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. Author manuscript; available in PMC: 2019 May 1.
Published in final edited form as: J Allergy Clin Immunol. 2017 Aug 30;141(5):1844–1853.e2. doi: 10.1016/j.jaci.2017.07.027

NOVEL STAT1 MUTATION DISRUPTS SMALL UBIQUITIN-RELATED MODIFIER (SUMO) CONJUGATION CAUSING GAIN OF FUNCTION

Elizabeth P Sampaio 1,*,#, Li Ding 1, Stacey R Rose 2,&, Phillip Cruz 3, Amy P Hsu 1, Anuj Kashyap 1, Lindsey B Rosen 1, Margery Smelkinson 4, Tatyana A Tavella 1, Elise M N Ferre 2, Meredith K Wierman 5, Christa S Zerbe 1, Michail S Lionakis 2, Steven M Holland 1
PMCID: PMC5832513  NIHMSID: NIHMS902572  PMID: 28859974

Abstract

Background

Sumoylation is a post-translational reversible modification of cellular proteins by the conjugation of SUMO (small ubiquitin-related modifier) and comprises an important regulator of protein function.

Objective

To characterize the molecular mechanism of a novel mutation at the SUMO motif on STAT1.

Methods

STAT1 sequencing and functional characterization were performed in transfection experiments using immunoblotting and immunoprecipitation in STAT1-deficient cell lines. Transcriptional response and target gene activation were also investigated in peripheral blood mononuclear cells.

Results

We identified a novel STAT1 mutation (c.2114A>T, p.E705V), within the SUMO motif (702IKTE705), in a patient with disseminated Rhodococcus infection, Norwegian scabies, chronic mucocutaneous candidiasis, hypothyroidism and esophageal squamous cell carcinoma. The mutation is located in the tail segment and is predicted to disrupt STAT1 sumoylation. Immunoprecipitation experiments performed in transfected cells confirmed absent STAT1 sumoylation for E705V, while it was present in wild type (WT) STAT1 cells, as well as the loss of function (LOF) mutants L706S and Y701C. Further, stimulation with IFN-γ led to enhanced STAT1 phosphorylation, enhanced transcriptional activity and target gene expression in the E705V transfected compared to WT transfected cells. Computer modeling of WT and mutant STAT1 molecules showed variations in the accessibility of phosphorylation site Y701 that corresponded to the LOF and GOF variants.

Conclusion

This is the first report of a mutation in the STAT1 sumoylation motif associated with clinical disease. These data reinforce sumoylation as a key post-translational regulatory modification of STAT1 and identify a novel mechanism for GOF STAT1 disease in humans.

Keywords: STAT1, gain of function, sumoylation, SUMO, IFN-γ, Rhodococcus infection

INTRODUCTION

Heterozygous dominant gain of function (GOF) mutations in STAT1 have been described associated with chronic mucocutaneous candidiasis (CMC), disseminated fungal infections, autoimmunity, arterial aneurysms, squamous cell carcinomas, viral and mycobacterial infections (1–5). The majority of GOF mutations reported to date are located in the DNA binding and coiled-coil domains, with two reported in the SH2 domain (1, 6). These mutations are mostly associated with excessive STAT1 phosphorylation, enhanced IFN-target gene expression and diminished IL-17 producing T cells (3–5).

IFN-γ activates STAT1 through Janus tyrosine kinase (JAK)-mediated phosphorylation of a single tyrosine (Y701) at the C-terminus. Post-translational modifications of STAT molecules regulate their function. Sumoylation is a post-translational reversible modification of cellular proteins by the conjugation of SUMO (small ubiquitin-related modifier), an ~11 kDa ubiquitin-like protein that is covalently attached through a cascade of enzymatic reactions. The conjugation of SUMO to a specific lysine residue in a protein occurs within the SUMO consensus motif ψKX[E/D] (ψ represents a large hydrophobic residue; K is the lysine to which SUMO is attached; and X represents any amino acid) (7–9). This post-translational modification affects a variety of biological processes such as regulation of transcription, intracellular trafficking, signal transduction, transcription regulators (IκBα), and others (8–11). On STAT1, SUMO conjugation occurs at a single lysine at 703, within the consensus motif 702IKTE705, at the C-terminal region. Previous studies have demonstrated that targeted mutations in this motif of STAT1 lead to enhanced response to IFN-γ and enhanced phosphorylation (12–15).

