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BS32028 > Nuclear Receptors > Flashcards

Nuclear Receptors Flashcards

1
Q

How do nuclear receptors exert their effect?

A
  • Ligand enters cell
  • Ligand binds to receptor in cytosol
  • Ligand-receptor complex enters nucleus
  • Gene expression is stimulated/inhibited by recruitment of co-activators/co-repressors
  • Protein synthesis
  • Cellular effects
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2
Q

What are the effects of NR activation?

A
  • Large scale changes (not local)
  • RNA, DNA, and protein levels start to increase (RNA first and continues to increase, the protein slowly increases, then DNA rapidly but only to 100% increase and then decreases again)
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3
Q

What is the effect of NR on neural stem cell (NSC) fate?

A

Can either promote or suppress functions when differentiating

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4
Q

What are Nuclear Receptors (NRs)?

A
  • Hormone sensing transcriptional factors
  • Roles in cell proliferation, differentiation, cellular homeostasis
  • Translate dietary/endocrine signals into changes in gene expression
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5
Q

What is the function of co-activators and co-repressors?

A

They are regulatory proteins

Co-activator: for ligand-induced transcription

Co-repressor: mediate active repression of unliganded nuclear receptors

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6
Q

What are the target genes of NRs?

A

Comprise a complex genetic network in which their coordinated activity defines the physiological hormonal responses

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7
Q

What are the structural and functional criteria for NRs?

A
  • Nuclear or cytosolic localisation in absence of ligands
  • Half site recognition
  • Homodimers vs heterodimers (vs monomers)
  • Sequence similarity in DBD (DNA-binding domain)
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8
Q

How many NRs do humans have?

A
  • 48 receptors
  • 4 families
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9
Q

What are orphan receptors?

A

Receptors with unknown ligands (haven’t found ligands for them yet)

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10
Q

Examples of nuclear receptors where the receptor in the absence of its ligand is localised in the nucleus

A
  • Vitamin D receptor (VDR) (ligand: vitamin D3)
  • Peroxisome proliferator-activated protein (PRAR) (fatty acids)
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11
Q

Examples of nuclear receptors where the receptor in the absence of its ligand is localised in the cytosol

A
  • Androgen receptor (AR) (ligand: testosterone)
  • Progesterone receptor (PR)
  • Mineralocorticoid receptor (MR) (ligand: aldosterone)
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12
Q

What are characteristics of NR ligands?

A
  • Small
  • Lipophilic
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13
Q

List the 3 different mechanisms of NR signalling

A
  • Homodimer receptors bind
  • Heterodimer receptor-RXR complex binds
  • Monomer binding
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14
Q

Receptors for what ligand typically bind to DNA as a homodimer?

A

Steroids and hormones

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15
Q

Receptors for what ligand typically bind to DNA complexed with RXR?

A

Non-steroids
Ligands that exert an affect on autocrine or paracrine responses
Also thyroid hormone (T3 and T4)

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16
Q

Receptors for what ligand typically bind to DNA as a monomer?

A

Possible mechanism for some orphan receptors

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17
Q

How does homodimeric nuclear receptor binding work?

A

Receptors are bound to hsp90 (heat shock protein 90)
Requires ligand binding to dissociate
Can then bind to DNA as dimers

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18
Q

How does heterodimeric nuclear receptor binding work?

A

Receptor binds to HRE (hormone response element) which is complexed with RXR
Ligand binding is required for activation

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19
Q

What is the structure of a NR?

A

N terminal - AF1 - DBD - Hinge - LBD/dimerisation - AF2 - C terminal

AF1 - ligand independent site for activation by several kinase/phosphorylation pathways
DBD - DNA binding domain. Zn finger interaction (2, spanning 60 aa)
LBD - ligand binding domain. Largely hydrophobic amino acids lining the cavity
AF2 - Ligand dependent site for interaction with other protein families, essential for activation or repression of transcription

20
Q

What are the functions of the P- and D-box in the NR?

