L10: Interleukin Signalling II

Question 1

Overview of JAK/STAT pathway following IL-3 binding (L9):

Answer 1

• IL-3 binds alpha chains in gp140 -> receptor activation
• Cytosolic STATs bind phosporylated beta chain -> JAK phosphorylates STAT, disrupting binding leading to dissociation
• pSTATs dimerise in various combination (7 different in mammals)
• Conformational change reveals NLS -> nuclear import

Question 2

How does STAT dimer effect gene transcription once imported into nucleus?

Answer 2

• Nuclear port transit (via importin binding)
• GAS binding (promoter in DNA)
• Recruits accessory transcription factors (also recognising specific DNA motifs)
• Nuclear phosphatases -> STAT dimer dissociates -> exportin binding sequence revealed

Question 3

Key ways in which specificity can be conferred in STAT signalling: -> multitude of different transcriptional responses

Answer 3

• Different components at various levels (IL ligands, receptors particularly gp140 group, STATs)
• STAT SH2 domain binds to particular receptor sequences containing a pTyr receptor specificity -> same STAT on different sequences -> differential transcription
• Beyond this, the 7 mammalian STATs can co-combine as hetero and homodimers and even tetramers
• Cell-specific epigenetic modifications of chromatin (e.g. DNA methylation, histone modification)

Question 4

Fast acting regulators of IL signalling:

Answer 4

• Protein tyrosine phosphatases (reversible)
• Protein inhibitors of activated STATs (PIAS)
• Occurring in cytoplasm in matter of minutes

Question 5

Slow acting regulators of IL signalling:

Answer 5

• CIS-SOCS family (generally permanent destruction)
• CIS = cytokine inducible SH2 containing protein
• SOCS = suppressor of cytokine signalling
• Occurring over a matter of hours or days

Question 6

Protein tyrosine phosphatases: Structure and key examples

Answer 6

• Contain SH2 domain -> bind to pTyr on beta-c chain or STAT
• 3 structured domains (2xSH2, 1x catalytic phosphatase) -> SH2 domain masks phosphatase domain in resting state but is unmasked upon activated receptor binding
• -> SHP1 then dephosphorylates JAK2 to undo receptor activation
• 4 members…
• SHP-1, 2
• TCPTP
• PTP1b

Question 7

PIAS: Structure + Function, Key examples

Answer 7

• Bind to SH1 domains of STATs -> preventing DNA binding in nucleus
• 4 genes encoding 7 proteins
• PIAS1/STAT1
• PIAS3/STAT3
• PIASX/STAT4
• PIASY/STAT1

Question 8

CIS-SOCS family:

Answer 8

• Carry out long term regulation of cytokine signalling (particularly crucial in regulating inflammation)
• 8 related intracellular proteins (CIS and SOCs 1-7)
• Trancriptional targets of STATs (negative feedback)
• All have SH2 domains and SOCS box, some (1 and 3) also have a kinase inhibitory region
• SOCS1 is a tumour supressor

Question 9

Degradation mechanisms and inhibitory mechanisms of CIS-SOCS family:

Answer 9

Degradation:

• SH2 domain and SOCS box
• SH2 domain recognises pTyr on receptor
• Recruits E3 ubiquitin ligases to receptor complex -> Ub tags for degradation

Inhibition:

• Only members with KIR (1 and 3)
• SH2 domain binds pTyr on receptors to block JAK activity via KIR

Question 10

Outline the impacts of interleukin signalling in SCID:

Answer 10

• X-linked condition
• Severe combined immune deficiency
• IL-2 Rgamma defects (T and B cells) -> Burkitt’s lymphoma
• Promising cure using gene therapy -> issue of economics resulted in cessation of treatment

Question 11

Further examples of conditions due to defective interleukin/related cytokine signalling: (x5)

Answer 11

• Compromised immunity (INFyRI mutations)
• Crohn’s disease (inflammation of gut epithelial cells due to constitutive activation mutations of STAT3)
• Atopic asthma/dermatitis (hypersensitive reaction of immune cells due to altered SOCS3 functioning)
• Myeloproliferative disorders (JAK2 mutations)
• AML (IL-3a overexpression, STAT1 activation, inactivation of SOCS1, hyperactivation of JAK2)

Question 12

+ How can cytokine signalling can be utilised in therapy for anemic patients?

Answer 12

• Kidney dialysis -> anemia (low RBC count)
• Treat with recombinant Epo (binds surface of erythroid progenitor to stimulate RBC production)
• Epo and G-CSF can also be used as adjuncts in cancer therapy to boost RBC and granulocyte production

Question 13

+ IL-2 in X-linked SCID; possible therapy?

Answer 13

• Many cases of SCID are due to a deficiency of the IL-2 receptor gamma chain, on the X chromosome
• Lentiviral vector can be used (gene therapy) to introduce a functional gamma chain into HSCs -> immune cells can thus be generated

Question 14

+ Outline how protein phosphatases oppose cytokine signalling (give an example):

Answer 14

• Remove (hydrolyse) phosphates from specific phosphotyrosines on specific target proteins
• e.g. SHP1 negatively regulates signalling by several types of cytokine receptors; binds cytokine receptor and inactivates the associated JAK protein
• It contains two SH2 domains that typically block the catalytic site until stimulation occurs (i.e. recognition of the pTyr on activated receptor by SH2)

Question 15

+ Attributes of cytokine signalling:

Answer 15

• Pleiotropy
• Redundancy
• Synergism
• Antagonism
• Cascade induction