Comprehensive lecture notes for Protein structures and function in cell signaling module covered in MCB3025F. These notes cover all content taught in lectures as well as additional materials (powerpoints, textbooks) required to succeed. These notes were created by a student who achieved a distincti...
Good notes, however, it is labelled wrong. This is cell signaling notes, not protein processing notes.
Par: ggauntlett • 5 mois de cela
Hi, sorry about that error! I have corrected this and let me know if you would still like the protein processing notes free of charge.
Par: enyasteyn • 5 mois de cela
Hi, yes, please. That would be much appreciated. Thanks!
Par: ggauntlett • 5 mois de cela
Not a problem, please send your email address.
Par: enyasteyn • 5 mois de cela
enyasteyn9@gmail.com :)
Vendeur
S'abonner
ggauntlett
Avis reçus
Aperçu du contenu
MCB3025F
Protein structures and function in cell signaling
Section 1: Receptor-ligand interactions
Lecture 1: Steroids: Important signaling molecules
- a steroid is an organic compound that consists of a four-ring structure, (four rings of carbon atoms) nots aliphatic not
aromatic and they are all made of cholesterol
- have sex hormones such as progesterone, estrogen and testosterone
- adrenocorticoid hormones are made in the adrenal gland and include cortisol, cortisone and aldosterone
- structure is very similar but slight difference affects the receptor and the precise interactions between the steroid and the
protein and are very specific interactions and one functional group can make a big difference to the affinity or specificity of
a particular ligand
Steroid receptors
- intracellular receptors: members of the nuclear receptor family
and mechanism of steroid receptor action
- nuclear receptor, in absence of ligand, located in the cytosol
- hormone binding to the nuclear receptor triggers dissociation
of heat shock proteins, dimerization and translocation to the
nucleus where the nuclear receptor binds to a specific sequence
of DNA known as a hormone response element (HRE)
- the NR DNA complex in turn recruits other proteins that are
responsible for transcription of downstream DNA into mRNA,
which is eventually translated into protein which results in a
change in cell function
- some reside in the cytoplasm and some reside in the nucleus and the glucocorticoid receptor for example, binds to cortisol,
lives in the cytoplasm and can easily enter the cell as its hydrophobic
- passes through the membrane, binds to the intracellular receptor, the hormone binds and kicks off the chaperones (heat
shock) as the receptor undergoes a conformational change
- receptor dimerizes, enters the nucleus via active transport through the nuclear pore, binds to DNA elements called
hormone response elements in the promoters of target genes and regulates transcription and can recruit components of
transcriptional machinery with as RNA polymerase, coactivators, corepressors, mediator complex, chromatin remodelers
- causes change in levels of expressions of mRNA which exists the nucleus, enters the ribosome, translated into protein
which changes cellular function
Concepts intracellular steroid receptors
- Ligand-activated transcription factors: transcription factors themselves but need to be activated via ligand binding
- protein-ligand interactions: ligand selectivity and affinity, agonists (endogenous hormone), partial agonists, antagonists,
protein-ligand degradation is also a mechanism of regulation
- protein-protein interactions: interact with several proteins such as chaperones, each other (dimerization), other
transcription factors, chromatin, cofactors, mediator and RNA polymerase II
- protein-DNA interactions: bind DNA sequence-specifically
- Post-translational modification: phosphorylation, removal of inhibitor protein, receptor turnover, chromatin remodeling
- are excellent models to study concepts and mechanisms of intracellular cell signaling and gene regulation
Physiological function of steroids
- Glucocorticoids (cortisol) via glucocorticoid receptor (GR): immune function (inflammation, immune response),
homeostasis, metabolism, bone density, stress response, ubiquitous (in all cells), knockout is lethal, however they do have
very bad side effects if taken for an extended period of time such as making bones brittle and can cause osteoporosis
- Mineralocorticoids (aldosterone) via mineralocorticoid receptor (MR): blood pressure, selective expression (kidneys)
- Estrogen via the estrogen receptor (ER) and progesterone via the progesterone receptor (PR): all aspects of female
reproduction (in development, menstrual cycle, pregnancy) also brain function
- Androgens (testosterone) via the androgen receptor (AR): all aspects of male reproduction (in development,
spermatogenesis), metabolism, muscle density hence athletes take it to try build up muscle, too much
can cause the individual to become sterile
Endogenous HPA (hypothalamic, pituitary, adrenal) steroids
- HPA is an endocrine axis and it is the hypothalamic pituitary adrenal axis
- brain controls everything, interprets the environment, hunger, stress and it decides when you need to
produce cortisol
,- sends a message in the form of a peptide hormone, corticotropic releasing hormone (CRH) from the hypothalamus to the
pituitary
- there is a specific cell surface receptor for this hormone in the pituitary which binds the hormone and causes synthesis and
release of adrenocorticotropin hormone (ACTH)
- ACTH is released by the pituitary, travels in the blood to the adrenal glands where there is a cell surface receptor which
binds ACTH
- binding causes synthesis and release of cortisol which changes metabolism, homeostasis, stress response, immune
functions etc.
