Receptors - Leid Flashcards
Steroid, thyroid hormones, PPAR-y and vitamin A receptors act in similar or dissimilar ways?
Similar, although the ligands are very diverse.
There is a centrally-located DNA binding domain. Recognize sequence of about 13-18 nucleotides in the genome, and bind to that sequence specifically.
If you compare human glucocorticoid receptors to vitamin A drosophila receptors there is 70% similarity.
Two points about conservation of binding domains on receptors.
- DNA binding domain is highly conserved
2. Ligand binding domain is less conserved
What is the basis for most drug interactions?
The ability of drugs to activate or inhibit SXR, which is related to 3A4 activity.
Where are hormone receptors located?
Nuclear receptors are located in the nuclear, and not associated with any membranes at all.
DNA binding domain
- Bind to DNA in a sequence-specific manner on the promoter region.
- crucial because we want estrogen receptor that estrogens are supposed to regulate. This is all encoded by the DNA binding domain.
- Specific base pair contact is required.
- Can also piggy-back on top of proteins that are bound to DNA.
What do hormone receptors bind to?
They bind to the promoter region of the gene, directly, to regulate transcription.
- Then we transcribe the nuclear mRNA into protein coding regions called exons, and non-coding regions called introns.
- During translation we take out the introns and make the proteins.
How do drugs affect gene expression?
The cause gene expression.
Compare the dimerization of estrogen and thyroid hormone.
Homodimers - 1 molecule of estrogen receptor and 1 molecule of estrogen receptor
Heterodimer - 1 molecule of thyroid receptor and 1 molecule of vitamin A receptor
How are the alpha helices situated in the hormone dimers?
They are situated perpendicular to each other.
There is a support helix and recognition helix in each monomer. The support helix interacts with recognition helix, but not the DNA.
PPARy and RXR are heterodimers with each other.
Each monomer interacts with their own DNA binding domains in a major groove.
How does the sequence differ depending on the dimer?
Estrogen receptor homodimer has a palindromic sequence that it binds to. The “half site” sequence is the same (the first half) and the second is a mirror image.
The heterodimers
The major dimerization interface is in the ligand binding domain.
So
What interacts more between the dimers, the DNA binding domain or the ligand binding domain?
The ligand binding domain. There are weak interactions in the DNA binding domain.
These receptors bind to DNA in a sequence-specific manner, and are able to pick out 13 nucleotides out of your genes. Very important.
TATA box
most eukaryotic genes do not have TATA boxes (<50%), but most prokaryotic genes have these.
Still requires TATA factors for transcription to occur
If it has, then it is within 30 bps
GC box
Rich in Gs and Cs
CAAT box
Enhancer protein binds to the CAAT box. These are farther upstream than TATA (w/i 200 bps)
Silencers and Enhancers
- Silence or enhance genes
- Work in a distance-dependent manner - they are way out there
- They may loop back and touch where mRNA is made
What do hormone receptors do?
They are going to affect the transcription of the gene in either a negative or positive way. mRNA will be made or not.
How do we get all of these proteins to come to the template and make mRNA when they are wrapped up in histones?
Activated nuclear receptors enhance the formation of PIC.
PIC
Pre-initiation complex, containing 44 different proteins.
Assemble PIC at the start site of transcription
RNA polymerase
What is the rate-limiting step of transcription?
Assembly of the PIC
What is DNA wrapped around?
Histone octamer
What is the function of H1?
It is the stabilizing histone of the histone octamer that binds like a staple to the DNA around the histones.
What are on the histone tails?
They are rich in lysine and arginines, which are subject to postranslational modifications such as acetylation and methylation.
Lysine charge normal, with acetylation.
Tails of histones
Positive charge at physiological pH because of amine
Amine, when acetylated, becomes an amid. Charge is 0.
DNA charge
Anionic polymer, with a negative charge.
What happens to the charge of the histones when the lysine are acetylated?
It becomes more neutral, which means that the DNA lets go of the histones.
What happens when lysine is methylated?
Nothing. The charge is still positive. No histone interactions.
Methylation forms a binding site for proteins that bind methylated lysines or arginines specifically (enhance recruitment of PIC to template)
Possibly more important than charge thing.
Hormone receptors will influence post-translational modifications, acetylation and methylation of histones that are associated with DNA and accelerate formation of the PIC.
A little more complicated because these modifications are reversible.
Acetyltransferases
Add acetyl group
Methyltransferases
Add methyl group
Deacetylases
Remove acetyl group
Demethylases
Removes methyl group
What modifications are negative for transcription?
Deacetylase
What modifications are negative for transcription?
Deacetylase
Epigenetic modulation
Changing the proteins associated with DNA to change how the DNA is read. This is crucial.
