SUMMARY Mitochondrial dysfunction and oxidative stress are common features of Down syndrome (DS). However, the underlying mechanisms are not known. We investigated the relationship between abnormal energy metabolism and oxidative stress with transcriptional and functional changes in DS cells. Impaired mitochondrial activity correlated with altered mitochondrial morphology. Increasing fusion capacity prevented morphological but not functional alterations in DS mitochondria. Sustained stimulation restored mitochondrial functional parameters but increased ROS production and cell damage, suggesting that reduced DS mitochondrial activity is an adaptive response to avoid injury and preserve basic cellular functions. Network analysis of genes overexpressed in DS cells demonstrated functional integration in pathways involved in energy metabolism and oxidative stress. Thus, while preventing extensive oxidative damage, mitochondrial downregulation may contribute to increased susceptibility of DS individuals to clinical conditions in which altered energy metabolism may play a role such as Alzheimer’s disease, diabetes, and some types of autistic spectrum disorders.

STAT6 participates in classical IL-4/IL-13 signaling and stimulator of interferon genes-mediated antiviral innate immune responses. Aberrations in STAT6-mediated signaling are linked to development of asthma and diseases of the immune system. In addition, STAT6 remains constitutively active in multiple types of cancer. Therefore, targeting STAT6 is an attractive proposition for treating related diseases. Although a lot is known about the role of STAT6 in transcriptional regulation, molecular details on how STAT6 recognizes and binds specific segments of DNA to exert its function are not clearly understood. Here, we report the crystal structures of a homodimer of phosphorylated STAT6 core fragment (STAT6 CF ) alone and bound with the N3 and N4 DNA binding site. Analysis of the structures reveals that STAT6 undergoes a dramatic conformational change on DNA binding, which was further validated by performing molecular dynamics simulation studies and small angle X-ray scattering analysis. Our data show that a larger angle at the intersection where the two protomers of STAT meet and the presence of a unique residue, H415, in the DNAbinding domain play important roles in discrimination of the N4 site DNA from the N3 site by STAT6. H415N mutation of STAT6 CF decreased affinity of the protein for the N4 site DNA, but increased its affinity for N3 site DNA, both in vitro and in vivo. Results of our structure-function studies on STAT6 shed light on mechanism of DNA recognition by STATs in general and explain the reasons underlying STAT6's preference for N4 site DNA over N3.STAT6 | N4 site DNA recognition | JAK-STAT pathway | antiviral innate immunity | crystal structure P roteins belonging to the STAT family mediate transmission of signals of numerous cytokines and growth factors from the cell membrane to the nucleus via the classical JAK-STAT pathway (1). Malfunctions in this pathway are known to result in immune system disorder and cancers. Therefore, the JAK-STAT pathway is considered to be of great importance in the development of therapeutic interventions (2). The mammalian STAT family is made up of seven structurally and functionally related proteins named STAT1, 2, 3, 4, 5a, 5b, and 6 (3). All of the STAT proteins share a conserved domain organization (Fig. 1A).STAT6, an important member of the STAT family, plays a crucial role in the differentiation of Th2 cells and has been implicated in the development of asthma (4). This STAT is primarily stimulated by IL-4 and IL-13. A recent study reported that STAT6 plays a pivotal role in antiviral signaling initiated by host cells in response to viral infections (5). STAT6 could be activated by the stimulator of interferon genes/TBK1 cascade via phosphorylation of Y641. Intriguingly, residue S407 located in the DNA-binding domain (DBD) of STAT6 has been shown to be phosphorylated by TBK1. However, its implication for biological function of STAT6 is currently unknown (5). Thus, structural studies on STAT6 and its complex with DNA are essential to address several unanswered q...

