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Metabolic Basis of Disease - Coggle Diagram

- - - - basic compounds e.g quinone, arent absrobed from stomach (low pH) -> are absorbed better from small intestine (pH 7-9) where they won't be ionised
      - weak acids e.g. salicyclic acid + neutral compounds, aren't ionised @ low pH -> can be absorbed from stomach (pH 1-15)
      - most ionisable groups: Carb. acids, amines, imides, phenols, thiols --> formation of ions depends on pH of body compartment -> polar ions don't cross non-polar cell membranes v.well and therefore are poorly absorbed
- - - - Oral Administration: 1.dissolved in aqueous medium of stomach and intestine
        
        pass from gut lumen through gut wall
        
        through liver before reaching systemic circulation
        
        in lumen before absorption or in gut wall during absorption
        
        A) Carrier Mediated Process
        
        Facilitated Transport: energy isn't consumed, e.g. glucose transport in human erythrocytes, v. specific i.e. only glucose D forms --> 1. D-glucose binds to carrer protein in a conformation open outside, 2. carrier protein shifts to a conformation open outside, 3. D.glucose is released inside+carrier shifts back to normal conformation
        
        Active Transport: consumes energy, Na/K pump -> more K+ into cell and Na+ out --> high Na+ outside / K+ inside levels meed to be inverted for some biological processes e.g. protein biosynthesis
        
        B) Passive Processes
        
        1) Filtration: if hydrostatic pressure or osmotic gradient - <100Da will mass through gradient but most drugs are >100Da -> blood-brain barrier
        
        water diffuses through membarnes -. solute conc across the membrane
        
        channels act like pores -> allow passage of small molecules in/out of cell
        
        2) Passive Diffusion: most important for uncharged and non-polar solutes -> lipid soluble drugs penetrate lipid membranes -> polar molecules can't pass through membranes
        
        trans membrane diffusion driven by the trans membrane conc gradient fo drug
        
        rate of diffusion determined by Flick's Law (see notes)
    - - Bioavailability = extent to which and @ which rate a drug reaches the circulation from plasma to the organs
      - rate + extent of distribution: chemical structure of drug, blood flow to each organ, tissue size, binding to plasma proteins and tissue compartments, permeability of tissue membranes
      - drug molecules bind to plasma proteins // most important protein: albumin, also glyco- and lipoproteins -> weakly acidic drugs bind to albumin, weakly basic drugs bind to glycoproteins
      - bound drug -> no effect // free drug -> more easily eliminated
    - - = when a drug is inactivated or active metabolites are formed
      - metabolites can also be drugs -> toxins responsible for secondary effects or the actual active drug (=prodrug)
      - Phase 1 Rxns: - oxidation: monooxygenanses enzymes (oxidases) - alcohol or aldehyde dehydrogenases, monamine oxidase // reduction: ;ess common, reductase enzymes // hydrolysis: of esters and amines in common, no specific esterases
        
        oxidations catalysed by monooxygenases e.g. in endoplasmic reticulum: cytochrome p450 // a major redox system (ER) catalyses the reductive cleavage of O2, transferring O to substrate, and transfers other O to form water
        
        cytochrome p450 is a monooxygenase -> monooxygenases catalyse oxidations
        
        oxidations catalysed by other oxidoreductases; alcohol dehydrogenases, aldehyde dehydrogenases, xanthine oxidas, monoamine oxidases
        
        Reductions at carbon atoms (carbonyl reductase or quinone reductase), at nitrogen atoms (nitro and azo reductases) <-- see notes for these mechanisms
      - Phase 2 rxn: compounds with OH, NH, COOH groups -> react with endogenous compounds (glucuronic, sulphate, glycine, acetate, glutathione) to form conjugates -> usually become inactive, greater hydrophilicity -> easily excreted
        
        conjugation rxns link an endogenous moiety to: the original drug (polar functions), a phase one metabolite)
        
        e.g. methylation, acylation, acetylation, sulfate conjugation
    - - Billiary Excretion (liver and pancreas)
        
        major route for drugs (metabolites from phase 2 glucuronidation) w/Mr of >300 + v.polar
        
        active transport of drugs and metabolites into bile -> then pass to intestine -> most secreted as faeces
      - Renal Excretion
        
        GLOMERUS: glomerular filtration: blood flow to kidneys filtered through glomeruli // pores of glomerular capilleries permit passage of most drugs but not blood cells or large molecules (>60kDa)
        
