Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone.

RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3'-to-5' unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our understanding of enteroviruses and the two types of RNA remodeling activities.

Stepwise high-throughput virtual screening of Rho kinase inhibitors from natural product library and potential therapeutics for pulmonary hypertension.

CONTEXT: Pulmonary hypertension (PH) is a devastating disease characterized by progressive elevation of pulmonary arterial pressure and vascular resistance due to pulmonary vasoconstriction and vessel remodeling. The activation of RhoA/Rho-kinase (ROCK) pathway plays a central role in the pathologic progression of PH and thus the Rho kinase, an essential effector of the ROCK pathway, is considered as a potential therapeutic target to attenuate PH. OBJECTIVE: In the current study, a synthetic pipeline is used to discover new potent Rho inhibitors from various natural products. MATERIALS AND METHODS: In the pipeline, the stepwise high-throughput virtual screening, quantitative structure-activity relationship (QSAR)-based rescoring, and kinase assay were integrated. The screening was performed against a structurally diverse, drug-like natural product library, from which six identified compounds were tested to determine their inhibitory potencies agonist Rho by using a standard kinase assay protocol. RESULTS: With this scheme, we successfully identified two potent Rho inhibitors, namely phloretin and baicalein, with activity values of IC50 = 0.22 and 0.95 µM, respectively. DISCUSSION AND CONCLUSION: Structural examination suggested that complicated networks of non-bonded interactions such as hydrogen bonding, hydrophobic forces, and van der Waals contacts across the complex interfaces of Rho kinase are formed with the screened compounds.

Increasing glutamate promotes ischemia-reperfusion-induced ventricular arrhythmias in rats in vivo.

BACKGROUND: Glutamate mediates cerebral ischemia injury via N-methyl-D-aspartate (NMDA) receptor-coupled ion channels, but the activities of glutamate in the heart remain unclear. AIMS: To investigate whether or not glutamate contributes to ischemia- and reperfusion (IR)-induced arrhythmias. METHODS: Myocardial IR was induced by occlusion of the left anterior descending coronary artery for 30 min and reperfusion for another 30 min. A score system was used to quantify arrhythmias. MK801 (a noncompetitive NMDA receptor antagonist), dihydrokainate (DHK, a glutamate transporter inhibitor) and gabapentin (GBP, a glutamate release inhibitor) were used before ischemia. Serum glutamate levels, Ca(2+)-ATPase activity, SERCA2a protein expression and myocardial mitochondrial Ca(2+) content were assayed. RESULTS: Myocardial IR caused a significant increase in serum glutamate and high incidences of ventricular arrhythmias. GBP and MK801 significantly ameliorated ventricular arrhythmias, improved SERCA2a expression and sarcoplasmic reticulum Ca(2+)-ATPase activity and reduced Ca(2+) accumulated in mitochondria. By contrast, DHK significantly exacerbated reperfusion-related arrhythmias and mitochondrial Ca(2+) overload while it decreased SERCA2a expression and activity. CONCLUSION: This study showed that glutamate mediates reperfusion arrhythmias, and the corresponding mechanism may be associated with Ca(2+) overload via the NMDA receptor. Reperfusion arrhythmias may be prevented by inhibiting the release of glutamate or by antagonizing NMDA receptors.

IL­6/STAT3/miR­34a protects against neonatal lung injury patients.

The present study aimed to investigate the protective role of the interleukin (IL)­6/signal transducer and activator of transcription 3 (STAT3)/microRNA (miR)­34a signaling pathway in patients with neonatal lung injury (NLI) and the underlying molecular mechanisms of these effects. It was demonstrated that miR­34a serum expression was significantly upregulated in patients and mice with NLI. Meanwhile, IL­6 and phosphorylated­STAT3 protein expression, and tumor necrosis factor (TNF)­α, IL­1ß and IL­18 activity levels in NLI mice were significantly induced compared with the normal control group. The promotion of IL­6 protein expression resulted in significantly increased TNF­α, IL­1ß and IL­18 activity levels, phosphorylated­STAT3 and p65 protein expression, and miR­34a expression in NLI mice compared with the corresponding normal control groups. In A549 cells treated with lipopolysaccharides, the promotion of miR­34a protein expression significantly increased TNF­α, IL­1ß and IL­18 activity levels, and induced transcription factor p65 protein expression compared with the corresponding negative control groups. Collectively, the data of the present study indicate that IL­6R/STAT3/miR­34a possesses a protective role in patients with neonatal lung injury.

Doxycycline ameliorates aggregation of collagen and atrial natriuretic peptide in murine post-infarction heart.

Heart failure after myocardial infarction (MI) is associated with the aggregation of collagen and some misfolded proteins. This study was aimed to assess the therapeutic efficacy of doxycycline (Dox) in MI-induced heart failure and elucidate the potential mechanisms involved. A heart failure model of animals was established by ligating the left anterior descending coronary artery of rats. The administration of Dox via drinking water (25mg/kg/day) was initiated after surgery and lasted for two weeks. After cardiac function evaluation by echocardiography, all animals were killed to assess the aggregation of type I collagen, atrial natriuretic peptide (ANP), and the activities of matrix metalloproteinases (MMPs), autophagosomes and microtubule-associated protein 1 light chain 3 (LC3). Dox treatment significantly improved cardiac function and attenuated cardiac hypertrophy. Histological observation revealed that Dox significantly reduced the expression of collagen and ANP in the heart. Further investigation showed that Dox significantly inhibited the activities of MMP-2 and MMP-9, increased autophagosomes and enhanced LC3-II in post-infarction hearts. This study revealed that Dox treatment could promote autophagy, reduce ANP aggregation in post-infarction hearts, and inhibit MMP-2 and MMP-9 activities. Dox might act as a potential therapeutic drug for preventing proteotoxicity and cardiac dysfunction.