Lectures 1-7 - signalling Flashcards
Explain features of cytokines
> 50 cytokines known
4 different classes
Small proteins 5-20kDa
Released by cells affect other cells
Especially important for cells of immune systems
Can be auto stimulatory or help with signaling between 2 cells
What are the four distinct structural families of cytokines
TNF tumor necrosis factor
interferon
chemokines
hematopoeitin
Explain features of the cytokine family TNF
TNF = Tumour necrosis factor
Oligamarisation of receptor, bring receptors together by binding of cytokine
Trimeric ligand, brings 3 receptors together (trimeric receptos)
Beta stranded
Explain features of the cytokine of the interferon family/ IFNγ interferon
Alpha helical
dimeric ligand (2 receptors)
Explain features of the cytokine family: Chemokines
Wound healing
Many different forms, trimer dimer, monomer
Explain how HIFa works in different conditions
normal oxygen levels:
prolyl hydroxylase hydroxylates Hif1α and marks it for ubiquitylation/destruction
hypoxic conditions:
asparagin hydroxylase hydroxylates Hif1α, and its activation results in target gene expression
Features of EPO
EPO stands for erytropoietin
stimulates RBC maturation
activates erythroid progenitor cells
a cytokine
Explain the process of RBC maturation from a hematopoietic stem cell
Hematopoietic stem cell producing both progenitprs od other types of blood cell and
erythroid progenitor (CFU-E - colony forming unit erythroid) with many EPO receptors present on surface
When EPO present
3-5 cell divisions, leading to 30-100 erythroids (with less/little EPO receptors)
Explain the trend for Kd for ligand binding and physiological/cellular response and how this relates ti EPO and erythroid progenitor division
50 % of the physiological response is reached by only ≈18 % of the receptors occupied (example)
At ligand concentration at the Kd, 50 % of the receptors will be occupied; this leads to 80% physiological response
generally ligand concentrations are below the Kds
In terms of EPO:
an erythroid progenitor cell has 1000 EPO receptors
only 100 need to be occupied to elicit a cellular response (division of the progenitor)
What are the average Hematocrit levels for men and women?
Women: 36.1% to 44.3%
Men: 40.7% to 50.3%
What does hematocrit mean?
proportion of RBCs when centrifuged (they go to bottom)
Explain the levels of hematocrit in children
High in newborns
broad range in two month olds
low in 3 month olds
slightly low/slightly broader range in age 6-12
boys age 12-18 - slightly lower than men
Girls age 12-18 very similar to women but higher top range
What are the causes of low hematocrit?
low RBC in cancers that cause elevated production of other types of blood cells:
-Lymphoma: increased B / T Lymphocyte WBC
-Hodgkin’s Lymphoma: increased Lymphocyte WBC
-Leukemia: increased immature WBC
-Multiple Myeloma: increased Myeloma cells (malignant plasma cells in the bone marrow) reduces production of several blood cells including RBC
Cancers use the blood producing stem cells on the expense of RBC production.
Explain the uses of EPO in disease states
treating anemia in these conditions:
chronic kidney disease
inflammatory bowel disease
(Crohn’s disease and ulcer colitis)
myelodysplasia (blood disorder), resulting from
exposure to chemicals (benzene)
treatment of cancer (chemotherapy and radiation)
Disadvantages of EPO treatment
increased risk of death, myocardial infarction, stroke, venous thromboembolism, tumor recurrence
What is JAK? What does it do?
JAK = kinase, senses signall from EPO-R
Describe the structure of an EPO-R
- Extracellular ligand binding domains
- Transmembrane a-helix
- Cytosolic tale
Explain JAK structure
number of different domains, one able to bind tale of EPO receptor, one is kinase (two lobes and lip)
Explain the JAK/STAT signalling cascade activation
- cytokine binding & cytokine receptor dimerisation
- phosphorylation of associated JAK kinase, leading to activation
- phosphorylation of additional residues (entire tale of receptor) (hyperphosphorylation) - resulting step needed for receptor to be shown to be activated
- recognition by inactive monomeric transcription factor STAT (a TF) (recognises hyperphosphorylated tales)
- phosphorylation and release of STAT
- STAT dimerisation (by SH2 domain recognising phosphprylation), diamarization leads to exposure of Nuclear localisation signal) therefore entry to nucleus
Explain what an SH2 domain recognises
phosphorylated tyrosine on the receptor
How do different SH2 domains recognise different sequences and proteins?
