Intermolecular disulfide bond influences unphosphorylated STAT3 dimerization and function

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 bond

A study on the risk of subaortic and pulmonic stenosis and on genetic aspects of echocardiography measurements in the Italian Boxer dog

Subaortic and pulmonic stenosis are congenital cardiac defects affecting several dog breeds (Jacobs, 1990; Buchanan, 1992; Tidholm, 1997). Subaortic stenosis (SAS) is characterized by a fibrous ring of tissue placed immediately below the aortic valve

Transient acceleration events in LISA Pathfinder data: properties and possible physical origin

We present an in depth analysis of the transient events, or glitches, detected at a rate of about one per day in the differential acceleration data of LISA Pathfinder. We show that these glitches fall in two rather distinct categories: fast transients in the interferometric motion readout on one side, and true force transient events on the other. The former are fast and rare in ordinary conditions. The second may last from seconds to hours and constitute the majority of the glitches. We present an analysis of the physical and statistical properties of both categories, including a cross-analysis with other time series like magnetic fields, temperature, and other dynamical variables. Based on these analyses we discuss the possible sources of the force glitches and identify the most likely, among which the outgassing environment surrounding the test-masses stands out. We discuss the impact of these findings on the LISA design and operation, and some risk mitigation measures, including experimental studies that may be conducted on the ground, aimed at clarifying some of the questions left open by our analysis.Peer ReviewedPostprint (author's final draft

Transient acceleration events in LISA Pathfinder data: Properties and possible physical origin

We present an in depth analysis of the transient events, or glitches, detected at a rate of about one per day in the differential acceleration data of LISA Pathfinder. We show that these glitches fall in two rather distinct categories: fast transients in the interferometric motion readout on one side, and true force transient events on the other. The former are fast and rare in ordinary conditions. The second may last from seconds to hours and constitute the majority of the glitches. We present an analysis of the physical and statistical properties of both categories, including a cross-analysis with other time series like magnetic fields, temperature, and other dynamical variables. Based on these analyses we discuss the possible sources of the force glitches and identify the most likely, among which the outgassing environment surrounding the test-masses stands out. We discuss the impact of these findings on the LISA design and operation, and some risk mitigation measures, including experimental studies that may be conducted on the ground, aimed at clarifying some of the questions left open by our analysis

S-Glutathionylation at Cys328 and Cys542 Impairs STAT3 Phosphorylation.

STAT3 is a latent transcription factor that promotes cell survival and proliferation and is often constitutively active in cancers. Although many reports provide evidence that STAT3 is a direct target of oxidative stress, its redox regulation is poorly understood. Under oxidative conditions STAT3 activity can be modulated by S-glutathionylation, a reversible redox modification of cysteine residues. This suggests the possible cross-talk between phosphorylation and glutathionylation and points out that STAT3 is susceptible to redox regulation. Recently, we reported that decreasing the GSH content in different cell lines induces inhibition of STAT3 activity through the reversible oxidation of thiol groups. In the present work, we demonstrate that GSH/diamide treatment induces S-glutathionylation of STAT3 in the recombinant purified form. This effect was completely reversed by treatment with the reducing agent dithiothreitol, indicating that S-glutathionylation of STAT3 was related to formation of protein-mixed disulfides. Moreover, addition of the bulky negatively charged GSH moiety impairs JAK2-mediated STAT3 phosphorylation, very likely interfering with tyrosine accessibility and thus affecting protein structure and function. Mass mapping analysis identifies two glutathionylated cysteine residues, Cys328 and Cys542, within the DNA-binding domain and the linker domain, respectively. Site direct mutagenesis and in vitro kinase assay confirm the importance of both cysteine residues in the complex redox regulatory mechanism of STAT3. Cells expressing mutant were resistant in this regard. The data presented herein confirmed the occurrence of a redox-dependent regulation of STAT3, identified the more redox-sensitive cysteines within STAT3 structure, and may have important implications for development of new drugs