Chemical proteomic mapping of redox signalling in the intracellular pathogen, Toxoplasma gondii

细胞内病原体弓形虫氧化还原信号的化学蛋白质组图谱

• 批准号: 1949152
• 金额: --
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 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=studentship-1949152
• 关键词: Chemical; proteomic; mapping; redox; signalling

BackgroundOf all protozoan parasitic infections, T. gondii is the most widespread, infecting 30-50% of the human population across 88 countries. Clinical toxoplasmosis is a serious health risk for immunodeficient individuals, and T. gondii is a significant veterinary pathogen, imposing economic burden on agricultural industries. However, current drugs suffer from issues with toxicity, prolonged treatment regimens and the emergence of resistance. Hence, there is a continuing need to discover novel drug targets.As an obligate intracellular pathogen, T. gondii requires the environment of a host cell to survive and propagate. Pathogenesis is driven by iterative lytic growth of asexual tachyzoites, a process associated with a range of molecular events that occur in both the parasite and host. Signalling molecules such as Ca2, K and cyclic nucleotides are key regulators of tachyzoite invasion, replication and egress. More recently, hydrogen peroxide (H2O2) and nitric oxide (NO) have been recognised as important signalling molecules in eukaryotes. While notorious transducers of stress, at low levels these reactive oxygen species (ROS) can modulate protein function via reversible, discrete and selective oxidation of redox-sensitive cysteine residues. For H2O2, cysteine oxidation can alter the activity and/or localisation of diverse protein classes including kinases, phosphatases, ion channels and metabolic enzymes. Indeed, T. gondii encounters ROS throughout its life cycle and is known to suppress the production of H2O2 during oxidative challenge by host macrophages. Despite this, the full impact of redox signals on the parasite's biology is unclear.As part of efforts to identify novel druggable nodes in the proteomes of intracellular pathogens such as T. gondii and Plasmodium, the overarching aim of this project is to molecularly map redox signalling in T. gondii. To achieve this, a combination of multidisciplinary techniques spanning biochemistry, molecular, cell and chemical biology will be used.Objectives1. Identify protein-associated reactive cysteines using chemical proteomicsRedox signalling is associated with the sensitivity of reactive cysteine thiols to oxidative post-translational modification. To first profile cysteine thiol reactivity in the T. gondii proteome, a quantitative mass-spectrometry-based chemical proteomic workflow will be established based on a published platform. A series of bioinformatics analyses will be performed on identified hits to gain insight into their biological importance and prioritise downstream molecular interrogation.2. Systematic genetic validation of reactive cysteinesTo systematically assess the contribution of the identified reactive cysteines to protein function and parasite fitness, a novel CRISPR/Cas9-based phenotypic screen will be undertaken. Those cysteines considered essential will be prioritised for validation using traditional genetic and biochemical approaches.3. Identification and characterisation of redox sensorsThe proteomic workflow described in Objective 1 will be modified to enable detection of reactive cysteines that are sensitive to H2O2 oxidation and thus have the capacity to transduce redox signals. Redox sensors will be validated biochemically, and reverse genetic approaches will be used to assess their contribution to essential cellular processes including host-cell invasion, replication and egress.Broad impactThe current resurgence of covalent drugs reflects their track-record of success in the clinic. Examples include the inhibitors Afatinib and Ibrutinib, which form covalent bonds with cancer-associated kinases. With global sales of Ibrutinib expected to reach $9 billion in 2020, there is sustained industrial interest in covalent inhibitors targeting cysteines. Our research has the potential to uncover new druggable hotspots in clinically-important parasites, and thus has broad impact on industry and health.

