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: 10703889
• 负责人: Achim Werner
• 金额: $ 155.89万
• 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/10703889
• 关键词: Affect; Alleles; Binding; Biochemical; Biogenesis; Biological Assay; Blood; Bone Marrow; Brain; CRISPR/Cas technology; CUL3 gene; Cell Differentiation process; Cell Fate Control; Cells; Clinical; Codon Nucleotides; Collaborations; Complex; Defect; Dermal; Development; Developmental Delay Disorders; Disease; Dysmyelopoietic Syndromes; Electrolytes; Embryonic Development; Enzymes; Erythroid Progenitor Cells; Exhibits; Face; Fever; Fibroblasts; Future; Genes; Genotype; Hematology; Hematopoietic; Hematopoietic stem cells; Human Development; Human Genetics; Hypertension; Impairment; Individual; Inflammation; Inflammatory; Initiator Codon; Kidney; Lead; Link; Lymphocyte; Malignant Neoplasms; Messenger RNA; Modeling; Molecular; Mosaicism; Multiple Myeloma; Mutation; Myelogenous; Myeloid Cells; Names; Neural Crest; Neurons; Pathogenesis; Pathogenicity; Patients; Phenotype; Polyarteritis Nodosa; Post-Translational Protein Processing; Protein Isoforms; Proteins; Proteomics; RNA Splicing; Recurrence; Regulation; Relapsing polychondritis; Reporter; Reporting; Research; Residual state; Rheumatism; Ribosomes; Risk; Role; Signal Transduction; Single Nucleotide Polymorphism; Somatic Mutation; Sweet' s Syndrome; Syndrome; Temperature; Temporal Arteritis; Translations; Ubiquitin; United States National Institutes of Health; Universities; Vacuole; Variant; Work; Zebrafish; autoinflammatory; base; blood pressure control; clinical diagnosis; clinical predictors; craniofacial; craniofacial development; cytopenia; disease prognosis; extracellular vesicles; familial hyperkalemic hypertension; genetic selection; human disease; human embryonic stem cell; in vitro Model; insight; kidney cell; male; member; mutant; novel; novel therapeutic intervention; pediatric patients; peripheral blood; programs; self-renewal; stem cells; survival prediction; ubiquitin ligase; urinary

To elucidate novel roles for specific CUL3-RING ubiquitin ligases during neural crest and craniofacial development a) Identification and molecular characterization of a novel CUL3 variant causing hypertension and craniofacial and brain anomalies (Chatrathi*, Collins* et al., Hypertension 2022) In collaboration with the Undiagnosed Diseases Program (UDP) we have identified a novel de novo heterozygous CUL3 variant (CUL3474477) in a pediatric patient with familial hyperkalemic hypertension and global developmental delay, neurodevelopmental defects, and dysmorphic facial features. Using patient-derived urinary extracellular vesicles and dermal fibroblasts, biochemical assays, and cultured kidney cells we determined the molecular mechanisms by which CUL3474477 leads to dysregulation of the CRL3KLHL3-WNK-SPAK/ORS1 signaling axis, which controls blood pressure through regulating the Na-Cl cotransporter NCC in the kidney. We show that CUL3474477 causes reduced total CUL3 levels due to increased autoubiquitylation. The CUL3474477 that escapes autodegradation exhibits increased formation of CRL3KLHL3 complexes that are impaired in ubiquitylating WNK, thus resulting in aberrant activation of signaling, electrolyte imbalances, and hypertension. Proteomic analysis of CUL3 complexes revealed that, in addition to increased KLHL3 binding, the CUL3474477 variant also exhibits increased interactions with many other BTB substrate adaptors (including the neural crest regulator KBTBD8 and the neuronal regulator KCTD13), providing a rationale for the patients diverse craniofacial and neurodevelopmental phenotypes. We conclude that the pathophysiological effects of CUL3474477 are caused by reduced CUL3 levels and formation of catalytically impaired CRL3 complexes. To determine the functions of spatially regulated E1 activity and ubiquitin activation during hematopoietic cell-fate decisions a) Identification and molecular basis of VEXAS syndrome (Beck et al., 2020, NEJM) Employing a genotype-first approach focusing on ubiquitylation genes, we identified 25 male patients with somatic variants at codon 41 in X-linked UBA1 (p.M41V, p.M41T, p.M41L). These individuals all presented with severe, late-onset autoinflammatory disease characterized by fevers, cytopenias, and vacuoles in myeloid and erythroid precursors cells. These patients fulfilled clinical criteria for inflammatory (relapsing polychondritis, Sweet