1. Genetics and Genomics
  2. Medicine

Allelic strengths of encephalopathy-associated UBA5 variants correlate between in vivo and in vitro assays

  1. Xueyang Pan
  2. Albert N Alvarez
  3. Mengqi Ma
  4. Shenzhao Lu
  5. Michael W Crawford
  6. Lauren C Briere
  7. Oguz Kanca
  8. Shinya Yamamoto
  9. David A Sweetser
  10. Jenny L Wilson
  11. Ruth J Napier
  12. Jonathan N Pruneda  Is a corresponding author
  13. Hugo J Bellen  Is a corresponding author
  1. Department of Molecular and Human Genetics, Baylor College of Medicine, United States
  2. Jan & Dan Duncan Neurological Research Institute, Texas Children’s Hospital, United States
  3. Department of Molecular Microbiology & Immunology, Oregon Health & Science University, United States
  4. Center for Genomic Medicine, Massachusetts General Hospital, United States
  5. Department of Neuroscience, Baylor College of Medicine, United States
  6. Division of Medical Genetics & Metabolism, Massachusetts General Hospital for Children, United States
  7. Division of Pediatric Neurology, Department of Pediatrics, Oregon Health & Science University, United States
  8. VA Portland Health Care System, United States
  9. Division of Arthritis & Rheumatic Diseases, Oregon Health & Science University, United States
https://doi.org/10.7554/eLife.89891.3
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Cite this article
  1. Xueyang Pan
  2. Albert N Alvarez
  3. Mengqi Ma
  4. Shenzhao Lu
  5. Michael W Crawford
  6. Lauren C Briere
  7. Oguz Kanca
  8. Shinya Yamamoto
  9. David A Sweetser
  10. Jenny L Wilson
  11. Ruth J Napier
  12. Jonathan N Pruneda
  13. Hugo J Bellen
(2023)
Allelic strengths of encephalopathy-associated UBA5 variants correlate between in vivo and in vitro assays
eLife 12:RP89891.
https://doi.org/10.7554/eLife.89891.3
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7 figures, 4 tables and 2 additional files

Figures

Figure 1
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Brain magnetic resonance imaging (MRI) images of the proband.

(A) A diagram showing the UFMylation pathway. Details of the biochemical processes in the pathway are described in the main text. In UBA5 proteins, only the adenylation domains are shown in the diagram. (B) A diagram of the UBA5:UFM1:UFC1 complex. In the complex, two copies of UBA5 form a homodimer that interacts with ubiquitin fold modifier 1 (UFM1) via a trans-binding mechanism. The activation of UFM1 requires the adenylation domain of one UBA5 subunit and the UFM1-interacting sequence (UIS) of the other UBA5 subunit in the complex. The opposing protomer of the UBA5 homodimer also contributes a UFC1-binding sequence (UBS) that is required for UFM1 transthiolation. (C) Axial T2 image showing periventricular T2 hyperintensity (blue arrow) resulting in prominence of the subcortical U-fibers (white arrows). (D) Sagittal flair showing mild thinning of the corpus callosum (red arrow). (E) Axial flair image demonstrating widening of the sylvian fissures (white asterisks).

Figure 2
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UFMylation pathway, conservation of UBA5, and generation of fly Uba5 loss of function (LoF) alleles.

(A) Alignment of the human UBA5 and fly Uba5 protein sequences. The functional domains of UBA5 are marked in colored boxes. The developmental and epileptic encephalopathy 44 (DEE44)-associated variants are marked in the protein topology diagram and the protein sequence alignment (letters in red). (B) Generation of the Uba5T2A-Gal4 allele and the uses of the allele in flippase (FLP)-mediated conversion. The expression of the GAL4 to drive a fluorescent protein allows assessment of gene expression, and humanization of the flies by expression of human UBA5 cDNA. (C) Generation of Uba5 null allele by CRISPR-mediated indel formation. (D) Loss of Uba5 causes lethality in early developmental stage. The lethality is rescued by a genomic rescue construct, the expression of FLP (Uba5T2A-Gal4 mutants only), and the expression of human UBA5 cDNA.

Figure 3 with 1 supplement
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Uba5 is expressed in a subset of neurons and glial cells in fly central nervous system (CNS).