We identified a novel STAT1 GOF mutation in the tail segment domain, E705V, in a patient who had disseminated Rhodococcus equi infection, Norwegian scabies, CMC, and fatal squamous cell carcinoma. This mutation is adjacent to leucine 706, where the well-characterized dominant negative LOF STAT1 mutation, L706S, is associated with disseminated mycobacterial disease (1, 16). The E705V mutant leads to decreased sumoylation of STAT1, excessive STAT1 phosphorylation, atypical nuclear paracrystal accumulation and enhanced IFN-γ target gene expression. This mutation disrupts a post-translational modification of STAT1 indicating a potential molecular mechanism for GOF STAT1 mutations.

METHODS

Cell lines and blood samples

COS-7 cells (ATCC, Manassas, VA) and the human fibrosarcoma STAT1 deficient cell line U3C (kindly donated by Dr. JL Casanova, Rockefeller University, NY) were maintained in culture in a humidified 5% CO2 incubator in DMEM and RPMI medium (Gibco Laboratories), respectively, supplemented with 10% FCS, 2mM L-glutamine and antibiotics. Transient transfections were performed using Lipofectamine 2000 transfection reagent (Life Technologies) according to manufacturer’s recommendation. In addition, peripheral blood mononuclear cells (PBMCs) from the patient and healthy donors were used for in vitro assays. Samples were collected under Institutional Review Board(IRB)-approved protocols. The patient provided written informed consent and healthy volunteer blood samples were obtained through the Department of Transfusion Medicine, NIH. Healthy controls were age and sex matched.

STAT1 sequencing

Primers spanning exons and flanking splice sites of human STAT1 and full-length cDNA were designed using Primer Select (DNAstar Lasergene). Genomic DNA and total RNA were extracted from primary blood cells and genomic amplification performed with Platinum PCR Supermix High Fidelity (Invitrogen). Sequencing was done with Big Dye Terminators v3.1 (Applied Biosystems, Foster City, CA), run on an Applied Biosystems 3730XL sequencer and aligned to the consensus sequence NM_007315.3 using Sequencer software (Gene Codes).

Constructs

Mutated STAT1 sequences and GFP-tagged constructs were created (BioInnovatise Inc., Rockville, MD) using a STAT1 expression vector (Origene Technologies, Rockville, MD). pSG5-His-SUMO1 and Flag-SENP1 constructs were obtained from Addgene (Cambridge, MA) (17, 18). All plasmids were isolated using the QIAprep kit (QIAGEN) according to manufacturer’s recommendations and mutations were verified by sequencing.

Evaluation of STAT1 activation

Tyrosine (Tyr701, Y701) STAT1 phosphorylation (pSTAT1) was assayed in transfected cells (WT, STAT1 mutants and SENP-1 constructs) stimulated with IFN-γ (1,000 IU/ml). IFN-γ was added to the cell culture medium for 30 min before the addition of the kinase inhibitor staurosporine (0.5μM; Calbiochem, Gibbstown, NJ), or the phosphatase inhibitor pervanadate (15), and incubation continued for 30 min. Cells were then lysed in lysis buffer (Cell Signaling Technology, Danvers, MA) containing protease and phosphatase inhibitors (Calbiochem) and analyzed by immunoblotting. Determination of DNA binding activity and of transcriptional activity were assayed through an ELISA-like colorimetric assay (TRANSAM; Active Motif, Carlsbad, CA) and a dual luciferase assay (Promega, Madison, WI), respectively, as described (4, 5).