        TUBULAR SECRETION: drug is transported against a conc gradient from the blood capilleries across tubular membranes to tubular fluid
        
        TUBULAR RE-ABSORPTION: >90% of protein-free filtrate is reabsorbed via the tubular cells // solutes and drugs dissolved in filtrate can be reabsorbed
  - - - CLASSICAL THEORY: drug action mediated by formation of drug-receptor complexes (equilibrium)
      - OCCUPANCY THEORY
        
        CLARK THEORY: 2 parts
        
        intrinsic activity: initiation of the biological effect (ability of drug-receptor complex to initiate a response)
        
        affinity: complexation of drug and receptor (capacity of drug to bind receptor)
        
        ARIEN'S THEORY: drug effect directly proportional to the no. of receptors occupied (dynamic equilibrium) - effect ceases when the drug-receptor complex dissociates - this theory doesn't account to partial effects
      - INDUCED FIT THEORY: for enzyme-substrate interactions - receptors don't need to be in the required conformation to accept drug - conformational chnage induced as drug interacts w/receptor - orienting the essential binding sites // conformational chnage - may initate biological effect
      - activation-aggregation theory: even when drug is absent, receptor is in dynamic equilibrium
    - - covalent bond: strongest, irreversible, not v common, e.g. alkylation (of DNA)I
      - ionic bond: @ physiologic pH: basic side chains of AAs (e.g. lysine, arginine) are protonated -> cationic environment // acidic groupse e.g. glutamic acid are deprotonated -> neg. charged groups
        
        rug and receptor can be attracted by opp. charges on their surfaces
        
        attraction decreases as the square of the distance between the charged sites increases
        
        operates in concert with H bonding
      - Ion-Dipole and Dipole-Dipole Interactions: diff electronegativities of other atoms to carbon - assymetric distribution of electrons that produce dipoles -> dipoles in a drug molecule interact to complementary dipole in the receptor / reversible / net charge of dipol is less than a full ion
      - Van der Waals / London Forces: some atoms on non-polar moelcules have temporoary non-symmetric distribution of electrons to cause a transient dipole -> atoms can appraoch each other - dipoles attract, forming an interaction
      - Hydrogen bonding: between H and O,N,S // H is the only atom taht can carry + charge at physiological pH while remaining covalently attached to a molecule // reversible //directional interaction, i.e. promotes specificity // supplemented by electrostatic forces // maintain the structural integrity of peptides/proteins
      - charge transfer complexes : transfer of an electron from a donor (groups that have pi bonds) to an acceptor (pi bond with an electron withdrawing group e.g. carbonyl) // reversible
      - hydrophobic binding: non-polar/non-polar interaction between receptor and drug - receptor and drug molecule's aliphatic chains surrounded by water molecules - as the chains approach, water moelcules are squeezed out to allow non-polar chains to align with each other side by side - this effect reduces the area of non-polar surface exposed to water - entropically driven
    - - A) Hormones: chemicals secreted into blood and carried by blood/fluid tissues to cells they act upon ( 2 classes)
        
        proteins, peptides and modified AA: HYDROPHILIC, BIND TO RECEPTORS ON SURFACE OF TARGET CELLS
        
        steroids: HYDROPHOBIC, DIFFUSE FREELY IN CELLS, THEIR TARGET CELLS CONTAIN CYTOPLASIC AND/OR NUCLEAR PROTEINS THAT SERVE AS RECEPTORS OF THE HORMONE
      - B) Neurotransmitters
        
        Released by axon terminals of a neuron that can stimulate/inhibit another neuron (post synaptic , e.g. muscle cell)
        
        It’s coordination is faster and more localised than that of a hormone
        
        Axon terminals between a neuron and receiving cell = synapse —> synaptic knob has vesicles containing neurotransmitter
        
        Arrival of nerve impulse - calcium channels open in membrane, exocytosis of some vesicles - neurotransmitter released into synaptic cleft and bind to receptors on post synaptic membrane
      - Both hormones and neurotransmitters are endogenous agents as they originate within the organism - drugs aren’t endogenous
      - Agonists = drugs that mimic the effects of endogenous regulatory compounds
        
        Partial agonist = can’t produce the same level of effect as the agonist
        
        Inverse agonist = elicits the opposite response than the of the agonist or endogenous compound
        
        Antagonist = bind to the receptor but have no intrinsic regulatory activity, thus prevent endogenous compound from binding to receptor
        
