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

National Institute Of Dental & Craniofacial Research

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Abstract

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.