Evaluation of gene expression and cytokine production

U3C transfected cells and frozen stored PBMCs from the patient and healthy donors were used. Gene expression was assayed in cells left unstimulated or stimulated with IFN-γ (1,000 IU/ml) for 6 h by qRT-PCR using Taqman expression assays (Applied Biosystems). GAPDH was used as normalization control. Evaluation of cytokine release was performed in cells stimulated with IFN-γ+LPS (200ng/ml) for 24 h. Culture supernatants were assayed further using a custom bead based cytokine assay (Bio-Plex, BioRad, Hercules, CA).

Immunoprecipitation and Immunoblotting

COS-7 and U3C or U3A cells were transiently transfected with WT or mutant STAT1 constructs, and SUMO1 construct. Cells were lysed in lysis buffer supplemented with N-ethylmaleimide (NEM, 20mM; Sigma) and protease inhibitors. For immunoprecipitation, whole cell lysates were incubated with anti-STAT1 antibody (or isotype control antibody; Santa Cruz Biotechnology) and protein G-Sepharose (Amersham Biosciences) overnight at 4°C. The beads were washed, heated, and the proteins resolved by immunoblotting for SUMO-STAT1 proteins. For analysis of the cell lysates, equal amounts of proteins were transferred to PVDF membranes and were incubated with the primary antibodies, anti-pSTAT1 Tyr701, anti-STAT1, anti-SENP1 (Cell Signaling), and anti-SUMO-1 (Abcam). Blots were stripped and re-probed with anti-STAT1 and anti-tubulin (Millipore) antibodies to assess protein loading.

Evaluation of Th17 response

Frozen PBMCs obtained from the patient and two healthy donors were thawed when cells (106) were either unstimulated or stimulated with PMA (20ng/mL; Sigma) and ionomycin (1μM; Sigma) in the presence of brefeldin A (10μg/mL; Sigma) at 37°C overnight. For detection of intracellular cytokines, cells were stained with Live/Dead Fixable Yellow Dead Cell Stain Kit (Invitrogen) as well as for extracellular CD3 (eVolve605), CD8 APC-eFluor780 (eBioscience), CD45RO PE-CF594 (BD Biosciences), and CD27 BV421 (BioLegend) for 30 min. Cells were fixed (paraformaldehyde 4%), permeabilized, and stained for intracellular antigens with CD4 PE-Cy7 (BD Biosciences), IL-17A PE, IFN-γ FITC, and TNF-α APC (eBioscience). Data were collected using an LSR Fortessa (BD Biosciences) and analyzed using FlowJo software (Treestar) by gating on lymphocytes (forward & side scatter) and on viable CD3+CD4+CD8-CD45RO+ CD27− cells.

Confocal microscopy

Transfected U3C cells carrying the WT-STAT1-GFP or the E705V-STAT1-GFP were fixed and assayed as described (15). Confocal microscopy on fixed cells was performed on a Leica SP5 microscope (Leica Microsystems, Mannheim, Germany) using a 63X/1.4NA oil immersion objective and hybrid HyD detectors. Fluorochromes were excited using diode 405nm and Argon 488nm lasers.

Molecular modeling and molecular dynamics

A homology model of the complete WT-STAT1 protein was produced using the I-TASSER server (19, 20), since existing crystal structures of STAT1 (PDB codes 1YVL and 1BF5) have missing residues. Initial models of the mutant proteins were generated in the program Chimera (21). Molecular dynamic simulations were performed on the nonphosphorylated WT structure and each of the mutant models with the CHARMM27 (22) force field under isobaric-isothermal conditions with periodic boundary conditions using the NAMD (23) program (v.2.7) on the Biowulf Linux cluster (NIH, Bethesda, MD). Each model was explicitly solvated with TIP3P water molecules and Na+ and Cl− neutralizing counterions using the VMD program (24). Electrostatic interactions were calculated using the Particle-Mesh Ewald summation. Prior to the start of the simulation, an energy minimization was performed, followed by slow warming to 310 K in 10 K increments of 5 psec each. Production runs were conducted at 310 K for 150 nsec. For all simulations, a two fsec integration time step was used along with a 12 Å non-bonded term cutoff. Langevin dynamics were used to maintain temperature and a modified Nosé-Hoover Langevin piston was used to control pressure. The average Solvent Accessible Surface Area for Tyr701 in each of the MD trajectories was calculated using VMD.