        Works through competition for active site but can sometimes also bind to other places on the receptor
      - Opioids
        
        opioid agonists: heroin, methadone, morphine
        
        All natural and synthetic opioids exhibit a T shape conformation - 2 broad hydrophobic surfaces that are at right angles - has a methylated nitrogen which is charged at a physiological pH - charged nitrogen is essential for activity and lies in one of the hydrophobic planes - hydroxyl at C3 in other plane is essentual <-- all opioids have this piperdine ring
        
        opioid agonist treatment: i.e. treatment for opioid agonist dependence - targets biological factors related to heroin addiction - steady state opioid maintenance prevents withdrawal and relieves craving - euphoric effects of heroin are clocked
        
        partial agonists have a limited intrinsic efficacy (ability to produce a desired result) - pain control plateaus as dose is increased and you may not receive sufficient pain relief, e.g. Temgesic (Buprenex) - lasts 6 hrs with pharamalogical peak after 1 hr - metabolised by liver - exerts its effect via high affinity binding to μ subclassopiate receptors in CNS - has slow rate of dissociation from its receptor - accounts for longer duration than morphine + its low level of phsyical dependence
        
        The μ (mu) opioid receptors (MORs) are a specific class of opioid receptors that play a central role in the effects of opioid agonists. They are part of the G-protein-coupled receptor (GPCR) family and are primarily responsible for mediating the therapeutic and adverse effects of opioids.
        
        narcotic antagonists are produced by a change on the N atom of an opioid agonist, e.g. naloxone and naltrexone
      - nuclear receptors = large family of structurally related ligand-inducible TFS, including estrogen receptors (ER + ERB)
        
        the phytoestrogen, genestein, is buried within the hydrophobic core but H12 doesn't adopt the distinctive 'agonist' position - H12 instead lies in a similiar orientation to that observed w/ER antagonists - the suboptimal alighnment of the transactivation helix results in genestein's partial agonist character w/ERB
        
        NUCLEIC ACIDS AS RECEPTORS: - few chemcial differences between healthy DNA and DNA from other species e.g. virus, cancer - drug specificty hard to achieve - no principal interactions by which dna-based drugs act
        
        Nucleotides of DNA: Base +Deoxyribose + Phosphate
        
        have a hydrophobic pocket -specific ligand binds to helix 12 (H12) - when agonist is bound, H12 is orientated anti-parallel to H11 (hydrophobic groove exposed for the binding of coregulator proteins) - when antagonist is bound - H12 moves outward, rotates and packs into a hydrophobic groove between helices 3,4+5 -> co-activators needed for transcription can't bind
        
        General interactions w/DNA: intercalators, alkylators + minor-groove binders
        
        intercalators: non-covalent interaction; drug held perpendicular to axis // drug = aromatic or heteroaromatic (flat) - between base pairs by stacking - to accomadate drug, base pairs distort and seperate vertically, stretching the sugar-phospahte backbone - energetically favoured - it can hold intercalated molecule and stabilise complex - normal base stacking - intercalation doesn't disrupt nromal DNA H-bonds but can destroy helical structure and interferes with H binding polymerases, topoisomerases - e.g.ethidium bromide
        
        alkylators: an irreversible binding to DNA - involves transfr of an alkyl group - rxn must take place at pH 7.4 - fucntional groups that serve as alkylating agents: CH3I, CH3CH2I, Micheal acceptors, small rings e.g. epoxides - nucleophiles can be modifies by alylating agents (Nucleophiles: thiolates, phosphates and guanine N7 in DNA) can be modified by an alkylating agent
        
        minor groove binders: compounds that show curvature to fit into the narrow groove, hold pos. charges to establish H bonds with dna bases, can form hydrophobic interactions to displace the wall of hydration in the minor groove e.g. netropsin
        
        H12 positioning allows the receptor to discriminate between agonists and antagonists
      - ezymatic inhibition: proteins are natural ligands - proteins are enzymes - enzymes are an interaction
        
        drugs targeting protein receptors - agonists, antagonists // drugs targeting enzymes - to inhibit their catalytic activity // enzyme substarte complex accurs by interactions discussed i.e. ionic bonding
        
        diseases - result of a deficiency or excess of metabolite/substrate in body - if deficiency can be corrected by controlling enzyme that produces the corresponding metabolite - develop dugs that target those enzymes // drugs that control enzyme action = inhibitors
        
        classes of Enzyme Inhibitors:
        
        reversible
        
        competitive inhibition: both substrate and inhibitor can fit binding site - inhibitor can't be processed by enzyme - stops catalytic activity --> inhibitor decreases amount of enzyme for catalysis by formation of EI complex - inhibitor can't effect the ES complex after its been formed - dissociation constant for I : Ki = [E][I]/[EI] --> consequences of competiitve equilibrium - Vmax is unchanged (at high levels of S all of I is displaced by S) - Km is increased (increased substrate required to reach max velocity - results in Michaelis-menton eqn)
        
