Nuclear (Hormone) Receptors Flashcards
Why is PPAR-alpha targeted therapeutically?
It is activated by drugs to treat human disorders of lipid metabolism
How can FAS-dependent activation of PPAR-alpha be used to find a ligand for PPAR-alpha?
The ligand probably produced by FAS. Binding of the ligand increased by increased FAS activity. Ligand displaced by PPAR-alpha agonist. To purify: mouse injected with AdFLAG-virus, liver harvested, hypotonic lysis and extract nuclear fraction, incubate with alpha-FLAG Ab, elute with 3 x FLAG peptide, mass spec
How do you find an endogenous ligand for a NR?
Exploit fatty acid synthase (FAS)-dependent activation of PPAR-alpha
What are nuclear receptors?
Ligand-modulated transcription factors that bind directly to DNA
Give 4 components of a transcriptional regulatory complex
- transcription factors
- chromatin remodelling factors
- co-activators and co-repressors
- basal transcription machinery
How do chromatin remodelling factors generally work?
Generally through PTMs of histones/ remodeling (bending) DNA
Nuclear receptors bind to specific sequences in DNA. What are these called?
Nuclear receptor response elements
The activity of gene regulatory proteins is modulated in several ways in eukaryotic cells. How are nuclear receptors primarily modulated?
- Ligand binding
2. Regulated nuclear entry
What are histones?
Small basic DNA packaging proteins associated with DNA
How does acetylation of lysine residues in histone tails affect transcription?
Increases transcription of that gene
There are approximately 40 NRs in total. Give a few examples of NRs that fall into:
1) Class I
2) Class II
3) Class III
1) Thyroid hormone receptors, Retinoic acid receptors, PPARs, Vitamin D receptors
2) RXR (retinoid X receptor)
3) Glucocorticoid receptor, Androgen receptor, Progesterone receptor, Estrogen receptor
What type of receptors are NRs?
Intracellular receptors for HYDROPHOBIC signal molecules (e.g. steroid/ thyroid hormones and retinoic acid receptor).
The receptor-ligand complex acts as a TF in the nucleus
What sort of ligands do NRs bind?
Hydrophobic
How does the Class II NR, Retinoid X Receptor (RXR) generally function?
As a heterodimer with other NRs, e.g. Class I NRs (TR, RAR, PPAR, VDR)
What are the 5 functional domains within a NR? (from N-terminal to C-terminal)
- AF-1 (ligand-INDEPENDENT transactivation domain)
- DBD (recognises specific DNA sequences)
- variable linker region
- LBD
- AF-2 (ligand-DEPENDENT transcriptional activation domain in C-terminal portion of LBD)
What is the function of the AF-1 domain in NRs?
It is a ligand-INDEPENDENT transactivation domain
What is the function of the AF-2 domain at the C-terminal end of NRs?
It is a ligand-DEPENDENT transcriptional activation domain
What are the three key domains in nuclear receptor co-activators such as CBPs?
- RID (NR-interacting domain): LxxLL
- KIX (CREB-binding domain)
- HAT
What are the three key domains of a nuclear receptor co-repressor such as NCoR or SMRT?
- RD1 (recruits HDACs indirectly via mSin3)
- RD2/3 (directly recruits HDACs)
- RID (receptor-interacting domain: LxxI/H IxxxI/L)
Which domain is found in both co-activators such as CBP/p300 and co-repressors such as NCoR and SMRT?
RID - the NR interacting domain
Which co-repressor interacts with retinoic and thyroid hormone receptors?
SMRT
What type of molecule are endogenous nuclear receptor ligands?
Small lipophilic molecules
How do Class III NRs (eg glucocorticoid receptor, androgen receptor, progesterone receptor, estrogen receptor) work?
1) Steroid hormone permeates plasma membrane and binds receptor (which is bound to HSP complex)
2) Nuclear receptor undergoes LIGAND-DEPENDENT RELEASE from cytoplasmic sequesteration
3) Liganded receptor DIMERISES & TRANSLOCATES to the nucleus
4) Receptor binds to DNA and recruits co-activators to enhance transcription of specific target genes
How do Class I NRs (e.g. thyroid receptor, retinoic acid receptor, PPAR and vitamin D receptor) work?
- Receptor dimer (typically heterodimer with RXR) is stably bound to DNA in an inactive state (often bound to corepressors)
- Lipophilic ligand permeates the plasma membrane and nucleus OR ligand precursor is converted intracellularly
- Ligand binds to receptor, allowing recruitment of co-activators and transcription of specific target genes
What is the likely signalling mechanism for ‘orphan’ nuclear receptors?
- Receptor monomer is stably bound to DNA in an inactive state.
- Ligand permeates the plasma membrane and nucleus OR other PTM of receptor results in its activation
- Receptor recruits co-activators and transcription of specific target genes
Name 2 diseases that are treated by targeting the Retinoid X Receptor
- Dermatoses
2. Cancer (as chemotherapy)
Name two PPAR agonists that are used as pharmacotherapeutics. What diseases do each of them treat?
1) Fibrates - PPAR-alpha agonists - treat Dyslipidemias
2) Thizolidenediones - PPAR-gamma agonists - treat type II diabetes
Which NR do thiazolidenediones (TZDs) target? How are they used to treat Type II diabetes?
PPAR-gamma (agonists). They restore insulin sensitivity
Which NRs do Fibrates target? How do they treat dyslipidemias?
PPAR-alpha (agonists). They lower TGCs and VLDL and increase HDL
How can the sex hormone receptors AR and ER be targeted? What is this used for?
By estradiol (ER) and testosterone (AR) for hormone replacement and contraception
How can NR activity be measured?
1) quantifying changes in the levels of expression of target genes
2) utilising promoter-reporter assays
Give an example of a promoter-reporter assay that can be used to measure nuclear receptor activity
Luciferase: light signal = luciferase expression = promoter activity = nuclear receptor binding
