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 interactions involved. Moreover, we propose to study the interplay of specificity and promiscuity between Nedd8 and ubiquitin.
We plan to search for new mono-ubiquitin and Nedd8 receptor domains, to isolate and characterize new neddylated/mono-ubiquitinated proteins and to define the specific Nedd8 contribution.

Nedd8 is the family component closest to ubiquitin (57% identity) and there is strong homology between the two conjugation system. This high degree of similarity may have been maintained because of a requirement to bind common partners. On the other hand, seven residues are conserved in all the NEDD8 but are totally different from ubiquitin where they are not conserved. Moreover, a notable difference in the electrostatic potential surface, the presence of a distinct positive region in the middle of the NEDD8 acidic face
suggest a potential for specific interactions. Moreover, NEDD8 has never been purified as a poly-chain and it can be considered a modification similar to mono-ubiquitin and therefore, by similarity, involved in endocytosis and down-regulation. We propose experiments aimed at distinguishing the contribution of the two modifying proteins, with a particular emphasys on EGF receptor endocytosis.
The experimental strategy, that we have developed and extensively used in the past years, is based on the screening of large repertoires of protein fragments in search of ligands for any bait protein of interest. Within the framework of an European integrated project we are now focusing on the characterization of the entire protein interaction network mediated by small protein recognition modules in human cells. Namely we have invested on those domain family that bind to proline rich peptides (SH3, WW, EVH, GYF)e and to peptides phosphorylated in Ser, Thr and Tyr (14-3-3, FHA, Brct, WD40, SH2, PTB ….).

The contribution of the presently proposed project should add informations on the protein-protein interactions, involving domains able to bind motifs modified (induced by growth factor) by addition of Nedd8 or mono-ubiquitin and functionally involved in protein traficking (VHS, UIM, UEV….).

Once the complete repertoire of intracellular receptors/ligands and their physical interactions is described, high resolution genetic, biochemical and cytological studies can be addressed to testing the functional wiring. <<<

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 the ordered synergic work of several modular proteins, containing domains involved in protein interaction (SH3, WW, coiled-coil, EH, VHS) or lipid recognition (ENTH, FYVE). Other domains that play a role in the ubiquitination/degradation pathway (UIM, UBA, HECT, RING) are often found in proteins that are involved in receptor downregulation. Some of these domains are also found in the proteins that deliver peptides of the biosynthetic pathway directly from the trans-Golgi network to the lysosome, for degradation.

- The Mono-Ubiquitin Binding Network-
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. Polyubiquitination is a mechanism for targeting a poly-ubiquitinated substrate to degradation by the proteasome. Differently, addition of a single mojety of ubiquitin either at a single or at multiple sites, is a signal dependent device establishing new specific protein interactions. 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 and in virus budding.
Numerous domains present in proteins involved in endocytosis and protein delivery (e.g., VHS, UIM, UEV) bind the small protein ubiquitin when it is conjugated to the protein to be delivered.
Ubiquitination regulates the internalization and MVB (multi vescicular body) sorting of many transmembrane proteins, such as receptor tyrosine kinases (RTKs), G-protein coupled receptors, ion channels, transporters, and permeases.
Ub-mediated endocytosis of RTKs ( e.g., EGFR) is the main mechanism for down-regulation of the activated receptors. Two recent reports demonstrate that in-frame fusion of ubiquitin to EGFR results in the constitutive internalization and enhanced degradation of the chimeric protein (1,2) .
Although mono-ubiquitination of RTKs is sufficient for endocytosis, several RTKs are monoubiquitinated at multiple sites, potentially changing the dynamics of endocytosis by increasing avidity for a single interactor or providing multiple binding sites.
According to a simple model, ubiquitin conjugated membrane proteins could be directed to coated pits by adaper proteins like Eps15 that contain both a clathrin and an ubiquitin binding domain. The role of ubiquitination in endocytosis is not limited to the initial internalization step. Once proteins are localized to endosomal membranes, ubiquitination serves to sort proteins into internal vesicles within the endosomes through inward budding-forming structures known as multivesicular bodies (MVBs). Here some proteins are destined for degradation, whereas others are recycled back to the plasma membrane via recycling endosomes. At this stage recognition of the ubiquitylated proteins is mediated by ESCRT-1 (endosomal sorting complex required for transport). Genetic studies indicate that removal of ubiquitin moieties is necessary for completion of the sorting at the MVBs. A deubiquitinating enzyme (in yeast DUB Doa4p), removes ubiquitin from cargo molecules and, in this way, rescues ubiquitin molecules from degradation and restores cellular ubiquitin homeostasis (3).
Other domains, like UBA or CUE (Coupling of Ubiquitin conjugation to ER degradation), also bind mono- and poly-ubiquitin, but they are mostly found in proteins involved in cell cycle control, protein degradation and DNA excision- repair (4).