        Non Competitive Inhibition: molecule binds to a 2nd site on enzyme (not active site) - modifies rxn rate - distorts enzyme / substrate and inhibitor can bind simultaneously / presence of inhibtor slows product formation rate / inhibitor binds to both E and ES -> both slope (Km/Vmax) + y intercept (1/Vmax) increase
        
        Vmax increase : at increased levels of substarte, Inhibitor still bound // Km increase at increased substarte to reach lower Vmax
        
        Uncompetitive Inhibiton : inhibitor binds only to the ES complex -> prevents catalysis // Vmax is reduced: amount of ESI formed depeneds on Inhibitor -> solution to the steady state eq'n results in the TBCCCC
        
        irreversible
        
        Active site-directed inhibitors: inhibitor binds covalently to the binding site - reduces conc of active enzyme - educed rate of formation of products
        
        suicide inhibitors: inhibior binds to the enzyme binding site where its modified _> reactive group that irreversible reacts w/the active site of the enzyme
        
        transition state inhibitors: transient but bind to active site of enzyme - structures compatible with active site // molecules resembling a TS (charge distribution and structure) = transition-state inhibitors // binding of substrate analogues to enzymes: fast // binding of TS analogues: slow
  - - - analogue based pharmacophore: ligand-receptor complex structure is unknown // key functional elements of ligand
        
        known ligands (agonists, antagonists, inhibitors) -> representation by modelling programme -> superposition of structures (place on top of each other) -> min. structure needed to obtain a response
      - structure based pharmacophore : ligand-receptor complex structure is known // key functional elements of ligand and/or receptor
      - structure based differs from analogue based in: ligand is present in the receptor bound conformation - important functional groups to bind the receptor are obvious - receptor excluded volume is obvious - other receptor recogniton points (pharmacophores) includded
      - structure based defined same way that analogues based: spatial relationship between pharmacophoric elements defined by distnaces, angles or cartesian co-ords - aromatic rings reduced to a centroid and a vector perpendicular to the plane of the aromatic ring - receptor excluded volume can be incorporated
    - - bioisosteres = groups of molecules w/chemical and physical similarities producing broadly similiar brological effects
      - requirements for new drug approval: drug is safe, is effective in treating disease, can be manufactured correctly and reproducibly each time
        
        new drug: design and synthesis (Target, Prototype, Optimisation), development (Preclinical phase, Clinical phases I, II, III, IV, Marketing)
        
        hit-to-lead optimisation: make compounds more effective and safer
        
        pharmacophore interacts w/receptor -> can't be removed to keep affinity/activity
        
        bioisosterism, salts -> play a role in ADMET - can be chnaged to improve activity (abssorption, solubiltiy etc)
        
        prediscovery: understand disease, idnetify biological targets, ensure target is involved in the disease
        
        hit compound (obtained after screening) - has shown activity in screening)
        
        lead compound = compound w/activity that would usefful (may have suboptimal structure) // prototype -> serendipity (accidental discovery)
        
        hit-to-lead optimisation: make compounds more effective and safer _. pharmacophore interacts w/receptor and thus can't be removed to keep affinity/activity --> bioisosterism, salts --> role in ADMET -> can be changed to improve activty (absorption, solubilty etc)
        
        pharmocokinetic behaviour can be modulated using salts - effects on solubility, stability etc
        
        1st class salt - enhance the aqueous solubility of poorly soluble drugs, thereby improving dissolution and absorption e.g. NaCl
        
        2nd class drug - improve the chemical or physical stability of the drug, protecting it from degradation during storage or in the gastrointestinal tract - not naturally occuring, low toxicity and good tolerability
        
        3rd class salt - modify the release profile of a drug, allowing for sustained, delayed, or targeted drug release - slow dissolution or adsorption for longer therapeutic effects
        
