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

阐明特异性CUL3-RING泛素连接酶在神经嵴和颅面发育中的新作用 a）作为外胚层细胞命运定型和神经管形成的必要调节剂的CRL 3-KLHL 4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CRL 3作为外胚层发育的候选调节因子。我们专注于一个CRL3含有底物适配器KLHL4，一个不好的特点BTB蛋白编码的X染色体上。通过将生物化学、蛋白质组学和成像方法与iPSC和鸡胚胎模型相结合，我们确定了CRL3KLHL4作为在发育中的脊椎动物头部外胚层中特异性运作的基于单倍性的开关机制的核心。这种调节协调在神经管形成和外胚层图案化到未来的皮肤，大脑和颅面骨骼的空间和转录不同的域的形态重排。从机制上讲，我们发现，CRL3 KLHL4的主要底物是I组p21激活的激酶（PAK），其典型地作为小GTdR CDC42的下游效应子。 有趣的是，我们发现通过CRL3KLHL4的PAKs的单泛素化将PAKs转化为CDC 42抑制剂，以限制脊椎动物头部发育期间的细胞骨架信号通路。有趣的是，CDC42激活复合物GIT-PIX作为CRL3KLHL4的底物特异性共衔接子，使PAK单泛素化，从而积极参与效应物-抑制剂转化。我们发现CUL3和KLHL4中与神经发育和颅面疾病相关的变异体，这些变异体减少了CRL 3 KLHL4复合物组装和PAK泛素化，但未能限制CDC 42的激活，这一事实突出了这种调节回路的重要性。这导致发育中的头部区域中的过度活化的CDC42信号传导，这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倍量化依赖性效应器至抑制剂开关的实施如何限制前部CDC 42信号传导，以确保忠实的外胚层结构域和神经管形成，解释了细胞命运和形态测量变化如何协调以建立未来的皮肤、大脑和颅面骨骼。 确定造血细胞命运决定过程中空间调控的E1活性和泛素活化的功能 与Peter Grayson博士（NIAMS）和丹尼尔卡斯特纳博士（NGHRI）的实验室合作，我们先前确定了一组在p.M41时具有UBA 1体细胞变异的患者，所有患者均表现为重度迟发性自身炎症性疾病，我们将其称为VEXAS综合征（Beck et al，NEJM 2020）。我们对这些突变的机制研究揭示了通过骨髓细胞中的异常同种型翻译导致的功能性细胞质UBA 1的丧失是这种新型疾病的主要原因，增加了由失调的泛素化引起的自身炎性疾病的例子的不断增长的列表（正如我们在Beck等人，Nat Rev Rheumol 2022）。我们的研究结果进一步阐明了UBA 1定位的意想不到的调节，表明空间调节的泛素激活对正常造血是必不可少的。该项目的主要目标是剖析细胞质UBA 1功能的丧失如何导致自身炎症，以及在血液和胚胎发育过程中，受调节的泛素激活如何驱动细胞命运的决定。 a）VEXAS综合征的临床预测因子的鉴定和作为疾病发病机制的贡献者的UBA 1b的残留翻译的测定（Ferrada等人，血，2022年） 我们与临床NIH合作者一起，试图确定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例在p.M41（p.M41V/T/L）（UBA 1胞质异构体（UBA 1b）翻译的起始密码子）处UBA 1体细胞致病变异的患者。我们发现p.M41V基因型是VEXAS综合征患者生存率降低的风险。使用体外模型和患者来源的细胞，我们证明了p.M41V变体比p.M41L或p.M41T支持更少的UBA1b翻译，为降低的存活率提供了分子基础（图4C）。我们进一步表明，这三个典型的VEXAS变异产生更多的UBA1b比任何其他六个可能的单核苷酸变异在这个密码子。最后，我们报告了一个临床诊断的VEXAS患者，两个新的UBA 1突变发生在顺式相同的等位基因。一个突变（c.121 A> T; p.M41L）在报告基因测定中导致UBA1b翻译严重减少，但与第二个突变（c.119 G> C; p.G40A）共表达将UBA1b水平恢复到典型突变水平。我们的结论是，残余UBA1b翻译的调节是根本的VEXAS综合征的发病机制，并有助于疾病的预后。

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.