Abstract
The subcellular localization of proteins is critical to their biological roles. Moreover, whether a protein is membrane-bound, secreted, or intracellular affects the usefulness of, and the strategies for, using a protein as a diagnostic marker or a target for therapy. We employed a rapid and efficient experimental approach to classify thousands of human gene products as either “membrane-associated/secreted” (MS) or “cytosolic/nuclear” (CN). Using subcellular fractionation methods, we separated mRNAs associated with membranes from those associated with the soluble cytosolic fraction and analyzed these two pools by comparative hybridization to DNA microarrays. Analysis of 11 different human cell lines, representing lymphoid, myeloid, breast, ovarian, hepatic, colon, and prostate tissues, identified more than 5,000 previously uncharacterized MS and more than 6,400 putative CN genes at high confidence levels. The experimentally determined localizations correlated well with in silico predictions of signal peptides and transmembrane domains, but also significantly increased the number of human genes that could be cataloged as encoding either MS or CN proteins. Using gene expression data from a variety of primary human malignancies and normal tissues, we rationally identified hundreds of MS gene products that are significantly overexpressed in tumors compared to normal tissues and thus represent candidates for serum diagnostic tests or monoclonal antibody-based therapies. Finally, we used the catalog of CN gene products to generate sets of candidate markers of organ-specific tissue injury. The large-scale annotation of subcellular localization reported here will serve as a reference database and will aid in the rational design of diagnostic tests and molecular therapies for diverse diseases.
Highlights
The subcellular localization of proteins critically affects their biological roles and functions
Identification of MS- and CN-Encoding Genes Based on previously reported microarray experiments [13], we chose a panel of 11 cell lines whose gene expression patterns would, in aggregate, encompass a large set of mRNAs (Table S1)
Genes encoding proteins inserted into cellular membranes or that are secreted were designated as the ‘‘MS reference set,’’ while genes encoding cytoplasmic or nuclear proteins were designated as the ‘‘CN reference set.’’ Limiting our search to the 24,365 UniGene clusters that were detectably expressed in at least three of the samples, we used empirical localization data reported in the Swiss-Prot [15] and LocusLink [16] databases to identify 2,701 known MS clusters and 2,744 known CN clusters
Summary
The subcellular localization of proteins critically affects their biological roles and functions. Some of these, including Trastuzumab (Herceptin), Rituximab (Rituxan), and Cetuximab (Erbitux), target TM proteins on the surface of malignant cells and have firmly established their value in the treatment of cancer. Secreted and shed proteins with tumor- or disease-specific expression patterns represent potential targets for diagnostic assays in biological fluids. Such assays are currently being used to screen patients for diagnosis or detection of recurrence of a number of malignancies, including prostate, ovarian, and liver cancer [2]. Large-scale identification of surface, secreted, and intracellular proteins that are specific to organs, tissues, or disease has great potential value in facilitating the further development of these therapeutic and diagnostic approaches
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