Protein Interaction Domains Flashcards
Using a specific study to illustrate your answer, discuss how a particular protein interaction technique has aided our understanding of signalling pathways
- What is FRET as a protein interaction technique? (general; process; advantages) (1) (1/6)
- distance-dependent
- energy is transferred non-radiatively from an excited molecular fluorophore (donor) to an acceptor fluorophore
- by intramolecular long-range dipole-dipole coupling
- widely used fluorophores = class of auto-fluorescent proteins “GFP”
- technique develops on prior studies (LM=cellular structures & limited functions) b/ FRET = ID cellular events & specific transduction pathways (that req assembly of protein complexes)
- intensity-based imaging = study of interactions inside living cells
- new imaging technologies & computer software analysis = protein functions & interactions
- if RET occurs, the donor signal is quenched & acceptor signal is sensitised & increased
- allows co-localisation of the donor- & acceptor-labelled probes to be seen = verification of interactions that occur @ close proximities (1-10 nm)
- several FRET microscopy tech = available, each optimised to the study of diff interactions
Using a specific study to illustrate your answer, discuss how a particular protein interaction technique has aided our understanding of signalling pathways
- Where has FRET aided our understanding of signalling pathways? (GFP; FRET-FILM; RASSF-MST) (1) (2/6)
- GFP spectral mutants w/ mut that inf the fluorescent prop of GFP by altering interactions b/w the chromophore & it’s enviro
- can be optimised & used for PI-studies i.e., CFP/YFP pair
- FRET-FILM (combination of 2 tech)
- FILM: monitors enviro by altering lifetime of the molecules
- FRET: monitors direct interaction of 2 fluorophores when they are in close proximity
- advantages of F-F = only req donor fluorescence lifetime to be measured - when in presence of acceptor, donor lifetime = shorter (b/c RET = happened)
- tech was exploited to inv interactions b/w Ras-association domain family (RASSF) & mammalian sterile 20-like kinases (MST);they provide a Kd for the interaction
- MSTs: major inhibitor of prolif (MST-1), inv in apoptosis, oncogenesis, & organ growth (via its ser-thr kinase)
- RASSF: 10 members, have a common Ras-association domain & SARAH domain - potential to bind Ras oncoproteins & mediate p-p int w/ other SARAH-DCP
- RASSF p = components of the MST/Hippo pathway - ≈ restrict proliferation, expected to play role in tumorigenesis
- ≈ oppose Raf/Mek/Erk stimulation of cell growth/prolif that also dep on Ras activation
Using a specific study to illustrate your answer, discuss how a particular protein interaction technique has aided our understanding of signalling pathways
- Where has FRET aided our understanding of signalling pathways? (study; constructs; cells) (1) (3/6)
- Margineanu et al., (2016) made fluorescent constructs (to assay RASSF-MST interaction; FRET)
- 10 RASSF proteins = modified by + EFGP to the N-T (donor)
- MST1 kinase & it’s isolated SARAH domain (SARAH_MST1) = labelled w/ mCherry @ N-T (acceptor)
- negative controls: evaluate FXs of non-specific int on FRET readouts
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COS7 cells = transfected w/ constructs
- cells = fibroblast morphology
- used for transfection XPMs - analyse actions of functional molecules
Using a specific study to illustrate your answer, discuss how a particular protein interaction technique has aided our understanding of signalling pathways
- Where has FRET aided our understanding of signalling pathways? (conditions; EGFP lifetime findings) (1) (4/6)
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dimerisation of RASSF w/ SARAH_MST1 domain using FRET:
- box plots: median EGFP lifetimes for each COS7 cell, averaged over 10 films of view using monoexponential analysis
- done in 3 conditions
1) EGFP-RASSF_1-10 only
2) EGFP-RASSF_1-10 + mCherry-SARAH_MST1
3) EGFP-RASSF_1-10 + mCherry-MST1ΔSARAH (≠SARAH domain) - RASSF_1-6: EGFP lifetimes (ps) = ↓when 2nd construct used (interaction ✓)
- RASSF_7-10: EGFP lifetimes was similar in all 3 constructs
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co-transfection of RASSF & SARAH_MST1 (1:1) average EGFP lifetime = ↓by 130-310 (ps) for RASSF_1-6
- for RASSF_7-10, reduction in mean lifetime = < 32 ps ≈ little/no interaction
- results suggest = specific interactions, based on dimerisation of SARAH domains b/w 1-6 & MST1
- little/no interaction b/w SARAH domain and 7-10 (≈ b/c these RASSFs = coiled-coil/unstructured C-T, ≠ a SARAH domain)
Using a specific study to illustrate your answer, discuss how a particular protein interaction technique has aided our understanding of signalling pathways
- Where has FRET aided our understanding of signalling pathways? (biochemical data; PM-RASSF) (1) (5/6)
- group also provided biochemical data that supported results
- most of interacting residues of SARAH_RASSF1-6 monomers = well-conserved & aligned to heterodimerise w/ SARAH_MST1 monomer
- contact interface involves side chains & n/polar residues for all 6 heterodimers that form
- also some polar/charged int b/w acidic/basic residues of each prot
- b/c of this, group chose 3 key n/polar residues (main helix) for mutational studies.