        1st class acids - e.g. HCl - form highly water-soluble salts with weakly basic drugs - increased solubility
        
        computational methods for optimisation:
        
        molecular mechanics (force fields) - atoms bounded by harmonic forces - force fields are eq'ns of potential energy - stretching, bending, torsion, electrostatic interactions - identify most stable conformation of the drug, can predict how drug would bind to a receptor
        
        quantum mechanics -
        
        'ab initio' methods - based on schrodinger eq'ns and LCAO - complete molecular hamiltonian used which considers Born-Oppenheimer (seperated movement of e- and nucleus), Hartree Fock ( approximate the electronic structure of atoms, molecules, and solids), AOs expressed as Gaussian functions - basis sets (sets of AOs to define each atom and generate MOs by linear combination
        
        density functional theory methods - properties of a compound can be computed by its electron densty - Hartre fock as described earlier is more to do with wavefucntions
        
        how to choose bwteeen MM and QM - MM: quick, larger structures, useful for energy minimisation // QM - accuracy, can do conformational analysis, mechanism of rxn
        
        modelling based on targets structure - docking techniques = approach drug to active site of target - does it fit - prediction of LS complex - many degress of freedom which can be an issue // molecular dynamics - search for min. energy conformation between drug and active site - based on newtons laws of movement - each atom is a point mass - simulates movement - search favorable energy interactions
        
        3D-QSAR (Three-Dimensional Quantitative Structure-Activity Relationship) - selection of molecules of known activity - search their biocative conformation - alignment of molecules in the conformation - calculate steric and electronic parameters - derivation of a function correlating parameters and actvity - auto-concsistency of predictability of the fucntion
        
        high throughput screening (HTS) - use of automated equipment to rapidly test millions of samples for biological activity at cellular, molecular level
        
        "Me Too" Drug Design - companies whihc produce off brand versions of drugs that are already v important - optimisation by searching analogues - increased development competitiion has led to a decrease on time that 1st class drugs have on market place - offers clinical options and price competition
        
        agonists (induce response) --> interact with target in same way // antagonist (block a response) --> range of possible building blocks
        
        auxophore = part of molecule not including pharmacophore --> some may interfere w/binding of pharamcophoric groups and must be excised -> some auxophorics can be modified w/effect on potency -> way to identify pharmacophore/auxophore --> cut away pieces of lead and measure effect
        
        screening -. combinational chemistry - library - simultaneous ynthesis of a large number of compounds - synthesised on a solid support (Polymer) or in solution
        
        early evaluation: understand lead compound effect on receptors, cells, animals etc - to predict behaviour in humans
      - DEVELOPMENT
        
        Pre clinical phase - research and cell and animal studies only - stability studies, metabolism, toxicology, characterisations, kinetics -> in-vitro ttests (cells, isolated tissues) / in-vivo tests (mouse, rat) --> IND (Investigational New Drug) Application - summary of all work done -filed and approved before human trials
        
        Clinical Trials - only 5 candidtates make it to Phase 1 clinical trials - hold can be imposed at any time for safety - most of the cost here
        
        phase 1: healthy volunteers receive single does of max tolerated dose in a non-blind trial
        
        phase 2: diseased patients: get info on efficacy, side effects, risks - can compare new drug w/placebo and current standard treatment - single blind trail (one inert placebo and standard drug)
        
        phase 3: diseased patients: double blind trials - can compare new drug w/placebo and current standard treatment - v. expensive - certain toxic elements may appear
        
        NDA (new drug application) - review process up to 18 mnths - if approval obtained - clinical phase 4 and post marketing surveillance - release of drug for perscription use - viglient marketing supervision
        
        patents have avg life of 11.5 yrs / generics after patent expired - no clinicl trials - must be bioequivalent
- - - - fate of dietary glucose: tissues need glucose (for energy - "blood sugar") - tissues up regulate glucose transporter (GLUT-4)
      - pancreas makes insulin - Helps lower blood glucose levels by promoting the uptake of glucose into cells, especially muscle, fat, and liver cells, where it can be used for energy or stored as glycogeninsulin signalling pathway has effects on lipid metabolism, growth and protein metabolism --> regulation of insulin central to regulating glucose metabolism and health - these processes b/down due to genetic and environmental factors
      - dietary choices -> circulate glucose -> increased circulating glucose = hyperglycemia -> affects multiple organs, eye, brain, kidneys
      - Type1 Diabetes: damage to pancreas - pancreatic beta cells die - decreased insulin production -'juvenile diabetes' - results in hyperglycemia (abnormally high levels of glucose (sugar) in the blood) - ketosis can occur = use of ketone bodies as a primary energy source when glucose levels low -> organ failure in the case of diabetic ketoacidosis - recombinant (synthetic) insulin as a revolutionaised treatment - presence of T cells which react with beta cell antigens suggests autoimmune disease (body’s immune system mistakenly attacks and destroys the insulin-producing beta cells in the pancreas - decrease in insulin) - CD8 T cells v. important in mediating destruction - CD4 provide cytokine help, TNF, IFN gamma - some genetic markers associated w/T1 dia - typical in children
      - Type2 Diabetes: adult onset diabetes- tissues irresponsive to insulin - insulin resistance develops in periphery - muscles cant use glucose - increased risk for CVD, obesity, infections, kidney disease (diabetic nephropathy) - diet induced
        
