Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

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

Biallelic UFM1 and UFC1 mutations expand the essential role of ufmylation in brain development Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 doi:10.1093/brain/awy135 BRAIN 2018: 141; 1934–1945 | 1934 Biallelic UFM1 and UFC1 mutations expand the essential role of ufmylation in brain development 1, 2, 3, 2 Michael S. Nahorski, * Sateesh Maddirevula, * Ryosuke Ishimura, * Saud Alsahli, 4 5 6 7 Angela F. Brady, Anaı¨s Begemann, Tsunehiro Mizushima, Francisco J. Guzma´n-Vega, 3 3 2 2 2 Miki Obata, Yoshinobu Ichimura, Hessa S. Alsaif, Shams Anazi, Niema Ibrahim, 2 2 2,8 2,8 Firdous Abdulwahab, Mais Hashem, Dorota Monies, Mohamed Abouelhoda, 2,8 9 9 5 5 Brian F. Meyer, Majid Alfadhel, Wafa Eyaid, Markus Zweier, Katharina Steindl, 5,10 7 1 3 Anita Rauch, Stefan T. Arold, C. Geoffrey Woods, Masaaki Komatsu and 2,8,11 Fowzan S. Alkuraya *These authors contributed equally to this work. The post-translational modification of proteins through the addition of UFM1, also known as ufmylation, plays a critical develop- mental 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 develop- ment. 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 wide- spread 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. 1 Cambridge Institute for Medical Research, Wellcome Trust MRC Building Addenbrookes Hospital, Hills Rd, Cambridge CB2 0QQ, UK 2 Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia 3 Department of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Chuo-ku, Niigata 951-8510, Japan 4 North West Thames Genetics Service, Level 8V, St Mark’s Hospital, Northwick Park Hospital Watford Road, Harrow, HA1 3UJ, UK 5 Institute of Medical Genetics, University of Zurich, 8952 Schlieren-Zurich, Switzerland 6 Picobiology Institute, Graduate School of Life Science, University of Hyogo, Ako-gun, Hyogo 678-1297, Japan 7 King Abdullah University of Science and Technology, Computational Bioscience Research Center, Division of Biological and Environmental Sciences and Engineering, Thuwal 23955-6900, Saudi Arabia Received December 25, 2017. Revised March 3, 2018. Accepted March 23, 2018. Advance Access publication June 2, 2018 The Author(s) (2018). Published by Oxford University Press on behalf of the Guarantors of Brain. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 UFM1 and UFC1 mutations in brain development BRAIN 2018: 141; 1934–1945 | 1935 8 Saudi Human Genome Program, King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia 9 King Abdullah International Medical Research Centre, King Saud bin Abdulaziz University for Health Sciences, Division of Genetics, Department of Pediatrics, King Abdullah Specialized Children Hospital, King Abdulaziz Medical City, Ministry of National Guard-Health Affairs (NGHA), Riyadh, Saudi Arabia 10 Neuroscience Center Zurich, University of Zurich and ETH Zurich, Zurich 8057, Switzerland 11 Department of Anatomy and Cell Biology, College of Medicine, Alfaisal University, Riyadh, Saudi Arabia Correspondence to: Fowzan S. Alkuraya Department of Genetics King Faisal Specialist Hospital and Research Center Riyadh Saudi Arabia E-mail: [email protected] Correspondence may also be addressed to: Masaaki Komatsu Department of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Chuo-ku, Niigata 951-8510, Japan E-mail: [email protected] C. Geoffrey Woods Cambridge Institute for Medical Research Wellcome Trust MRC Building Addenbrookes Hospital, Hills Rd Cambridge CB2 0QQ, UK E-mail: [email protected] Keywords: ufmylation; UFM1; UFC1; encephalopathy; epilepsy (Tatsumi et al., 2010). The physiological context of ufmy- Introduction lation remains incompletely understood. The literature on the relevance of ufmylation to human health was originally Post-translational modification greatly expands the func- limited to cancer and, to a much lesser extent, complex tional repertoire of proteins beyond the confines of their coding sequence, and serves a diverse array of regulatory diseases such as diabetes, ischaemic heart diseases and al- roles in protein turnover, localization and interactions coholic liver disease (Liu et al., 2014; Yoo et al., 2015; Wei and Xu, 2016). As with other biochemical processes, how- (Hitosugi and Chen, 2014; Tompa et al., 2014). ever, Mendelian diseases involving deficiency of the individ- Ubiquitination is a well-established post-translational modi- ual components of ufmylation offered a unique opportunity fication involving a series of enzymatic reactions that add to reveal the extent to which ufmylation influences human ubiquitin to its target protein (Hershko and Ciechanover, physiology. We and others have reported hypomorphic mu- 1998). These reactions involve a few E1 activating enzymes, tations in UBA5 in children with severe infantile onset epi- dozens of E2 conjugating enzymes and hundreds of E3 ligat- leptic encephalopathy (Colin et al., 2016; Duan et al., ing enzymes. More than a dozen ubiquitin-like (UBL) pro- 2016; Muona et al., 2016). Additionally, Hamilton et al. teins have been found to similarly modify their target (2017) reported that a homozygous mutation in UFM1 proteins (Schulman and Harper, 2009). UBL are classified caused a severe early-onset encephalopathy with progres- into those that are activated and conjugated to substrates sive microcephaly, although the mechanism remained un- (type I) e.g. SUMO, NEDD8, ATG8, ATG12, URM1, clear. In this report, we provide evidence that it is the UFM1, FAT10, and ISG15, and those that do not undergo process of ufmylation that is essential for normal nervous conjugation (type II) (Cappadocia and Lima, 2018). system development and function, based on the identifica- UFM1 (ubiquitin-fold modifier 1), a 9.1-kDa protein tion of UFM1 and UFC1 biallelic mutations, which we with a tertiary structure similar to ubiquitin, is among the show lead to a remarkably similar neurological phenotype most recently identified type I UBL proteins (Komatsu and accompanying impairment in ufmylation. et al., 2004). The process of covalently attaching UFM1 to its target proteins, termed ufmylation, requires E1 acti- vating enzymes (UBA5), E2 conjugating enzymes (UFC1), and an E3 ligase (UFL1) (Daniel and Liebau, 2014). UFM1 Materials and methods is activated by UBA5, forming a high-energy thioester bond. Activated UFM1 is then transferred to UFC1, in a Human subjects similar thioester linkage (Komatsu et al., 2004). Ufmylation is completed by the catalysis of UFM1 transfer to the sub- Patients and available relatives were recruited with informed strate protein through the action of the E3 enzyme UFL1 consent as part of IRB-approved research protocols (RAC# Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 1936 | BRAIN 2018: 141; 1934–1945 M. S. Nahorski et al. 2080006 and 2121053; StV 11/09). Clinical data, including in pull-down assay buffer (20 mM Tris-Cl, pH 7.5, 150 mM laboratory and imaging studies, were collected from all par- NaCl, 1 mM EDTA, 1 mM DTT, 0.05% Nonidet P-40). The ticipants. Blood was collected in EDTA tubes for DNA extrac- pulled-down protein complexes were extensively washed with tion and in Na-heparin tubes for the establishment of pull-down assay buffer, and the obtained samples were sub- lymphoblastoid cell lines. jected to NuPAGE (4–12% acrylamide gradient) and Coomassie brilliant blue staining. Autozygome analysis Cell culture Genomewide single nucleotide polymorphism (SNP) genotyp- HEK293T cells were grown in Dulbecco’s modified Eagle ing using Axiom SNP Chip (Affymetrix) from all available medium (DMEM) containing 10% foetal bovine serum patients and relatives was pursued to determine the candidate (FBS), 5 U/ml penicillin, and 50mg/ml streptomycin. Lympho- autozygome as described before (Alkuraya, 2010, 2012). blasts were cultured in RPMI 1640 supplemented by 10% Runs of homozygosity 42 Mb were considered surrogates of FBS, 5 U/ml penicillin, and 50mg/ml streptomycin. To generate autozygosity given the consanguineous nature of the study UFM1- and UFC1-knockout cells, each UFM1 and UFC1 families as determined by AutoSNPa. Homozygosity mapping guide RNA designed using the CRISPR Design tool (http:// was performed on all available family members using crispr.mit.edu/) was subcloned into pX330-U6-Chimeric_BB- HomozygosityMapper (http://www.homozygositymapper.org/). CBh-hSpCas9 (Addgene #42230), a human codon-optimized SpCas9 and chimeric guide RNA expression plasmid. Exome sequencing and variant HEK293T cells were co-transfected with the pX330 and pEGFP-C1 (#6084-1, Clontech Laboratories) vectors, and cul- filtering tured for 2 days. Thereafter, the GFP-positive cells were sorted Exome capture was performed using TruSeq Exome and expanded. Loss of UFM1 and of UFC1 was confirmed by Enrichment kit (Illumina) following the manufacturer’s proto- heteroduplex mobility assay followed by immunoblot analysis col. Samples were prepared as an Illumina sequencing library, with anti-UFM1 and anti-UFC1 antibodies, respectively. and in the second step, the sequencing libraries were enriched for the desired target using the Illumina Exome Enrichment Immunoblot analysis protocol. The captured libraries were sequenced using Illumina HiSeq 2000 Sequencer. The reads were mapped Cells were lysed with ice-cold TNE buffer (10 mM Tris-Cl, pH against UCSC hg19 (http://genome.ucsc.edu/) by BWA (http:// 7.5, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, and pro- bio-bwa.sourceforge.net/). The SNPs and indels were detected tease inhibitors). The samples were separated using the by SAMtools (http://samtools.sourceforge.net/). Variants from NuPAGE system (Invitrogen) on 12% Bis-Tris gels in whole exome sequencing (WES) were filtered such that only NuPAGE MOPS SDS Running Buffer, and transferred to novel (or very low frequency 50.1%), coding/splicing, homo- polyvinylidene difluoride (PVDF) membranes. Antibodies zygous variants that are within the candidate autozygome against FLAG (Medical & Biological Laboratories Co., Ltd., (autozygous intervals exclusive to the affected individuals) M185-3L), UFC (Abcam, ab189251) and UFM1 (Abcam, and are predicted to be pathogenic were considered as likely ab109305) were purchased from the indicated suppliers. causal variants (Alkuraya, 2013, 2016). Frequency of variants Anti–UBA5 and UFM1 polyclonal antibodies were described was determined using publicly available variant databases previously (Komatsu et al., 2004). The immunoreactive bands (1000 Genomes, Exome Variant Server and ExAC) as well were detected by LAS-4000 (GE Healthcare UK Ltd.). The as a database of 2369 in-house ethnically-matched exomes. quantitative densitometric analyses of FLAG-UBA5-MYC- Pathogenicity is likely if the mutation is loss-of-function (spli- UFM1, FLAG-UFC1-MYC-UFM1, endogenous UBA5-UFM1 cing/truncating) or, in the case of missense/in-frame indels, re- intermediate, and endogenous UFC1-UFM1 intermediate rela- moves a highly conserved amino acid and is predicted to be tive to free FLAG-UBA5, FLAG-UFC1, endogenous UBA5, pathogenic by the three in silico prediction modules PolyPhen, and endogenous UFC1 were carried out using Multi Gauge SIFT and CADD. Version 3.2 Image software (Fuji Film, Tokyo, Japan). Statistical analysis was performed using an unpaired t-test (Welch test). The data represent the means  standard error In silico modelling (SE) of three separate experiments. 