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Christine Flashcards

1
Q

Molecular signalling

A

The transmission of information, often from the external surface of a cell to the nucleus
Determines the biological output after the activation/engagement of receptors

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2
Q

How do cells signal?

A

Activation of intracellular proteins
Activation of second messengers
Protein-protein interactions
Enzymatic cascades

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3
Q

Mechanisms of signal transmittance

A

Conformational coupling (pre-formed complex) e.g. channels, receptors
Conformational coupling (diffusion-dependent complex formation) e.g. cAMP, cGMP, Ca, proteins
Post-translational modifications e.g. phosphorylation, acetylation, oxidation
Protein degradation e.g. ubiquitin-proteasome, proteases

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4
Q

How are signals measured?

A

Enzyme activity
Changes in gene expression
Fluorescent probe e.g. Ca mobilisation, phosphoinositides
Measure phosphorylated substances (Ab-based methods) e.g. immunoblotting, ELISA, flow cytometry

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5
Q

Key features of molecular signalling

A

Specificity (of response)
Coordination (of signals)
Location (within the cell)
Regulation (signal must be down-regulated)

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6
Q

Protein interaction domains

A

Sequence- and structural-specific regions within proteins that enable them to bind to/interact with other cellular molecules

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7
Q

SH2

A

Src homology 2 domain
Binds to phosphotyrosine residues via a ‘positively-charged pocket’
100 AA
Present in a diverse range of cytosolic proteins
High “off” rate - interactions are dynamic, rapid on/off
Has an extended surface that binds to residues at the C-terminal of the phosphotyrosine - variability in the AA sequence of this determines specificity
e.g. Src SH2 binds to pYEEI, Grb2 SH2 binds to pYVNV

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8
Q

PTB

A

Phosphotyrosine binding domain
Present in scaffolding proteins e.g. Shc, IRS, FRS2
Originally thought to bind NPXpY units, but Shc PTB can also bind to PIP2 and FRS2 PTB can bind to a non-phosphorylated FGFR ligand

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9
Q

14-3-3

A

Binds to phosphoserine
Dimer
Important in the regulation of DNA repair and apoptosis (can bind to Bad)

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10
Q

WD40

A

Involved in ubiquitination/degradation pathways and G1 to S phase transition

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11
Q

FHA

A

Forkhead-associated domain
Binds to phosphothreonine (pTXXD motif)
Important in DNA repair and protein trafficking

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12
Q

SH3

A 
Src homology 3 domain
Binds to proline-rich motifs e.g. XPnXP, where is a hydrophobic AA)
50 AA
3 pocket structure
Constitutive binding
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13
Q

WW

A

Binds to proline-rich motifs

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14
Q

EVHI

A

Binds to proline-rich motifs

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15
Q

PH

A

Pleckstrin homology domain
Binds to phospholipids/phosphoinositides e.g. PIP2, PIP3
“Protein-lipid interaction”
120 AA
Present in scaffolding proteins e.g. Gab, PKB, PDK1, kinases
Enables specific recruitment to the membrane
3 variable loops, positively charged surface

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16
Q

PDZ

A

Primarily involved in anchoring receptor proteins to the cytoplasm - plays a role in localising cellular elements
Binds to the C-terminals of target proteins via a specific motif

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17
Q

FYVE

A

Bind to PIP3

Regulate membrane traffic in endosomes

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18
Q

PX

A

Bind to phosphoinositides and SH3 domains

Diverse function

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19
Q

FRET

A

Fluorescence Resonance Energy Transfer
A mechanism describing the energy transfer between 2 light sensitive molecules
A donor fluorophore (in its electronically excited state) may transfer energy to an acceptor fluorophore through non-radiative dipole-dipole coupling
The efficiency of this energy transfer is inversely proportional to the 6th power of the distance between the fluorophores
Measurement of FRET efficiency can be used to determine if 2 fluorophores are within a certain distance of one another

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20
Q

Roles of protein phosphorylation

A

Control enzyme activity e.g. by changing enzyme conformation
Regulate interactions between proteins
Control protein localisation within the cell
Initiation, regulation and coordination of signalling pathways