        insulin resistance (IR) = metabolic overload - molecular mechanism of defective IR signalling - a variety of obesity-associated factors impinge on homeostatic IR signalling and GLUT4 expression - Loss of GLUT-4 translocation drives hyperglycemia - IRS made inactive which contains Ser-P and Tyr-P - Tyr P needed for glucose uptake
    - - inflammation triggers insulin resistance - inflammation due to expansion of adipose tissues due to obesity -> increased proinflammatory cytokines
      - adipose tissue expansion is due to a calorie excess which can cause healthy and unhealthy expansion
        
        healthy expansion: increased adipogenesis, decreased cell volume, increased insulin sensitivity, decreased inflammation
        
        unhealthy expansion: decreased adipogenesis, increased cell hypertrophy, decreased insulin sensitivity, increased low grade inflammation, increased macrophages inflammation
        
        leads to lipotoxicity : ectopic lipid deposition, NAFLD, instulin resistance, T2Dia, CVD
        
        adipogenesis = fat-laden cells (adipocytes) develop and accumulate as adipose tissue at various sites in the body, both as subcutaneous fat and as depots.
        
        Small adipocytes (fat ladden cell) have multilocular lipid droplets, organised cortical actin, intact GLUT4 translocation
        
        heterotrophic adipocyte - large cell size, uniocular-like (1 eye) lipid droplet, disorganised cortical actin, impaired GLUT4 translocation
        
        becomes a dying adipocyte and releases fatty acids - inflamed adipose tissue - adipocyte death and lipotoxicity
      - obesity = response to increased lipid intake, regulated by inflammatory response
        
        insulin sensitive = anti-inflammatory // insulin resistance = proinflammatory // immune cells mediate inflammation in adipose tissue
- - - - macrophage foam cell = in vitro generated - upregulated uptake of deposited lipids - intracellular stoarge of excess lipids (lipid droplets) - poor efflux (flowing of substance) - inflammatory activation (cytokine, chemokine production), recruitment of new monocytes, new macrophages foster and promote inflammation
      - Regulation of intracellular cholesterol is dysfunctional in macropahge foam cells: foam cells are the hallmark of atherosclerosis
        
        hyperlipidemia : increased LDL - continual exposure and uptake / decreased HDL - defecctive efflux
        
        systemic inflammation promotes macrophage recruitment - macrophage phagocytosis is unregulated - defective resolution of inflammation - no macrophage egress
        
        modification of LDL -'response to retention theory' - how LDL cholesterol LDL particles are modified by oxidative stress, EC enzymes - new DAMPs (Damage-associated molecular patterns ) revleaed - Hijack innate immuen system - cells lack enzymes to degrade or efflux - innapropriate trafficking to intracellular compartments
        
        in context of atherosclerosis - LDL particles become retained in arterial walls - can undergo modification due to local environment
        
        LDL particle - phospholipid monolayer - cholesteryl esters, FREE cholesterol, triglyceride core, apolipoproteins embedded // sensitive to oxidation and other modifications - particularly in sub-endothelial soaces - inflammation enhances this (Free radicals) // alters physical and chemical properies including receptor specificity!
        
        modified forms are internalised faster - uptake is unregulated (doesn't reach a maximum) - it is receptor mediated (block it with inhibitors) - receptor system regulated v differntly from LDL-R // i.e. modified LDL are strcuturally v different so require differnt receptors to recognise them otherwise they will accumulate in arterties - these diff receptors are = scavemger receptors
        
