Protein post-translational modifications (PTMs) are major players in regulating a broad range of cellular processes. Among these, ubiquitination, i.e. the covalent attachment of one or more ubiquitin (Ub) to a lysine of the substrate, is involved in the regulation of several events, such as cell division, differentiation, signal transduction and protein trafficking. Because of the large numbers of Ub moieties that can be conjugated to a single substrate and the widespread number of proteins that can be involved in the reaction, large scale identification of modified proteins can be helpful in understanding how Ub works in intracellular pathways. Proteomics technologies for global detection and quantitation of proteins are election tools to gain insights into PTMs-mediated network and have already been used for the characterizations of Ub-conjugated and Ub–associated proteome in human and yeast cells. Despite this bulk of informations, little is known about the inducibility of the process and the pathways to which modified proteins belong. The aim of this work was to obtain a comprehensive map of ubiquitinated proteins after EGF (Epidermal Growth Factor) stimulation in mammalian cells, using affinity purification and advanced mass spectrometry. An experimental strategy based on an inducible cell line expressing FLAG-His-Ub was used. By combining stable isotope labelling in cell culture (SILAC) with tandem affinity purification (TAP) techniques, I identified and quantified proteins whose ubiquitination is modulated by EGF stimulation. Interestigly, a subset of them are not directly involved in EGFR endocytosis/signaling but in rather different cellular pathways such as translation and transcription. These results will be instrumental in reconstructing the signaling cascade mediated by Ub downstream the EGFR activation.

Proteomic profiling of EGF-induced ubiquitinated proteins : phd degree in molecular medicine / E. Argenzio ; S. Polo. - : . DIPARTIMENTO DI MEDICINA, CHIRURGIA E ODONTOIATRIA, 2008 Sep 29. ((20. ciclo, Anno Accademico 2006/2007.

Proteomic profiling of EGF-induced ubiquitinated proteins : phd degree in molecular medicine

E. Argenzio
2008-09-29

Abstract

Protein post-translational modifications (PTMs) are major players in regulating a broad range of cellular processes. Among these, ubiquitination, i.e. the covalent attachment of one or more ubiquitin (Ub) to a lysine of the substrate, is involved in the regulation of several events, such as cell division, differentiation, signal transduction and protein trafficking. Because of the large numbers of Ub moieties that can be conjugated to a single substrate and the widespread number of proteins that can be involved in the reaction, large scale identification of modified proteins can be helpful in understanding how Ub works in intracellular pathways. Proteomics technologies for global detection and quantitation of proteins are election tools to gain insights into PTMs-mediated network and have already been used for the characterizations of Ub-conjugated and Ub–associated proteome in human and yeast cells. Despite this bulk of informations, little is known about the inducibility of the process and the pathways to which modified proteins belong. The aim of this work was to obtain a comprehensive map of ubiquitinated proteins after EGF (Epidermal Growth Factor) stimulation in mammalian cells, using affinity purification and advanced mass spectrometry. An experimental strategy based on an inducible cell line expressing FLAG-His-Ub was used. By combining stable isotope labelling in cell culture (SILAC) with tandem affinity purification (TAP) techniques, I identified and quantified proteins whose ubiquitination is modulated by EGF stimulation. Interestigly, a subset of them are not directly involved in EGFR endocytosis/signaling but in rather different cellular pathways such as translation and transcription. These results will be instrumental in reconstructing the signaling cascade mediated by Ub downstream the EGFR activation.
POLO, SIMONA LAURA ANNA
Ubiquitin ; proteomics ; SILAC ; EGFR signaling
Settore MED/04 - Patologia Generale
Proteomic profiling of EGF-induced ubiquitinated proteins : phd degree in molecular medicine / E. Argenzio ; S. Polo. - : . DIPARTIMENTO DI MEDICINA, CHIRURGIA E ODONTOIATRIA, 2008 Sep 29. ((20. ciclo, Anno Accademico 2006/2007.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/59661
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