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What is the genomic and functional structure of the glucocorticoid receptor?
- N-terminal - Exon 2
- DNA binding domain
- Hinge
- Ligand binding domain
- C-terminal
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What's unique about glucocorticoid receptors compared to other steroid receptors?
- Different areas indicate different functions
- HSP90 binding
- Activation function
- Dimerization
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Describe glucocorticoid cellular signaling.
- Ligand (cortisol) normally sitting in the cytoplasm
- Comes in plasma
- Need to have a carrier protein b/c hydrophobic
- Crosses membrane b/c looks like a steroid
- Binds to GC receptor
- Changes conformation of protein
- Displaces chaperone protein HSP90
- Dimerization and Nuclear localization signal (NLS) now exposed
- Induces dimerization of GR's
- Dimer works as transcription factor
- Binds to DNA
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What is the NLS?
- Nuclear localization signal
- Short sequence within protein, like a tag
- Sends protein to appropriate location in cell
- Sends through nuclear pore, into the nucleus
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Why are the GR's not already in the nucleus?
- NLS signal is actually obscured/protected/covered/not seen regularly
- Hidden from view by chaperone protein HSP90
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What are the two ways to locate proteins?
- Stain it with antibody
- Tag it with fluorescence to see in real time
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What is this image showing us? (GR nuclear translocation)
- How much of the nucleus gets filled with GR's
- 2 minutes, filling
- 10 minutes, pretty concentrated in nucleus
- Lowers after that
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Why does the GR nuclear localization lower after about 10 minutes?
- Diffuses out again
- Temporary response to stress
- Ligand interaction with GR is on and off
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If a protein is changing, why is it pretty long lasting?
Transcription and translation
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What is the downstream effects of GR activation?
- DNA binds to specific areas of protein in the promotor
- DNA -> RNAs -> Membrane Proteins/channels/pumps etc.
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What is a promotor?
- Regulatory element that sits near the start of protein
- Inert, needs to be activated
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What are the 2 ways to study gene expression?
- By analyzing expression of single genes
- By analyzing the whole transcriptome
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What are examples of analyzing expression of single genes?
- RT-PCR
- In situ hybridization
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What are examples of analyzing the whole transcriptome?
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What is a transcriptome?
The set of all RNA molecules, produced in one or a population of cells
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What does transcriptome analysis reveal?
Widespread, selective and cell-specific gene regulation by GR
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What kind of transcription factor is GR?
Zinc factor
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What are the two ways in which GR genomic effects are mediated?
- GR can interact with cis-DNA elements
- GR can affect transcription via interactions with other transcription factors
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What are cis-DNA elements?
Elements that are next to each other on the DNA
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What are examples of cis-DNA elements?
- Glucocorticoid Response Elements
- GRE and nGRES
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What does the "n" stand for in nGRE?
Negative
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What is the Hormone Response Element?
- Short sequence of DNA within the promotor of a gene
- Sequence is most commonly a pair of inverted repeats, separated by three nucleotides
- Location where receptor binds as a dimer
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What does the Hormone Response Element do?
Able to bind a specific hormone receptor complex and regulate transcription
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How is the HRE generally written?
- 5'AGAACAnnnTGTTCT3'
- 3'TCTTGTnnnACAAGA5'
- where n represents any nucleotide.
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What are some examples of other transcription factors that GR can interact with?
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What are the 2 ways GR-induced gene expression is activated?
- Basal transcription machinery
- Via co-activators
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What are the different ways GR-induced gene expression is repressed?
- Displacement of activating transcription factors
- Competitive interactions with activating TF's
- Inhibitory interactions with activating TF's
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What are some examples of repressing gene expression by displacement of activating TF's?
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What does the genetic organization of GR predict?
Alternative splicing sites
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What is alternative splicing?
- One gene - many proteins
- Different parts of the DNA are spliced and turned into mRNA in different combinations
- Make different proteins
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How much of the human genome may undergo alternative splicing?
Up to 75%
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What is asthma?
A disease in which inflammation of the airways causes airflow into and out of the lungs to be restricted
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What happens when an asthma attack occurs?
- Mucus production is increased
- Muscles of the bronchial tree become tight
- Lining of the air passage swells
- Reduces airflow and produces the characteristic wheezing sound
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How is chronic severe asthma (and allergy) treated?
- Synthetic steroid prednisone
- Suppresses inflammation
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What happens to asthma patients after chronic prolonged treatment with prednisone?
- Become steroid-resistant
- Do not respond to the prednisone treatment anymore
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What is the common hypothesis and treatment for asthma patients becoming steroid resistant?
- Hypothesis: Adaptation
- Treatment: Increase the steroid dose
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What really happens to asthma patients who become steroid-resistant after prolonged treatment with prednisone?
- mRNA from the lung epithelium of those patients have revealed high levels of GR-beta isoform
- GR-beta dimerizes with GR-alpha but doesn't bind to GREs and can't modulate transcription
- It was discovered that GR-beta antagonizes and competes with GR-alpha, hence blocking the GC effects on the target cells
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What was the real conclusion and treatment for asthma patients who have become steroid-resistant?
- Conclusion: Those patients have a switch in alternative splicing in their lungs, and therefore become resistant to GC treatment
- Treatment: Stop steroids!
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