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Harnish - 3 1 Cellular and Molecular Biology Legacy Summary - Part One Welcome to the Bachelor of Health Sciences (Honours) program here at McMaster University. When you arrive in September, you’ll take a course called Cellular and Molecular Biology (HTH SCI 1I06), which covers a variety of topics pertaining to intracellular and extracellular communication mechanisms via signaling molecules. Below is a brief outline of some of the items that will be covered; we hope you will find it useful in your studies. Figure 2. The Big Picture... Figure 1. The connection between gene expression and cell signaling. Response requires proteins, which are mediated by gene expression. Components of these processes will be discussed in further detail. Gene Expression Molecular Biology With the help of numerous enzymes, cells are able to replicate their DNA. These double stranded helical structures are transcribed into single stranded RNA, which is translated into a peptide chain. The structure of the peptide chain is altered in accordance to four different stages, corresponding to primary, secondary, tertiary, and quaternary structures. Proteins, the outputs of genes, are the final product of this structural change. The ordering of this process is known as the “central dogma” of molecular biology - the steps will never naturally occur in reverse order. Details of these processes will be further discussed over the course of Cellular and Molecular Biology 1I06, and are briefly outlined in Figure 2. Gene expression is the process of deriving information from DNA. Each set of genes has its own set of control functions for where and when it is expressed. There are two types of gene expression: constitutive expression and inducible expression. Constitutive expression is constant and low-level expression. Inducible Expression occurs when the genes are stimulated to do so, thus they can be turned on and off. Gene expression can be controlled at numerous points, however it is most efficient to target gene expression early. Direct DNA interaction and transcriptional control are the most effective methods (however there are others). Gene Regulatory Sequences (GRS): binding sites for gene regulatory proteins (GRP) to change what is expressed. • GRP-GRS Complex can act as a transcriptional activator or transcriptional repressor Promoter: DNA sequence where general transcription factors (TF) and polymerase assemble. • general TFs that assemble at the promoter are similar for all polymerase II transcribed genes • GRPs are specific and locations of binding sites relative to the promoter are different for each gene 2 Figure 3. Interactions between a GRS and promoter sequence near a gene. Examples include: dopamine, acetylcholine, and norepinephrine. Receptor Theory Post-Translational Modification: • proteins must be modified after they are made • most go through the endoplasmic reticulum, where they undergo structural modifications ex. folding, addition of sugar groups (glycosylation) • move into the golgi bodies - further maturation occurs proteins are moved to intended destination, ex. receptors are moved to the membrane • see Figure. 2 Signaling Molecules Figure 4. General Features: A receptor is a protein that signaling molecules bind to, which triggers a certain effect. yhA protein can exist in many different shapes at different times and equilibrium exists between active and inactive conformations of a receptor. They can be inductive (activated by a ligand) or constitutive (spontaneously activated). The process of a molecule binding to any one receptor is stochastic (random) and contains a probability distribution. The Law of Mass Action states that equilibrium is reached when half the available receptors are occupied and the other half is free (D + R --> DR Complex) Agonists An agonist binds to a receptor to elicit a response. Type Preferred Conformation Equilibrium Shift Full Agonist Active More active Partial Agonist Active and Inactive Partially active Inverse Agonist Inactive Inactive Example: Histamine Synthesis • messenger molecules are assembled via enzymes • synthesized from histidine (amino acid) using histamine decarboxylase Storage (optional) • may be stored in vesicles and are released when the vesicles fuse with the cell membrane • stored in granules, various stimuli (such as allergic reactions) cause the granule to fuse to the cell membrane, releasing histamine Response • three steps: recognition of signaling molecule, signal transduction, amplification • dependent on type of receptor • several types of histamine receptors, various effects Termination • either through degradation by enzymes or re-uptake by other cells • enzymatic degradation or reuptake by other nearby cells Neurotransmitters: signaling molecules released by neurons (brain cells) - follow the general pathway. Antagonists An antagonist binds to a receptor and does not elicit a response; it prevents an agonist from functioning. Type Interaction Impact Competitive Antagonist • orthosteric (same receptor site as ligand) • surmountable: effects of antagonist can be overcome by adding more ligand Potency (more potent = requires less drug for same effect) Noncompetitive Antagonist • allosteric (different receptor site than ligand) • insurmountable: maximum response can never be seen Efficacy (more efficient = higher response from same amount of drug) 3 Pharmacological antagonism describes two molecules acting on the