Regulatory roles of ubiquitin
c/o IFOM-IEO Campus
Via Adamello, 16 - 20139 Milan, Italy
Tel: +39-02-574303242; FAX: +39-02-574303231
Our research interests focus on understanding the molecular basis and the regulatory pathways of ubiquitination, a process whereby one or more ubiquitin (Ub) monomers or chains are covalently attached to target proteins.
Fig1: The ubiquination process [+zoom]
One of the most intriguing features of the ubiquitin pathway is its emerging involvement in functions not directly related to protein degradation. The variety of structurally distinct Ub modifications, the dynamic and reversible nature of the ubiquitination cascade and the ability of Ub binding domains (UBDs) to decipher, transduce and amplify the Ub-based signal, make Ub a highly versatile intracellular messenger (Fig.1).
The existence of a vast and dynamic array of Ub signals raises the central question of how specificity is achieved during conjugation, recognition, and signal transduction. Indeed, the molecular requirements for generating and recognizing various Ub signals are poorly understood and form a central unresolved issue in the field. We are interested in understanding the mechanism of Ub-chain transfer to the substrate. To investigate the E3 catalysis we are pursuing a biochemical approach coupled to X-ray crystallography studies.
The covalent attachment of Ub to proteins can alter their localization, activities, and ultimate fate. Of particular interest in signaling is the ability of the Ub modification to induce, similarly to phosphorylation, de novo protein:protein interactions, through the recognition of ubiquitinated proteins by proteins harboring UBDs, a mechanism that sits at the heart of several signaling cascade. We have previously identified, solved the structures and characterized two novel UBDs present in Rabex-5 , a GEF (guanine nucleotide exchange factor) for Rab5. We are now investigating other proteins harboring novel UBDs.
This process is tightly controlled within the cell by endogenous signals, such as DNA damage, and exogenous signals such as growth factor stimulation. In this latter instance, one of the best-characterized model systems is represented by the epidermal growth Factor (EGF). Upon EGF stimulation a variety of proteins are subject to the Ub modification. These include the EGF receptor (EGFR) and the components of the downstream endocytic machinery .
In the past we contributed to clarify the impact of ubiquitination on receptor internalization, intracellular sorting, and ultimate metabolic fate.
Now we wish to draw a complete physical and functional map of the molecular machinery involved in the Ub-based endocytosis and signaling of EGFR. Our idea is to integrate a wide proteomic approach to Ub as a signaling device with high-resolution investigations into how Ub can modulate the function of endocytic proteins. We recently obtained the EGF-regulated Ubiproteome by two complementary purification strategies coupled to quantitative proteomics. Our results unveil a complex impact of growth factor signaling on Ub-based signaling networks, which extends well beyond present knowledge. In addition to endocytic proteins, the EGF-regulated Ubiproteome includes a large number of signaling proteins, ubiquitin ligases and deubiquitinating enzymes, transporters and aminoacid permeases, and proteins involved in translation and transcription. Next, we would like to understand how the signal from activated RTKs is delivered to the ubiquitination machinery and to investigate in details the new effectors found by proteomics.