ERS07 Mechanism Of Action Of Steroid Hormones Flashcards
Steroid hormones
Steroidogenesis: ALL derived from Cholesterol (C27)
—> progressive **loss of carbons + **hydroxylation (oxidation)
Key features:
- Small hydrophobic (∵ benzene ring)
- Subtle structural differences between them
C18 steroid / Estrane
- Estrogen —> Estradiol
C19 steroid / Androstane
- Androgens —> Testosterone
C21 steroid / Pregnane
- Progestogens —> Progesterone
- Glucocorticoids —> Cortisol
- Mineralocorticoids —> Aldosterone
MOA of steroid hormones
Bound to carrier protein in bloodstream —> diffuse through plasma membrane —> bind to Cytosolic receptor (e.g. Glucocorticoid receptor) / Nuclear receptor (e.g. Estrogen receptor) —> Conformational change of receptor —> Bind to DNA —> Turn on transcription
***Steroid hormone receptors
ALL contain 5 common regions
- Ligand-binding / Inhibitor-binding domain (LBD) (e.g. Heat-shock protein HSP: an inhibitor)
- mediate ligand-binding
- ***mediate regulation of NLS - Dimerisation sites
- 橫跨 LBD, DBD - Nuclear localisation signal (NLS)
- 屬於LBD
- hinge between LBD and DBD
—> allow dimerised protein to go into nucleus - DNA-binding domain (DBD)
- Zinc finger domain
- bind to HRE - Transactivation domain (TAD) / Variable / Regulatory domain
- mediate activating effect of receptor
- ***offer specificity
***MOA of Glucocorticoid receptor (GR)
HSP bound to GR (also block DBD from binding to DNA)
—> retained in cytoplasm when without hormone binding
—> Cortisol bind to GR
—> HSP dissociate from GR
—> GR conformational change
—> Dimerisation of GR
—> Exposing DBD, TAD, NLS
—> GR dimer go through nuclear pore into nucleus by NLS
—> DBD mediate binding of GR to Glucocorticoid Response Element (GRE) (a DNA sequence within **promoter of gene)
—> TAD mediate interaction of GR with **Co-activator proteins (Co-activator communicate with Basal transcription complex bound to TATA box)
—> Turn on gene transcription, expression
- Early findings indicating regulation of gene expression at transcriptional level by steroid hormones
(結論: Steroid hormone regulate Puffing —> mRNA synthesis)
***Ecdysone (steroid prohormone by insects)
—> regulate “puffing” of insect salivary polytene chromosomes
Polytene chromosomes: Large chromosomes undergone multiple cycles of DNA synthesis without cell division (2^10 = 1024)
- contain invariant bands (85% DNA)
—> allow us to follow what happen to a particular gene locus
Add Ecdysone to salivary gland preparation
—> Changes in pattern of bands
—> Deconsensation and Recondensation of bands (**Puffing)
—> Characteristic puffing patterns follow precise temporal sequence
—> Newly synthesised RNAs labelled by **3H-uridine localised to puffs (presence of **mRNA synthesis)
—> **Conclusion: Ecdysone coordinates specific programs of gene expression + Puff is location of active gene transcription / mRNA synthesis
Ashburner hypothesis: Primary + Delayed secondary responses
Primary response:
Steroid hormone binds to Steroid hormone receptor (in salivary gland cells)
—> bind to promoter of gene / activate primary response genes
—> first puffing
—> synthesis of primary response proteins
Delayed secondary response:
—> primary response protein shut off primary response gene + turn on secondary response gene
—> late puffing
—> synthesis of secondary response proteins
Later (1970), use of radioactive labelled hormones to detect steroid hormone receptors and their association with high-affinity binding sites in chromatin
—> Labels seen under microscope to **move from cytoplasm to nucleus suggesting effect on gene expression
—> Specific sites labelled on chromatin (~50-100 sites / cell = number of genes regulated)
—> **Different specific chromatin sites in different cells acted on by different hormones
- Biochemical analysis and purification of Hormone-receptor complexes
(結論: 要有Conformational change)
Use of 3H-labelled hormones + High affinity analogs e.g. Dexamethasone
—> Able to detect hormone-binding proteins (intracellular receptors)
Lysate preparation (e.g. liver cell) (contain glucocorticoid receptor) + 3H-Dexamethasone
1. Incubate at 4oC
—> No binding to chromatin on filter, Low mobility in ion-exchange column
2. Incubate at 37oC
—> Binding to chromatin on filter, Increased mobility in column
**Conclusion:
Steroid hormone receptors after binding to hormone
—> undergo **“transformation” / “conformational change” (that **require energy + change surface charge of protein)
—> before Hormone-receptor complexes can bind to Chromatin
—> **Model of steroid hormone proposed
Purification of glucocorticoid receptor with help of radiolabelled binding analogs (e.g. Dexamethasone)
Glucocorticoid receptor will be labelled with radiolabel once bound to Dexamethasone
—> purify through many steps (based on charge, size, shape, affinity etc.)