3D experimental structures were retrieved from the Protein Data Bank (PDB) and analysed using PYMOL (www.pymol. In vitro thioester formation assay org). In vitro thioester formation assay was conducted as previously reported (Komatsu et al., 2004). Briefly, recombinant GST- Pull-down assay R81C Gly83 UFM1C2, GST-UFM1C2 , GST- UFM1 , GST- R23Q T106I Recombinant proteins were purified with glutathione S-trans- UBA5, GST-UFC1, GST-UFC1 and GST-UFC1 were ferase (GST) affinity purification system according to the produced in Escherichia coli and recombinant proteins were manufacturer’s protocol (GE Healthcare). The GST moiety of purified by chromatography on Glutathione Sepharose 4B all purified proteins without GST-UBA5 was removed on (GE Healthcare). After digestion of GST by PreScission column by PreScission protease (GE Healthcare). GST-UBA5 Protease (GE Healthcare), the recombinant proteins were dia- bound to Glutathione Sepharose 4B (GE Healthcare) was lyzed against 50 mM BisTris (pH 6.5), 100 mM NaCl, 10 mM incubated for 20 min at 4 C with indicated purified proteins MgCl , and 0.1 mM DTT (reaction buffer). Thioester 2 Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 UFM1 and UFC1 mutations in brain development BRAIN 2018: 141; 1934–1945 | 1937 formation reactions contained reaction buffer with 0.8mg R81C Gly83 UFM1C2, UFM1C2 or UFM1 and some of the following: 5 mM ATP, 0.08 (for UFC1-UFM1 thioester forma- tion assay) or 0.8mg (for UBA5-UFM1 thioester formation R23Q T106I assay) UBA5 and 0.8mg UFC1, UFC1 or UFC1 . Reactions were incubated for 5 min at 25 C and stopped by the addition of NuPAGE LDS Sample Buffer lacking reducing agent, followed by a 10-min incubation at 37 C, NuPAGE (4–12% acrylamide gradient) and Coomassie brilliant blue staining. Data shown are representative of three separate experiments. Assessment of apoptosis in response to endoplasmic reticulum stress HeLa or SH-SY5Y cells were plated into six-well plates at 7.5  10 cells/ml. After 18 h they were transfected with either 2mgGFP, 1mg UFM1WT/1mg GFP or 1mg UFM1R81C/1mg TM GFP using FuGENE HD or X-tremeGENE HP, respectively according to the manufacturer’s protocols. Media was changed after 24 h and tunicamycin added after 48 h. For endoplasmic reticulum stress marker analysis, cells were incubated with 5mg/ ml tunicamycin for 8 h. mRNA was extracted from cells using the RNeasy Plus Mini Kit (Qiagen) according to the manufac- turer’s instructions. The concentration of the mRNA was calcu- TM lated and 1mg converted to cDNA using the qScript cDNA synthesis kit (Quanta Biosciences). Levels of HSPA5, DDIT3 and GAPDH were assayed using pre-designed TaqMan Gene Expression Assays (ThermoFisher Scientific). The level of gene expression was normalized to that of GAPDH for each sample assessed. For assessment of apoptosis in response to endoplas- mic reticulum stress, cells were incubated in different concentra- tions of tunicamycin for 48 h. They were then, trypsinized and stained with Annexin V, Alexa Fluor 647 conjugate (ThermoFisher Scientific) and DAPI to assess early and late stages of apoptosis, respectively. Cells were analysed on a BD TM LSR Fortessa flow cytometer, selecting only those expressing GFP. Data were analysed using FlowJo software, and cells ex- pressing either DAPI and/or Annexin V were considered apoptotic. Results Identification of novel severe early infantile encephalopathy phenotypes Two Sudanese families were recruited independently by C.G.W. and F.S.A., each with two children presenting with the core phenotype of profound global developmental delay, failure to thrive, progressive microcephaly and re- fractive epilepsy. Salient clinical features include subtle facial dysmorphism, severe axial hypotonia and appendicu- lar hypertonia. Available brain imaging revealed dysmyeli- nation and volume loss. Hypsarrhythmia was documented on EEG. The severity of the presentation is evidenced by the premature death of two of the four patients at ages 9 months and 8.5 years, respectively (Table 1, Fig. 1 and Supplementary Tables 1 and 2). Table 1 Summary of the clinical features of patients with UFM1 and UFC1 mutations ID 12DG0178 12DG1577 14DG0050 16DG1614 MDL-17-3196 MDL-17-3892 17DG0828 ID76366 UK1 UK2 10DG0945 10DG0946 Gene UFC1 UFC1 UFC1 UFC1 UFC1 UFC1 UFC1 UFC1 UFM1 UFM1 UFM1 UFM1 Mutation c.317C4T c.317C4T c.317C4T c.317C4T c.317C4T c.317C4T c.317C4T c.68G4A c.241C4T c.241C4T c.241C4T c.241C4T p.(Thr106Ile) p.(Thr106Ile) p.(Thr106Ile) p.(Thr106Ile) p.(Thr106Ile) p.(Thr106Ile) p.(Thr106Ile) p.(Arg23Gln) p.(Arg81Cys) p.(Arg81Cys) p.(Arg81Cys) p.(Arg81Cys) Age 16 years 23 years 3 years 5 years 5 years 31 months 8 years 4 years 13 months 13 months 2 years 1 year Gender F F F F F F F M F M M M Microcephaly+ + - + + + + + +++ + Short Stature + + - + + + + + + + + + Underweight + + + + + + + + + + + + GDD +++++ + +++ + + + Seizures + - - + + - - + + + + + GDD = global developmental delay; F = female; M = male. Microcephaly was always secondary i.e. postnatal. Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 1938 | BRAIN 2018: 141; 1934–1945 M. S. Nahorski et al. Figure 1 UFM1-related clinical phenotype. (A) Pedigree of Family 1 with UFM1 mutation. (B and C) Images from Family 1 exhibiting lack of major facial dysmorphism. (D and E) Pes cavus as a result of abnormal tone in Patients 10DG0945 and 10DG0946, respectively (Family 1). (F) Genome-wide homozygosity mapping revealed a single critical locus. Blue box indicates haplotype (Chr13:36292810-42302880) identical by descent. (G) Pedigree of Family 2 with UFM1 mutation. (H) Facial image of Patient UK1 (Family 2) showing full cheeks. (I and J) Peripheral oedema of Patients UK1 and UK2 (Family 2). (K and L) Brain MRI of Patient UK1 showing cerebellar hypoplasia and thin corpus callosum and frontal cortical polymicrogyria. (M) Schematic diagram of UFM1 protein representing ubiquitin-fold modifier 1 domain with the mutation indicated. The Arg81 at the mutation site is highly conserved from humans to C. elegans. Independently, we encountered a highly similar phenotype sequencing of the index in each of the two families revealed in three Saudi families and one Swiss family with eight af- the same sole novel homozygous variant within this interval: fected members (one of which was previously described, albeit UFM1: NM_016617.3:c.241C4T:p.(Arg81Cys) (Fig. 1). briefly) (Anazi et al., 2016). They all presented with severe Similarly, genotyping of the Saudi families with a similar early infantile encephalopathy, progressive microcephaly, axial encephalopathy phenotype revealed that they all share the hypotonia, appendicular hypertonia and refractory epilepsy. same ancestral haplotype (Chr1:158957700-162094700), Although brain MRI findings were largely non-specific, again supporting a recessive founder mutation (Fig. 2). some had evidence of basal ganglia involvement (Table 1, Indeed, exome sequencing on four of the seven patients Fig. 2 and Supplementary Tables 1 and 2). Phenotypic com- revealed a single novel homozygous variant within the crit- parison of our patients with UFM1 and UFC1 mutations to ical locus: UFC1: NM_016406.3:c.317C4T:p.(Thr106Ile), previously reported patients with UFM1 and UBA5 mutations which segregated with the phenotype in all four families as canbefoundinTable 2. revealed by targeted Sanger sequencing. Exome sequencing on a Swiss patient revealed a novel homozygous variant in UFC1: NM_016406.3:c.68G4A:p.(Arg23Gln) inherited UFM1 and UFC1 define novel loci for from the heterozygous parents. Mutations at Thr106 and severe infantile encephalopathy with Arg23 involve highly conserved residues (Fig. 2). progressive microcephaly Although the two Sudanese families are not known to be Hypomorphic effect of UFM1 muta- related, they originate from the same village in Sudan. tion on the UFM1-system Indeed, autozygome analysis revealed a single shared auto- zygous interval (Chr13:36292810-42302880) between the The UFM1 variant is predicted pathogenic by PolyPhen affected members with the same ancestral haplotype, (0.538/possibly damaging), SIFT (0/ deleterious) and CADD strongly supporting a recessive founder mutation. Exome (35), as are the UFC1 variants c.317C4T:p.(Thr106Ile) Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 UFM1 and UFC1 mutations in brain development BRAIN 2018: 141; 1934–1945 | 1939 Figure 2 UFC1-related clinical phenotype. (A) Pedigrees of the families with UFC1 mutation. (B) Genome-wide homozygosity plot high- lighting a single critical locus from Families 1–3, who harbour the p.(Thr106Ile) mutation. Blue box indicates haplotype (Chr1:158957700- 162094700) identical by descent. (C) Clinical images showing multiple joint contractures and emaciation (Patient 17DG0828). (D and E) Clinical images of younger patients suggest a progressive nature of emaciation (Patients 14DG0050 and 16DG1614, respectively). (F) Brain MRI of Patient 16DG1614 showing bilateral hyperintense signals in the basal ganglia. (G and H) Brain MRI of Patient MDL-17-3196 showing bilateral hyperintense signals in the basal ganglia in the first year that resolved in a repeated brain MRI in the fourth year of age. (I) Brain MRI of Patient ID76366 showing markedly delayed myelination. (J) Schematic diagram of UFC1 protein showing UFC1 domain and mutation site. Mutations Arg23Gln and Thr106Ile are highly conserved from humans to C. elegans. Table 2 Comparison of phenotype between this study patients with UFM1 and UFC1 mutations and those previously reported with mutations in UFM1 and UBA5 Comparison between UFM1-UBA5-UFC1 pathway reported cases Current cohort UFM1-related cases UBA5-related cases Gene UFC1 UFM1 UFM1(PubMed: 28931644) UBA5 (PubMed: 27545681, 27545674, 28965491) Mutation c.317C4T; c.68G4A c.241C4T c.-273_-271delTCA c.1111G4A c.904C4T c.971_972insC c.778G4A c.1165G4T c.169A4G c.503G4A c.164G4A c.684G 4 A Number of cases 8 4 16 19 Failure to thrive 100% (8/8) 100% (4/4) 63% (10/16) 89% (8/9) Short stature 88% (7/8) 100% (4/4) 75% (12/16) 77% (10/13) Microcephaly 88% (7/8) 100% (4/4) 100% (16/16) 100% (18/18) Global developmental delay 100% (8/8) 100% (4/4) 100% (16/16) 100% (19/19) Seizures 50% (4/8) 100% (4/4) 75% (12/16) 84% (16/19) Brain MRI Basal ganglia abnormality 33% (2/6) 0% (0/4) 100% (16/16) 0% (0/17) Delayed CNS myelination 17% (1/6) 75% (3/4) 100% (16/16) 24% (4/17) Cerebellar hypoplasia 0% (0/6) 75% (3/4) 81% (13/16) 24% (4/17) Mortality 0% (0/8) 100% (4/4) 56% (9/16) 24% (4/19) Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 1940 | BRAIN 2018: 141; 1934–1945 M. S. Nahorski et al. R81C Figure 3 Hypomorphic effect of the UFM1 mutation on the UFM system. (A) Molecular basis for the effect of the UFM1 mutation. Top: Crystal structure of the heterodimeric complex between UFM1 (magenta and yellow) and UBA5 (cyan and green), taken from PDB id 5IAA. Bottom: Magnification of the boxed region. The view is tilted horizontally by 30 compared to the top panel. For clarity, only side chains 81 81 of Arg and of Arg -interacting residues are shown. (B) In vitro pull-down assay. Pull-down assay with GST-UBA5 and UFM1, UFM1 mutants or LC3B. GST-UBA5 conjugated with Glutathione Sepharose 4B was incubated with purified recombinant UFM1, UFM1 mutants or LC3B. LC3B is known to interact with UBA5. The pulled-down complexes were subjected to NuPAGE (4–12% acrylamide gradient) and Coomassie brilliant C250S blue staining. GST-UBA5, LC3B, UFM1 and UFM1 mutants are indicated. (C and D) Immunoblot assay. Indicated constructs (0.1mg for UBA5 C116S 0.5mg for UFC1 , and 2mg for UFM1 or mutants) were expressed in UFM1-deficient HEK293T cells. Twenty-four hours after transfection, the (continued) Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 UFM1 and UFC1 mutations in brain development BRAIN 2018: 141; 1934–1945 | 1941 C116S [PolyPhen (0.998/probably damaging), SIFT (0/deleterious), intermediate of UFC1 with MYC-UFM1 (Fig. 3D). CADD (32)] and c.68G4A:p.