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4.1 Transfusion and Transplant Immunology > Lymphocyte Activation - Gene Expression > Flashcards

Lymphocyte Activation - Gene Expression Flashcards

1
Q

What are the three groups of induced gene products of T cel activation?

A

Immediate genes - expressed within 30 mins

Early genes - expressed within 1-2 hours

Late genes - expressed after 2 days

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2
Q

What are the immediate genes, give five examples?

A

These tend to be transcription factors
- c-Fos
- c-Jun
- c-Myc
- NFAT
- NF-kB

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3
Q

What are five examples of early genes?

A

IL-2
IL-2R
IL-3
IL-6
IFN-gamma

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4
Q

What are the late genes?

A

Adhesion molecules

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5
Q

Write about the immediate genes
(NB - coming up on exam)
(3)

A

Transcription factors c-Fos and c-Jun come together toform a master transcription factor known as AP-1 (activator-protein 1)

c-Fos, c-Jun, c-Myc are wild type transcription factors that control proliferation in cells -> they are proto-oncogenese

Can be mutated to be constantly switched on and become cancer

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6
Q

What is the start point of signal transduction, what are the two most common forms of this?
(3)

A

Interaction between a signal and its receptor

Signals that cannot penetrate the cell membrane bind to cell surface receptors

Hydrophobic signals e.g. steroids diffuse through the cell membrane and bind intracellular receptors

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7
Q

What molecule are signals often transducted through?

A

G-proteins such as Ras

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8
Q

What are G proteins?

A

Membrane-linked molecules whose activites are controlled by the binding of GTP (on) and GDP (off)

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9
Q

What swithces Ras on and off?

A

Ras bound to guanine triphosphate is swithed on

Ras bound to guanine diphosphate is swthced off

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10
Q

How do we activate G proteins such as Ras, how do we get from GDP to GTP?
(4)

A

In the resting state G proteins are bound to GDP

Signalling activates guanine-nucleotide exchange factors (GEFs) to displace GDP from small G proteins and allow GTP to bind

Over time, the small G protein hydrolyzes GTP to GDP i.e. GTP looses a phosphate group to become inactivated

*you would think a phosphate is added on like other molecules but this is not the case - do not get mixed up

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11
Q

What is a second messenger?

A

A molecule or ion that can diffuse to other cellular sites and evoke changes

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12
Q

Give five examples of second messengers

A

cAMP
cGMP
Ca2+
DAG
IP3

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13
Q

What are the three most important second messengers?

A

Ca2+
DAF
IP3 - inositol triphosphate

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14
Q

When is Ca2+ released, what does it do?
(2)

A

Ca2+ is released after co-stimulator signal

Calcium rapidly diffuses throughout the cell and induces conformational changes in calmodulin

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15
Q

What are adaptor/scaffold molecules?

A

Molecules that allow all the tiny molecules involved in cell activation to come together and find each other at the right time

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16
Q

How is a scaffold built up?
(3)

A

Start of with an unphosphorylated scaffold protein

Acivation of protein kinase results in phosphorylation of a scaffold

The phosphorylated scaffold recruits signalling proteins that bind to it

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17
Q

Give three examples of adaptor/scaffold molecules

A

SLP76
LAT -> linked or activated T cells
GADS

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18
Q

What amplifies up the original signal?

A

Enzyme cascades

In particular kinase cascades

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19
Q

What are the three main points in the kinase cascade?

A

Raf -> Mek -> Erk

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20
Q

What are the steps of T cel activation so far?

