Biochemistry Exam Study Notes (Flinders University) PDF

lOMoARcPSD|47861184 EXAM- Study - Lecture notes ALL Biochemistry Of Human Disease (Flinders University) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study ALCOHOL CASE Describe the pathways of alcohol metabolism including the role of alcohol and aldehyde dehydrogenases and the cytochrome P450- containing microsomal oxidizing system (MEOS). Outline how these pathways are regulated. Ethanol is a small water and lipid-soluble molecule, readily absorbed via passive absorption. Up to 5% of ingested ethanol enters the gastric mucosal cells of the upper GI tract, the rest gets absorbed in the intestine and enters the blood. 2-10% is excreted via lungs and kidneys, 85-98% is metabolised in the liver. - The major route of ethanol metabolism in the liver (around 80%) is through alcohol dehydrogenase (ADH), a cytosolic enzyme that oxidizes ethanol to acetaldehyde with reduction of NAD+ to NADH - Acetaldehyde is a highly reactive compound, causing damage to the liver and other tissues. Around 90% is rapidly metabolised (via ALDH) to acetate (non-toxic). - Acetate enters the bloodstream and is converted to Acetyl CoA in the liver, muscles and other tissues of the body before entering the TCA cycle to fuel production of ATP and ETC substrates (eg. NADH) - Metabolism of ethanol leads to accumulation of NADH and a resultant increase in the NADH/NAD+ ratio of the cell. This leads to inhibition of alternative energy systems (gluconeogenesis, glycolysis > pyruvate, FA > fatty acyl CoA) with consequent accumulation of free fatty acids, pyruvate and intermediate products of glycolysis (eg. DHAP). - Accumulated products are eliminated through conversion to VLDLs (FA + DHAP > hyperlipidaemia) and lactate (leading to lactic acidosis) - Approximately 10% to 20% of ingested ethanol is oxidized through a microsomal ethanol oxidizing system (MEOS), comprising cytochrome P450 enzymes in the endoplasmic reticulum (especially CYP2E1). CYP2E1 has a high Km for ethanol and is inducible by ethanol. Therefore, the proportion of ethanol metabolized through this route is greater at high ethanol concentrations and greater after chronic consumption of ethanol. - Chronic alcohol consumption causes reduction in gastric ADH and upregulation of ethanol-inducible enzyme pathways (Microsomal Ethanol Oxidising System, MEOS incl. CYP2E1), with 5 - 10x increases in enzyme expression. The induction of CYP2E1 by alcohol appears to be through translational, post-translational (protein Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study stabilization), and transcriptional mechanisms. Increased activity of this pathway leads to increased production of toxic reactive oxygen species, exacerbating alcohol-related damage to the liver and other tissues (cirrhosis, hepatic steatosis, alcohol-induced hepatitis etc). - This can be taken too far however, as years of heavy drinking can damage the liver (cirrhosis). This can negatively affect the liver enzymes ADH and CYP2E1, therefore people with liver disease will have slower rates of alcohol metabolism. Explain how alcohol metabolism affects cellular NADH/NAD+ ratio and describe how this may affect carbohydrate metabolism. Refer to textbook Describe the most common liver function tests, understand which enzyme activities are commonly analyzed and how the results help in interpretation of the patient’s condition. Refer to exercise book Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study Mr De Koning has an extremely high plasma triacylglycerol level at 5.8 mmol/L. (Ref interval: 0.3 - 2.0). This indicates that his lipid metabolism is heavily inhibited. Explain how alcohol metabolism can perturb lipid metabolism, increase fatty acid accumulation and triglyceride synthesis leading to changes in lipid profile in plasma. Outline the mechanisms underlying alcohol-induced steatosis. Effects of high NADH/NAD+ ratio: 1) Inhibition of fatty acid oxidation The metabolic purpose of fatty acid oxidation is to generate NADH for ATP generation by oxidative phosphorylation, but an alcohol consumer's NADH needs are met by ethanol metabolism. Therefore, Acetyl - CoA oxidation in the TCA cycle and hence FA oxidation are inhibited → FA increases < Additionally, chronic alcohol consumption causes mitochondrial damage, significantly reducing the rate of electron transport → oxidative phosphorylation becomes uncoupled → fatty acid oxidation decreases further > Altered lipid function leads to ROS having effects on lipids and altering them in the cell, affecting cell structures. 2) Favours formation of TAG Body attempts to reduce fatty acid levels through re-esterification of FA into TAG by fatty acyl transferase in the endoplasmic reticulum. High NADH levels → glycolysis inhibited → High dihydroxyacetone phosphate (DHAP) levels → high glycerol-3-phosphate (G3P) levels → increased TAG levels Glycolysis is inhibited due to high NADH/NAD+ ratio. Body attempts to reduce NADH by reversing the pathway. This involves the conversion of DHAP to G3P (intermediate of glycolysis), leading to overall increase in TAG → causes incorporation of TAG into VLDL that accumulate in the liver. Source of fatty acid can be increased after ethanol consumption, because of release of adrenaline, which increases adipose tissue lipolysis. Outline how products of alcohol metabolism may result in hepatitis, fibrosis and cirrhosis (including likely contributions from acetaldehyde and free radicals) and discuss the involvement Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study of modifications of cellular proteins and lipids, mitochondrial damage and triggering of inflammatory processes). Refer to exercise book for hepatitis. - Cirrhosis is the end stage of alcoholic liver disease and is characterised by scarring (fibrosis) of the hepatic tissue which interferes with normal functions - Generally irreversible and can be fatal if not treated - Scarring occurs as the result of inflammation which in turn results from the persistent injury and death of the liver cells and steatohepatitis (inflammation resulting from the fatty liver) present due to excessive alcohol intake - Fibrosis occurs by overstimulation of fibroblasts and stellate cells that synthesise and deposit excessive extracellular matrix material (collagen) - Acetaldehyde, a toxic metabolite of alcohol, results in further stimulation of stellate cells - There are four generally recognised stages of liver cirrhosis. The first two are considered to be compensated cirrhosis (where the liver still retains a reasonable level of functionality) and the latter two, respectively, decompensated (where the liver struggles, the symptoms appear and immediate medical attention is necessary)  Stage 1 cirrhosis involves some scarring of the liver, but few symptoms. This stage is considered compensated cirrhosis, where there are no complications. Compensated cirrhosis means that the liver is still able to perform many of its functions despite the scarring.  Stage 2 cirrhosis includes worsening portal hypertension and the development of varices. This stage is still considered to be compensated.  Stage 3 cirrhosis involves the development of swelling in the abdomen and advanced liver scarring. This stage marks decompensated cirrhosis, with serious complications and possible liver failure.  Stage 4 cirrhosis can be life threatening and people can develop end-stage liver disease (ESLD), which is fatal without a transplant. Outline the major consequences of impaired liver function in alcoholic liver disease including effects on portal blood pressure, Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study disposal of “waste products” via the bile and the ability of the liver to secrete normal amounts of plasma protein. Refer to exercise book Demonstrate a basic understanding of normal liver function including the role of the liver in carbohydrate and lipid metabolism and protein synthesis. Demonstrate an understanding of the consequences of alcohol- induced induction of MEOS and how this may affect metabolism of other drugs DIABETES CASE 1. The mechanisms of insulin secretion in response to glucose 2. Normal metabolic responses to dietary glucose mediated by changes in insulin and glucagon levels including: insulin-stimulated glycogen synthesis in muscle and liver insulin-stimulated lipogenesis in liver and adipose tissue suppression of glucagon regulated gluconeogenesis in the liver insulin-stimulated insertion of glucose transporters into muscle and adipose tissue cell membranes leading to increased glucose uptake (including an understanding of the tissue specific differences in glucose uptake). Rise in blood glucose level above above set point (4.6-5.5 mmol/L) is detected by pancreatic β cells Blood glucose enters pancreatic β cells via GLUT 2 transporters Triggers secretion of insulin by exocytosis → High insulin:glucagon ratio Insulin stimulates glucose uptake by Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study o Muscle and adipose tissue through increased expression of GLUT 4 transporters o Liver through increased expression of glucokinase (high affinity for glucose) Insulin also initiates protein kinase cascades to stimulate conversion of excess glucose to glycogen (for storage in liver and muscle) and TAGs (for storage in adipose tissue): o Suppresses gluconeogenesis by the liver and stimulates glycolysis o Insulin secretion activates PFK-2 by dephosphorylation. PFK-2 converts Fructose-6-phosphate to Fructose 2,6 bisphosphate. Increased levels of F26BP activate PFK-1. o PFK-1 is one of the most important regulatory enzymes of glycolysis, catalyzing the commitment step for Glycolysis to occur. o Stimulates glycogen synthesis in both muscle and the liver  Activates glycogen synthase and deactivates glycogen phosphorylase: o Stimulates TAG synthesis in liver:  excess acetyl-CoA used for fatty acid synthesis → TAGs packaged into VLDL and exported to adipose tissue o The entry of glucose into adipose tissue also provides glycerol 3-phosphate for the synthesis of TAG The main target organ of glucagon is the liver. stimulates glycogen breakdown (glycogenolysis) and inhibits glycogen synthesis bind to cell surface receptors → trigger the cyclic AMP cascade → activation of glycogen phosphorylase and the inactivation of glycogen synthase: inhibits glycolysis and stimulates gluconeogenesis ○ prevent a further drop in blood glucose levels ○ inhibits glycolytic enzymes pyruvate kinase, PFK-1 and glucokinase ○ Glucagon is released during fasting creating a signaling cascade, increasing c-AMP. This result in the phosphorylation of FBPase-2 and its activation. FBPase-2 catalyzes the conversion of F26BP to fructose 6-phosphate resulting in Gluconeogenesis. Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study Normal mechanism of insulin action in tissues (eg in controlling membrane insertion of glucose transporters in muscle) following stimulation of insulin release from b-cells in the pancreas including: binding of insulin to the insulin receptor and activation of receptor tyrosine kinase activity, phosphorylation of insulin receptor substrates (IRSs) and activation of PI 3- kinase Akt activation leading to membrane insertion of GLUT-4 Role of GSK-3 alternate signalling pathways in insulin action (e.g. MAP kinase pathway-no details required). Refer to exercise book Have an understanding of how reduced insulin signalling in insulin resistance and Type 2 Diabetes Mellitus affects the metabolic response to dietary glucose. Glucose uptake in adipose tissue Insulin sensitive cell: Insulin stimulates translocation of GLUT4 containing vesicles to plasma membrane, allowing glucose to enter the adipocyte. In an insulin resistant cell there is prolonged exposure to high concentrations of insulin → down regulation of insulin receptors → fewer insulin receptors per unit of surface area than adipocytes from normal individuals → decreased sensitivity of adipose tissue cells to insulin → decreased glucose uptake Additionally, Impaired insulin action in adipose tissue allows for increased lipolysis which will promote re-esterification of lipids in other issues (e.g. liver) and further exacerbates insulin resistance. Glucose uptake in the muscle Insulin sensitive cell: Insulin stimulates translocation of GLUT4 containing vesicles to plasma membrane, allowing glucose to enter the myocyte. Insulin resistant cell: Accumulation of diacylglycerol (DAG) → activates PKCθ and other stress kinases → serine phosphorylation of IRS1→ impairs GLUT4 translocation to the membrane. → glucose uptake reduced Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study Glucose uptake in the liver Normal person: Insulin inhibits gluconeogenesis and activates glycogen synthesis → decreasing hepatic glucose production Insulin resistance: Liver uses GLUT2 transporters which are not insulin dependant → can still facilitate uptake of glucose Glucose enters liver cells as entry into muscle cells are impared → lipogenesis and increased hepatic TAG accumulation and secretion as VLDL Fatty acid metabolites such as DAG accumulate in liver ○ DAG activates PKCε → impairs insulin signalling by phosphorylating insulin receptor → impaired suppression of glucose production (aka increased gluconeogenesis) ○ Akt2 (Protein kinase B - one of the 3 closely related serine/threonine-protein kinases) not activated through the PI13K/Akt pathway: activation of FOXO (forkhead box) transcription factors (FOXO1 and FOXA2) → Stimulates conversion of pyruvate to G6P. Increased G6P concentration suppresses further glucose uptake + promotes its conversion into glucose for release into the bloodstream How does this affect circulating blood glucose levels? As a result, circulating blood glucose levels increase due to decreased glucose uptake by skeletal muscle, adipose tissue and the liver. Coupled with a decline in pancreatic B-cell function, hyperglycaemia develops. How does insulin resistance affect glycogen synthesis in muscle and liver? Insulin resistance diminishes the ability of insulin to activate glycogen synthesis in both muscle and liver cell. Lack of glucose transport into myocytes via GLUT4 transporters decreases intracellular G6P available for conversion into glycogen, thus decreasing glycogen synthesis in the muscle. As explained before, in an insulin resistant liver cell, Akt2 is not activated via the PI13K/Akt pathway: Glycogen synthase kinase (GSK3) remains active → inhibits glycogen synthase → glycogen synthesis is reduced Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study Likely mechanisms by which exercise improves control of glucose and lipid metabolism. Normally exercise promotes: increased demand for energy in the muscle increased glucose uptake into muscle use of glycogen initially increase use of FA oxidation and flux through the ETC to produce energy (ATP). Increase in lipolysis in adipose (due to increased hormone sensitive lipase activity stimulated by adrenalin). In the liver gluconeogenesis is used to produce more glucose to meet the demand of muscle Glycogenolysis is increased and lipogenesis is decreased. Insulin resistance: There is increased FA in muscle cells. This drives an increase in mitochondrial beta-oxidation that exceeds the capacity of the Krebs cycle leading to an accumulation of byproducts of FA oxidation, FA metabolites (eg DAG), ROS etc. Results in activation of stress kinases and inhibition of insulin signalling Exercise in Insulin resistance: Muscle Increased use of FA in muscle lowers the production of FA metabolites such as DAG and therefore improves insulin receptor signaling. This in turn improves Glut4 translocation and glucose uptake, which improves blood glucose levels. Exercise increases FA uptake into muscle from the circulation. Increased demand for energy will use up fatty acids and thus increase the demand for glucose as an energy source. Exercise leads to increased expression of genes involved in metabolic processes such as betaoxidation and mitochondrial function via the transcription factors PPARa and PGC1. AMPK is activated (increased AMP:ATP ratio) leading to increased FA uptake into mitochondria and beta-oxidation. Exercise also reduces ROS production through improving mitochondrial function Exercise in Insulin resistance: Adipose FA are sourced from adipose tissue leading to decreased adipose tissue and less uptake of FFA into liver. Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study There is also increase activity of lipoprotein lipase, lecithin cholesterol acyltransferase (LCAT) and hepatic lipase. Hormone sensitive lipase is also stimulated by adrenaline to promote lipolysis. In the long-term exercise promotes loss of weight/fat from adipose tissue. Decreased adipose tissue reduces release of inflammatory cytokines, which affects liver and pancreas function. Role of diet in slowing the progression of Type 2 Diabetes (in general terms). It was discovered that the dietary fibre increased post-meal satiety or decreased subsequent hunger. Therefore, suggesting that soluble dietary fibres delay absorption of dietary carbs related to viscous, gel-forming properties of these fibers and, as such, reducing postprandial glucose excursions. Thus, it is vital that the GI is considered when designing a diet plan for an individual with type 2 diabetes, as this individual would need to be avoiding blood glucose spikes. For long term success diabetes self- management education is critical. Furthermore, short-chain fatty acids are produced as a result of fermentation of indigestible dietary fibre within the colon. This may also regulate glucose homeostasis by inhibiting hepatic glucose production, stimulating hepatic glucose storage via glycogen synthesis and ultimately increasing peripheral insulin sensitivity. Basically, IR can ultimately be reversed by replacing simple sugars with complex carbohydrates and fibre. The main reasons behind why health professionals would suggest this dietary change is due to the longer digestion requirement, thus, blood glucose spikes can be avoided. Blood glucose spikes are avoided because unlike complex carbs, simple sugars raise blood sugar levels very soon after eating as they’re easily broken down. Role of AMP kinase as a master regulator of metabolism and glucose control (in general terms) and how the drug Metformin regulates circulating glucose levels. When taken, Metformin acts as a regulator of blood glucose levels and ultimately improves insulin sensitivity. In type 2 diabetic patients gluconeogenesis is usually active due to the liver’s resistance to the effects of insulin and increased levels of cAMP, in response to glucagon, which activates CREB which in combination of TORC2, leads to transcription of genes required for gluconeogenesis. Therefore this gluconeogenesis pathway remains stimulated in type 2 diabetic patients, contributing to high blood glucose levels. Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study At the molecular level, metformin inhibits the mitochondrial respiratory chain in the liver, leading to activation of AMPK via an upstream activation of protein kinase LKB1, enhancing insulin sensitivity (via effects on fat metabolism) and lowering cAMP, thus reducing the expression of gluconeogenic enzymes. Activation of AMPK phosphorylates and reduces the activity of acetyl CoA (which is required for fatty acid synthesis) leading to reduced fatty acid synthesis and increased fatty acid oxidation. AMPK activation inhibits the transcription of SREBP-1, which regulates transcription of HMG CoA reductase, responsible for transcription of other lipogenic enzymes to synthesise cholesterol leading to reduced the biosynthesis of cholesterol. Activation of AMPK phosphorylates a co-activator of the CREB transcription factor called TORC2, when TORC2 is phosphorylated it is isolated in the cytoplasm and CREB becomes very inefficient at transcribing a gene that is required to upregulate genes that code for enzymes involved in gluconeogenesis. This important transcriptional coactivator is PGC1alpha responsible for transcriptional activation of glucose 6 - phosphotase and phosphoenolpyruvate carboxykinase (PEPCK) which are key. Metformin increases AMPK activity which is associated with higher rates of glucose disposal increasing GLUT 4 translocation and muscle glycogen concentrations. General understanding of how hyperglycemia contributes to development of long-term diabetes complications. Possible mechanisms resulting in insulin resistance in obesity including roles of fatty acids and protein kinase C. Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study CYSTIC FIBROSIS CASE Understand the key structural features of the cystic fibrosis transmembrane conductance regulator (CFTR) protein and how they relate to the function of the CFTR protein as a chloride ion channel. The CFTR protein is part of the ATP-binding cassette (ABC) or traffic ATPase gene family. The CFTR is an ATP-dependent, cyclic-AMP dependent Protein Kinase-A (PKA) regulated chloride channel. Although there is no full length, high resolution structure for CFTR, other ABC transporters give us an insight into the structure and function of the CFTR. The core ABC transporter architecture is comprised of two transmembrane spanning domains (TMDs) and two nucleotide binding domains (NBDs). Both transmembrane spanning domains are comprised of 6 membrane spanning alpha helices each. The CFTR protein also contains a regulatory domain (R domain) which regulates the activity of the ligand-gated channel when it is phosphorylated. Overall, the CFTR protein is located in the atypical membrane lining of cells in the airways and can also be found in the sweat glands, intestinal, reproductive, hepatic, and renal epithelia. The CFTR protein is 1480 amino acids in length and comprised of 5 domains which each play a role in the closing and opening of the channel to allow the movement of anions. There is also a sixth domain called the PDZ domain, found at the C-terminus of the protein, which helps anchor the CFTR protein to the cytoskeleton. Opening 1. The regulatory domain is first phosphorylated by Protein kinase C which changes the conformation of the R domain. 