(Arg23Gln) [PolyPhen (0.901/ Conversely, the formation of the UFM1-UFC1 intermediate R81C probably damaging), SIFT (0.02/deleterious) and CADD (35)]. was almost abolished when MYC-UFM1 or MYC- Gly83 C116S We therefore carried out experiments to assess if and in which UFM1 were expressed together with UFC1 way these mutations affect ufmylation. The C-terminal tail of (Fig. 3D). In good agreement with those analyses, an UFM1 (residues 79–83) is essential for its adenylation by the in vitro thioester formation assay revealed that while E1 enzyme UBA5 and subsequent thioester formation with wild-type UFM1 forms an intermediate with UBA5, R81C UBA5 (Oweis et al., 2016). The crystallographic structure of UFM1 slightly had the ability to form the intermediate R81C UBA5 in the complex with UFM1 (PDB ID 5IAA) shows that (Fig. 3E). We also observed that UFM1 is hardly trans- the tail region, encompassing Arg directly binds to UBA5 ferred to UFC1 (Fig. 3F). In the next series of experiments, R81C (Fig.3A).Since Arg interacts with the negatively charged we tested the effect of the UFM1 mutation on the 241 209 183 residues Glu ,Glu and Asp of UBA5 (Fig. 3A). The intermediate formation with endogenous UBA5 and UFC1 substitution of Arg with a shorter and hydrophobic Cys is as well as UFM1-conjugation, using lymphoblasts derived expected to markedly reduce the strength of the interaction from an affected individual. Expression of free UFM1 pro- between UFM1 and UBA5. Accordingly, an in vitro pull- tein in lymphoblasts from an affected individual was com- down assay revealed that the mutant has a lower binding parable to control cells (Fig. 3G, left panel). By affinity to UBA5 than wild-type UFM1 (Fig. 3B). Next, we immunoblot analysis with non-reducing samples, we de- R81C investigated whether UBA5isabletoactivateUFM1 .To tected the intermediates of UFM1-UBA5 and of UFM1- do this, we used a UBA5-mutant whose active site, cysteine UFC1. As predicted, the level of UFM1-UBA5 intermedi- 250 C250S (Cys ) was substituted with a serine (termed UBA5 ). ates was significantly lower in lymphoblasts of affected in- When thecysteineresidue at theactivesiteofE1and E2 dividual compared to control cells (Fig. 3G, middle panel). enzymes is replaced by a serine, an O-ester bond instead of Likewise, the formation of the UFM1-UFC1 intermediate a thioester bond is formed with its respective modifier pro- markedly declined (Fig. 3G, right panel). We also found teins, which become stable even under reducing conditions that the level of two UFM1-conjugates with cellular pro- (Komatsu et al., 2004). To exclude an effect of endogenous teins in patient-derived lymphoblasts was significantly UFM1, we generated UFM1-deficient HEK293T cells reduced compared to control lymphoblasts (Fig. 3G, left (Supplementary Fig. 1A). As expected, when we co-expressed R81C panel). Taken together, our data show that UFM1 C250S FLAG-UBA5 and wild-type MYC-UFM1 into UFM1-de- has a hypomorphic effect on the UFM1-system and provide ficient HEK293T cells, MYC-UFM1 formed a stable inter- a plausible mechanistic explanation. C250S mediate with FLAG-UBA5 (Fig. 3C). Such intermediate C250S formation was completely abrogated when UBA5 was Gly83 83 co-expressed with MYC-UFM1 ,whose Gly essential Hypomorphic effect of UFC1 muta- for the adenylation and activation through UBA5 is deleted tions on the UFM1-system (Fig. 3C). Though we still observed the intermediate in the R81C The UFC1 structure consists of the catalytic core domain case of MYC-UFM1 ,its levelwas only 75% of that of conserved in all E2-like enzymes and an additional N-ter- wild-type UFM1 (Fig. 3C). R81C minal helix (Mizushima et al., 2007). Thr on the UFC1 The defective activation of UFM1 by the UBA5 E1 structure is on the opposite site of the proposed UBA5 enzyme is expected to also hamper the subsequent step, binding site (involving helix 2) (Liu et al., 2009), suggest- namely the formation of the intermediate with its cognate ing that mutation of this residue does not affect the inter- E2-like enzyme UFC1. Therefore, we investigated whether R81C action with UBA5. Indeed, an in vitro pull-down assay UFM1 forms an intermediate with UFC1 in cells. showed that the Thr106Ile mutation does not have any When wild-type MYC-UFM1 was expressed together with C116S 116 106 effect on binding to UBA5 (Supplementary Fig. 2). Thr FLAG-UFC1 in which the active site, cysteine (Cys ), is located in a coiled region close to the catalytic Cys , was substituted with serine, we clearly detected the Figure 3 Continued cell lysates were subjected to immunoblot analysis with anti-FLAG antibody. Bar graphs indicate the quantitative densitometric analyses of FLAG- UBA5-MYC-UFM1 and FLAG-UFC1-MYC-UFM1 intermediates relative to free FLAG-UBA5 and FLAG-UFC1, respectively. Statistical analyses were performed using the unpaired t-test (Welch test). The data represent the means  SE of three separate experiments. **P5 0.01 and ***P5 0.001. (E and F) In vitro thioester formation assay of UFM1 by UBA5 (E) and of UFM1 by UFC1 (F). The assay was conducted as described in the ‘Materials and methods’ section. Data shown are representative of three separate experiments. (G) Immunoblot analysis in case (P1: 10DG0945, Individual V1 in Fig. 1A) and control (C1: a healthy Sudanese young female) lymphoblasts. Reducing (DTT plus) and non-reducing (DTT minus) samples were prepared from lymphoblasts and subjected to immunoblot analysis for UFM1 (left), UBA5 (middle), and UFC1 (right). R81C We used a hand-made anti-UFM1 antibody in this experiment since the commercial antibody did not recognize UFM1 . Bar graphs indicate the quantitative densitometric analyses of UBA5-UFM1 and UFC1-UFM1 intermediates relative to free UBA5 and UFC1, respectively. Statistical analysis was performed using the unpaired t-test (Welch test). The data represent the means  SE of three separate experiments. **P5 0.01 and ***P5 0.001. Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 1942 | BRAIN 2018: 141; 1934–1945 M. S. Nahorski et al. Figure 4 Hypomorphic effect of UFC1 mutants on the UFM system. (A) Localization of the key binding sites on UFC1. Binding sites for E1 and E3 are indicated. Boxed region shows the active site, and the coiled region for binding site to UFM1 is coloured in orange. The mutated 23 106 residues Arg and Thr are highlighted in yellow and green, respectively. Encircled region shows the proximity of R23 with regions involved in 90 91 130 106 binding to E1 (helix 2) and E3 (Tyr ,Pro ,Pro ; cyan). (B) Bottom: Magnification of Thr . Residues involved in hydrophobic or polar 106 106 interactions with Thr are shown as stick models. Colouring as in A, except for the mutant Ile , which is shown as light grey stick model, with 23 23 positions of minor clashes indicated as discs. Top: Magnification of Arg . Colouring as in A, except for the mutant Gln , which is shown as C116S C116S/T106I C116S/R23Q magenta stick model. (C) Immunoblot assay. Indicated constructs (0.5mg for UFC1 , UFC1 or UFC1 and 2mg for UFM1) were expressed in UFC1-deficient HEK293T cells. Twenty-four hours after transfection, the cell lysates were subjected to immunoblot analysis with indicated antibodies. Bar graphs indicate the quantitative densitometric analyses of FLAG-UFC1-MYC-UFM1 intermediates relative to free FLAG-UFC1. Statistical analyses were performed using the unpaired t-test (Welch test). The data represent the means  SE of three separate experiments. *P5 0.05. (D) In vitro thioester formation assay of UFM1 by UFC1. The assay was conducted as described in the ‘Materials and methods’ section. Data shown are representative of three separate experiments. (E) Immunoblot analysis in case (P2, P3, P4: V:1) and control (C1: a healthy Sudanese young females) lymphoblasts. Reducing (DTT plus) and non-reducing (DTT minus) samples were prepared from lymphoblasts and subjected to immunoblot analysis for UFC1 (left), and UFM1 (right). Bar graph indicates the quantitative densitometric analysis of UFC1-UFM1 intermediates relative to free UFC1. Statistical analysis was performed using the unpaired t-test (Welch test). The data represent the means  SE of three separate experiments. **P5 0.01. Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 UFM1 and UFC1 mutations in brain development BRAIN 2018: 141; 1934–1945 | 1943 and within the site mapped to be required for interactions 5mg/ml tunicamycin (Supplementary Fig. 3). Neither did with UFM1 (Liu et al., 2009) (orange segment in Fig. 4A). we note appreciable difference in endoplasmic reticulum In the crystal structure of unliganded UFC1, Thr inter- stress-induced apoptosis in cells expressing UFM1 wild- type or UFM1 p.R81C, after 48 h incubation in tunicamy- acts with the hydrophobic core of the protein and is mostly cin at various concentrations. excluded from the solvent (Fig. 4B). Replacing the polar threonine with the slightly larger and completely hydropho- bic isoleucine is expected to change the anchoring, stereo- chemistry and dynamics of this region, affecting the Discussion intermediate formation of UFM1 with UFC1. Similarly, 23 90 91 ˚ Our understanding of the pathogenesis of infantile encephal- Arg is close (10 A) to the residues Tyr , Pro and opathy at the molecular level has greatly expanded in recent Pro that form a substructure and is potentially involved years due in large part to the rapidly growing use of gen- in E3-binding (Liu et al., 2009) (Fig. 4A). Arg is also near omics to diagnose and classify this highly heterogeneous to the helix 2, which is involved in binding to E1 group of disorders. The remarkable diversity and breadth (Mizushima et al., 2007). Consequently, the substitution of implicated molecular pathways are consistent with the of the positively charged Arg with a shorter and un- highly complex nature of the brain (Hamdan et al., 2017). charged glutamine may affect binding of UFC1 to E1 or T106I Indeed, the observation that the majority of these conditions E3 (Fig. 4B). Therefore, we tested whether the UFC1 R23Q are not associated with significant systemic findings, further and UFC1 variants impact on the ufmylation. To do supports the notion that brain vulnerability is inherent to its this, HEK293T cells deleting UFC1 were generated complexity. This is especially remarkable when one considers (Supplementary Fig. 1B), and MYC-UFM1 together with C116S T106I/C116S the fundamental and ubiquitous nature of the many biolo- FLAG-UFC1 , FLAG-UFC1 or FLAG- R23Q/C116S gical processes that are impaired in the various genetic forms UFC1 was expressed in the UFC1-deficient of infantile encephalopathy, including post-translational mod- HEK293T cells. While an UFC1-UFM1 intermediate was C116S ification (Alazami et al., 2015; Anazi et al., 2016, 2017). clearly detected in FLAG-UFC1 -expressing UFC1-defi- Ufmylation is a highly conserved post-translational mod- cient HEK293T cells, only a faint intermediate formation T106I/C116S ification with each of its components having a corresponding was observed in the case of FLAG-UFC1 and R23Q/C116S orthologue in all multicellular organisms. In this study, we FLAG-UFC1 (Fig. 4C). Our in vitro thioester for- show that this pathway is critically required for normal mation assay also showed that while wild-type UFC1 T106I brain development and function in humans consistent with formed an intermediate with UFM1, UFC1 and R23Q suggestive data from model organisms. For example, the UFC1 substantially reduced this ability (Fig. 4D). In fruitfly models of UBA5 and UFM1 deficiency display lymphoblasts isolated from three affected individuals with T106I increased mortality, locomotive defects, and abnormal UFC1 , the intermediate formation with endogenous neuromuscular junctions (Duan et al.,2016).Similarly, defi- UFM1 was significantly suppressed in comparison with ciency of the UBA5 orthologue in Caenorhabditis elegans that in control lymphoblasts (Fig. 4E, left panel). Similar R81C results in increased susceptibility to induced seizures and to the case of UFM1 , the UFM1-conjugate formation pharynx grinder paralysis as well as abnormal sensorial be- with cellular proteins was impaired in patient-derived haviour (Colin et al., 2016). Impaired motility and seizure- lymphoblasts (Fig. 4E, right panel). Taken together, we R81C like activity were also observed in zebrafish uba5 morphants concluded that although the mutations UFM1 