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CMB2001 - Eukaryotic Control of Gene Expression > Lectures 5-6: Transcriptional responses to stress and infection > Flashcards

Lectures 5-6: Transcriptional responses to stress and infection Flashcards

1
Q

Which environmental challenge does NF-κB respond to and what are the responses?

A

Environmental challenge - infection
Response - gene expression, repair, programmed death and immune response

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2
Q

What does NF-κB stand for?

A

Nuclear Factor κ immunoglobulin light chain in B cells

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3
Q

What does the family of NF-κB transcription factors regulate?

A

Inflammation, DNA damage, cell death, cell adhesion and proliferation

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4
Q

Which domain is homologous in the mammalian NF-κB family?

A

Rel Homology Domain (RHD)

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5
Q

How do different types of NF-κB arise?

A

Dimers are formed in different arrangements

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6
Q

How do p100 & 105 function and why?

A

They function as I-κB like inhibitors because they contain ankyrin repeats

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7
Q

How are p52 & 50 processed?

A

Proteolytically from precursor proteins P100 & 105

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8
Q

Where did NF-κB evolve from?

A

From eukaryotic immune cells

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9
Q

What is the ubiquitin - proteasome pathway?

A

A protein substrate is added to ubiquitin (Ub) using ATP
A chain of Ub molcules become attached to the protein substrate which is recognised by a 26S proteasome
Ub is then removed and the protein is linearised and injected into the core of the proteasome where it is digested
The peptides are degraded into amino acids or used in antigen presentation

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10
Q

How is NF-κB activated?

A

A ligand binds to an active site on the CSM which forms a cascade producing IKK kinase which stimulates the IκB complex
NF-κB is in an inactive cytoplasmic complex as (e.g. p50 and RelA)
IκB becomes phosphorylated then ubiquinated (proteolytically degraded)
NF-κB translocates to the nucleus which binds to the target allowing transcription

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11
Q

What is NF-κB induced by?

A

Inflammatory cytokines
Bacterial products
Viral protein & infection
DNA-damage
Cell stress

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12
Q

What does NF-κB regulate?

A

The immune and inflammatory responses
Stress responses
Cell survival and cell death
Cell adhesion
Proliferation

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13
Q

What is the mammalian IκB kinase (IKK) family?

A

It is a complex made up of 3 subunits, IKKα and IKKβ and IKKγ/NEMO (a regulatory subunit called the NF-κB essential modifier)
CC1 and CC2, coiled coil regions 1 and 2, zinc finger domain, helix-loop-helix domain and NEMO-binding domain

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14
Q

What is the mammalian IκB family?

A

They are inhibitors of NF-κB consisting of IκBα, IκBβ and IκBε and Bcl-3
They contain ankyrin repeats motifs in their C-termini, PEST, domain rich in proline, glutamate, serine and threonine

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15
Q

How does NF-κB provide a rapid response to environmental challenges?

A

It is a pre synthesised complex that is held in an inactive complex bound to an inhibitor, this allows a rapid release

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16
Q

What are the different pathways NF-κB can be released and how do they differ?

A

Canonical (classical) pathway - involves both IKKα and IKKβ leads to production of p50
Non-canonical (alternative) pathway - involves IKKα only and produces p52

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17
Q

What are the different responses NF-κB and IKK produce?

A

Inflammation, proliferation, cell death and anti-proliferative effects, survival, angiogenesis and tumour promotion and metastasis

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18
Q

What are the different genes that are used as knockouts and what phenotype is produced?

A

rela - apoptosis
c-rel - immunity
relb - immunity/inflammation
nfkb1 - immunity/neurological
nfkb1/relb - inflammation
nfkb1/c-rel - immunity
c-rel/rela - apoptosis
ikbα - inflammation
ikbε - immunity
bcl-3 - immunity
IKKα - immunity
IKKβ - apoptosis
IKKα/IKKβ - apoptosis
IKKγ - apoptosis

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19
Q

What are the disadvantages of NF-κB/IKK regulating different processes?

A

Mistakes can be made which can lead to diseased cells

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20
Q

How are diseased cells stopped in NF-κB processes?