A

TCR complex and MHC/peptide engagement occurs

p56-Lck is associated with CD4 and CD8

In resting T cells Lck is sequestered from the TCR complex in lipid rafts

The TCR complex moved into lipid rafts on engagment with the MHC.peptide complex

Lck phosphorylates the ITAMS of CD3 ans in the cytoplasmic tails of the zeta chains

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21
Q

What are membrane/lipid rafts?
(4)

A

Specialised regions of the cell membrane enriched for saturated lipids and cholesterol

GPI-linked proteins and acylated proteins such as Src family kinases e.g. Lck are found in lipid rafts

Lipid rafts are dynamic structures that can change size and protein content

Some proteins migrate into lipid rafts when they are oligomerised by binding ligand e.g. TCR

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22
Q

What is the function of the lipid rafts?
(3)

A

They prevent the inappropriate activation of the TCR complex

TCR cannot move into lipid raft until it has engaged with antigen on MHC

Only then can Lck phosphorylate the ITAMS in the zeta chains of CD3 (and other chains of CD3)

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23
Q

Talk about the structure of the zeta chain of CD3, what binding sites are present?

A

Each zeta chain has 3 ITAMS

Each ITAM has 2 tyrosine residues for phosphorylation (thing of ITAM structure)

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24
Q

After TCR has moved into lipid raft, and after signal 1 and 2, what binds to zeta chain of Cd3 and where?
(3)

A

ZAP-70 binds one of the three ITAMS of zeta chain of CD3

ZAP-70 does so by using its two tandem SH2 domains to bind to the two phosphorylated tyrosines of the ITAM

Basically ZAP-70 can bind to CD3 in the same way p56-Lck can

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25
Q

What is ZAP-70 and how is ZAP-70 activated?

A

Its a tyrosine kinase

Its activated by phosphorylation by Lck/autophosphorylation

I think its also activated after the work of p56Lck on CD3 -> once CD3 is activated then ZAP-70 can bind and initiate next step

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26
Q

What is another cytoplasmic tyrosine kinase found in physical association with the TCR other than ZAP-70, what does it do?

A

Membrane-associated Fyn

It is thought to play a similar role to p56Lck

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27
Q

What can ZAP-70 phosphorylate other than ITAMS, what do these do?

A

It can bind to a number of membrane-associated adaptor molecules:
- SLP-76
- LAT
- GADS

These act as anchor points for several intracellular signal transduction pathways

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28
Q

What kind of structure/binding domain is required for ITAMs?

A

2 consequtive SH2 domains

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29
Q

What is ZAP-70?

A

Zeta associated protein of 70 kilodaltons

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30
Q

Name a molecule with 2 consecutive SH2 domains suitable for binding to the Zeta chains of ITAMs?

A

ZAP-70 -> zeta chain associated protein

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31
Q

How is Zap-70 activated?

A

Activated by Lck or by autophosphorylation i.e. it can activate itself

addition of a phosphate activates Zap-70

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32
Q

Explain in your own words the steps required to activate Zap-70 and what comes after this?
(4)

A

TCR complex and coreceptors are clustered within membrane lipid rafts by antigen recognition

Lck phosphorylates tyrosines in ITAMs of zeta chains of CD3 -> activating CD3

Zap-70 binds to phophotyrosines of Z chains and phosphorylates adapter/scaffold proteins such as LAT

Assembly of adapter protein and enzyme scaffolds occurs, multiple signaling pathways are activated

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33
Q

Explain in your own words the steps required to activate Zap-70 and what comes after this?
(4)

A

TCR complex and coreceptors are clustered within membrane lipid rafts by antigen recognition

Lck phosphorylates tyrosines in ITAMs of zeta chains of CD3 -> activating CD3

Zap-70 binds to phophotyrosines of Z chains and phosphorylates adapter/scaffold proteins such as LAT

Assembly of adapter protein and enzyme scaffolds occurs, multiple signaling pathways are activated

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34
Q

What domains allow for ZAP-70 binding to ITAMS of CD3?

A

2 tandem SH2 domains
-> these bind to phosphorylated tyrosines found in the zeta chains of an activated/phosphorylated ITAM (phosphorylated by Lck previously)

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35
Q

What does ZAP-70 binding to ITAMS activate?

