Ubiquitin-dependent cell-fate decisions during human development and disease

人类发育和疾病过程中泛素依赖性细胞命运决定

• 批准号: 10920191
• 负责人: Achim Werner
• 金额: $ 178.55万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10920191
• 关键词: Alleles; Anterior; Biochemical; Biological Assay; Blood; Brain; CDC42 gene; CUL3 gene; Cell Differentiation process; Cell Fate Control; Cells; Chick Embryo; Clinical; Codon Nucleotides; Collaborations; Complex; Cytoplasm; Cytoskeleton; Data; Defect; Development; Disease; Dissection; Ectoderm; Ectoderm Cell; Embryonic Development; Ensure; Enzymes; Future; Genetic Transcription; Genotype; Goals; Head; Hematopoiesis; Hematopoietic; Human Development; Human Genetics; Inflammatory; Initiator Codon; Lead; Ligation; Modeling; Molecular; Monomeric GTP-Binding Proteins; Morphology; Mutation; Myeloid Cells; National Institute of Arthritis, and Musculoskeletal, and Skin Diseases; Neural Crest; Neural Tube Closure; Neural tube; Neurons; Pathogenesis; Pathogenicity; Patients; Pattern; Post-Translational Protein Processing; Protein Isoforms; Proteins; Proteomics; Regulation; Reporter; Reporting; Research; Residual state; Risk; Role; Signal Pathway; Signal Transduction; Single Nucleotide Polymorphism; Skeleton; Skin; Syndrome; Translations; Ubiquitin; United States National Institutes of Health; Variant; X Chromosome; autoinflammation; autoinflammatory diseases; clinical diagnosis; clinical predictors; cohort; craniofacial; craniofacial development; developmental disease; disease prognosis; functional loss; human disease; human embryonic stem cell; imaging approach; in vitro Model; induced pluripotent stem cell; inhibitor; insight; member; novel; novel therapeutic intervention; p21 activated kinase; programs; screening; self-renewal; stem cells; survival prediction; ubiquitin ligase

To elucidate novel roles for specific CUL3-RING ubiquitin ligases during neural crest and craniofacial development a) Identification and mechanistic dissection of CRL3-KLHL4 as an essential regulator of ectodermal cell-fate commitment and neural tube formation (Asmar et al., Nat Comm 2023) We employed systematic biochemical screening of human disease variants to identify several CRL3s as candidate regulators of ectodermal development. We focused on a CRL3 containing the substrate adaptor KLHL4, a poorly characterized BTB protein encoded on the X-chromosome. By combining biochemical, proteomic, and imaging approaches with iPSC and chick embryo models we identified CRL3KLHL4 as central to a monoubiquitylation-based switch mechanism that specifically operates in the developing vertebrate head ectoderm. This regulation coordinates morphological rearrangements during neural tube formation and ectodermal patterning into spatially and transcriptionally distinct domains of the future skin, brain, and craniofacial skeleton. Mechanistically, we show that the major substrates of CRL3KLHL4 are group I p21-activated kinases (PAKs) that canonically act as downstream effectors of the small GTPase CDC42. Intriguingly, we find that monoubiquitylation of PAKs by CRL3KLHL4 converts PAKs into CDC42 inhibitors to restrict cytoskeletal signaling pathways during vertebrate head development. Interestingly, the CDC42 activating complex GIT-PIX acts as a substrate-specific co-adaptor for CRL3KLHL4 to monoubiquitylate PAKs, thus actively participating in the effector-to-inhibitor conversion. The importance of this regulatory circuit is highlighted by the fact that we identify neurodevelopmental and craniofacial disease-associated variants in CUL3 and KLHL4 that reduce CRL3KLHL4 complex assembly and PAK ubiquitylation and fail to limit CDC42 activation. This causes hyperactivated CDC42 signaling in the developing head region, which results in ectodermal domain formation and neural tube closure defects. Our data thus reveals how implementation of a monoubiquitylation-dependent effector-to-inhibitor switch restricts anterior CDC42 signaling to ensure faithful ectodermal domain and neural tube formation, explaining how cell-fate and morphometric changes are coordinated to establish the future skin, brain, and craniofacial skeleton. To determine the functions of spatially regulated E1 activity and ubiquitin activation during hematopoietic cell-fate decisions In collaboration with the labs of Dr. Peter Grayson (NIAMS) and Dr. Daniel Kastner (NGHRI) we previously identified a cohort of patients with somatic variants in UBA1 at p.M41, all presenting with a severe, late-onset autoinflammatory disease, we have termed VEXAS syndrome (Beck et al, NEJM 2020). Our mechanistic studies of these mutations have revealed loss of functional cytoplasmic UBA1 through aberrant isoform translation in myeloid cells as a major cause of this novel disorder, adding to the growing list of examples of autoinflammatory disorders caused by dysregulated ubiquitylation (as we have reviewed in Beck et al., Nat Rev Rheumatol 2022). Our results have further elucidated unexpected regulation of UBA1 localization suggesting that spatially regulated ubiquitin activation is essential for normal hematopoiesis. The main goal of this project is to dissect how loss of cytoplasmic UBA1 function leads to autoinflammation and how regulated ubiquitin activation may drive cell-fate decisions during blood and embryonic development. a) Identification of clinical predictors of VEXAS syndrome and determination of residual translation of UBA1b as a contributor to disease pathogenesis (Ferrada et al., Blood, 2022) Together with our clinical NIH collaborators we sought to determine independent predictors of survival in VEXAS and to understand the mechanistic basis for these factors. We analyzed 83 patients with somatic pathogenic variants in UBA1 at p.M41 (p.M41V/T/L), the start codon for translation of the cytoplasmic isoform of UBA1 (UBA1b). We found the p.M41V genotype to be a risk for decreased survival in VEXAS syndrome. Using in vitro models and patient-derived cells, we demonstrate that p.M41V variant supports less UBA1b translation than either p.M41L or p.M41T, providing a molecular rationale for decreased survival (Fig. 4C). We further show that these three canonical VEXAS variants produce more UBA1b than any of the six other possible single nucleotide variants within this codon. Finally, we report a clinically diagnosed VEXAS patient with two novel UBA1 mutations occurring in cis on the same allele. One mutation (c.121 A>T; p.M41L) caused severely reduced translation of UBA1b in a reporter assay, but co-expression with the second mutation (c.119 G>C; p.G40A) rescued UBA1b levels to those of canonical mutations. We conclude that regulation of residual UBA1b translation is fundamental to the pathogenesis of VEXAS syndrome and contributes to disease prognosis.