What are the effects of epigenetic modifications?
They change the way the genome is read
They are passed down from generation to generation
Changes in the histone code is a major mechanism that leads to the development of disease
Same as histone code
What is the only covalent modification of DNA that we know of?
Methylation
What are the three processes of histone modifications?
Write the code (writers)
Read the code (readers)
Erase the code (erasers)
Acetyltransferase
Writer
Place acetyl group on lysine
Methyltransferase
Writer
Methylate lysine or arginines
Kinase
Writer
Phosphorylate serine residues on histone tails
Ub E3 ligases
Writer
Place ubiquitin on lysine
Bromodomains
Reader - A protein with a bromodomain sequence
Bind to acetylated lysines. These are part of the PIC
Chromodomains
Reader
Proteins that contain chromodomains bind to methylated lysines
Tudor domains
Reader
WD40
Reader
Deacetylases
Eraser
Remove acetyl groups
Demethylases
Eraser
Remove methyl groups
Phosphatases
Eraser
Remove phosphate groups
DUbs
Eraser
Remove ubiquitin.
Where do writers come from?
Proteins that are recruited to the template by activated hormone receptors to write the code.
What are readers?
They are proteins that bind to the code. When they bind to their specific sequences, they accelerate the rate of formation of the PIC, which is rate-limiting for transcription of the code.
After the readers and writers do their business, how do we reset the code?
Recruit erasers to “erase” the marks that were written.
Nuclear receptors will affect what?
Writing
Erasing
When a lysine is acetylated, what happens to the charge?
The positive charge in lysine holds the histone tail to negatively charged DNA. The acetylation changes the charge to neutral, which destabilizes the chromatin to a certain extent.
What does methylation do to the charge of lysine/arginine?
Nothing. It is about providing a binding domain for PIC. The barcode.
Different proteins bind to the different methylated species of lysine.
How does methylation of lysine compare to methylation of arginine?
Lysine - mono, di, or tri-methylate. Different proteins bind to different methylated forms.
Arginine - mono methyl, dimethyl symmetrically or asymmetrically. Each of these is a template for a protein to bind to it.
Which proteins do the methylation?
The writers. They are recruited by the activated hormone receptors.
Which proteins sit down on the acetylated or methylated DNA?
The bromodomain (acetyl) or chromodomain-containing (methyl) proteins.
What are the most famous erasers?
Histone-deacetylases, which remove the acetyl groups on lysine to regenerate the positive charge.
The whole process is reversible.
Unbound receptors
Some interact with erasers - promote transcriptional silencing.
Lysine acetylation is always associated with transcriptional activation
Lysine deacetylation is always associated with transcriptional silencing or suppression.
Unactivated receptors or those with antagonists bound…
Some interact with erasers - promote transcriptional silencing.
Lysine acetylation is always associated with transcriptional activation
Lysine deacetylation is always associated with transcriptional silencing or suppression.
TATA box
Not always there
TATA-binding protein (TBP)
Have two bromodomains that are binding to adjacent acetylated lysine
Have some groups that are binding to methylated groups
This assembly causes formation of the PIC to facilitate transcription
What is important with modification?
That modification occurs, but also that it occurs in a specific pattern. This pattern of methylation and acetylation is like the Winco barcode to silence or express a gene.
What are readers a part of?
The initiation of transcription by PIC recruitment
Where is the ligand-binding domain on the receptor?
A big section near the C terminus.
When a hormone binds to the hormone receptor, what happens to helix?
It locks into place, which creates a shallow hydrophobic groove for the writer (green) to bind.
What mediates dimerization?
The ligand-binding domain
What receptors generally homodimerize? Heterodimerize?
Steroid receptors like estrogen homo.
Non-steroid receptors heterodimerize.
What is the apo-enzyme?
The receptor without drug or ligand, unactivated.
What is the activated form of the receptor?
The diethylstilbestrol form. Helix 12 is stabilized on the surface or face of the receptor.
If 4-hydroxy tamoxifen binds to a receptor, what would the conformation of the receptor be?
It is a SERM, the helix 12 would be flipped around and stabilized in a different conformation than in an agonist form, off the face of the receptor.
In breast cancer, there are mutations that happen due to genetics and promoted by the drugs that these people are given throughout their life. What do these mutations do to the structure of the protein?
The mutations cause the receptor to be in the agonist conformation without an agonist present.
What does Fulvestrant do?
SERD -
Selective estrogen receptor destroyer or degrader
Non steroid receptors are sitting on DNA in the absence of hormone agonists.
