RICERCA ITALIANA     

The Nedd8 and mono-ubiquitin binding network

Università degli Studi di Roma "Tor Vergata"

Abstract

Covalent modifications of proteins, by addition of the 76 amino-acid ubiquitin, encompass the addition of polymeric chains (poly-ubiquitination) and the decoration of the substrate with a single ubiquitin monomer. Mono-ubiquitination is promoted both by intracellular or extracellular stimuli and regulates a variety of functions. For instance, it appears to regulate the transport/sorting of many membrane proteins both in the endocytic/biosynthetic pathway.
Nedd8 is the family component closest to ubiquitin (60% identity) and there is strong homology between the two conjugation system. On the other end, seven exposed residues and charge distribution differ in the two molecules suggesting a potential for specific interactions.
Our goal is to contribute to the description of the protein interaction network based on substrates modified by mono-ubiquitin and/or Nedd8 and their receptors. We propose to concentrate on the ubiquitin-network to reveal the complete repertoire of physical interactions involved. Moreover, we propose to study the interplay of specificity and promiscuity between Nedd8 and ubiquitin.
We plan to search for new Nedd8 and mono-ubiquitin receptor domains, to isolate and characterize new mono-ubiquitinated / neddylated proteins (after EGF stimulation) and to define the specific Nedd8 contribution.

Principal Investigator

Luisa CASTAGNOLI Università degli Studi di ROMA "Tor Vergata"

Research Objectives

Protein interactions preside over cell physiology and some of us are confident that a complete and quantitative understanding of the protein interaction mesh could eventually result in being able to predict the cell response to any given stimulus. This task is complicated by the recognition that the relation between genotype and phenotype is mediated by an intricate network of physical and functional interactions between the protein products of the approximately 25.000 genes in the genome of a complex organism. An essential intermediate step toward this long term objective is the determination of the complete protein interaction web where all the tiles of the cell puzzle are correctly put into place. Initially, a more informative description of protein interaction networks can be achieved by considering explicitly the modular nature of proteins. Protein networks are therefore represented as modular domains, covalently linked to form proteins, and binding to their targets in partner proteins. Families of conserved modules binding to short extended peptides mediate a relatively large fraction of the non-covalent interactions linking different proteins in the network.

Our goal is to contribute to the description of the protein interaction network based on substrates, post-translationally modified by conjugation to Nedd8 or mono-ubiquitin, and their receptor domains. We propose to concentrate on the ubiquitin-network to reveal the complete repertoire of physical >>>

Timescale

24 months

National and international background

Cells are exposed to external stimuli and the signals are propagated by spatially and temporally ordered physical interactions among proteins. Many of these interconnections depend on protein domains specifically binding short peptides or lipids. Signaling research has recently achieved a deep understanding of the protein interactions and initial biochemical events following the activation of receptor tyrosine kinases, the recruitment of phosphotyrosine binding proteins, adaptors and the cascades of kinases involved in signal transduction. On the contrary, the mechanisms involved in the subsequent signal desensitization are only partially unraveled. Signal desensitation requires endocytosis, down regulation and degradation.
Activated receptor proteins are captured in clathrin coated pits or in caveolae and their fate can be either to be recycled to the membrane or down regulated and finally removed via the lysosome or the proteasome pathway. Numerous protein interactions involved in this process are constitutive, while some respond to particular stimuli and induce post-translational modifications orginating new protein-protein interactions.
Sorting of plasma membrane proteins into the lumen of lysosomes is a process that requires a specific post-translational modification, the mono-ubiquitination of cargo proteins, as well as dedicated protein-sorting complexes that specifically recognize ubiquitinated cargo. This highly regulated delivery process relies on >>>