DESCRIPTION: The long-term goal of this research is to understand how the ubiquitin domain is recognized as a targeting signal. The ubiquitin domain is a diverse and widely used targeting signal that is post- translationally attached to a variety of cellular proteins. The ubiquitin pathway is implicated in growth control (cancer and apoptosis), signal transduction (inflammation and transcriptional control), and the stress response (protein quality control, neurodegeneration, and genome integrity). Conjugation of the domain serves to modify the localization or activities of the target protein. Conjugation of a single ubiquitin to histones is involved in chromatin and DMA transactions and as a sorting signal in endosomal sorting pathways. NEDD8 conjugation regulated ubiquitin E3 liganses while SUMO-1 (small ubiquitin-like modifier) conjugation is widely involved in nuclear protein metaboslism. Poly ubiquitination (or SUMOylation) of target proteins is achieved by attachment of one ubiquitin to another through lysine residues. K48-Iinked polyubiquitin is a signal for delivery of the target protein to the proteasome for proteolysis while K63-linked chains are involved in assembling signaling complexes in the NFkB pathway and play some undetermined role in DNA repair. Linkages through lysines 6,11, 29, and 33 are also observed, although the function of these chains, as well as that of polySUMO-2/3 chains is completely unknown. The unifying hypothesis is that there are specific receptors or adapters that specifically recognize the different ubiquitin domains and contexts and that subsequently direct the conjugated protein to the appropriate cellular fate. This study proposes to define some of the potential roles of the different chain linkages by studying polyubiquitin recognition and defining the receptors and binding proteins that distinguish among different version of the ubiquitin domain. Two approaches are used here. First, a directed approach uses deubiquitinating enzymes as a model for specific recognition of polyubiquitin, either by direct recognition (USP5/isopeptidase T, Aim 1) or by adapter-assisted recognition of specific substrates (BAP1, Aim 2). Second, a modern systems biology approach takes advantage of our synthesis of polyubiquitin chain analogs to identify ubiquitin-binding proteins with specificity for different ubiquitin domains and different architectures (Aim 3).