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Statements

Subject Item
n2:RIV%2F00209805%3A_____%2F13%3A%230000420%21RIV14-GA0-00209805
rdf:type
skos:Concept n16:Vysledek
rdfs:seeAlso
http://onlinelibrary.wiley.com/doi/10.1002/pro.2299/abstract;jsessionid=F8B1804D27F005381295F370CA40BB77.f04t04
dcterms:description
Many regulatory proteins are homo-oligomeric and designing assays that measure self-assembly will provide novel approaches to study protein allostery and screen for novel small molecule modulators of protein-interactions. We present an assay to begin to define the biochemical determinants that regulate dimerization of the cancer-associated oncoprotein AGR2. A two site-sandwich microtiter assay (2S MTA) was designed using a DyLight800-labeled monoclonal antibody that binds to an epitope in AGR2 to screen for synthetic self-peptides that might regulate dimer stability. Peptides derived from the intrinsically disordered N-terminal region of AGR2 increase in trans oligomer stability as defined using the 2S MTA assay. A DSS-crosslinking assay that traps the AGR2 dimer through K95-K95 adducts confirmed that Δ45-AGR2 was a more stable dimer using denaturing gel electrophoresis. A titration of wt-AGR2, Δ45-AGR2 (more stable dimer), and monomeric AGR2E60A revealed that Δ45-AGR2 was more active in binding to Reptin than either wt-AGR2 or the AGR2E60A mutant. Our data have defined a functional role for the AGR2 dimer in the binding to its most well characterized interacting protein, Reptin. The ability to regulate AGR2 oligomerization in trans opens the possibility for developing small molecules that regulate its' biochemical activity as potential cancer therapeutics. The data also highlight the utility of this oligomerization assay to screen chemical libraries for ligands that could regulate AGR2 dimer stability and its' oncogenic potential. Many regulatory proteins are homo-oligomeric and designing assays that measure self-assembly will provide novel approaches to study protein allostery and screen for novel small molecule modulators of protein-interactions. We present an assay to begin to define the biochemical determinants that regulate dimerization of the cancer-associated oncoprotein AGR2. A two site-sandwich microtiter assay (2S MTA) was designed using a DyLight800-labeled monoclonal antibody that binds to an epitope in AGR2 to screen for synthetic self-peptides that might regulate dimer stability. Peptides derived from the intrinsically disordered N-terminal region of AGR2 increase in trans oligomer stability as defined using the 2S MTA assay. A DSS-crosslinking assay that traps the AGR2 dimer through K95-K95 adducts confirmed that Δ45-AGR2 was a more stable dimer using denaturing gel electrophoresis. A titration of wt-AGR2, Δ45-AGR2 (more stable dimer), and monomeric AGR2E60A revealed that Δ45-AGR2 was more active in binding to Reptin than either wt-AGR2 or the AGR2E60A mutant. Our data have defined a functional role for the AGR2 dimer in the binding to its most well characterized interacting protein, Reptin. The ability to regulate AGR2 oligomerization in trans opens the possibility for developing small molecules that regulate its' biochemical activity as potential cancer therapeutics. The data also highlight the utility of this oligomerization assay to screen chemical libraries for ligands that could regulate AGR2 dimer stability and its' oncogenic potential.
dcterms:title
Development of a fluorescent monoclonal antibody-based assay to measure the allosteric effects of synthetic peptides on self-oligomerization of AGR2 protein Development of a fluorescent monoclonal antibody-based assay to measure the allosteric effects of synthetic peptides on self-oligomerization of AGR2 protein
skos:prefLabel
Development of a fluorescent monoclonal antibody-based assay to measure the allosteric effects of synthetic peptides on self-oligomerization of AGR2 protein Development of a fluorescent monoclonal antibody-based assay to measure the allosteric effects of synthetic peptides on self-oligomerization of AGR2 protein
skos:notation
RIV/00209805:_____/13:#0000420!RIV14-GA0-00209805
n16:predkladatel
n17:ico%3A00209805
n3:aktivita
n15:P
n3:aktivity
P(ED2.1.00/03.0101), P(GAP301/11/1678)
n3:cisloPeriodika
9
n3:dodaniDat
n14:2014
n3:domaciTvurceVysledku
n6:5395488 Murray, Euan n6:9637419
n3:druhVysledku
n18:J
n3:duvernostUdaju
n4:S
n3:entitaPredkladatele
n9:predkladatel
n3:idSjednocenehoVysledku
69076
n3:idVysledku
RIV/00209805:_____/13:#0000420
n3:jazykVysledku
n13:eng
n3:klicovaSlova
oligomerization; allostery; protein interactions; monoclonal antibody
n3:klicoveSlovo
n5:oligomerization n5:monoclonal%20antibody n5:allostery n5:protein%20interactions
n3:kodStatuVydavatele
US - Spojené státy americké
n3:kontrolniKodProRIV
[A3CAFE2C109C]
n3:nazevZdroje
Protein Science
n3:obor
n20:EB
n3:pocetDomacichTvurcuVysledku
3
n3:pocetTvurcuVysledku
8
n3:projekt
n7:GAP301%2F11%2F1678 n7:ED2.1.00%2F03.0101
n3:rokUplatneniVysledku
n14:2013
n3:svazekPeriodika
22
n3:tvurceVysledku
Vojtěšek, Bořivoj Murray, Euan Müller, Petr
n3:wos
000323410100011
s:issn
0961-8368
s:numberOfPages
13
n12:doi
10.1002/pro.2299