2. This then allows Protein Kinase A to phosphorylate the R domain. The phosphorylation of the R domain by PKA does NOT open the channel but is required if the channel is to be opened. 3. ATP binds to the Nucleotide Binding Domain 1 (NBD1). This leads to both NBD1 and NBD2 forming a weak NBD1-NBD2 complex. Ie. They form a heterodimer. 4. Binding of the NBD1 and NBD2 brings the transmembrane domains together, however, they are not close enough to form a channel yet. 5. The binding of a second ATP molecule to the NBD1-NBD2 complex leads to a strong NBD1-NBD2 heterodimer being formed. 6. The formation of a strong NBD1-NBD2 heterodimer along with the phosphorylation of the R domain by PKC and PKA leads to the opening of the CFTR channel and the conductance of anions across the cell membrane. Closing 1. Membrane bound phosphatase removes the phosphate groups on the R domain. This does NOT lead to the closing of the channel. Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study 2. The hydrolysis of the 2nd ATP molecule in the NBD1-NBD2 complex leads to the closure of the channel. Be able to relate the different classes of CFTR mutations and how they affect CFTR function to how protein mutations can affect folding and trafficking. There are 6 main commonly occurring classes of mutations found in the CFTR gene, each of which have an effect on the protein’s ability to function properly. Class I mutations lead to the formation of a premature stop codon which results in the CFTR protein not being synthesised. Mutations of this class comprise approximately 12% of those found in cystic fibrosis patients. Class II mutations are the most common form of mutation found in cystic fibrosis patients. The deletion of phenylalanine at position 508 is the most common defect found in cystic fibrosis patients, present in approximately 70% of patients. The defective protein can still function as a chloride channel in cell free lipid membranes. However, when synthesised by normal cellular mechanisms it is recognised as misfolded and is degraded shortly after being synthesised before it can reach the surface of the cell membrane. Class III mutations possess little to no chloride channel function in vivo because of abnormalities in the nucleotide binding domain resulting in disordered regulation. This type of mutation leads to a channel opening defect such that anions cannot pass through the cell membrane. Class IV mutations lead to defects in ion transport and a reduced conductance of the protein CFTR channel. These mutations lead to a less severe pulmonary phenotype. Class V mutations result in reduced numbers of CFTR transcripts and Class VI causes instability of CFTR protein at the cell surface and a reduced half life of the protein. Understand that perturbed protein folding is the underlying basis of many diseases. Be able to outline the processes of normal protein folding. Be able to outline the steps required for the mature CFTR protein to reach the plasma membrane after its translation, including folding and the role of the ubiquitin proteasome system, and trafficking to the membrane. Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study Describe the effect of the delF508 mutation on CFTR with respect to the folding process and CFTR activity. There should be an understanding of the fate of delF508 CFTR protein within a cell. What part of the CFTR protein is affected by the delF508 mutation? The most common mutation, called delta F508 (delF508), is a deletion of one amino acid at position 508 in the CFTR protein. The mutation is a deletion of three nucleotides spanning positions 507 and 508 of the CFTR gene on chromosome 7, which ultimately results in the loss of a single codon for the amino acid phenylalanine. A person with the delF508 mutation will produce an abnormal CFTR protein that lacks this phenylalanine residue and which results in a temperature sensitive folding defect. This mutant peptide is more sensitive to denaturation and ultimately affects it efficacy as a polypeptide. A fully denatured protein lacks both tertiary and secondary structure and exists as a so-called random coil. This leads to the retention of the protein in the endoplasmic reticulum, and subsequent degradation by the proteasome. Why is this important for folding? When properly folded, it is shuttled to the cell membrane, where is becomes a transmembrane protein responsible for opening channels which release chloride ions out of cells; it also simultaneously inhibits the uptake of sodium ions by another channel protein to maintain a specific intracellular sodium ion concentration. Both of these functions help to maintain an ion gradient that causes osmosis to draw water out of the cells. How is protein folding determined? As the polypeptide chain is being synthesised by a ribosome, the linear chain begins to fold into its three-dimensional structure. Folding begins to occur even during translation of the polypeptide chain. Amino acids interact with each other to produce a well- defined three-dimensional structure, this is the folded protein, known as the native state. The resulting three-dimensional structure is determined by the amino acid sequence or primary structure. A group of proteins known as chaperones, may assist in protein folding by binding to stabilise an otherwise unstable structure of a protein in its folding pathway, but chaperones do not contain the necessary information to know the correct native structure of the protein they are aiding; rather, chaperones work by preventing incorrect folding conformations. Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study These chaperones are supplied with ATP in order to perform their function. However, due to the deletion of three nucleotides spanning positions 507 and 508 of the CFTR gene on chromosome 7, the regular folding of the protein is interrupted, hence the production of this dysfunction protein. Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study Describe what is meant by “correctors” and “potentiators” of CFTR function. Be able to describe how the delF508 mutation could be corrected and how potentiators can improve function of the G551D CFTR mutant. CFTR modulators are drugs which improves the function of defective CFTR proteins. There are 3 main types of modulators: correctors, potentiators and amplifiers. CFTR correctors: Correct the CFTR protein to form the right 3D shape, preventing them from being degraded and increasing their stability. Generally targeted at delF508 mutations Act as: - chaperones by binding directly to the F508del-CFTR protein - proteostasis regulators, which work by creating conditions in the cell so that higher concentrations of CFTR are made Improves trafficking of functional CFTR protein and hence channel density at the cell membrane --> chloride transport is enhanced, partially restoring the F508del-CFTR in human airway epithelial cells and reducing chloride levels in sweat. A popular chemical corrector for F508del-CFTR is VX-809, a.k.a. Lumacaftor CFTR potentiators: Improve gating of CFTR proteins. Binds to a specific binding site on the CFTR protein, causing the gate to remain open for improved chloride flow. VX-770 a.k.a. Ivacaftor is the most popular allosteric potentiator. G551D mutations are: Found in Class lll mutations and are known as gating mutations. Expressed at the cell surface membrane but are unable to transport enough chloride ions due to impaired nucleotide binding and stops ATP activation and regulation of the CFTR protein. Only occurs in an estimated 6% of Cystic Fibrosis cases Missense mutations: - Usually located in the NBD1 of CFTR and lead to an exchange of glycine for aspartate at position 551. - Exchange of glycine for aspartic acid (an alpha amino acid responsible for the biosynthesis of proteins) Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study o Introduction of a large negatively charged side chain within the binding site for ATP is thought to disrupt the nucleotide- binding pocket of the NBD1. Hence, binding of ATP to the NBD1 and succeeding dimerization of the NBDs is impaired so that the channel fails to be activated by ATP and to exhibit normal gating. Ivacaftor potentiator: Binds directly to CFTR, possibly on membrane spanning domains and improves channel gating for chloride transport. Promotes ATP-independent gating and stabilizing the protein while its channel is open to cause a delay in channel closure. Be able to outline subsequent health implications resulting from perturbed CFTR function in these organs. The pancreatic duct cells secrete bicarbonate into the duct lumen via an apical membrane Cl2/HCO3 2 (chloride/bicarbonate) exchanger, while the H+ ions produced is exchanged for extracellular Na+ on the basolateral side of the cell. The H+ enters the pancreatic capillaries to eventually meet up in portal vein blood with the HCO3 2 produced by the stomach during the generation of luminal H+. As with most transport systems, the energy for secretion of HCO3 2 is ultimately provided by Na+ /K+ -ATPase pumps on the basolateral membrane. Cl2 normally does not accumulate within the cell because these ions are recycled into the lumen through the cystic fibrosis transmembrane conductance regulator (CFTR) via a paracellular route. Na+ and water move into the ducts due to the electrochemical gradient established by chloride movement through the CFTR. The Cl2/HCO3 2 (chloride/bicarbonate) exchanger on the apical membrane plays a key role in bringing bicarbonate into the cell. When Cl2 accumulates in the cell due to the impaired CTFR function in cystic fibrosis, pancreatic HCO3 2 secretions are decreased. In addition, the lack of normal water movement into the lumen leading to a thickening of pancreatic secretions. This results in: The pancreatic duct becoming plugged with thick mucus preventing secretion The pancreatic digestive secretions accumulate behind the blocked duct, fluid-filled cysts form in the pancreas Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study The affected pancreatic tissue gradually degenerating and becoming fibrotic Partial or complete loss of exocrine pancreatic function results in a distinct reduction of fat digestibility Malnourishment eventually results Understand the basis of inheritance of cystic fibrosis and the postnatal screening methods used to diagnose the disease or for genetic pre-pregnancy counselling. Be able to outline what role the CFTR protein plays in the lungs, pancreas and stomach in maintaining ion balance and thus hydration at these sites. Be able to describe what is happening to the water and ion balance within the lung epithelium and in the airways. Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study CHOLESTEROL CASE Recognise that cholesterol: (i) is a 27-carbon molecule; (ii) is mostly non-polar (or hydrophobic) consisting of a four-ring structure with a hydrocarbon tail; (iii) contains a single polar hydroxyl group. (Full details of the structure do not have to be learnt.) Describe the distribution and major functions of cholesterol and cholesteryl esters in cells. Briefly describe the sources of dietary cholesterol, the digestion of this cholesterol and the processes resulting in transport to the liver. Cholesterol is an alicyclic compound, in its “free” form, the cholesterol molecule contains: 27 carbon atoms A simple hydroxyl group at C3 A double bond between C5 and C6 An eight-membered hydrocarbon chain attached to carbon 17 in the D-ring A methyl group (carbon 19) attached to carbon 10 A second methyl group (carbon 18) attached to carbon 13 Cholesterol is a normal component of most body tissues, especially those of the brain, nervous system, liver, and blood. Most of cholesterol ends up in the cells, where it performs vital structural and metabolic functions. Cell membranes contain cholesterol in the phospholipid bilayer which makes it stronger, more flexible but less fluid and less permeable to water-soluble substances such as ions and monosaccharides. The polar hydroxyl group of cholesterol is orientated towards the surface. The hydrocarbon tail and the steroid nucleus lie in the hydrophobic core. A cis double bond in the fatty acyl chain of phospholipid bends the chain to create a hydrophobic binding site for cholesterol. Cholesterol is also needed to form sex hormones and adrenal hormones, vitamin D and bile salts. Some cholesterol is transformed into vitamin D and some are coupled with proteins to become lipoproteins. Around one-third of plasma cholesterol exists in the free (or unesterified) form. The remaining two-thirds exist as cholesterol esters in which a long- chain fatty acid (usually linoleic acid) is attached by ester linkage to the hydroxyl group at C3 of the A-ring. Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study A small portion of hepatic cholesterol is used for the synthesis of hepatic membranes, but the bulk of synthesized cholesterol is secreted from the hepatocyte as either: Cholesterol esters Biliary cholesterol (cholesterol found in the bile) Bile acids Cholesterol esters are more hydrophobic than free cholesterol and its production in the liver is catalyzed by acylCoA–cholesterol acyl transferase (ACAT). ACAT catalyzes the transfer of a fatty acid from coenzyme A to the hydroxyl group on carbon 3 of cholesterol. Foods derived from animals contain cholesterol and cholesteryl esters. These components are not found in food derived from plants which have different sterols. Major sources of dietary cholesterol include meats and poultry (beef, chicken, pork, lamb), seafood (squid, salmon, tuna), and dairy products (eggs, ice cream, cheese, milk, butter). After consumption and absorption, triglycerides and cholesterol are re- esterified in the intestinal mucosal cells and then coupled with various apoproteins, phospholipids, and unesterified cholesterol into lipoprotein particles called chylomicrons. The composition of chylomicrons is 90% TAG, 5% cholesterol, 3% phospholipids, and 2% protein. The major apolipoproteins of chylomicrons are apolipoprotein B48 (apoB48), apoCII, and apoE. The apoproteins not only add to the hydrophilicity and structural stability of the particle, they have other functions as well: 1) They activate certain enzymes required for normal lipoprotein metabolism 2) They act as ligands on the surface of the lipoprotein that target specific receptors on peripheral tissues that require lipoprotein delivery for their innate cellular functions. Chylomicrons are secreted into intestinal lymph, entering the bloodstream through the thoracic duct, and bind to the wall of capillaries in adipose and skeletal muscle tissue. The apoCII activates lipoprotein lipase (LPL), an enzyme that projects into the lumen of capillaries in adipose tissue, cardiac muscle, skeletal muscle, and the acinar cells of mammary tissue. This activation allows LPL to hydrolyze the chylomicrons, leading to the release of free fatty acids derived from core triacylglycerides of the lipoprotein into these target cells. The muscle cells then oxidize the fatty Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study acids as fuel, whereas the adipocytes and mammary cells store them as triacylglycerols After removal of the triglyceride core, remnant chylomicron particles are formed. These are high in cholesterol esters and characterized by the presence of apoproteins B, CIII, and E. These remnants are cleared from the circulation by binding of their E apoprotein to a receptor present only on the surface of hepatic cells. Subsequently, the bound remnants are taken to the inside of hepatic cells by endocytosis. The endocytosed vesicle then fuses with a lysosome, and the apolipoproteins, cholesteryl esters, and other components of the remnant are hydrolytically degraded, releasing amino acids, free cholesterol, and fatty acids. The receptor is recycled. This process liberates cholesterol, which is then either converted into bile acids, excreted in bile, or incorporated into lipoproteins originated in the liver (VLDL). Outline the pathway for synthesis of cholesterol and describe major features of regulation via modification of the activity of HMGCoA reductase. Cholesterol synthesis is broken down to various main steps: Stage 1: Synthesis of HMG-CoA  In the cytoplasm, Cholesterol synthesis is initiated by 2 x Acetyl CoA molecules condensing to form Acetoacetyl CoA, this then condenses with a third acetyl-CoA  These then follow through HMG-CoA synthase to produce HMG-CoA (3-Hydroxy-3-Methyl-Glutaryl-CoA)  This HMG-CoA synthase reaction is present in the cytosol Stage 2: Synthesis of Mevalonate  HMG-CoA is then reduced to produce Mevalonic acid (Mevalonate), catalysed by HMG-CoA Reductase, an enzyme embedded in the membrane of the Endoplasmic Reticulum (ER)  This step uses 2 NADPH as a reducing agent, and releases CoA and NADP+  The HMG-CoA reductase is a rate limiting step, which means it is heavily regulated in multiple ways - and is an important step in cholesterol synthesis Stage 3: Synthesis of Cholesterol  Once Mevalonic acid is produced, it goes through a mevalonate pathway (which requires 3 ATP) to produce isophentylprophosphate Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study  It takes 6 x isophentylprophate to then produce Squalene  Through various steps, Squalene is converted to Lanosterol, and through additional steps finally forming Cholesterol What regulates this process: There are a few different factors that regulate cholesterol synthesis As mentioned before, HMG-CoA Reductase is a major control point for cholesterol biosynthesis, and is subject to different kinds of metabolic control. SREBP / SCAP  HMG-CoA Reductase is controlled the transcription factor SREBP (Sterol Regulatory Element Binding Protein) and SCAP (SREBP- Cleavage and Activating Protein), which are both embedded in the ER membrane  The SREBP increases transcription of gene coding for HMG-CoA Reductase  SCAP senses and responds to cholesterol levels  When cholesterol levels are low, SCAP joins with SREBP (SCAP:SREBP complex) and migrates to the Golgi for modification  SCAP then undergoes cleavage of SREBP (activating it) which leads to increased synthesis of HMG-CoA and therefore increased synthesis of cholesterol.  When cholesterol levels are high, SCAP prevents the activation of SREBP, which therefore leads to the down regulation of cholesterol synthesis AMP  AMP activates enzyme AMPK, which phosphorylates HMG-CoA, and therefore causes inhibition of cholesterol synthesis. Therefore, cholesterol synthesis decreases when ATP levels are low, and increases when ATP levels are high. Hormonally  An increase of insulin favours the upregulation of HMG CoA reductase  Elevated glucagon leads to the phosphorylation of HMG CoA Reductase, which therefore inactivates it Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study Drugs  Statins (commonly used for high cholesterol) acts as a competitive inhibitor to HMG-CoA Reductase, therefore inhibiting cholesterol synthesis Cholesterol  When cholesterol is present, it binds to SCAP and inhibits it, leading to the inhibition of SREPB2 and therefore negatively regulates HMG- CoA reductase  Therefore, cholesterol negatively regulates its own production Describe the general features of lipoproteins. Structure of lipoproteins: Lipoprotein is an assembly of lipids (cholesterol and triglyceride) and protein (apoproteins). They are classified based on their density. Because lipids are lighter than proteins, particles that contains more lipids are larger in size but have a lower density. Different types of lipoproteins have different size of proteins on their surface. These proteins serve as address tags that determines their destination and hence functions of each protein. Lipoproteins are composed of a neutral lipid core containing triglycerides and cholesterol esters surrounded by a shell of amphipathic apolipoproteins, phospholipid and free cholesterol. These amphipathic compounds are water soluble. Polar phospholipids form ionic bonds with the negatively charged hydroxyl molecules in the blood. The TAG and cholesterol carried by the lipoproteins are obtained from endogenous source. Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study Understand what is meant by VLDL, LDL cholesterol and HDL cholesterol and describe the roles of VLDLs, LDLs and HDLs in the transport of cholesterol between cells and tissues. Describe the function of LDL receptors and the effects of uptake of LDL on cholesterol storage and synthesis and on the synthesis of new LDL receptors. Chylomicrons  largest and least dense of lipoproteins  High TAG content  Synthesized from dietary lipids within epithelial cells of small intestine, secreted into lymphatic vessels and enter bloodstream via the left subclavian vein  Major apolipoproteins: apolipoprotein B48 (apoB48), apoCII, and apoE  ApoCII activates lipoprotein lipase (LPL), an enzyme which projects into the lumen of capillaries in adipose tissue, cardiac muscle, skeletal muscle, and the acinar cells of mammary tissue o LPL hydrolyses chylomicrons --> release fee fatty acids (FFA) o FFA is...  