阐明特定 CUL3-RING 泛素连接酶在神经嵴和颅面发育过程中的新作用 a) CRL3-KLHL4作为外胚层细胞命运承诺和神经管形成的重要调节因子的鉴定和机制剖析（Asmar等人，Nat Comm 2023） 我们对人类疾病变异进行了系统的生化筛选，以确定几种 CRL3 作为外胚层发育的候选调节因子。我们重点关注包含底物接头 KLHL4 的 CRL3，这是一种编码在 X 染色体上的特征不明确的 BTB 蛋白。通过将生化、蛋白质组学和成像方法与 iPSC 和鸡胚模型相结合，我们确定 CRL3KLHL4 是基于单泛素化的开关机制的核心，该机制专门在发育中的脊椎动物头部外胚层中发挥作用。这种调节在神经管形成和外胚层模式形成未来皮肤、大脑和颅面骨骼的空间和转录上不同区域的过程中协调形态重排。从机制上讲，我们表明 CRL3KLHL4 的主要底物是 I 组 p21 激活激酶 (PAK)，其典型地充当小 GTPase CDC42 的下游效应器。 有趣的是，我们发现 CRL3KLHL4 对 PAK 的单泛素化会将 PAK 转化为 CDC42 抑制剂，从而限制脊椎动物头部发育过程中的细胞骨架信号传导途径。有趣的是，CDC42 激活复合物 GIT-PIX 作为 CRL3KLHL4 的底物特异性共适配器来单泛素化 PAK，从而积极参与效应子到抑制剂的转化。我们在 CUL3 和 KLHL4 中发现了与神经发育和颅面疾病相关的变异，这些变异减少了 CRL3KLHL4 复合物的组装和 PAK 泛素化，并且无法限制 CDC42 的激活，这一事实凸显了该调节回路的重要性。这会导致发育中的头部区域 CDC42 信号过度激活，从而导致外胚层结构域形成和神经管闭合缺陷。因此，我们的数据揭示了单泛素化依赖性效应器到抑制剂开关的实现如何限制前部 CDC42 信号传导，以确保忠实的外胚层结构域和神经管形成，解释细胞命运和形态变化如何协调以建立未来的皮肤、大脑和颅面骨骼。 确定空间调节的 E1 活性和泛素激活在造血细胞命运决定过程中的功能 通过与 Peter Grayson 博士 (NIAMS) 和 Daniel Kastner 博士 (NGHRI) 的实验室合作，我们之前鉴定了一组在 p.M41 存在 UBA1 体细胞变异的患者，所有患者均表现为严重的迟发性自身炎症性疾病，我们将其称为 VEXAS 综合征（Beck 等人，NEJM 2020）。我们对这些突变的机制研究表明，髓系细胞中异常异构体翻译导致功能性细胞质 UBA1 的丧失，是导致这种新型疾病的主要原因，这增加了因泛素化失调引起的自身炎症性疾病的例子不断增多（正如我们在 Beck 等人，Nat Rev Rheumatol 2022 中所评论的那样）。我们的结果进一步阐明了 UBA1 定位的意外调节，表明空间调节的泛素激活对于正常造血至关重要。该项目的主要目标是剖析细胞质 UBA1 功能的丧失如何导致自身炎症，以及受调节的泛素激活如何驱动血液和胚胎发育过程中的细胞命运决定。 a) 鉴定 VEXAS 综合征的临床预测因素并确定 UBA1b 的残留翻译作为疾病发病机制的贡献者（Ferrada 等人，Blood，2022） 我们与 NIH 临床合作者一起寻求确定 VEXAS 生存的独立预测因素，并了解这些因素的机制基础。我们分析了 83 名在 p.M41 (p.M41V/T/L) 处存在 UBA1 体细胞致病性变异的患者，UBA1 是 UBA1 (UBA1b) 细胞质亚型翻译的起始密码子。我们发现 p.M41V 基因型存在导致 VEXAS 综合征生存率降低的风险。使用体外模型和患者来源的细胞，我们证明p.M41V变体比p.M41L或p.M41T支持更少的UBA1b翻译，为生存率降低提供了分子原理（图4C）。我们进一步表明，这三个典型的 VEXAS 变体比该密码子内其他六种可能的单核苷酸变体产生更多的 UBA1b。最后，我们报告了一名临床诊断的 VEXAS 患者，其同一等位基因上有两种新的 UBA1 顺式突变。在报告基因检测中，一种突变（c.121 A>T；p.M41L）导致 UBA1b 翻译严重降低，但与第二种突变（c.119 G>C；p.G40A）共表达可将 UBA1b 水平恢复到典型突变的水平。我们得出的结论是，残余 UBA1b 翻译的调节是 VEXAS 综合征发病机制的基础，并有助于疾病预后。

