Atomistic details of effect of disulfide bond reduction on active site of Phytase B from Aspergillus niger: A MD Study

The molecular integrity of the active site of phytases from fungi is critical for maintaining phytase function as efficient catalytic machines. In this study, the molecular dynamics (MD) of two monomers of phytase B from Aspergillus niger, the disulfide intact monomer (NAP) and a monomer with broken disulfide bonds (RAP), were simulated to explore the conformational basis of the loss of catalytic activity when disulfide bonds are broken. The simulations indicated that the overall secondary and tertiary structures of the two monomers were nearly identical but differed in some crucial secondary–structural elements in the vicinity of the disulfide bonds and catalytic site. Disulfide bonds stabilize the β-sheet that contains residue Arg66 of the active site and destabilize the α-helix that contains the catalytic residue Asp319. This stabilization and destabilization lead to changes in the shape of the active–site pocket. Functionally important hydrogen bonds and atomic fluctuations in the catalytic pocket change during the RAP simulation. None of the disulfide bonds are in or near the catalytic pocket but are most likely essential for maintaining the native conformation of the catalytic site.

Abbreviations

PhyB - 2.5 pH acid phophatese from Aspergillus niger, NAP - disulphide intact monomer of Phytase B, RAP - disulphide reduced monomer of Phytase B, Rg - radius of gyration, RMSD - root mean square deviation, MD - molecular dynamics.     

Structure modeling and antidiabetic activity of a seed protein of Momordica charantia in non-obese diabetic (NOD) mice

Momordica charantia is a well known medicinal plant used in the traditional medicinal system for the treatment of various diseases including diabetes mellitus. Recently, a novel protein termed as ADMc1 from the seed extract of M. charantia has been identified and isolated showing significant antihyperglycemic activity in type 1 diabetic rats in which diabetes was induced. However, the structure of this protein has not yet been analyzed. Homology modeling approach was used to generate a high quality protein 3D structure for the amino acid sequence of the ADMc1 protein in this study. The comparative assessment of secondary structures revealed ADMc1 as an all-alpha helix protein with random coils. Tertiary structure predicted on the template structure of Napin of B. Napus (PDB ID: 1SM7) with which the ADMc1 showed significant sequence similarity, was validated using protein structure validation tools like PROCHECK, WHAT_CHECK, VERIFY3D and ProSA. Arrangement of disulfide bridges formed by cysteine residues were predicted by the Dianna 1.1 server. The presence of multiple disulfide bond confers the stable nature of the ADMc1 protein. Further, the biological activity of the ADMc1 was assessed in non-obese diabetic (NOD) mice which are spontaneous model of type 1 diabetes. Significant reduction in the blood glucose levels of NOD mice was observed up to 8 h post administration of the rADMc1 protein. Overall, the structural characterizations with antihyperglycemic activity of this seed protein of Momordica charantia demonstrate its potential as an antidiabetic agent.     

Minocycline,levodopa and MnTMPyP induced changes in the mitochondrial proteome profile of MPTP and maneb and paraquat mice models of Parkinson's disease

Mitochondrial dysfunction is the foremost perpetrator of the nigrostriatal dopaminergic neurodegeneration leading to Parkinson's disease (PD). However, the roles played by majority of the mitochondrial proteins in PD pathogenesis have not yet been deciphered. The present study investigated the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and combined maneb and paraquat on the mitochondrial proteome of the nigrostriatal tissues in the presence or absence of minocycline, levodopa and manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP). The differentially expressed proteins were identified and proteome profiles were correlated with the pathological and biochemical anomalies induced by MPTP and maneb and paraquat. MPTP altered the expression of twelve while combined maneb and paraquat altered the expression of fourteen proteins. Minocycline, levodopa and MnTMPyP, respectively, restored the expression of three, seven and eight proteins in MPTP and seven, eight and eight proteins in maneb- and paraquat-treated groups. Although levodopa and MnTMPyP rescued from MPTP- and maneb- and paraquat-mediated increase in the microglial activation and decrease in manganese-superoxide dismutase expression and complex I activity, dopamine content and number of dopaminergic neurons, minocycline defended mainly against maneb- and paraquat-mediated alterations. The results demonstrate that MPTP and combined maneb and paraquat induce mitochondrial dysfunction and microglial activation and alter the expression of a bunch of mitochondrial proteins leading to the nigrostriatal dopaminergic neurodegeneration and minocycline, levodopa or MnTMPyP variably offset scores of such changes.     