Thyroid, vit D, retinoic acid, PPARy
Steroid receptors are also sitting on DNA in the absence of agonist, but are in equilibrium with a form of the receptor which is interacting with heat-shock protein. What agonist is does is bind to the receptor, dissociate heat-shock protein, and hormone-bound receptor complex then binds to DNA.
Blah
What is the classic steroid receptor?
Glucocorticoid receptors
Mostly in the cytosol associated with heat-shock protein. (The oddball - most steroid hormones are not associated with heat-shock protein while agonist is absent).
What is the last transcriptional factor to come to the complex?
TF2H
Then Pol II is off
How long does this signaling pathway take?
Hours, to days, to weeks.
Hormones take a while, not like using a g-protein coupled receptors with a beta agonist that works in seconds.
What is the other type of protein that can bind to the receptor that is larger than the writer?
An eraser
Where do the readers bind?
To the modified histones as part of the PIC
Where do writers bind?
To the receptor
Where do the erasers bind?
To the receptor
When is the receptor degraded?
The receptor has to be degraded to work, for the DNA to be read. It happens before RNA polymerase reads the code.
What causes a receptor to be in different conformations?
The structure of the drug. The different conformations have different pharmacological activity.
Writers
Interact with hormone-activated hormone receptors
Why doesn’t a writer interact with an apo receptor?
Because it is interacting with helix 12, which isn’t in the right conformation in the apo form.
What are TIFs?
Transcriptional intermediary factors, which are cofactors necessary to recruit writers and the PIC. Doesn’t help with further transcription once template and PIC is already in place. RNA polymerase can re-initiate from template once it has occurred. There is some timer that turns off transcription, but once the template is open, transcription can occur several times.
What helices are important in the binding of a writer to an agonist-bound receptor?
3,4,5, and 12
The writer is really a complex with multiple writers on board, bound to dimeric nuclear receptors. What are the proteins involved in the complex, and what do they do?
Agonist bound receptor interacting with complex that contains writers.
p160 is a protein which contain the leucine-rich sequences that fit in the shallow groove in the the nuclear receptor. There are 3 HATs (acetyltransferases) PRMT (methyltransferase writer) NUMAC or SWI/SNF (remodels nucleosomes) CBP/p300 (writer with HAT) P/CAF (writer with HAT)
Want to modify epigenome (acetylate, methylate) and move nucleosomes around using NUMAC
3 times a writer
This complex assembly is entirely dependent on the hormone or drug
Erasers
Cannot be bound to agonist-bound form of the receptor
Interact with apo form (no agonist) of the receptor because of steric hindrance
Bound to the SERM/antagonist or unactivated form of the receptor
Steroid hormone receptors
In the absence of agonist, can be bound to heat shock protein. Heat shock protein is absolutely essential to hold the receptor in a conformation that agonist can bind to. When the ligand binds, then heat shock proteins dissociate.
Non-steroid hormone receptors
Type II
In absence of agonist, exist in co-repressor complex. Complex has protein that couples receptor other other proteins which are erasers (HDAC’s)
This co-repressor complex binds to the apo and possibly the SERM form of the receptor (not the agonist-bound form)
The co-repressor complex has a bunch of HDAC’s, which remove acetyl groups from histones and therefore are called erasers.
What is the difference between a writer binding to the receptor domain and an eraser binding to the ligand-binding domain?
The sequence that they bind to looks similar, but the eraser-binding motif is a little more “stretched” out.
The writer can only bind when TIFs are present as coactivators and the agonist is bound.
Erasers bind non-steroid hormone receptors in the absence of agonists and sometimes when the receptor is in the SERm/antagonist form.
NCoR
Nuclear co-repressor
Binds to erasers to actively silence genes
What are HDAC I’s used for
They are mainstays in certain types of cancer
Can be useful for certain CNS conditions
What are types of HDAC I’s?
Valproic acid
What are the class I HDACs?
1,2,3, &8
What are the class II HDACs
4,5,6,7,9,10
What can inhibit caspase 8?
cFLIP. This means that cell death pathway cannot be carried out completely.
Are the death domain pathways like TNFalpha, FAS, and DR4/5 intrinsic or extrinsic?
Extrinsic
What are the decoy receptors?
The have the external machinery of the death receptors, but do not have the intracellular death domains. They are a way for a cancer cell to suck up death signals so the cells do not die.
What type of bone loss occurs in primary osteoporosis?
In Postmenopausal osteoporosis, cancellous (or trabecular) bone decreases. Jaw is affected, radius, vertebral fractures.
In senile osteoporosis, both trabecular and cortical bone mass decreases. Leads to hip, pelvic, and vertebral fractures.
What are causes of secondary osteoporosis?
