ユビキチン様修飾システムUFM1システムの生物学的意義の解明

学位の種別: 課程博士審査委員会委員 : （主査）東京大学客員教授 田中 啓二, 東京大学教授 上田 卓也, 東京大学教授 井上 純一郎, 東京大学准教授 深井 周也, 東京大学教授 村田 茂穂University of Tokyo(東京大学

Biallelic Variants in UBA5 Link Dysfunctional UFM1 Ubiquitin-like Modifier Pathway to Severe Infantile-Onset Encephalopathy

The ubiquitin fold modifier 1 (UFM1) cascade is a recently identified evolutionarily conserved ubiquitin-like modification system whose function and link to human disease have remained largely uncharacterized. By using exome sequencing in Finnish individuals with severe epileptic syndromes, we identified pathogenic compound heterozygous variants in UBAS, encoding an activating enzyme for UFM1, in two unrelated families. Two additional individuals with biallelic UBAS variants were identified from the UK-based Deciphering Developmental Disorders study and one from the Northern Finland Intellectual Disability cohort. The affected individuals (n = 9) presented in early infancy with severe irritability, followed by dystonia and stagnation of development. Furthermore, the majority of individuals display postnatal microcephaly and epilepsy and develop spasticity. The affected individuals were compound heterozygous for a missense substitution, c.1111G>A (p.A1a371Thr; allele frequency of 0.28% in Europeans), and a nonsense variant or c.164G>A that encodes an amino acid substitution p.Arg5SHis, but also affects splicing by facilitating exon 2 skipping, thus also being in effect a loss-of-function allele. Using an in vitro thioester formation assay and cellular analyses, we show that the p.A1a371Thr variant is hypomorphic with attenuated ability to transfer the activated UFM1 to UFC1. Finally, we show that the CNS-specific knockout of Ufml in mice causes neonatal death accompanied by microcephaly and apoptosis in specific neurons, further suggesting that the UFM1 system is essential for CNS development and function. Taken together, our data imply that the combination of a hypomorphic p.A1a371Thr variant in trans with a loss-of-function allele in UBAS underlies a severe infantile-onset encephalopathy.Peer reviewe

The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3

The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3</p

Biallelic UFM1 and UFC1 mutations expand the essential role of ufmylation in brain development

The post-translational modification of proteins through the addition of UFM1, also known as ufmylation, plays a critical developmental role as revealed by studies in animal models. The recent finding that biallelic mutations in UBA5 (the E1-like enzyme for ufmylation) cause severe early-onset encephalopathy with progressive microcephaly implicates ufmylation in human brain development. More recently, a homozygous UFM1 variant was proposed as a candidate aetiology of severe early-onset encephalopathy with progressive microcephaly. Here, we establish a locus for severe early-onset encephalopathy with progressive microcephaly based on two families, and map the phenotype to a novel homozygous UFM1 mutation. This mutation has a significantly diminished capacity to form thioester intermediates with UBA5 and with UFC1 (the E2-like enzyme for ufmylation), with resulting impaired ufmylation of cellular proteins. Remarkably, in four additional families where eight children have severe early-onset encephalopathy with progressive microcephaly, we identified two biallelic UFC1 mutations, which impair UFM1-UFC1 intermediate formation with resulting widespread reduction of cellular ufmylation, a pattern similar to that observed with UFM1 mutation. The striking resemblance between UFM1- and UFC1-related clinical phenotype and biochemical derangements strongly argues for an essential role for ufmylation in human brain development. The hypomorphic nature of UFM1 and UFC1 mutations and the conspicuous depletion of biallelic null mutations in the components of this pathway in human genome databases suggest that it is necessary for embryonic survival, which is consistent with the embryonic lethal nature of knockout models for the orthologous genes

A Polarized Raman Spectroscopic Method for Advanced Analyses of the Osteon Lamellar Structure of Human Bone

Raman spectroscopy has recently been used for quantitative analyses of cortical bone tissue and related materials, such as dentin and enamel. While those analyses have proven useful as potential diagnostic tools, the Raman spectrum of bone encrypts a wealth of additional molecular scale details about structure and crystal arrangement, which are yet to be unfolded. Such details directly link to both bone physiology and pathology. In this work, a triple monochromator spectrometer with high spectral resolution, employed in polarized light configurations, was used to extract quantitative details about the preferential crystallographic orientation of apatite and collagen components in a human proximal femoral cortical bone sample. This body of information was then used to model the bone structure at the nanometric scale through a methodology that could be key in assessments of bone structure in health and disease