- is an endocrine system as hormone is made in one gland and travels somewhere else to its target
- carefully regulated by negative and positive feedback loops
Endogenous HPG (hypothalamic, pituitary, gonadal) steroids
- testes are the male gonads and the ovaries are the female gonads and the HPG is the gonadal axis
where brain sends out messages from the hypothalamus (GnRH peptide hormone) which binds to the
GnRH receptor in the pituitary gonadotropins which produce gonadotropins which travel to the
gonads, luteinizing hormone (LH) and follicle stimulating hormone (FSH) which regulate the synthesis
and release of testosterone in the testes and estrogen and progesterone in females
- are also regulated by feedback mechanisms
- some contraceptives change hormone levels such as causing low estrogen levels and estrogen protects
against HIV infection so can increase susceptibility to HIV
Receptor-ligand (or protein-ligand) interactions
- ligand binding causes a change in the conformation of the receptor to expose surfaces for interaction with other proteins,
usually referred to as activation (protein is the receptor and ligand is the hormone)
- ligand binding causes a change in the conformation which exposes surfaces that then enable the receptor to interact with
other proteins, called activation
- steroid receptors get activated by steroids
Importance of these interactions
- affinity and specificity of a ligand/hormone for a receptor, the receptor and ligand concentrations, and the way the ligand
changes the receptor conformation, are critical parameters that determine physiological responses to endogenous hormones
and drugs, most drugs target receptors
- receptor and ligand concentrations and the way they change the receptor conformation are critical to determine
physiological response to endogenous hormones and drugs
- if designing a drug and want to target a specific receptor, want to make it cause an increase in transcription so that it binds
to a coactivator, understanding physiological responses is critical
Lecture 2: Ligand interactions, Kd, Fractional occupancy, Ki
- receptor ligands bind with high affinity and specificity
- affinity: strength of binding to a specific receptor
- specificity: ability of a hormone to distinguish between related receptors, or of a receptor to distinguish between related
hormones
Kd, the equilibrium dissociation constant
- is a measure of affinity, and is the range of the concentration of circulating hormones
- is approximately equal to the concentration of ligand required to saturate half the receptors
- changes in concentration of circulating hormones around the Kd allow large changes in fractional occupancy and hence
physiological response
- units are molar, greater Kd means lower affinity and smaller Kd means higher affinity
Fractional occupancy or saturation
- fractional occupancy or fractional saturation is the fraction of total receptors that are occupied by hormone or ligand
- it depends on Kd and hormone concentrations
- as more hormones added, more receptor is occupied and how much more depends on the affinity of hormone for receptor
- fractional occupancy or fractional saturation = Y
- can work out fractional occupancy if we determine Kd in a binding experiment and if we know the
hormones concentration and this will determine the response
[P] = concentration of free receptor protein (unbound by hormone)
[L] = concentration of free ligand or hormone (when ligand is in large excess, [Lfree] >> [PT] this is approximately equal to
concentration of total ligand)
[PL] = concentration of protein-ligand or receptor-hormone complex
Kd = dissociation equilibrium constant (units M)
[PT] = total protein or receptor concentration
, - ligand concentration and fractional occupancy is not a linear relationship
- equations only hold when you have large excess of ligand over the amount of receptor
Protein (P) binding to ligand (L) P + L PL
- the dissociation constant Kd is equal to the concentration of ligand at which
50% of the protein is bound
- the smaller the Kd, the higher the affinity of the protein for the ligand
- relationship between the concentration of ligand or hormone versus
fractional saturation, get a hyperbolic graph
- we know Kd is more or less equal to the concentration of ligand for 50%
occupation
- where ligand concentration is around the Kd, it is the steepest part oof the
curve hence why the circulating hormone has a concentration around the Kd
value, if change the concentration up or down, get the biggest change in
fractional occupancy
Relationship between fractional occupancy and physiological response
- not linear, and depends on receptor and cell
- maximal physiological response to many external signals occurs when only
a fraction of the receptor molecules are occupied by ligand
- in example: 50% of the maximal physiological response is induced at a
ligand concentration at which only 18% of the receptors are occupied
- likewise, 80% of the maximal response is induced when the ligand
concentration equals the Kd value, at which 50% of the receptors are
occupied
- response all depends on all the proteins and pathways downstream of the
receptor that are mediating the response and that will reach a maximal level,
cannot exceed this level
- blue is the response and the red is the fractional occupancy
- as you increase the fraction of receptors bound by a ligand you increase the response
- both are saturable and reach a maximum but don’t necessarily correspond numerically as it depends on downstream
signaling molecules
Sample calculations with Kd and fractional occupancy
Sample questions: What percentage of receptors are occupied?