        SCAVENGER RECEPTORS: fam of strcuturally unrelated receptors - bind a wide range of modified self and non self ligands - trigger uptake by receptor mediated internalisation = 'scavenge' dead cells, lipids, bacterial componenets, amyloid - original PRRs - discriminate infectious self and non-infectious self
        
        scavenger receptor expression is unregulated // SR mediated loading can impair efflux
        
        plaque formation and macrophage foam cells: SR-activation triggers TLR signalling - actiavtes NKkB-induced interleukins -> Lipid deposition leads to extracellular cholesterol crystal formation -> frustrated phagocytosis - SR mediated uptake of cholesterol triggers crystal formation at lysosome - disrupts lysosomal inetgraity - full inflammation activation with IL-1 secretion - IL1 can trigger plaque formation and promote cytokines, e.g. IL18 - Further plaque progression can lead to a plaque complication i.e. atherothrombosis (blood clots in arteries) - thus IL-1 is the key cullrpit in CVD
    - - regulation of LDL receptor - Srebp2 = trc factor promotes expression of sterol response genes including LDL-R - normally bound to SCAP at ER (inactive) - SCAP = cholesterol sensitive (through interaction with INSIG) // high cellular cholesterol levels maintain this interaction, SREBP at ER // low cholesterol levels alters SCAP-INSIG interaction and SREBP-SCAP moves to golgi - SREBP2 activated by proteolytic cleavage, liberates bHLH-zipper N term fragment - in in nucleus, binds SRE and increases LDL-R expression to promote lipid uptake
      - Regulation of LDL receptor - Pcsk9 = PCSK9 regulates LDL-R protein activity - secreted on plasma membrane - binds LDL-C/LDL-R complex - after internalisation, PCSK9-binfing promotes degredation of LDL-R - blocks recycling of LDL-R to plasma membrane - after serum LDL levels (less LDL-R, increased serum LDL-C)
      - cholesterol efflux and reverse cholesterol transport (RCT) pathway: lipid-loaded cells need to 'unload' (to avoid inflammation) - cholesterol needs to be removed from peripheral tissues and from foam cells to be delivered to liver to be broken down : HDL act as cholesterol acceptors, facilitating its removal from cells - lipid droplets within cells store cholesterol as cholesterol esters (CE) - CE are hydrolysed bacl to free cholesterol (FC) by cellular enzymes so they can be transported to apolipoproteins associated with HDL - results in newly syntehsised HDL or modification of existing HDL particles to accept more cholesterol - HDL particles carry the cholesterol through blood to liver where it is then recycled to form new very low density lipoprotein (VLDL) or LDL particles, OR excreted into bile and elimanated as feces
        
        this process helps reduce choelsterol accumulation in macrophages and foam cells - prveneting plague formation
      - cholesterol efflux at the plasma membrane - ABC transporters: choelsterol must get out of cell - DIFFUSION or ACTIVE TRANSPORT - active transport uses energy and requires TRANSPORTER (ABC proteins) - recquires ACCEPTOR (HDL/apolipoprotein)
        
        ABC proteins - family of memebrane bound transporters - ATP-Binding Cassette transporters - many involved in transporting cholesterol out of cell - transport protein engaged affects acceptor used - thus modifies HDL particle as its generated
        
        ABCA1 - membrane loacted, transports FC (free cholesterol) to extracellular apoA1 - generates new HDL
        
        ABCG1 - ER located, binds FC and oxysterols (ER - ENDOPLASMIC RETICULUM) - transports to plasma membrane - effluxes to HDL particles - makes them lipid rich
        
        SR-B1 - scavenger receptor family - facilitated diffusion of FC - can work both ways and accept cholesterol inside cell
        
        regulation of effluxL LXR System: ABC transporters are transcriptionally regulated by LXR system - Liver-X-Receptor active when cellular cholesterol rises - generates oxysterols - bind LXRs in nucleus - promote activation and binding target genes - promote activation and binding taregt genes - includes ABCA1/ABCG1 - often work in concert wth RXR partners
      - coordinated regulation of cellular cholesterol: SREBP SYSTEM (promote upatke and syntehsis genes - activated by low cellular cholesterol content - de-activated by increased cellular cholesterol) // LXRs SYSTEM (promote efflux genes - actiavted by increased cellular cholesterol content)
  - - - adverse effects of hypercholesterolemia: blood vessels contain plaque formation and atherosclerosis -> CVD // bone marrow and spleen experience HSC (hematopoietic stem cells) expansion and skews their differentiation toward the myeloid lineage, increasing granulocyte and monocyte production // brain: blood/brain barrier function, amyloid deposition (AD) // lung: alveolar macrophage (AM) proliferation and remodeling -> asthma
- - - - ligand binding to RTKs - most RTKs are monomeric - ligand binding to EC domain induces dimerization - some ligands are diemric - effectively 'crosslinks' two recpetor molecules - insulin receptors occur naturally as a dimer - activation is due to the conformational change of thereceptor upon ligand binding
        
        consequences of receptor dimerization : autophosphorylation (kinase in one subunit phosphorylates one or more tyrosine residues on the other) - phosphorylation within the catalytic domain increase intrinsic kinase activity - phosphorylation outside the catalytic domain creates specific binding sites for other proteins - autophosphorylated receptors bind to signalling proteins that have SH2 domains
        
