New tools for ubiquitin signaling.

Protein ubiquitination is rapidly emerging as a key regulatory mechanism of many cellular processes. Important roles in proteasomal degradation are contrasted by various non-degradative functions of ubiquitin in intracellular trafficking, signal transduction and the DNA damage response. This versatility arises from eight structurally and functionally distinct polyubiquitin chains that co-exist in cells.1,2 Biochemical characterization identified proteins and enzymes that specifically assemble, recognize and hydrolyze polyubiquitin in a linkagespecific fashion, and structural analysis starts to reveal mechanisms of linkage recognition. What has been much more challenging is to understand the cellular roles of individual chain types and the interplay of the different forms of ubiquitination. A particularly fascinating area of ubiquitin research evolves around cytokine signaling pathways, for example, the response to tumor necrosis factor (TNF)-α. In addition to canonical roles of Lys48-linked chains as a degradation signal, the first described instances of non-degradative Lys63-linked ubiquitination in nuclear factor κB (NFκB) signaling more than 10 years ago opened the door for a vast number of discoveries. Recently, the picture became even more complex with reports implicating Lys11 chains and linear (or Met1linked) chains in TNFα signaling. The idea that linear chains cooperate with Lys63-linkages in activating cytokine-triggered kinases including inhibitor of κB kinase has resulted in some debate. However, the recent identification of SHARPIN as a component of the linear ubiquitin chain assembly complex, LUBAC, and its important roles in inflammation further supported that linear chains are key players in NFκB signaling. Despite this biochemical and genetic evidence, reagents that directly interrogate the roles of linear versus other polyubiquitin chains in vivo have been lacking. In this issue of Journal of Molecular Biology, Matsumoto et al. report the engineering of a linkage-specific antibody against linear ubiquitin chains and show its application in Western blotting, immunoprecipitation and immunofluorescence studies.12 A strategy that combined phage display