Poly‐ubiquitin chains paradox: development of novel and selective poly‐ubiquitin binding domains (LB184)

Abstract

Ubiquitylation occurs through isopeptide linkage between the C‐terminus of Ub and the ε‐amino group of a lysine residue on the target substrate. Ub itself has seven Lys residues (K6, K11, K27, K29, K33, K48, and K63), each of which can participate in further ubiquitylation, generating poly‐Ub chains. The ability of Ub to form polymers through various lysines as well as the NH2‐terminus appears to be central to the versatility of this system in regulating cellular processes. The most extensively characterized of these polymers are linked through either K48 or K63. K48‐linked polyUb predominantly targets proteins for proteasomal degradation, whereas K63‐linked polyUb appears to regulate protein function, subcellular localization, or protein‐protein interactions. A growing body of evidence now implicates K11‐linked polyUb in mitotic regulation and endoplasmic reticulum associated degradation (ERAD). It is apparent from the above, that different poly‐ubiquitin linkages convey different information to the cell and suggests strongly that the cell contains elements capable of decoding this information, i.e. linkage specific ubiquitin binding domains. Development of Poly‐Ubiquitin selective tools will revolutionize the field of ubiquitin. Most UBDs described to date show little ability to discriminate between different linkages; although, it is possible to construct linkage specific tandem ubiquitin binding entities (TUBEs) by manipulating the spacing and rigidity of the linkers between tandem UBDs. We and our collaborators have constructed a series of K63‐specific UBDs and demonstrated their utility for Far Western blotting and “pull‐down” type experiments for evaluating the levels and identities of proteins bearing K63‐linked polyubiquitin chains under a variety of treatments. We will describe the results of proteomic experiments in which we identified new classes of UBDs. When validated, this information will have a major impact on our understanding of the functioning of the Ubiquitin‐Proteasome Pathway and suggest new targets for intervention.Grant Funding Source: Supported by R43CA165561