- Protein domains receptors of conjugated ubiquitin-

VHS Domain: Present in VPS-27, Hrs and STAM).
The VHS domain is a ~140 residues long domain. Resolution of the crystal structure of the VHS domain of Drosophila Hrs and human Tom1 revealed that it consists of eight helices arranged in a double-layer superhelix.
VHS and ENTH domains have a similar fold but different primary sequences and functions. Some authors have also suggested a distant folding homology between the N-terminal deubiquitinating Josephin domain of ataxin-3, and the VHS and ENTH domains (5). ENTH
(Epsin N-terminal homology domain) occupies the N-terminal parts of many endocytic proteins and binds to lipids. The VHS domain is found at the N- terminus of proteins and it is considered to have a cargo recognition role in vesicular trafficking.
VHS is present in 15 proteins of the human proteome:
GGA proteins (Golgi-localized, gamma ear-containing, ARF-binding protein) and TOM (Target of myb-like 1 protein) proteins, display a domain composition VHS-GAT-GAE homology domain (GAT: GGA and TOM; GAE: gamma-adaptin ear homology) (6). TOM proteins are involved in intra-Golgi transport and recently TOM1 was shown to be targeted to endosomes through its C-terminal interaction with the FYVE domain containing protein Endofin. The function of the GGAs in mediating Golgi to lysosomal trafficking is well established, GGAs mediate the sorting of ubiquitinated proteins at the TGN.
STAM (signal transducing adaptor molecule) STAM/EAST/Hbp (Hrs binding protein), which all share the domain composition VHS-UIM-SH3-ITAM and carry one or two ubiquitin-interacting motifs (UIM)
Hgs (hepatocyte growth factor-regulated tyrosine kinase .substrate). Proteins with a FYVE domain C-terminal to VHS. They also carry one or two ubiquitin-interacting motifs. Domain composition: VHS-FYVE-UIM
Hgs/Hrs binds clathrin, eps15, STAMs and, through its PSAP motif, interacts with Tgs101 that is part of the ESCRTI protein complex.
Although the VHS of GGA has been clearly implicated in the targeting of some trans Golgi receptor to the late endosomes the function of the remaining VHS domains remains elusive. The VHS domains of GGA proteins are known to recognize the C-terminal acidic cluster of dileucine signal (DxxLL), present in some TGN-associated receptors.
On the other hand, the remaining VHS, lacking the critical binding residues in helices 6 and 8, do not bind the dileucine signal. Mizuno et al. have shown that STAM proteins bind ubiquitinated proteins through their UIM and VHS domains and Yamakami et al. have shown binding of TOM1 to ubiquitinated-proteins, mediated by the VHS and the GAT domains (7,8).

UIM Motif
The Ubiquitin Interacting Motif (UIM) was first described in the 26S proteasome subunit PSD4/RPN-10. It occurs in many proteins in multiple copies and it is known to bind, with a dissociation constant in the high micromolar range, to a hydrophobic patch of mono- or poly- ubiquitin. It is found in many proteins involved in the endocytic pathway, including Hrs, Stam, Eps15, the S.cerevisiae VPS27 vacuolar sorting protein and ataxin-3, a protein involved in ataxia disease.

UBC domain
There are several different E2 ubiquitin conjugating enzymes (over 30 in humans), which are broadly grouped into four classes, all of which have a core catalytic domain (UBC domain) containing the active site cysteine), and some of which have short N- and C-terminal amino acid extension. E2 enzymes carry a covalently bound Ubiquitin protein and associate with an E3 enzyme , Ub-ligase. This interaction leads to ubiquitination of the specific substrate.

UEV domain
( ubiquitin-conjugating enzyme E2 variant domain).
This domain has an E2-like fold but it is devoid of catalytic activity. UEVs are homologous to E2 ubiquitin conjugating enzymes but lack the conserved cysteine residue at the active site, required for catalytic activity.
The N-terminal UEV domain of the yeast ESCRT I protein,Vps23, is required for sorting ubiquitinated proteins into the internal vesicles of multivesicular bodies. Comparing the crystal structure of the yeast Vps23 UEV in a complex with ubiquitin with the solution structure of the human Tsg101 UEV (the tumor susceptibility gene, part of a 350-kDa complex that binds Ub and mediates the sorting of ubiquitinated proteins into vesicles) in the absence of Ub, two loops that are conserved are shown to move toward each other to grip the Ub in a pincer-like grasp. The contacts with the UEV encompass two adjacent patches on the surface of the Ub, one containing several hydrophobic residues, including Ile-8, Ile-44, and Val-70, and the second containing a hydrophilic patch including residues Asn-60, Gln-62, Glu-64. The hydrophobic Ub patch interacting with the Vps23 UEV overlaps the surface of Ub interacting with the Vps27 UIM motif as well as with the VHS domain ( L8, I44, V70), suggesting a sequential model for ubiquitinated cargo binding by these proteins. In contrast, the hydrophilic patch encompasses residues uniquely interacting with the ESCRT-I UEV.

