权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Mechanistic and Structural Insights into NO sensing by Iron-Sulfur Cluster Regulators

铁硫簇调节器对 NO 传感的机理和结构见解

基本信息

批准号：
BB/P006140/1
负责人：
Nicolas Le Brun
金额：
$ 51.41万
依托单位：
University of East Anglia
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FP006140%2F1
关键词：
Mechanistic Structural Insights into NO

项目摘要

Nitric oxide (NO) is a toxic molecule that is generated by soil bacteria and in our bodies as a defence against pathogenic organisms trying to establish infection. One of the major ways by which NO exerts its toxic effects is through reaction with a widespread group of proteins that bind a type of cofactor containing both iron and sulfur arranged as a cluster. Members of this group play crucial roles in a very wide range of processes, including respiration and protein synthesis. To avoid NO toxicity, pathogenic (as well as harmless) organisms have evolved protective systems that detoxify NO by removing it through chemical reaction. The fact that iron-sulfur clusters are particularly sensitive to NO (and their modification is a major route by which NO exerts its toxic effects) has been exploited in nature, through the evolution of a number of regulatory proteins that themselves contain an iron-sulfur cluster and which function as biological switches, turning on the cellular detoxification response in the presence of NO. Despite the importance and widespread nature of the reaction of iron-sulfur clusters with NO, we still know relatively little about this process. Some important progress has been made in recent years, but the difficulties associated with working with iron-sulfur proteins, which are fragile and must be handled in O2-free environments, and with detecting and unambiguously identifying intermediates and products of the cluster reaction with NO have, up to now, been major obstacles.The project described in this proposal will lead to a major advance in our understanding of how NO-responsive iron-sulfur cluster-containing regulators function. The major subject of our proposed study is an iron-sulfur cluster regulator that is a member of a large and not well understood family of regulators found in a wide range of pathogenic and non-pathogenic bacteria, in which it functions as a primary NO sensor by controlling the cellular response to NO toxicity. We will also study a second regulatory protein that belongs to a family found only in a small number of bacteria, but which includes the pathogen that causes tuberculosis, one of the world's major killers, and the bacterium that is the source of many of the antibiotics currently in use in the clinic. Members of this family play key roles in cell developmental processes associated with stress response, including sporulation and dormancy, which is important for the ability of the tuberculosis pathogen to survive in the inhospitable environment of a human host for years, in a state that is highly resistant to antibiotics.The project will build on three important recent breakthroughs. Firstly, we have established novel mass spectrometry methodologies that enable us to detect iron-sulfur cluster regulators with their clusters intact. This now provides the opportunity to follow by mass spectrometry the reaction of the cluster with NO by detecting and identifying intermediates and products formed. Secondly, we have developed novel ways of studying the same proteins using vibrational spectroscopy, providing characteristic signatures according to the iron-NO complexes formed. Finally, working with a group in France, we have determined the high resolution structure of one of the regulators with its iron-sulfur cluster bound. This is a first for this family of iron-sulfur cluster regulators and provides the ideal basis on which to understand how the cluster promotes DNA binding and how it reacts with NO. We will exploit these recent advances to explore using a range of approaches the biochemistry of the reaction of NO with these proteins, revealing unprecedented mechanistic insight into how NO-sensing regulatory proteins function, and providing clues about how NO sensing, and therefore survival, of pathogens could be disrupted/prevented.

一氧化氮（NO）是一种有毒分子，由土壤细菌产生，在我们的身体中作为防御病原体试图建立感染。NO发挥其毒性作用的主要方式之一是通过与一组广泛存在的蛋白质反应，这些蛋白质结合一种含有铁和硫的辅因子，这些辅因子排列成簇。这一组的成员在非常广泛的过程中发挥着关键作用，包括呼吸和蛋白质合成。为了避免NO毒性，致病（以及无害）生物已经进化出通过化学反应去除NO来解毒NO的保护系统。铁硫团簇对NO特别敏感的事实（并且它们的修饰是NO发挥其毒性作用的主要途径）已经在自然界中被利用，通过许多调节蛋白的进化，所述调节蛋白本身含有铁-硫簇并且充当生物开关，在NO存在下开启细胞解毒反应。尽管铁-硫簇与NO反应的重要性和广泛性，我们对这个过程仍然知之甚少。近年来已经取得了一些重要的进展，但是与铁硫蛋白相关的困难，铁硫蛋白是脆弱的，必须在无O2环境中处理，以及检测和明确识别与NO的簇合反应的中间体和产物，到目前为止，本提案中描述的项目将导致我们对NO响应性含铁硫簇调节剂如何起作用的理解的重大进展。我们提出的研究的主要课题是铁硫簇调节剂，这是一个大的，并没有很好地理解在广泛的致病性和非致病性细菌中发现的调节剂家族的成员，其中它作为一个主要的NO传感器通过控制细胞对NO毒性的反应。我们还将研究第二种调节蛋白，它属于一个只在少数细菌中发现的家族，但其中包括导致结核病的病原体，结核病是世界上主要的杀手之一，并且是目前临床使用的许多抗生素的来源。该家族的成员在与应激反应相关的细胞发育过程中发挥关键作用，包括孢子形成和休眠，这对于结核病病原体在人类宿主的恶劣环境中生存多年的能力至关重要，并且对抗生素具有高度耐药性。该项目将建立在最近三项重要突破的基础上。首先，我们已经建立了新的质谱方法，使我们能够检测铁硫簇调节剂与他们的集群完整。这现在提供了通过质谱法检测和鉴定形成的中间体和产物来跟踪簇与NO的反应的机会。其次，我们已经开发了新的方法来研究相同的蛋白质使用振动光谱，根据铁-NO复合物形成提供特征签名。最后，与法国的一个小组合作，我们已经确定了其中一个具有铁硫簇结合的调节剂的高分辨率结构。这是第一次为这个家庭的铁硫簇调节剂，并提供了理想的基础上，了解如何集群促进DNA结合，以及它如何与NO反应。我们将利用这些最新的进展，探索使用一系列的方法的生物化学反应的NO与这些蛋白质，揭示前所未有的机械洞察如何NO传感调节蛋白质的功能，并提供关于如何干扰/阻止病原体的NO感测以及因此的存活的线索。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The Molecular Bases of the Dual Regulation of Bacterial Iron Sulfur Cluster Biogenesis by CyaY and IscX.

DOI：
10.3389/fmolb.2017.00097
发表时间：
2017
期刊：
Frontiers in molecular biosciences
影响因子：
5
作者：
Adinolfi S;Puglisi R;Crack JC;Iannuzzi C;Dal Piaz F;Konarev PV;Svergun DI;Martin S;Le Brun NE;Pastore A
通讯作者：
Pastore A

The Di-Iron Protein YtfE Is a Nitric Oxide-Generating Nitrite Reductase Involved in the Management of Nitrosative Stress.

DOI：
10.1021/jacs.1c12407
发表时间：
2022-04-27
期刊：
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
影响因子：
15
作者：
Crack, Jason C.;Balasiny, Basema K.;Bennett, Sophie P.;Rolfe, Matthew D.;Froes, Afonso;MacMillan, Fraser;Green, Jeffrey;Cole, Jeffrey A.;Le Brun, Nick E.
通讯作者：
Le Brun, Nick E.

Mass spectrometric detection of iron nitrosyls, sulfide oxidation and mycothiolation during nitrosylation of the NO sensor [4Fe-4S] NsrR.

NO传感器[4FE-4S] NSRR的亚硝基化过程中亚硝基素，硫化物氧化和霉菌性的质谱检测。