Cell Signalling in Health and Disease Flashcards
Toll-Like Receptor Signalling
what is needed for a foreign protein to induce an immune response
an adjuvant
what is the immune system dogma
only responds to foreign molecules
what is toll-like receptor 4 activated by
lipopolysaccharides
function and description of LPS-binding protein (LBP)
is an acute-phase protein released by the liver
binds to LPS
what is the function of the LPS-LBP
transfers LPS onto CD14 on the surface of phagocytes
what occurs once LPS is bound to CD14 on phagocytes
CD14 interacts with TLR4
leads to activation of NFkappaB in the nucleus
what binds to the LPS bound TLR4
the adapter protein - Myd88
binds to the TIR domain of TLR4
what does TIR bound Myd88 accomplish
activates IRAK
IRAK phosphorylates TRAF6
what is the function of phosphorylated TRAF6
activates MAPKKK and TAK1 which become a complex
function of activated MAPKKK-TAK1
degrades IkB
leads to NFkB entry into nucleus
what are the 2 TLR4 signalling pathways
Myd88 dependent
Myd88 independent, TRIF dependent
what does TLR4 siganlling via the TRIF dependent pathway lead to
IFN-beta response
what are the general features of TLR Myd88 - dependent pathway
activated by most TLR’s
leads to activation of MAP kinases and NFkB
expression of inflammatory cytokines
up-regulation of co-stimulatory molecules and MHC on cell surface
what are the general features of the TLR Myd88-independent pathway
activated by TLR4/3
leads to activation of IRF-3 and production of type I interferon
increased co-stimulation and MHC on cell surface
expression of inflammatory cytokines
maturation of dendritic cells
what is NFkB
it is an inducible transcription factor expressed in all mammalian cells
why is NFkB usually inactive
because inhibitory transcription factors are usually bound
how is NFkB liberated and activated
phosphorylation of IkB leads to its ubiquitination
leads to release of active NFkB
what is the NFkB family composed of
5 related transcription factors:
p50
p52
RelA
c-Rel
RelB
what is required for NFkB mediated transcription
formation of heterodimers with activation domains
macrophages have stimulant-dependent responses, what is the difference in response to a LPS and glucan-beta
LPS - leads to tolerant macrophage
- desensitized/immune tolerance
-decrease in IL6/TNFalpha
glucan-beta - leads to trained macrophage
- sensitized/trained immunity
- increase in IL6/TNFalpha
how does the dose of LPS affect the macrophage response
high dose of LPS - results in tolerant macrophage
low dose - doesn’t result in tolerant macrophage
how does an LPS stimulus affect chromatin formation
leads to acetylation - subsequent loosening of chromatin for gene transcription
how does a second LPS stimulus affect chromatin formation
leads to deacetylation - no transcription
the cell has become tolerant
how can nucleic acids be a PAMP, when our own cells contain them in RNA/DNA
toll-like receptors bind to features of nucleic acid specific to microbes absent in mammalian cells
what is TLR9 specific for and why
an unmethylated CpG DNA
bacterial CpGs are unmethylated whereas mammalian CpGs are usually methylated
what does TLR3 recognise
long double stranded RNA - common in microbes, rare in mammals
what is the NOD protein structure composed of
CARD - Caspase activating and recruitment domain
NBD - nucleotide binding domain
LRR’s - leucine rich repeats
what is the difference between NOD - 1/2
NOD-1 - one CARD domain
NOD-2 - two CARD domains
what are the ligands of the NOD receptors and where are they found
peptidoglycans
found in gram+ bacteria in cell wall
also found in thin layer of periplasmic space in gram - bacteria
what do NOD1/2 receptors bind to
NOD1 - Meso-DAP domain of peptidoglycan
NOD2 - MDP domain of a peptidoglycan
how does NOD1/2 activation leads to activation of NFkB
NOD1/2 signal via serine/threonine RIP2 kinase (RICK)
RICK mediates ubiquitination of NEMO/IKKgamma
leads to activation of NFkB
besides NFkB what else does NOD signalling activate
activation of MAPK
what does LPS-TL4 signalling increased expression of NFkB lead to the increased expression of what
leads to the increased expression of NLR3P and IL-1b
what does the increased expression of NLR3P/IL-1b lead to
forms a complex with ASC - preventing the ubiquitination of ASC
what does ASC/NLR3P/IL-1b form
forms an inflammasome
NLR3P forms a star shape around stacks of ASC
what does an inflammasome form
the caspase activity
how do phagocytes sabotage TLR signalling
1 - camouflaging or changing the molecules that interact TLR
2 - interference with downstream TLR signalling
3 - hijacking lymphocytes to hide
function of the glycoprotein hemagglutinin (HA)
it is a lectin that mediates binding of the virus to target cells and entry of the viral genome