(A) The expression of nuclear localized mCherry (mCherry.nls) driven by the Uba5T2A-Gal4 allele (Uba5T2A-Gal4>mCherry.nls) shows that Uba5 is expressed in L3 larvae and adult flies. (B, C) The larval CNS and adult brain of Uba5T2A-Gal4>mCherry.nls animals were immunostained with a neuronal (Elav, Panel B) or glial marker (Repo, Panel C). Maximum projections of confocal z-stack images are shown. Single plane, high magnification images of the regions indicated by the dashed squares are shown on the right to visualize the colocalizations between mCherry and the immunostaining signals. Arrows indicate cells that colocalize both markers. Scale bar, 100 μm.

Figure 3—figure supplement 1
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Single-cell gene expression pattern of Uba5 and the rescue of Uba5 mutants by tissue-specific UBA5 expression.

The expression pattern of Uba5 in whole adult fly (Li et al., 2022) (A) and adult brain tissue (Davie et al., 2018) (B) revealed by single-cell RNA sequencing profiles. The expression patterns of neuronal marker Elav and glial marker Repo are also shown. (C) The lethality of Uba5 hemizygous mutants is rescued by ubiquitous expression, but not by any tissue-specific expression of human UBA5 cDNA. Overexpression of UBA5 in Uba5 heterozygous mutants does not cause any obvious phenotype.

Figure 4 with 1 supplement
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Developmental and epileptic encephalopathy 44 (DEE44)-associated variants exhibit different rescuing abilities in flies.

(A) The DEE44-associated UBA5 variants rescued the lethality of Uba5 mutant flies with varying efficiency. Uba5T2A-Gal4/FM7 females were crossed with UAS-UBA5 males and the viability of Uba5T2A-Gal4/Y; UAS-UBA5/+ progenies were measured by Mendelian ratio and indicated by color codes: red, zero viability; yellow, partial viability (<90% of expected number); green, full viability (90% and above). (B) Three variants caused developmental delay in Uba5T2A-Gal4/Y; UAS-UBA5/+ flies. The embryos were collected within 6 hr and the number of eclosed adult flies was counted at the same time every day. Three replicates were performed in each group. (C) Five variants caused reduced lifespan in Uba5T2A-Gal4/Y; UAS-UBA5/+ flies. (D) Five variants caused progressive climbing defects in Uba5T2A-Gal4/Y; UAS-UBA5/+ flies. Flies were tested on Days 7 and 30. The climbing activity of CantonS wildtype flies is shown as reference. Numbers of animals (n values) in each group are indicated under the bars. (E) Three variants caused a bang-sensitive phenotype in Uba5T2A-Gal4/Y; UAS-UBA5/+ flies. Flies were tested on Day 30. The bang sensitivity of CantonS wildtype flies is shown as reference. Numbers of animals (n values) in each group are indicated under the bars. (B–E) Flies were cultured under 25°C. The results of DEE4 variant-expressing flies are compared with the result of reference UBA5-expressing flies. Results are presented as means ± standard error of the mean (SEM). Statistical analyses were performed via two-sided, unpaired Student’s t-test. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4—source data 1

Source data for Figure 4A–E.

https://cdn.elifesciences.org/articles/89891/elife-89891-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
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The Group II UBA5 variants do not cause obvious synaptic growth defects.

(A) Images of NMJ4 in segments A2–A4 stained with anti-horseradish peroxidase (HRP) and anti-cysteine string protein (CSP) in humanized flies expressing reference UBA5 or Group II variants. Scale bar, 10 μm. Quantification of the bouton number (B) and bouton size (C) of neuromuscular junctions (NMJs). Results are presented as means ± standard error of the mean (SEM). Statistical analyses were performed via two-sided, unpaired Student’s t-test. n = 8-10 biological replicates. ns, not significant.

Figure 4—figure supplement 1—source data 1

Source data for Figure 4—figure supplement 1B, C.

https://cdn.elifesciences.org/articles/89891/elife-89891-fig4-figsupp1-data1-v1.xlsx
Figure 5
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Structural analysis of UBA5 variants.

(A) Composite model of a UBA5 homodimer (green and blue) bound to ATP (gray sticks), ubiquitin fold modifier 1 (UFM1; magenta), and UFC1 (gold). The model was built using a series of UBA5 complex structures with UFM1 and UFC1 (PDB 6H77, 7NW1, and a modeled UBA5:UFC1 complex; Kumar et al., 2021; Soudah et al., 2019). Functional residues comprising the active site cysteines of UBA5 and UFC1, as well as the C-terminus of UFM1 are shown in yellow spheres. UBA5 variants are shown in red spheres and are labeled with their predicted structural effects. (B) Close-up view of variants (red sticks) within the UBA5 active site (yellow sphere), ATP-binding pocket, and homodimerization interface. (C) Close-up view of variants (red sticks) expected to impact UBA5 protein stability (results shown in the following figures).