A

This binding phosphorylates adapter proteins such as LAT and SLP-76

This starts the assembly of adapter protein and enzyme scaffolds

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36
Q

How is a LAT/SLP-76 scaffold made?
(7)

A

Activated ZAP-70 phosphorylates LAT and SLP-56

GADS then brings SLP-76 and LAT together

GADS:SLP-76:LAT complex recruits PLC-y

Phospholipase Cy (PLCgamma) and Itk is recruited to the membrane by PIP3

PLCy is then recruited by GADS:LAT:SLP-76 complex

Itk recruited to membrane by PIP3 where it is now phosphorylated by Lck

Activated Itk then associated with LAT/SLP-76 complex to phosphorylate PLCy1

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37
Q

What scaffold molecule brings SLP-76 and LAT together to start a scaffold, what is this molecule?

A

GADS

GADS is an adapter protein which may promote cross-talk between LAT and SLP-76 thereby coupling membrane-proximal events to downstream signaling pathways

38
Q

What does GADS:SLP-76:LAT complex do?

A

PLC-y has previously ben recruited to the cell membrane by PIP3

GADS:SLP-76:LAT then recruits PLC-y

PLC-y sits into a cradle like structure made by this complex

Scaffold acts as a site for Phohorylated Itk to phosphorylate PLC gamma

39
Q

What kind of scaffold does GADS:SLP-76:LAT form?

A

A cradle like structure for PLC-y to sit into

40
Q

How does PIP3 recruit moleucles to the cell membrane?

A

PIP3 draws in any molecule with a plextrin homology/ PH domain:
- PLC-y
- Itk

41
Q

Explain in your own words how PLCy-1 is activated
(8)

A

Signal 1 and Signal 2

ZAP-70 activation by Lck or autophosphorylation

ZAP-70 phosphorylates LAT and SLP-76

GAD:LAT:SLP-76

PIP3 recruits both Itk and PLC-y to the cell membrane

Itk is phosphorylated by Lck

Both PLC-y and activated Itk associate with the GADS:SLP-76:LAT complex, with PLc-y sitting into the scaffold cradle

Finally Itk phosphorylates PLC-y to activate it

42
Q

What exactly is Itk, what does it do?

A

Itk is a Tec kinase (not an Src kinase like Lck)

Itk is responsible for phosphorylating PLC-y

43
Q

How is PCL-y1 different from PLC-y2?

A

These are isoforms of the same molecule

PLC-y1 is found in T cells

PLC-y2 is found in B cells

44
Q

What is the role of activated PLCy1?

A

PLCy1 hydrolyses PIP2 (the starting substrate to making PIP3)

This cleaves PIP2 into two components: IP3 and Diacylglycerol

45
Q

If PIP2 is the starting substrate for PIP3, how does it make sense for PLC-y1 to hydrolyse PIP2?
(4)

A

There are multiply molecules of PIP2 (and PIP3) int he cell membrane of the T cell, the same molecule does not have to be used throughout the process

Multiply molecules of PIP2 and PIP3 are often used

PIP3 molecules present in cell membrane constantly recruit PLC-y and Itk molecules

PIP2 molecules are constantly being converted into PIP3 and being hydrolysed into IP3 and DAG at the same tim

46
Q

What is meant by PLC-y1 hydrolysing PIP2?

A

PLC-y1 uses water molecules to separate PIP2 into IP3 and DAG

47
Q

What does PIP2 stand for?

A

Phosphoinositol biphosphate

48
Q

What does IP3 stand for, what does IP3 do after its formation?

A

Inositol 1,4,5-triphosphate

It moves into the cytoplasm

49
Q

What does DAG stand for, what does it do after its formation?

A

Diacylglycerol

It remains in the cell membrane

50
Q

What acts on PIP2 to form PIP3?

A

PI3 kinase

51
Q

What does IP3 do once it moves into the cytoplasm?
(4)

A

IP3 opens calcium channels

This allows flow of Ca2+ from the Endoplasmic Reticulum into the cytosol

Depletion of Ca2+ in the ET leads to opening of CRAC channels in the plasma membrane

i.e. Crac channels in outer membrane of T cells open so that calcium can be taken from external T cell environment to replenish those in the ER

These channels allow entry of extracellular calcium into the cell

52
Q

What happens when extracellular Ca2+ enters the cytoplasm?
(3)

A

Ca2+ binds calmodulin

This complex activates an enzyme calcineurin

Calcineurin is a phosphatase essential the dephosphorylation of NFAT and therefore its movement from the cytoplasm to the nucleus

53
Q

How is transcription controlled on a NFAT level?