Background The dual Na + and cardiac Ca 2+ -release channel inhibitor, Flecainide (FLEC) is effective in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by mutations in cardiac Ca 2+ -release channels (RyR2), calsequestrin (Casq2), or calmodulin. FLEC suppresses spontaneous Ca 2+ waves in Casq2-knockout (Casq2 −/− ) cardiomyocytes, a CPVT model. However, a report failed to find FLEC efficacy against Ca 2+ waves in another CPVT model, RyR2-R4496C heterozygous mice (RyR2 R4496C+/− ), raising the possibility that FLEC efficacy may be mutation dependent. Objective To address this controversy, we compared FLEC in Casq2 −/− and RyR2 R4496C+/− cardiomyocytes and mice under identical conditions. Methods After 30 min exposure to FLEC (6 μM) or vehicle (VEH), spontaneous Ca 2+ waves were quantified during a 40 s pause after 1 Hz pacing train in the presence of isoproterenol (ISO, 1 μM). FLEC efficacy was also tested in vivo using a low dose (LOW: 3 mg/kg ISO + 60 mg/kg caffeine) or a high dose catecholamine challenge (HIGH: 3 mg/kg ISO + 120 mg/kg caffeine). Results In cardiomyocytes, FLEC efficacy was dependent on extracellular [Ca 2+ ]. At 2 mM [Ca 2+ ], only Casq2 −/− myocytes exhibited Ca 2+ waves, which were strongly suppressed by FLEC. At 3 mM [Ca 2+ ] both groups exhibited Ca 2+ waves that were suppressed by FLEC. At 4 mM [Ca 2+ ], FLEC no longer suppressed Ca 2+ waves in both groups. Analogous to the results in myocytes, RyR2 R4496C+/− mice ( n = 12) had significantly lower arrhythmia scores than Casq2 −/− mice ( n = 9), but the pattern of FLEC efficacy was similar in both groups (i.e., reduced FLEC efficacy after HIGH dose catecholamine challenge). Conclusion FLEC inhibits Ca 2+ waves in RyR2 R4496C+/− cardiomyocytes, indicating that RyR2 channel block by FLEC is not mutation-specific. However, FLEC efficacy is reduced by Ca 2+ overload in vitro or by high dose catecholamine challenge in vivo , which could explain conflicting literature reports.

Introduction Cytosolic protein, p62/SQSTM1, regulates the protein turnover rate, and it interacts with various stress signaling proteins in cells. Accumulations of p62/SQSTM1 have been reported in hearts with cardiomyopathy. We recently reported that p62/SQSTM1 protein deletions reduced beta‐adrenergic response in myocytes and improved chronic ischemia‐induced Calcium (Ca) defect. Here, we studied acute and chronic stress‐induced Ca handling in myocytes genetically suppressed p62/SQSTM1 protein. Methods Myocytes (n=3 per group) isolated from Wild‐type (WT), and p62/SQSTM1+/‐ (p62 HET) at Base, 5 days, and 10 days after Isoproterenol (ISO; beta‐agonist) injections (150 mg/kg). We measured myocytes contractility (Fraction shortening of sarcomeres), systolic Ca release (CaT), SR Ca contents (CaffT), and Fractional Ca release, (CaT/CaffT) in field‐stimulated single myocytes. Myocytes loaded with fluorescent Ca indicators (Fura‐2AM; cytosolic Ca loading). Heart tissues in each group were processed for western blot (n=3 per group). Results The baseline myocytes contractility and Ca regulation were not different among the groups. P62 HET myocytes significantly increased contractility and fractional Ca release (the function of SR, F/F0; WT base, 0.58±0.07, WT ISO, 0.84±0.01, p62 HET ISO, 0.79±0.04) after acute ISO stimulation (1 uM, p<0.05), similar to WT myocytes. Interestingly, p62 HET myocytes preserved SR Ca contents, whereas WT myocytes increased SR Ca contents. Chronic ischemia‐induced p62 HET myocytes improved fractional Ca release; however, both systolic Ca release (F/F0; WT base, 0.24±0.03, WT ISO 10days, 0.29±0.06, p62 HET ISO 10 days, 0.17±0.02) and SR Ca contents (F/F0; WT base, 0.43±0.05, WT ISO, 0.49±0.10, p62 HET ISO, 0.33±0.04) were proportionally reduced (p<0.05). Conclusion Taken together, suppression of p62/SQSTM1 protein restores stress‐induced Ca dysregulation in myocytes. p62/SQSTM1 protein may directly contribute to systolic Ca release via beta‐adrenergic signaling.