在所有原生动物寄生虫感染中，弓形虫分布最广，感染了88个国家30-50%的人口。临床弓形虫病是免疫缺陷个体的严重健康风险，弓形虫是一种重要的兽医病原体，给农业带来了经济负担。然而，目前的药物存在毒性、治疗方案延长和耐药性出现等问题。因此，持续需要发现新的药物靶点。作为一种专性细胞内病原体，弓形虫需要宿主细胞的环境才能生存和繁殖。发病机制是由无性速殖子的反复裂解生长驱动的，这一过程与发生在寄生虫和宿主体内的一系列分子事件有关。信号分子如Ca2， K和环核苷酸是速殖子入侵，复制和退出的关键调节因子。最近，过氧化氢（H2O2）和一氧化氮（NO）被认为是真核生物中重要的信号分子。虽然众所周知的应激转导器，但在低水平下，这些活性氧（ROS）可以通过氧化还原敏感的半胱氨酸残基的可逆、离散和选择性氧化来调节蛋白质功能。对于H2O2，半胱氨酸氧化可以改变多种蛋白质类别的活性和/或定位，包括激酶、磷酸酶、离子通道和代谢酶。事实上，弓形虫在其整个生命周期中都会遇到ROS，并且已知在宿主巨噬细胞的氧化挑战中会抑制H2O2的产生。尽管如此，氧化还原信号对寄生虫生物学的全面影响尚不清楚。作为鉴定细胞内病原体（如弓形虫和疟原虫）蛋白质组中新的可药物节点的努力的一部分，本项目的总体目标是绘制弓形虫氧化还原信号的分子图谱。为了实现这一目标，将结合生物化学、分子生物学、细胞生物学和化学生物学等多学科技术。利用化学蛋白质组学鉴定与蛋白质相关的活性半胱氨酸氧化还原信号与活性半胱氨酸硫醇对氧化翻译后修饰的敏感性有关。为了首次分析弓形虫蛋白质组中的半胱氨酸硫醇反应性，将基于已发表的平台建立基于质谱的定量化学蛋白质组工作流程。一系列的生物信息学分析将对确定的hit进行，以深入了解其生物学重要性，并优先进行下游分子询问。为了系统地评估已鉴定的活性半胱氨酸对蛋白质功能和寄生虫适应性的贡献，将进行一种新的基于CRISPR/ cas9的表型筛选。那些被认为必需的半胱氨酸将优先使用传统的遗传和生化方法进行验证。氧化还原传感器的鉴定和表征目标1中描述的蛋白质组学工作流程将被修改，以能够检测对H2O2氧化敏感的活性半胱氨酸，从而具有转导氧化还原信号的能力。氧化还原传感器将被生化验证，反向遗传方法将被用来评估它们对基本细胞过程的贡献，包括宿主细胞的入侵、复制和退出。广泛的影响当前共价药物的复苏反映了它们在临床中的成功记录。例子包括抑制剂阿法替尼和伊鲁替尼，它们与癌症相关激酶形成共价键。Ibrutinib的全球销售额预计到2020年将达到90亿美元，业界对靶向半胱氨酸的共价抑制剂持续感兴趣。我们的研究有可能在临床重要的寄生虫中发现新的药物热点，从而对工业和健康产生广泛的影响。

项目成果

Cellular barcoding of protozoan pathogens reveals the within-host population dynamics of Toxoplasma gondii host colonization

原生动物病原体的细胞条形码揭示了弓形虫宿主定植的宿主内群体动态

• DOI: 10.1101/2020.08.06.239822
• 发表时间: 2020
• 作者: Wincott C

Activity- and reactivity-based proteomics: Recent technological advances and applications in drug discovery.

• DOI: 10.1016/j.cbpa.2020.06.011
• 发表时间: 2021-02-01
• 期刊: Current opinion in chemical biology
• 影响因子: 7.8
• 作者: Benns, Henry James;Wincott, Ceire Joanne;Child, Matthew Andrew
• 通讯作者: Child, Matthew Andrew

An extracellular redox signal triggers calcium release and impacts the asexual development of Toxoplasma gondii

细胞外氧化还原信号触发钙释放并影响弓形虫的无性发育

• DOI: 10.1101/2021.02.04.429728
• 发表时间: 2021
• 作者: Alves E

Prioritization of antimicrobial targets by CRISPR-based oligo recombineering

• DOI: 10.1101/2021.02.04.429737
• 发表时间: 2021-02
• 期刊: bioRxiv
• 作者: H. Benns;M. Storch;J. Falco;F. Fisher;E. Alves;C. Wincott;J. Baum;G. Baldwin;E. Weerapana;E. Tate;M. Child