Recognition signal has variation, can have different types of SH2 domains
Summarise the protein-protein events of the JAK/STAT pathway (EPO receptors)
EPO binds EpoR
dimerisation of EpoR
activates cytosolic JAK-Kinase
receptor phosphorylation
Receptor(P) recognised by SH2
activates STAT pathway
exposes NLS, pathway continues in the nucleus
How is the EPO signalling cascade switched off?
short term adaptation (SHP1)
long term adaptation (SOCS)
Explain short term adaptation to switch off the EPO signalling cascade
Need a phosphatase to take off phosphates on the lip domain of JAK
SHIP1 needs an SH2 domain to become associated (has 2 SH2 domains)
Normally inactive in structure, when binds, unravels and nibbles off particular phosphate
Explain long term adaptation to switch off the EPO signalling cascade
SOCS Recognizes with SH2 domain, binds to phosphate
SOCS box, has an E3 ubiquitin ligase system (marks for destruction)
Receptor is destroyed, would need to make new receptors to activate
Explain how signals are differentiated in the EPO signalling cascade
several pathways activated at once, EPO signals in more than one way:
- STAT - most direct pathway (Transcriptional activation TA)
- GRB/Shc adaptor regulate the Ras/MAP kinase pathway (TA and T repression)
- phospholipase Cγ regulate Ca2+ levels (TA and TR, and modification of other cellular proteins)
- PI-3 kinase acivate the protein kinase B pathway (TA and TR, and modification of other cellular proteins)
Explain what the different STATS in the JAK/STAT pathway activate gene expression of
STAT-1: IFNγ
STAT-1, -2: IFNα/β
STAT-3, -5:
growth hormones
STAT-4: IL8 receptor (IL18R)
STAT-5: EPO
Explain when the different interferons are produced
- Virus infected cells produce the type I interferon IFNα (dendritic cells) and IFNβ (fibroblasts) that are
1. autocrine: towards the infected cell
2. paracrine: towards uninfected neighbours
-interferons signal through the Jak-STAT pathway to produce > 300 gene products, including cytokines
- T-cells & NK cells produce type II interferon INFγ
–INFγ can kill virus infected cells or cancerous cells
–autocrine: inhibition of virus replication, apoptosis
–paracrine: up-regulation of MHC-I and NK-cell activation
Explain the different types of interferons and how they signal
IFNα/β
- type I interferon, produced by dendritic cells (IFNα) and fibroblasts (IFNβ) in response to virus infection
- via Jak1/Tyk2 & STAT1/2 heterodimers
IFNγ
- type II interferon, produced by T-cells & NK cells
- via Jak1/Jak2 and STAT1 homodimers
What Jaks and STATs do Erytropoietins signal through
via Jak2 and STAT5 homodimers
What does wound healing mean in terms of cytokines
converting an inflammatory cytokine response to an anti-inflammatory response
Explain the phases of wound healing
platelet activation: blood platelets in the wound produce PDGF
inflammatory phase: production of cytokines to clear infections
proliferative phase (anti-inflammatory) : production of CXCL8, IL-1a, IL-1b, CCL2, VEGF, TGF-b and TNF
Name the acute inflammatory responders
G-CSF, PDGF, IL-1a
Name the chronic inflammatory cytokines
MMPs, TGF-b1, TGFb-2, IL-5
Name the neutral growth factors
IGF-1, KGF, EGF
Name the anti-inflammatory cytokines
IL1-RA, IL-10, IL-11, HGF, IL-4, IL6, IL-16, IL-13, IL18-RA, TGF-b3
explain TGFb signalling
Transforming Growth Factor TGF is involved in embryo development
TGFβ is released from macrophages and affects numerous cells types
The TGFβ receptor has kinase activity
TGFβ phosphorylates SMAD
SMAD enters the nucleus
activates transcription of genes that lead to growth, adhesion, invasion, inflammation, proliferation
Give an overview of he EGF pathway
EGF (epidermal growth factor) binds to an RTK (receptor tyrosine kinase)
The RTK auto-phosphorylates and then activates cytosolic kinases
The kinases enter the nucleus and phosphorylate TFs
transcriptional activation
NB: Kinase itself enters the nucleus (active in both cytosol and nucleus)
What makes the EPO receptor and RTK different
In RTK Kinase is part of the receptor (has a kinase domain) whereas EPO receptor kinase just associates
Explain the structure of the receptor tyrosine kinase (RTK) and what binds to it
Extracellular receptor, a-helix transmembrane domain, tale with a kinase domain
2EGF monomers bind
Explain the first step of the EGF pathway
ligand binding (EGF) causes receptor dimerisation and activation of intrinsic kinase
Explain the second step in the EGF pathway after receptor dimerisation