Statistical analysis

Results are mean ± standard deviation (SD). Differences between groups were assessed by the Student’s t-test using GraphPad Prism Software (San Diego, CA). Statistical significance level was p<0.05.

RESULTS

Patient clinical description

At age 34 a Caucasian man presented with subacute fever and respiratory symptoms. Bilateral cavitary lung lesions were seen on chest computed tomography (Figure 1A), and Rhodococcus equi was recovered from his blood and bronchoalveolar lavage. He was successfully treated with a combination of vancomycin and ciprofloxacin. He had a history of recurrent sinopulmonary infections since childhood as well as hypothyroidism, hypogonadism and CMC. His CMC was with an azole resistant Candida albicans and involved the oral mucosa, esophagus, toenails and fingernails (Figure 1B). Due to refractory esophageal candidiasis, he had developed esophageal strictures and dysphagia, which required repeated esophageal dilations. Dermatotomal zoster recurred during adolescence. Severe Norwegian scabies involving his extremities, face and torso was successfully treated with ivermectin and topical permethrin. The patient was T lymphopenic with hypergammagloulinemia: CD3+ 348/μl (reference value 615–2348/μl); CD4+ 176/μl(334–1556/μl); CD8+ 129/μl(149–787/μl); CD19+ 28/μl (81–493/μl); CD16+/CD56+ 36/μl(109–607/μl). Serum IgG 1986 mg/dl (700–1600); IgA 29 mg/dl (70–400); IgM 214 mg/dl (40–230 mg/dl); IgE 22.9 mg/dl (0–90). Positive anti-nuclear (ANA) antibodies (6.4 EU [normal range, 0–0.9 EU)] was detected, whereas others were negative including anti-extractable nuclear antigens (ENA), anti-thyroglobulin antibody, thyroid peroxidase antibody, thyrotropin receptor antibody, anti-parietal antibody, intrinsic factor antibody, anti-liver-kidney-microsomal antibody, tissue transglutaminase IgA antibody, GAD65 antibody, anti-mitochondrial antibody and anti-smooth muscle antibody. At age 36 he was diagnosed with stage IIIA squamous carcinoma of the esophagus, for which he underwent chemotherapy. Rhodococcus equi pneumonia recurred at age 36 as did CMC. The patient died due to further complications of his squamous carcinoma and no further material is available for testing. His father had had hypothyroidism, diabetes, CMC, recurrent pulmonary infections and died from Pseudomonas pneumonia. His mother and sister were healthy.

Figure 1. Patient.

Figure 1

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(A) Chest tomography. Lesions are visible both in the right and left lung due to Rhodococcus equi; (B) Onychomycosis caused by azole-resistant Candida albicans (arrows).

STAT1 mutation

His severe pulmonary infections and CMC led to genetic investigation of STAT1. Sequencing of STAT1 genomic DNA and cDNA identified the novel heterozygous mutation c.2114 A>T, p.E705V, in the tail segment domain. Interestingly, the adjacent residue 706, is the site of the autosomal dominant LOF mutation L706S (16), and the nearby mutant LOF Y701C associated with disseminated mycobacterial disease and ostemyelitis (25). The mutation c.2114 A>T was not found in dbSNP 147, 1000 Genomes Project or EXAC (26). The patient’s parents were not available for screening.