A

Normally i.e. a cell without a signal holds NFAT in the cytosol

This is done by phosphorylating NFAT which prevents it from moving from the cytosol into the nucleus

Thus preventing NFAT signalling gene transcription

54
Q

How is NFAT held by phosphorylation?

A

The serine and threonine residues of NFAT are phosphorylated

This keeps NFAT in the cytoplasm of unstimulated cells

55
Q

Talk about calcineurin, what is it, how is it formed?

A

It is a serine phosphatase enzyme

It is a dumbbell-shaped protein

It is formed by the binding of Ca2+ to calmodulin

It has 2x C2+ binding sites i.e. can bind a total 4 Ca2+ ions

56
Q

When might we want to prevent NFAT activation?

A

In a transplant patient - want to prevent NFAT activated gene trancription to prevent rejection

57
Q

What molecules/drugs do we use to prevent NFAT activation?
(2)

A

Immunophilins:
- Cyclophilins
- FK-binding proteins

58
Q

What are immunophilins?

A

Distinct peptidyl-prolyl isomerases such as cyclophilins and FK-binding proteins

59
Q

Explain how cyclophilins are used to stop activation of NFAT
(3)

A

Cyclophilins (enzyme) bind Cyclosporin A (drug)

CsA:Cyp complex binds to calcineurin preventing it from working as a phosphatase i.e. it cannot dephosphorylate NFAT

Prevents NFAt moving into nucleus and thus preventing transcription of genes and thus rejection

60
Q

Explain how tacrolimus is used to prevent NFAT activiation to prevent rejection
(3)

A

Tacrolimus (drug) binds FK-binding proteins

Tacrolimus:FKBP complex binds to calcineurin preventing it from working as a phosphatase i.e. it cannot dephosphorylate NFAT

Prevents NFAt moving into nucleus and thus preventing transcription of genes and thus rejection

61
Q

What is tacrolimus also called?

A

FK506

62
Q

From the hydrolysation of PIP2, IP3 and DAG are formed, what does DAG go on to do?

A

DAG activates PKCtheta

63
Q

What does activation of PKCtheta by DAG to?

A

This leads to the assembly of a membrane-bound complex consisiting of CARMA1, BCL-10 and MALT1

64
Q

What does assembly of CARMA1:BCL-10:MALT1 complex activate?

A

This activates a mulitprotein enzyme complex (IKKa, IKKB, IKKy), inhibitor of KB kinase (IKK)

65
Q

What does IKK/inhibitor of KB kinase do?

A

IKK phosphorylates the inhibitor of kb (IKB)

66
Q

What does IKB do in a resting cell, what does its phosphorylation result in?

A

IKB normally binds NF-KB -> this keeps it in the cytosol preventing transcription

When IkB is phosphorylated It is degraded, releasing NFKB

NFKB then migrated to the nucleus resulting in gene transcription of early genes such as IL-2

67
Q

How does the end resulting signal of IP3 differ from that of DAG?

A

IP3 -> NFAT -> transcription factors

DAG -> NFKB -> early genes such as IL2

68
Q

Explain in your own words how DAG results in the activation of early gene transcription through NF-KB?

A

DAG recruits PKC-theta to the membrane

PKC-theta phosphorylates a scaffold protein CARMA1

Phosphorylated CARMA1 recruits other scaffolds such as BCL10 and MALT1

CARMA1:BCL10:MALT1 complex activates IKK (inhibitor of IkB)

Ikb holds NFkB in the resting state

IKK phosphorylates IkB causing it to release NF-KB

NF-kB migrates to the nucleus

69
Q

Explain in your own words how DAG results in the activation of early gene transcription through NF-KB?

