Homology modeling, docking, molecular dynamics simulation, and structural analyses of coxsakievirus B3 2A protease: an enzyme involved in the pathogenesis of inflammatory myocarditis

2A protease of the pathogenic coxsackievirus B3 is key to the pathogenesis of inflammatory myocarditis and, therefore, an attractive drug target. However lack of a crystal structure impedes design of inhibitors. Here we predict 3D structure of CVB3 2A^pro based on sequence comparison and homology modeling with human rhinovirus 2A^pro. The two enzymes are remarkably similar in their core regions. However they have different conformations at the N-terminal. A large number of N-terminal hydrophobic residues reduce the thermal stability of CVB3 2A^pro, as we confirmed by fluorescence, western blot and turbidity measurement. Molecular dynamic simulation revealed that elevated temperature induces protein motion that results in frequent movement of the N-terminal coil. This may therefore induce successive active site changes and thus play an important role in destabilization of CVB3 2A^pro structure.     

High CO2 Levels Cause Skeletal Muscle Atrophy via AMP-activated Kinase (AMPK), FoxO3a Protein,and Muscle-specific Ring Finger Protein 1 (MuRF1)

Patients with chronic obstructive pulmonary disease, acute lung injury, and critical care illness may develop hypercapnia. Many of these patients often have muscle dysfunction which increases morbidity and impairs their quality of life. Here, we investigated whether hypercapnia leads to skeletal muscle atrophy. Mice exposed to high CO₂ had decreased skeletal muscle wet weight, fiber diameter, and strength. Cultured myotubes exposed to high CO₂ had reduced fiber diameter, protein/DNA ratios, and anabolic capacity. High CO₂ induced the expression of MuRF1 in vivo and in vitro, whereas MuRF1^−/− mice exposed to high CO₂ did not develop muscle atrophy. AMP-activated kinase (AMPK), a metabolic sensor, was activated in myotubes exposed to high CO₂, and loss-of-function studies showed that the AMPKα2 isoform is necessary for muscle-specific ring finger protein 1 (MuRF1) up-regulation and myofiber size reduction. High CO₂ induced AMPKα2 activation, triggering the phosphorylation and nuclear translocation of FoxO3a, and leading to an increase in MuRF1 expression and myotube atrophy. Accordingly, we provide evidence that high CO₂ activates skeletal muscle atrophy via AMPKα2-FoxO3a-MuRF1, which is of biological and potentially clinical significance in patients with lung diseases and hypercapnia.     

Latent Autoimmune Diabetes in Adults in the United Arab Emirates: Clinical Features and Factors Related to Insulin-Requirement

Aims

To describe and to characterize clinical features of latent autoimmune diabetes in adults (LADA) compared to type 1 and type 2 diabetes in the UAE.

Methods

In this cross-sectional study a dataset including 18,101 subjects with adult-onset (>30 years) diabetes was accessed. 17,072 subjects fulfilled the inclusion/exclusion criteria. Data about anthropometrics, demographics, autoantibodies to Glutamic Acid Decarboxylase (GADA) and to Islet Antigen 2 (anti-IA2), HbA1c, cholesterol and blood pressure were extracted. LADA was diagnosed according to GADA and/or anti-IA2 positivity and time to insulin therapy.

Results

437 (2.6%) patients were identified as LADA and 34 (0.2%) as classical type 1 diabetes in adults. Mean age at diagnosis, BMI, waist circumference, systolic blood pressure and HbA1c significantly differed between, LADA, type 2 and type 1 diabetes, LADA showing halfway features between type 2 and type 1 diabetes. A decreasing trend for age at diagnosis and waist circumference was found among LADA subjects when subdivided by positivity for anti-IA2, GADA or for both antibodies (p=0.013 and p=0.011 for trend, respectively). There was a gradual downward trend in autoantibody titre in LADA subjects requiring insulin within the first year from diagnosis to subjects not requiring insulin after 10 years of follow-up (p<0.001).

Conclusions

This is the first study describing the clinical features of LADA in the UAE, which appear to be different from both type 1 and type 2 diabetes. Furthermore, we showed that the clinical phenotype of LADA is dependent on different patterns of antibody positivity, influencing the time to insulin requirement.     

Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfusion-induced lung apoptosis

Heme oxygenase-1 (HO-1) is emerging as an important cytoprotective enzyme system in a variety of injury models. To optimize future therapeutic applications of HO-1, it is necessary to delineate the precise functions and mechanisms as well as modes of externally regulating HO-1 expression. Investigations have been limited by difficulties with the generation of HO-1 null mice and the lack of specific HO-1 inhibitors. Lung ischemia-reperfusion (I-R) injury is the inciting event in acute lung failure following transplantation, surgery, and shock. To study the function of HO-1 in I-R-induced lung injury, we designed small interfering RNA (siRNA) sequences that effectively suppress HO-1 expression both in vitro and in vivo in an organ-specific manner. In this study we show that there is enhanced apoptosis, via increased Fas expression and caspase 3 activity, in the presence of HO-1 siRNA in endothelial cells and mouse lung during I-R injury, whereas HO-1 overexpression attenuates apoptosis. To the best of our knowledge, we are the first to demonstrate that lung-specific siRNA delivery can be achieved by intranasal administration without the need for viral vectors or transfection agents in vivo, thereby obviating potential concerns for toxicity if siRNA technology is to have clinical application in the future.     

IQGAP1-Dependent Signaling Pathway Regulates Endothelial Cell Proliferation and Angiogenesis

Background

Vascular endothelial growth factor receptor-2 (VEGFR-2) signaling is an obligate requirement for normal development and pathological angiogenesis such as cancer and age-related macular degeneration. Although autophosphorylation of tyrosine 1173 (Y1173) of VEGFR-2 is considered a focal point for its angiogenic signal relay, however, the mechanism of phosphorylation of Y1173, signaling proteins that are recruited to this residue and their role in angiogenesis is not fully understood.

Methodology/Principal Findings

In this study we demonstrate that c-Src kinase directly through its Src homology 2 (SH2) domain and indirectly via c-Cbl binds to phospho-Y1057 of VEGFR-2. Activation of c-Src kinase by a positive feedback mechanism phosphorylates VEGFR-2 at multi-docking site, Y1173. c-Src also catalyzes tyrosine phosphorylation of IQGAP1 and acts as an adaptor to bridge IQGAP1 to VEGFR-2. In turn, IQGAP1 activates b-Raf and mediates proliferation of endothelial cells. Silencing expression of IQGAP1 and b-Raf revealed that their activity is essential for VEGF to stimulate angiogenesis in an in vivo angiogenesis model of chicken chorioallantoic membrane (CAM).

Conclusions/Significance

Angiogenesis contributes to the pathology of numerous human diseases ranging from cancer to age-related macular degeneration. Determining molecular mechanism of tyrosine phosphorylation of VEGFR-2 and identification of molecules that are relaying its angiogenic signaling may identify novel targets for therapeutic intervention against angiogenesis-associated diseases. Our study shows that recruitment and activation of c-Src by VEGFR-2 plays a pivotal role in relaying angiogenic signaling of VEGFR-2; it phosphorylates VEGFR-2 at Y1173, facilitates association and activation of IQGAP1 and other signaling proteins to VEGFR-2. IQGAP1-dependent signaling, in part, is critically required for endothelial cell proliferation, a key step in angiogenesis. Thus, Y1057 of VEGFR-2 serves to regulate VEGFR-2 function in a combinatorial manner by supporting both diversity of recruitment of angiogenic signaling proteins to VEGFR-2, and its ability to promote angiogenesis.     

Nerve growth factor protects against 6-hydroxydopamine-induced oxidative stress by increasing expression of heme oxygenase-1 in a phosphatidylinositol 3-kinase-dependent manner

The survival signal elicited by the phosphatidylinositol 3-kinase (PI3K)/Akt1 pathway has been correlated with inactivation of pro-apoptotic proteins and attenuation of the general stress-induced increase in reactive oxygen species (ROS). However, the mechanisms by which this pathway regulates intracellular ROS levels remain largely unexplored. In this study, we demonstrate that nerve growth factor (NGF) prevents the accumulation of ROS in dopaminergic PC12 cells challenged with the Parkinson's disease-related neurotoxin 6-hydroxydopamine (6-OHDA) by a mechanism that involves PI3K/Akt-dependent induction of the stress response protein heme oxygenase-1 (HO-1). The effect of NGF was mimicked by induction of HO-1 expression with CoCl(2); by treatment with bilirubin, an end product of heme catabolism; and by infection with a retroviral expression vector for human HO-1. The relevance of HO-1 in NGF-induced ROS reduction was further demonstrated by the evidence that cells treated with the HO-1 inhibitor tin-protoporphyrin or infected with a retroviral expression vector for antisense HO-1 exhibited enhanced ROS release in response to 6-OHDA, despite the presence of the neurotrophin. Inhibition of PI3K prevented NGF induction of HO-1 mRNA and protein and partially reversed its protective effect against 6-OHDA-induced ROS release. By contrast, cells transfected with a membrane-targeted active version of Akt1 exhibited increased HO-1 expression, even in the absence of NGF, and displayed a greatly attenuated production of ROS and apoptosis in response to 6-OHDA. These observations indicate that the PI3K/Akt pathway controls the intracellular levels of ROS by regulating the expression of the antioxidant enzyme HO-1.