Functional assays

We performed direct testing of STAT1 mediated functions on frozen patient PBMC. Stimulation of PBMCs with IFN-γ and LPS+IFN-γ led to markedly enhanced expression of IP10 and CXCL9 genes (Figure 2A) and to higher cytokine levels in the supernatants of patient cells (IP10, mean 30,901pg/ml vs. 10,393pg/ml in controls; TNF-α, 47,332 vs. 21,452pg/ml, Figure 2B), respectively. TH17 levels following stimulation with PMA/ionomycin in patient T cells were decreased (0.4% positive cells) compared to controls (1.66 ± 0.2%, Figure 2C). Intracellular expression of TNF-α and IFN-γ were preserved. Somewhat surprisingly, IFN-γ driven STAT1 phosphorylation in thawed patient PBMCs was essentially normal. However, following treatment with the kinase inhibitor staurosporine, patient pSTAT1 levels were 1.5 to 2 fold higher than normal in the patient samples (Figure S1).

Figure 2. Enhanced pSTAT1 in transfected cells.

Figure 2

Figure 2

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STAT1-deficient cell lines transfected with WT, E705V and E705Q STAT1 constructs were stimulated or not (NS) with IFN-γ (1,000 IU/ml) and treated further with (A–C) staurosporine (n=5), or (D) pervanadate (one representative experiment out of two). Cell lysates were immunoblotted for pSTAT1, STAT1 and tubulin.

Since it was clear from clinical, sequence and functional testing that this patient had severe GOF STAT1 disease, we pursued further molecular characterization of the identified mutation. We transfected E705V-STAT1 into STAT1-deficient U3C and U3A cells and used immunoblotting following IFN-γ stimulation. In selected experiments, we used staurosporine to block further phosphorylation and the phosphatase inhibitor pervanadate to block dephosphorylation. Following treatment with staurosporine, STAT1 phosphorylation (pSTAT1) was increased in cells transfected with E705V compared to WT-STAT1 (Figures 3A–3C). Similar results were observed when cells were transfected with the previously described sumoylation deficient STAT1 mutant E705Q (Figure 3C) (15, 27). In addition, treatment of cells with pervanadate increased the phosphorylation in WT, as expected, and to a greater extent for the E705V mutant (Figure 3D).

Figure 3. Enhanced response in E705V transfected cells.

Figure 3

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(A) DNA binding GAS activity, (B) transcriptional response and (C) gene expression were evaluated in cells transfected with WT and STAT1 mutants following IFN-γ stimulation. Data are mean fold (± SD) relative to WT non-stimulated (NS) from two independent experiments performed in triplicate. *p<0.05 when compared to WT; **p<0.01when compared to LOF mutants.

In order to further evaluate the importance of SUMOylation on STAT1 phosphorylation we co-transfected COS-7 cells with WT-STAT1 and a construct expressing the deSUMOylating enzyme SENP1. Following IFN-γ stimulation, SENP1 co-transfected cells showed higher STAT1 phosphorylation than cells transfected with WT-STAT1 alone (Figure S2).

DNA binding activity to the GAS motif following IFN-γ activation was enhanced for E705V-STAT1 compared to WT-STAT1 (Figure 4A). Evaluation of GAS-luciferase reporter transcriptional activity was significantly elevated for E705V-STAT1 compared to WT (Figure 4B), but absent in cells transfected with the LOF mutants, L706S or Y701C. Gene expression assayed in transfected cells showed that expression of the IFN-γ induced genes (IP10/CXCL10, CIITA, CXCL11) was higher with both E705V and E705Q mutants (Figure 4C). Moreover, co-transfection of E705V and WT-STAT1 led to enhanced transcriptional response and gene expression (Figures 4B and 4C), consistent with the dominant GOF activity of the mutant allele.

Figure 4. Functional response in primary cells.

Figure 4

Figure 4

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(A) PBMCs from the patient and two normals were stimulated with IFN-γ and assayed by RT-PCR. Data are mean fold increase relative to NS; (B) Cytokine production (pg/ml) in response to LPS+IFN-γ. Assays were performed in triplicate. *p<0.05 compared to normals; (C) TH17 response in PBMCs stimulated with PMA/ionomycin. Dot plots are representative for each individual.