Figure 6
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Preparation and stability of UBA5 variant proteins.

(A) Coomassie-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of all purified UBA5 variant proteins. (B) Thermal shift assay measuring the melting temperature (Tm) of all UBA5 variant proteins, with the exception of p.Gly168Glu and p.Cys303Arg which could not be produced. The p.Gln312Leu variant displayed two melting curves. Experiments were performed in triplicate over three biological replicates. (C) Change in melting temperature for all UBA5 variants in the presence of 5 mM ATP. Upon ATP addition, the p.Gln312Leu variant transitioned to a single melting curve. Experiments were performed in triplicate over three biological replicates. (B, C) Statistical analyses were performed via unpaired Student’s t-test. n = 3 biological replicates. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 6—source data 1

Unedited gel image of Figure 6A.

https://cdn.elifesciences.org/articles/89891/elife-89891-fig6-data1-v1.zip
Figure 6—source data 2

Source data for Figure 6B, C.

https://cdn.elifesciences.org/articles/89891/elife-89891-fig6-data2-v1.xlsx
Figure 7
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Measuring ubiquitin fold modifier 1 (UFM1) activation and transthiolation with UbiReal.

(A) Cartoon schematic illustrating the complexes formed during UFM1 activation and transthiolation, as well as their expected molecular weights. The fluorescent group attached to UFM1 is denoted by an orange star. The expected molecular weight for the UBA5–UFM1 intermediate is based on a UBA5 homodimer with one UFM1 molecule. (B) Proof-of-concept UbiReal assay monitoring the fluorescence polarization (FP) of Alexa488-labeled UFM1 alone (species 1), following addition of UBA5 (species 2), and following addition of UFC1 (species 3). (C) Fluorescence scan of samples described in (B) separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), illustrating the formation of activated UFM1 complexes. Each species is labeled with the analogous cartoon schematic presented in (A). (D) UbiReal assay tracking UFM1 activation by reference and variant UBA5 proteins over time. (E) Area under the curve quantification of UFM1 activation performed at 22°C. Experiments were performed in triplicate over three biological replicates. Statistical analyses were performed using a Welch’s t-test with comparison to the reference UBA5 data. (F) As in (E), for reactions performed at 37°C. (G) UbiReal assay tracking UFM1 transthiolation for reference UBA5 and variants that showed little or no effect on activation. (H) Area under the curve quantification of UFM1 transthiolation performed at 22°C. Experiments were performed in triplicate over three biological replicates. Statistical analyses were performed using a Welch’s t-test with comparison to the reference UBA5 data. (I) As in (H), for reactions performed at 37°C. (E, F, H, I) Statistical analyses were performed via Welch’s t-test. n = 3 biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 7—source data 1

Unedited gel image of Figure 7C.

https://cdn.elifesciences.org/articles/89891/elife-89891-fig7-data1-v1.zip
Figure 7—source data 2

Source data for Figure 7B, E, F, H, I.

https://cdn.elifesciences.org/articles/89891/elife-89891-fig7-data2-v1.xlsx

Tables

Table 1
Summary of genotypes of the reported cases.
ReferencesFamilyAllele #1Allele #2
Colin et al., 2016Ap.Ala371Thr (IA*)p.Gln302*
Bp.Ala371Thr (IA)p.Lys324Asnfs*14
Cp.Asp389Tyr (IA)p.Val260Met (II)
Dp.Met57Val (II)p.Gly168Glu (III)
Muona et al., 2016Ap.Ala371Thr (IA)p.Arg55His (III)
Bp.Ala371Thr (IA)p.Tyr285*
C, Ep.Ala371Thr (IA)p.Arg188*
Dp.Ala371Thr (IA)p.Arg61*
Arnadottir et al., 2017p.Ala371Thr (IA)p.Ala288 = (splicing variant)
Daida et al., 2018p.Tyr72Cys (IB)Deletion
Mignon-Ravix et al., 2018p.Tyr53Phe (II)†p.Tyr53Phe (II)
Low et al., 2019p.Asp389Gly (IA)Deletion
Briere et al., 2021Ap.Ala371Thr (IA)p.Cys303Arg (III)
Bp.Ala371Thr (IA)p.Arg188*
Cp.Ala371Thr (IA)p.Leu254Pro (III)
Dp.Ala371Thr (IA)p.Cys303Arg (III)
This studyp.Met57Val (II)p.Gln312Leu (IB)
  1. *

    The variant classification using fly phenotypic assays (results shown in Figure 4).

