Investigations on the RNA binding and phosphorylation of groundnut bud necrosis virus nucleocapsid protein

Groundnut bud necrosis virus belongs to the genus Tospovirus, infects a wide range of crop plants and causes severe losses. To understand the role of the nucleocapsid protein in the viral life cycle, the protein was overexpressed in E. coli and purified by Ni-NTA chromatography. The purified N protein was well folded and was predominantly alpha-helical. Deletion analysis revealed that the C-terminal unfolded region of the N protein was involved in RNA binding. Furthermore, the N protein could be phosphorylated in vitro by Nicotiana benthamiana plant sap and by purified recombinant kinases such as protein kinase CK2 and calcium-dependent protein kinase. This is the first report of phoshphorylation of a nucleocapsid protein in the family Bunyaviridae. The possible implications of the present findings for the viral life cycle are discussed.

Mycobacterium leprae RecA is structurally analogous but functionally distinct from Mycobacterium tuberculosis RecA protein

Mycobacterium leprae is closely related to Mycobacterium tuberculosis, yet causes a very different illness. Detailed genomic comparison between these two species of mycobacteria reveals that the decaying M. leprae genome contains less than half of the M. tuberculosis functional genes. The reduction of genome size and accumulation of pseudogenes in the M. leprae genome is thought to result from multiple recombination events between related repetitive sequences, which provided the impetus to investigate the recombination-like activities of RecA protein. In this study, we have cloned, over-expressed and purified M. leprae RecA and compared its activities with that of M. tuberculosis RecA. Both proteins, despite being 91% identical at the amino acid level, exhibit strikingly different binding profiles for single-stranded DNA with varying GC contents, in the ability to catalyze the formation of D-loops and to promote DNA strand exchange. The kinetics and the extent of single-stranded DNA-dependent ATPase and coprotease activities were nearly equivalent between these two recombinases. However, the degree of inhibition exerted by a range of ATP:ADP ratios was greater on strand exchange promoted by M. leprae RecA compared to its M. tuberculosis counterpart. Taken together, our results provide insights into the mechanistic aspects of homologous recombination and coprotease activity promoted by M. lepare RecA, and further suggests that it differs from the M. tuberculosis counterpart. These results are consistent with an emerging concept of DNA-sequence influenced structural differences in RecA nucleoprotein filaments and how these differences reflect on the multiple activities associated with RecA protein. (C) 2011 Elsevier B.V. All rights reserved.

Catabolite repression of phosphoenolpyruvate carboxykinase by a zinc finger protein under biotin- and pyruvate carboxylase-deficient conditions in Pichia pastoris

We have identified a methanol- and biotin-starvation-inducible zinc finger protein named ROP [repressor of phosphoenolpyruvate carboxykinase (PEPCK)] in the methylotrophic yeast Pichia pastoris. When P. pastoris strain GS115 (wild-type, WT) is cultured in biotin-deficient, glucose-ammonium (Bio(-)) medium, growth is suppressed due to the inhibition of anaplerotic synthesis of oxaloacetate, catalysed by the biotin-dependent enzyme pyruvate carboxylase (PC). Deletion of ROP results in a strain (Delta ROP) that can grow under biotin-deficient conditions due to derepression of a biotin- and PC-independent pathway of anaplerotic synthesis of oxaloacetate. Northern analysis as well as microarray expression profiling of RNA isolated from WT and Delta ROP strains cultured in Bio(-) medium indicate that expression of the phosphoenolpyruvate carboxykinase gene (PEPCK) is induced in Delta ROP during biotin- or PC-deficiency even under glucose-abundant conditions. There is an excellent correlation between PEPCK expression and growth of Delta ROP in Bio(-) medium, suggesting that ROP-mediated regulation of PEPCK may have a crucial role in the biotin- and PC-independent growth of the Delta ROP strain. To our knowledge, ROP is the first example of a zinc finger transcription factor involved in the catabolite repression of PEPCK in yeast cells cultured under biotin- or PC-deficient and glucose-abundant conditions.

