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B301 > L7: TGF-B 1 > Flashcards

L7: TGF-B 1 Flashcards

1
Q

TGF-beta ligands: Overview of role and family

A
  • TGF-beta 1, 2 and 3 in humans
  • Part of a large superfamily of cytokines including TGF-betas, activins, nodal, BMPs etc (connected by a large degree of structural homology)
  • Control gene expression, especially in development and morphogenesis
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2
Q

Pleiotropic role of TGF-beta in humans:

A
  • Mainly inhibiting proliferation
  • Can also control wound healing, ECM formation, apoptosis, differentiation and immunosuppression
  • Affecting expression of caretaker, gatekeeper and landscaper genes
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3
Q

TGF-beta structure and expression:

A
  • Ubiquitous; one or more isoforms expressed in most cells
  • Synthesised as precursor proteins -> sequestered in E-C matric where inactive form binds ECM components
  • Provides a reservoir for rapid signalling events
  • Very stable structure due to cysteine knots (3x disulphide bonds) which are hard to denature
  • Other key domains: LAP (Pro-domain), LSKL/RPKP in LAP and TGFB respectively which bind together, finger turn domains (beta sheets able to interact with receptor)
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4
Q

How are TGF-betas activated?

A
  • Pro-protein: prodomain and TGF-beta domain
  • Cleaved by proteolysis (e.g. furin)
  • LAP dimers and prodomain dimers formed (disulphide bridges)
  • LTBP binds prodomain
  • Thrombospondin binds LSKL sequence
  • To activate, TSP-1 competitively inhibits for prodomain -> proteolysis -> mature dimeric ligand formed
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5
Q

TGFB-RI vs TGFB-RII:

A
  • Both TM homodimers
  • RI: Inactive until ligand binds
  • RII: constitutively active kinase activity
  • Both serine/threonine kinases
  • TGB-RIII specifically required for TGFB2 signalling, increasing the local concentration of ligand
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6
Q

Receptor binding in TGF-beta pathway:

A
  • Constitutively active RII recruits TGFB dimer
  • Stimulates recruitment of RI dimer to complex
  • RII transphosphorylates RI (at GSGS sequence in GS domain; Ser165 crucial) -> determines intensity of signalling
  • Conformational change in RI displaces immunophilin (FKBP12 site)
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7
Q

What are Smads?

A
  • Mediate signal transduction from receptor to nucleus
  • R-smads: Receptor -> Smads 2 and 3 which can be phosphorylated
  • Co-smads: common mediator aka Smad 4 -> not phosphorylated
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8
Q

Smad signal transduction: Nuclear translocation

A
  • MH2 domain of Smad2/3 binds P-ser (adjacent to kinase domain of RI) -> phosphorylation by RI at MH2
  • -> dissociation of MH1 and MH2 domain within molecule, revealing the nuclear localisation sequence (NLS)
  • MH1/NLS able to bind importin-beta
  • Simultaneously, MH2 of Smad4 binds pMH2 domain of Smad2/3 and association of a second Smad 2/3 at MH2
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9
Q

Smad signalling within nucleus:

A
  • Smad/importin complex encounters Ran GTP
  • When activated, Ran binds / displaces importin-B from complex
  • Complex then able to bind TFs
  • MH1 domains of Smads and TF recognise DNA target sequence
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10
Q

How is specificity conferred in Smad signalling within the nucleus:

A
  • Co-activators and co-repressors also bind smads -> control accessibility of DNA (HATs/HDACs)
  • The right sequences / order / spacing in smads confers specificity of regulation
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11
Q

Smad structure:

A
  • MH1, linker and MH2 domains
  • NLS (binds imp B) and DNA binding part of MH1
  • Other binding sites: Smurf (at PPXY in linker) and SARA (Smad anchor of receptor activation)
  • Smurfs: E3 ubiquitin ligases
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12
Q

Receptor internalisation in TGF-beta signalling;

A
  • TGF-beta receptors continuously internalise and recycle
  • Smad signalling requires internalisation by clathrin-mediated endocytosis
  • They can, however, enter caveolae
  • Here, SARA interacts with PLs in PM and stabilises Smad 2/3 binding to complex
  • Targets complex to CCPs
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13
Q

+ Example of TF that Smads interact with and impact on gene expression (bone formation)

A
  • Runx 2 transcription factor
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14
Q

+ How are TGFB-receptors post translationally modified?