syndrome, polyarteritis nodosa, giant cell arteritis) and hematologic (myelodysplastic syndrome or multiple myeloma) conditions. We have named this disease VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. VEXAS syndrome is defined by somatic mosaicism: in affected patients, the recurrent UBA1 p.M41 mutation occurs in more than half of hematopoietic stem cells in the bone marrow, yet in the peripheral blood, it is found only in myeloid cells but not in lymphocytes, suggesting genetic selection. These findings highlight a novel paradigm for rheumatic diseases in which, similar to malignancy, somatic variants drive inflammation. Our mechanistic analyses revealed that VEXAS syndrome mutations at p.M41 lead to a reduction in translation of normally active cytoplasmic UBA1b (initiated from M41) and emergence of catalytically impaired UBA1c (initiated from M67). Using patient-cell based assays and CRISPR/Cas9-edited zebrafish models, we showed that this isoform swap results in loss of ubiquitylation and induction of inflammatory signaling specifically in mutant peripheral blood myeloid cells. Taken together, our ubiquitylation-focused genotype-first approach allowed the discovery of VEXAS syndrome, a novel autoinflammatory disorder caused by somatic mutations in UBA1. Through studying these mutations, we have identified loss of cytoplasmic ubiquitin activation through aberrant translation of UBA1 isoforms in myeloid cells as a major cause of this disease. In addition, our findings uncover an unexplored regulatory layer of ubiquitin signaling at its apex, which we predict to contribute to developmental cell-fate decision. b) Novel UBA1 mutations causing VEXAS syndrome (Poulter*, Collins* et al., Blood, 2021) In collaboration with the lab of Dr. Sinisa Savic at the University of Leeds, we identified two novel somatic UBA1 mutations leading to VEXAS syndrome. First, a splice-altering variant (c.118-1G>C) that results in aberrant UBA1 messenger RNAs lacking regions around p.M41 required for translation of the cytoplasmic UBA1b isoform. Second, p.S56F, a variant that, unlike p.M41 mutations, does not result in loss of cytoplasmic UBA1b translation but rather causes a temperature-dependent loss in UBA1 activity. Thus, our results reveal the existence of different molecular mechanisms by which mutations can inactivate UBA1 function to cause VEXAS syndrome, an observation that we are dissecting in ongoing/future work. c) 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. 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) 导致高血压以及颅面和大脑异常的新型 CUL3 变体的鉴定和分子特征（Chatrathi*、Collins* 等人，Hypertension 2022） 我们与未确诊疾病计划 (UDP) 合作，在一名患有家族性高钾性高血压、全身发育迟缓、神经发育缺陷和畸形面部特征的儿科患者中发现了一种新的从头杂合 CUL3 变异 (CUL3474477)。利用患者来源的尿液细胞外囊泡和真皮成纤维细胞、生化检测和培养的肾细胞，我们确定了 CUL3474477 导致 CRL3KLHL3-WNK-SPAK/ORS1 信号轴失调的分子机制，该信号轴通过调节肾脏中的 Na-Cl 协同转运蛋白 NCC 来控制血压。我们发现，CUL3474477 由于自身泛素化增加而导致总 CUL3 水平降低。逃避自降解的 CUL3474477 表现出 CRL3KLHL3 复合物的形成增加，这些复合物在泛素化 WNK 中受损，从而导致信号传导异常激活、电解质失衡和高血压。 CUL3 复合物的蛋白质组学分析表明，除了 KLHL3 结合增加外，CUL3474477 变体还表现出与许多其他 BTB 底物接头（包括神经嵴调节器 KBTBD8 和神经元调节器 KCTD13）的相互作用增加，为患者不同的颅面和神经发育表型提供了理论依据。我们得出的结论是，CUL3474477 的病理生理效应是由 CUL3 水平降低和催化受损的 CRL3 复合物形成引起的。 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 a) VEXAS 综合征的识别和分子基础（Beck 等人，2020，NEJM） 采用关注泛素化基因的基因型优先方法，我们鉴定了 25 名男性患者，其 X 连锁 UBA1 密码子 41 处存在体细胞变异（p.M41V、p.M41T、p.M41L）。这些个体均患有严重的迟发性自身炎症性疾病，其特征为发烧、血细胞减少以及骨髓和红系前体细胞中出现空泡。这些患者符合炎症（复发性多软骨炎、Sweet综合征、结节性多动脉炎、巨细胞动脉炎）和血液学（骨髓增生异常综合征或多发性骨髓瘤）病症的临床标准。我们将这种疾病命名为 VEXAS（空泡、E1 酶、X 连锁、自身炎症、体细胞）综合征。 VEXAS 综合征的定义是体细胞嵌合：在受影响的患者中，复发性 UBA1 p.M41 突变发生在骨髓中一半以上的造血干细胞中，但在外周血中，仅在骨髓细胞中发现，而在淋巴细胞中未发现，这表明遗传选择。这些发现凸显了风湿性疾病的一种新模式，在这种疾病中，与恶性肿瘤类似，体细胞变异会导致炎症。我们的机制分析表明，p.M41 处的 VEXAS 综合征突变导致正常活跃的细胞质 UBA1b（从 M41 开始）翻译减少，并出现催化受损的 UBA1c（从 M67 开始）。使用基于患者细胞的检测和 CRISPR/Cas9 编辑的斑马鱼模型，我们发现这种异构体交换会导致泛素化的丧失和炎症信号的诱导，特别是在突变的外周血骨髓细胞中。 总而言之，我们以泛素化为重点的基因型优先方法发现了 VEXAS 综合征，这是一种由 UBA1 体细胞突变引起的新型自身炎症性疾病。通过研究这些突变，我们发现骨髓细胞中 UBA1 亚型的异常翻译导致细胞质泛素活化丧失是导致这种疾病的主要原因。此外，我们的研究结果揭示了泛素信号在其顶端的一个未经探索的调节层，我们预计这将有助于发育细胞命运的决定。 b) 导致 VEXAS 综合征的新 UBA1 突变（Poulter*、Collins* 等人，Blood，2021） 我们与利兹大学 Sinisa Savic 博士的实验室合作，发现了两种导致 VEXAS 综合征的新型体细胞 UBA1 突变。首先，剪接改变变体 (c.118-1G>C) 导致异常的 UBA1 信使 RNA 缺乏细胞质 UBA1b 同种型翻译所需的 p.M41 周围区域。其次，p.S56F 是一种变异体，与 p.M41 突变不同，它不会导致细胞质 UBA1b 翻译丧失，而是导致 UBA1 活性随温度依赖性丧失。因此，我们的结果揭示了存在不同的分子机制，通过这些机制突变可以使 UBA1 功能失活从而导致 VEXAS 综合征，这是我们在正在进行/未来的工作中剖析的观察结果。 c) 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。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。