Structure and dynamics of protein excited states with millisecond lifetimes

An in-depth understanding of biological processes often requires detailed atomic resolution structures of the molecules involved. However in solution where most of these processes occur the conformation of biomolecules like RNA, DNA and proteins is not static but fluctuates. Routinely used structural techniques like X-ray crystallography, NMR spectroscopy and cryo-electron microscopy have almost always been used to determine the structure of the dominant conformation or obtain an average structure of the biomolecule in solution with very little detailed information regarding the dynamics of these molecules in solution. Over the last few years, NMR based methods have been developed to study the dynamics of these biomolecules in solution in a site-specific manner with the aim of generating structures of the different conformations that these molecules can adopt in solution. One powerful technique is the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiment, which can be used to detect and characterize protein excited states that are populated for as less as 0.5% of the time with ∼0.5–10 millisecond lifetimes. Due to recent advances in NMR pulse sequences and labeling methodology, it is now possible to determine the structures of these transiently populated excited states with millisecond lifetimes by obtaining accurate chemical shifts, residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs) of these excited states. In these excited states the dynamics of some methyl containing residues can also be studied.

Nonspecific Interaction between DNA and Protein allows for Cooperativity: A Case Study with Mycobacterium DNA Binding Protein

Different DNA-binding proteins have different interaction modes with DNA. Sequence-specific DNA protein interaction has been mostly associated with regulatory processes inside a cell, and as such extensive studies have been made. Adequate data is also available on nonspecific DNA protein interaction, as an intermediate to protein's search for its cognate partner. Multidomain nonspecific DNA protein interaction involving physical sequestering of DNA has often been implicated to regulate gene expression indirectly. However, data available on this type of interaction is limited. One such interaction is the binding of DNA with mycobacterium DNA binding proteins. We have used the Langmuir-Blodgett technique to evaluate for the first time the kinetics and thermodynamics of Mycobacterium smegmatis Dps 1 binding to DNA. By immobilizing one of the interacting partners, we have shown that, when a kinetic bottleneck is applied, the binding mechanism showed cooperative binding (n = 2.72) at lower temperatures, but the degree of cooperativity gradually reduces (n = 1.38) as the temperature was increased We have also compared the kinetics and thermodynamics of sequence-specific and nonspecific DNA protein interactions under the same set of conditions.

Functional regulation of PVBV Nuclear Inclusion protein-a protease activity upon interaction with Viral Protein genome-linked and phosphorylation

Regulation of NIa-Pro is crucial for polyprotein processing and hence, for successful infection of potyviruses. We have examined two novel mechanisms that could regulate NIa-Pro activity. Firstly, the influence of VPg domain on the proteolytic activity of NIa-Pro was investigated. It was shown that the turnover number of the protease increases when these two domains interact (as: two-fold; trans: seven-fold) with each other. Secondly, the protease activity of NIa-Pro could also be modulated by phosphorylation at Ser129. A mutation of this residue either to aspartate (phosphorylation-mimic) or alanine (phosphorylation-deficient) drastically reduces the protease activity. Based on these observations and molecular modeling studies, we propose that interaction with VPg as well as phosphorylation of Ser129 could relay a signal through Trp143 present at the protein surface to the active site pocket by subtle conformational changes, thus modulating protease activity of NIa-Pro. (C) 2011 Elsevier Inc. All rights reserved.

Biologically triggered exploding protein based microcapsules for drug delivery

Biologically triggered exploding microcapsules were synthesized by layer-by-layer assembly of biopolymers. The microcapsules showed controlled rupturing behaviour upon exposure to a pathologically relevant biomolecule, trypsin. These microcapsules offer significant potential for clinical applications.

Injection-molded high-density polyethylene-hydroxyapatite-aluminum oxide hybrid composites for hard-tissue replacement: Mechanical, biological, and protein adsorption behavior

In this article, we report the mechanical and biocompatibility properties of injection-molded high-density polyethylene (HDPE) composites reinforced with 40 wt % ceramic filler [hydroxyapatite (HA) and/or Al2O3] and 2 wt % titanate as a coupling agent. The mechanical property measurements revealed that a combination of a maximum tensile strength of 18.7 MPa and a maximum tensile modulus of about 855 MPa could be achieved with the injection-molded HDPE20 wt % HA20 wt % Al2O3 composites. For the same composite composition, the maximum compression strength was determined to be 71.6 MPa and the compression modulus was about 660 MPa. The fractrography study revealed the uniform distribution of ceramic fillers in the semicrystalline HDPE matrix. The cytocompatibility study with osteoblast-like SaOS2 cells confirmed extensive cell adhesion and proliferation on the injection-molded HDPE20 wt % HA20 wt % Al2O3 composites. The cell viability analysis with the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed a statistically significant difference between the injection-molded HDPE20 wt % HA20 wt % Al2O3 composites and sintered HA for various culture durations of upto 7 days. The difference in cytocompatibility properties among the biocomposites is explained in terms of the difference in the protein absorption behavior. (C) 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

