Nuclear Cunts Part 2 Flashcards
What is Chromosome Immunoprecipitation (ChIP) used for and how is it done?
Used for: mapping the DNA target of DNA-binding proteins and mapping the DNA target of histone modifying enzymes.
Method: Take a group of cells and cross-link the DNA and protein (or leave in its native bound form). Lyse the cells and then do sonication or enzyme digestion to fragment the chromatin Immunoprecipitate the DNA-protein complexes with antibodies directed towards the protein of interest. Purify the DNA and analyze the bound DNA using PCR, qPCR, Microarray, or sequencing.
What are SERMS and what are they useful in treating?
SERMs are neither pure agonists nor pure antagonists. It depends on what tissue they are acting in. Useful for a variety of treatments, especially in cancer treatments.
How do structurally similar NR proteins regulate different genes in different cells?
Ligand:
- Different specificity for ligand binding by NR.
- Metabolic control of ligand availability (used to discriminate specificity between aldosterone and cortisol).
- Nuclear Receptor:
1. Recognize different DNA sequences (hormone response elements).
2. Different expression patterns of NRs in cells (some may have higher levels of estrogen receptors than others, and so they respond differently to the ligands).
3. Activate different coactivators/ coregulators . So the type of effect we get from the cell depends not only on the presence of the receptor, but also the assemblage of the coregulators that are also involved
in regulating DNA.
Does ligand specificity always explain receptor specificity?
Nope. In some cases it doesn’t. Worst case scenario is comparing MR and GR. The affinity of mineralocorticoid receptor is almost the same for both cortisol and aldosterone. Similarly, looking at the glucocorticoid receptor, it also has similar affinities for cortisol and aldosterone. So how does the cell discriminate and respond appropriately to the ligand. For the glucocorticoid its pretty easy because cortisol is in a high abundance in the body compared to aldosterone, so the glucocorticoid receptor can stand for being regulated by aldosterone. And so the main regulator for the glucocorticoid receptor is cortisol just because of its high
abundance in the body (its about 10 times higher). This creates a problem for the mineralocorticoid receptor.
How does the mineralocorticoid receptor distinguish between cortisol and aldosterone if they have similar affinities for the receptors and cortisol is at a much higher concentration?
The cells in the collecting duct of the kidney have an enzyme that selectively degrades cortisol. This allows the mineralocorticoid receptor to just respond toaldosterone. This enzyme is 11 beta hydroxysteroid dehydrogenase. Located fairly exclusively in the colellcting duct, and selectively degrades cortisol so that it is no longer recognized by the mineralocorticoid receptor. So the specificity doesn’t stem from the ligand (ligand specificity), and doesn’t stem from the ligandbinding to the receptor (receptor specificity). It comes from the availability of the ligand because the ligand not being used is degraded. Converts cortisol to cortisone (inactive).
How can you study nuclear receptor function?
If you have the coding region of a receptor, transfect the plasmid into the cell. At the same time transfect another plasmid that may have a response element you want to test and evaluate for its activation or inhibition of transcription via action with the transfected nuclear receptor. Downstream of the element put a reporter gene. Typically luciferase. If the ligand binds then it allows for the transcription of luciferase which can be measured using a luminometer. Could also use Chloramphenicol Acetyl Transferase reporter gene to measure acetylation of chloramphenicol but this is an older and more difficult assay.
What are the functional domains of nuclear receptors?
N-terminus AF1 Co-activator region (isoform specific): This region is variable between receptors. Called AF-1 for Activator Functions.
- DNA binding domain with “zinc fingers’: small region (about 62 aa) that binds to the response element.
- Ligand binding domain (hormone binding domain): Hormone domain not only binds ligand to activate the nuclear receptor, but also has the co-activator binding region to, and thats the region thats going to be binding the histone acetyltransferase in the presence of ligand, or the histone deacetylase in the absence of ligand. Also binds HSP90 in the absence of receptor to inhibit the receptor action when ligand is not present and keep it in the cytosol.
- Also usually a hinge region in between DNA binding domain and ligand binding domain.
Could one swap out different regions of nuclear receptor domains easily?
