Dynamic evolution of ubiquitin and of ubiquitin-like proteases

Ubiquitin is the most conserved protein in eukaryotes, and its role as a post-translational modifier, its effect on the functions of other cell proteins, is under intensive study. The aim of this dissertation is to shed light on functional and evolutionary aspects of ubiquitin itself, by employing bioinformatics, comparative genomics, structural biology and biochemistry. The following questions will be addressed: What is the specificity of ubiquitination? How promiscuous are ubiquitin proteases? When did pathogenic prokaryotes acquire such proteases? Why is ubiquitin expressed as different precursor proteins? Where do we find non-conserved ubiquitins, and what is their function?; Ubiquitination is governed by at least two layers of specificity, one of which being the transfer of ubiquitin to a specific lysine of the substrate. By analyzing preferred ubiquitination sites, I have found features likely to influence the transfer reaction, possibly explaining the function of some degrons.; Deubiquitinating enzymes remove ubiquitin from substrate proteins, but can they also remove ubiquitin-like proteins such as ISG15? Conducting a proteomic screen, I have discovered bispecificity in several proteases, most notably in a proteasome-associated deubiquitinating enzyme. Further analyses identify ISG15 as a post-translational modifier of proteins destined for proteasomal degradation.; Ubiquitin is unique to eukaryotes; yet pathogenic Chlamydiae express deubiquitinating enzymes of eukaryotic origin. Phylogenetic and biochemical studies suggest environmental fungi as the most likely donors for this lateral gene transfer across kingdoms. The evolutionary timing of the gene transfer indicates a possible connection to the emergence of pathogenicity in Chlamydiae.; Ubiquitin is encoded by a multi-gene family. The coevolution of these ubiquitin genes suggests frequent ectopic homologous recombinations in which the polyubiquitin gene acts as donor for gene conversion. Conservation of all ubiquitin genes might depend on such recombinatorial maintenance, and this might explain why one of the three genes is encoded in form of a concatemer.; However, some species fail to conserve all their respective ubiquitin genes. One of these variants, the "ubiquitin" moiety of a ribosomal subunit in G. lamblia, has no sequence similarity to ubiquitin. In the final part, the structure and chimeric function of this polypeptide will be discussed.