A homozygous UBA5 pathogenic variant causes a fatal congenital neuropathy

[Background] UBA5 is the activating enzyme of UFM1 in the ufmylation post-translational modification system. Different neurological phenotypes have been associated with UBA5 pathogenic variants including epilepsy, intellectual disability, movement disorders and ataxia.[Methods and results] We describe a large multigenerational consanguineous family presenting with a severe congenital neuropathy causing early death in infancy. Whole exome sequencing and linkage analysis identified a novel homozygous UBA5 NM_024818.3 c.31C>T (p.Arg11Trp) mutation. Protein expression assays in mouse tissue showed similar levels of UBA5 in peripheral nerves to the central nervous system. CRISPR-Cas9 edited HEK (human embrionic kidney) cells homozygous for the UBA5 p.Arg11Trp mutation showed reduced levels of UBA5 protein compared with the wild-type. The mutant p.Arg11Trp UBA5 protein shows reduced ability to activate UFM1.[Conclusion] This report expands the phenotypical spectrum of UBA5 mutations to include fatal peripheral neuropathy.MC-S was supported by ISCIII (JR15/00042) and Junta de Andalucia-Consejeria de Salud (B-0005-2017). This work was supported by the NHMRC, grants to NGL and GR (APP1002147, APP1035955, APP1080587). MK is supported by a Grant-in-Aid for Scientific Research on Innovative Areas (19H0506), a Grant-in-Aid for Scientific Research (B) (18H02611), the Japan Society for the Promotion of Science (an A3 foresight program) and the Takeda Science Foundation (to MK). RI is supported by a Grant-in-Aid for Young Scientists (B) (18K15061)

Novel therapeutic strategy for cervical cancer harboring FGFR3-TACC3 fusions.

We previously found that therapeutic targetable fusions are detected across various cancers. To identify therapeutic targetable fusion in uterine cervical cancer, for which no effective gene targeted therapy has yet been clinically applied, we analyzed RNA sequencing data from 306 cervical cancer samples. We detected 445 high confidence fusion transcripts and identified four samples that harbored FGFR3-TACC3 fusion as an attractive therapeutic target. The frequency of FGFR3-TACC3-fusion-positive cervical cancer is also 1.9% (2/103) in an independent cohort. Continuous expression of the FGFR3-TACC3 fusion transcript and protein induced anchorage-independent growth in the cervical epithelial cell line established from the ectocervix (Ect1/E6E7) but not in that from endocervix (End1/E6E7). Injection of FGFR3-TACC3 fusion-transfected-Ect1/E6E7 cells subcutaneously into NOG mice generated squamous cell carcinoma xenograft tumors, suggesting the association between FGFR3-TACC3 fusion and squamous cell carcinogenesis. Transfection of a FGFR3-TACC3 fusion transcript into four cervical cancer cell lines (SiHa, ME180, HeLa, and Ca Ski) induced activation of the MAPK pathway and enhancement of cell proliferation. Transcriptome analysis of the FGFR3-TACC3 fusion-transfected cell lines revealed that an IL8-triggered inflammatory response was increased, via activation of FGFR3-MAPK signaling. Continuous expression of FGFR3-TACC3 fusion led to activation of the PI3K-AKT pathway only in the two cell lines that harbored PIK3CA mutations. Sensitivity to the FGFR inhibitor, BGJ398, was found to depend on PIK3CA mutation status. Dual inhibition of both FGFR and AKT showed an obvious synergistic effect in cell lines that harbor mutant PIK3CA. Additionally, TACC3 inhibitor, KHS101, suppressed FGFR3-TACC3 fusion protein expression and showed antitumor effect against FGFR3-TACC3 fusion-transfected cell lines. FGFR3-TACC3 fusion-positive cancer has frequent genetic alterations of the PI3K/AKT pathway and selection of appropriate treatment based on PI3K/AKT pathway status should be required. Oncogenesis 2018 Jan 23; 7(1):4

Biallelic UFM1 and UFC1 mutations expand the essential role of ufmylation in brain development.

The post-translational modification of proteins through the addition of UFM1, also known as ufmylation, plays a critical developmental role as revealed by studies in animal models. The recent finding that biallelic mutations in UBA5 (the E1-like enzyme for ufmylation) cause severe early-onset encephalopathy with progressive microcephaly implicates ufmylation in human brain development. More recently, a homozygous UFM1 variant was proposed as a candidate aetiology of severe early-onset encephalopathy with progressive microcephaly. Here, we establish a locus for severe early-onset encephalopathy with progressive microcephaly based on two families, and map the phenotype to a novel homozygous UFM1 mutation. This mutation has a significantly diminished capacity to form thioester intermediates with UBA5 and with UFC1 (the E2-like enzyme for ufmylation), with resulting impaired ufmylation of cellular proteins. Remarkably, in four additional families where eight children have severe early-onset encephalopathy with progressive microcephaly, we identified two biallelic UFC1 mutations, which impair UFM1-UFC1 intermediate formation with resulting widespread reduction of cellular ufmylation, a pattern similar to that observed with UFM1 mutation. The striking resemblance between UFM1- and UFC1-related clinical phenotype and biochemical derangements strongly argues for an essential role for ufmylation in human brain development. The hypomorphic nature of UFM1 and UFC1 mutations and the conspicuous depletion of biallelic null mutations in the components of this pathway in human genome databases suggest that it is necessary for embryonic survival, which is consistent with the embryonic lethal nature of knockout models for the orthologous genes