and T106I R23Q (Colin et al., 2016). More relevant to the phenotype of the UFC1 UFC1 affect different proteins, they all patients we described in this study is our recently published impact ufmylation. Hence our analysis explains the similar brain-specific conditional knockout of Ufm1 under the patient phenotype by showing that these mutations cause nestin promoter (Muona et al., 2016), which allowed us to phenotypically similar hypomorphic effects on the UFM1- directly observe the brain pathology associated with im- system. paired ufmylation in vivo. Although these mice appeared normal at birth, they uniformly died in the first day after Mutation in UFM1 does not affect birth. Histopathological examination revealed that their brains were microcephalic with evidence of increased apop- endoplasmic reticulum stress- tosis, consistent with the proposed role of ufmylation in mediated apoptosis neuronal development and survival (Muona et al., 2016). Studies have reported that the pathogenesis of ufmylation- Similar to our previous work on UBA5-related severe related encephalopathy is associated with endoplasmic re- infantile encephalopathy, we show that mutations in ticulum stress-mediated apoptosis. Thus we tested UFM1 UFM1 itself as well as in UFC1, encoding the sole E2 mutation (p.R81C) effect on endoplasmic reticulum stress. conjugating enzyme for UFM lead to widespread impair- We found no significant impact of UFM1 wild-type or ment of ufmylation. Our results show a reduction rather mutant expression on the induction of endoplasmic reticu- than abrogation of ufmylation with the activity of UFM1 lum stress markers CHOP (DDIT3) and BIP (HSPA5) in and UFC1 mutants at 60–75% of their wild-type counter- either HeLa or SH-SY5Y cells, after 8 h treatment with parts. A recently reported promoter mutation in UFM1 in Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 1944 | BRAIN 2018: 141; 1934–1945 M. S. Nahorski et al. the context of infantile encephalopathy with or without Acknowledgements epilepsy is similarly predicted to only reduce but not elim- inate transcription of an otherwise normal transcript We thank the study families for their enthusiastic partici- (Hamilton et al., 2017). The clinical phenotype of previ- pation. We also thank the Sequencing and Genotyping ously reported patients with UBA5 (Colin et al., 2016; Core Facilities at KFSHRC for their technical support. Duan et al., 2016; Muona et al., 2016) and UFM1 (Hamilton et al., 2017) mutations are similar to our UFM1 and UFC1 patients, particularly regarding failure Funding to thrive, short stature, microcephaly, GDD, seizures, basal ganglia abnormality, delayed CNS myelination, and M.N. is supported by the Wellcome Trust. R.I. is supported cerebellar hypoplasia (Table 2). These observations strongly by Grant-in-Aid for JSPS Research Fellows (JP16J07037). argue for a minimum threshold required for embryonic via- C.G.W. acknowledges support from the NIHR Cambridge bility, and this would be consistent with the mouse embry- Biomedical Research Campus. M.K. is supported by Grant- onic lethal phenotype observed in the complete knockout of in-Aid for Scientific Research on Innovative Areas Uba5, Ufbp1, Ufl1 or Ufm1 (M.K., unpublished data) (JP25111006 and 15K21749 to M.K.), a Japan Society (Tatsumi et al., 2011; Cai et al., 2015; Zhang et al., 2015). for the Promotion of Science (an A3 foresight program, The pathogenesis of ufmylation-related encephalopathy to M.K.), and the Takeda Science Foundation (to M.K.). remains unclear e.g. is the disease process a progressive F.S.A. is supported by King Salman Center for Disability apoptosis of selected neuron classes, or a neuronal dysfunc- Research and King Abdulaziz City for Science and tion that becomes increasingly evident during development? Technology (13-BIO1113-20, and Saudi Human Genome The known localization of the ufmylation cascade and Program). S.T.A. and F.J.G.V. are supported by funding target proteins to the luminal side of the endoplasmic re- from King Abdullah University of Science and ticulum and the proposed role in regulating the unfolded Technology (KAUST). A.R. is supported by radiz—Rare protein response (UPR) and endoplasmic reticulum stress- Disease Initiative Zu¨ rich, Clinical Research Priority mediated apoptosis, suggest a potential mechanistic link Program for Rare Diseases of the University of Zurich. (Lemaire et al., 2011; Zhang et al., 2015; Ishimura et al., 2017). Expanded endoplasmic reticulum network and increased endoplasmic reticulum volume in response to Supplementary material tunicamycin, both markers of endoplasmic reticulum Supplementary material is available at Brain online. stress, have indeed been observed in fibroblasts from pa- tients with UBA5 mutations (Colin et al., 2016). Long-term endoplasmic reticulum stress driven by mutation of specific disease-related genes is known to cause various adult-onset References neurodegenerative phenotypes by overwhelming the UPR Alazami AM, Patel N, Shamseldin HE, Anazi S, Al-Dosari MS, and inducing apoptosis (Hetz and Saxena, 2017). While Alzahrani F, et al. Accelerating novel candidate gene discovery in we found no evidence of UFM1 wild-type or p.R81C over- neurogenetic disorders via whole-exome sequencing of prescreened multiplex consanguineous families. Cell Rep 2015; 10: 148–61. expression affecting endoplasmic reticulum stress induction Alkuraya FS. Autozygome decoded. Genet Med 2010; 12: 765–71. or apoptosis in response to tunicamycin (Supplementary Alkuraya FS. Discovery of rare homozygous mutations from studies of Fig. 3), further work should serve to investigate the mech- consanguineous pedigrees. Curr Protoc Hum Genet 2012; Chapter anisms by which defects in ufmylation lead to early-onset 6: Unit 6.12. encephalopathy in humans. However, it may be that the Alkuraya FS. The application of next-generation sequencing in the autozygosity mapping of human recessive diseases. Hum Genet ufmylation cascade, rather than a global effect on endoplas- 2013; 132: 1197–211. mic reticulum or UPR, could disrupt neuron function Alkuraya FS. Discovery of mutations for Mendelian disorders. Hum through effects on neural-essential proteins; the neuronal Genet 2016; 135: 615–23. cell adhesion molecule (NCAM) interacts and co-localizes Anazi S, Maddirevula S, Faqeih E, Alsedairy H, Alzahrani F, with UFC1, and CDK5 activity is controlled by Shamseldin H, et al. Clinical genomics expands the morbid CDK5RAP3, which aggregates with UFL1 and UFSP2 at genome of intellectual disability and offers a high diagnostic yield. Mol Psychiatry 2016; 22: 615–24. the endoplasmic reticulum membrane (Homrich et al., Anazi S, Maddirevula S, Salpietro V, Asi YT, Alsahli S, Alhashem A, 2014). et al. Expanding the genetic heterogeneity of intellectual disability. In conclusion, our study suggests that impaired ufmyla- Hum Genet 2017; 136: 1419–29. tion leads to a recognizable syndrome of severe infantile Cai Y, Pi W, Sivaprakasam S, Zhu X, Zhang M, Chen J, et al. UFBP1, encephalopathy and progressive microcephaly with or with- a key component of the Ufm1 conjugation system, is essential for ufmylation-mediated regulation of erythroid development. PLoS out epilepsy. Further studies are needed to discern the exact Genet 2015; 11: e1005643. pathomechanism of ufmylation-related neurodevelopmental Cappadocia L, Lima CD. Ubiquitin-like protein conjugation: struc- disorder, which may lead to possible therapies especially tures, chemistry, and mechanism. Chem Rev 2018; 118: 889–918. when one considers the hypomorphic nature of the Colin E, Daniel J, Ziegler A, Wakim J, Scrivo A, Haack TB, et al. observed mutations. Biallelic variants in UBA5 reveal that disruption of the UFM1 Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 UFM1 and UFC1 mutations in brain development BRAIN 2018: 141; 1934–1945 | 1945 cascade can result in early-onset encephalopathy. Am J Hum Genet conservation in the metazoan UFM1-UBA5-UFC1 ubiquination 2016; 99: 695–703. pathway. J Struct Funct Genomics 2009; 10: 127–36. Daniel J, Liebau E. The ufm1 cascade. Cells 2014; 3: 627–38. Liu H, Li J, Tillman B, French B, French S. Ufmylation and Duan R, Shi Y, Yu L, Zhang G, Li J, Lin Y, et al. UBA5 mutations FATylation pathways are downregulated in human alcoholic and cause a new form of autosomal recessive cerebellar ataxia. PLoS nonalcoholic steatohepatitis, and mice fed DDC, where Mallory– One 2016; 11: e0149039. Denk bodies (MDBs) form. Exp Mol Pathol 2014; 97: 81–8. Hamdan FF, Myers CT, Cossette P, Lemay P, Spiegelman D, Laporte Mizushima T, Tatsumi K, Ozaki Y, Kawakami T, Suzuki A, AD, et al. High rate of recurrent de novo mutations in developmen- Ogasahara K, et al. Crystal structure of Ufc1, the Ufm1-conjugating tal and epileptic encephalopathies. Am J Hum Genet 2017; 101: enzyme. Biochem Biophys Res Commun 2007; 362: 1079–84. 664–85. Muona M, Ishimura R, Laari A, Ichimura Y, Linnankivi T, Keski- Hamilton EM, Bertini E, Kalaydjieva L, Morar B, Dojcˇa´kova´ D, Liu J, Filppula R, et al. Biallelic variants in UBA5 link dysfunctional et al. UFM1 founder mutation in the Roma population causes re- UFM1 ubiquitin-like modifier pathway to severe infantile-onset en- cessive variant of H-ABC. Neurology 2017; 89: 1821–8. cephalopathy. Am J Hum Genet 2016; 99: 683–94. Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem Oweis W, Padala P, Hassouna F, Cohen-Kfir E, Gibbs DR, Todd EA, 1998; 67: 425–79. et al. Trans-binding mechanism of ubiquitin-like protein activation Hetz C, Saxena S. ER stress and the unfolded protein response in revealed by a UBA5-UFM1 complex. Cell Rep 2016; 16: 3113–20. neurodegeneration. Nat Rev Neurol 2017; 13: 477–91. Schulman BA, Harper JW. Ubiquitin-like protein activation by E1 en- Hitosugi T, Chen J. Post-translational modifications and the Warburg zymes: the apex for downstream signalling pathways. Nat Rev Mol effect. Oncogene 2014; 33: 4279–85. Cell Biol 2009; 10: 319–31. Homrich M, Wobst H, Laurini C, Sabrowski J, Schmitz B, Diestel S. Tatsumi K, Sou YS, Tada N, Nakamura E, Iemura S, Natsume T, Cytoplasmic domain of NCAM140 interacts with ubiquitin-fold et al. A novel type of E3 ligase for the Ufm1 conjugation system. modifier-conjugating enzyme-1 (Ufc1). Exp Cell Res 2014; 324: J Biol Chem 2010; 285: 5417–27. 192–9. Tatsumi K, Yamamoto-Mukai H, Shimizu R, Waguri S, Sou YS, Ishimura R, Obata M, Kageyama S, Daniel J, Tanaka K, Komatsu M. Sakamoto A, et al. The Ufm1-activating enzyme Uba5 is indispens- A novel approach to assess the ubiquitin-fold modifier 1-system in able for erythroid differentiation in mice. Nat Commun 2011; 2: cells. FEBS Lett 2017; 591: 196–204. 181. Komatsu M, Chiba T, Tatsumi K, Iemura Si, Tanida I, Okazaki N, Tompa P, Davey NE, Gibson TJ, Babu MM. A million peptide motifs et al. A novel protein-conjugating system for Ufm1, a ubiquitin-fold for the molecular biologist. Mol Cell 2014; 55: 161–9. modifier. EMBO J 2004; 23: 1977–86. Wei Y, Xu X. UFMylation: a unique & fashionable modification for Lemaire K, Moura RF, Granvik M, Igoillo-Esteve M, Hohmeier HE, life. Genomics Proteomics Bioinformatics 2016; 14: 140–6. Hendrickx N, et al. Ubiquitin fold modifier 1 (UFM1) and its target Yoo HM, Park JH, Jeon YJ, Chung CH. Ubiquitin-fold modifier 1 acts UFBP1 protect pancreatic beta cells from ER stress-induced apop- as a positive regulator of breast cancer. Front Endocrinol 2015; 6: 36. tosis. PLoS One 2011; 6: e18517. Zhang M, Zhu X, Zhang Y, Cai Y, Chen J, Sivaprakasam S, et al. Liu G, Forouhar F, Eletsky A, Atreya HS, Aramini JM, Xiao R, et al. RCAD/Ufl1, a Ufm1 E3 ligase, is essential for hematopoietic stem NMR and X-RAY structures of human E2-like ubiquitin-fold modi- cell function and murine hematopoiesis. Cell Death Differ 2015; 22: fier conjugating enzyme 1 (UFC1) reveal structural and functional 1922–34. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Brain Oxford University Press

Loading next page...