A

There are regulatory mechanisms that give specificity to its activation

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21
Q

How is the phosphorylation and degradation of IκB, α, β or ε regulated?

A

Selective activation of different NF-κB homo and heterodimers

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22
Q

How do dimers help diversify the activation of NF-κB?

A

They have different properties and functions as they have different subunits
Meaning they have different specificity to target different genes and positioning
They have a different orientation meaning they are not palindromic

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23
Q

How does translocation of NF-κB to nucleus and the modification of NF-κB subunits give transcriptional specificity?

A

After being translocated to the nucleus they are subject to post-translational modifications or by interactions with other nuclear transcriptional regulators
Determine which gene targets are regulated by NF-κB

24
Q

How does DNA binding and gaining access to promoter/enhancer give transcriptional specificity?

A

They can be activated very quickly by NF-κB for some genes or NF-κB can recruit chromatin remodellers (Swi/Snf) or can rely on other proteins to do this (HATs)

25
Q

How was IFNβ involved in the SARS-CoV-2 immune response?

A

An imbalanced response was produced as proteins were manipulated to produce a higher inflammatory response and a low anti-viral response
It also contained a number of precisely arranged and orientated TF binding sites (including NF-κB)

26
Q

Why does IFNβ need to be tightly controlled?

A

As production takes place during viral infection which leads to a signal cascade leading to the activation of over 50 anti-viral genes
This is due to the recruitment of the basal transcription complex and gene transcription by RNA polymerase II

27
Q

How does transactivation (basal transcription complex and coactivators) give transcriptional specificity?

A

An enhancesome complex is created allowing an interaction interface which recruits coactivators (p300/CBP)
These interactions are weak and only favoured appropriately

28
Q

What is the role of NF-κB in transactivation (BTC and coactivators)?

A

NF-κB participates in a combination lock
p50/RelA works at the beta interferon enhancer, other combinations do not due to varying ability to interact with other TFs and conformation binding

29
Q

How can each of the levels of regulation be regulated to give transcriptional specificity?

A

Using signalling pathways which can act co-operatively or antagonistically

30
Q

What is hypoxia?

A

It is a lowering of the O2 concentrations compared to sea level ± 20.9% O2 or the normal levels cells are exposed to

31
Q

What are the different states of oxygen concentrations?

A

Pure oxygen
Hyperoxia
Normoxia
Hypoxia
Anoxia

32
Q

How do the different levels of oxygen differ in tissues?

A

There are lower O2 levels in most other tissues

33
Q

What physiological processes is hypoxia involved in?

A

Development: Placenta, heart, bone and vasculature
Adaptation: high altitude living, intense muscle exercise
Medical: Cancer, high altitude diseases, stroke, etc.

34
Q

How did hypoxia impact COVID-19?

A

Rapid shallow breathing was due to a severe lack of O2 and sub-normal CO2

35
Q

How do cells react to low O2?

A

Translational block (saves energy)
Transcriptional program
Chromatin structure changes
microRNA signature
DNA replication block
Restoration of O2 homeostasis
Cell survival
Cell death

36
Q

What is HIF?

A

Hypoxia Inducible Factor
A heterodimeric transcription factor (HIF-α, HIF-β)

37
Q

What are the properties of HIF-α and HIF-β?

A

HIF-α: responds to O2
HIF-1α: ubiquitously expressed in all tissues
HIF-2α: similar to HIF-1α, expression restricted to certain areas
HIF-3α: expression restricted, lacks C-terminous transactivation domain. Dominant negative inhibitor for 1α & 2α, activates a set of genes in hypoxia

38
Q

How does the structure of the HIF proteins differ?

A

HIF-1&2α contain: basic helix-loop-helix(HLH), per/ARNT/Sim domain(PAS), oxygen dependent degradation domain(ODD), nuclear localisation signal(NLS) and C-terminal transactivation domain(CTD)
HIF-3α: HLH, PAS, ODD, leucine zipper
HIF-1β: HLH, PAS and pass-associated C-terminal domain

39
Q

How are HIFs regulated to require oxygen function?

