Flashlight into the Function of Unannotated C11orf52 using Affinity Purification Mass Spectrometry.

For an enhanced understanding of the biological mechanisms of human disease, it is essential to investigate protein functions. In a previous study, we developed a prediction method of gene ontology (GO) terms by the I-TASSER/COFACTOR result, and we applied this to uPE1 in chromosome 11. Here, to validate the bioinformatics prediction of C11orf52, we utilized affinity purification and mass spectrometry to identify interacting partners of C11orf52. Using immunoprecipitation methods with three different peptide tags (Myc, Flag, and 2B8) in HEK 293T cell lines, we identified 79 candidate proteins that are expected to interact with C11orf52. The results of a pathway analysis of the GO and STRING database with candidate proteins showed that C11orf52 could be related to signaling receptor binding, cell-cell adhesion, and ribosome biogenesis. Then, we selected three partner candidates of DSG1, JUP, and PTPN11 for verification of the interaction with C11orf52 and confirmed them by colocalization at the cell-cell junctions by coimmunofluorescence experiments. On the basis of this study, we expect that C11orf52 is related to the Wnt signaling pathway via DSG1 from the protein-protein interactions, given the results of a comprehensive analysis of the bioinformatic predictions. The data set is available at the ProteomeXchange consortium via PRIDE repository (PXD026986).

Bioinformatic Prediction of Gene Ontology Terms of Uncharacterized Proteins from Chromosome 11.

In chromosome 11, 71 out of its 1254 proteins remain functionally uncharacterized on the basis of their existence evidence (uPE1s) following the latest version of neXtProt (release 2020-01-17). Because in vivo and in vitro experimental strategies are often time-consuming and labor-intensive, there is a need for a bioinformatics tool to predict the function annotation. Here, we used I-TASSER/COFACTOR provided on the neXtProt web site, which predicts gene ontology (GO) terms based on the 3D structure of the protein. I-TASSER/COFACTOR predicted 2413 GO terms with a benchmark dataset of the 22 proteins belonging to PE1 of chromosome 11. In this study, we developed a filtering algorithm in order to select specific GO terms using the GO map generated by I-TASSER/COFACTOR. As a result, 187 specific GO terms showed a higher average precision-recall score at the least cellular component term compared to 2413 predicted GO terms. Next, we applied 65 proteins belonging to uPE1s of chromosome 11, and then 409 out of 6684 GO terms survived, where 103 and 142 GO terms of molecular function and biological process, respectively, were included. Representatively, the cellular component GO terms of CCDC90B, C11orf52, and the SMAP were predicted and validated using the overexpression system into 293T cells and immunofluorescence staining. We will further study their biological and molecular functions toward the goal of the neXt-CP50 project as a part of C-HPP. We shared all results and programs in Github (https://github.com/heeyounh/I-TASSER-COFACTOR-filtering.git).