E705V mutation leads to defective sumoylation of STAT1

To determine whether E705V affected STAT1 sumoylation, equal amounts of protein were immunoprecipitated with anti-STAT1 antibody and immunoblotted with anti-STAT1 and anti-SUMO1 antibodies. SUMO-associated STAT1, identified as a slower migrating band (~110–120 kDa), was detected in unstimulated WT transfected cells, confirming that STAT1 is sumoylated at steady state; the SUMO band was absent in E705V-STAT1 (Figures 5A–5D), as well as in K703R (Figures 5A and 5C) and E705Q (Figures 5C and 5D) transfectants. Cells co-transfected with WT and E705V also had absent SUMO staining bands (Figures 5B and 5D), consistent with the dominant nature of the STAT1 mutant construct. In contrast, the LOF STAT1 mutations at the adjacent residue, L706S (Figures 5A and 5C), and the nearby tyrosine mutated, Y701C (Figure 5D), showed preserved SUMO-associated STAT1 in immunoprecipitates. SUMO1 was also detected in immunoprecipitated lysates obtained from U3C transfected WT-STAT1 cells but it was absent in the E705V-STAT1 samples (not shown). Specificity of the assay was reinforced by the absence of STAT1 and SUMO bands following immunoprecipitation with the isotype antibody.

Figure 5. E705V STAT1 failed to conjugate SUMO.

Figure 5

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(A–D) Transfected COS-7 cells show SUMO1 and the slower migrating SUMO-STAT1 conjugate bands in the WT-STAT1, (A, B) L706S and (D) Y701C cells, but absent in (A–D) E705V, (A, B) K703R, (B, D) E705Q and (C, D) E705V+WT-STAT1; (D) Bands are absent following IP with the isotype antibody. Experiments were performed at least thrice.

Detection of nuclear paracrystals

The previous description of the assembly of nuclear paracrystals in SUMO-free STAT1 transfected cells following activation with IFN-γ (15, 28), prompted us to examine whether the STAT1 mutant E705V led to the assembly of similar paracrystals in activated cells. Confocal microscopy confirmed the ability of E705V-STAT1 to translocate to the nucleus following in vitro stimulation of cells with IFN-γ and showed accumulation of SUMO-free E705V-STAT1 protein into punctate structures (Figure 6). These structures were absent in IFN-γ activated WT-STAT1 cells.

Figure 6. Accumulation of nuclear paracrystals.

Figure 6

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Following IFN-γ stimulation (1,000 IU/mL), SUMO-free STAT1 accumulates in the nucleus as assembly particles. Transfected U3C cells (WT-STAT1-GFP; E705V-GFP) were stimulated or not for 1 h and fixed. Confocal microscopy shows accumulation of punctuate particles (arrows) in the E705V-STAT1 cells, which were absent in the WT-STAT1. One representative experiment out of four is presented.