 
/lp/ou_press/biallelic-ufm1-and-ufc1-mutations-expand-the-essential-role-of-nMajPkKKVe

References (35)

Publisher
Oxford University Press
Copyright
Copyright © 2022 Guarantors of Brain
ISSN
0006-8950
eISSN
1460-2156
DOI
10.1093/brain/awy135
Publisher site
See Article on Publisher Site

Abstract

Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 doi:10.1093/brain/awy135 BRAIN 2018: 141; 1934–1945 | 1934 Biallelic UFM1 and UFC1 mutations expand the essential role of ufmylation in brain development 1, 2, 3, 2 Michael S. Nahorski, * Sateesh Maddirevula, * Ryosuke Ishimura, * Saud Alsahli, 4 5 6 7 Angela F. Brady, Anaı¨s Begemann, Tsunehiro Mizushima, Francisco J. Guzma´n-Vega, 3 3 2 2 2 Miki Obata, Yoshinobu Ichimura, Hessa S. Alsaif, Shams Anazi, Niema Ibrahim, 2 2 2,8 2,8 Firdous Abdulwahab, Mais Hashem, Dorota Monies, Mohamed Abouelhoda, 2,8 9 9 5 5 Brian F. Meyer, Majid Alfadhel, Wafa Eyaid, Markus Zweier, Katharina Steindl, 5,10 7 1 3 Anita Rauch, Stefan T. Arold, C. Geoffrey Woods, Masaaki Komatsu and 2,8,11 Fowzan S. Alkuraya *These authors contributed equally to this work. The post-translational modification of proteins through the addition of UFM1, also known as ufmylation, plays a critical develop- mental 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 develop- ment. 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 wide- spread 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. 1 Cambridge Institute for Medical Research, Wellcome Trust MRC Building Addenbrookes Hospital, Hills Rd, Cambridge CB2 0QQ, UK 2 Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia 3 Department of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Chuo-ku, Niigata 951-8510, Japan 4 North West Thames Genetics Service, Level 8V, St Mark’s Hospital, Northwick Park Hospital Watford Road, Harrow, HA1 3UJ, UK 5 Institute of Medical Genetics, University of Zurich, 8952 Schlieren-Zurich, Switzerland 6 Picobiology Institute, Graduate School of Life Science, University of Hyogo, Ako-gun, Hyogo 678-1297, Japan 7 King Abdullah University of Science and Technology, Computational Bioscience Research Center, Division of Biological and Environmental Sciences and Engineering, Thuwal 23955-6900, Saudi Arabia Received December 25, 2017. Revised March 3, 2018. Accepted March 23, 2018. Advance Access publication June 2, 2018 The Author(s) (2018). Published by Oxford University Press on behalf of the Guarantors of Brain. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 UFM1 and UFC1 mutations in brain development BRAIN 2018: 141; 1934–1945 | 1935 8 Saudi Human Genome Program, King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia 9 King Abdullah International Medical Research Centre, King Saud bin Abdulaziz University for Health Sciences, Division of Genetics, Department of Pediatrics, King Abdullah Specialized Children Hospital, King Abdulaziz Medical City, Ministry of National Guard-Health Affairs (NGHA), Riyadh, Saudi Arabia 10 Neuroscience Center Zurich, University of Zurich and ETH Zurich, Zurich 8057, Switzerland 11 Department of Anatomy and Cell Biology, College of Medicine, Alfaisal University, Riyadh, Saudi Arabia Correspondence to: Fowzan S. Alkuraya Department of Genetics King Faisal Specialist Hospital and Research Center Riyadh Saudi Arabia E-mail: [email protected] Correspondence may also be addressed to: Masaaki Komatsu Department of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Chuo-ku, Niigata 951-8510, Japan E-mail: [email protected] C. Geoffrey Woods Cambridge Institute for Medical Research Wellcome Trust MRC Building Addenbrookes Hospital, Hills Rd Cambridge CB2 0QQ, UK E-mail: [email protected] Keywords: ufmylation; UFM1; UFC1; encephalopathy; epilepsy (Tatsumi et al., 2010). The physiological context of ufmy- Introduction lation remains incompletely understood. The literature on the relevance of ufmylation to human health was originally Post-translational modification greatly expands the func- limited to cancer and, to a much lesser extent, complex tional repertoire of proteins beyond the confines of their coding sequence, and serves a diverse array of regulatory diseases such as diabetes, ischaemic heart diseases and al- roles in protein turnover, localization and interactions coholic liver disease (Liu et al., 2014; Yoo et al., 2015; Wei and Xu, 2016). As with other biochemical processes, how- (Hitosugi and Chen, 2014; Tompa et al., 2014). ever, Mendelian diseases involving deficiency of the individ- Ubiquitination is a well-established post-translational modi- ual components of ufmylation offered a unique opportunity fication involving a series of enzymatic reactions that add to reveal the extent to which ufmylation influences human ubiquitin to its target protein (Hershko and Ciechanover, physiology. We and others have reported hypomorphic mu- 1998). These reactions involve a few E1 activating enzymes, tations in UBA5 in children with severe infantile onset epi- dozens of E2 conjugating enzymes and hundreds of E3 ligat- leptic encephalopathy (Colin et al., 2016; Duan et al., ing enzymes. More than a dozen ubiquitin-like (UBL) pro- 2016; Muona et al., 2016). Additionally, Hamilton et al. teins have been found to similarly modify their target (2017) reported that a homozygous mutation in UFM1 proteins (Schulman and Harper, 2009). UBL are classified caused a severe early-onset encephalopathy with progres- into those that are activated and conjugated to substrates sive microcephaly, although the mechanism remained un- (type I) e.g. SUMO, NEDD8, ATG8, ATG12, URM1, clear. In this report, we provide evidence that it is the UFM1, FAT10, and ISG15, and those that do not undergo process of ufmylation that is essential for normal nervous conjugation (type II) (Cappadocia and Lima, 2018). system development and function, based on the identifica- UFM1 (ubiquitin-fold modifier 1), a 9.1-kDa protein tion of UFM1 and UFC1 biallelic mutations, which we with a tertiary structure similar to ubiquitin, is among the show lead to a remarkably similar neurological phenotype most recently identified type I UBL proteins (Komatsu and accompanying impairment in ufmylation. et al., 2004). The process of covalently attaching UFM1 to its target proteins, termed ufmylation, requires E1 acti- vating enzymes (UBA5), E2 conjugating enzymes (UFC1), and an E3 ligase (UFL1) (Daniel and Liebau, 2014). UFM1 Materials and methods is activated by UBA5, forming a high-energy thioester bond. Activated UFM1 is then transferred to UFC1, in a Human subjects similar thioester linkage (Komatsu et al., 2004). Ufmylation is completed by the catalysis of UFM1 transfer to the sub- Patients and available relatives were recruited with informed strate protein through the action of the E3 enzyme UFL1 consent as part of IRB-approved research protocols (RAC# Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 1936 | BRAIN 2018: 141; 1934–1945 M. S. Nahorski et al. 2080006 and 2121053; StV 11/09). Clinical data, including in pull-down assay buffer (20 mM Tris-Cl, pH 7.5, 150 mM laboratory and imaging studies, were collected from all par- NaCl, 1 mM EDTA, 1 mM DTT, 0.05% Nonidet P-40). The ticipants. Blood was collected in EDTA tubes for DNA extrac- pulled-down protein complexes were extensively washed with tion and in Na-heparin tubes for the establishment of pull-down assay buffer, and the obtained samples were sub- lymphoblastoid cell lines. jected to NuPAGE (4–12% acrylamide gradient) and Coomassie brilliant blue staining. Autozygome analysis Cell culture Genomewide single nucleotide polymorphism (SNP) genotyp- HEK293T cells were grown in Dulbecco’s modified Eagle ing using Axiom SNP Chip (Affymetrix) from all available medium (DMEM) containing 10% foetal bovine serum patients and relatives was pursued to determine the candidate (FBS), 5 U/ml penicillin, and 50mg/ml streptomycin. Lympho- autozygome as described before (Alkuraya, 2010, 2012). blasts were cultured in RPMI 1640 supplemented by 10% Runs of homozygosity 42 Mb were considered surrogates of FBS, 5 U/ml penicillin, and 50mg/ml streptomycin. To generate autozygosity given the consanguineous nature of the study UFM1- and UFC1-knockout cells, each UFM1 and UFC1 families as determined by AutoSNPa. Homozygosity mapping guide RNA designed using the CRISPR Design tool (http:// was performed on all available family members using crispr.mit.edu/) was subcloned into pX330-U6-Chimeric_BB- HomozygosityMapper (http://www.homozygositymapper.org/). CBh-hSpCas9 (Addgene #42230), a human codon-optimized SpCas9 and chimeric guide RNA expression plasmid. Exome sequencing and variant HEK293T cells were co-transfected with the pX330 and pEGFP-C1 (#6084-1, Clontech Laboratories) vectors, and cul- filtering tured for 2 days. Thereafter, the GFP-positive cells were sorted Exome capture was performed using TruSeq Exome and expanded. Loss of UFM1 and of UFC1 was confirmed by Enrichment kit (Illumina) following the manufacturer’s proto- heteroduplex mobility assay followed by immunoblot analysis col. Samples were prepared as an Illumina sequencing library, with anti-UFM1 and anti-UFC1 antibodies, respectively. and in the second step, the sequencing libraries were enriched for the desired target using the Illumina Exome Enrichment Immunoblot analysis protocol. The captured libraries were sequenced using Illumina HiSeq 2000 Sequencer. The reads were mapped Cells were lysed with ice-cold TNE buffer (10 mM Tris-Cl, pH against UCSC hg19 (http://genome.ucsc.edu/) by BWA (http:// 7.5, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, and pro- bio-bwa.sourceforge.net/). The SNPs and indels were detected tease inhibitors). The samples were separated using the by SAMtools (http://samtools.sourceforge.net/). Variants from NuPAGE system (Invitrogen) on 12% Bis-Tris gels in whole exome sequencing (WES) were filtered such that only NuPAGE MOPS SDS Running Buffer, and transferred to novel (or very low frequency 50.1%), coding/splicing, homo- polyvinylidene difluoride (PVDF) membranes. Antibodies zygous variants that are within the candidate autozygome against FLAG (Medical & Biological Laboratories Co., Ltd., (autozygous intervals exclusive to the affected individuals) M185-3L), UFC (Abcam, ab189251) and UFM1 (Abcam, and are predicted to be pathogenic were considered as likely ab109305) were purchased from the indicated suppliers. causal variants (Alkuraya, 2013, 2016). Frequency of variants Anti–UBA5 and UFM1 polyclonal antibodies were described was determined using publicly available variant databases previously (Komatsu et al., 2004). The immunoreactive bands (1000 Genomes, Exome Variant Server and ExAC) as well were detected by LAS-4000 (GE Healthcare UK Ltd.). The as a database of 2369 in-house ethnically-matched exomes. quantitative densitometric analyses of FLAG-UBA5-MYC- Pathogenicity is likely if the mutation is loss-of-function (spli- UFM1, FLAG-UFC1-MYC-UFM1, endogenous UBA5-UFM1 cing/truncating) or, in the case of missense/in-frame indels, re- intermediate, and endogenous UFC1-UFM1 intermediate rela- moves a highly conserved amino acid and is predicted to be tive to free FLAG-UBA5, FLAG-UFC1, endogenous UBA5, pathogenic by the three in silico prediction modules PolyPhen, and endogenous UFC1 were carried out using Multi Gauge SIFT and CADD. Version 3.2 Image software (Fuji Film, Tokyo, Japan). Statistical analysis was performed using an unpaired t-test (Welch test). The data represent the means  standard error In silico modelling (SE) of three separate experiments. 