- The EGFR from membrane to lysosome-
Stimulation with epidermal growth factor induces activation of EGFR/ErB-1, autophosphorylation and recruitment of Cbl, among other proteins necessary for signal propagation. Cbl is then phosphorylated by the kinase domain of the receptor and in turn ubiquitylates the EGFR at multiple sites. The multi-ubiquitination of EGFR could either serve to increase avidity or, if executed in a temporal hierarchy, to create binding sites for sequentially intervening adaptors. Ubiquitylated receptors are sorted into clathrin-coated pits by a multiprotein complex including coat adaptors such as Epsin and Eps15. Fission of clathrin-coated vesicles is mediated by a GTPase, dynamin. Progression through the endocytic pathway is characterized by the shedding of clathrin, a decrease in the internal pH and the accumulation of hydrolytic enzymes. EGFR trafficking from early to late endosomes/MVB is dependent on its continued association with Cbl and its sustained ubiquitylation. MVB sorting is regulated through recognition of ubiquitylated cargo by Hgs/Hrs/Vps27p, Tsg101/Vps23p and other components of ESCRT complexes. Invagination of the limiting membrane of the MVB forms internal vesicles. This process,in yeast, is coupled to substrate de-ubiquitylation by Doa, and dissociation of the ESCRT complex from endosomes, mediated by the AAA ATPase Vps4. Fusion of the MVB with the lysosome results in cargo degradation (9).

- Nedd8, an ubiquitin like protein: alternative or overlapping function?-
Monomeric ubiquitin and ubiquitin binding domains play a role in the endocytosis that could be compared to the pivotal role played in signaling pathway by the phosphorylation of tyrosine and phosphotyrosine binding domains. The potentiality of interactions of such a bulky modification is intriguing. Ubiquitin, once conjugated to phosphorylated receptors, is recognized by adaptors containing specific ubiquitin binding domains thereby regulating receptor endocytosis as well as sorting to the lysosome. In human, EnsEMBL shows at least one gene containing the InterPro domain IPR000626 (Ubiquitin) in each human chromosome. These multiple polypeptides that are distinct from, but related to, ubiquitin are also enzymatically coupled to target macromolecules, and participate in diverse biological processes such as DNA repair, autophagy and signal transduction.
Addition of SUMO/Sentrin to a target protein (sumoylation) changes the localization of this protein or its interactions with other proteins. The first identified target of SUMO, the trafficking protein RanGAP1 (which helps to shuttle proteins from the cytoplasm to the nucleus) requires the addition of SUMO before it can be localized to the nuclear pores. Localization of several proteins to discrete sites within the nucleus (called PML nuclear bodies) also depends on their sumoylation.

Nedd8 (Neural Precursor Cell Expressed,Developmentally Downregulated 8) shares 60% amino-acid identity with ubiquitin. NEDD8-specific protease NEDP1 processes preNEDD8 to its mature form and deconjugates NEDD8 from substrates such as p53 and cullins. A single-residue difference in the C-terminus of NEDD8 and ubiquitin contributes significantly to the ability of NEDP1 to discriminate between them (10). Biochemical analysis demonstrates that Ala-72 of NEDD8 (Arg-72 in Ub), performs a key role in preventing the interaction of NEDD8 with the ubiquitin E1 enzyme thereby acting to prevent the inappropriate diversion of NEDD8 into ubiquitin-specific pathways (11).
Till recently,the only known Nedd8/ RUB1-modified proteins were the cullins. Cullins are common subunits of a large group of multisubunit E3 ligase, the so-called SCF (Skp1/cullin-1/F-box) and CBC (cullin-2/elongin BC) E3s, which add ubiquitin to their target proteins (12). The neddylation of cullins is promoted by a RING finger protein (Roc1) that recruits Ubc12, the E2-like. NEDD8 modification of Cul-1 enhances recruitment of Ub-conjugating enzyme Ubc4 (E2) to the SCF complex (E3) and accelerating the formation of the E2–E3 complex, stimulates protein polyubiquitylation. Recently, p53 was identified as a target of neddylation by MDM2 that was identified as an E3 ligase common to the ubiquitin and NEDD8 conjugation pathway (13) . Nedd8 is the family component closest to ubiquitin (60% identity) and there is strong homology between the two conjugation system. This high degree of similarity may have been maintained because of a requirement to bind common partners. On the other hand, seven residues are conserved in all the NEDD8 but differ from ubiquitin suggesting a potential for specific interactions. In the same time, NEDD8 has never been purified as a poly-chain and it can be considered a modification similar to mono-ubiquitin. We propose experiments aimed at distinguishing the contribution of the two modifying proteins, with a particular emphasys on receptor endocytosis. <<<