A

DAG recruits PKC-theta to the membrane

PKC-theta phosphorylates a scaffold protein CARMA1

Phosphorylated CARMA1 recruits other scaffolds such as BCL10 and MALT1

CARMA1:BCL10:MALT1 complex activates IKK (inhibitor of IkB)

Ikb holds NFkB in the resting state

IKK phosphorylates IkB causing it to release NF-KB

NF-kB migrates to the nucleus

70
Q

What three proteins form the multiprotein enzyme complex known as IKK/inhibitor of kB kinase?

A

IKKa
IKKB
IKKy (NEMO)

71
Q

DAG recruits PKCtheta to the cell membrane, it can recruit one other molecule, what is this?

A

RasGRP/Ras guanyl-releasing protein

72
Q

What is RasGRP, what does it do?

A

RAS guanyl-releasing protein

Its a guanine nucleotide exchange factor

It activates Ras

73
Q

What is RasGRP, what does it do?

A

RAS guanyl-releasing protein

Its a guanine nucleotide exchange factor

It activates Ras

74
Q

What is a guanine nucleotide exchange factor?

A

These take away GDPs away from a G protein and allow replacement with GTP to activate a G protein

75
Q

What is a guanine nucleotide exchange factor?

A

These take away GDPs away from a G protein and allow replacement with GTP to activate a G protein

76
Q

What is the Ras/MAP kinase pathway?

A

LAT (scaffold) binds GRb-2 (adaptor) and Sos

This converts Ras GDP to Ras GTP

This initiates the mitogen-activated protein kinase pathway (MAP kinase pathway)

77
Q

What is Ras, what does it do?
(4)

A

A small G protein

It consists of a single polypeptide chain

Its a pivotal component of the signal transduction pathway

When activated by GTP it initiates MAP

78
Q

What does MAP kinase pathway stand for?

A

Mitogen-activated protein kinase pathway

79
Q

What activates the Ras/MAP pathway?

A

When LAT binds to Grb-2 and Sos and not SLP-76 (to activate PLCy1)

80
Q

What are the three main steps in the MAP pathway?

A

Ras
Mek
Erk

81
Q

What are the three main steps in the MAP pathway?

A

Ras
Mek
Erk

82
Q

What is ERK, how is it activated, what does this do?

A

ERK is the end product of the MAP kinase pathway

It is also known as MAP kinase

It is activated by phosphorylation

Phosphorylated ERK enters the nucleus where it can phosphorylate/activate Elk

83
Q

What is ERK also called?

A

MAP kinase

84
Q

What is Elk, what does it do?

A

A transcription factor necessary for the expression of Fos

85
Q

What is Fos needed for?

A

c-Fos comes together with c-Jun to form AP-1 -> a master transcription factor needed to regulate the transcription of IL-2

86
Q

How is AP-1 produced?

A

MAP kinase (JNK) -> dont know what this JNK is -> is activated/phosphorylated by PKCtheta

This allows JNK to phosphorylate c-Jun

Activated C-Jun translocates to the nucleus where it can dimerise with c-Fos to form AP-1

87
Q

How is AP-1 produced?

A

MAP kinase (JNK) -> dont know what this JNK is -> is activated/phosphorylated by PKCtheta

This allows JNK to phosphorylate c-Jun

Activated C-Jun translocates to the nucleus where it can dimerise with c-Fos to form AP-1

88
Q

What is KSR and what does it do?

A

KSR is a scaffold protein that localises MAP kinases to the membrane where ras can angage with Raf

89
Q

Talk about the function of AP-1
(3)

A

Is physically associates with other transcription factors in the nucleus including NFAT

It works best in combination with NFAT

It requires NFAT for the expression of IL-2, IL-4, TNF and other cytokine genes

90
Q

Talk about the function of AP-1
(3)

A

Is physically associates with other transcription factors in the nucleus including NFAT

It works best in combination with NFAT

It requires NFAT for the expression of IL-2, IL-4, TNF and other cytokine genes

4.1 Transfusion and Transplant Immunology (24 decks)

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