Oxidised as fuel in muscle cells  Stored as TAG in adipocytes and mammary cells o Partially hydrolysed chylomicrons remaining (chylomicron remnants) lost apoCII protein, but retain apoE and apoB 48 o ApoE binds to receptors on plasma membranes of liver cells --> taken up by receptor mediated endocytosis Side note: ApoE is expressed in many cells, but highly expressed in liver and CNS Very Low-Density Lipoproteins  Intake of excess carbohydrates which exceed fuel requirement of liver --> formation of TAG  Nascent VLDL is formed from TAG combined with free and esterified cholesterol, phospholipids, and the major apoprotein apoB100  Secreted from the liver into bloodstream Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study  Nascent VLDL accepts apoCII and apoE from circulating HDL --> form mature VLDL  VLDL transported from the hepatic veins to capillaries in skeletal and cardiac muscle and adipose tissue, as well as lactating mammary tissues, where LPL is activated by apoCII in the VLDL particles. o LPL release FA and glycerol  FA oxidised as fuel in muscle cells  FA used as resynthesis of TAG in fat cells o Remaining VLDL in bloodstream (VLDL remnants)  ~ 50% taken up by liver cells: ApoE binds to receptors on plasma membranes of liver cells --> taken up by receptor mediated endocytosis  ~ 50% have additional core TAG removed --> formation of IDL Intermediate-Density Lipoproteins and Low-Density Lipoproteins  IDL formed by removal of TAG from VLDL  LDL formed by removal of TAG from IDL through action of hepatic TAG lipase  LDL are rich in cholesterol and cholesterol esters  ~60% LDL transported back to liver o ApoB100 binds to specific apoB100 receptors in the liver cell plasma membranes --> endocytosed into the hepatocyte o ApoB100 receptors are mainly expressed in the liver  ~40% LDL transported to extrahepatic tissues (e.g. adrenocortical and gonadal cells) o ApoB100 binds to apoB100 receptors --> LDL particles internalised, and cholesterol is used:  synthesis of steroid hormones Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study  Membrane synthesis  Vitamin D synthesis  Remnant IDL particles which contain apoE can be taken up by liver via the apoE/remnant receptor  Side note: Excess LDL saturate receptor mediated uptake of LDL --> more LDL available for uptake by macrophages near endothelial cells of arteries --> inflammatory response and increase risk of atherosclerosis High density Lipoproteins  3 ways nascent HDL can be formed o Synthesis by the liver o Budding of apoproteins from chylomicrons and VLDL as they are digested by LPL o Free apoAI shed from circulating lipoproteins acquiring phospholipids and lipoproteins from cell membranes  Nascent HDL contains: o Predominant apoproteins are: apoAI, apoAII, apoCI, and apoCII o Very low levels of TAG and cholesterol esters in its core  Nascent HDL accumulates phospholipids and cholesterol from cells lining the blood vessels --> forms mature HDL o Cells which contain protein ATP binding cassette protein 1 (ABCA1) moves cholesterol to outer leaflet of the membrane o Cholesterol accepted by HDL and are esterified by lecithin- cholesterol acyl transferase (LCAT) to prevent escape via the same route o Central hollow core progressively fills with cholesterol esters  Primary pathway of HDL cholesterol: o Uptake of HDL by scavenger receptor SR-B1 on many cells --> transfer cholesterol and cholesterol esters into cells --> HDL dissociates from receptors and re-enters circulation  HDL then returns the cholesterol to the liver via reverse cholesterol transport Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study o HDL transfers cholesterol esters directly to liver cells via apoE receptor o HDL transfers cholesterol esters to chylomicrons and VLDL and VLDL remnants in exchange for TAG  Facilitated by cholesterol ester transfer protein (CETP)  Increases amount of cholesterol returned to liver via VLDL and its remnants Understand what is meant by atherosclerosis and outline key cellular changes involved in its development. LDL is taken up into cells via apo B-100 receptor-mediated endocytosis. Intracellular cholesterol intake is regulated via three feedback mechanisms; LDL-receptor expression, ACAT-mediated storage of cholesterol and HMG CoA reductase (regulates endogenous production of cholesterol). Defects in the LDL receptor are associated with familial hypercholesterolaemia. This is exacerbated by upregulated intracellular production of cholesterol due to reduced inhibition of HMG CoA reductase When present in excess, LDL can infiltrate and accumulate within the vascular endothelium (particularly at sites of endothelial damage) and become oxidised (ox-LDL) at the apoprotein B-100 by reactive oxygen species in the subendothelial space. Accumulation of ox-LDL in turn promotes migration of circulating monocytes into the vascular wall, where they become active macrophages and phagocytose ox-LDL in the sub- endothelial layer. Upon phagocytosis of ox-LDLs, macrophages form atherogenic foam cells with reduced immune function. Importantly, macrophages recognise LDLs via a scavenger receptor, which (unlike typical LDL receptors) is not down-regulated by incoming cholesterol. Consequently, foam cells will continue to ingest ox-LDL until they eventually undergo lysis, releasing their contents (including cholesterol and pro-inflammatory mediators) into the subendothelial space. As cholesterol is hydrophobic, it is precipitated, and forms diffuse atheromatous plaques within the arterial tunica intima (innermost layer of the arteries). Over time, this can contribute to the development of an atheroma, which expands into the luminal space and effectively reduces the diameter of the vessel (i.e. stenosis). This process is exacerbated by deposition and crystallization of circulating calcium in atheromatous regions, as well as accumulation of fibrogenic substances (eg. collagen), further contributing to sclerosis and narrowing of the vessel wall. Reduced Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study luminal diameter and increased arterial resistance in turn restricts blood flow to the target tissue, resulting in ischemia and functionaldecline of downstream tissues and organs Atherosclerosis and stenosis typically remain asymptomatic until an atheroma ulcerates and exposes its highly thrombogenic core (containing collagen, tissue factors etc.) to the luminal contents. This leads to activation of circulating platelets, setting off the coagulation cascade and resulting in formation of a thrombus (blood clot). This will often manifest clinically as a myocardial infarction, stroke or other ischemic event. HIGH-DENSITY LIPOPROTEIN  HDL transports cholesterol, triglycerides and phospholipids from the peripheral tissues back to the liver and steroidogenic organs. Serum HDL concentration is inversely associated with the risk of developing atherosclerosis (i.e. increased HDL decreases the risk of plaque formation).  HDL has the smallest diameter and highest density of the lipoprotein molecules, due to its high protein:lipid ratio. HDL is synthesized in the liver and released into the circulation, where it acts to remove cholesterol from peripheral tissues via the ATP-binding cassette transporter A1 (ABCA1).  HDL contains a plasma enzyme, lecithin-cholesterol acyltransferase (LCAT), which esterifies free cholesterol to an entirely hydrophobic cholesteryl ester (CE) that is sequestered into the nonpolar HDL core.  HDL particles increase in size as they circulate through the bloodstream and incorporate more cholesterol and phospholipid molecules from cells and other lipoproteins. Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study  HDL transports cholesterol predominantly to the liver, adrenals, ovary, and testes. These target organs express HDL receptors (such as scavenger receptor BI, SR-BI) which recognize and bind HDL apoprotein A-I, mediating the selective uptake of esterified cholesterol from HDL (direct pathway).  Cholesterol is also removed from HDL indirectly via cholesteryl ester transfer protein (CETP), which exchanges VLDL triglycerides with HDL cholesteryl esters. VLDLs are in turn processed to LDL and removed from the circulation via the LDL receptor pathway. ATHEROSCLEROSIS  LDL is the smaller, denser remnant molecule of VLDL that has lost triglyceride through the action of lipoprotein lipase (LPL).  LDL performs an essential role in transporting lipids (including cholesterol) to the tissues of the body, though chronic elevation of serum LDL is associated with a significantly increased risk of developing atherosclerosis.  Circulating LDL can become deposited within the vascular wall at sites of endothelial damage, oxidised by ROS and engulfed by macrophages, producing lipid-rich foam cells.  Foam cells undergo lysis, releasing cholesterol and inflammatory mediators into the subendothelial space. Atheromatous plaques, comprising cholesterol, calcified tissue, extracellular matrix, inflammatory and smooth muscle cells, form within the endothelial intima. Over time, these processes lead to fibrosis and narrowing of the vessel wall, contributing to the development of an atheroma.  As these plaques build, the luminal diameter of the vessel narrows (stenosis), obstructing blood flow to downstream organs and tissues.  Complications include arterial occlusion, plaque rupture (resulting in embolization and/or thrombosis), aneurysm (due to weakening of the vessel wall) and ischemic tissue or organ damage at downstream sites. Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study Describe the relationship between LDL cholesterol and HDL cholesterol in the blood and the likelihood of cardiovascular disease. Recognise that many genetic factors can influence the risk of cardiovascular disease. Understand that mutations in the LDL receptor or in ApoB100 can dramatically increase this risk and describe the underlying mechanism. Describe the mechanism of action of statins in reducing cholesterol in the blood Refer to exercise book Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study CANCER CASE There are two main known types of hereditary colorectal cancer (HNPCC and FAP). HNPCC involves microsatellite instability and mutation of mismatch repair genes. FAP involves mutation of APC. The role of APC in wnt signalling in normal colon cells and the consequences of APC mutation on the wnt signalling pathway in colon cancer. APC is a huge 2843-amino acid protein involved in wnt signalling or beta- catenin signalling pathway. β-Catenin is usually (in the absence of growth stimulating Wnt stimulation of the cell) is bound by a series of other proteins (i.e. dvl, axin, CK1, GSK3, APC and β-TrCP) that form a destruction complex and are vital in keeping the level of β-Catenin low by targeting it for proteasomal degradation via its phosphorylation and ubiquitination (a modification by adding ubiquitin, a regulatory protein) - APC is an integral part of the destruction complex (binds β-Catenin to the destruction complex) and inhibits β-Catenin's movement into the nucleus - CKI (Casein Kinase I) performs initial phosphorylation of β-Catenin which allows GSK3 (Glycogen Synthase Kinase 3) to act - GSK3 additionally phosphorylates β-Catenin - β-TrCP ubiquinates β-Catenin when it's phosphorylated and this is what targets it for degradation - Ultimately, it prevents β-Catenin from accumulating in the cell When Wnt binds a frizzled receptor on the surface of the cell it leads to phosphorylation of the LRP protein - This leads to translocation of the destruction complex to the membrane where LRP is - Dvl protein (a part of the destruction complex) binds to LRP and gets activated and inhibits the destruction complex - β-Catenin does not get phosphorylated & ubiquitinated and ths does not get degraded - When β-Catenin levels subsequently rise, it enters the nucleus and upregulates the transcription of its target genes via TCF transcription factor (normally inhibited by groucho protein in the absence of β-Catenin) - This leads to cell proliferation Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study 800 germline mutations have been identified in patients with FAP. 95% of those are nonsense or frameshift mutations that cause early termination of translation and result in a smaller truncated protein unable to function properly. As for cancer-causing somatic mutations, over 60% of them occur in a rather small region called MCR, mutational cluster region, that comprises less than 10% of the coding sequence of the gene. These mutations also often lead to production of a truncated protein that is nonunctional. These truncations result in a loss of APC's ability to bind beta-catenin and axin, which disrupts the destruction complex and prevents the protein from doing its job in the wnt pathway and leads to accumulation of beta- catenin Another function of APC protein is thought to be stabilisation of microtubules, which are involved in chromosome segregation by spindles. In a study I looked at it was observed that APC accumulates near the chromosomes during mitosis and when APC protein is mutated as it does in FAP, the microtubules bind the kinetochores with less effectiveness. The conclusion that the researchers reached was that loss of the sequences near the end of the protein contributes to chromosomal instability in colorectal cancer. HNPCC is associated with microsatellite instability (MSI). What are micro- satellites and what is MSI? How does MSI arise? What is the significance of its detection in the adenoma? What are MicroSatellites? MicroSatellites are regions present throughout the genome where a nucleotide (or a unit of 2 or more) is repeated. Usually Cytosine-Adenine (C-A) Mostly found in introns, though they can be found in promoter regions, untranslated regions and even axons The number of nucleotide repeats, normally, is the same in every cell What is MSI? MSI is present if a cell has one or two alleles with different number of repeats Microsatellite instability is a change that occurs in the DNA of certain cells (such as tumour cells) in which the number of repeats of microsatellites is different than the number of repeats that was in the inherited DNA. Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study Microsatellite instability may be caused by impaired DNA mismatch repair protein (MMR) which relates to defects in the ability of the cell to repair mistakes in newly synthesised DNA. So MSIs can be thought of as genetic hypermutability (predisposition to mutation) resulting from impaired DNA MMR The presence of MSI is evidence that MMR is not functioning normally. How does MSI arise? During replication, DNA polymerase can make mistakes in adding the correct nucleotide To fix this, the MMR binds to the new DNA, removes the mistake and allows DNA polymerase to try again. Therefore, mutations in one of the four MMR (MSH2, MLH, MSH6 and PMS2) proteins can impair their function and the mistakes are left in the new strands of DNA What is the significance of its detection in the adenoma? The presence of MSI in these cells provides evidence that there is impaired function of MMRs. Hereditary Non-Polyposis Colon Cancer (HNPCC) or Lynch Syndrome: it is likely that the HNPCC is hereditary as over 90% of HNPCC are associated with MSI (10-15% from sporadic colorectal cancers) BAT(Big Adenine Tract)25 and BAT26 are mononucleotide microsatellites used as markers in screening of MSI. MSI analysis is a useful method of pre-screening colorectal adenoma patients for HNPCC which is relevant to Denzel due to his familial history of CRC. Adenomas in HNPCC increases risk for carcinomas progression DNA mismatch repair Mechanism of DNA mismatch repair (outline) Significance of the mismatch repair system for minimising incidence of mutations, including microsatellite instability Significance of inheriting a defect in one allele of MLH1 (or other mismatch repair proteins) – relationship to Hereditary Non-Polyposis Colon Cancer (HNPCC) In both bacteria and eukaryotic cells:  MutS protein binds specifically to a mismatched base pair (Figure 1,2 & 3) Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study o A dimer  MutSa: MSH2 associated with MSH6 – recognises single base mismatches  MutSb: MSH2 associated with MSH3 - recognises ≥1 bp insertion/deletion o Scans the DNA for mismatches by testing for sites that can be readily kinked - areas with abnormal base pairs (Normal pairing with hydrogen bonds: A-T, G-C) o When the MutS complexes (“sliding clamp”) bind DNA double helix, they exchange ADP for ATP. o Kinks the DNA  MutS complex binds to MutL complex  MutL scans the nearby DNA for a nick. (Figure 2 & 3) o A dimer  MutLa: MLH1 interacts with PMS2  MutLb: MLH1 interacts with PMS1  MutLg: MLH1 interacts with MLH3 o Once MutL finds a nick, it triggers the degradation of the nicked strand all the way back through the mismatch. o Because nicks are largely confined to newly replicated lagging strands in eukaryotes, replication errors are selectively removed. o In eukaryotes, MutL contains a DNA nicking activity that aids in the removal of the damaged strand. o Excision of the mismatch is performed by proteins such as exonuclease 1 and proliferating cell nuclear antigen (PCNA) o Followed by resynthesis and religation of the DNA strand with DNA polymerase and DNA ligase. For the MMR proteins to be released from DNA, ATP is hydrolyzed to ADP.  HNPCC (Hereditary Nonpolypsos Colorectal Cancer)/ Lynch Syndrome is the most common hereditary colorectal cancer (CRC). Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study  It is linked to germline mutations in one of four DNA mismatch repair genes (MMR) - MLH1, MSH2, MSH6 and PMS2  Mutations of MMR will therefore lack the ability to repair errors in DNA sequencing. HNPCC is an autosomal dominant syndrome. People who inherit one defective copy of a mismatch repair gene (along with a functional gene on the other copy of the chromosome) - have a higher risk of certain types of cancers. HNPCC can occur if a spontaneous mutation of the remaining functional gene can produce clone cells without the mismatch proofreading, and therefore accumulate the mutated cells unusually rapidly. When there is damage to the MMR genes, microsatellites become unstable and lead to Microsatellite Instability (MSI). MSI can lead to a “mutator phenotype” within the cell, and when these errors occur in growth regulators, tumours can start to develop ===> HNPCC. MSI occurs in around 15% of all CRC tumors in white populations. It arises as a result of defective MMR caused by the failure of one of the four main MMR genes, MSH2, MLH1, MSH6, or PMS2. On the cellular level, the mechanism is recessive. There are two different types of MMR gene failure: 1. HNPCC - caused by an inherited germline mutation in one allele followed by somatic inactivation of the wild-type allele in a colonic mucosa cell. These account for 3% to 5% of all CRCs. 2. Sporadic - failure caused by somatic inactivation of both alleles. These account for 10% to 15% of all CRCs. Step 1: Identifying MMR gene mutations Microsatellite Instability (MSI) and/or immunohistochemistry (IHC) testing of MMR genes. Mutations in MMR genes typically lead to truncated, non-functional proteins that do not stain when tested by IHC. Therefore, if a stain does not appear this will indicate that the protein is absent and not being expressed in the cell. This missing MLH1 gene can be an indicator of HNPCC, however, it is not definitive - as a missing MLH1 gene can also indicate sporadic colon cancers. It is important to distinguish between the hereditary form and the Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study sporadic form, as those with HNPCC have a high risk of developing other cancers. Step 2: Confirmation of HNPCC/Lynch Syndrome 3 methods to identify Lynch syndrome: 1) Excluding HNPCC/ Lynch Syndrome through testing for mutations in the BRAF gene  The common somatic V600E mutation in BRAF is present in 40-60% of MSI positive tumors and in 69% of tumors with absence of MLH1 on IHC but virtually never in Lynch syndrome.  Finding the mutation allows Lynch syndrome to be excluded; however, a negative result has no predictive value. 2) Mutation analysis of the MLH1 gene the presence or absence of germline mutations signaling Lynch syndrome can be determined. Presence of germline mutations in one of several DNA mismatch repair genes (MLH1, MSH2, MSH6, PMS2) or loss of expression of MSH2 due to deletion in the EPCAM gene (previously called TACSTD1) confirms NHPCC/Lynch syndrome 3) ***Not the most accurate method: Excluding HNPCC/ Lynch Syndrome through testing for MLH1 promoter methylation Directly assess MLH1 promoter methylation (e.g. by Methylation-specific PCR a.k.a. MSP).  If MLH1 promoter methylation present/ positive MSP test: highly suggestive of a sporadic tumor  If MLH1 promoter methylation absent/negative MSP test: Likely to be NHPCC/Lynch syndrome. Limitation: the test is only approximately 80% specific so it cannot completely rule out a diagnosis of Lynch syndrome since some MLH1 methylation has been reported in up to 46% of Lynch syndrome tumors. Recognise the following points about control and function of p53: In controlling the cell cycle: “activation” of p53 protein in response to DNA damage involves activation of protein kinases that phosphorylate p53, reducing p53 interaction with the MDM-2 protein Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study p53 protein levels depend on its rate of degradation which involves p53 binding to MDM2 proteins (autoregulatory feedback loop) p53 expression increases in response to aberrant growth signals or other cell damage Function of activated p53 protein as a transcription factor, binding to specific DNA sequences to increase expression of genes including p21, (an inhibitor of cyclin-dependent kinases) which can block G1 to S and G2 to mitosis transitions in the cell cycle. When chromosomes are damaged a repair is necessary before the cell can duplicate and segregate. This process occurs at checkpoints where a signalling cascade will occur. Low level DNA damage occurs in normal cell life, if this damage accumulates and the damage checkpoints are not functioning it leads to increased frequency of cancer – promoting mutations. Certain signals control the outcome at these checkpoints. Positive signals for the cell to progress and divide come from cyclins, cyclin dependent kinases and phosphatases. Negative signals for the cell to halt and not further divide if DNA damage is detected come from p35 gene, p21 protein. Checkpoint G1 prevents entry into S phase by inhibiting the activation of G1/S-Cdk and S-Cdk complexes. This inhibition can occur by the activation of gene regulatory protein p53 which is responsible for the transcription of several genes. One of the genes encode protein p21 which binds to G1/S- Cdk and S-Cdk complex and blocking entry into S phase. (Alberts, 2002) P53 gene may be the most important gene in human cancer it governs the cell’s responses to DNA damage. In particular the control of apoptosis (programed cell death). P53 can work in two ways in response to DNA damage 1. It can drive the damage or mutant cell to commit suicide. 