- residues align to L444, 448, 451 in SARAH_MST1 (highly conserved; dimer formation & stability)
- L → P = kinks to a-helices (≠ dimerise w/ SARAH_MST1 domain)
- FRET assays showed results of interaction b/w isolated WT-SARAH_MST1 domain and PM-RASSF1/5 (2 of the best characterised RASSF prot in lit)
- average EGF lifetimes from all 3 PM-RASSF showed ablation of interaction (≠ dimerisation)
- PM-RASSF5 showed reduction in average EGFP lifetime compared to -ive controls; dimerisation still occurs
- reduction of mean EGFP lifetime in cells XPSing mutants = smaller than WT-RASSF5; fraction of bound molecules = reduced ≈ reduced binding affinity/surrounding residues around mutation compensate
- current data = RASSF may = an adaptor for assembly of multiple protein complexes
- facilitates further functional interactions that involve MST kinases & other SARAH-DCP, wh/ could be regulated by Ras.
Using a specific study to illustrate your answer, discuss how a particular protein interaction technique has aided our understanding of signalling pathways
- Implication in disease (Ras effectors; methylation) (1) (6/6)
- Ras proteins ≈ direct causal role in human cancer w/ activating mut in Ras occurring in ~30% of tumours
- RASSF = Ras effectors
- RASSF1a = Isoform of RASSF1 = silenced in tumours
- RASSF members ≈ function as tumour suppressors by reg cell cycle & apoptosis
- investigating dimerisation & interactions w/ partners may reveal something about dysregulation
- RASSF1 promotor methylation silences it in cancer
- loss of RASSF1A XPSion = 1 of most common events in cancer
- aberrant promotor methylation reported in ~37 tumour types (breast, cervical, head & neck, colon)
- RASSF1A methylation freq = higher in cancer cell lines vs primary tumours
Using a specific example to illustrate your answer, discuss the use of specific molecular techniques in investigating and/or modelling a dysregulated molecule implicated in the development of a disease.
Molecular techniques (crystal structures; MSP:RON interaction; pathways) (2) (1/4)
- crystal structure analysis was used to confirm that:
- 1 MSP molecules interacts with the SEMA domain of 2 RON molecules
- induces dimerisation & activation
- activates Akt & Erk1/2 pathways
Using a specific example to illustrate your answer, discuss the use of specific molecular techniques in investigating and/or modelling a dysregulated molecule implicated in the development of a disease.
What they found about the dysregulated molecule (interaction; cell-specific changes caused by pathway activation) (2) (2/4)
Interaction
- β chain of MSP binds to TM protein (RON), via RONs SEMA domain
- 1 MSP: 2 RON = dimerisation & activation of Akt & Erk1/2 pathways
Behavioural changes
- Macrophages:
- activation of Akt wh/ promotes phagocytosis of complement C3-bicoated RBC via CR3 & ICAM-1 (XPS’ed on macro/monocytes/NK cells)
- Epstein-Barr virus infection
- Latent Membrane Protein-1 (oncoprotein) promotes binding of NF-kB to the RON promotor = ↑ RON XPS’ion in B-cells → tumour cell prolif (more RON to activate ∴ its downstream pathways = activated more too)
- STK/RON transfected Pro-B-cells
- prolif when stimulated by MSP
- STK/RON transfected mouse erythroleukemia cells
- apoptosis when stimulated
Using a specific example to illustrate your answer, discuss the use of specific molecular techniques in investigating and/or modelling a dysregulated molecule implicated in the development of a disease.
