Department of Psychosomatic Medicine and Psychotherapy, University Medical Center Göttingen, Göttingen, Germany.
Institute of Medical Microbiology, University Medical Center Göttingen, Göttingen, Germany.
Mol Immunol. 2019 Oct;114:30-40. doi: 10.1016/j.molimm.2019.07.008. Epub 2019 Jul 20.
Heterozygous gain-of-function (GOF) mutations in the cytokine-regulated transcription factor STAT1 (signal transducer and activator of transcription 1) lead to chronic mucocutaneous candidiasis (CMC). However, the molecular basis of these pathogenic missense mutations is largely unknown. In this study, we characterized in more detail the CMC-associated GOF substitution mutation of arginine-to-tryptophan at position 274 (R274W) and, in addition, the adjacent glutamine-to-alanine mutation at position 275 (Q275A). Both mutants displayed elevated tyrosine phosphorylation levels, prolonged nuclear accumulation, and increased transcriptional responses to interferon-γ (IFNγ) stimulation. No difference was observed between wild-type (WT) and mutant STAT1 in DNA sequence-specificity or dissociation kinetics from high-affinity DNA-binding elements known as gamma-activated sites (GAS). Furthermore, all variants exhibited similar cooperative DNA binding. Unexpectedly, in vitro dephosphorylation rates using the recombinant STAT1-inactivating Tc45 phosphatase in both the absence and presence of double-stranded GAS elements were similar in all STAT1 variants. Likewise, the rate of tyrosine phosphorylation by Janus kinase 2 (JAK2) was unaltered as compared to the WT molecule, excluding that the phenotype of these mutants is caused by either defective Tc45-catalyzed dephosphorylation or JAK2-induced hyper-activation. Interestingly, within 10 min of IFNγ exposure, the majority of R274W and Q275A molecules had entered the nucleus, whereas the wild-type protein remained predominantly cytosolic. Thus, the exchange of critical residues located at the binding interface in the antiparallel dimer conformer led to a premature accumulation of phospho-STAT1 in the nuclear compartment. In summary, our data show that the hyper-activity of the GOF mutations results, at least in part, from the premature nuclear import of the tyrosine-phosphorylated molecules and not from alterations in their phosphorylation or dephosphorylation rates.
细胞因子调节转录因子 STAT1（信号转导和转录激活因子 1）中的杂合获得性功能（GOF）突变导致慢性黏膜皮肤念珠菌病（CMC）。然而，这些致病性错义突变的分子基础在很大程度上尚不清楚。在这项研究中，我们更详细地描述了与 CMC 相关的 GOF 取代突变精氨酸到色氨酸 274 位（R274W），以及相邻的谷氨酰胺到丙氨酸 275 位（Q275A）突变。这两种突变体均显示出较高的酪氨酸磷酸化水平、延长的核积累以及对干扰素-γ（IFNγ）刺激的转录反应增加。野生型（WT）和突变 STAT1 之间在 DNA 序列特异性或与称为γ激活位点（GAS）的高亲和力 DNA 结合元件的解离动力学方面没有差异。此外，所有变体均表现出相似的协同 DNA 结合。出乎意料的是，在用重组 STAT1 失活 Tc45 磷酸酶进行体外去磷酸化时，在不存在和存在双链 GAS 元件的情况下，所有 STAT1 变体的去磷酸化速率均相似。同样，与 WT 分子相比，Janus 激酶 2（JAK2）的酪氨酸磷酸化速率没有改变，排除了这些突变体的表型是由 Tc45 催化的去磷酸化缺陷或 JAK2 诱导的过度激活引起的。有趣的是，在 IFNγ 暴露 10 分钟内，大多数 R274W 和 Q275A 分子已进入细胞核，而野生型蛋白仍主要位于细胞质中。因此，位于平行二聚体构象结合界面上的关键残基的交换导致磷酸化 STAT1 过早积累在核区室中。总之，我们的数据表明，GOF 突变的高活性至少部分归因于酪氨酸磷酸化分子的过早核输入，而不是由于其磷酸化或去磷酸化速率的改变。
