Transcriptional responses to stress and infection Flashcards
How does the cell/organism to respond to environmental threats?
The NF-κB, p53 and HIF pathways
What is the basic steps of response to environmental challenges?
Environmental challenge
Sensor
Signalling pathway
Effector
Cellular response
What is NF-kB?
Nuclear Factor of the kappa Immunoglobulin light chain in B cells
What is the role of The NF-κB transcription factor pathway?
Regulating:
Inflammation
DNA damage
Cell death
Cell adhesion
Proliferation
What is p65?
RelA
What is the function of the The Rel Homology Domain (RHD)?
Encodes the DNA binding and dimerisation functions of NF-kB.
How are p50 and p52 formed?
p50 and p52 are proteolytically processed from their precursor proteins p105 and p100 (p105 generally constitutive, p100 inducibly)
What is the function of the ankyrin repeats in the C-termini within p100 and p105?
Allow them to function as IkB-like inhibitors
What is LZ?
leucine zipper like domain
What is the function of the dimers that NF-kB forms?
Dimers bind DNA
What is the structure of RelA (p65)?
Rel homology domain
TA2
TA1
What is the structure of RelB?
LZ
The Rel Homology Domain
TAD
What is the structure of c-Rel?
The Rel Homology Domain
SDI
SDII
What is E3 Ubiquitin ligase?
A protein that facilitates the attachment of ubiquitin chains to a target protein
How does the ubiquitin (Ub)-proteasome pathway (UPP) of protein degradation work?
Ub is attached to proteins that are destined
for degradation.
A chain of five Ub molecules attached to
the protein substrate
Recognized by the 26S proteasome.
Ub is removed.
The protein is linearized and injected into the central core of the proteasome
It is digested to peptides.
The peptides are degraded to amino acids by peptidases in the cytoplasm or used in antigen presentation.
What is NF-κB is induced by?
Inflammatory cytokines
Bacterial products
Viral proteins & infection
DNA-damage
Cell Stress
What does NF-κB regulate?
The immune and inflammatory responses
Stress responses
Cell survival and cell death
Cell adhesion
Proliferation
How does the Activation of NF-kB work?
Ligand binds.
In unstimulated cell types, NF-kB is retained within the cytoplasm in an inactive form, bound to its inhibitor protein, IkB.
Upon cellular stimulation, IkB becomes phosphorylated which results in its ubiquitination and subsequent degradation by the 26S proteasome.
NF-kB is then free to translocate to the nucleus.
What is the structure of IkB kinase (IKK) complex?
*The catalytic subunits IKKa and IKKb
*The regulatory subunit called the NF-kB essential modifier NEMO also known as IKKg.
*CC1 and CC2, coiled coil regions 1 and 2;
*ZF, zinc finger domain;
*HLH, helix–loop–helix domain;
*NBD, NEMO-binding domain.
What are the inhibitors of NF-κB?
IκBα
IκBβ
IκBε
Bcl-3.
What is the structure of inhibitors of NF-κB (IκB) family?
Ankyrin repeat motifs (ANK) in their C termini. PEST, domain rich in proline (P), glutamate (E), serine (S) and threonine (T).
What is the function of anchored repeats?
Form a stacked alpha helix structure.
What is the importance of p100 and p105 having anchored repeats?
Can act as inhibitors, this means they need to be processed to p50 and p52 which don’t have them.
What is the importance of anchored repeats domain to inhibit NF-kB?
Binds to the NF-kB and causes a conformational change, this inhibits its DNA binding and keep it in the cytoplasm.
What are the Multiple levels of regulation that combine to give transcriptional specificity?
Phosphorylation and Degradation of IkB α, β or ε
Translocation of NF-κB to the Nucleus
Modification of NF-kB subunits
DNA Binding and Gaining Access to the Promoter/Enhancer
Transactivation
What is transactivation?
Interaction with the Basal Transcription Complex and Coactivators
What does a dimer do for you?
Different subunits have subtly different DNA binding specificity and so can target different genes and position themselves on DNA.
What is a HAT?
Histone acetyl transferase
What is Swi/Snf?
ATP dependent chromatin remodeller (can move/remove nucleosomes)
What is IRF?
Interferon response factor - multigene family
What is c-Jun?
Member of AP-1 family.
What is AFT2?
bZIP protein similar to c-Jun, heterodimerises with it
What is the function of beta interferon.?
a and b interferon bind specific cell surface receptors which results in a signalling cascade leading to the activation of over 50 anti-viral genes.
What role does the organisation of the beta interferon promoter play?
If the individual transcription factor binding sites in the b IFN enhancer are taken out of context and multimerised they can act as a viral inducible promoter - but they have varying basal levels of activity and can respond to other
inducers.