  2. †

    Consanguineous family.

Table 2
Bioinformatic predictions of the pathogenicity of reported UBA5 variants.
Variant 1Variant 2
Genomic position (GRCh38)3:132665830A>G3:132394214A>T
Amino acid changep.Met57Valp.Gln312Leu
Allele frequency in gnomADAbsentAbsent
CADD score25.329.1
SIFTDamagingDamaging
PolyPhen21.000 (probably damaging)0.999 (probably damaging)
MutationTasterDisease causingDisease causing
PROVEAN−3.18 (deleterious)−6.59 (deleterious)
Table 3
Summary of phenotypes of humanized flies expressing UBA5 variants.
VariantsSurvival rateDev. delayLifespanClimbing defectsBang sensitivity
Group IAp.Ala371ThrNormalNoNormalNoNo
p.Asp389GlyNormalNoNormalNoNo
p.Asp389TyrNormalNoNormalNoNo
Group IBp.Arg72CysNormalNoDecreasedYesNo
p.Gln312LeuNormalNoDecreasedYesNo
Group IIp.Tyr53PheDecreasedYesDecreasedYesYes
p.Met57ValDecreasedYesDecreasedYesYes
p.Val260MetDecreasedYesDecreasedYesYes
Group IIIp.Arg55HisLethalN.A.N.A.N.A.N.A.
p.Gly168GluLethalN.A.N.A.N.A.N.A.
p.Leu254ProLethalN.A.N.A.N.A.N.A.
p.Cys303ArgLethalN.A.N.A.N.A.N.A.
Table 4
Summary of protein stability and functions of UBA5 variants.
VariantsThermal stabilityATP-binding defect ‡UFM1 activationUFM1 transthiolation
Group IAp.Ala371ThrDecreasedNo defectNormalDecreased
p.Asp389GlyNormalNo defectDecreasedDecreased
p.Asp389TyrNormalNo defectDecreasedNormal
Group IBp.Arg72CysDecreasedNo defectDecreasedNormal
p.Gln312LeuLocal destabilization†No defectNormalNormal
Group IIp.Tyr53PheNormalStrongDecreasedN.A.
p.Met57ValNormalIntermediateNormalNormal
p.Val260MetDecreasedIntermediateDecreasedN.A.
Group IIIp.Arg55HisNormalStrongNear baselineN.A.
p.Gly168GluN.A.*N.A.N.A.N.A.
p.Leu254ProDecreasedStrongNear baselineN.A.
p.Cys303ArgN.A.*N.A.N.A.N.A.
  1. *

    Insoluble in protein purification.

  2. †

    See Results and Figure 6B.

  3. ‡

    Strong, 4–5°C in Tm shift; intermediate, 6–8°C in Tm shift. Reference UBA5 shows 13 °C in Tm shift upon ATP addition.

Additional files

MDAR checklist
https://cdn.elifesciences.org/articles/89891/elife-89891-mdarchecklist1-v1.pdf
Supplementary file 1

Summarizes the clinical features of all reported individuals with UBA5-associated developmental and epileptic encephalopathy 44 (DEE44) as well as the proband reported in this study.

https://cdn.elifesciences.org/articles/89891/elife-89891-supp1-v1.xlsx

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  1. Xueyang Pan
  2. Albert N Alvarez
  3. Mengqi Ma
  4. Shenzhao Lu
  5. Michael W Crawford
  6. Lauren C Briere
  7. Oguz Kanca
  8. Shinya Yamamoto
  9. David A Sweetser
  10. Jenny L Wilson
  11. Ruth J Napier
  12. Jonathan N Pruneda
  13. Hugo J Bellen
(2023)
Allelic strengths of encephalopathy-associated UBA5 variants correlate between in vivo and in vitro assays
eLife 12:RP89891.
https://doi.org/10.7554/eLife.89891.3