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UBASH3A Mediates Risk for Type 1 Diabetes Through Inhibition of T-Cell Receptor–Induced NF-Kb Signaling

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UBASH3A Mediates Risk for Type 1 Diabetes Through Inhibition of T-Cell Receptor–Induced NF-Kb Signaling Diabetes Volume 66, July 2017 2033 UBASH3A Mediates Risk for Type 1 Diabetes Through Inhibition of T-Cell Receptor–Induced NF-kB Signaling Yan Ge,1,2 Taylor K. Paisie,1,2,3 Jeremy R.B. Newman,2,4 Lauren M. McIntyre,2,4 and Patrick Concannon1,2 Diabetes 2017;66:2033–2043 | https://doi.org/10.2337/db16-1023 Although over 40 type 1 diabetes (T1D) risk loci have been study focuses on one such locus, on chromosome 21q22.3, mapped in humans, the causative genes and variants for containing two genes, TMPRSS3 and UBASH3A,ofwhich GENETICS/GENOMES/PROTEOMICS/METABOLOMICS T1D are largely unknown. Here, we investigated a candi- the latter is generally considered the most likely candidate date gene in the 21q22.3 risk locus—UBASH3A,whichis for this T1D risk locus (2–9). Besides T1D, single nucleotide primarily expressed in T cells where it is thought to play a polymorphisms (SNPs) in the 21q22.3 chromosomal region largely redundant role. Genetic variants in UBASH3A have are associated with several other autoimmune diseases, sug- been shown to be associated with several autoimmune gesting that this locus plays a broad role in autoimmunity diseases in addition to T1D. However, the molecular mech- (10–12). anism underlying these genetic associations is unresolved. UBASH3A (also known as STS-2, TULA,andCLIP4)is Our study reveals a previously unrecognized role of UBA- expressed primarily in T cells (13) and encodes a protein SH3A in human T cells: UBASH3A attenuates the NF-kB called ubiquitin-associated and SH3 domain–containing A signal transduction upon T-cell receptor (TCR) stimulation (UBASH3A). The UBASH3A protein has three functional by specifically suppressing the activation of the IkBki- nase complex. We identify novel interactions of UBASH3A domains: the N-terminal UBA (ubiquitin-associated), SH3 with nondegradative polyubiquitin chains, TAK1 and (SRC homology 3), and the COOH-terminal histidine phos- NEMO, suggesting that UBASH3A regulates the NF-kB phatase (also referred to as phosphoglycerate mutase-like signaling pathway by an ubiquitin-dependent mechanism. [PGM]) domains. It has been shown that the UBA domain Finally, we show that risk alleles at rs11203203 and binds to monoubiquitin (Ub); the SH3 domain interacts with rs80054410, two T1D-associated variants in UBASH3A, CBL—an E3 ubiquitin ligase—and dynamin; and the PGM increase UBASH3A expression in human primary CD4+ domain mediates self-dimerization (14–18). UBASH3A has T cells upon TCR stimulation, inhibiting NF-kB signaling four identified ubiquitination sites at lysine residues 15, via its effects on the IkB kinase complex and resulting in 202, 309, and 358. Monoubiquitination at Lys 202 causes reduced IL2 gene expression. UBASH3A to adopt a closed conformation, which prevents the binding of the UBA domain to substrates in trans (17). UBASH3A has a paralogue, UBASH3B (also known as Type 1 diabetes (T1D) is an autoimmune disease arising from STS-1 and TULA-2), which shares the same domain structure the destruction of the insulin-producing pancreatic b-cells. as UBASH3A. UBASH3B differs from UBASH3A in several T1D is a common, complex disease with multiple genetic significant ways. UBASH3B is ubiquitously expressed and and environmental risk factors. Although genome-wide has not been associated with any autoimmune or immune- association studies have discovered over 40 chromosomal mediated disorder in genome-wide association studies. regions where there is significant statistical evidence of UBASH3B displays significant protein tyrosine phosphatase association with T1D (1), the causative genes and variants activity both in vitro and in vivo and suppresses T-cell re- located in most of these regions have yet to be identified ceptor (TCR) signaling by dephosphorylating ZAP-70 and and their mechanisms of action determined. The current Syk (19–23). In contrast, UBASH3A exhibits very weak, 1Department of Pathology, Immunology and Laboratory Medicine, University of This article contains Supplementary Data online at http://diabetes Florida, Gainesville, FL .diabetesjournals.org/lookup/suppl/doi:10.2337/db16-1023/-/DC1. 2 Genetics Institute, University of Florida, Gainesville, FL © 2017 by the American Diabetes Association. Readers may use this article as 3 Genetics & Genomics Graduate Program, University of Florida, Gainesville, FL long as the work is properly cited, the use is educational and not for profit, and the 4 Department of Molecular Genetics & Microbiology,UniversityofFlorida,Gainesville,FL work is not altered. More information is available at http://www.diabetesjournals Corresponding author: Patrick Concannon, patcon@ufl.edu. .org/content/license. Received 22 August 2016 and accepted 20 March 2017. 