Intracellular peptides as natural regulators of cell signaling.
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 microm) 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 alpha-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.
Cunha
Fernanda M
FM
Department of Cell Biology and Development, University of São Paulo, São Paulo, SP 05508-900, Brazil.
Berti
Denise A
DA
Ferreira
Zulma S
ZS
Klitzke
Clécio F
CF
Markus
Regina P
RP
Ferro
Emer S
ES
D033965:Adaptor Protein Complex alpha Subunits / Q000235:genetics / Q000378:metabolism; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D034284:Dynamin I / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000235:genetics; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014162:Transfection; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D033965:Adaptor Protein Complex alpha Subunits; D009842:Oligopeptides; D044140:Receptor, Angiotensin, Type 1; D025801:Ubiquitin; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; D046988:Proteasome Endopeptidase Complex; D034284:Dynamin I