3D experimental structures were retrieved from the Protein Data Bank (PDB) and analysed using PYMOL (www.pymol. In vitro thioester formation assay org). In vitro thioester formation assay was conducted as previously reported (Komatsu et al., 2004). Briefly, recombinant GST- Pull-down assay R81C Gly83 UFM1C2, GST-UFM1C2 , GST- UFM1 , GST- R23Q T106I Recombinant proteins were purified with glutathione S-trans- UBA5, GST-UFC1, GST-UFC1 and GST-UFC1 were ferase (GST) affinity purification system according to the produced in Escherichia coli and recombinant proteins were manufacturer’s protocol (GE Healthcare). The GST moiety of purified by chromatography on Glutathione Sepharose 4B all purified proteins without GST-UBA5 was removed on (GE Healthcare). After digestion of GST by PreScission column by PreScission protease (GE Healthcare). GST-UBA5 Protease (GE Healthcare), the recombinant proteins were dia- bound to Glutathione Sepharose 4B (GE Healthcare) was lyzed against 50 mM BisTris (pH 6.5), 100 mM NaCl, 10 mM incubated for 20 min at 4 C with indicated purified proteins MgCl , and 0.1 mM DTT (reaction buffer). Thioester 2 Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 UFM1 and UFC1 mutations in brain development BRAIN 2018: 141; 1934–1945 | 1937 formation reactions contained reaction buffer with 0.8mg R81C Gly83 UFM1C2, UFM1C2 or UFM1 and some of the following: 5 mM ATP, 0.08 (for UFC1-UFM1 thioester forma- tion assay) or 0.8mg (for UBA5-UFM1 thioester formation R23Q T106I assay) UBA5 and 0.8mg UFC1, UFC1 or UFC1 . Reactions were incubated for 5 min at 25 C and stopped by the addition of NuPAGE LDS Sample Buffer lacking reducing agent, followed by a 10-min incubation at 37 C, NuPAGE (4–12% acrylamide gradient) and Coomassie brilliant blue staining. Data shown are representative of three separate experiments. Assessment of apoptosis in response to endoplasmic reticulum stress HeLa or SH-SY5Y cells were plated into six-well plates at 7.5  10 cells/ml. After 18 h they were transfected with either 2mgGFP, 1mg UFM1WT/1mg GFP or 1mg UFM1R81C/1mg TM GFP using FuGENE HD or X-tremeGENE HP, respectively according to the manufacturer’s protocols. Media was changed after 24 h and tunicamycin added after 48 h. For endoplasmic reticulum stress marker analysis, cells were incubated with 5mg/ ml tunicamycin for 8 h. mRNA was extracted from cells using the RNeasy Plus Mini Kit (Qiagen) according to the manufac- turer’s instructions. The concentration of the mRNA was calcu- TM lated and 1mg converted to cDNA using the qScript cDNA synthesis kit (Quanta Biosciences). Levels of HSPA5, DDIT3 and GAPDH were assayed using pre-designed TaqMan Gene Expression Assays (ThermoFisher Scientific). The level of gene expression was normalized to that of GAPDH for each sample assessed. For assessment of apoptosis in response to endoplas- mic reticulum stress, cells were incubated in different concentra- tions of tunicamycin for 48 h. They were then, trypsinized and stained with Annexin V, Alexa Fluor 647 conjugate (ThermoFisher Scientific) and DAPI to assess early and late stages of apoptosis, respectively. Cells were analysed on a BD TM LSR Fortessa flow cytometer, selecting only those expressing GFP. Data were analysed using FlowJo software, and cells ex- pressing either DAPI and/or Annexin V were considered apoptotic. Results Identification of novel severe early infantile encephalopathy phenotypes Two Sudanese families were recruited independently by C.G.W. and F.S.A., each with two children presenting with the core phenotype of profound global developmental delay, failure to thrive, progressive microcephaly and re- fractive epilepsy. Salient clinical features include subtle facial dysmorphism, severe axial hypotonia and appendicu- lar hypertonia. Available brain imaging revealed dysmyeli- nation and volume loss. Hypsarrhythmia was documented on EEG. The severity of the presentation is evidenced by the premature death of two of the four patients at ages 9 months and 8.5 years, respectively (Table 1, Fig. 1 and Supplementary Tables 1 and 2). Table 1 Summary of the clinical features of patients with UFM1 and UFC1 mutations ID 12DG0178 12DG1577 14DG0050 16DG1614 MDL-17-3196 MDL-17-3892 17DG0828 ID76366 UK1 UK2 10DG0945 10DG0946 Gene UFC1 UFC1 UFC1 UFC1 UFC1 UFC1 UFC1 UFC1 UFM1 UFM1 UFM1 UFM1 Mutation c.317C4T c.317C4T c.317C4T c.317C4T c.317C4T c.317C4T c.317C4T c.68G4A c.241C4T c.241C4T c.241C4T c.241C4T p.(Thr106Ile) p.(Thr106Ile) p.(Thr106Ile) p.(Thr106Ile) p.(Thr106Ile) p.(Thr106Ile) p.(Thr106Ile) p.(Arg23Gln) p.(Arg81Cys) p.(Arg81Cys) p.(Arg81Cys) p.(Arg81Cys) Age 16 years 23 years 3 years 5 years 5 years 31 months 8 years 4 years 13 months 13 months 2 years 1 year Gender F F F F F F F M F M M M Microcephaly+ + - + + + + + +++ + Short Stature + + - + + + + + + + + + Underweight + + + + + + + + + + + + GDD +++++ + +++ + + + Seizures + - - + + - - + + + + + GDD = global developmental delay; F = female; M = male. Microcephaly was always secondary i.e. postnatal. Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 1938 | BRAIN 2018: 141; 1934–1945 M. S. Nahorski et al. Figure 1 UFM1-related clinical phenotype. (A) Pedigree of Family 1 with UFM1 mutation. (B and C) Images from Family 1 exhibiting lack of major facial dysmorphism. (D and E) Pes cavus as a result of abnormal tone in Patients 10DG0945 and 10DG0946, respectively (Family 1). (F) Genome-wide homozygosity mapping revealed a single critical locus. Blue box indicates haplotype (Chr13:36292810-42302880) identical by descent. (G) Pedigree of Family 2 with UFM1 mutation. (H) Facial image of Patient UK1 (Family 2) showing full cheeks. (I and J) Peripheral oedema of Patients UK1 and UK2 (Family 2). (K and L) Brain MRI of Patient UK1 showing cerebellar hypoplasia and thin corpus callosum and frontal cortical polymicrogyria. (M) Schematic diagram of UFM1 protein representing ubiquitin-fold modifier 1 domain with the mutation indicated. The Arg81 at the mutation site is highly conserved from humans to C. elegans. Independently, we encountered a highly similar phenotype sequencing of the index in each of the two families revealed in three Saudi families and one Swiss family with eight af- the same sole novel homozygous variant within this interval: fected members (one of which was previously described, albeit UFM1: NM_016617.3:c.241C4T:p.(Arg81Cys) (Fig. 1). briefly) (Anazi et al., 2016). They all presented with severe Similarly, genotyping of the Saudi families with a similar early infantile encephalopathy, progressive microcephaly, axial encephalopathy phenotype revealed that they all share the hypotonia, appendicular hypertonia and refractory epilepsy. same ancestral haplotype (Chr1:158957700-162094700), Although brain MRI findings were largely non-specific, again supporting a recessive founder mutation (Fig. 2). some had evidence of basal ganglia involvement (Table 1, Indeed, exome sequencing on four of the seven patients Fig. 2 and Supplementary Tables 1 and 2). Phenotypic com- revealed a single novel homozygous variant within the crit- parison of our patients with UFM1 and UFC1 mutations to ical locus: UFC1: NM_016406.3:c.317C4T:p.(Thr106Ile), previously reported patients with UFM1 and UBA5 mutations which segregated with the phenotype in all four families as canbefoundinTable 2. revealed by targeted Sanger sequencing. Exome sequencing on a Swiss patient revealed a novel homozygous variant in UFC1: NM_016406.3:c.68G4A:p.(Arg23Gln) inherited UFM1 and UFC1 define novel loci for from the heterozygous parents. Mutations at Thr106 and severe infantile encephalopathy with Arg23 involve highly conserved residues (Fig. 2). progressive microcephaly Although the two Sudanese families are not known to be Hypomorphic effect of UFM1 muta- related, they originate from the same village in Sudan. tion on the UFM1-system Indeed, autozygome analysis revealed a single shared auto- zygous interval (Chr13:36292810-42302880) between the The UFM1 variant is predicted pathogenic by PolyPhen affected members with the same ancestral haplotype, (0.538/possibly damaging), SIFT (0/ deleterious) and CADD strongly supporting a recessive founder mutation. Exome (35), as are the UFC1 variants c.317C4T:p.(Thr106Ile) Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 UFM1 and UFC1 mutations in brain development BRAIN 2018: 141; 1934–1945 | 1939 Figure 2 UFC1-related clinical phenotype. (A) Pedigrees of the families with UFC1 mutation. (B) Genome-wide homozygosity plot high- lighting a single critical locus from Families 1–3, who harbour the p.(Thr106Ile) mutation. Blue box indicates haplotype (Chr1:158957700- 162094700) identical by descent. (C) Clinical images showing multiple joint contractures and emaciation (Patient 17DG0828). (D and E) Clinical images of younger patients suggest a progressive nature of emaciation (Patients 14DG0050 and 16DG1614, respectively). (F) Brain MRI of Patient 16DG1614 showing bilateral hyperintense signals in the basal ganglia. (G and H) Brain MRI of Patient MDL-17-3196 showing bilateral hyperintense signals in the basal ganglia in the first year that resolved in a repeated brain MRI in the fourth year of age. (I) Brain MRI of Patient ID76366 showing markedly delayed myelination. (J) Schematic diagram of UFC1 protein showing UFC1 domain and mutation site. Mutations Arg23Gln and Thr106Ile are highly conserved from humans to C. elegans. Table 2 Comparison of phenotype between this study patients with UFM1 and UFC1 mutations and those previously reported with mutations in UFM1 and UBA5 Comparison between UFM1-UBA5-UFC1 pathway reported cases Current cohort UFM1-related cases UBA5-related cases Gene UFC1 UFM1 UFM1(PubMed: 28931644) UBA5 (PubMed: 27545681, 27545674, 28965491) Mutation c.317C4T; c.68G4A c.241C4T c.-273_-271delTCA c.1111G4A c.904C4T c.971_972insC c.778G4A c.1165G4T c.169A4G c.503G4A c.164G4A c.684G 4 A Number of cases 8 4 16 19 Failure to thrive 100% (8/8) 100% (4/4) 63% (10/16) 89% (8/9) Short stature 88% (7/8) 100% (4/4) 75% (12/16) 77% (10/13) Microcephaly 88% (7/8) 100% (4/4) 100% (16/16) 100% (18/18) Global developmental delay 100% (8/8) 100% (4/4) 100% (16/16) 100% (19/19) Seizures 50% (4/8) 100% (4/4) 75% (12/16) 84% (16/19) Brain MRI Basal ganglia abnormality 33% (2/6) 0% (0/4) 100% (16/16) 0% (0/17) Delayed CNS myelination 17% (1/6) 75% (3/4) 100% (16/16) 24% (4/17) Cerebellar hypoplasia 0% (0/6) 75% (3/4) 81% (13/16) 24% (4/17) Mortality 0% (0/8) 100% (4/4) 56% (9/16) 24% (4/19) Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 1940 | BRAIN 2018: 141; 1934–1945 M. S. Nahorski et al. R81C Figure 3 Hypomorphic effect of the UFM1 mutation on the UFM system. (A) Molecular basis for the effect of the UFM1 mutation. Top: Crystal structure of the heterodimeric complex between UFM1 (magenta and yellow) and UBA5 (cyan and green), taken from PDB id 5IAA. Bottom: Magnification of the boxed region. The view is tilted horizontally by 30 compared to the top panel. For clarity, only side chains 81 81 of Arg and of Arg -interacting residues are shown. (B) In vitro pull-down assay. Pull-down assay with GST-UBA5 and UFM1, UFM1 mutants or LC3B. GST-UBA5 conjugated with Glutathione Sepharose 4B was incubated with purified recombinant UFM1, UFM1 mutants or LC3B. LC3B is known to interact with UBA5. The pulled-down complexes were subjected to NuPAGE (4–12% acrylamide gradient) and Coomassie brilliant C250S blue staining. GST-UBA5, LC3B, UFM1 and UFM1 mutants are indicated. (C and D) Immunoblot assay. Indicated constructs (0.1mg for UBA5 C116S 0.5mg for UFC1 , and 2mg for UFM1 or mutants) were expressed in UFM1-deficient HEK293T cells. Twenty-four hours after transfection, the (continued) Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 UFM1 and UFC1 mutations in brain development BRAIN 2018: 141; 1934–1945 | 1941 C116S [PolyPhen (0.998/probably damaging), SIFT (0/deleterious), intermediate of UFC1 with MYC-UFM1 (Fig. 3D). CADD (32)] and c.68G4A:p.