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. 2017 Sep 4:6:1636.
doi: 10.12688/f1000research.11878.1. eCollection 2017.

Functional characterizations of rare UBA1 variants in X-linked Spinal Muscular Atrophy

Chris D Balak  1 , Jesse M Hunter  1   2 , Mary E Ahearn  1 , David Wiley  3 , Gennaro D'urso  3 , Lisa Baumbach-Reardon  1   4
Affiliations

Functional characterizations of rare UBA1 variants in X-linked Spinal Muscular Atrophy

Chris D Balak et al. F1000Res. .

Abstract

Background: X-linked spinal muscular atrophy (XL-SMA) results from mutations in the Ubiquitin-Like Modifier Activating Enzyme 1 ( UBA1). Previously, four novel closely clustered mutations have been shown to cause this fatal infantile disorder affecting only males. These mutations, three missense and one synonymous, all lie within Exon15 of the UBA1 gene, which contains the active adenylation domain (AAD). Methods: In this study, our group characterized the three known missense variants in vitro. Using a novel Uba1 assay and other methods, we investigated Uba1 adenylation, thioester, and transthioesterification reactions in vitro to determine possible biochemical effects of the missense variants. Results: Our data revealed that only one of the three XL-SMA missense variants impairs the Ubiquitin-adenylating ability of Uba1. Additionally, these missense variants retained Ubiquitin thioester bond formation and transthioesterification rates equal to that found in the wild type. Conclusions: Our results demonstrate a surprising shift from the likelihood of these XL-SMA mutations playing a damaging role in Uba1's enzymatic activity with Ubiquitin, to other roles such as altering UBA1 mRNA splicing via the disruption of splicing factor binding sites, similar to a mechanism in traditional SMA, or disrupting binding to other important in vivo binding partners. These findings help to narrow the search for the areas of possible dysfunction in the Ubiquitin-proteasome pathway that ultimately result in XL-SMA. Moreover, this investigation provides additional critical understanding of the mutations' biochemical mechanisms, vital for the development of future effective diagnostic assays and therapeutics.

Keywords: SMAX2; UBA1; X-linked spinal muscular atrophy; disease mechanisms; ubiquitin proteasome system; ubiquitination.

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Conflict of interest statement

Competing interests: No competing interests were disclosed.

Figures

Figure 1.
Figure 1.. Uba1 conservation, domains, and modeling of XL-SMA mutations.
( a) XL-SMA variant residues show strict amino acid sequence conservation in nearly all eukaryotes from yeast to modern man. ( b) Schematic of the inferred domains of Uba1. Pathogenic variants and key residues are labeled. IAD = inactive adenylation domain, FCCH = first catalytic cysteine half domain, 4HB = 4 helix bundle, AAD = active adenylation domain, SCCH = second catalytic half domain. UFD = Ub fold domain. Known pathogenic variants marked with asterisk. Critical Adenylation and Thiolester residues are underlined. ( c) 3D protein modeling of S. cerevisiae Uba1 using Jmol modeling software. XL-SMA variants labeled in red, thiolestered Ub in yellow and non-covalently bound Ub-adenylate in blue. This figure has been reproduced and modified from Protein Data Bank public domain content (PDB ID: 4NNJ) under original permission from Schäfer et al. (2014). “Structure of the ubiquitin-activating enzyme loaded with two ubiquitin molecules.” Acta Crystallogr D Biol Crystallogr 70(Pt 5): 1311–1320.
Figure 2.
Figure 2.. Expression and purification of Uba1.
SDS-PAGE image of the purification of Uba1. Bands at ~118kDa indicate full length, active WT, p.M539I, p.S547G, p.E557V, Uba1 as purified by a thiolester-linkage capture column. As predicted, the p.D576A did not bind to the Ub-agarose. The p.D576A was purified by Co 2+ affinity column as seen in the far right lane.
Figure 3.
Figure 3.. Uba1 adenylation activity plotted as a function of Uba1 enzyme amount.
Kinetic assays were run in triplicate under saturating conditions (Mg 2+ (10mM), ATP (2mM), Ub (100µM)) at 37°C. Wild-type (WT) and XL-SMA variants were assayed with a range of Uba1 amounts in 50mM HEPES buffer pH 8.0. Linear regression of WT slope compared to each variant shows statistical significance for p.M539I (*P = 0.028) and p.E557V (***P<0.0001).
Figure 4.
Figure 4.. Uba1 adenylation activity as a function of Ub or ATP concentration.
Kinetic assays (150µL) were run in duplicate or triplicate with 100nM WT Uba1 and XL-SMA variants in 50mM HEPES buffer at 37°C to determine initial linear rates from the first 10–20 minutes of the reaction. A) Michaelis-Menten graph of Uba1 adenylation activity as a function of Ub concentration with saturating ATP(2000nM). B) Michaelis-Menten graph of Uba1 adenylation activity as a function of ATP concentration with saturating Ub (100µM). Data was normalized to control reactions and graphed in GraphPad Prism. Km and Vmax values as well as 95% confidence intervals were also calculated in GraphPad Prism (see Table 2). Only p.E557V Km (ATP) did not have overlapping confidence intervals with WT values.
Figure 5.
Figure 5.. Uba1 transthioesteration of Ube2e1.
WT and mutant forms of Uba1 were incubated with Ub and Ube2e1 in the presence of ATP and Mg2+ for the indicated time. Reactions were quenched with Laemli buffer, separated by SDS-PAGE and quantitated with ImageJ software. A) Gel images are representative of total protein images from three independent experiments. Inset for visualization of Uba1-S-Ub complex gel shift. Note the lack of E2-S-Ub formation in the p.D576A mutant control. B) Quantitation of the E2-S-Ub gel shift bands. Y-axis values are integrated Ube2e1-Ub band densities (in millions) normalized to total Uba1 band densities. Error bars represent the SEM. Symbols indicate statistical significance (#P<0.0001, **P<0.01, *P<0.05) compared to WT using 2-way ANOVA.
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Grants and funding

This study was funded by a research grant (MDA186435) from the Muscular Dystrophy Association of America, Arizona Department of Health Services Arizona Biomedical Research Commission (ADHS16-110501), and the Translational Genomics Research Institute Bridge Funding. CDB was supported, in part, by a fellowship from Freeport-McMoRan Copper and Gold Foundation, and the Helios Foundation.