Pharmacologic enhancement of UBA1 activity in models of VEXAS syndrome

VEXAS 综合征模型中 UBA1 活性的药理学增强

• 批准号: 10705749
• 负责人: Shengyun Fang
• 金额: $ 39.46万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-16 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10705749
• 关键词: Acceleration; Adult; Auranofin; Binding; Biochemical; Bioinformatics; Biological Assay; CRISPR/Cas technology; Cell Line; Cell model; Cells; Cellular biology; Charge; Clinical; Co-Immunoprecipitations; Codon Nucleotides; Cryoelectron Microscopy; Data; Defect; Disease; Dose; Endoplasmic Reticulum; Enhancers; Enzymes; Exhibits; Gene Expression; Genes; Goals; Hematopoietic stem cells; Immune; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Innate Immune Response; Interferon Type II; Interferons; Interleukin-6; Investigation; Link; Longevity; Mass Spectrum Analysis; Mediating; Missense Mutation; Modeling; Molecular Conformation; Mutation; Myelogenous; Myeloid Cells; NF-kappa B; Natural Immunity; Pathway interactions; Patients; Pharmaceutical Preparations; Process; Protein Secretion; Proteins; Proteome; Proteomics; Quantitative Reverse Transcriptase PCR; Recommendation; Refractory; Reporting; Rheumatoid Arthritis; Serum; Signal Transduction; Spectrometry; Stress; Syndrome; TNF gene; Testing; Text; Therapeutic; Therapeutic Effect; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; United States National Institutes of Health; Zebrafish; attenuation; autoinflammatory diseases; biological adaptation to stress; cell type; cytokine; effective therapy; endoplasmic reticulum stress; in vitro testing; misfolded protein; monocyte; mutant; neutrophil; novel; peripheral blood; pharmacologic; protein expression; proteostasis; public health relevance; reconstitution; response; therapeutic evaluation; treatment strategy; ubiquitin-protein ligase

SUMMARY VEXAS syndrome is a newly discovered adult-onset autoinflammatory disease that is refractory to treatment and often fatal. It is caused by somatic missense mutations of the X-linked E1 ubiquitin-like modifier-activating enzyme 1 gene (uba1) in hematopoietic stem cells. UBA1 protein catalyzes the initiation of most ubiquitination processes in cells. Mutant UBA1 proteins have reduced enzymatic activity. Uba1 mutant myeloid cells exhibit decreased ubiquitination and activated innate immune and stress pathways. Thus, enhancing UBA1 activity could be an effective treatment strategy for VEXAS syndrome. In preliminary studies, we found that auranofin, a clinical drug for rheumatoid arthritis, binds to UBA1, enhances UBA1 interactions with E2 ubiquitin-conjugating enzymes. As a result, auranofin has a general activity in promoting E3 ubiquitin ligase activities and accelerates the degradation of proteasomal substrate proteins. Significantly, auranofin also markedly promotes the activity of the major VEXAS-causing mutant UBA1 protein. This novel activity of auranofin requires concentrations about 5 to 18 times lower than the maximum serum concentration (Cmax) achieved by the therapeutic dose for rheumatoid arthritis. Thus, auranofin, as a UBA1 activity enhancer, may have tremendous therapeutic potential for VEXAS syndrome. In this proposal, our goals are to investigate 1) the mechanism by which auranofin enhances UBA1 activity in ubiquitination and 2) the potential of auranofin as a much-needed drug for VEXAS syndrome. To facilitate the investigation, we have created U937 monocyte-like cell lines that harbor the primary VEXAS-causing mutation (p.Met41 codon mutation) in uba1 (U937Duba1b) by CRISPR/cas9 technology. Our preliminary studies showed that U937Duba1b cells exhibit the similar pro-inflammatory and stress responses to that reported in patient-derived uba1 mutant monocytes, particularly the highly activated inflammatory signatures, including TNFa, IL-6, and IFN-g, and ER stress. We have obtained the zebrafish model (Duba1b) of VEXAS syndrome from Dr. David Beck (NIH) to assess the therapeutic effects of auranofin. Three specific Aims are proposed to accomplish our goals. Aim 1: To define the mechanism of how auranofin enhances UBA1 activity in ubiquitination. We will test the hypothesis that auranofin binding changes UBA1 conformation and enhances UBA1 interaction with E2s, resulting in increased ubiquitin charging to E2s and increased activities of E3s. We will use ubiquitination of misfolded proteins in the endoplasmic reticulum as an example to validate the mechanism in cells. Aim 2: To determine whether auranofin inhibits the major pro-inflammatory signaling (TNFa via NFkB, IL-6, and IFN-g) identified in patient-derived uba1 mutant monocytes. U937Duba1b cells will be used as a cellular model of VEXAS syndrome to determine whether auranofin inhibits the pro-inflammatory cytokine expression and their signaling. Aim 3: To profile ubiquitination reduction and proteostasis disturbance in U937Duba1b cells and determine whether auranofin alleviates the changes. Decreased ubiquitination (ubiquitinome) is likely the cause for VEXAS syndrome. Therefore, we will use a combination of quantitative mass spectrometry-based proteomics and bioinformatics to profile the changes in ubiquitinome and proteome in U937Duba1b cells. We will assess whether auranofin reverses the changes and inhibits pro- inflammatory responses and evaluate the therapeutic potential of auranofin in the zebrafish model of VEXAS.

