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 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.