Accelerated discovery of novel protein function in cultured human cells.
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.
Hodges
Emily
E
Center for Genomics and Bioinformatics, Karolinska Institute, SE-171 77 Stockholm, Sweden.
Redelius
Jenny Stjerndahl
JS
Wu
Weilin
W
Höög
Christer
C
D015415:Biomarkers / Q000097:blood; D014408:Biomarkers, Tumor; D020126:Brefeldin A / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D000939:Epitopes; D016650:Fluorescein-5-isothiocyanate; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007211:Indoles; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D040081:Protein Array Analysis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D020543:Proteome / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D015415:Biomarkers; D014408:Biomarkers, Tumor; D002843:Chromatin; D000939:Epitopes; D005456:Fluorescent Dyes; D007211:Indoles; D008565:Membrane Proteins; D011500:Protein Synthesis Inhibitors; D020543:Proteome; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; C007293:DAPI; D020830:rac1 GTP-Binding Protein; D016650:Fluorescein-5-isothiocyanate