Effects of CaMKII-Mediated Phosphorylation of Ryanodine Receptor Type 2 on Islet Calcium Handling,Insulin Secretion,and Glucose Tolerance

Altered insulin secretion contributes to the pathogenesis of type 2 diabetes. This alteration is correlated with altered intracellular Ca²⁺-handling in pancreatic β cells. Insulin secretion is triggered by elevation in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) of β cells. This elevation in [Ca²⁺]_cyt leads to activation of Ca²⁺/calmodulin-dependent protein kinase II (CAMKII), which, in turn, controls multiple aspects of insulin secretion. CaMKII is known to phosphorylate ryanodine receptor 2 (RyR2), an intracellular Ca²⁺-release channel implicated in Ca²⁺-dependent steps of insulin secretion. Our data show that RyR2 is CaMKII phosphorylated in a pancreatic β-cell line in a glucose-sensitive manner. However, it is not clear whether any change in CaMKII-mediated phosphorylation underlies abnormal RyR2 function in β cells and whether such a change contributes to alterations in insulin secretion. Therefore, knock-in mice with a mutation in RyR2 that mimics its constitutive CaMKII phosphorylation, RyR2-S2814D, were studied. This mutation led to a gain-of-function defect in RyR2 indicated by increased basal RyR2-mediated Ca²⁺ leak in islets of these mice. This chronic in vivo defect in RyR2 resulted in basal hyperinsulinemia. In addition, S2814D mice also developed glucose intolerance, impaired glucose-stimulated insulin secretion and lowered [Ca²⁺]_cyt transients, which are hallmarks of pre-diabetes. The glucose-sensitive Ca²⁺ pool in islets from S2814D mice was also reduced. These observations were supported by immunohistochemical analyses of islets in diabetic human and mouse pancreata that revealed significantly enhanced CaMKII phosphorylation of RyR2 in type 2 diabetes. Together, these studies implicate that the chronic gain-of-function defect in RyR2 due to CaMKII hyperphosphorylation is a novel mechanism that contributes to pathogenesis of type 2 diabetes.     

Targeting metabolic pathways proteins of Orientia tsutsugamushi using combined hierarchical approach to combat scrub typhus

Orientia tsutsugamushi (Ott) is a causative agent of chigger‐borne zoonosis, scrub typhus which is life threatening and highly pervasive illness in humans. In this report, we have mined and classified the proteins involved in pathways unique to Ott by using high‐throughput computational techniques. The 12 metabolic pathways were found to be unique to the pathogen. Forty‐six proteins were reported to be essential for the pathogen's survival and non‐homologous to the humans. The proteins were categorized into different classes, ie, enzymes, transporters, DNA‐binding, secretory, and outer membrane proteins. Further, in silico analysis of 46 proteins showed that 25 proteins were suitable therapeutic targets with known druggable properties. The structural modeling of B3CSG3 (MurA) protein was carried out and catalytic site essential for its functioning was analyzed. Virtual screening of chemical compounds was performed against modeled structure. The docking study by AutodockVina reported compound from PubChem with CID: 16036947 as best and potential inhibitor by means of docking score and binding affinity. The reliability and stability of the MurA‐16036947 complex were confirmed with molecular dynamics simulation. The report will provide insight to understand the mechanism of pathogenesis of Ott and instigate the development of effective treatment strategies against this disease.     

Application of filamentous phages in environment: A tectonic shift in the science and practice of ecorestoration

Theories in soil biology, such as plant–microbe interactions and microbial cooperation and antagonism, have guided the practice of ecological restoration (ecorestoration). Below‐ground biodiversity (bacteria, fungi, invertebrates, etc.) influences the development of above‐ground biodiversity (vegetation structure). The role of rhizosphere bacteria in plant growth has been largely investigated but the role of phages (bacterial viruses) has received a little attention. Below the ground, phages govern the ecology and evolution of microbial communities by affecting genetic diversity, host fitness, population dynamics, community composition, and nutrient cycling. However, few restoration efforts take into account the interactions between bacteria and phages. Unlike other phages, filamentous phages are highly specific, nonlethal, and influence host fitness in several ways, which make them useful as target bacterial inocula. Also, the ease with which filamentous phages can be genetically manipulated to express a desired peptide to track and control pathogens and contaminants makes them useful in biosensing. Based on ecology and biology of filamentous phages, we developed a hypothesis on the application of phages in environment to derive benefits at different levels of biological organization ranging from individual bacteria to ecosystem for ecorestoration. We examined the potential applications of filamentous phages in improving bacterial inocula to restore vegetation and to monitor changes in habitat during ecorestoration and, based on our results, recommend a reorientation of the existing framework of using microbial inocula for such restoration and monitoring. Because bacterial inocula and biomonitoring tools based on filamentous phages are likely to prove useful in developing cost‐effective methods of restoring vegetation, we propose that filamentous phages be incorporated into nature‐based restoration efforts and that the tripartite relationship between phages, bacteria, and plants be explored further. Possible impacts of filamentous phages on native microflora are discussed and future areas of research are suggested to preclude any potential risks associated with such an approach.