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

Nikolaus Machuy; Bernd Thiede; Krishnaraj Rajalingam; Christiane Dimmler; Oliver Thieck; Thomas F. Meyer; Thomas Rudel

doi:10.1074/mcp.M400089-MCP200

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

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

‖ To whom correspondence should be addressed: Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21/22, D-10117 Berlin, Germany. Tel.: 49-30-28460-415; Fax: 49-30-28460-401; E-mail: rudel{at}mpiib-berlin.mpg.de

Abstract

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 TNFα-induced release of pro-apoptotic factors from mitochondria. The strategy described here may be used for hypothesis-free, global gene function analysis.

Footnotes

Published, MCP Papers in Press, November 26, 2004, DOI 10.1074/mcp.M400089-MCP200
↵1 The abbreviations used are: 2-DE, two-dimensional gel electrophoresis; HDGF, hepatoma-derived growth factor; IAP, inhibitor of apoptosis proteins; IFN, interferon; LoF, loss of function; q-PCR, quantitative real-time PCR; RNAi, RNA interference; siLuc, siRNA directed against luciferase; siRNA, small interfering RNA; TNF/CHX, TNFα/cycloheximide; PSMA3, proteasome subunit α type 3; eIF3-p42, eukaryotic initiation factor 3 subunit 4.
↵* This work was supported by Grants 0312260 and 0313029A of the Bundesministerium für Bildung und Forschung (BMBF) (to T. R.). The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵S The on-line version of this manuscript (available at http://www.mcponline.org) contains supplemental material.
↵§ N. M. and B. T. contributed equally to this work.
- Received July 12, 2004.
- Revision received November 26, 2004.