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21
Q

JAK1 knockout

A

Perinatal lethality caused by impaired lymphopoiesis

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22
Q

JAK2 knockout

A

Embryonically lethal (no erythropoiesis)

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23
Q

JAK3 knockout

A

SCID (same as no common gamma chain)

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24
Q

STAT1 in cancer

A

Involved in IFNy response to infection
Tumour suppressor
STAT1 KO = no immune response, no apoptosis

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25
STAT3 in cancer
Required for the self-renewal of ESCs (recruited by LIF binding). KO = embryonically lethal Constitutively active in many tumours
26
STAT5 in cancer
Regulates apoptosis, promotes proliferation and cell cycle progression Mediator of BCR-Abl signalling - higher STAT5 levels seen in CML cells Constitutively phosphorylated/active in cancer cells
27
SOCS1-/- mice
Runted, die within 3 weeks due to liver degeneration Similar phenotype to mice that overexpress IFNy - because no SOCS1 = excessive IFNy, STAT1/6, IL-4 signalling Blocking IFNy or cross IFNy-/- mice with SOCS1-/- mice prevents disease
28
SOCS2-/- mice
Increased growth | Because SOCS2 negatively regulates cytokine receptors that drive growth
29
SOCS3-/- mice
Too many RBCs | SOCS3 regulates erythropoiesis
30
CIS-/- mice
Normal
31
SHP1
PTPN6
32
SHP2
PTPN11
33
Tetracycline-controlled transcriptional activation
A method of inducible gene expression, where transcription is reversibly turned on or off in the presence of tetracycline
34
Difference between Tet-Off and Tet-On inducible expression systems
Difference relates to their respective responses to tetracycline (not whether the transactivator turns a gene on or off) Tet-Off induces expression in the absence of tetracycline Tet-On induces expression in the presence of tetracycline
35
Plasmid system in tetracycline-controlled transcriptional activation
2 plasmid system Plasmid 1 = contains CMV promoter and TTa gene Plasmid 2 = contains TTa-dependent promoter and gene of interest
36
CMV promoter
Strong promoter, drives the constitutive expression of genes under its control
37
TTa
Tetracycline transactivator protein
38
TTa-dependent promoter
What TTa binds to Tet-Off system = TTa can only bind if it is not bound to tetracycline Tet-On system = TTa can only bind if it is bound to tetracycline
39
Mechanism of JAK/STAT inhibition by SOCS1
Binds to JAKs to inhibit their catalytic activity
40
Mechanism of JAK/STAT inhibition by SOCS3/CIS
Bind to cytokine receptors and block their association with other proteins
41
Mechanism of JAK/STAT inhibition by SOCS in general
Increase receptor degradation through recruitment of E3 ubiquitin ligase via SOCS box domain Bind to other signalling proteins e.g. IRS1
42
Yeast two-hybrid
Protein interaction technology A protein-fragment complementation assay, a method for the identification and quantification of protein-protein interactions
43
What is the premise behind the yeast two-hybrid assay?
The transcription of the reporter gene (GFP) relies on the binding of a TF to an UAS Y2H relies on the fact that most eukaryotic TFs are modular - they can be split into 2 parts and reconstituted non-covalently to form a functional TF However, the 2 fragments have low affinity for one another and must be brought together by interacting to which they are fused
44
Y2H method
TF split into 2 fragments: 1. DNA-binding domain (fused to target protein) 2. Activating domain (fused to potential binding partner of target protein) The fragments are introduced into yeast cells If the 2 proteins interact with one another, they will bring the BD and AD of the TF into close proximity, forming a functional transcriptional unit and activating/initiating transcription of the reporter gene
45
Protein microarrays
Can track large numbers of proteins in parallel Highly sensitive Automated
46
Similarities and differences between growth factor and cytokine receptors
``` Receptor chains Ligand Activation Signalling molecule recruitment Changes in disease ```
47