function of the glycoprotein neuraminidase (NA)
involved in the release of progeny virus from infected cells
how does neuraminidase achieve its function
cleaves the sugars that bind the mature viral particle
what is the RNA that is largely responsible for a viruses’ virulence
NS1
what do virus infected cells release to halt viral infection
IFN-alpha
IFN-beta
what is the function of IFN-alpha/beta
induces resistance to viral replication in all cells
increases MHC class I and increases antigen presentation
activate dendritic/macrophages
activate NK-cells to kill infected cells
what must be done to the CARD domain of RIG-1 for IFN induction
it must be ubiquitinated
what is responsible for ubiquitinating RIG-1
TRIM25
what is the difference between normal cells and cells infected with influenza in terms of RIG-1
in influenza infected cells the CARD domain of RIG-1 is not ubiquitinated
prevents IFN response
function of TRIM19
inhibits replication of many DNA/RNA viruses
function of TRIM5alpha
blocks replication of HIV
what is the function of TRIM21
key in initiating antibody response to influenza
outline the difference between autocrine/endocrine/paracrine function
autocrine - cell signals are expressed that effect the cell that released them
paracrine - cell signals released affect nearby cells
endocrine - cell signals released into bloodstream to effect distant cells
what does the release of IL-4 from T helper cells effect
B-cells - activation/proliferation/differentiation
thymocyte - proliferation
mast cells - proliferation
how do activated T helper cells effect macrophages
via release of IFN-gamma
what interleukins induce proliferation in B cells and what are the released from
IL-2
IL-4
IL-5
T helper cells
what does the release of IL-4 and IL-5 from Th cells to B cells cause
causes a class switch to IgE antibodies
how does IFN-gamma released from Th cells affect B-cells
inhibits class switch to IgE
how are Th cells activated
IL-12 released by macrophages
why do cytokines only effect cells in close proximity
most cytokines have a very short half-life
what is needed for cytokines to induce an effect on cells
high concentration of cytokines
may require cell-cell interaction
what are the cytokine receptor families
1 - immunoglobulin (IL-1)
2 - Class I cytokine (haematopoietin)
3 - Class II cytokine (interferons)
4 - chemokine family
5 - tumour necrosis family
what are the 3 domains of cytokine receptor families
extracellular domain
transmembrane domain
cytoplasmic domain
what is the GM-CSF receptor subfamily and how does it effect cytokine affinity
it is a common beta-subunit of the receptor
when absent it leads to low affinity
when present alongside the alpha-subunit it leads to high affinity
describe the majority of cytokine receptor chains
alpha chain - specific cytokine binding site
beta/gamma chain - required for high binding affinity
what is the IL-2 receptor subfamily
a common gamma subunit required for high cytokine binding affinity
what is X-linked severe combined immunodeficiency
an due to a defect in the gamma-chain on the X chromosome (aka - bubble boy)
highly compromised immune system
lack of T cells/NK cells and functional B cells
what is the Class I/II receptors transduction similar to
JAK/STAT pathway
what does the TNF receptor family activation lead to
caspase and NFkB activation
what type of receptor is the chemokine receptor family
G-protein associated receptors
outline the JAK section of the JAK/STAT transduction mechanism
JAK - is a Janus kinase bound to the tyrosine kinase receptor
once a cytokine binds to the TK, JAK is activated and phosphorylates the TK and phosphorylates STAT
outline the STAT section of the JAK/STAT transduction mechanism
STAT - signal transducers and activators of transcription
once STAT is activated (phosphorylated) by JAK it dimerises
once dimerised they enter the nucleus and initiate transcription
what happens when IFNy binds to a IFNy receptor (IFN type II signalling)
activation of STAT1 via phosphorylation to form a homodimer (opposite of dimerise)
what is the function of a STAT1 homodimer and what does it lead to
recognises GAS elements in the promoter region of target genes
release of inflammatories
release of macrophage activators
release of chemokines
what happens when IFNa binds to a IFNa receptor (IFN type I signalling)
a heterodimer of STAT1-STAT2 is formed via phosphorylation
the STAT1-STAT2 heterodimer along with IFN-9 binds to the IFN-stimulated response element (ISRE)
what does IFN type 1 signaling cause
release of antivirals
release of antiproliferative factors
release of chemokines
what is the effect of IL-6 binding to its receptor
homodimer of STAT3 is formed via phosphorylation
also binds to GAS on promotor regions of target genes