—> 10^3-10^4 / cell (trace amounts) among 10^10 protein molecules per cell
—> ***SDS polyacrylamide gel electrophoresis
—> Crude extract (a smear) —> Purified GR (single band on gel electrophoresis)
- Identification of glucocorticoid-responsive genes and cis-acting sequences required for hormonal activation of transcription - “Hormone response elements”
(結論: 研究咩係HRE)
- Question: What are glucocorticoid responsive genes?
- MMTV (Mouse mammary tumour virus) genes first identified as being glucocorticoid-responsive
—> stimulated by glucocorticoid
—> replication - Question: Where does GR bind within regulatory sequences (promoter) of responsive gene?
- Direct binding of GR to MMTV DNA demonstrated in **EM studies
- Binding region defined by **deletion analysis e.g. -84 to -305 of MMTV transcription-control region; region sufficient to specify glucocorticoid responsiveness
- HRE further defined by DNAase I ***footprinting analysis + sequence comparison
Deletion analysis
Deletion of certain DNA sequence
—> Disappearance of RNA transcript
DNAase I footprinting analysis
DNAase I = DNA endonuclease —> cut DNA at random places
Add DNAase I to DNA
—> random cutting at multiple places
—> all sorts of DNA fragments
—> uniform ladder on electrophoresis
If GR added, ***GR-bound-DNA regions are protected from DNAase I
—> gaps appear on electrophoresis (some fragments missing —> lighter area: “footprint”)
—> number of gaps = number of binding sites
—> called “Hormone Response Element” (HRE)
- Same HRE for Glucocorticoid receptor, Aldosterone receptor, Androgen receptor, Progesterone receptor
- Special HRE for Estrogen receptor
- **Features of HRE:
1. Double binding sites / 2 half sites
2. Dyad (Rotational) symmetry —> receptors bind as dimers —> stronger interaction with HRE
3. HREs can be placed in front of other genes to make them hormone-responsive
- Cloning and functional analysis of steroid hormone receptors
(結論: identify到唔同region既DNA sequence)
GR cDNA isolated by expression cloning
—> ALL steroid hormone receptors cloned similarly / by low stringency hybridisation
—> cDNA sequence allow prediction of protein structure and function
—> Conserved domains defined based on sequence homology
- DNA binding domain / Domain C (small domain, binding to HRE) —> **highest homology (最conserved)
- GR, MR, PR have same HRE —> 94% similar in cDNA sequence
- GR, **ER recognise different HRE —> 54% similar in cDNA sequence
DBD:
—> contains 2 copies of C4 zinc finger motif (2 sets of 4 Cysteine residues)
—> chelate Zinc ion (Zinc help mediate DNA binding)
—> 1 Zinc finger carry **NRS (nucleotide recognition signal) for HRE binding
—> 1 Zinc finger carry **Dimerisation domain (dimerise with the other monomer)
- Ligand binding domain / Domain E (C-terminal) (larger domain) —> ***less conserved (15-57% homology)
- Others:
- Variable domain (N-terminal) (i.e. ***not conserved) e.g. Transactivation domain
—> for specificity (turn on specific genes)
—> ∴ even though binding to same HRE but still are different in action
Technique of expression cloning:
- Purified GR —> injected into mouse —> Ab raised against GR —> 攞翻d Ab 出黎做Expression cloning / Library screening
- Identify mRNA sequence from liver cells
—> convert mRNA into cDNA by reverse transcriptase from retrovirus
—> allow mRNA sequence to be cloned into vectors
—> vectors carried in bacteria
—> multiple colonies formed by GR-expressing bacteria
—> bacteria lifted onto a nitrocellulose filter
—> **Ab recognise GR expressed by bacteria
—> staining technique
—> **pinpoint a particular colony of bacteria with expression of GR
—> repeated screening
—> get a **pure colony expressing only a particular cDNA
—> **sequence cDNA
—> determine a.a. sequence, protein structure, function
Estrogen: Genomic (Traditional) pathway vs Non-genomic (Nongenotropic) pathway
Genomic pathway: Estrogen bind to Estrogen receptor in ***nucleus —> conformational change —> bind to HRE / DNA —> turn on gene expression
Non-genomic pathway (Estrogen receptor NOT need to interact with DNA):
Estrogen bind to Estrogen receptor on **plasma membrane
—> Estrogen receptor interact with kinase
—> **Kinase activate **pre-existing protein function / go into nucleus to **modify transcription factor
—> turn on gene expression
—> ∴ require only the LBD of receptor and its extranuclear localisation (NO need DBD)
—> faster action
Non-genomic pathway for Estrogen / Androgen receptor
Mainly found in bone cells
- protect osteoblasts / osteocytes from dying (Anti-apoptotic effect)
- Osteoporosis: Non-genomic pathway not strong enough / activated enough
- mediated through ***Serine/Threonine protein kinase (e.g. Akt, ERK, RSK) by direct phosphorylation of proteins
- Inactivating Bad protein (mediate cell death) —> Bcl2 protein freed from Bad protein —> Antiapoptosis
- Activating transcription of some target genes via Elk-1/CREB —> Antiapoptosis
Bisphosphonate: act on these pathways to prevent bone loss
Raloxifene: SERM —> ↓ osteoclasts-mediated bone resorption, ↑ osteoblast-mediated bone formation
