or lymphomas (from lymphocytes).
4) Neural cells: cells of the nervous system having two main types; neurones (carry electrical signals) and glial cells (support cells). Neural cell cancers are neuroblastomas (from neurones) or gliomas (from glial cells).
5) Epithelial cells: cells forming continuous layers, these layers line surfaces and separate tissue compartments and have a variety of other functions. Epithelial cancers are carcinomas.
Outline epithelial organisation
Epithelial cells make organised, stable cell-cell junctions to form continuous, cohesive layers. Epithelial layers line internal and external body surfaces and have a variety of functions (e.g. transport, absorption, secretion, protection). Cell-cell junctions are key to the formation and maintenance of epithelial layers.
Outline epithelial classification
The two main criteria of epithelial classification are: >their shape: •squamous - flattened plate-shape •cuboidal - cuboid •columnar - arranged in columns > Their layering: •single layer - simple epithelium •multi-layered - stratified epithelium This classification is related to types of epithelial function.
Outline the single squamous epithelium
These arrangements are found in the lung alveolar (air sac) epithelium, mesothelium (lining major body cavities), endothelium lining blood vessels and other blood spaces). They form a thin epithelium that allows exchange to occur (e.g. gas exchange in the alveoli).
Outline the simple cuboidal epithelium
These epithelial cell arrangements are typical of the linings found in ducts (e.g. those lining the kidney collecting ducts).
Outline the simple columnar epithelium
These epithelial cell arrangements are typical of surfaces involved in absorption and secretion of molecules (e.g. enterocytes lining the gut, involved in the take up of the breakdown products of digestion).
Outline the 2 main types of the stratified squamous epithelium
1) Keratinizing: Epithelial cells which produce keratin and in doing so die becoming thicker, stronger, protective structures (e.g. epidermis - skin epithelium). Such cells lose their cellular organelles and nuclei, which are not visible under light microscopy. Keratinizing epithelium can form thick layers that protect underlying tissues for various physical and chemical insults (e.g. heat, cold, solvents (alcohol), abrasion, etc).
2) Non-keratinizing: Epithelial cells which do not undergo keratinisation. They retain their nuclei and organelles (e.g. epithelium lining the mouth, oesophagus, anus, cervix and vagina).
In the various layers, the cell shapes vary. The squamous classification relates to the surface cells.
Outline the pseudo-stratified epithelium
This epithelium appears to be multi-layered and are found in: the airway (trachea and bronchi) epithelium, various ducts in the urinary and reproductive tracts. On close examination, the surface cells have contact with the basal lamina.
Outline epithelial cell polarity
In a typical epithelial cell, the membrane can be seen to be organised into discrete domains by the formation of junctions. This membrane polarity is the key to generating a distinct polarity, with an apical domain at the lumenal (open) surface and a basolateral domain. The basal surface in contact with the extracellular matrix. The membrane between these two surfaces, where membranes of adjacent cells appose each other, is the lateral membrane. Most epithelial functions are directional, e.g. secretion, fluid and solute transport and absorption. These processes are not random but are highly organised. Epithelial polarity is required to give the directionality needed for epithelial function. Polarity in epithelial cells is is seen as different regions of the cell surface being different from one another, with discretely organised cellular contents.
Outline polarity in transporter epithelial
The pumps and channels involved in transporting ions and fluids across epithelial layers, need to be polarised. When they’re not polarised, that means that they’re present in all parts of the plasma membrane, so they’re pumping apically and basal laterally. Consequently, the direction flow is in all directions meaning that there is no desired net directional flow. If the transporters and channels are polarised, then directionality can’t be achieved because only one aspect of the plasma membrane is being pumped on, allowing the flow to be in the desired direction.
Outline the function of polarity in secretion
Most epithelial secrete in one direction only, either to the apical aspect into a lumen, or to a basal aspect into the interstitial space. In order to do that, the secretory machinery has to be polarised. If it were unpolarised, epithelial cells would secrete into both the apical and basal compartments, which could be catastrophic if secreting digestive enzymes into the basal aspect, as the body would digest its own tissues.