Lip domain on the kinase domain of the tyrosine kinase receptor is phosphorylated, followed by other tyrosine residues of the receptor
The SH2 domain of the adaptor protein GRB2 recogmises the phosphorylation and recruits it to the receptor
Explain the 3rd step of EGF pathway (involving sos and GTPase)
SH3 domain of GBR2 recruits Sos, which binds to the SH3 domain and also to the membrane bound Ras GTPase
Explain the 4th step in the EGF pathway, activation of Ras GTPase
Sos is a GEF and converts the Ras GTPase from the GDP (inactive) to the GTP state (active) (GTP is more abundant in cell than GDP)
Now a signaling pathway due to Ras being a signaling molecule (now onto the Ras/MAP cascade)
Explain the 5th step in the EGF pathway, activation of Raf kinase
N-terminal domain of Raf recognises Ras GTP (active form)
Raf kinase is inactive kept inactive by 14-3-3 protein (14-3-3 binds on the phosphrylated amino acids and holds it in inactive state)
when active Ras GTP, loss of block from this, therefore Raf/Ras active
Explain the 6th step of the EGF pathway (MAP kinase cascade)
Raf starts the MEK/MAP kinase cascade; ultimately, MAP translocates to the nucleus where it phosphorylates and activates many transcription factors
Lots of kinases in a row, amplifies the signal and allows more points of intervention for regulation
What in particular does the SH3 domain of GBR2 recognise about Sos
Proline rich sequence/peptide
Explain kinase activation in the EGF pathway
Low activity of cyclin A/Cdk2 = Thr160 unphosphorylated
High/active Cyclin A/Cdk2 = Thr160 phosphorylated
Explain phosphorylation and removal of a phosphate group
A protein kinase puts phosphate on – uses ATP
A protein phosphatase uses water to cleave off and release the phosphate
These things DO NOT synthesise ATP
What is one thing to note about phosphorylation
Phosphorylated doesn’t always mean active, phosphorylation is a switch but not necessarily in one direction
How is the GTPase switch different from phosphorylation
Only occurs in one direction
what do the following stand for:
GEFs, GAPs, RGSs, GDIs
GEFs: guanosine exchange factors e.g. SOS
GAPs: GTPase activating proteins
RGSs: regulators of G-protein signaling
GDIs: guanine nucleotide dissociation inhibitors
Explain how different types of molecules influence the GTPase switch
Active to inactive:
+GAPs
+RGSs
-GDIs
Inactive to active:
GTP converted to GDP
+GEFs
Explain features of GTPases
GTPases are also called GTP-binding-proteins
They are 160-180 amino acids in length
They have a conserved α,β-topology
Explain the structure of GTPase
b strands: 1-6
a-helix: 1-5
N terminus starts with b1, between b1 and alpha 1 is the G1 loop - P-loop phosphate binding motif (GxxxxGKS/T)
Between a1 and b2 G2 BUT G2 (T) and G3 (DxxG) overlap with the switches
b2 and b3 strands connected, G3 between b3 and a2 (swII connected to a2)
a2 - b4 - a3 - b5 (G4) - a4 - b6 (G5) a5 - C terminus
G4 (NKxD) and G5 (SAK) recognise guanine base
What parts of the GTPase recognise nucleotide?
Conserved G-elements:
- The switches interact with the GTP phosphate
- G2 (T) and G3 (DxxG) overlap with the switches
- The gamma phosphate positions the peptide
Explain the role of switch I and II in GTPases
The switches interact with the GTP phosphate
G2 (T) and G3 (DxxG) overlap with the switches
The gamma phosphate positions the peptide
Switch I and II change in structure (string model - imagine cutting string, switches open up)
Give an example of a GEF and how it interacts with a GTPase to perform its function
SOS proteins are guanosine nucleotide exchange factors (GEF)
activates Ras by:
disrupting tight interaction of nucleotides with Ras in 2 ways:
(Boriack-sjodin 1998)
- alpha-helix of Sos into Ras causes displacment of Switch 1 region of Ras
- side chains helix and distorted conformation of switch 2 region alter chemical environment of binding sites of phosphate groups of the nucleotide and Mg ion - binding no longer favoured
Explain the structure of Raf
N terminal regulatory domain
C terminal kinase domain
Explain the activation of Raf
active Ras GTP triggers activation of Raf kinase
When Raf is bound to the 14-3-3 protein the kinase is inactive
14-3-3 binds to two phosphoserine residues on Raf to keep inactive (One phosphoserine residue 14-3-3 binds to is on the kinase domain of Raf)
when Ras GTP interacts with Raf, causes release of 14-3-3 protein
Is Raf kept active or inactive in the cytosol?