same system. Physiological antagonism describes two molecules acting on different systems to reduce and nullify each other’s effects Drugs require affinity (ability to bind to receptors) and efficacy (ability to induce a response) Example of Receptor-Ligand: Nervous System. The parasympathetic and sympathetic nervous systems target different organs in the body simultaneously using different signaling molecules and pathways. The predominant tone refers to the pathway exerting a greater effect on the target tissue at resting stage. This is an example of physiological antagonism. For example, epinephrine is used in the sympathetic nervous system while acetylcholine is used in the parasympathetic. An example of a larger system that uses this GPCR pathway is the autonomic nervous system. Examples of Clinical Applications • Antihistamines act as antagonists to histamine receptors, which in turn are classified as g-protein coupled receptors (GPCRs) • Inhibit the effect of histamine on tissues in which the receptor being acted upon is found • For example, H1 antagonists (e.g. diphenhydramine) affect vasodilatation • Sarin is an organophosphate that binds covalently to acetylcholinesterase, an enzyme that breaks down the neurotransmitter acetylcholine in the synapse • Pralidoxime can be administered and effectively breaks this bond, allowing a sarin-pralidoxime complex to be excreted Research Methodologies Signal Transduction The basic pathway of signal transduction is: signal > reception > transduction > amplification > response. First, a cell surface receptor is activated by a ligand and the signal is transmitted into the cell by a second messenger. This is followed by signal amplification via a phosphorylation cascade, and physiological response. The signal transduction pathway involving cyclic adenosine monophosphate (cAMP): 1.Signal: a ligand binds to a GPCR 2.Reception: the GPCR dissociates into two parts and activates adenylyl cyclase (AC) 3.Transduction: AC converts ATP to cAMP, a second messenger which then binds to protein kinase A (PKA) 4.Amplification: the binding of cAMP to PKA allows the release of PKA’s catalytic subunit, which continues amplification by phosphorylating other proteins (phosphorylation cascade) 5.Response: physiological response depending on the type of ligand *Note that cAMP levels are regulated by phosphodiesterases (PDEs), enzymes degrade cAMP. PDEs are activated by PKA through phosphorylation and deactivated by phosphatases through dephosphorylation. Different research methods are used to analyze different types of molecules - certain methods are most effective with respect to certain molecules. Methodology What it does Fluorescence • glowing proteins used for tagging • can tell if target is there and how much • use to see if treatment meant to reduce target is working Gel Electrophoresis • separate molecules based on size and/or electrical charge • “blots” can be used to find presence of genes, expression of genes, or sequence proteins Organ bath • looks at response of tissue to combinations of substances at various concentrations Harnish - 3 1 Cellular and Molecular Biology Legacy Summary - Part Two Misunderstanding One concept we had difficulty understanding was why ligands may prefer one conformation over another in their applicable receptor(s), though this process is entirely stochastic (random). Since receptors are proteins, their structure can be changed. Initially, we struggled with the concept of how there can be different conformations of receptors, since we had previously believed that only active and inactive forms existed. Explanation In order to better understand this concept, we looked to the following analogy: • hungry Health Sci’s always eat pizza for lunch • each selects the type of pizza (s)he likes best • this represents a situation in which each Health Sci (ligand) that consumes (binds to) a pizza (receptor) prefers a different topping (conformation) • some conformations are more popular than others • ex. first years (the ligand) like (have an affinity for) pepperoni pizza (one conformation), so they always eat (bind to) the pepperoni pizza • as the first years enter the cafeteria and consume the pepperoni pizza, the cafeteria must replenish their supply of the pepperoni pizza - this corresponds to how the receptor changes to the conformation the ligand prefers When the first-years initially enter the cafeteria, any amount of the various different types of pizzas may be available for consumption. This represents the stochastic nature of receptors - they may exist as one of many possible conformations at any given time. When a receptor is not bound to a ligand, it is more likely to be found in its inactive form. When bound to an agonist, the receptor is more likely to be active. Although this aspect is stochastic, an equilibrium is established between the more and less active conformations of receptors. Shifts in equilibrium occur as ligands bind to receptors - the ligandreceptor complex is treated as an entirely different species, and is effectively removed from the equation. Now, receptors must be replaced according to demand. Conclusion The analogy of the “conformational cafeteria” helped us understand how each ligand has a preferred conformation, and how the binding of ligands to receptors affects the equilibrium of receptor conformations. The comparison of ligandreceptor interactions to personal preferences and product replacement helped to clarify this concept.