what is STAT-3 activated by
cytokines that bind to their corresponding receptor which contains a gp130 chain
how are STATS affected directly after ligand binding
activated rapidly
recruited to the intracellular domain of the receptor via binding of SH2 and phosphotyrosine residues
what does STAT-1/3 promote
STAT-1 - promotes apoptosis and anti-proliferatives
STAT-3 - promotes anti-apoptotic effect and cellular proliferation
they are antagonistic
what are the similarities between IL-12/23
share a p40 subunit and their receptors both possess a IL-12Rbeta1 subunit
what STAT’s are involved in IL-12/23
IL-12 - STAT-4
IL-23 - STAT-1/3/5
what are Th1 cells involved in
cell-mediated immunity
intracellular pathogens:
- viruses and certain bacterias
what are Th2 cells involved in
antibody-mediated immunity
needed to control extracellular pathogens including yeast and worms
what are Th1 cells activated by and what do they release
activated by IL-12
release IFNy, IL-2, TNF-b
what are Th2 cells activated by and what do they release
activated by IL-4
release IL-4/5/10/13
release of IL-4 leads to a positive feedback loop
how can some viruses and bacteria trick NK/dendritic cells
can induce IL-12 secretion to induce IFN-y production
naiive CD4+ T cells activated via IFN-y and IL-12 are committed to become Th1 cells
how can some pathogens such as some worms trick NK cells
induce IL-4 secretion
naiive CD4+ T cells activated by IL-4 are committed to become Th2 cells
what is the predominant activity of dendritic cells in the absence of infections
high production of TGF-b
low production of IL-6
what are CD4+ T cells in the absence of infection induced to express
express Foxp3
present with a regulatory phenotype
what do dendritic cells produce during the early stages of infection and why
high production of IL-6
CD4+ activated by IL-6 express RORyt and become Th17 cells
what is IL-4 employed in the defence against
parasitic worms
allergy
asthma
what is IL-12 employed in the defence against
extracellular bacteria
cancer
autoimmunity
what is IFN-y employed in the defence against
intracellular pathogens
what is TGF-b involved in
immunosuppression
what is macrophage polarisation driven by and what does it induce
driven by cues in the tissue microenvironment
induces cytokines, growth factors and microorganism associated molecular patterns
what cytokines are involved in the Th1 response within a tumour microenvironment
IFN-y
TNF-a
what cytokines are involved in the Th2 response within a tumour microenvironment
IL-10
IL-4
TNF-b
how is regulation of cytokine signaling performed
receptor internalisation
protein tyrosine phosphatases
dephosphorylation of activated STAT’s
recycling of STAT monomers to cytoplasm
how are JAKS inhibited
SOCS protein
how are STATS inhibited
PIAS binds to STAT dimers
prevent DNA recognition
what is a cytokine inhibitor
IL-1RA
binds to IL-1 receptor but does not initiate a response
what is the function of the TCR
allows T-cells to identify infected or malignant cells
how do TCR’s recognise infected/malignant cells
interactions with major histocompatibility molecules on the surface of APC’s
what is the structure of the TCR
a heterodimer
each chains consists of a variable and constant domain
what are the APC’s that express both MHC I/II
macrophages
dendritic cells
B-cells
function of CD4 and CD8
they are co-receptors found on T-cells that also bind to MHC to boost TCR signalling
what do CD4/8 each target
CD4 - MHC class II
CD8 - MHC class I
what is the structure of the immunological synapse
dSMAC - beneath layer of bulky molecules
pSMAC - middle layer of cell adhesion molecules
cSMAC - uppermost layer of TCR, accessory and siganlling molecules
give a summary of how a immunological synapse is formed
first pSMAC adhesions molecules bind loosely to the cell, allowing TCR to inspect
if TCR recognises a pMHC clusters of signalling molecules are formed
the T cells polarises and the mTOC moves underneath the synapse
what are the pros and cons of using imaging cell-cell interactions to study the synapse
pros - optimum image quality
multiple antibodies can be studied at once
cons - cells need to be fixed, so only one time point
what are the pros and cons of using GFP tagging to study the synapse
pros - multiple time points
cons - large GFP molecules can change the activity of the subject of study
what is are the features of the zeta chain of the TCR
intracellular chain
has the most binding sites for signalling molecules
what are the major SRC kinases in T cells
Fyn and Lck
what are the first 3 steps of in the synapse formation
1 - synapse forms as T cell approached the APC
2 - the TCR recognises the peptide in the MHC and binds
3 - initial signals are sent by phosphorylation
outline step 4 in the formation of the synapse