Ligand-induced activation of RTKs/cytokine receptors
The cytosolic domain of RTKs contains a protein tyrosine kinase catalytic site, whereas the cytosolic domain of cytokine receptors is closely associated with a separate JAK In both types of receptor, ligand binding causes a conformational change that promotes dimerisation, bringing together the intrinsic (RTK)/associated (cytokine) kinases The kinases phosphorylate each other on a Tyr residue in the activation lip, causing the lip to move out of the kinase catalytic site and allowing ATP/a protein substrate to bind Activated kinase then phosphorylates other Tyr residues in the receptor's cytosolic domain The resulting phosphotyrosines function as docking sites for signal transduction proteins with SH2/PTB domains
48
Structure of JAKs
H2N-receptor binding-pseudo kinase-kinase-COOH
49
Deletion of JAK pseudo kinase domain
Affects JAK function but currently unsure what the actual function of the domain is
50
How is the specificity of the JAK/STAT pathway determined?
Multiple cytokines activate the same JAK | The specificity of the response is determined by receptor recruitment of STATs
51
Mutations in JAK1/Tyk2
No response to IFNa/b
52
Mutations in JAK1/JAK2
No response to IFNy
53
JAK expression profiles
Jak1, Jak2, Tyk2 = ubiquitous | Jak3 = restricted expression to WBCs
54
Ser/Thr kinases
5 major families | Main families = MAPK, PKB
55
MAPK
Mitogen-activated protein kinases e.g. ERK1/2, JNK, p38 Evolutionarily conserved Part of phosphorelay system where a series of 3 protein kinases phosphorylate and activate one another (Raf --> MEK --> ERK) Activated by dual phosphorylation of Thr and Tyr "Proline-directed kinases" - phosphorylate PXS and TP motifs
56
Tyrosine phosphatases
Can be positive and negative regulators of molecular signalling 2 classes - transmembrane and cytoplasmic
57
Common features of tyrosine phosphatases
Phosphotyrosine-specific phosphohydrolase activity Conserved active site motif containing: Cys - required for catalysis, nucleophilic attack on P to remove phosphate group Asp - protonates reformed Tyr group after loss of phosphate Arg - stabilises pY in pocket so reaction can occur
58
Insulin receptor
RTK
59
Insulin receptor pathway
Phosphorylation of receptor Tyr residues generates a binding site for IRS that are then activated by phosphorylation Activated IRS1 can bind to/activate PI3K and Shc/Grb2 Both of these pathways lead to glucose/lipid metabolism
60
How is the insulin signalling pathway switched off?
By PTP1B | PTP1B also regulates leptin signalling
61
Leptin
Hunger hormone (inhibits hunger, generates feeling of satiety) Signals through JAK2/STAT3 Leptin-activated JAK2 is a substrate for PTP1B
62
SHP structure
2 tandem SH2 domains in N-terminus follows by a PTP domain SHP kept inactive by intramolecular binding of N-terminal SH2 to PTP domain (auto-inhibition) Inhibition relieved by binding of SH2 domain to a pY
63
Moth-eaten mice
Moth-eaten appearance as a result of spontaneous mutation in SHP-1 gene Mice die with 3 weeks of birth due to pneumonitis (lung inflammation due to massive macrophage infiltration) SHP-1 involved in regulation of STAT5 signalling
64
Discovering SHP-1 substrates
Use substrate-trapping mutants - want to bind substrate and phosphatase for long enough so that complex can be coimmunoprecipitated Cys --> Ser = no nucleophilic attack, catalytically dead, substrate is trapped Asp --> Ala = no protonation of leaving group so cannot dissociate, catalytically dead, substrate trapping Arg --> Met = substrate not stabilised in pocket
65
Catalytically inactive SHP-1
Increases IL-3-mediated cellular growth
66
SHP-2
Dual function: phosphatase and adaptor molecule with no phosphatase activity
67
Investigating catalytic activity of SHP-2
Cys --> Ser mutant is catalytically inactive and substrate trapping, reduces activation of MAPKs by many stimuli It is believed that SHP-2 acts as an adaptor molecule to activate the pathway then dissociates and carries out its phosphatase activity to down-regulate the pathway

Molecular Signalling (6 decks)

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