Outline 4 main types of of cell-cell junctions in epithelia (top to bottom)
1) Tight junction: form a belt, usually around the apical lateral membrane. They seal the gaps between the cells.
2) Adherens junction: essentially the master junction which controls the formation of all of the other junctions.
3) Desmosomes: spot junctions, scattered throughout the lateral membrane, that form mechanically tough junctions between cells. They are important in tissues that require to resist mechanical stresses.
4) Gap junction: a channel forming junction, that form pores between cells and allow cells to exchange and share materials. These act as communicating junctions to allow cells to form communities and synchronise a number of activities.
Outline the structure and function of transporting epithelia
In these epithelia, the plasma membranes contain high concentrations of ion transporters. Typically, mitochondria are closely associated with extensive basal membrane infoldings, providing energy for active transport across the abundant membranes. The infoldings increase the amount of basal membrane that can pump ions and water. Mitochondria are concentrated in the basal aspect of the cell, close to the basal infoldings which contain the active transporters. Because active transport is mainly confined to the basal membranes, ion and water transport will move directionality.
Outline the structure and function of the absorptive epithelium
The interior surface of the wall of the small intestine is folded into numerous finger-like processes that point into the interior, called villi. The villi are covered with intestinal epithelial cells. The dense-microvillus brush-border, contains large amounts of active transporters and channels for the uptake of nutrients from the lumen of the gut. As the concentration of nutrients increases in the cytoplasm of the absorptive cells, it diffuses down its concentration gradient into the basal interstitial space to be collected in the capillaries and distributed in the circulation.
Outline the structure and function of secretory epithelium
In tissues whose main purpose is secretion, the epithelium is often arranged in tubules and glands of varying complexity. However, in many epithelial tissues, individual, dispersed secretory cells can be present in the epithelium. There are two mains types of secretion: exocrine (into a duct or lumen) and endocrine (into the bloodstream). Endocrine and exocrine cells can be seen to have distinct arrangements of their organelles. In an exocrine secretory cell, the organelles are arranged for secretion from the apical plasma membrane. Endocrine cells secrete their contents to the basal aspect. The basal aspects of endocrine secretory cells surround a thin-walled capillary. The secretory vesicles are positioned so that when their contents are released, they have close access to the blood circulation.
Outline the 2 epithelial classifications based on the way cells secrete
1) Constitutive – secretory vesicles, as they are formed, move directly to the plasma membrane and release their contents, e.g. production of plasma proteins by hepatocytes (constitutive endocrine secretion).
2) Stimulated – secretory vesicles are stored in the cytoplasm and only fuse with the plasma membrane to release their contents, e.g. the release of adrenaline from cells of the adrenal medulla after a fight-or-flight stimulus (stimulated endocrine secretion); when stomach contents enter the duodenum, pancreatic acinar cells are stimulated to release their digestive enzymes into ducts (stimulated exocrine secretion).
Outline epithelial turnover in the small intestine
As a cell migrates up the villus epithelium, new cells are constantly being produced by the crypt stem cells, to replace the cells constantly being lost from the villus tip.
What does the inhibition of the proliferation of intestinal crypt fells lead to?
This occurs in cancer chemotherapy and results in loss of the finger-like intestinal villi and flattening of the intestinal mucosa. This is responsible for many of the gastro-intestinal disturbances that are side-effects of chemotherapy. Cell loss from the villus tips continues as normal, but the failure to produce new cells to replace the lost cells results in a loss of tissue and the villi shorten.
Outline epithelial turnover in the epidermis
The epidermis is the keratinising stratified squamous epithelium of the body’s surface. Surface cells are constantly being lost, but are replaced by new cells being formed in the basal layer which migrate up while undergoing a programme of differentiation that eventually leads to them flattening out and keratinising. Each layer replaces the one above as the layers are lost from the surface.
What is hyperproliferation?
In contrast to a loss of proliferation, hyperproliferation of epithelial cells results in increased cell numbers and a thickening of cell layers. This can be in response to repeated or constant pressure. If the increase in cell production is greater than the cell loss from the surface, cells will accumulate creating an increased thick hard layer e.g. pressure and abrasion to areas of the skin results in local hyperproliferation leading to “hard skin” or “corns”. Infectious agents such as papilloma virus can also induce hyperproliferation. They do this by hijacking the cellular machinery of stratified squamous epithelia and inducing increased cell proliferation, which results in a surface growth, e.g. a wart as shown below.