2. It can trigger a mechanism that stops the cell from dividing if the damage remain unrepaired. P53 in undamaged cells are in low concentrations and unstable due to interaction with Mdm2, a ligase that targets p53 for destruction by proteasome. In cells with DNA damage, the damaged DNA activates protein kinases that phosphorylate p53 and prevent it binding to Mdm2. Degradation is ceased and p53 concentration increases and gene transcription increases. If p53 gene is defective it cannot encode protein p21, so there is no binding to G1/S-Cdk and S-Cdk complex and blocking entry into S phase. Therefore continuation into S phase will continue with the DNA damage. Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study M-Cdk is activated leads to early mitosis where the cell assembles a mitotic spindle and prepares for segregation of duplicated DNA damaged chromosomes. The G1 checkpoint is ineffective without the p53 gene. These cells defective in p53 gene carry on dividing and DNA replication without pausing at checkpoints to repair the breaks and other DNA lesions. Once a chromosome carrying a duplication and lacking a telomere has been generated, repeated rounds of replication, chromatid fusion and unequal breakage (chromosomes become fragmented and incorrectly re- joined) can increase the number of copies and therefore lead to further mutants in which the gene is amplified to a high copy number and a high disruption to the genome. Checkpoint G2 prevents entry into mitosis when incomplete DNA replication is detected. Damaged DNA sends a signal to a series of protein kinases that phosphorylate and inactivate the phosphatase Cdc25 (which is the activator of Cdk1 at the onset of mitosis). This blocks the dephosphorylation and activation of M-Cdk, thereby blocking entry into mitosis. When the DNA damage is repaired, the inhibitory signal is turned off, and cell-cycle progression resumes. At least half of all human cancers involve a mutation of p53. Cancer cells accumulate mutations due to cell proliferation and avoidance of cell death. Most human tumours have either a TP53 mutation of the gene itself and 95% of these lie in the DNA core binding site. Missense mutations result in tumour - associated form of p53 being predominantly full length, with a single amino acid change in the core domain. Point mutations are made of 2 classes: structural which disrupt structure and destabilize the whole protein leading to the negative activity association with the wild allele and new oncogenic properties. The other is DNA contact which has little effect on p53 folding but interfere with residues involved with DNA binding. Other p53 tumours are via inactivation or by signal transduction pathway. Inactivation via over- expression of MDM2 (ubiquitin ligand) negative regulators or loss of inhibitor ARF which controls p53 stability - tumour suppressor by interaction with MDM2 ( which inhibits MDM2 activation) and inhibits p53 degradation therefore stabilizing it. Current drug trials and development: p53 pathway frustration Research and progress ocurring in current drug trials and development 2 areas: Molecules that activate p53 by blocking protein - protein interactions with MDM2 are in early development. Protein - protein interaction sites are usually large and shallow and affinity is achieved through the summation Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study of many weak interactions. A small molecule must mimic these interactions while maintaining good drug like quality and be shape complementary. Molecules that bind and stabilize mutant p53 that can rescue the function of certain p53 mutants have been discovered by both structure - based design and cell - based screens. The challenge of reactivating mutant p53 with peptides, antibodies or small molecules is whether or not they will be able to target all the mutation classes observed in p53. Drugs: Nutlin (MDM2 binding) phase 1 clinical testing, Ml-219 (MDM2 binding) phase 1, PRIMA-1 protein folding - phase 1 In controlling apoptosis: existence of extrinsic and intrinsic pathways of apoptosis, and basic concepts of their activation the role p53 protein in the intrinsic pathway including its role as a transcription factor that increases the expression of Bax (a pro-apoptotic protein). Briefly describe the features of an apoptotic cell? Apoptosis is a mechanism by which a cell undergoes controlled cell death. It is an energy dependent process and key features include; cell shrinkage, chromatin condensation, and nuclear fragmentation. The formation of apoptotic bodies is a key part of apoptosis, with phosphatidylserine being expressed on the outside of these bodies. It is one of the markers recognised by macrophages and other nearby phagocytotic cells that engulf the apoptotic bodies. Explain the role of p53 in apoptosis and more specifically within the extrinsic (death receptor) and intrinsic (mitochondrial) apoptotic pathways. Extrinsic pathway: The extrinsic pathway of apoptosis involves death receptors. Death receptors are transmembrane proteins, which consists of three primary domains: an extracellular ligand binding domain, transmembrane domain and intracellular death domain. Death receptors belong to the TNF receptor family, which includes the receptor for TNF itself and the Fas death receptor. Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study When a death receptor binds a ligand, adaptor proteins are recruited by the intracellular death domain of the death receptor. The adaptor proteins recruit two molecules of procaspases 8, 10 or both. Bringing them into close contact allows for their activation when they autocatalytically cleave each other to form active caspase 8, or 10. These cleaved and stabilised proteases are now considered to be active initiator caspases which can go on to activate executioner caspases 3, 6, and 7, producing a caspase cascade. The caspase cascade results in the formation of apoptotic fragments involved in apoptosis. Unlike the intrinsic pathway, the extrinsic is activated independently of p53. It is normally activated in an immune response by interaction of a ligand expressed on cytotoxic T cells with death receptors on target cell membranes. However, p53 can upregulate the expression of some pro- apoptotic receptors, such as those encoding death receptor 5 (DR5) and Fas, by inducing the transcription of associated genes. Therefore, p53 is capable of transcribing and activating the death receptors essential for the initiation of the extrinsic apoptotic pathway. Furthermore, p53 also directly activates caspase 8, which is a key protein involved in the activation of executioner caspases to induce apoptosis. Mutations to p53 reduces the expression of receptor proteins, namely Fas and DR5, necessary for the activation of the apoptotic pathway. Furthermore, p53 is responsible for the activation of caspase 8, therefore mutations to p53 would result in insufficient activation of caspase 8, thereby halting the extrinsic apoptotic pathway in the early stages and resulting in insufficient direction of the cell through apoptosis. Intrinsic pathway: Cells can also activate their apoptosis program from inside the cell, usually in response to injury or other stresses, such as the accumulation of p53 in cytosol, DNA damage or lack of oxygen, nutrients, or extracellular survival signals such as growth factors. These are referred to as ‘death signals’. The receival of an environmental stress or death signal by a cell results in the release of cytochrome c from the mitochondria via the activation of pro-apoptotic members of the Bcl2 family. These proapoptotic members can be divided into two major classes: BH3-only members (i.e. Bid) and pro-death ion-channel forming members (i.e. Bax and Bak). The activation of Bid subsequently activates Bax, which then forms an ion- channel in the mitochondrial membrane, stimulating the release of cytochrome c. Once it has been released into the cytosol, cytochrome c binds to cytosolic factor, apaf. Normally, anti-apoptotic protein, Bcl2 is Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study bound to apaf, maintaining apaf in an inactive state. However, at the same time that Bid is activating Bax, Bid also binds Bcl2, therefore disrupting the Bcl2-apaf complex, freeing apaf to bind to cytosolic cytochrome c and form an apaf-cytochrome c complex. The formation of the apaf-cytochrome c complex binds initiator caspase 9, forming an active complex known as the apoptosome. The apoptosome cleaves and activates execution caspases 3, 6 and 7. P53 is a pivotal stimulator of the intrinsic apoptosis pathway in response to severe cellular stresses such as DNA Damage and hypoxia. The action of p53 on regulating the intrinsic pathway is important as p53 stimulates the transcription of pro-apoptotic factors Puma and Noxa. These factors are important in apoptosis as they inhibit the activity of the antiapoptotic members of the Bcl family such as Bcl2. The upregulation of Puma and Noxa also indirectly activate pro apoptotic genes Bax and Bid by releasing them from inhibition, allowing them to promote apoptosis by the mitochondrial pathway as described above. P53 also directly induces apaf- 1 expression, up-regulating the formation of the apoptosome. Mutations to p53 results in the persistent inhibition of the intrinsic apoptotic pathway by antiapoptotic factors such as Bcl-2. Furthermore, intrinsic apoptosis cannot be stimulated as pro-apoptotic genes Bax and Bid will be inhibited. Moreover, p53 promotes the transcription and expression of death receptor proteins Fas and Dr5 and upregulates the activation of caspase 8; thereby p53 also stimulates the