TZDs - more fat, less bone
phenobarbital, phenytoin, anti-estrogens
Glucocorticoids!!! Almost 50% of those on these get fractures
What do glucocorticoids do to the bone?
They increase osteoclasts for a short amount of time, but quickly this decreases as well. There is a decrease in osteoblasts. Osteoclasts are dependent on osteoblasts.
What is FRAX?
10 year risk for developing osteoporosis
T-score is how your BMD is compared to young, healthy adults
Z-score is how your BMD is compared to age, race, and gender appropriate. (Weight is an advantage)
What is the therapeutic objective for medications for osteoporosis?
To maintain BMD, rather than increase it.
Osteopenic
-1 to -2.5 should consider medication
80 million people, a lot of people
What are the four phases of bone remodeling?
Activation - osteoblast signals (RANKL) cause osteoclasts to form and differentiate at the bone lining cell layer
Resorption - osteoclasts chomp chomp
Reversal - osteoblasts signals osteoclasts to stop chomping. Osteoblasts get ready
Formation - Osteoblasts lay down new ostioid (unmineralized matrix), hydroxyapatite is deposited to make it hard
Paget’s disease
- can get anywhere
- painful
- making too much, poor quality bone
- excessive osteoclast activity
- osteoblasts fill in too quickly and do a poor job
- bone is structurally disorganized
- more susceptible to fracture
- treat using bisphosphonates
Hypercalcemia of malignancy
Why do cancer patients often develop high calcium levels?
20% have bone metastasis
80% have PTH look-alike secreted from tumors
About 50% die within 30 days
How do you treat osteoporosis?
- physical activity
- Calcium supplementation (1-1.2g elemental calcium/day
- strontium
- vitamins
- Hormones
- Cinacalcet
- Bisphosphonates
What does strontium do?
Only in Europe, induces osteoblast proliferation and differentiation, stops reabsorption of osteoblasts, has anabolic effect on bone, and inhibits osteoclasts
Hormones
Calcitonin- from salmon, may cause cancer in lab animals, not huge effect on bone fractures. Not very effective
Teriparatide - PTH pharmacologically has an anabolic effect on bone formation, increases BMD. Only 2 years. When you stop, BMD goes down. Start someone on bisphosphonates before stopping to maintain high levels
Estrogens and SERMs (Raloxifiene) - estrogen receptors in osteoblasts to decrease expression of RANKL. Suppress osteoclast activation.
Cinacalcet - Rarely used. Increases sensitivity to calcium sensor in PT gland. Less likely to secrete PTH. Makes it think that calcium levels are high. Decreases bone resorption.
Bisphosphonates - These are cheap and they work to maintain BMD. Have problems. Inhibit osteoclasts: either ATP analogs (1st gen) to form a toxic ATP derivative in the cell. Very specific to bone, especially osteoclasts. N-containing ones will disrupt cell metabolism.
Bisphosphonates
N-containing - disrupt cholesterol metabolism
Non-N containing - Toxic ATP derivatives in osteoclasts
What do you have to worry about if you profoundly inhibit osteoclast activity?
Osteonecrosis of the jaw (ONJ), especially with surgery or some type of injury
Who is at risk for ONJ?
Mostly cancer patients on IV bisphosphonates, less for osteoporosis patients on bisphosphonates. Also using Denosumab can cause this.
How should you decrease your risk of ONJ?
- Take care of dental stuff before starting therapy
- Avoid dental procedures if you are on IV bisphosphonates for cancer
- Stop bisphosphonates 3 months before procedure, 3 months after
What is OPG?
It is secreted by osteoblasts to bind RANKL before RANKL binds to RANK on the osteoclasts. It is an inhibitor
What it Denosumab?
It it a monoclonal antibody that binds to RANK instead of RANKL.
What does estrogen/SERMs do to RANKL expression?
They repress RANKL to inhibit osteoclast formation and action.
They also stimulate OPG expression, which inhibit RANKL by binding to it instead of RANK.
WNT signalling
WNT binds to frizzled, activates disheveled, which inhibits GSK3.
GSK3 is a kinase which is normally phosphorylating beta-katinin to degrade it. When it the phosphorylation is inhibited, then beta-katinin goes into the nucleus to regulate genes to drive mesenchymal stem cells to bone formation.
SOST binds to LRP5/6, which is part of the frizzled complex to inhibit WNT signaling.
How do you make bone?
Need to inhibit SOST (because it is inhibiting Wnt
Two antibodies to inhibit SOST
Romosozumab and blosozumab
These will be available soon
What is Dikkopf?
Acts like SOST. When we inhibit SOST, Dikkopf takes over.
How do we inhibit both SOST and Dikkopf?
There is a chimeric antibody that can bind both SOST and Dikkopf.
