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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
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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)
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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
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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.