PRINCIPLES OF COMPLEX POLYUBIQUITIN SIGNALING, THE STRUCTURAL BASIS FOR UBIQUITIN-UBISTATIN INTERACTIONS, AND NOVEL ASSAYS FOR THE CHARACTERIZATION OF DEUBIQUITINASES

Abstract

Ubiquitination is the most versatile and is certainly one of the most difficult post-translation modifications to understand in eukaryotic life. In the process of ubiquitination the C-terminus of ubiquitin (Ub), a small 8.65 kDa protein is covalently attached to εNH2 groups of lysine side chains on target proteins. Once attached, additional Ubs can be added to the original Ub at eight unique linkage sites (M1, K6, K11, K27, K29, K33, K48, or K63) to form polyUb chains. This internal Ub-Ub linkage dictates the structural conformation of the polyUb chain, which in turn governs the receptors that can recognize a given chain. PolyUb chains were thought to be homogeneously linked until very recently when mixed linkage polyUb chains were detected on several cellular pathways. This observation implied that instead of having just eight distinct polyUb signals, there were now potentially quadrillions of unique chains. The results presented within represent the first in depth studies of mixed linkage polyUb chains, focusing on the structural impact of linkage mixing. For mixed K48 and K63 linked chains the findings support that their individual linkage properties are preserved regardless of linkage mixing. However, simulations for mixed linkage chains containing different linkages imply that many novel polyUb signals are possible.

The ubiquitin-proteasome pathway is the primary mechanism to degrade short lived proteins in the cell and has also emerged as a top therapeutic target. Ubistatins, a class of small molecules bring about the same effects as existing proteasome inhibitor drugs by directly binding the polyUb chain. However, virtually nothing is known about the structural properties for any ubistatin/Ub complex. Here is provided the first structure of a ubistatin/Ub complex along with data that overwhelmingly validates the structure. Other important factors regarding the ubistatin/Ub interaction including the stoichiometry and dual hydrophobic / electrostatic binding mechanism are also uncovered.

Proteomic analysis of polyUb conjugates has been limited to determining which linkage types are present. A novel method for K63 linked polyUb conjugates, which can measure consecutive K63 linkages is described here. This method allows the proteomics community to gain unprecedented information on cellular pathways utilizing K63 linkages.