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

基本信息

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

项目摘要

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.

正如面部识别必须在看似相似的个体之间挑出较小的差异一样，细胞也必须辨别极度相似但不同的蛋白质之间的微小差异。该研究项目的重点是两种蛋白质，大小，形状和一般特征非常相似，但行为的行为却大不相同。两种蛋白质都是例外的，因为它们附着在其他蛋白质上以改变其命运。以这种方式，这两个“蛋白质修饰剂”的影响扩展到细胞生物学的各个方面。然而，与这两个兄弟姐妹蛋白相似，一种是混杂的 - 众所周知的泛素所暗示的，在整个细胞中都存在普遍存在 - 而另一个“类似于泛素1”（rub1）（rub1）害羞而降级。这是怎么发生的？哪些分子识别设备决定使整个细胞中无泛素的漫游，但是将其内向的兄弟姐妹rub1带到更本地化的笔中？相应地，它们的琐事也有所不同：而泛素会导致无数蛋白质发送到蜂窝垃圾场，但RUB1实际上鼓励其（精心选择的）目标正常运行。在联合团队合作中，马里兰州大学的Fushman教授和Technion-IIT教授的实验室发现，泛素和Rub1并不像一般认为的那样隔离。通过分子欺骗，一些rub1似乎是化妆为泛素，反之亦然。这是否反映了分子识别安全检查中的效率低下，还是在各自任务中的某些模糊性在细胞存活中起积极作用？该项目使用一系列生物物理，生化和分子细胞生物学工具，旨在了解Rub1和泛素的独特特性，以及这两种蛋白质如何以明显的细胞结局信号，尽管它们具有压倒性的相似性。设计独特的突变，该项目将研究细胞内Rub1和泛素交换作用时会发生什么。一个人可以在细胞防御措施之前推动分子欺骗，并克服侵入者，或者像在某些病理情况下一样，显然会放弃？该研究项目的结果将阐明如何通过使用泛素修饰来循环旧蛋白质，有助于保持细胞的健康状况。该项目旨在详细比较泛素和rub1的详细比较，其物理化学特性，它们的结合靶标和它们的独特属性，以使其具有独特的属性，并具有这些特性，以使其具有独特的属性，并具有这些特质的信号，并且它们的属性构成了这些属性，并且它们的属性构成了这些特性，并相似之处。这项研究将检验以下假设：尽管两种蛋白质作为整个Eukarya的独立修饰符都存在，但它们并不能保持为完全不可交流的信号，并且细胞机械允许一些交叉激活，主要是RUB1进入泛素信号系统。这种交叉激活的机制，流行率和结果是该项目的重点。为了实现这些目标，已经制定了三个目标。首先是将RUB1和泛素的结构，生物物理和生物化学特征与单体及其形成聚合物（均匀和混合）的能力进行比较。通过为每个信号使用已建立的酶促测定和结合测定，本研究将在原子/残基级别映射，将RUB1和泛素定义为单独信号的区别特性。第二个目的是绘制“ Rubylome”（即Rub1共轭靶标的曲目）的范围，以及如何与更广泛研究的“泛素组”进行比较。对于正确区分Rub1和泛素结合位点的目前方法，对Rubylome的研究已受到显着阻碍。通过使用创新的质谱法，该项目将获得独特的和共享共轭靶标的完整图片，特别是混合rub1-泛素聚合物存在的程度。第三个目的是揭示泛素和rub1在体内的单独信号以及它们交叉激活的细胞结果是什么。通过工程rub1和泛素变体能够在细胞和重构的酶级联反应中穿透彼此的信号通路，这项研究将表征所得的扰动。获得的信息将用于了解RUB1和泛素的独特特性，从而使这两种蛋白质充当不同的细胞信号。这些全面的研究和泛素与RUB1的比较以及它们交叉激活的结果将（i）确定RUB1的独特细胞共轭靶标和受体，（ii）揭示了RUB1信号和泛素信号的固有决定因素，以及（III）更好地理解了什么使ubiquitin ubiquitin的理解。美国国家科学基金会和以色列双原则科学基金会的支持。该奖项反映了NSF的法定任务，并使用该基金会的知识分子优点和更广泛的影响审查标准来反映NSF的法定任务。