A

Proline hydroxylases (PHD) are used to for hydroxyproline that can carry out thus function

40
Q

How is HIF regulated?

A

Transcription produces mRNA which is translated into the HIFα protein
The protein is then regulated post translationally in the presence of oxygen using PHDs to add hydroxyl groups
This is recognised by VHL (Von Hippel Lindae protein) causing ubiquination to take place degrading the protein

41
Q

What happens to HIF regulation when oxygen is not present?

A

HIF1α mRNA evades block on translation and lack of oxygen inactivates PHD proteins so HIF1α is not degraded through ubiquination

42
Q

How is factor inhibiting HIF (FIH) used in regulation of HIF?

A

It uses the O2 to hydroxylate key residues within the HIF1α subunit

43
Q

What are the different pathways HIF controls?

A

Oxygen supply
Transcription
Cellular metabolism
Cell growth
HIF control
Cell death

44
Q

How does hypoxia impact cancer?

A

Many tumours have a poor blood supply in specific areas of the mass
Activation of HIF stimulates growth of new blood vessels which brings nutrients
It also regulates other tumour characteristics such as increased evasion and metastasis (spreads the tumour to other areas of the body) enhances growth and survival and increases glycolysis

45
Q

What is the key structure of the p53 tumour supressor?

A

Transactivation domain (TAD), proline rich domain, DNA binding domain, nuclear localisation sequence, tetramerisation domain and C-terminal

46
Q

Which processes does p53 regulate?

A

Apoptosis, autophagy, cell cycle arrest, DNA repair, embryo implantation, inhibition of angiogenesis, inhibition of ROS/survival, innate immunity, metabolism, p53 regulation and senescence
Tumour suppression, development, stem cell modulation and fertility

47
Q

Which other proteins can p53 regulate?

A

NF-κB and HIF

48
Q

How is p53 regulated?

A

Environmental stress/damage produces mdm2 and activates p53 then producting ATM kinase which leads to dissociation of the mdm2 and p53 complex
Once activated p53 induces cell cycle arrest allowing repair and survival or apoptosis to discard damaged cells
Leads to cellular and genetic stability

49
Q

What is the role of alternative reading frame?

A

It is another tumour suppressor induced by oncogenes, it also disrupts mdm2 and p53, ARF binds to mdm2 inhibiting ubiquitin
Activation of mdm2 results in increased levels of transcriptionally active p53

50
Q

What is the role of mdm2?

A

It is an E3 ubiquitin ligase, promotes ubiquination of p53 leading to degradation, keeping p53 levels low in undamaged cells

51
Q

What is the mdm2 feedback loop?

A

p53 stimulates mdm2 gene expression limiting the extent of p53 activation
Overexpression of Mdm2 in cancer inactivates p53 so cancer can thrive

52
Q

What is the response to DNA damage?

A

p53 becomes phosphorylate at S15 by ATM/ATR kinases, mdm2 is also phosphorylated and have an effect of disrupting the interaction between mdm2 and p53

53
Q

What role does negative feedback play in transcription factor pathways?

A

A mechanism to limit the period of response to a stimulus activating these pathways
Failure leads to death or disease
p53 induces mdm2 (inhibitor) causes proteolytic degradation
HIF1α induces PHD which causes proteolytic degradation
NF-κB induces IκBα (inhibitor) removes it from the nucleus and retains it in the cytoplasm

54
Q

How do cancer cells impact p53 activity?

A

They find ways to inactivate p53, even when p53 is not mutated
Regulators are either mutated, inactivated or altered so p53 activity is inhibited

55
Q

Which part of p53 is highly mutated in cancer?

A

The DNA binding domain

56
Q

What is an example of a syndrome that is effected by p53 mutation?

A

Li-Fraumen syndrome a hereditary cancer predisposition that is most commonly caused by a mutation to a gene called TP53 which has the same genetic blueprint as p53
Takes the genes ability to function correctly

57
Q

What are the key differences between NF-κB, HIF and p53?

A

NF-κB activation involves degradation of an inhibitor but HIF and p53 are continuously degraded in unstimulated cells
When stimulated they become stable

CMB2001 - Eukaryotic Control of Gene Expression (4 decks)

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