        RTK is inactive - kinase activity stimulated by trans-phosphorylation - activated RTK - can now interact with intracellular signaling proteins.
        
        binding domains for phosphotyrosine residues : phospholipase Cy1 - downstream partner showing link between phosphatidyl turnover, Ca+2 mobilization and actiavtion of PKC
        
        SH2 domains : interact w/phospho-tyr residues // SH3 domains: interact w/proline rich seuquences
        
        SH2 Domain: 100 AA, as well as interacting with phospho-Tyr, it also mediates interactions w/ 1-6 AA on C terminus of phospho-Tyr residue
        
        3 major SH2 domains:
        
        Src-type SH2 domain - recognises charged residues in positions +1, +2 as well as a hydrophobic residue in position +3 C terminally from phospho-Tyr
        
        PLC-type SH2 domains: interact w/aliphatic residues in positions +1 to +5
        
        Grb2-type SH2 domains: recognise an Asp residue in position +2
      - Signal Transduction through EGFR
        
        EGFR -prototypic RTK (mutated version) - 4 family members: ErbB 1-4
        
        EGFR (ErbB1): binds several ligands including EGF Transforming Growth Factor-alpha (TGF-α) - Cell proliferation and differentiation
        
        HER2 (ErbB2): lacks a direct ligand but is the prferred heterodimerization partner for the other fam members - overexpressed in cancers such as HER-2 positive breast cancer
        
        HER3 (ErbB3): impaired kinase activity but can dimerize with ErbB1, 2 or 4
        
        HER4 (ErbB4): Fully functional kinase domain and ligand-binding capability
        
        downstream signalling: 4 EGFRs exhibit 90 potential Tyr phosphorylation sites - can interact w/approx. 80 partner proteins
        
        intracellular signalling pathways: e.g. Ras-MAPK Pathway
        
        Ras is a GTPase switch protein - its activation is enhanced by Guanidine Exchnage Factor (GEF) - a GDP-GTP exchange // deactivation of Ras-GTP complex requires GAP which increases intrinsic GTPase activity
        
        mutant Ras proteins associated w/many cancers - mutant Ras can bind GTP but cannot hydrolyse it - remains in its 'on' state constantly - most oncogenic Ras proteins contain a mutation in codon 12 (Gly) - most common are Gly -> Asp , Gly -> Val - blocks the binding of GAP to Ras and prevents GTP hydrolysis - this blocks binding of GAP to Ras and prevents GTP hydrolysis
        
        Linking Ras to RTKs : experimental evidence -fibroblasts were induced to proliferate with FGF and EGF - anti-Ras antibody microinjected: cell proliferation arrest - injection of mutant Ras proteins allows cell to proliferate in absence of growth factors (demonstrates Ras is essential for transmitting the mitogenic signal downstream of RTKs)
        
        molecular mechanism: 2 cytosolic proteins involved: GRB2, Sos - SH2 domain in GRB2 binds to a P*-tyrosine residue in activated receptor - two SH3 domains of GRB2 bind to and actiavte Sos ( a GEF protein) - Sos can convert inactive GDP-Ras into active GTP-Ras - c terminus of Sos inhibits its nucleotide exchange activity - binding of GRB2 relieves this inhibition
        
        Ras is activated when GTP bound (inactive when GDP bound) - facilitated by GEF portion of Sos
        
        MAP-kinase phosphorylation pathway activated by Ras
        
        LIGANDS: EGF or EGF-like growth factors (13 hormone-like proteins) // RESPONSES: cell proliferation, migration, differentiation, cell death // OUTCOME: dependent on cell type, physiological context, ligand receptor isoform // OVEREXPRESSION: hyperactivity associated w/human malignancies
        
        Ligand binding induces receptor dimerization (HER2 is preferred dimerization partner - thus HER2 targeted therapies) - Dimerized receptors autophosphorylate tyrosine residues on their intracellular domains - phosphotyrosines serve as docking sites for signaling proteins containing SH2 domains, activating pathways such as Ras-MAPK (proliferation), PI3K-Akt (survival), JAK-STAT (transcription)