Numerous studies have shown that the intracellular inclusions seen in several major human neurodegenerative diseases, including Alzheimer's disease, contain ubiquitin. This has led to speculation that a defect or alteration in the ubiquitin-mediated protein degradation pathway may be important in the pathogenesis of these diseases. For example, these ubiquitinated inclusions may be the result of cellular attempts to eliminate pathogenic insults by the activation of both extralysosomal (ubiquitin-mediated) and lysosomal mechanisms of intracellular protein degradation. However, the precise role of ubiquitin in the pathogenesis of these diseases is still not known. Although the structure of ubiquitin has been known for several years, the proteins involved in the ubiquitin-mediated protein degradation pathway, including both enzymes and products, have been difficult to isolate in large amounts. However, now that the genes for some of these proteins have been cloned, there are potentially a number of them that can be studied in more detail than was previously possible. The objective of this research is to expand our basic understanding of ubiquitin-mediated protein degradation by examining three components of the system, using protein X-ray crystallographic techniques. Determination of the structures of these proteins and correlation with their function should expand considerably our knowledge of the ubiquitin-mediated system of protein degradation. One of these proteins is that bind to ubiquitin and participate in a number of diverse cellular processes. This protein has been crystallized, and X-ray data have been collected for native and derivative crystals using an electronic area detector. The other two proteins have recently been purified in sufficient quantities to initiate crystallographic studies. The first is a di-ubiquitin conjugate joined through an isopeptide bond involving the COOH-terminus of one molecule and Lys48 of the other. This conjugate will serve as a model for the structure of ubiquitin- protein conjugates destined for degradation. The second is a ubiquitin carboxyl-terminal hydrolase, an enzyme that cleaves the isopeptide bond of ubiquitin-protein conjugates. Determination of this structure will provide important information about the mechanisms involved in the generation of free ubiquitin from conjugates.
