Pier Paolo Di Fiores's group - Simona Polo (»Add to Infobox)

General description: The monoubiquitin (monoUb)-network is emerging as a major regulator of protein function beyond its well-established role in protein degradation. While initial observations in various labs project a physiological relevance of monoubiquitination comparable to that of phosphorylation, the characterization of the system has only just begun. We are trying to integrate a wide proteomic approach to monoUb as a signaling device with high-resolution studies of how monoUb can modulate the function of endocytic/signaling proteins. Presently three major levels of study are being pursued in the lab: 1.  Elucidating the monoUb proteome. 2.  Dissecting the role of monoubiquitination in endocytosis 3.  Elucidating the molecular mechanisms of coupled monoubiquitination Role in RUBICON: We plan to identify the intracellular repertoire of proteins that are modified by monoubiquitination following growth factor treatment. The major problem is that most Ub-modified proteins are poly-Ub proteins destined for degradation, whose isolation is not desirable. We have developed an efficient protocol, based on digestion of cellular lysates with exogenously added proteasome, that get rid of the greatest majority of polyUb-containing proteins, while leaving MonoUb-proteins unaffected. Proteasome-treated lysates (from cells stimulated or not with growth factors) will be then subjected to various affinity purification steps. The protocol includes purification by anti-Ub antibodies and by Ub-interacting domains (first and foremost the UIM motif), followed by identification by mass spectrometry.

MEMBERS (5/5)

Polo Simona (Group leader / Pier Paolo Di Fiore's Group)