What is the role of the coactivator interaction surface complex?
This complex forms an “interaction interface” to allow the high affinity recruitment of transcriptional coactivators such as p300/CBP. Individual transcription factor: coactivator interactions are generally weak favouring the formation of coactivator complexes only at the promoters and enhancers where an appropriate interaction interface is created and the coactivator activity is required.
What is BTM?
Basal Transcription machinery
What is hypoxia?
Hypoxia can be defined as a lowering of the O2 concentrations compared to sea level ± 20.9% O2
What are the Cellular responses to hypoxia?
Restoration of oxygen homeostasis
Cell survival
Cell death
What is HIF?
Hypoxia Inducible Factor
Heterodimeric transcription factor: HIF-α and HIF-1β
What is HIF-1α?
Ubiquitously expressed in all tissues
What is
HIF-2α, expression restricted to certain tissues.
What is HIF-3α?
Expression restricted to certain tissues and lacks C-terminus transactivation domain.
Functions as a dominant negative inhibitor for HIF-1α and HIF-2α.
Activates a different set of genes in hypoxia
What is the structure of HIF-1α?
CTAD, C-terminal transactivation domain
NLS, nuclear localization signal
NTAD, N-terminal transactivation domain
PAS, Per/ARNT/Sim domain
HLH – basic Helix-loop-helix (DNA binding)
ODD, oxygen dependent degradation domain
What is the structure of HIF-2α
CTAD, C-terminal transactivation domain
NLS, nuclear localization signal
NTAD, N-terminal transactivation domain
PAS, Per/ARNT/Sim domain
HLH – basic Helix-loop-helix (DNA binding)
ODD, oxygen dependent degradation domain
What is the structure of HIF-3α?
LZIP, leucine zipper
NTAD, N-terminal transactivation domain
PAS, Per/ARNT/Sim domain
HLH – basic Helix-loop-helix (DNA binding)
ODD, oxygen dependent degradation domain
What is the structure of HIF-1β?
PAS, Per/ARNT/Sim domain
HLH – basic Helix-loop-helix (DNA binding)
PAC, PAS-associated C-terminal domain
What is the Regulation of HIF-1α in normoxia?
Proline hydroxylases (PHDs) and FIH enzymes use O2 to hydroxylate key residues within the HIF-1α subunit.
Hydroxylation of the ODD signals for VHL (a ubiquitin E3 ligase) binding and ubiquitination occurs, leading to proteasomal degradation
What is the Regulation of HIF-1α in hypoxia?
PHDs and FIH are inhibited, and HIF-1α is
stabilized and able to dimerize with HIF-1β and activate target gene transcription through recruitment of co-activators.
What is the structure of p53?
The domain structure typical for transcription factors, with distinct DNA binding and multimerisation domains.
What are the domains within the p53 tumour suppressor?
Trans – transactivation domain (TAD)
P – proline rich domain
NLS – nuclear localisation sequence
Tet – tetramerisation domain
How is the p53 activity regulated?
*p53 is inactivated by its negative
regulator, Mdm2.
*Upon DNA damage lead to the dissociation of the p53 and mdm2 complex.
*Once activated, p53 will induce a cell cycle arrest or apoptosis.
What are the role of Mdm2?
It’s major role is to promote the ubiquitination of p53, leading to its degradation by the proteasome.
This keeps p53 levels low in undamaged cells.
What is mdm2?
Mdm2 is an E3 ubiquitin ligase.
What is the action of mdm2?
In response to DNA damage p53 becomes phosphorylated at serine 15 by the ATM or ATR kinases.
Mdm2 is also phosphorylated.
These phosphorylation events have the effect of disrupting the interaction between p53 and Mdm2.
What is the action of the mdm2 feedback loop?
p53 also stimulates Mdm2 gene expression, thus forming a negative feedback loop that limits the extent of p53 activation.
Overexpression of Mdm2 inactivates the p53 pathway, preventing cell cycle arrest and the induction of apoptosis in response to oncogene activation or DNA damage.
What is ARF?
p14ARF is another tumour suppressor whose expression is induced by oncogenes as a result of increased cellular proliferation.
What is the action of ARF?
It disrupts the interaction between the p53 tumour suppressor and Mdm2.
ARF binds Mdm2 and inhibits its ubiquitin ligase activity.
Inactivation of Mdm2 by ARF therefore results in increased levels of transcriptionally active p53.
What is Li-Fraumeni syndrome?
Hereditary cancer predisposition
syndrome.
What causes LFS syndrome?
LFS is a hereditary genetic condition.
This condition is most commonly caused by a mutation (alteration) in a gene called TP53, which is the genetic blueprint for a protein called p53. The mutation takes away the gene’s ability to
function correctly.
