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

NSF/MCB-BSF：泛素和 Rub1 的交叉激活以了解它们作为单独的蛋白质修饰剂的作用

• 批准号: 1818280
• 负责人: David Fushman
• 金额: $ 60万
• 依托单位: University of Maryland, College Park
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1818280&HistoricalAwards=false
• 关键词: NSF; MCB; BSF; Cross; activation

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.

就像面部识别必须找出看似相似的个体之间的微小差异一样，细胞也必须分辨出极其相似但不同的蛋白质之间的微小差异。这项研究的重点是两种在大小、形状和一般特征上极其相似的蛋白质，但它们的行为却截然不同。这两种蛋白质都是特殊的，因为它们附着在其他蛋白质上改变自己的命运；在这种方式下，这两种“蛋白质修饰物”的影响几乎延伸到细胞生物学的各个方面。然而，尽管这两个兄弟蛋白很相似，但一个是混杂的--正如它的名字泛素所暗示的，在整个细胞中无处不在--而另一个“类似泛素1”(Rub1)是害羞的，被降级了。这是怎么发生的？是什么分子识别设备决定让泛素在细胞中自由漫步，而又将其更内向的兄弟姐妹Rub1关在更本地化的笔中？相应地，他们的工作也不同：泛素导致无数蛋白质被送到细胞垃圾场，而Rub1实际上鼓励其(精心挑选的)靶标正常工作。在联合团队合作中，马里兰大学的富什曼教授和理工学院的格利克曼教授的实验室发现，泛素和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低效，或者它们各自任务中的一些模糊性对细胞生存起到了积极作用？使用一系列生物物理、生化和分子细胞生物学工具，该项目旨在了解Rub1和泛素的独特性质，以及这两种蛋白质如何发出信号，指示不同的细胞结果，尽管它们有着惊人的相似之处。通过设计独特的突变，这个项目将研究当Rub1和泛素在细胞内互换角色时会发生什么。在细胞防御开始流行，或者克服入侵者，或者像在某些病理情况下，显然放弃之前，一个人可以把分子诡计推到什么程度？这一研究项目的结果将阐明如何通过泛素修饰来回收旧蛋白质，帮助保持细胞年轻和健康。本项目旨在详细比较泛素和Rub1的物理化学性质、结合靶标/景观和它们的结合伙伴，以了解它们的独特性质，以及这些蛋白质如何在极其相似的情况下传递不同的细胞结果信号。这项研究将检验这样一种假设，即尽管这两种蛋白质作为单独的修饰物存在于真核生物中，但它们并不是作为完全不可互换的信号保持的，而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、流行率和结果是本项目的重点。为了实现这些目标，制定了三个目标。第一个是比较Rub1和Ubiquitin作为单体的结构、生物物理和生化特征以及它们形成聚合物(均相和混合)的能力。通过对每个信号使用已建立的酶分析和结合分析，这项研究将在原子/残基水平上映射将Rub1和泛素定义为独立信号的区别性质。第二个目的是绘制“Rubylome”(即，Rub1结合靶标的谱系)的范围，以及它与更广泛研究的“泛素素”的比较情况。由于目前的方法不能正确区分Rub1和泛素结合位点，对Rubylome的研究受到了很大的阻碍。通过使用创新的质谱学方法，该项目将全面了解独特与共享的结合靶标，特别是Ru1-Ubiquitin混合聚合物的存在程度。第三个目标是揭示泛素和Rub1在多大程度上作为独立的信号在体内保持，以及它们交叉激活的细胞结果是什么。通过设计能够穿透彼此信号通路的Rub1和Ubiquitin变体，无论是在细胞中还是在重组的酶级联中，这项研究将表征由此产生的扰动。所获得的信息将被用来理解Rub1和泛素的独特属性，这些属性使这两种蛋白质充当不同的细胞信号。这些对泛素和Rub1及其交叉激活结果的全面研究和比较将(I)确定Rub1独特的细胞结合靶点和受体，(Ii)揭示Rub1信号和泛素信号的内在决定因素，以及(Iii)更好地理解是什么导致泛素泛素。这一美国/以色列合作项目得到了美国国家科学基金会和以色列双国科学基金会的支持。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

A novel recognition site for polyubiquitin and ubiquitin-like signals in an unexpected region of proteasomal subunit Rpn1.

• DOI: 10.1016/j.jbc.2021.101052
• 发表时间: 2021-09
• 期刊: The Journal of biological chemistry
• 作者: Boughton AJ;Liu L;Lavy T;Kleifeld O;Fushman D
• 通讯作者: Fushman D