Inactive - becomes active when:
The N-terminal domain of Raf can bind to Ras-GTP (but not to Ras-GDP)
The Ras-GTP/Raf interaction releases the 14-3-3 protein
Ras-GTP/Raf interaction triggers activation of Raf kinase
14-3-3 structure
30kDa
Perfect dimer, two fold axis (hetero and homodimers)
positive charged pockets recognise the two phosphate residues on protein
One will be bind to the N terminal domain and one to the kinase domain of Raf
14-3-3 involved in…
signal transduction e.g.
cell division
apoptosis
differentiation
classes of 14-3-3
mammals have α, β, γ, δ, ε, η, σ, τ, and ζ classes
what is cRafs recognition motif
RSx-pS-xP - (pS = phosphoserine)
do all 14-3-3 interacting proteins have the conserved c-Raf recognition motif?
NO
Explain the method for selecting good 14-3-3 binders
Start with (MA)xxx-pS-xxxAKK as unbiased start
therefor sequence has 6 variable positions
Round 1:
-immobilise 14-3-3 on beads
-add peptide library
-Wash – everything that’s unbound, sequence retained peptides left
Arganine seen at -1 and -3 but nit high enough for deinate
Round 2:
-Proline was fixed at +2
-use sequence MAxxxx-pS-xPxAKK
- test all AAs apart from Cys
2 motifs found:
Motif 1: ARSHpSYPA
Motif 2: RLYHpSLPA
When 2 motifs were found when finding 14-3-3 binders, what was done/discovered?
crystal analysis:
Superposition of two peptides – proline identified in two different positions:
Motif 1: cis-Pro
Motif 2: trans-Pro
peptides can bind to 14-3-3 proteins in two different modes
Motif 1: Raf-like motif
Motif 2: Cdc25-like motif
Explain what is used to check for affinity of 14-3-3 binders
spr experiment RW
Explain the activation of raf/downstream signalling (simple)
Active Ras GTP triggers activation of Raf
Active Raf triggers MAP kinase pathway
If Raf carries the linear sequence motif, where is the second motif for Raf to bind to the GTPase?
in the switch domain of Ras – highly conserved, read out by the N-terminal domain of active Raf
What are the recognition motifs of c-Ras and c-Raf
c-Raf recognition motif on c-Ras: DEYDPTIED
14-3-3 recognition motif on c-Raf: RSx(pS)xP
Explain HIV Nefs structure and the reasoning behind it
Central helix - the rest are segments of extended peptides
Has to expose a number of peptide motifs needed for its cell hijacking abilities
Explain the positions of the different motifs on Nef and what they do/induce
SEE PHOTO FOR LOCATION
MGxxxS - Myristolation
DD - AP2/V1H ATPase - regulates ph stopping acidification
EE - b-COP- endosomal sorting
ExxxLL - AP1/2/3- entry into cell
DDPxxE - Raf
twist 180:
PxxPxR - SH3 (lck, hck) - modulates T-cell response
FPD - Thioesterase - needed for endocytosis- regulator for enzymatic activity
WLL - CD4 - downregulates this - modulate immune response as well as stopping further virus infection of the cell
EEEE - PACS1 - inteferes with Golgi trafficking
RR - Pak1/2 - kinases, play a role in entry, replication and spread (effects apoptosis and cytoskeleton organisation)
Explain which motifs of Nef are involved in protein modulation, trafficking and signalling
Protein modulation:
MGxxxS - myristolation
Trafficking:
WL - CD4
EEEE - PACS1
FPD- Thioesterase
EE - b-COP
ExxxLL - AP1/2/3
DD - VH1 ATPase
Signalling:
PxxPxR - SH3 (lck, hck)
RR - Pak1/2
DDPxxE - Raf
What are all the subversion mechanisms of Nef based on?
Peptide recognition and therefore mimicry of host peptide signals
3 things Nef regulates and how?
- Cell cycle:
Stimulates MAP cascade by Raf interaction - Host immunity:
Down regulates CD4 avoiding superinfection
SH3 interaction interferes with T cell signalling - Trafficking:
- AP1/2/3 signals regulate sorting into clathrin coat
- PACS1 - interferes with Golgi trafficking
- Thioesterase needed for endocytosis
- PAK - play major role in entry, replication and spread