Protein Model Discrimination Using Mutational Sensitivity Derived from Deep Sequencing

A major bottleneck in protein structure prediction is the selection of correct models from a pool of decoys. Relative activities of similar to 1,200 individual single-site mutants in a saturation library of the bacterial toxin CcdB were estimated by determining their relative populations using deep sequencing. This phenotypic information was used to define an empirical score for each residue (Rank Score), which correlated with the residue depth, and identify active-site residues. Using these correlations, similar to 98% of correct models of CcdB (RMSD <= 4 angstrom) were identified from a large set of decoys. The model-discrimination methodology was further validated on eleven different monomeric proteins using simulated RankScore values. The methodology is also a rapid, accurate way to obtain relative activities of each mutant in a large pool and derive sequence-structure-function relationships without protein isolation or characterization. It can be applied to any system in which mutational effects can be monitored by a phenotypic readout.

Distinct Roles of FANCO/RAD51C Protein in DNA Damage Signaling and Repair Implications for Fanconi Anemia and Breast Cancer Susceptibility

RAD51C, a RAD51 paralog, has been implicated in homologous recombination (HR), and germ line mutations in RAD51C are known to cause Fanconi anemia (FA)-like disorder and breast and ovarian cancers. The role of RAD51C in the FA pathway of DNA interstrand cross-link (ICL) repair and as a tumor suppressor is obscure. Here, we report that RAD51C deficiency leads to ICL sensitivity, chromatid-type errors, and G(2)/M accumulation, which are hallmarks of the FA phenotype. We find that RAD51C is dispensable for ICL unhooking and FANCD2 monoubiquitination but is essential for HR, confirming the downstream role of RAD51C in ICL repair. Furthermore, we demonstrate that RAD51C plays a vital role in the HR-mediated repair of DNA lesions associated with replication. Finally, we show that RAD51C participates in ICL and double strand break-induced DNA damage signaling and controls intra-S-phase checkpoint through CHK2 activation. Our analyses with pathological mutants of RAD51C that were identified in FA and breast and ovarian cancers reveal that RAD51C regulates HR and DNA damage signaling distinctly. Together, these results unravel the critical role of RAD51C in the FA pathway of ICL repair and as a tumor suppressor.

Withania somnifera reverses Alzheimer's disease pathology by enhancing low-density lipoprotein receptor-related protein in liver

A 30-d course of oral administration of a semipurified extract of the root of Withania somnifera consisting predominantly of withanolides and withanosides reversed behavioral deficits, plaque pathology, accumulation of beta-amyloid peptides (A beta) and oligomers in the brains of middle-aged and old APP/PS1 Alzheimer's disease transgenic mice. It was similarly effective in reversing behavioral deficits and plaque load in APPSwInd mice (line J20). The temporal sequence involved an increase in plasma A beta and a decrease in brain A beta monomer after 7 d, indicating increased transport of A beta from the brain to the periphery. Enhanced expression of low-density lipoprotein receptor-related protein (LRP) in brain microvessels and the A beta-degrading protease neprilysin (NEP) occurred 14-21 d after a substantial decrease in brain A beta levels. However, significant increase in liver LRP and NEP occurred much earlier, at 7 d, and were accompanied by a rise in plasma sLRP, a peripheral sink for brain A beta. In WT mice, the extract induced liver, but not brain, LRP and NEP and decreased plasma and brain A beta, indicating that increase in liver LRP and sLRP occurring independent of A beta concentration could result in clearance of A beta. Selective down-regulation of liver LRP, but not NEP, abrogated the therapeutic effects of the extract. The remarkable therapeutic effect of W. somnifera mediated through up-regulation of liver LRP indicates that targeting the periphery offers a unique mechanism for A beta clearance and reverses the behavioral deficits and pathology seen in Alzheimer's disease models.