Outline the structure of cholesterol
Cholesterol is a steroid composed of 27 carbon atoms. It is composed of cyclic rings with a hydrophobic tail. The steroid ring structure is planar. Apart from the hydroxyl group at position 3, the molecule is very hydrophobic, consisting only of carbon and hydrogen atoms.
Outline the function of cholesterol
Cholesterol is a vital component of cell membranes, indeed, more than 90% of the cholesterol in our bodies is found in cell membranes. A key property of cholesterol is that it can increase and decrease membrane stiffness, depending on the temperature and the nature of the membrane.
How are cholesterol requirements met in humans?
Dietary cholesterol uptake in humans is limited to around 500mg/day. Given the great need for cholesterol as a membrane component, all physiological requirements for cholesterol are supplied by the liver through de novo synthesis of cholesterol from acetyl-CoA.
Outline the pathway by which cholesterol can be synthesised
1) Synthesis of isopentenyl pyrophosphate, an activated isoprene unit which serves as a key building block (in the cytoplasm).
2) Condensation of six molecules of isopentenyl pyrophosphate to form squalene (in the cytoplasm).
3) Cyclisation and demethylation of squalene by monooxygenases to give cholesterol (in the ER).
Outline phase 1 of cholesterol biosynthesis
2 molecules of Acetyl-CoA condense to form the molecule Acetoacetyl CoA under the catalysis of the enzyme beta-ketothiolase.
Outline phase 2 of cholesterol biosynthesis
Another molecule of Acetyl CoA condenses with Acetoacetyl CoA, under the catalysis of HMG-CoA synthase, to form 3-Hydroxy-3methylglutaryl CoA (HMG-CoA).
Outline phase 3 of cholesterol biosynthesis
HMG CoA is reduced, under the catalysis of HMG-CoA reductase, to generate Mevalonate. HMG-CoA reductase is under negative feed back control by the end product cholesterol, the intermediate mevalonate and bile salts.
Outline phase 4 of cholesterol biosynthesis
Mevalonate undergoes sequential phosphorylation at the hydroxyl groups at position 3 and 5, followed by decarboxylation to form 3-Isopentenyl pyrophosphate. This activated isoprene unit is a useful building block for further synthesis.
Outline how Isoprene units confer lipophilicity to biomolecules
Dolichol phosphate is a specialized lipid molecule located in the ER membrane and involved in N-linked glycosylation of proteins. Likewise, proteins can undergo lipid modifications such as prenylation (addition of farnesyl or a geranyl-geranyl moiety to C-terminal cysteine residues) which gives them affinity for lipid bilayers. The same lipophilic properties of the isoprene unit confine ubiquinone to the inner membrane of mitochondria.
Outline phase 5 of cholesterol biosynthesis
Via an isomerization reaction, Dimethylallyl pyrophosphate can be produced from isopentyl PP. This can condense with a unit of Isopentenyl-PP to form the C10 compound Geranyl-PP. A third isopentenyl-PP molecule is added to form the C15 intermediate farnesyl-PP.
Outline phase 6 of cholesterol biosynthesis
Two farnesyl-PP molecules condense to form C30 squalene plus 2 molecules of pyrophosphate.
Outline phase 7 of cholesterol biosynthesis
Squalene is cyclized to cholesterol, in 3 steps (2 which are in phase 7):
1) Squalene is first reduced in the presence of oxygen and NADPH to form squalene epoxide which has a different C=C bond distribution priming the molecule for carbon ring fusion.
2) The enzyme squalene epoxide lanosterol-cyclase catalyses the formation of Lanosterol. A series of 1,2-methyl group and hydride shifts along the chain of the squalene molecule result in the formation of the four rings.
Outline phase 8 of cholesterol biosynthesis
3) Lanosterol is subsequently reduced and three methyl units removed (demethylated) to generate cholesterol.
