Abstract
Experimental approaches that enable direct investigation of human protein function are necessary for comprehensive annotation of the human proteome. We introduce a cell-based platform for rapid and unbiased functional annotation of undercharacterized human proteins. Utilizing a library of antibody biomarkers, the full-length proteins are investigated by tracking phenotypic changes caused by overexpression in human cell lines. We combine reverse transfection and immunodetection by fluorescence microscopy to facilitate this procedure at high resolution. Demonstrating the advantage of this approach, new annotations are provided for two novel proteins: 1) a membrane-bound O-acyltransferase protein (C3F) that, when overexpressed, disrupts Golgi and endosome integrity due likely to an endoplasmic reticulum-Golgi transport block and 2) a tumor marker (BC-2) that prompts a redistribution of a transcriptional silencing protein (BMI1) and a mitogen-activated protein kinase mediator (Rac1) to distinct nuclear regions that undergo chromatin compaction. Our strategy is an immediate application for directly addressing those proteins whose molecular function remains unknown.
Highlights
Experimental approaches that enable direct investigation of human protein function are necessary for comprehensive annotation of the human proteome
The pursuit of whole genome annotation has led to the development of a variety of high throughput (HTP)1 methods with the objective to study gene function en masse and the capacity to deliver a spectrum of data ranging from transcriptional information at the RNA level to identifying interaction partners at the protein level [1, 2]
Utilizing the SFINX tool, which displays nine different methods for predicting membrane topology and two methods for signal peptide prediction, we were able to estimate with confidence topological features such as transmembrane (TM) helices and signal peptide (SP) cleavage sites from the amino acid sequence of the selected proteins (Table I) [12, 19]
Summary
Membrane Topology Predictions and Domain AssignmentsâNine methods for predicting membrane topology were consulted and displayed graphically by the SFINX web server from plots generated for each protein sequence [12]. Transmembrane prediction programs included TMHMM2.0 [13] and Phobius [14]. Supplementary KyteDoolittle plots for analyzing hydrophobicity were generated [15].
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