Project Summary Post-translational signaling through ubiquitination is essential to all eukaryotic life, and dysregulation of this process can lead to severe forms of disease. The far-reaching importance of ubiquitin signaling across many cellular processes stems from its ability to form a diverse set of polymeric chains that signal for distinct outcomes. The complexity of ubiquitin signaling vastly outweighs our understanding of its regulation and cellular outcomes. While the signaling roles for some ubiquitin chain types are known (e.g. protein degradation or cell cycle regulation), the functions of many so-called `atypical' chains have remained a mystery despite decades of research. As an alternative approach to studying fundamental human biology, we study the interactions between invading pathogenic bacteria and the host ubiquitin signaling network. In addition to a range of other ubiquitin-targeted activities, to support infection bacteria have evolved secreted effector proteins to assemble and remove host ubiquitin signals, in some cases with exquisite specificity toward discrete ubiquitin chain types. We propose that ubiquitin-targeted bacterial effectors represent a rich opportunity to study human ubiquitin signaling from an `outside-in' perspective. To explore this opportunity, we have developed a multipronged approach that has identified novel ubiquitin-targeted activities among important bacterial pathogens. Using structural and biochemical approaches we will explain the mechanisms and specificities of these bacterial enzymes, at which point they will be used as case studies and tools to extend our work toward deciphering the regulatory and signaling complexities of the human ubiquitin system. Our innovative approach to studying ubiquitin biology plays to our strengths in the biochemical mechanisms of its regulation, and leverages the strong evolutionary pressure placed on bacteria to usurp specific aspects of host ubiquitin signaling. Over the next five years we will demonstrate the breadth of bacterial ubiquitin-targeted activities and their utility for dissecting the intricacies of ubiquitin signaling, which feeds into our larger vision of understanding the motivations and ramifications of bacterial interference in host signaling processes.