Involvement in disease (MSP; RON; M-R signalling in chronic inflammation; cancer) (2) (3/4)
- Macrophage-stimulating protein (MSP) = soluble protein made mostly in the liver
- only known ligand for receptor d’origine Nantais (RON)
- RON (memb of the MET proto-oncogene family of RTKs)
- activation of RON in macro results in inhibition of NO synthesis & elicits LPS-induced inflam resp (severe consequences if over-stimulated)- aberrant RON XPS’ion & activation exists in various cancer types
- MSP-RON sig may be involved in cancer and immune system disease, b/c ass w/ chronic inflamm resp ∴ may be a novel regulator of inflammation
- MSP-RON (M-R) axis involvement in inflammation
- M-R activates PI3K/Akt pathway → inhibits XPS’ion of NO-synthase
- LPS-induced production of NO strongly inhibits mRNA & protein XPS’ion of RON in mouse peritoneal macrophages
- This is an auto-inhibition: inflammation doesn’t get out of control by too much RNS production b/c other immune cells respond to NO (T-cells, APC, neutrophils, mast- and NK-cells) - reg immune & inflam resp
- M-R activates LKB1-AMPK pathway → induces SHP TC → inhibits TRAF6 poly-Ub → inhibits TLR signalling
- ↑ SHP XPS’ion inhibits assembly of NLRP3 inflammasome & maturation of IL-1β
- Inflammasome: large cytosolic multi-protein complexes that assemble in resp to infection/stress-ass stimuli
- leads to activation of caspase-1 mediated inflam resp → cleavage & secreting of pro-inflam cytokines (IL-1β/-18)
- causes induction of pyroptosis (inflam form of cell death) to defend against infection
- RON XPS’ion ≈ marker of terminal differentiation of resident macrophages
- another example of how changes are cell-specific
- breast cancer bone metastasis:
- breast cancer cells secrete more MSP
- M-R pathway = activated in osteoclasts (bone-T-resident macrophages)
- induces bone destruction, permissive of metastasis
Using a specific example to illustrate your answer, discuss the use of specific molecular techniques in investigating and/or modelling a dysregulated molecule implicated in the development of a disease.
Molecular techniques used (expression techniques; immunohistochemical analyses; phosphotyrosine assays; western blotting) (2) (4/4)
Expression
- aberrant RON activation achieved via over-XPS’ion of WT RON through generation of active isoforms i.e., RONΔ160 & RONΔ155 (latter was discovered in the specific study)
- persistent activation of downstream pathways
- naturally occurring isoforms
- have cell-transforming & tumour growth activities
Immunohistochemical analyses
- used to detect RON XPS’ion in adenocarcinoma samples, using anti-RON ab that recog different regions of RON
Phosphotyrosine assays
- determine if RON is constitutively active in vivo
- cellular proteins from frozen tumour samples were tested
- RON was immunoprecipitated with mAB, ID2, & detected in western blotting with mAB, 4G10, to pY
- in all samples pRON was detected
- indicates that increased RON XPS’ion in certain cancers is accompanied by constitutive phos in vivo
Western blotting
- showed that RON was XPS’ed in all 11 samples tested
- mostly an 180 kDa single-chain pro-RON and an 145 kDa RON β-chain
- in certain cancer samples, 3 proteins were detected, the third was also not present in control samples
- 3-different size proteins are likely produced by certain cancer cell lines
- variants were cloned using RT-PCR using total RNA isolated from tumour samples
- discovery of a novel RON variant that had not been ID before - RONΔ155
- splicing of RON mRNA @ different regions occurs in different cancers, resulting in expression of different RON variants that are constitutively active