2034 Novel Role for UBASH3A in T1D Susceptibility Diabetes Volume 66, July 2017 possibly acid-dependent, phosphatase activity in vitro; Stimulation and Lysis of Cells in vivo, knockout of the murine homolog of UBASH3A For stimulation longer than 30 min, cells were stimu- results in only a modest increase in phosphorylation of lated with 5 mg/mL plate-bound anti-CD3 (clone OKT3; ZAP-70 (19,24). BioLegend) with or without 5 mg/mL soluble anti-CD28 Mice lacking either Ubash3a or Ubash3b alone, or in antibody (clone CD28.2; BioLegend). For stimulation shorter combination, exhibit no overt defects without immune chal- than 30 min, cells were starved of serum for 4 h, and then 2/2 2/2 lenge (13). However, T cells from Ubash3a Ubash3b incubated for 30 min on ice with 10 mg/mL soluble anti- double-knockout mice are hyperresponsive to TCR stim- CD3 and 10 mg/mL soluble anti-CD28. Next, 10 mg/mL ulation compared with T cells from wild-type (WT) mice, goat anti-mouse IgG (SouthernBiotech) was added to the 2/2 2/2 whereasTcellsfromUbash3a and Ubash3b single- cells, followed by incubation at 37°C for the indicated pe- knockout mice display only a modest increase in prolif- riods of time. Mock stimulation was performed with only eration (19). A similar hierarchical response is seen in cell culture medium. the trinitrobenzene sulfonic acid–induced colitis model, Whole-cell lysates were extracted as previously described where knockout of either Ubash3a or Ubash3b increases (27). For some experiments as indicated in the figure leg- both inflammation and T-cell responses, but the ends, whole-cell lysates were extracted with cell lysis buffer 2/2 2/2 Ubash3a Ubash3b double-knockout mice display a containing 25 mmol/L HEPES, pH 7.0, 150 mmol/L NaCl, more severe phenotype than either of the single-knockout 0.5% NP-40, 1 mmol/L EDTA, protease and phosphatase mice (25). These findings suggest that Ubash3a, in combi- inhibitors, and 5 mmol/L N-ethylmaleimide (NEM), a deu- nation with Ubash3b, acts to inhibit T-cell activation and biquitinase inhibitor. function, albeit by an as-yet-unresolved mechanism. Nuclear extracts were prepared using the NE-PER Nuclear In the current study, we define the roles of UBASH3A and Cytoplasmic Extraction Reagents (Thermo Fisher Scien- and its genetic variants in T1D. Our study reveals novel tific), according to the manufacturer’sprotocol. interactions between UBASH3A, TAK1, and NEMO, which regulate TCR-induced NF-kB signaling. T1D risk alleles Transfection of HEK293T Cells in UBASH3A are shown to be associated with increased AcDNAofthefull-lengthtranscriptofUBASH3A was cloned UBASH3A expression and decreased IL2 expression in acti- into the pcDNA3.1 vector (Thermo Fisher Scientific). Expres- vated human primary CD4+ T cells. sion constructs encoding WT (Addgene plasmid #17608), lysine-48 (K48)-only (Addgene plasmid #17605), and lysine-63 (K63)-only Ub (Addgene plasmid #17606) tagged with hem- RESEARCH DESIGN AND METHODS agglutinin (HA) were gifts from Ted Dawson (Johns Hopkins Sample Information University) (28). HEK293T cells were transfected using the Frozen viable peripheral blood mononuclear cells (PBMCs) X-tremeGENE HP DNA Transfection Reagent (Roche). from healthy subjects of European ancestry were obtained Coimmunoprecipitation and Immunoblotting from the Type 1 Diabetes Genetics Consortium (T1DGC) Coimmunoprecipitation and immunoblotting were performed UBASH3A and from STEMCELL Technologies. genotyping as previously described (27), and antibodies used for these data used in this study were either obtained from T1DGC experiments are provided in Supplementary Table 1. or generated by PCR and Sanger sequencing. All biospeci- mens and data were represented by only nonidentifying Quantitative PCR codes. This study was approved by the University of Florida Frozen PBMCs from healthy subjects were thawed, and Institutional Review Board. primary CD4+ T cells were negatively selected using the Human CD4+ T Cell Isolation kit and LS MACS columns Generation of UBASH3A2/2 and (Miltenyi). Cells were stimulated as described above for 6 h, UBASH3A-Overexpressing Cell Clones and then total RNA was extracted using the RNeasy Plus To knockout UBASH3A in Jurkat cells, a CRISPR construct Mini kit (QIAGEN). First-strand cDNA was synthesized us- targeting exon 2 of the UBASH3A gene was generated (26) ing oligo(dT)20 primer and the iScript Select cDNA Synthesis using the guide sequence 59-CACGGGGAGGAAGACGGC kit (Bio-Rad). PCRs containing SYBR Green I were performed GG-39 and the pSpCas9n(BB)-2A-Puro plasmid (Addgene on a LightCycler 480 II real-time PCR instrument (Roche). All plasmid #48141, a gift from Feng Zhang, Massachusetts samples were tested in duplicate, and CT values were gener- Institute of Technology). To overexpress UBASH3A in ated by the second derivative maximum method provided
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