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BIOL3013 - Molecular recognition > Lectures 1-7 - signalling > Flashcards

Lectures 1-7 - signalling Flashcards

1
Q

Explain features of cytokines

A

> 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

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2
Q

What are the four distinct structural families of cytokines

A

TNF tumor necrosis factor

interferon

chemokines

hematopoeitin

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3
Q

Explain features of the cytokine family TNF

A

TNF = Tumour necrosis factor
Oligamarisation of receptor, bring receptors together by binding of cytokine
Trimeric ligand, brings 3 receptors together (trimeric receptos)
Beta stranded

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4
Q

Explain features of the cytokine of the interferon family/ IFNγ interferon

A

Alpha helical
dimeric ligand (2 receptors)

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5
Q

Explain features of the cytokine family: Chemokines

A

Wound healing

Many different forms, trimer dimer, monomer

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6
Q

Explain how HIFa works in different conditions

A

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

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7
Q

Features of EPO

A

EPO stands for erytropoietin

stimulates RBC maturation

activates erythroid progenitor cells

a cytokine

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8
Q

Explain the process of RBC maturation from a hematopoietic stem cell

A

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)

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9
Q

Explain the trend for Kd for ligand binding and physiological/cellular response and how this relates ti EPO and erythroid progenitor division

A

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)

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10
Q

What are the average Hematocrit levels for men and women?

A

Women: 36.1% to 44.3%

Men: 40.7% to 50.3%

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11
Q

What does hematocrit mean?

A

proportion of RBCs when centrifuged (they go to bottom)

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12
Q

Explain the levels of hematocrit in children

A

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

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13
Q

What are the causes of low hematocrit?

A

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.

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14
Q

Explain the uses of EPO in disease states

A

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)

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15
Q

Disadvantages of EPO treatment

A

increased risk of death, myocardial infarction, stroke, venous thromboembolism, tumor recurrence

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16
Q

What is JAK? What does it do?

A

JAK = kinase, senses signall from EPO-R

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17
Q

Describe the structure of an EPO-R

A
  • Extracellular ligand binding domains
  • Transmembrane a-helix
  • Cytosolic tale
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18
Q

Explain JAK structure

A

number of different domains, one able to bind tale of EPO receptor, one is kinase (two lobes and lip)

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19
Q

Explain the JAK/STAT signalling cascade activation

A
  1. cytokine binding & cytokine receptor dimerisation
  2. phosphorylation of associated JAK kinase, leading to activation
  3. phosphorylation of additional residues (entire tale of receptor) (hyperphosphorylation) - resulting step needed for receptor to be shown to be activated
  4. recognition by inactive monomeric transcription factor STAT (a TF) (recognises hyperphosphorylated tales)
  5. phosphorylation and release of STAT
  6. STAT dimerisation (by SH2 domain recognising phosphprylation), diamarization leads to exposure of Nuclear localisation signal) therefore entry to nucleus
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20
Q

Explain what an SH2 domain recognises

A

phosphorylated tyrosine on the receptor

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21
Q

How do different SH2 domains recognise different sequences and proteins?

A

Recognition signal has variation, can have different types of SH2 domains

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22
Q

Summarise the protein-protein events of the JAK/STAT pathway (EPO receptors)

A

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

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23
Q

How is the EPO signalling cascade switched off?

A

short term adaptation (SHP1)

long term adaptation (SOCS)

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24
Q

Explain short term adaptation to switch off the EPO signalling cascade

A

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

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25
Q

Explain long term adaptation to switch off the EPO signalling cascade

A

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

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26
Q

Explain how signals are differentiated in the EPO signalling cascade

A

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)
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27
Q

Explain what the different STATS in the JAK/STAT pathway activate gene expression of

A

STAT-1: IFNγ

STAT-1, -2: IFNα/β

STAT-3, -5:
growth hormones

STAT-4: IL8 receptor (IL18R)

STAT-5: EPO

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28
Q

Explain when the different interferons are produced

A
  • 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

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29
Q

Explain the different types of interferons and how they signal

A

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
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30
Q

What Jaks and STATs do Erytropoietins signal through

A

via Jak2 and STAT5 homodimers

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31
Q

What does wound healing mean in terms of cytokines

A

converting an inflammatory cytokine response to an anti-inflammatory response

32
Q

Explain the phases of wound healing

A

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

33
Q

Name the acute inflammatory responders

A

G-CSF, PDGF, IL-1a

34
Q

Name the chronic inflammatory cytokines

A

MMPs, TGF-b1, TGFb-2, IL-5

35
Q

Name the neutral growth factors

A

IGF-1, KGF, EGF

36
Q

Name the anti-inflammatory cytokines

A

IL1-RA, IL-10, IL-11, HGF, IL-4, IL6, IL-16, IL-13, IL18-RA, TGF-b3

37
Q

explain TGFb signalling

A

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

38
Q

Give an overview of he EGF pathway

A

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)

