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Single Chain Antibodies as Tools to Study transforming growth factor--Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. YUMAB GmbH, Rebenring 33, D-38106 Braunschweig, Germany..
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
Tech Univ Carolo Wilhelmina Braunschweig, Inst Biochem Biotechnol & Bioinformat, Dept Biotechnol, Spielmannstr 7, D-38106 Braunschweig, Germany..
Tech Univ Carolo Wilhelmina Braunschweig, Inst Biochem Biotechnol & Bioinformat, Dept Biotechnol, Spielmannstr 7, D-38106 Braunschweig, Germany.;YUMAB GmbH, Rebenring 33, D-38106 Braunschweig, Germany..
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2016 (Engelska)Ingår i: Molecular & cellular proteomics (online), ISSN 1535-9476, E-ISSN 1535-9484, Vol. 15, nr 6, s. 1848-1856Artikel i tidskrift (Refereegranskat) Published
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Abstract [en]

The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF- signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF- signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF- signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA. Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling.

Ort, förlag, år, upplaga, sidor
2016. Vol. 15, nr 6, s. 1848-1856
Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Identifikatorer
URN: urn:nbn:se:uu:diva-299590DOI: 10.1074/mcp.M115.055756ISI: 000377822900006PubMedID: 26929218OAI: oai:DiVA.org:uu-299590DiVA, id: diva2:949709
Forskningsfinansiär
Knut och Alice Wallenbergs StiftelseEU, Europeiska forskningsrådet, 222635; 241481; 294409VetenskapsrådetVetenskapsrådet, NT-E0383401 MH-K2013-66x-14436-10-5Tillgänglig från: 2016-07-22 Skapad: 2016-07-22 Senast uppdaterad: 2017-11-28Bibliografiskt granskad

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Zieba, AgataMorén, AnitaChen, LeiSöderberg, OlaMoustakas, AristidisLandegren, Ulf

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Zieba, AgataMorén, AnitaChen, LeiSöderberg, OlaMoustakas, AristidisLandegren, Ulf
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Institutionen för immunologi, genetik och patologiMolekylära verktygLudwiginstitutet för cancerforskningInstitutionen för medicinsk biokemi och mikrobiologi
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Molecular & cellular proteomics (online)
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)

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