Butturini Elena, Gotte Giovanni, Dell'Orco Daniele, Chiavegato Giulia, Marino Valerio, Canetti Diana, Cozzolino Flora, Monti Maria, Pucci Piero, Mariotto Sofia
Department of Neuroscience, Biomedicine and Movement, Biochemistry Section, University of Verona, Verona, Italy.
CEINGE Biotecnologie Avanzate and Department of Chemical Science, University of Naples 'Federico II', Naples, Italy Istituto Nazionale di Biostrutture e Biosistemi (INBB), Rome, Italy.
Biochem J. 2016 Oct 1;473(19):3205-19. doi: 10.1042/BCJ20160294. Epub 2016 Aug 2.
Signal transducer and activator of transcription 3 (STAT3) is a transcription factor activated by the phosphorylation of tyrosine 705 in response to many cytokines and growth factors. Recently, the roles for unphosphorylated STAT3 (U-STAT3) have been described in response to cytokine stimulation, in cancers, and in the maintenance of heterochromatin stability. It has been reported that U-STAT3 dimerizes, shuttles between the cytoplasm and nucleus, and binds to DNA, thereby driving genes transcription. Although many reports describe the active role of U-STAT3 in oncogenesis in addition to phosphorylated STAT3, the U-STAT3 functional pathway remains elusive.In this report, we describe the molecular mechanism of U-STAT3 dimerization, and we identify the presence of two intermolecular disulfide bridges between Cys367 and Cys542 and Cys418 and Cys426, respectively. Recently, we reported that the same cysteines contribute to the redox regulation of STAT3 signaling pathway both in vitro and in vivo The presence of these disulfides is here demonstrated to largely contribute to the structure and the stability of U-STAT3 dimer as the dimeric form rapidly dissociates upon reduction in the S-S bonds. In particular, the Cys367-Cys542 disulfide bridge is shown to be critical for U-STAT3 DNA-binding activity. Mutation of the two Cys residues completely abolishes the DNA-binding capability of U-STAT3. Spectroscopic investigations confirm that the noncovalent interactions are sufficient for proper folding and dimer formation, but that the interchain disulfide bonds are crucial to preserve the functional dimer. Finally, we propose a reaction scheme of U-STAT3 dimerization with a first common step followed by stabilization through the formation of interchain disulfide bonds.
信号转导与转录激活因子3（STAT3）是一种转录因子，在响应多种细胞因子和生长因子时，其酪氨酸705位点会发生磷酸化而被激活。最近，未磷酸化的STAT3（U-STAT3）在细胞因子刺激、癌症以及异染色质稳定性维持方面的作用已被报道。据报道，U-STAT3会形成二聚体，穿梭于细胞质和细胞核之间，并与DNA结合，从而驱动基因转录。尽管许多报道描述了U-STAT3除了磷酸化的STAT3之外在肿瘤发生中的积极作用，但U-STAT3的功能途径仍然难以捉摸。在本报告中，我们描述了U-STAT3二聚化的分子机制，并确定分别在Cys367与Cys542以及Cys418与Cys426之间存在两个分子间二硫键。最近，我们报道了相同的半胱氨酸在体外和体内对STAT3信号通路的氧化还原调节都有作用。这些二硫键的存在在此被证明在很大程度上有助于U-STAT3二聚体的结构和稳定性，因为二聚体形式在二硫键还原后会迅速解离。特别是，Cys367-Cys542二硫键对U-STAT3的DNA结合活性至关重要。这两个半胱氨酸残基的突变完全消除了U-STAT3的DNA结合能力。光谱研究证实，非共价相互作用足以实现正确折叠和二聚体形成，但链间二硫键对于维持功能性二聚体至关重要。最后，我们提出了U-STAT3二聚化的反应方案，首先是一个共同步骤，然后通过形成链间二硫键来实现稳定。