STAT1 modeling

After initial minimization, the WT STAT1 model generated by I-TASSER displays the phosphorylation site, Tyr701, extending up into the solution phase and presumably more available for phosphorylation, similar to the 1BF5 crystal structure (Figure 7A). Leu706 is in a mostly hydrophobic pocket, as shown in the ribbon drawing of the C-terminal domain of STAT1 (Figure 7B). While the MD simulation shows a fair amount of flexibility in the C-terminal loop, Leu706 tends to remain in the hydrophobic pocket and “anchor” the C-terminal end of the protein. This keeps Tyr701 mostly pointing into the solvent phase, though not necessarily in the same orientation as the crystal structure or the initial stages of the MD run. The L706S mutation leads to loss of a hydrophobic residue to anchor the C-terminal strand to the rest of the protein. During the MD simulation, it quickly opens up and rearranges so that Tyr701 now anchors the C-terminal region to the rest of the protein by forming hydrogen bonds with nearby acidic amino acids. Figure 7B shows the C-terminal domain from a representative snapshot in the simulation, and depicts a hydrogen bond between Asp646 and Tyr701. In this state Tyr701 is mostly buried and presumably unavailable for phosphorylation (Figure 7C) in which Tyr701 is colored red and is on the interior of the protein. While sumoylation still occurs for this mutant, since the sumoylation consensus sequence is not affected, the lower accessibility of the phosphorylation site likely also decreases its phosphorylation and activity. In contrast, the E705V mutation makes the area around L706 even more hydrophobic, thereby helping to more tightly anchor the C-terminal strand to the rest of the protein. In particular, Val705 interacts with the alpha helix residues Thr635-Ala641, in addition to the parts of the hydrophobic pocket around Leu706 seen in the wild-type model. The C-terminal tail is much more rigid throughout the MD run, resulting in Tyr701 staying predominantly pointing into solution phase. This mutant also rather consistently displays the same “up” orientation seen in the 1BF5 crystal structure relative to the wild-type-model (Figure 7D). This model suggests that the Tyr701 site is more available for phosphorylation, as illustrated in figure 7D, which shows the C-terminal domain from a representative snapshot from the MD simulation of E705V. The hydrophobic residues appear as line drawings surrounding V705 (in red stick representation). This figure also shows Tyr 701 as sticks pointing “up”, and the location of L706 (also in stick representation) is shown nearby in the extended hydrophobic pocket. The average solvent accessible surface area of Tyr701 throughout each of the three molecular dynamics runs (WT, 23 Å2; E705V, 29 Å2; L706S, 12 Å2) illustrates the differences in the availability of the phosphorylation site, which occurs in the absence of SUMO. The dominant negative LOF mutant L706S has a significantly lower accessibility for Tyr701, which is predominantly buried during the MD run (Figure 7C). In contrast, the increase in pSTAT1 observed for E705V can correlate with more available Tyr701 for phosphorylation.

Figure 7. Molecular dynamics simulations on STAT1 and mutants.

Figure 7

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(A) The WT C-terminal region model shows L706 in a mostly (not entirely) hydrophobic pocket, with Tyr701 extending up into solution; (B) In the L706S, Tyr701 is mostly buried and unavailable, (C) its surface representation shown in red; (D) E705V makes the area around L706 more hydrophobic, allowing Tyr701 to point “up”.

DISCUSSION

This is the first pathologic identification of the critical role of sumoylation in STAT1 disease. The mutation at position 705, which lies at the C-terminal end of STAT1, in the consensus motif for sumoylation, disrupts SUMO conjugation to STAT1 and is associated with the occurrence of many of the typical STAT1 GOF clinical manifestations including CMC, fatal squamous cell carcinoma, and endocrinopathies.

In addition, this is the first identified association of Rhodococcus equi, a gram positive coccobacillus, in GOF STAT1 disease. Rhodococcus equi is a facultative intracellular opportunistic pathogen encountered in immunocompromised hosts such as those with HIV/AIDS or transplantation. It is closely related to both Mycobacteria and Nocardia, a fact that is likely relevant in its occurrence in this disease. A recent report of immune reconstitution around Rhodococcus equi leading to cavitary pneumonia in an HIV infected patient with Burkitt lymphoma (29), confirms a link of this organism to immune-mediated pathology.

Post-translational modifications shape the response and fate of transcription factors following cell stimulation. SUMO proteins are covalently attached to proteins, a phenomenon that is rapidly reversed by deSUMOylating enzymes or SENPs (sentrin-specific proteases), which ultimately lead to detection of low levels of in vivo sumoylated proteins (9–11). Proteomic studies point to ~3,600 SUMO targeted proteins about 66% of which are thought to be nuclear (30).

Studies have shown that sumoylation of Interferon regulatory factors (IRF) 3, 7 and 8 attenuates their function (31, 32), whereas sumoylation of retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein (MDA5) positively regulates their function, leading to increased expression of IFN-β (33–35).