A

P box residues make base-specific contacts with DNA
D box ridues nvolved in dimerization and half site spacing

21
Q

How do receptors bind to response elements (RE)?

A

Homodimers: palindrom
Heterodimers: palindrome, direct repeat, or inverted palindrome

22
Q

Can do a mobility shift assay to examine Protein-DNA interactions

A

Can do acrylamide gel electrophoresis
One labelled DNA fragment is used without protein, and one with a protein bound
In the electrophoresis the free DNA moves further than the DNA-protein complex

Mutating the DNA (A-t and T-A) doesn’t affect protein binding, but amino acid changes in the protein does

23
Q

What is the structural basis of NR ligand binding?

A

‘Mousetrap model’
Ligand binds to trap > more stable conformational change prevents ligand’s exit (structural change varies)

Without a ligand, the structure of the receptor is fit for a corepressor
With the ligand, the strcuture of the receptor is fit for a coactivator

A common motif mediates binding of NR activators and repressors (leusine residues at certain positions)

Structural considerations discriminate between corepressor and coactivator binding (c-terminal tail has different position)

24
Q

Name multiple coactivator complexes and their function

A

CBP/p160/PCAF - histone acetyltransferase activity
SWI/SNF complex - possesses ATP-dependent chromatin remodelling activity
TRAP/DRIP complex - recruits RNA polymerase II (RNAPII) holoenzyme

Possibly, chromatin remodelling occurs first to relieve repression imposed by high-order chromatin structure, then acetylation, and finally activation using TRAP/DRIP

25
Q

When do corepressors bind?

A

Bind to NRs in the absence of ligand or the presence of NR antagonists

26
Q

Why are Nuclear Hormone Receptors (NHRs) a good drug target?

A

Control key cellular functions
Bind a wide variety of ligands

27
Q

Explain the GR (glucocorticoid receptor) signalling pathways

A

Glucocorticoid binds to the GR-hsp90 complex in the cytoplasm
HSP90 dissociates and GR is hyperphosphorylated and activated (changes conformation)
GR translocates into the nucleus
GR binds as homodimer to GRE (either directly, by binding to other transcription factors, or through direct binding an interacting with neighbouring transcription factors)
Can then enhance or repress transcription

28
Q

What are glucocorticoids responsible for once bound to GR?

A

Are immunosuppressant
Inhibits activation of transcription factors (ie protein-1, NFkappaB) > decreased transcription of cytokine genes (ie IL-2, TNF-alpha, IFN-gamma, IL-1 etc) > less IL-2 means less proliferation of Th cells

They also increase synthesis and release of anti-inflammatory proteins (ie annexin, protease inhibitors)

29
Q

What are the desirable and undesirable effects of glucocorticoid binding to GR?

A

Desirable: dissociated GR ligand inhibits inflammatory transcription factors without inducing GRE-mediated transcription

Undesirable: activation of GRE-mediated transcription (weight gain, hypertension, muscle weakness, diabetes, osteoporosis)

30
Q

What is a SNuRM?

A

Selective Nuclear Receptor Modulator

31
Q

GR SNuRMs

A

GR ligands that selectively inhibit pro-inflammatory transcription factors and not GRE-mediated transcription

Dissociated glucocorticoids or dissociated GR ligands

32
Q

What are SARMs?

A

Selective androgen receptor modulators

33
Q

Androgen and its side effects

A

Androgen - male sex. development, muscoskeletal development
Androgens: testosterone, dihydrotestosterone (DHT)

Prostate cancer initiation and progression is uniquely dependent on AR

Synthetic steroidal AR ligands may be used for treatment in men -> can lead to benign prostate hyperplasia (BPH) and prostate cancer

Despite androgen deprivation therapy with luteinizing-hormone-releasing hormone (LHRH), most get castrate-resistant prostate cancer (CRPC)

CRPC may be causally related to continued transactivation of AR

34
Q

What is the process of testosterone binding to AR?