(Arg23Gln) [PolyPhen (0.901/ Conversely, the formation of the UFM1-UFC1 intermediate R81C probably damaging), SIFT (0.02/deleterious) and CADD (35)]. was almost abolished when MYC-UFM1 or MYC- Gly83 C116S We therefore carried out experiments to assess if and in which UFM1 were expressed together with UFC1 way these mutations affect ufmylation. The C-terminal tail of (Fig. 3D). In good agreement with those analyses, an UFM1 (residues 79–83) is essential for its adenylation by the in vitro thioester formation assay revealed that while E1 enzyme UBA5 and subsequent thioester formation with wild-type UFM1 forms an intermediate with UBA5, R81C UBA5 (Oweis et al., 2016). The crystallographic structure of UFM1 slightly had the ability to form the intermediate R81C UBA5 in the complex with UFM1 (PDB ID 5IAA) shows that (Fig. 3E). We also observed that UFM1 is hardly trans- the tail region, encompassing Arg directly binds to UBA5 ferred to UFC1 (Fig. 3F). In the next series of experiments, R81C (Fig.3A).Since Arg interacts with the negatively charged we tested the effect of the UFM1 mutation on the 241 209 183 residues Glu ,Glu and Asp of UBA5 (Fig. 3A). The intermediate formation with endogenous UBA5 and UFC1 substitution of Arg with a shorter and hydrophobic Cys is as well as UFM1-conjugation, using lymphoblasts derived expected to markedly reduce the strength of the interaction from an affected individual. Expression of free UFM1 pro- between UFM1 and UBA5. Accordingly, an in vitro pull- tein in lymphoblasts from an affected individual was com- down assay revealed that the mutant has a lower binding parable to control cells (Fig. 3G, left panel). By affinity to UBA5 than wild-type UFM1 (Fig. 3B). Next, we immunoblot analysis with non-reducing samples, we de- R81C investigated whether UBA5isabletoactivateUFM1 .To tected the intermediates of UFM1-UBA5 and of UFM1- do this, we used a UBA5-mutant whose active site, cysteine UFC1. As predicted, the level of UFM1-UBA5 intermedi- 250 C250S (Cys ) was substituted with a serine (termed UBA5 ). ates was significantly lower in lymphoblasts of affected in- When thecysteineresidue at theactivesiteofE1and E2 dividual compared to control cells (Fig. 3G, middle panel). enzymes is replaced by a serine, an O-ester bond instead of Likewise, the formation of the UFM1-UFC1 intermediate a thioester bond is formed with its respective modifier pro- markedly declined (Fig. 3G, right panel). We also found teins, which become stable even under reducing conditions that the level of two UFM1-conjugates with cellular pro- (Komatsu et al., 2004). To exclude an effect of endogenous teins in patient-derived lymphoblasts was significantly UFM1, we generated UFM1-deficient HEK293T cells reduced compared to control lymphoblasts (Fig. 3G, left (Supplementary Fig. 1A). As expected, when we co-expressed R81C panel). Taken together, our data show that UFM1 C250S FLAG-UBA5 and wild-type MYC-UFM1 into UFM1-de- has a hypomorphic effect on the UFM1-system and provide ficient HEK293T cells, MYC-UFM1 formed a stable inter- a plausible mechanistic explanation. C250S mediate with FLAG-UBA5 (Fig. 3C). Such intermediate C250S formation was completely abrogated when UBA5 was Gly83 83 co-expressed with MYC-UFM1 ,whose Gly essential Hypomorphic effect of UFC1 muta- for the adenylation and activation through UBA5 is deleted tions on the UFM1-system (Fig. 3C). Though we still observed the intermediate in the R81C The UFC1 structure consists of the catalytic core domain case of MYC-UFM1 ,its levelwas only 75% of that of conserved in all E2-like enzymes and an additional N-ter- wild-type UFM1 (Fig. 3C). R81C minal helix (Mizushima et al., 2007). Thr on the UFC1 The defective activation of UFM1 by the UBA5 E1 structure is on the opposite site of the proposed UBA5 enzyme is expected to also hamper the subsequent step, binding site (involving helix 2) (Liu et al., 2009), suggest- namely the formation of the intermediate with its cognate ing that mutation of this residue does not affect the inter- E2-like enzyme UFC1. Therefore, we investigated whether R81C action with UBA5. Indeed, an in vitro pull-down assay UFM1 forms an intermediate with UFC1 in cells. showed that the Thr106Ile mutation does not have any When wild-type MYC-UFM1 was expressed together with C116S 116 106 effect on binding to UBA5 (Supplementary Fig. 2). Thr FLAG-UFC1 in which the active site, cysteine (Cys ), is located in a coiled region close to the catalytic Cys , was substituted with serine, we clearly detected the Figure 3 Continued cell lysates were subjected to immunoblot analysis with anti-FLAG antibody. Bar graphs indicate the quantitative densitometric analyses of FLAG- UBA5-MYC-UFM1 and FLAG-UFC1-MYC-UFM1 intermediates relative to free FLAG-UBA5 and FLAG-UFC1, respectively. Statistical analyses were performed using the unpaired t-test (Welch test). The data represent the means  SE of three separate experiments. **P5 0.01 and ***P5 0.001. (E and F) In vitro thioester formation assay of UFM1 by UBA5 (E) and of UFM1 by UFC1 (F). The assay was conducted as described in the ‘Materials and methods’ section. Data shown are representative of three separate experiments. (G) Immunoblot analysis in case (P1: 10DG0945, Individual V1 in Fig. 1A) and control (C1: a healthy Sudanese young female) lymphoblasts. Reducing (DTT plus) and non-reducing (DTT minus) samples were prepared from lymphoblasts and subjected to immunoblot analysis for UFM1 (left), UBA5 (middle), and UFC1 (right). R81C We used a hand-made anti-UFM1 antibody in this experiment since the commercial antibody did not recognize UFM1 . Bar graphs indicate the quantitative densitometric analyses of UBA5-UFM1 and UFC1-UFM1 intermediates relative to free UBA5 and UFC1, respectively. Statistical analysis was performed using the unpaired t-test (Welch test). The data represent the means  SE of three separate experiments. **P5 0.01 and ***P5 0.001. Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 1942 | BRAIN 2018: 141; 1934–1945 M. S. Nahorski et al. Figure 4 Hypomorphic effect of UFC1 mutants on the UFM system. (A) Localization of the key binding sites on UFC1. Binding sites for E1 and E3 are indicated. Boxed region shows the active site, and the coiled region for binding site to UFM1 is coloured in orange. The mutated 23 106 residues Arg and Thr are highlighted in yellow and green, respectively. Encircled region shows the proximity of R23 with regions involved in 90 91 130 106 binding to E1 (helix 2) and E3 (Tyr ,Pro ,Pro ; cyan). (B) Bottom: Magnification of Thr . Residues involved in hydrophobic or polar 106 106 interactions with Thr are shown as stick models. Colouring as in A, except for the mutant Ile , which is shown as light grey stick model, with 23 23 positions of minor clashes indicated as discs. Top: Magnification of Arg . Colouring as in A, except for the mutant Gln , which is shown as C116S C116S/T106I C116S/R23Q magenta stick model. (C) Immunoblot assay. Indicated constructs (0.5mg for UFC1 , UFC1 or UFC1 and 2mg for UFM1) were expressed in UFC1-deficient HEK293T cells. Twenty-four hours after transfection, the cell lysates were subjected to immunoblot analysis with indicated antibodies. Bar graphs indicate the quantitative densitometric analyses of FLAG-UFC1-MYC-UFM1 intermediates relative to free FLAG-UFC1. Statistical analyses were performed using the unpaired t-test (Welch test). The data represent the means  SE of three separate experiments. *P5 0.05. (D) In vitro thioester formation assay of UFM1 by UFC1. The assay was conducted as described in the ‘Materials and methods’ section. Data shown are representative of three separate experiments. (E) Immunoblot analysis in case (P2, P3, P4: V:1) and control (C1: a healthy Sudanese young females) lymphoblasts. Reducing (DTT plus) and non-reducing (DTT minus) samples were prepared from lymphoblasts and subjected to immunoblot analysis for UFC1 (left), and UFM1 (right). Bar graph indicates the quantitative densitometric analysis of UFC1-UFM1 intermediates relative to free UFC1. Statistical analysis was performed using the unpaired t-test (Welch test). The data represent the means  SE of three separate experiments. **P5 0.01. Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 UFM1 and UFC1 mutations in brain development BRAIN 2018: 141; 1934–1945 | 1943 and within the site mapped to be required for interactions 5mg/ml tunicamycin (Supplementary Fig. 3). Neither did with UFM1 (Liu et al., 2009) (orange segment in Fig. 4A). we note appreciable difference in endoplasmic reticulum In the crystal structure of unliganded UFC1, Thr inter- stress-induced apoptosis in cells expressing UFM1 wild- type or UFM1 p.R81C, after 48 h incubation in tunicamy- acts with the hydrophobic core of the protein and is mostly cin at various concentrations. excluded from the solvent (Fig. 4B). Replacing the polar threonine with the slightly larger and completely hydropho- bic isoleucine is expected to change the anchoring, stereo- chemistry and dynamics of this region, affecting the Discussion intermediate formation of UFM1 with UFC1. Similarly, 23 90 91 ˚ Our understanding of the pathogenesis of infantile encephal- Arg is close (10 A) to the residues Tyr , Pro and opathy at the molecular level has greatly expanded in recent Pro that form a substructure and is potentially involved years due in large part to the rapidly growing use of gen- in E3-binding (Liu et al., 2009) (Fig. 4A). Arg is also near omics to diagnose and classify this highly heterogeneous to the helix 2, which is involved in binding to E1 group of disorders. The remarkable diversity and breadth (Mizushima et al., 2007). Consequently, the substitution of implicated molecular pathways are consistent with the of the positively charged Arg with a shorter and un- highly complex nature of the brain (Hamdan et al., 2017). charged glutamine may affect binding of UFC1 to E1 or T106I Indeed, the observation that the majority of these conditions E3 (Fig. 4B). Therefore, we tested whether the UFC1 R23Q are not associated with significant systemic findings, further and UFC1 variants impact on the ufmylation. To do supports the notion that brain vulnerability is inherent to its this, HEK293T cells deleting UFC1 were generated complexity. This is especially remarkable when one considers (Supplementary Fig. 1B), and MYC-UFM1 together with C116S T106I/C116S the fundamental and ubiquitous nature of the many biolo- FLAG-UFC1 , FLAG-UFC1 or FLAG- R23Q/C116S gical processes that are impaired in the various genetic forms UFC1 was expressed in the UFC1-deficient of infantile encephalopathy, including post-translational mod- HEK293T cells. While an UFC1-UFM1 intermediate was C116S ification (Alazami et al., 2015; Anazi et al., 2016, 2017). clearly detected in FLAG-UFC1 -expressing UFC1-defi- Ufmylation is a highly conserved post-translational mod- cient HEK293T cells, only a faint intermediate formation T106I/C116S ification with each of its components having a corresponding was observed in the case of FLAG-UFC1 and R23Q/C116S orthologue in all multicellular organisms. In this study, we FLAG-UFC1 (Fig. 4C). Our in vitro thioester for- show that this pathway is critically required for normal mation assay also showed that while wild-type UFC1 T106I brain development and function in humans consistent with formed an intermediate with UFM1, UFC1 and R23Q suggestive data from model organisms. For example, the UFC1 substantially reduced this ability (Fig. 4D). In fruitfly models of UBA5 and UFM1 deficiency display lymphoblasts isolated from three affected individuals with T106I increased mortality, locomotive defects, and abnormal UFC1 , the intermediate formation with endogenous neuromuscular junctions (Duan et al.,2016).Similarly, defi- UFM1 was significantly suppressed in comparison with ciency of the UBA5 orthologue in Caenorhabditis elegans that in control lymphoblasts (Fig. 4E, left panel). Similar R81C results in increased susceptibility to induced seizures and to the case of UFM1 , the UFM1-conjugate formation pharynx grinder paralysis as well as abnormal sensorial be- with cellular proteins was impaired in patient-derived haviour (Colin et al., 2016). Impaired motility and seizure- lymphoblasts (Fig. 4E, right panel). Taken together, we R81C like activity were also observed in zebrafish uba5 morphants concluded that although the mutations UFM1 and T106I R23Q (Colin et al., 2016). More relevant to the phenotype of the UFC1 UFC1 affect different