PEPTIDE THERHOLD SCAFFOLD MEANING SOFTWARE

The microarray slides with AXIN1 peptide library were incubated with the following purified recombinant proteins: CK1Пµ ( A), c-Myc ( B), p53 ( C), and Pin1 ( D). 2, AвЂ“D).ДЇigure 2 AXIN1-binding epitopes for CK1Пµ, c-Myc, Pin1, and p53 identified by peptide array approach.

All the remaining binding sites of the aforementioned candidate proteins were located in the central IDR of AXIN1 ( Fig. 2 B) as they were located predominantly neither on the surface nor on the вЂњlinearвЂќ О±-helix (see вЂњExperimental proceduresвЂќ for more details about quantification). Peptides mapping to the DIX domain were not considered as putative binding sites of c-Myc (see Fig. However, this information can be still relevant because the epitopes identified within the RGS domain were located on the О±-helices and exposed to the solvent. All tested proteins showed interaction with at least one of two linear peptides forming solvent-exposed О±-helix (aa 133вЂ“152 and aa 154вЂ“175 in human AXIN1) from the RGS domain here, however, the interpretation is not so straightforward because the linear peptides may not truly recapitulate the situation in the folded RGS domain. S2) revealed that each interaction partner of AXIN1 can efficiently interact with at least three linear peptide motifs. These candidates were chosen as the representative AXIN1-binding partners from individual signaling pathways ( Fig. To identify the peptide epitopes required for direct interaction with AXIN1, the following candidates were used for a peptide microarray experiment: CK1Пµ, c-Myc, p53, and Pin1.

PEPTIDE THERHOLD SCAFFOLD MEANING SOFTWARE

The signal was subsequently detected by a high-resolution fluorescence scanner and analyzed using spot-recognition software (schematized in Fig. To perform a single peptide microarray experiment, the glass slide with immobilized peptides was incubated with the recombinant protein of interest followed by antibody staining and visualization using a fluorescence marker. S1) that were immobilized via a linker molecule on a microscopic slide. We designed a peptide microarray containing 13-mer peptides overlapping by 10 residues covering the entire human AXIN1 sequence ( Fig. To understand better how AXIN1 achieves this, we decided to map binding epitopes within the IDRs of AXIN1 with high resolution. It is obvious, just simply for steric reasons, that only a very limited number of proteins can be associated with the AXIN1 scaffold at the same time, and it is likely that one AXIN1 complex is dedicated to only one biological function. Not surprisingly, these proteins regulating distinct biochemical processes bind to the central IDR of AXIN1. The binding regions for biologically very important AXIN1 interaction partners, namely CK1Пµ, c-Myc, p53, and Pin1, have been mapped earlier by deletion mutagenesis (for schematics see Fig. On the basis of these results, we propose a model in which AXIN1 competes with dishevelled (DVL) for CK1Пµ and regulates CK1Пµ-induced phosphorylation of DVL and activation of Wnt/ОІ-catenin signaling. We functionally validated the specific AXIN1вЂ“CK1Пµ interaction identified here with epitope-mimicking peptides and with AXIN1 variants having deletions of short binding epitopes. casein kinase 1 Пµ (CK1Пµ) c-Myc peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1 (Pin1) and p53, we found at least three different epitopes, predominantly in the central IDR of AXIN1. For each of the AXIN1-binding partners tested, i.e. Here, we report the use of a peptide microarray based on the human scaffold protein AXIN1 for high-throughput and -resolution mapping of binding sites for several AXIN1 interaction partners in vitro. This approach either provides information of low resolution (large deletions) or very laboriously tries to precisely define the binding epitope via single amino acid substitutions.

The classical approach for identification of these interaction interfaces is based on mutagenesis combined with biochemical techniques such as coimmunoprecipitation or yeast two-hybrid screening. IDRs represent >40% of the eukaryotic proteome and play a crucial role in proteinвЂ“protein interactions. Intrinsically disordered regions (IDRs) are protein regions that lack persistent secondary or tertiary structure under native conditions. Glycobiology and Extracellular Matrices.

Peptide therhold scaffold meaning