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J. Biol. Chem., Vol. 283, Issue 36, 24448-24459, September 5, 2008

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Intracellular Peptides as Natural Regulators of Cell Signaling^*

Fernanda M. Cunha¹, Denise A. Berti, Zulma S. Ferreira^¶, Clécio F. Klitzke^||2, Regina P. Markus^¶1, and Emer S. Ferro¹³

From the Departments of Cell Biology and Development and ^¶Physiology, Biomedical Science Institute, University of São Paulo, São Paulo, SP 05508-900, the Department of Biochemistry, Federal University of São Paulo, São Paulo, SP 04044-020, and the ^||Center for Applied Toxinology, CAT/CEPID, Butantan Institute, São Paulo, SP 05503-900, Brazil

Protein degradation by the ubiquitin proteasome system releases large amounts of oligopeptides within cells. To investigate possible functions for these intracellularly generated oligopeptides, we fused them to a cationic transactivator peptide sequence using reversible disulfide bonds, introduced them into cells, and analyzed their effect on G protein-coupled receptor (GPCR) signal transduction. A mixture containing four of these peptides (20–80 µM) significantly inhibited the increase in the extracellular acidification response triggered by angiotensin II (ang II) in CHO-S cells transfected with the ang II type 1 receptor (AT1R-CHO-S). Subsequently, either alone or in a mixture, these peptides increased luciferase gene transcription in AT1R CHO-S cells stimulated with ang II and in HEK293 cells treated with isoproterenol. These peptides without transactivator failed to affect GPCR cellular responses. All four functional peptides were shown in vitro to competitively inhibit the degradation of a synthetic substrate by thimet oligopeptidase. Overexpression of thimet oligopeptidase in both CHO-S and HEK293 cells was sufficient to reduce luciferase activation triggered by a specific GPCR agonist. Moreover, using individual peptides as baits in affinity columns, several proteins involved in GPCR signaling were identified, including -adaptin A and dynamin 1. These results suggest that before their complete degradation, intracellular peptides similar to those generated by proteasomes can actively affect cell signaling, probably representing additional bioactive molecules within cells.

Received for publication, February 15, 2008 , and in revised form, June 20, 2008.

^* This work was supported by São Paulo State Research Foundation Grant 04/04933-2 and grants from Financiadora de Estudos e Projetos and the National Laboratory of Synchrotron Light (São Paulo Proteome Network). The costs of publication of this article were defrayed in part by the payment 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1–3.

¹ Supported by research fellowships from the Brazilian National Council of Research.

² Supported by Fórum Permanente das Relaçôes Universidade-Empresa (Instituto UNIEMP).

³ To whom correspondence should be addressed: Av. Prof. Lineu Prestes, 1524, Sala 431, Campus Universitário, São Paulo, 05508-900, SP, Brazil. Tel.: 55-11-30917310; Fax: 55-11-3091-7402; E-mail: eferro{at}usp.br.

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