A

Testostrone enters the cell > 5alpha-reductase converts T into dihydrotestosterone > binds to AR > HSP is released from AR > dimerisation and phosphorylation of AR > enter nucleus > bind to androgen response element of DNA > coactivators (ARA70) and coreppresors activated > biological response (growth, survival, production of prostate-specific antigen (PSA))

35
Q

5 possible ways to androgen independence

A
  • More AR or enhanced AR sensitivity, or more testosterone/DHT/more T to DHT​
  • AR is non-specific and activated by non-androgenic molecules present in the circulation
  • Receptor tyrosine kinases (RTKs) are activated, and AR shows ligand-independent activation. ​
  • Changes in other apoptotic pathways obviate the need for AR or its ligand. ​
  • Androgen-independent cancer cells that are always present may be selected for by therapy
36
Q

Characteristics of an ideal SARM

A

Non-steroidal AR ligand
High specificity
Improved oral activity (steroidal antigens can’t be delivered orally due to the risk of liver toxicity)
Desirable tissue selectivity (muscle > prostate)

37
Q

What are SERMs?

A

Selective estrogen receptor modulators

38
Q

What is the role of oestrogen and its side effects?

A

Necessary for female reproductive system
- Proliferation/ differentiation of heathy breast tissue
- Heart + bone
- Influence brain + mood
- Vaginal lining to stay thick and lubricated

Loss of oestrogen during menopause:
- Hot flashes and night sweats
- Rise in LDL cholesterol (increased risk of coronary disease + heart attacks)
- Increase in bone loss

Hormone replacement therapy (HRT) is effective in reducing osteoporotic fractures and improving severe menopausal symptoms
BUT promotes cancer of breast and uterine lining

39
Q

What do SERMs do?

A

Oestrogens are mainly agonists
Anti-oestrogens are mainly antagonists

SERMs show tissue selectivity; agonists in some tissues (bone, liver, cardiovascular system), antagonist in other tissues (brain, breast), mixed agonist/antagonist in uterus

40
Q

What can Oestrogen Receptors be targeted with to prevent cancerous growth?

A

Aromatase inhibitors - inhibit synthesis of ER ligands

Peptides and small molecules - inhibit coactivators

Antiestrogens, SERMs - binds to ER but doesn’t activate the molecule

Small molecule inhibitors - can either block the activation of the receptor by the actual ligand, or target the ER for degradation

41
Q

What are the 3 most well known SERMs?

A

Tamoxifen
Raloxifene
Toremifene

42
Q

Characteristics of Tamoxifen

A

A SERM
- Reduces probability of breast cancer in high risk women by 50%
- Preserves bone density, some reduction in fractures
- Long term usage: higher risk of endometrial cancer
- Potent hepatocarcinogen (liver cancer) in rodents (not proven in humans)

43
Q

Characteristics of Raloxifene

A

A SERM
- Unlikely to react with DNA
- No proliferative effects on uterus
- No increased risk of endometrial cancer
- Study of Tamoxifen And Raloxifene (STAR) proved Raloxifene had less side effects

44
Q

Characteristics of Toremifene

A

A SERM
- Newer
- Similar properties and side effects to Tamoxifen

45
Q

How do you test if a molecule is the endogenous ligand for an oprhan receptor?

A

Test in transient transfections
- Demonstrate physical binding
- Demonstrate that ligand and receptor are present in the same cell (at appropriate concentrations)
- Find target genes and show ligand and receptor dependent regulation in vivo

46
Q

What is a transfection?

A

Deliberately introducing naked or purified nucleic acids into eukaryotic cells

47
Q

What are characteristics of an endogenous ligand of an orphan receptor?

A
  • Nuclear availability
  • High binding potency (nM range)
  • Ability to induce conformational change in the protein structure
  • NR-related physiological function as a hormone

The same NR may have distinct endogenous ligands in distinct tissues or cell types

BS32028 (4 decks)

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