CD45 (in dSMAC) is a phosphatase that can remove inhibitory phosphates from kinases - first is Fyn
Structure of SMAC is important for bridging these molecules together - clustering
outline step 5 in the formation of the synapse
the co-receptors (CD4/8) have another kinase - Lck - on their tail
this is also brought in when the co-receptor binds MHC
what are the benefits of using phosphorylation in signalling cascades
extremely rapid
no protein synthesis/degradation required
creates binding sites on proteins
rapidly reversed using phosphatase - quick responses
what is the function of Fyn/Lck and what does it lead to
phosphorylate the ITAMs in the zeta chain
allows ZAP70 to bind
ZAP70 is then phosphorylated
leads to downstream substrate activation
what are steps 6/7/8 of synapse formation
6 - these kinases (Fyn/Lck) phosphorylate ITAMs on the zeta chain
7 - the pITAMs are bound by ZAP70
8 - ZAP70 is then phosphorylated and activated
what is the final step - 9 - of the synapse formation
ZAP70 phosphorylates LAT
helps assemble signalling complexes
what is LAT
a linker for T cell activation
what does activation of LAT and SLP76 allow
allows the recruitment and activation of phopsholipase C gamma (PLCy) and guanine exchange factors
what are the critical mediators of the next wave of signalling after phosphorylation
PLCy
guanine exchange factors
how does activation of the PLCy pathway occur
PLCy has 2 SH2 domains which allow the recruitment to phosphotyrosine residues
what is the function of PLCy
cleaves PIP2 into IP3 and diacylglycerol (DAG)
function of IP3
opens Ca2+ channels - allows calcium entry from the ER and extracellular fluid
Ca2+ activates calmodulin
what does calmodulin along with DAG activate
protein kinase C (PKC)
what are the intracellular calcium levels compared to extracellular and how is maintained
100,000x lower than extracellular M
pumped out of the cell or into the ER
what is the major use for Ca2+ ions in T cells
activation of the phospholipase C pathway
what do we use to study calcium signalling
indo-1
function of indo-1 to study calcium signalling
when indo-1 binds to calcium it emits a wavelength of 390nm (violet) and when unbound it emits a wavelength of 500nm (green)
function of ionomycin
it is a calcium ionophore
increases calcium concentration in the cytoplasm
what is a small G-protein signal that gets amplified - most important one
Ras
what occurs to Ras and what is its function
recruited and held in place by adapter proteins
adapter proteins are assembled by phosphoLAT
Ras activates the MAP kinase siganlling pathway
if an antigen triggering disease cannot be deleted as it is an important protein what can be done
we can try to disrupt its binding to the TRC by injecting altered peptide ligands
what are altered peptide ligands
T-cell epitopes of the peptide with one or two amino acid variations that bind TCR but do not initiate T cell proliferations
how do APL’s perform their function
once bound to TCR they recruit less src kinases leading to incomplete phosphorylation of ITAM’s
partial activation of ITAM’s leads to activation of only some pathways
do APL work in patients
no
only really works in vitro
what was used to prevent T cells targeting donor organs (immunosuppression)
inhibition of calcineurin
first prototype was cyclosporin
what is calcineurin
Ca2+ dependent serine/threonine protein phosphatase that activates NFAT - key to T cell activation
how do calcineurin inhibitors (CNI’s) perform their function
CNI’s bind to cytosolic proteins called immunophilins
CNI-immunophilin complex binds calcineurin to prevent its phosphatase activity
NFAT is not dephosphorylated - no T-cell activation - IL-2 release is inhibited
do CNI’s work
yeah
what is belatecept and how is it used to prevent T-cell co-stimulation
it is a CTLA4 antibody
extracellular domain of CLTA4 is fused to the Fc domain of an antibody
this Ig binds to B7 at higher affinity than endogenous C28
B7 is not available to interact T cells
does belatecept work
yes
but cannot respond to actual threats to immune system
what is one way in which tumour cells hide from the immune system
they upregulate PD1L/2 and CTLA4
meaning that when T-cells come to kill the tumour they recognise the CTLA4 and do not attack (exhausted)
the tumour uses negative co-stimulation
what are some drugs that are used to try and prevent tumour cells from using negative co-stimulation to hide from the immune system
PDL-1inhibitors
PD-L1inhibitors
CTLA4 inhibitors
do negative co-stimulatory inhibitors work
yes
but they do not work when the T cells cannot find the tumour - so inhibiting negative co-stimulation does not help - breast cancer
what is a side-effect of negative co-stimulation inhibitors
autoimmune diseases
cytokine storm
