NSF/MCB-BSF: Cross-activation of ubiquitin and Rub1 to understand their roles as separate protein modifiers

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Just as facial recognition must pick out minor differences between seemingly similar individuals, cells too must discern minute differences between extremely similar, yet different proteins. At the focus of this research project are two proteins that are extremely similar in size, shape, and general features, yet behave quite differently. Both proteins are exceptional in that they attach to other proteins to alter their fate; in this manner, the influence of these two "protein modifiers" extends to essentially every aspect of cell biology. Yet, as similar as these two sibling proteins are, one is promiscuous - and as its name Ubiquitin implies, is ubiquitous throughout the cell - whereas the other "Resembles Ubiquitin 1" (Rub1) is shy and relegated. How does this happen? What are the molecular recognition devices that decide to give Ubiquitin free rein to roam throughout the cell, yet corral its more introvert sibling, Rub1, to a more localized pen? Correspondingly, their chores differ too: whereas Ubiquitin causes countless proteins to be sent to the cellular dump yards, Rub1 actually encourages its (carefully selected) targets to function properly. In joint teamwork, the laboratories of Prof Fushman at University of Maryland and Prof Glickman at Technion-IIT have found that Ubiquitin and Rub1 are not quite as segregated as generally thought. By molecular trickery some Rub1 appears to masquerade as ubiquitin and vice versa. Does this reflect inefficiencies in molecular recognition security checks, or does some fuzziness in their respective tasks play a positive role in cellular survival? Using a slew of biophysical, biochemical and molecular cell biology tools at their disposal, this project aims at understanding the unique properties of Rub1 and Ubiquitin and how these two proteins signal for distinct cellular outcomes despite their overwhelming similarities. Designing unique mutations, this project will study what happens when Rub1 and Ubiquitin swap roles inside cells. How far can one push molecular trickery before the cellular defenses catch on and either overcome the trespassers, or as in some pathological cases, apparently give up? The results of this research project will clarify just how recycling of old proteins through the use of Ubiquitin modification, helps keep cells young and healthy.This project aims at detailed comparison of Ubiquitin and Rub1, their physico-chemical properties, the conjugation targets/landscapes, and their binding partners in order to understand their unique properties and how these proteins signal for distinct cellular outcomes despite their overwhelming similarities. The research will test the hypothesis that although the two proteins are present as separate modifiers across eukarya, they are not maintained as completely non-interchangeable signals, and the cellular machinery allows for some cross-activation, primarily of Rub1 into the Ubiquitin signaling system. The mechanisms, prevalence, and the outcomes of such cross-activation are in the focus of this project. To achieve these goals, three Aims have been formulated. The first is to compare structural, biophysical, and biochemical characteristics of Rub1 and Ubiquitin as monomers and their ability to form polymers (homogeneous and mixed). By using established enzymatic and binding assays for each signal, this research will map at the atomic/residue level the distinguishing properties that define Rub1 and Ubiquitin as separate signals. The second Aim is to chart the extent of the "Rubylome" (i.e., the repertoire of Rub1 conjugation targets) and how it compares with the more extensively studied "Ubiquitinome". Studies of the Rubylome have been significantly hampered by the inability of current approaches to properly distinguish Rub1 and Ubiquitin conjugation sites. By using an innovative mass spectrometry approach, this project will obtain a full picture of unique versus shared conjugation targets, in particular the extent to which mixed Rub1-Ubiquitin polymers exist. The third Aim is to reveal to what extent Ubiquitin and Rub1 are maintained as separate signals in vivo, and what is the cellular outcome of their cross-activation. By engineering Rub1 and Ubiquitin variants capable of penetrating each other's signaling pathways, both in cells and in reconstituted enzymatic cascades, this research will characterize the resulting perturbations. Information garnered will be used to understand the unique properties of Rub1 and of Ubiquitin that enable these two proteins to act as distinct cellular signals. These comprehensive studies and comparison of Ubiquitin to Rub1 and of the outcomes of their cross-activation will (i) identify unique cellular conjugation targets and receptors for Rub1, (ii) reveal the inherent determinants of the Rub1 signal and the Ubiquitin signal, and (iii) provide a better understanding of what makes ubiquitin Ubiquitin. This collaborative US/Israel project is supported by the US National Science Foundation and the Israeli Binational Science Foundation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.