extrinsic apoptotic pathway and mutations to p53 will downregulate this effect. Features that distinguish a metastatic cancer cell from a cancer cell in the original tumour. The molecular components of adherens junction important for cell-cell adhesion An outline of the steps required for a cancer cell to metastasise to another site. An overview of the role of integrins in migration Outline the process of EMT Describe the molecular components of adherens junction important for cell-cell adhesion? Adherens junctions serve to couple individual cells into various arrangements required for tissue structure and function. The central structural components of adherens junctions are transmembrane adhesion receptors, and their associated actin-binding/regulatory proteins. Adherens junctions are anchorage sites for actins filaments connecting Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study one actin filament bundle in one cell with the next. Adherens junctions consist of two basic adhesive units: the cadherin/catenin and nectin/afadin complexes. These adhesive complexes link a homophilic recognition event with the underlying actin cytoskeleton. Adherens junction performs multiple functions including initiation and stabilization of cell-cell adhesion, regulation of the actin cytoskeleton, intracellular signalling and transcriptional regulation. The cadherin/catenin “core adhesive” complex contains components that mediate homophilic recognition across the intercellular cleft (cadherin), actin association (α-catenin) and regulate actin dynamics and stabilization of the complex at the cell surface. The cadherins are coupled indirectly to actin filaments via β-catenin and other anchor proteins. α-Catenin, vinculin and plakoglobin (which is a relative of β-catenin also called γ- catenin) are probably also present in the linkage or involved in control of its assembly however the details of the anchorage are not well understood. Another intracellular protein called p 120-catenin also binds to the cadherin cytoplasmic tail and regulates cadherin function. Similar to the cadherin/catenin adhesive unit, the nectin-afadin complex contains components that can mediate intercellular adhesion and actin association. Describe the role played by integrins: Integrins are principle receptors used by cells for binding to the extracellular matrix via proteins, they function as transmembrane linkers between the extracellular matrix and the cytoskeleton  Integrins are transmembrane heterodimers comprised of two non- covalently bound glycoprotein subunits – alpha & beta, they can switch between an active state where it readily forms attachments and to an inactive state where it does not  Both subunits span the membrane with short intracellular C-terminal tails and large N-terminal in the extracellular domain  These large extracellular domains of the subunits combine to create a binding site for extracellular proteins  The extracellular portion of the integrin dimer binds specific amino acid sequences in the extracellular matrix such as laminin, fibronectin or to ligands on the surface of other cells. The binding of ligands can result in dramatic changes in conformation of the integrin  The intracellular portion binds to the cytoskeleton via a complex of proteins, this intracellular linkage is to actin filaments via talin  The binding of a matrix component to an integrin can send a message into the interior of the cell, and conditions in the cell Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study interior can send a signal outward to control binding of the integrin to matrix  Tension applied to an integrin can cause it to tighten its grip on intracellular and extracellular structures, and loss of tension can loosen its hold, so that molecular signalling complexes fall apart on either side of the membrane. In this way, integrins can also serve not only to transmit mechanical and molecular signals, but also to convert the one type of signal into the other  Structural changes at one end is coupled with structural changes at the other end so that any signalling can be communicated in either direction across the cell membrane  Complex assemblies of proteins organise themselves around the intracellular tails of the integrins and produce intracellular signals that influence most aspects of cell behaviour including proliferation and survival How is adhesion disrupted in order for a normal cell to migrate? When integrins cluster at sites of matrix contact, they help trigger the assembly of cell–matrix junctions called focal adhesions. Among the many proteins recruited into these junctions is a cytoplasmic tyrosine kinase called focal adhesion kinase (FAK). FAK is recruited to focal adhesions by intracellular anchor proteins such as talin, which binds to the integrin β subunit, or paxillin, which binds to one type of integrin α subunit. FAK then binds to the cytosolic tail of one of the integrin subunits with the assistance of other proteins. The clustered FAK molecules cross- phosphorylate each other, creating phosphotyrosine-docking sites where the Src kinase can bind. Src and FAK now phosphorylate each other and other proteins that assemble in the junction, including many of the signaling proteins used by receptor tyrosine kinases. In this way, the two tyrosine kinases signal to the cell that it has adhered to a suitable foundation, where the cell can now survive, grow, divide, migrate, and so on. FAK helps disassemble focal adhesions and this loss of adhesions is required for normal cell migration. By interacting with both conventional signaling receptors and focal adhesions, FAK can couple migratory signals to changes in cell adhesion. Many cancer cells have elevated levels of FAK, which may help explain why they are often more motile than their normal counterparts. Outline the process of epithelial to mesenchymal transition (EMT). Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study Epithelial-mesenchymal transition (EMT) is a process that allows a polarized epithelial cell, which normally interacts with the basement membrane via its basal surface, to undergo multiple biochemical changes that enable it to transition into a mesenchymal cell and detach from the basement membrane and enter the circulation and travel to a secondary site where they may form micro- and macro-metastases. Distinct molecular processes mediate EMT and enable it to reach completion such as the activation of transcription factors, expression of specific cell-surface proteins, reorganization and expression of cytoskeletal proteins, production of ECM-degrading enzymes, and changes in the expression of specific microRNAs. Epithelial cells are closely connected to each other by tight junctions, gap junctions and adherens junctions, have an apico-basal polarity, polarization of the actin cytoskeleton, are bound by a basal lamina at their basal surface and express E-cadherin. Conversely, mesenchymal cells, lack this polarization, have a spindle-shaped morphology and interact with each other only through focal points. Mesenchymal cells possess enhanced migratory capacity, invasiveness, resistance to apoptosis, increased production of extracellular matrix (ECM) components and express N-cadherin, fibronectin and vimentin. EMTs occur in three distinct biological settings and are responsible for different functions and therefore are classified as type 1, type 2 and type 3. Type 1 is involved in embryonic development, type 2 is involved in fibrosis and wound healing and type 3 involves the secondary epithelia transforming into cancer cells enabling invasion and metastasis. Steps of epithelial–mesenchymal transition (EMT):  Break down of epithelial cell–cell contacts such as tight junctions, adherens junctions, desmosomes and gap junctions.  Loss of cell polarity through the disruption of the Crumbs, partitioning defective (PAR) and Scribble (SCRIB) polarity complexes leading to detachment from the basement membrane.  downregulation of an epithelial genes and activation of mesenchymal genes by transcription factors SNAIL1, zinc-finger E- box-binding (ZEB) and basic helix–loop–helix (bHLH). This facilitates the reorganization of the cytoskeletal actin architecture and changes in cell shape to that of mesenchymal phenotype.  cells acquire motility and invasive abilities Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study  express matrix metalloproteinases (MMPs) that can degrade extracellular matrix (ECM) proteins further enabling invasive behaviour.  Additionally, cells that have undergone EMT acquire resistance to senescence and apoptosis. Describe the process of metastasis. Metastasis involves a complex series of steps in which cancer cells leave the original/primary tumour site and migrate to other parts/a secondary site of the body via the bloodstream and/or the lymphatic system. To do so, malignant cells break away from the primary tumour and attach to and degrade proteins that make up the surrounding extracellular matrix (ECM), which separates the tumour from adjoining tissues. By degrading these proteins, cancer cells are able to breach the ECM and escape. The location of the metastases is not always random, with different types of cancer tending to spread to particular organs and tissues. This specificity is mediated by soluble signal molecules such as chemokines, and transforming growth factor beta TGF-β. Initiation of metastasis requires invasion, which is enabled by EMT. Carcinoma cells in a primary tumour lose cell-cell adhesion mediated by E- cadherin repression and break through the basement membrane with increased invasive properties and enter the bloodstream. Later, when these circulating tumour cells (CTCs) exit the bloodstream to form micro- metastases, they undergo MET for clonal outgrowth at these metastatic sites. Platelets in the blood have the ability to initiate the induction of EMT in cancer cells. When platelets are recruited to a site in the blood vessel, they can release a variety of growth factors and cytokines including the EMT inducer TGF-β. The release of TGF-β by platelets in blood vessels near primary tumours enhances invasiveness and promotes metastasis of cancer cells in the tumour. Process of metastasis:  Begins with EMT thereby allowing the cell to leave the original tumour site  Enter the blood stream/lymph (migration)  Establish growth in a new site Take home messages: Downloaded by Tia Efthimiou ([email protected]) lOMoARcPSD|47861184 Biochemistry exam study  The main function of adherens junctions is intercellular adhesion, they consist of two basic adhesive units: the cadherin/catenin and nectin/afadin complexes.  Adherens junction performs multiple functions including initiation and stabilization of cell-cell adhesion, regulation of the actin cytoskeleton, intracellular signalling and transcriptional re

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