总结 VEXAS综合征是一种新发现的成人发病的自身炎症性疾病， 往往是致命的。它是由X连锁的E1泛素样修饰物激活酶1基因的体细胞错义突变引起的 （uba1）在造血干细胞中。UBA 1蛋白催化细胞中大多数泛素化过程的起始。突变体 UBA1蛋白具有降低的酶活性。Uba1突变型髓样细胞表现出泛素化降低和活化 先天免疫和应激途径。因此，增强UBA 1活性可能是VEXAS的有效治疗策略 综合征在初步研究中，我们发现金诺芬，一种治疗类风湿关节炎的临床药物，与UBA 1结合， UBA1与E2泛素结合酶的相互作用。结果，金诺芬具有促进E3的一般活性， 泛素连接酶的活性和加速降解的蛋白酶体底物蛋白。值得注意的是，金诺芬还 显著促进主要VEXAS引起的突变体UBA 1蛋白的活性。金诺芬的这种新活性 需要比治疗药物达到的最大血清浓度（Cmax）低约5至18倍的浓度 类风湿关节炎的治疗方法因此，金诺芬作为UBA 1活性增强剂，可能具有巨大的治疗潜力， Vexas综合征。在这项提案中，我们的目标是研究1）金诺芬增强UBA 1的机制 泛素化活性和2）金诺芬作为VEXAS综合征急需药物的潜力。方便 在研究中，我们已经建立了U937单核细胞样细胞系，其具有主要的VEXAS引起的突变（p.Met41 通过CRISPR/cas9技术检测uba1（U937Duba1b）中的密码子突变。我们的初步研究表明，U937Duba1b 细胞表现出与在患者来源的UBA 1突变单核细胞中报道的相似的促炎和应激反应， 特别是高度活化的炎症信号，包括TNF α、IL-6和IFN-γ，以及ER应激。我们有 从大卫贝克博士（NIH）处获得了VEXAS综合征的斑马鱼模型（Duba1b），以评估治疗效果 金诺芬为实现我们的目标，提出了三个具体目标。目的1：确定金诺芬如何 增强UBA 1在泛素化中的活性。我们将检验金诺芬结合改变UBA 1构象的假设 并增强UBA 1与E2 s的相互作用，导致泛素对E2 s的充电增加和E3 s活性增加。 我们将使用内质网中错误折叠蛋白的泛素化作为一个例子来验证这种机制， 细胞目的2：确定金诺芬是否抑制主要的促炎信号传导（TNF α通过NF κ B、IL-6和TNF α介导的炎症反应）。 IFN-g）在患者来源的uba1突变单核细胞中鉴定。U937Duba1b细胞将用作VEXAS的细胞模型 综合征，以确定金诺芬是否抑制促炎细胞因子表达及其信号传导。目标3： 分析U937Duba1b细胞中泛素化减少和蛋白质稳态紊乱，并确定金诺芬是否 确认更改。泛素化（ubiquitinome）的减少可能是VEXAS综合征的原因。所以我们 将使用基于定量质谱的蛋白质组学和生物信息学相结合的方法来描述 U937Duba1b细胞中的泛素组和蛋白质组。我们将评估金诺芬是否逆转了这些变化并抑制了前- 在VEXAS的斑马鱼模型中，研究金诺芬对炎症反应的影响，并评估金诺芬的治疗潜力。

项目成果

Auranofin targets UBA1 and enhances UBA1 activity by facilitating ubiquitin trans-thioesterification to E2 ubiquitin-conjugating enzymes.

• DOI: 10.1038/s41467-023-40537-x
• 发表时间: 2023-08-09
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Yan, Wenjing;Zhong, Yongwang;Hu, Xin;Xu, Tuan;Zhang, Yinghua;Kales, Stephen;Qu, Yanyan;Talley, Daniel C. C.;Baljinnyam, Bolormaa;LeClair, Christopher A. A.;Simeonov, Anton;Polster, Brian M. M.;Huang, Ruili;Ye, Yihong;Rai, Ganesha;Henderson, Mark J. J.;Tao, Dingyin;Fang, Shengyun
• 通讯作者: Fang, Shengyun