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Molecular & Cellular Proteomics 4:44-55, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

A Global Approach Combining Proteome Analysis and Phenotypic Screening with RNA Interference Yields Novel Apoptosis Regulators^*,S

Nikolaus Machuy^,, Bernd Thiede^,¶, Krishnaraj Rajalingam^¶, Christiane Dimmler^¶, Oliver Thieck^¶, Thomas F. Meyer^¶ and Thomas Rudel^¶,||

From RNAx GmbH, Breddiner Weg 5, 13591 Berlin, Germany; and the ^¶ Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21/22, D-10117 Berlin, Germany

Global approaches like proteome or transcriptome analyses have been performed extensively to identify candidate genes or proteins involved in biological and pathological processes. Here we describe the identification of proteins implicated in the regulation of apoptosis using proteome analysis and the functional validation of targets by RNA interference. A high-throughput platform for the validation of synthetic small interfering RNAs (siRNAs) by quantitative real-time PCR was established. Genes of the identified factors were silenced by automated siRNA transfection, and their role in apoptotic signaling was investigated. Using this strategy, nine new modulators of apoptosis were identified. A subsequent detailed study demonstrated that hepatoma-derived growth factor (HDGF) is required for TNF-induced release of pro-apoptotic factors from mitochondria. The strategy described here may be used for hypothesis-free, global gene function analysis.

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