Yes. The receptors are very modular in domain. So one could take a glucocorticoid
receptor and swap out a region (maybe the ligand binding domain) for another
receptor (estrogen receptor) and you can switch its specificity very easily. So the
domains are very separate from one another and can be switched. Can do the same
with the DNA binding domain.
Describe the homology of nuclear receptors.
The two classes have the same types of domains, but the receptors within a class are more similar to eachother than they are to receptors of a different class. So class I is like andother class I receptor and same goes with class II.
What do the DNA-binding domains of nuclear receptors look like and how do they bind DNA?
DNA binding domaine has cystein-cystein zinc fingers. Two sets of cysteins on each finger that coordinate the zinc ion that forms the finger. These regions bind DNA. It was thought that these fingers stuck down into the DNA, but this is not true. Instead, the first zinc finger asssumes a structure in which this part of the zinc finger forms an alpha helix and sits in the major groove of DNA and reads the nucleotides. The second zinc finger does not bind DNA at all, but is involved in dimerization between the two sets of zinc fingers in the two subunits of the homodimer. In general, proteins that bind DNA have some sort of a zinc finger. In the nuclear receptors, there are always 2 zinc fingers on each of the monomeric regions, but in proteins like transcription factors there can be more.
What is the important region in the ligand binding domain of nuclear receptors?
Helix 12 region on the C terminal end. Upon receptor binding, helix 12 changes its conformation and moves to another location. Agonist binds in a hydrophobic pocket which moves helix 12 in such a way to allow co-activators to bind to the receptor. Co-activators can then recruit enzymes such as HATs which acetylate the histones and allow for access of transcription factors and RNA pol. When antagonist binds helix 12 moves in such a way that blocks co-activators from binding and instead allows for co-repressors to bind. Histone deacetylases can then bind to the nuclear receptor and work to deacetylate the histones resulting in a tighter interaction between DNA and histones and tightens the winding of nucleosomes to repress transcription.
Do nuclear receptors only have the agonist bound state or antagonist bound states?
No. Not only can nuclear receptors transition between a state that favors co- activators and another state that favors co-repressors, but they can actually undergo intermediate transitions depending on the ligand thats bound. Selective receptor modulators dont put the receptors in a stricly agonist or antagonist state, but they put it in an intermediate state. So you can have different effects depending on the nature of the ligand.
What are some effects of estrogen?
Estrogen has a lot of different effects on the body depending on age and maturity of the individual. In young individuals its involved in stimulating growth and female characteristics. As the individual ages it will be involved in stimulating reproductive function like ovulation and menstration. LH and FSH stimulate estrogen and progesterone production in ovaries. Reproductive function: ovulation, menstrual cycle, pregnancy. Development of female characteristics in puberty. Bone health, prevention of osteoporosis. Increase amount of “good” HDL cholesterol; decrease “bad” LDL cholesterol; decrease risk of heart disease, Estrogen-dependent cancers (breast, ovarian); cell proliferation, Thrombosis (blood clots), Prevent menopausal symptoms (hot flashes, mood swings, fatigue)
How does estrogen plus progesterone do as a hormone replacement therapy? What are some of the effects?
- Estrogen given by itself was found to increase breast cancers, so they tried to give it with progesterone, but there were still some bad effects accompanying the good effects. The bad effects tended to outweigh the bad effects. Good effects: strengthens bones, decreases risk of colon cancer, reduces menopausal symptoms (hot flashes). Bad effects: increases invasive breast cancer risk, increases heart attacks, increases strokes, increases blood clots. The hormone replacement therapy was called Prempro. Because of these effects, women are much less often prescribed estrogen replacement meds, and if they are its for a much shorter time.
What is the main nautral agonist to estrogen receptors, what is it used as, and why is there interest to target the estrogen receptor?
Estrogen. Used as a contraceptive, typically combined with progesterone. Can also establish an ovulation cycle in women who don’t have one. Used as hormone replacement therapy. 80% of breast cancers have estrogen receptors on them and are responsive to estrogen. So one might want to inhibit the estrogen receptor.