1) Q: Kd = 1 nM, [H] = 0.1 nM
ANS: [RH]/[RT] = 0.1/1.1 = 9.09%
2) Q: Kd = 1 nM, [H] = 1 nM
ANS: ½ = 50%
3) Kd = 1 nM, [H] = 10 nM
ANS: 10/11 = 90.9%
- if ligand concentration increased 10 fold times, the Ks stays the same (is a constant for a particular receptor and ligand)
- when 10 fold increase the ligand concentration, change the occupancy only from 9 50 50% (don’t change the occupancy
10 times), not a linear relationship but have changed occupancy so get a bigger response as more receptors are occupied
- don’t know how much the response will change as not a linear relationship
- if increase by another 10 fold, increase from 50% to 90% so not a 10 fold in occupancy again but a substantial increase
which will probably increase response unless maxed out at 50%, depending on the system
Sensitivity of a cell to external signals is determined by the number of surface receptors
- the cellular response to a particular signaling molecule depends on the number of receptor-ligand complexes
- change is receptor levels is a mechanism of regulation (as well as change of hormone levels)
- the fewer receptors present on the surface of a cell, the less sensitive the cell is to that ligand, and as a consequence, the
higher the ligand concentration necessary to induce the usual physical response
- a lot of signaling pathway s are regulated by changing receptor levels up or down, for example steroids cortisol
downregulates its own receptor as a way of blunting the response
- become tolerant to drugs because the more drugs taken, become insensitive and have to make more and more to get the
same effect, that’s because the receptors are downregulated
- as receptors get downregulated, need more ligand to get same fractional occupancy to get same response
Les avantages d'acheter des résumés chez Stuvia:
Qualité garantie par les avis des clients
Les clients de Stuvia ont évalués plus de 700 000 résumés. C'est comme ça que vous savez que vous achetez les meilleurs documents.
L’achat facile et rapide
Vous pouvez payer rapidement avec iDeal, carte de crédit ou Stuvia-crédit pour les résumés. Il n'y a pas d'adhésion nécessaire.
Focus sur l’essentiel
Vos camarades écrivent eux-mêmes les notes d’étude, c’est pourquoi les documents sont toujours fiables et à jour. Cela garantit que vous arrivez rapidement au coeur du matériel.
Foire aux questions
Qu'est-ce que j'obtiens en achetant ce document ?
Vous obtenez un PDF, disponible immédiatement après votre achat. Le document acheté est accessible à tout moment, n'importe où et indéfiniment via votre profil.
Garantie de remboursement : comment ça marche ?
Notre garantie de satisfaction garantit que vous trouverez toujours un document d'étude qui vous convient. Vous remplissez un formulaire et notre équipe du service client s'occupe du reste.
Auprès de qui est-ce que j'achète ce résumé ?
Stuvia est une place de marché. Alors, vous n'achetez donc pas ce document chez nous, mais auprès du vendeur ggauntlett. Stuvia facilite les paiements au vendeur.
Est-ce que j'aurai un abonnement?
Non, vous n'achetez ce résumé que pour €2,65. Vous n'êtes lié à rien après votre achat.