项目成果

Novel CUL3 Variant Causing Familial Hyperkalemic Hypertension Impairs Regulation and Function of Ubiquitin Ligase Activity.

• DOI: 10.1161/hypertensionaha.121.17624
• 发表时间: 2022-01
• 期刊: Hypertension (Dallas, Tex. : 1979)
• 作者: Chatrathi HE;Collins JC;Wolfe LA;Markello TC;Adams DR;Gahl WA;Werner A;Sharma P
• 通讯作者: Sharma P

Nix interacts with WIPI2 to induce mitophagy.

Nix 与 WIPI2 相互作用诱导线粒体自噬。

• DOI: 10.15252/embj.2023113491
• 发表时间: 2023
• 期刊: The EMBO journal
• 作者: Bunker,EricN;LeGuerroué,François;Wang,Chunxin;Strub,Marie-Paule;Werner,Achim;Tjandra,Nico;Youle,RichardJ
• 通讯作者: Youle,RichardJ

TNIP1 inhibits selective autophagy via bipartite interaction with LC3/GABARAP and TAX1BP1.

• DOI: 10.1016/j.molcel.2023.02.023
• 发表时间: 2023-03
• 期刊: Molecular cell
• 影响因子: 16
• 作者: François Le Guerroué;Eric N Bunker;William M. Rosencrans;J. Nguyen;Mohammed A. Basar;Achim Werner

A ubiquitin-based effector-to-inhibitor switch coordinates early brain, craniofacial, and skin development.

• DOI: 10.1038/s41467-023-40223-y
• 发表时间: 2023-07-26
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Asmar, Anthony J.;Abrams, Shaun R.;Hsin, Jenny;Collins, Jason C.;Yazejian, Rita M.;Wu, Youmei;Cho, Jean;Doyle, Andrew D.;Cinthala, Samhitha;Simon, Marleen;van Jaarsveld, Richard H.;Beck, David B.;Kerosuo, Laura;Werner, Achim
• 通讯作者: Werner, Achim

Control of craniofacial and brain development by Cullin3-RING ubiquitin ligases: Lessons from human disease genetics.

• DOI: 10.1016/j.yexcr.2020.112300
• 发表时间: 2020-11-15
• 期刊: EXPERIMENTAL CELL RESEARCH
• 影响因子: 3.7
• 作者: Asmar, Anthony J.;Beck, David B.;Werner, Achim
• 通讯作者: Werner, Achim