39
Q

What makes the EPO receptor and RTK different

A

In RTK Kinase is part of the receptor (has a kinase domain) whereas EPO receptor kinase just associates

40
Q

Explain the structure of the receptor tyrosine kinase (RTK) and what binds to it

A

Extracellular receptor, a-helix transmembrane domain, tale with a kinase domain

2EGF monomers bind

41
Q

Explain the first step of the EGF pathway

A

ligand binding (EGF) causes receptor dimerisation and activation of intrinsic kinase

42
Q

Explain the second step in the EGF pathway after receptor dimerisation

A

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

43
Q

Explain the 3rd step of EGF pathway (involving sos and GTPase)

A

SH3 domain of GBR2 recruits Sos, which binds to the SH3 domain and also to the membrane bound Ras GTPase

44
Q

Explain the 4th step in the EGF pathway, activation of Ras GTPase

A

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)

45
Q

Explain the 5th step in the EGF pathway, activation of Raf kinase

A

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

46
Q

Explain the 6th step of the EGF pathway (MAP kinase cascade)

A

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

47
Q

What in particular does the SH3 domain of GBR2 recognise about Sos

A

Proline rich sequence/peptide

48
Q

Explain kinase activation in the EGF pathway

A

Low activity of cyclin A/Cdk2 = Thr160 unphosphorylated

High/active Cyclin A/Cdk2 = Thr160 phosphorylated

49
Q

Explain phosphorylation and removal of a phosphate group

A

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

50
Q

What is one thing to note about phosphorylation

A

Phosphorylated doesn’t always mean active, phosphorylation is a switch but not necessarily in one direction

51
Q

How is the GTPase switch different from phosphorylation

A

Only occurs in one direction

52
Q

what do the following stand for:
GEFs, GAPs, RGSs, GDIs

A

GEFs: guanosine exchange factors e.g. SOS

GAPs: GTPase activating proteins

RGSs: regulators of G-protein signaling

GDIs: guanine nucleotide dissociation inhibitors

53
Q

Explain how different types of molecules influence the GTPase switch

A

Active to inactive:
+GAPs
+RGSs
-GDIs

Inactive to active:
GTP converted to GDP
+GEFs

54
Q

Explain features of GTPases

A

GTPases are also called GTP-binding-proteins

They are 160-180 amino acids in length

They have a conserved α,β-topology

55
Q

Explain the structure of GTPase

A

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

56
Q

What parts of the GTPase recognise nucleotide?

A

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

57
Q

Explain the role of switch I and II in GTPases

A

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)

58
Q

Give an example of a GEF and how it interacts with a GTPase to perform its function

A

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

59
Q

Explain the structure of Raf

A

N terminal regulatory domain
C terminal kinase domain

60
Q

Explain the activation of Raf

A

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

61
Q

Is Raf kept active or inactive in the cytosol?

A

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

62
Q

14-3-3 structure

A

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

63
Q

14-3-3 involved in…

A

signal transduction e.g.

cell division

apoptosis

differentiation

64
Q

classes of 14-3-3

A

mammals have α, β, γ, δ, ε, η, σ, τ, and ζ classes

65
Q

what is cRafs recognition motif

A

RSx-pS-xP - (pS = phosphoserine)

66
Q

do all 14-3-3 interacting proteins have the conserved c-Raf recognition motif?

A

NO

67
Q

Explain the method for selecting good 14-3-3 binders

A

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

68
Q

When 2 motifs were found when finding 14-3-3 binders, what was done/discovered?

A

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

69
Q

Explain what is used to check for affinity of 14-3-3 binders

A

spr experiment RW

70
Q

Explain the activation of raf/downstream signalling (simple)

A

Active Ras GTP triggers activation of Raf
Active Raf triggers MAP kinase pathway

71
Q

If Raf carries the linear sequence motif, where is the second motif for Raf to bind to the GTPase?

A

in the switch domain of Ras – highly conserved, read out by the N-terminal domain of active Raf

72
Q

What are the recognition motifs of c-Ras and c-Raf

A

c-Raf recognition motif on c-Ras: DEYDPTIED

14-3-3 recognition motif on c-Raf: RSx(pS)xP

73
Q

Explain HIV Nefs structure and the reasoning behind it

A

Central helix - the rest are segments of extended peptides

Has to expose a number of peptide motifs needed for its cell hijacking abilities

74
Q

Explain the positions of the different motifs on Nef and what they do/induce

A

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)

75
Q

Explain which motifs of Nef are involved in protein modulation, trafficking and signalling

A

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

76
Q

What are all the subversion mechanisms of Nef based on?

A

Peptide recognition and therefore mimicry of host peptide signals

77
Q

3 things Nef regulates and how?

A
  1. Cell cycle:
    Stimulates MAP cascade by Raf interaction
  2. Host immunity:
    Down regulates CD4 avoiding superinfection
    SH3 interaction interferes with T cell signalling
  3. 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

BIOL3013 - Molecular recognition (6 decks)

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