A
  • Phosphorylation -> Smad activation, MAPK pathways
  • Ubiquitylation -> Receptor degradation
  • Sumoylation -> Facilitating Smad activation
  • (All enzymatically reversible)
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15
Q

+ How might receptor PTMs facilitate MAPK activation?

A
  • TGFBRII phosphorylation by Src (on tyrosine)
  • Enables TGBRII to recruit Grb2 and Shc through their SH2 domains
  • TGFB-induced activation o fp38 mitogen-activated proteins kinase pathway (MAPK)
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16
Q

+ How are Smurfs activated?

A
  • N-terminal domain of Smad7 stimulates activity of Smurf1 or Smurf2 by recruiting the ubiquitin-conjugating E2 enzyme ‘UbcH7’ to the HECT domain of the E3 ligase (smurf)
17
Q

+ What is sumoylation?

A
  • Covalent attachment of a SUMO polypeptide that resembles a ubiquitin
  • Process requires sequential activation of E1, E2 and E3 SUMO ligases
  • Regulates subcellular localisation of perinuclear and nuclear proteins (i.e. TFs)
18
Q

+ How is ubiquitylation-induced degradation inhibited in TGFBRI?

A
  • UCH37 (deubiquitylation enzyme) associates
  • Site is distinct from Smurf interacting motif (PY)
19
Q

+ Additional function of iSmads beyond direct inhibition:

A
  • Acting as a scaffold for recruitment for further inhibitory factors
  • e.g. PP1 (via GADD34 subunit) -> dephosphorylation and subsequent inactivation of TGFBRI
  • Also recruit HECT-type E3 ubiquitin ligases (Smurfs are a member of this family) -> degradation via ubiquitination
20
Q

+ What is the normal role of Dapper proteins? Extra role in TGFB signalling?

A
  • First identified as suppressors or activators of Wnt signalling
  • Dapper2 (Dpr2) is able to inhibit TGFB and nodal signalling by binding type I receptors and promoting degradation in lysosomes (not via ubiquitylation)
21
Q

+ Purported role of WD-repeat containing proteins on TGFB signalling (name 2):

A
  • Several WD-repeat containing proteins have been implicated as negative regulators of TGFB signalling
  • e.g. STRAP
  • e.g. B-alpha subunit of PP2A
22
Q

+ Purported role of FYVE-domain containing proteins on TGFB signalling (name 2):

A
  • Facilitating Smad interactions with receptors and activation
  • e.g. SARA (stabilises Smad2, 3 binding to receptor complex; also directs the activated receptor to clathrin-mediated endocytosis)
  • e.g. Endofin can interact with Smad1 (as in BMP signalling); possibly fulfilling a similar role to SARA. Thought to have multiple complex interactions since it also interacts with type I receptors and Smad4
23
Q

+ Give 2 examples of non-Smad signalling by TGFB with physiological effects:

A
  • TGBRI-induced, dissociation of tight junctions -> Par6-Smurf1 complex forms, confers ubiquitylation of RhoA -> key role in EMT
  • Activation of MAPK signalling (typically weaker than that of the Smad leg of the pathway) (requires association of Grb2 and pShc)
24
Q

+ What are the 2 routes for endocytosis of TGFB-bound TGFBR complexes?

A
  • Clathrin-mediated endocytosis
  • Caveolin-1 and lipid-raft mediated-endocytosis
25
Q

+ Outline clathrin-mediated endocytosis (key domain):

A
  • Receptors internalised constitutively through clathrin coated pits
  • Mediated by a di-leucine internalisation motif in TBRII cytoplasmic domain
  • They then enter EEA-1 and Rab5-positive early endosomes
  • Able to recycle back to PM (Rab11 dependent)

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