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LIBRIS Formathandbok  (Information om MARC21)
FältnamnIndikatorerMetadata
00005305naa a2200421 4500
001oai:DiVA.org:uu-299590
003SwePub
008160722s2016 | |||||||||||000 ||eng|
0247 a https://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-2995902 URI
0247 a https://doi.org/10.1074/mcp.M115.0557562 DOI
040 a (SwePub)uu
041 a engb eng
042 9 SwePub
072 7a ref2 swepub-contenttype
072 7a art2 swepub-publicationtype
1001 a Blokzijl, Andriesu Uppsala universitet,Institutionen för immunologi, genetik och patologi,YUMAB GmbH, Rebenring 33, D-38106 Braunschweig, Germany.4 aut
2451 0a Single Chain Antibodies as Tools to Study transforming growth factor--Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens
264 1c 2016
338 a print2 rdacarrier
520 a 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.
650 7a MEDICIN OCH HÄLSOVETENSKAPx Medicinsk bioteknologix Medicinsk bioteknologi0 (SwePub)304012 hsv//swe
650 7a MEDICAL AND HEALTH SCIENCESx Medical Biotechnologyx Medical Biotechnology0 (SwePub)304012 hsv//eng
7001 a Zieba, Agatau Uppsala universitet,Molekylära verktyg4 aut0 (Swepub:uu)agazi628
7001 a Hust, Michaelu Tech Univ Carolo Wilhelmina Braunschweig, Inst Biochem Biotechnol & Bioinformat, Dept Biotechnol, Spielmannstr 7, D-38106 Braunschweig, Germany.4 aut
7001 a Schirrmann, Thomasu 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.4 aut
7001 a Helmsing, Saskiau Tech Univ Carolo Wilhelmina Braunschweig, Inst Biochem Biotechnol & Bioinformat, Dept Biotechnol, Spielmannstr 7, D-38106 Braunschweig, Germany.4 aut
7001 a Grannas, Karinu Uppsala universitet,Institutionen för immunologi, genetik och patologi4 aut0 (Swepub:uu)kargr691
7001 a Hertz, Ellenu Uppsala universitet,Institutionen för immunologi, genetik och patologi4 aut
7001 a Morén, Anitau Uppsala universitet,Ludwiginstitutet för cancerforskning4 aut0 (Swepub:uu)anitmore
7001 a Chen, Leiu Uppsala universitet,Molekylära verktyg4 aut0 (Swepub:uu)leich344
7001 a Söderberg, Olau Uppsala universitet,Molekylära verktyg4 aut0 (Swepub:uu)olasoder
7001 a Moustakas, Aristidisu Uppsala universitet,Institutionen för medicinsk biokemi och mikrobiologi,Ludwiginstitutet för cancerforskning4 aut0 (Swepub:uu)arimo287
7001 a Dubel, Stefanu Tech Univ Carolo Wilhelmina Braunschweig, Inst Biochem Biotechnol & Bioinformat, Dept Biotechnol, Spielmannstr 7, D-38106 Braunschweig, Germany.4 aut
7001 a Landegren, Ulfu Uppsala universitet,Molekylära verktyg4 aut0 (Swepub:uu)ulfland
7102 a Uppsala universitetb Institutionen för immunologi, genetik och patologi4 org
7730 t Molecular & Cellular Proteomicsg 15:6, s. 1848-1856q 15:6<1848-1856x 1535-9476x 1535-9484
8564 8u https://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-299590
8564 8u https://doi.org/10.1074/mcp.M115.055756

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