proteins, they all patients we described in this study is our recently published impact ufmylation. Hence our analysis explains the similar brain-specific conditional knockout of Ufm1 under the patient phenotype by showing that these mutations cause nestin promoter (Muona et al., 2016), which allowed us to phenotypically similar hypomorphic effects on the UFM1- directly observe the brain pathology associated with im- system. paired ufmylation in vivo. Although these mice appeared normal at birth, they uniformly died in the first day after Mutation in UFM1 does not affect birth. Histopathological examination revealed that their brains were microcephalic with evidence of increased apop- endoplasmic reticulum stress- tosis, consistent with the proposed role of ufmylation in mediated apoptosis neuronal development and survival (Muona et al., 2016). Studies have reported that the pathogenesis of ufmylation- Similar to our previous work on UBA5-related severe related encephalopathy is associated with endoplasmic re- infantile encephalopathy, we show that mutations in ticulum stress-mediated apoptosis. Thus we tested UFM1 UFM1 itself as well as in UFC1, encoding the sole E2 mutation (p.R81C) effect on endoplasmic reticulum stress. conjugating enzyme for UFM lead to widespread impair- We found no significant impact of UFM1 wild-type or ment of ufmylation. Our results show a reduction rather mutant expression on the induction of endoplasmic reticu- than abrogation of ufmylation with the activity of UFM1 lum stress markers CHOP (DDIT3) and BIP (HSPA5) in and UFC1 mutants at 60–75% of their wild-type counter- either HeLa or SH-SY5Y cells, after 8 h treatment with parts. A recently reported promoter mutation in UFM1 in Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 1944 | BRAIN 2018: 141; 1934–1945 M. S. Nahorski et al. the context of infantile encephalopathy with or without Acknowledgements epilepsy is similarly predicted to only reduce but not elim- inate transcription of an otherwise normal transcript We thank the study families for their enthusiastic partici- (Hamilton et al., 2017). The clinical phenotype of previ- pation. We also thank the Sequencing and Genotyping ously reported patients with UBA5 (Colin et al., 2016; Core Facilities at KFSHRC for their technical support. Duan et al., 2016; Muona et al., 2016) and UFM1 (Hamilton et al., 2017) mutations are similar to our UFM1 and UFC1 patients, particularly regarding failure Funding to thrive, short stature, microcephaly, GDD, seizures, basal ganglia abnormality, delayed CNS myelination, and M.N. is supported by the Wellcome Trust. R.I. is supported cerebellar hypoplasia (Table 2). These observations strongly by Grant-in-Aid for JSPS Research Fellows (JP16J07037). argue for a minimum threshold required for embryonic via- C.G.W. acknowledges support from the NIHR Cambridge bility, and this would be consistent with the mouse embry- Biomedical Research Campus. M.K. is supported by Grant- onic lethal phenotype observed in the complete knockout of in-Aid for Scientific Research on Innovative Areas Uba5, Ufbp1, Ufl1 or Ufm1 (M.K., unpublished data) (JP25111006 and 15K21749 to M.K.), a Japan Society (Tatsumi et al., 2011; Cai et al., 2015; Zhang et al., 2015). for the Promotion of Science (an A3 foresight program, The pathogenesis of ufmylation-related encephalopathy to M.K.), and the Takeda Science Foundation (to M.K.). remains unclear e.g. is the disease process a progressive F.S.A. is supported by King Salman Center for Disability apoptosis of selected neuron classes, or a neuronal dysfunc- Research and King Abdulaziz City for Science and tion that becomes increasingly evident during development? Technology (13-BIO1113-20, and Saudi Human Genome The known localization of the ufmylation cascade and Program). S.T.A. and F.J.G.V. are supported by funding target proteins to the luminal side of the endoplasmic re- from King Abdullah University of Science and ticulum and the proposed role in regulating the unfolded Technology (KAUST). A.R. is supported by radiz—Rare protein response (UPR) and endoplasmic reticulum stress- Disease Initiative Zu¨ rich, Clinical Research Priority mediated apoptosis, suggest a potential mechanistic link Program for Rare Diseases of the University of Zurich. (Lemaire et al., 2011; Zhang et al., 2015; Ishimura et al., 2017). Expanded endoplasmic reticulum network and increased endoplasmic reticulum volume in response to Supplementary material tunicamycin, both markers of endoplasmic reticulum Supplementary material is available at Brain online. stress, have indeed been observed in fibroblasts from pa- tients with UBA5 mutations (Colin et al., 2016). Long-term endoplasmic reticulum stress driven by mutation of specific disease-related genes is known to cause various adult-onset References neurodegenerative phenotypes by overwhelming the UPR Alazami AM, Patel N, Shamseldin HE, Anazi S, Al-Dosari MS, and inducing apoptosis (Hetz and Saxena, 2017). While Alzahrani F, et al. Accelerating novel candidate gene discovery in we found no evidence of UFM1 wild-type or p.R81C over- neurogenetic disorders via whole-exome sequencing of prescreened multiplex consanguineous families. Cell Rep 2015; 10: 148–61. expression affecting endoplasmic reticulum stress induction Alkuraya FS. Autozygome decoded. Genet Med 2010; 12: 765–71. or apoptosis in response to tunicamycin (Supplementary Alkuraya FS. Discovery of rare homozygous mutations from studies of Fig. 3), further work should serve to investigate the mech- consanguineous pedigrees. Curr Protoc Hum Genet 2012; Chapter anisms by which defects in ufmylation lead to early-onset 6: Unit 6.12. encephalopathy in humans. However, it may be that the Alkuraya FS. The application of next-generation sequencing in the autozygosity mapping of human recessive diseases. Hum Genet ufmylation cascade, rather than a global effect on endoplas- 2013; 132: 1197–211. mic reticulum or UPR, could disrupt neuron function Alkuraya FS. Discovery of mutations for Mendelian disorders. Hum through effects on neural-essential proteins; the neuronal Genet 2016; 135: 615–23. cell adhesion molecule (NCAM) interacts and co-localizes Anazi S, Maddirevula S, Faqeih E, Alsedairy H, Alzahrani F, with UFC1, and CDK5 activity is controlled by Shamseldin H, et al. Clinical genomics expands the morbid CDK5RAP3, which aggregates with UFL1 and UFSP2 at genome of intellectual disability and offers a high diagnostic yield. Mol Psychiatry 2016; 22: 615–24. the endoplasmic reticulum membrane (Homrich et al., Anazi S, Maddirevula S, Salpietro V, Asi YT, Alsahli S, Alhashem A, 2014). et al. Expanding the genetic heterogeneity of intellectual disability. In conclusion, our study suggests that impaired ufmyla- Hum Genet 2017; 136: 1419–29. tion leads to a recognizable syndrome of severe infantile Cai Y, Pi W, Sivaprakasam S, Zhu X, Zhang M, Chen J, et al. UFBP1, encephalopathy and progressive microcephaly with or with- a key component of the Ufm1 conjugation system, is essential for ufmylation-mediated regulation of erythroid development. PLoS out epilepsy. Further studies are needed to discern the exact Genet 2015; 11: e1005643. pathomechanism of ufmylation-related neurodevelopmental Cappadocia L, Lima CD. Ubiquitin-like protein conjugation: struc- disorder, which may lead to possible therapies especially tures, chemistry, and mechanism. Chem Rev 2018; 118: 889–918. when one considers the hypomorphic nature of the Colin E, Daniel J, Ziegler A, Wakim J, Scrivo A, Haack TB, et al. observed mutations. Biallelic variants in UBA5 reveal that disruption of the UFM1 Downloaded from https://academic.oup.com/brain/article/141/7/1934/5032368 by DeepDyve user on 12 July 2022 UFM1 and UFC1 mutations in brain development BRAIN 2018: 141; 1934–1945 | 1945 cascade can result in early-onset encephalopathy. Am J Hum Genet conservation in the metazoan UFM1-UBA5-UFC1 ubiquination 2016; 99: 695–703. pathway. J Struct Funct Genomics 2009; 10: 127–36. Daniel J, Liebau E. The ufm1 cascade. Cells 2014; 3: 627–38. Liu H, Li J, Tillman B, French B, French S. Ufmylation and Duan R, Shi Y, Yu L, Zhang G, Li J, Lin Y, et al. UBA5 mutations FATylation pathways are downregulated in human alcoholic and cause a new form of autosomal recessive cerebellar ataxia. PLoS nonalcoholic steatohepatitis, and mice fed DDC, where Mallory– One 2016; 11: e0149039. Denk bodies (MDBs) form. Exp Mol Pathol 2014; 97: 81–8. Hamdan FF, Myers CT, Cossette P, Lemay P, Spiegelman D, Laporte Mizushima T, Tatsumi K, Ozaki Y, Kawakami T, Suzuki A, AD, et al. High rate of recurrent de novo mutations in developmen- Ogasahara K, et al. Crystal structure of Ufc1, the Ufm1-conjugating tal and epileptic encephalopathies. Am J Hum Genet 2017; 101: enzyme. Biochem Biophys Res Commun 2007; 362: 1079–84. 664–85. Muona M, Ishimura R, Laari A, Ichimura Y, Linnankivi T, Keski- Hamilton EM, Bertini E, Kalaydjieva L, Morar B, Dojcˇa´kova´ D, Liu J, Filppula R, et al. Biallelic variants in UBA5 link dysfunctional et al. UFM1 founder mutation in the Roma population causes re- UFM1 ubiquitin-like modifier pathway to severe infantile-onset en- cessive variant of H-ABC. Neurology 2017; 89: 1821–8. cephalopathy. Am J Hum Genet 2016; 99: 683–94. Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem Oweis W, Padala P, Hassouna F, Cohen-Kfir E, Gibbs DR, Todd EA, 1998; 67: 425–79. et al. Trans-binding mechanism of ubiquitin-like protein activation Hetz C, Saxena S. ER stress and the unfolded protein response in revealed by a UBA5-UFM1 complex. Cell Rep 2016; 16: 3113–20. neurodegeneration. Nat Rev Neurol 2017; 13: 477–91. Schulman BA, Harper JW. Ubiquitin-like protein activation by E1 en- Hitosugi T, Chen J. Post-translational modifications and the Warburg zymes: the apex for downstream signalling pathways. Nat Rev Mol effect. Oncogene 2014; 33: 4279–85. Cell Biol 2009; 10: 319–31. Homrich M, Wobst H, Laurini C, Sabrowski J, Schmitz B, Diestel S. Tatsumi K, Sou YS, Tada N, Nakamura E, Iemura S, Natsume T, Cytoplasmic domain of NCAM140 interacts with ubiquitin-fold et al. A novel type of E3 ligase for the Ufm1 conjugation system. modifier-conjugating enzyme-1 (Ufc1). Exp Cell Res 2014; 324: J Biol Chem 2010; 285: 5417–27. 192–9. Tatsumi K, Yamamoto-Mukai H, Shimizu R, Waguri S, Sou YS, Ishimura R, Obata M, Kageyama S, Daniel J, Tanaka K, Komatsu M. Sakamoto A, et al. The Ufm1-activating enzyme Uba5 is indispens- A novel approach to assess the ubiquitin-fold modifier 1-system in able for erythroid differentiation in mice. Nat Commun 2011; 2: cells. FEBS Lett 2017; 591: 196–204. 181. Komatsu M, Chiba T, Tatsumi K, Iemura Si, Tanida I, Okazaki N, Tompa P, Davey NE, Gibson TJ, Babu MM. A million peptide motifs et al. A novel protein-conjugating system for Ufm1, a ubiquitin-fold for the molecular biologist. Mol Cell 2014; 55: 161–9. modifier. EMBO J 2004; 23: 1977–86. Wei Y, Xu X. UFMylation: a unique & fashionable modification for Lemaire K, Moura RF, Granvik M, Igoillo-Esteve M, Hohmeier HE, life. Genomics Proteomics Bioinformatics 2016; 14: 140–6. Hendrickx N, et al. Ubiquitin fold modifier 1 (UFM1) and its target Yoo HM, Park JH, Jeon YJ, Chung CH. Ubiquitin-fold modifier 1 acts UFBP1 protect pancreatic beta cells from ER stress-induced apop- as a positive regulator of breast cancer. Front Endocrinol 2015; 6: 36. tosis. PLoS One 2011; 6: e18517. Zhang M, Zhu X, Zhang Y, Cai Y, Chen J, Sivaprakasam S, et al. Liu G, Forouhar F, Eletsky A, Atreya HS, Aramini JM, Xiao R, et al. RCAD/Ufl1, a Ufm1 E3 ligase, is essential for hematopoietic stem NMR and X-RAY structures of human E2-like ubiquitin-fold modi- cell function and murine hematopoiesis. Cell Death Differ 2015; 22: fier conjugating enzyme 1 (UFC1) reveal structural and functional 1922–34.

Journal

BrainOxford University Press

Published: Jul 1, 2018

Keywords: mutation; encephalopathy; brain development; enzymes; phenotype; protein processing, post-translational

There are no references for this article.