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

就像面部识别必须识别出看似相似的个体之间的微小差异一样，细胞也必须识别出极其相似但不同的蛋白质之间的微小差异。这个研究项目的重点是两种蛋白质，它们在大小、形状和一般特征上极其相似，但行为却截然不同。这两种蛋白质的特殊之处在于它们附着在其他蛋白质上，从而改变它们的命运；以这种方式，这两种“蛋白质修饰剂”的影响基本上延伸到细胞生物学的每一个方面。然而，尽管这两个兄弟蛋白很相似，但其中一个是混杂的——正如它的名字Ubiquitin所暗示的那样，它在整个细胞中无处不在——而另一个“类似于Ubiquitin 1”（Rub1）则是害羞的和被降格的。这是怎么发生的？是什么分子识别装置决定让泛素在细胞中自由漫游，而把它更内向的兄弟Rub1关在一个更局限的笼子里？相应地，它们的任务也不同：泛素导致无数的蛋白质被送到细胞垃圾场，而Rub1实际上鼓励它（精心挑选的）目标正常工作。在马里兰大学的富什曼教授和理工学院的格利克曼教授的联合团队合作中，他们发现Ubiquitin和Rub1并不像人们通常认为的那样完全分离。通过分子诡计，一些Rub1似乎伪装成泛素，反之亦然。这是否反映了分子识别安全检查的效率低下，或者它们各自任务中的一些模糊性在细胞存活中发挥了积极作用？利用大量的生物物理、生化和分子细胞生物学工具，该项目旨在了解Rub1和Ubiquitin的独特特性，以及这两种蛋白质如何发出不同的细胞结果信号，尽管它们具有压倒性的相似性。设计独特的突变，该项目将研究当Rub1和泛素在细胞内交换角色时会发生什么。在细胞防御系统发现并战胜入侵者之前，或者像在某些病理病例中那样，显然放弃之前，分子诡计能推进到什么程度？这个研究项目的结果将阐明如何通过使用泛素修饰来回收旧蛋白质，帮助细胞保持年轻和健康。该项目旨在详细比较Ubiquitin和Rub1，它们的物理化学性质，偶联靶点/景观，以及它们的结合伙伴，以了解它们的独特性质，以及尽管它们具有压倒性的相似性，但这些蛋白质如何发出不同细胞结果的信号。这项研究将验证这样一个假设，即尽管这两种蛋白质在真核生物中作为单独的修饰物存在，但它们并不是作为完全不可互换的信号来维持的，并且细胞机制允许一些交叉激活，主要是Rub1进入泛素信号系统。这种交叉激活的机制、流行程度和结果是本项目的重点。为了实现这些目标，我们制定了三个目标。首先是比较Rub1和Ubiquitin作为单体的结构、生物物理和生化特性及其形成聚合物（均质和混合）的能力。通过对每个信号使用已建立的酶和结合分析，本研究将在原子/残基水平上绘制将Rub1和Ubiquitin定义为独立信号的区分特性。第二个目的是绘制“Rubylome”的范围（即Rub1偶联靶标库），并将其与更广泛研究的“泛素组”进行比较。由于目前的方法无法正确区分Rub1和泛素结合位点，对红素的研究受到了很大的阻碍。通过使用创新的质谱分析方法，该项目将获得独特的与共享的偶联目标的全貌，特别是混合rub1 -泛素聚合物存在的程度。第三个目的是揭示Ubiquitin和Rub1在体内作为独立信号维持的程度，以及它们交叉激活的细胞结果。通过工程设计Rub1和泛素变体能够穿透彼此的信号通路，无论是在细胞中还是在重组的酶级联中，本研究将描述由此产生的扰动。所获得的信息将用于了解Rub1和泛素的独特特性，使这两种蛋白质能够作为不同的细胞信号。这些全面的研究和Ubiquitin与Rub1及其交叉激活结果的比较将(i)确定Rub1独特的细胞偶联靶点和受体，（ii）揭示Rub1信号和Ubiquitin信号的内在决定因素，以及（iii）更好地理解泛素是什么使泛素成为泛素。这个美国/以色列合作项目由美国国家科学基金会和以色列两国科学基金会支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.