Prior work identified STAT1 sumoylation as an important negative regulator of IFN-γ signaling (12–14, 27, 36). While the C-terminal sumoylation sequence in STAT1, 702KXTE705, is highly conserved across species, it is not conserved among the other STAT molecules (12). Disruption of the sumoylation motif with loss of SUMO conjugation to STAT1 has been described for the engineered mutants K703R, E705Q, and E705A (12–14). The E705V-STAT1 disrupts SUMO conjugation to STAT1 and results in enhanced transcriptional response to IFN-γ, both in primary cells from the patient and in transfected cells.

Consistent with previous publications (15, 37), we observed accumulation of punctate STAT1-containing paracrystals in the nuclei of E705V transfected cells following IFN-γ stimulation but not in the nuclei of those transfected with WT-STAT1. It is postulated that these crystals are less soluble but transcriptionally active (15, 28). An additional study in mice expressing SUMO-free STAT1 (37) also evidenced that lack of SUMO led to paracrystal formation of activated STAT1. Strikingly, the analogous sumoylation motif, ψKTE, is naturally altered in STAT3 (706LKTK709), and following stimulation, WT-STAT3 can also form dimers that polymerize and assemble into punctate structures in the nuclei of cells (15).

Interestingly, there appear to be several levels of modulation exerted by sumoylation: (i) Zimnik et al. (38) have shown phosphorylation and sumoylation of STAT1 to be mutually exclusive. SUMO is bulky and precludes phosphorylation of the nearby Tyr701 residue; (ii) Our modeling experiments indicate that in E705V the Tyr701 phosphorylation site is modestly more exposed than in WT. Tyr701 accessibility is probably not the primary mechanism for the increased activity of E705V-STAT1, rather we believe it may be secondary to the elimination of the blocking effect of SUMO; (iii) Lack of SUMO, also seen in E705V, is associated with nuclear paracrystal formation. The observation of enhanced pSTAT1 in response to IFN-γ following co-transfection with WT-STAT1 and SENP1 corroborate that disobstruction of Tyr701 by loss of SUMO is bound to enhanced phosphorylation and crystal formation, leading consequently to enhanced transcriptional response and play a major role in GOF; (iv) Grönholm et al. (27) showed that E705Q-STAT1 disrupted sumoylation and exposed the site of interaction at the surface toward the DNA, leading to persistent DNA binding and enhanced activation, as compared to WT-STAT1. In addition, overexpression of the deSUMOylating enzyme, SENP1, reduced SUMO conjugation and led to enhanced DNA binding activity.

Our results reinforce the main impact of this mutant to be on the nuclear mechanisms of STAT1 activation. This is manifest by the multiple routes of clinical disease, mRNA transcription, protein expression and transfection experiments. Despite the clear evidences of GOF STAT1 physiology, pSTAT1 levels in response to IFN-γ in primary cells assayed in vitro were normal. These data suggest that pSTAT1 levels detected by flow cytometry may be insensitive for GOF STAT1 detection in some cases.

The combination of availability, paracrystals, nuclear retention, enhanced DNA binding and gene expression conspire to prolong STAT1 activation. SUMO conjugation is a critical component of cellular immune regulation following IFN-γ activation and points to a novel regulatory mechanism for the development of the GOF STAT1 phenotype. Moreover, the ability of SUMO to modulate STAT1 activation suggests new targets for therapeutic exploitation.

Supplementary Material

Clinical Implications.

This mutation is the first pathologic identification of the critical role of sumoylation in STAT1 function and disease. The current data indicate a potential target for STAT1 therapeutic modulation in GOF disease.

Acknowledgments

Funding support: This study was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIAID, NIH and has been funded in part with federal funds from the National Cancer Institute, NCI, NIH, under Contract No. HHSN 261200800001E.

We are grateful to the Biological Imaging Facility/RTB, NIAID/NIH for assistance with the confocal experiments. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Abbreviations

STAT1

Signal transducer and activator of transcription 1

GAS

Gamma activating sequence

SUMO

Small ubiquitin-related modifier

GOF

Gain of function

LOF

Loss of function

PVDF

Polyvinylidene difluoride

Footnotes

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