2D-DIGE based urinary proteomics and functional enrichment studies to reveal novel Mycobacterium tuberculosis and human protein biomarker candidates for pulmonary tuberculosis.

In the absence of a sensitive and specific diagnostic modality capable of detecting all forms of tuberculosis (TB), proteomics may identify specific Mycobacterium tuberculosis (M.tb) proteins in urine, with a potential as biomarkers. To identify candidate biomarkers for TB, proteome profile of urine from pulmonary TB patients was compared with non-disease controls (NDC) and disease controls (DC, Streptococcus pneumonia infected patients) using a combination of two-dimensional difference gel electrophoresis (2D-DIGE) and liquid chromatography tandem mass spectrometry (LCMS/MS). Eleven differentially expressed host proteins and Eighteen high abundant M.tb proteins were identified. Protein-protein interactome (PPI) and functional enrichment analyses like Gene Ontologies, Reactome pathway etc. demonstrated that the human proteins mainly belong to extracellular space and show physiological pathways for immune response and hematological disorders. Whereas, M.tb proteins belong to the cell periphery, plasma membrane and cell wall, and demonstrated catalytic, nucleotide binding and ATPase activities along with other functional processes. The study findings provide valuable inputs about the biomarkers of TB and shed light on the probable disease consequences as an outcome of the bacterial pathogenicity.

Identification of invasion proteins of Cryptosporidium parvum.

Host cell interactions and invasion by Cryptosporidium is a complex process mediated by zoites ligand-host cell receptors. Knowledge of proteins involved in this process will enable entry level inhibitors to be tried as therapeutic agents. In the present study, invasion proteins of Cryptosporidium parvum were studied in vitro. Cryptosporidium sporozoites membrane proteins were isolated and Cy5 dye labelled. They were then allowed to interact with the intact host cells. The interacting proteins were identified using 2-dimensional gel electrophoresis followed by mass spectrometry analysis. Sixty-one proteins were identified including twenty-seven previously reported invasion proteins. The newly identified proteins such as serine/threonine protein kinase, PI4 kinase, Hsp105 and coiled coil may have their roles in the parasitic invasion process. Thus, a new approach was used in the study to identify the probable proteins involved in invasion and/or host-parasite interactions. The advantage of this method is that it takes only a months' time instead of decades to identify these proteins involved in invasion process.

Crystal structure of the VapBC-15 complex from Mycobacterium tuberculosis reveals a two-metal ion dependent PIN-domain ribonuclease and a variable mode of toxin-antitoxin assembly.

Although PIN (PilT N-terminal)-domain proteins are known to have ribonuclease activity, their specific mechanism of action remains unknown. VapCs form a family of ribonucleases that possess a PIN-domain assembly and are known as toxins. The activities of VapCs are impaired by VapB antitoxins. Here we present the crystal structure of the VapBC-15 toxin-antitoxin complex from Mycobacterium tuberculosis determined to 2.1Å resolution. The VapB-15 and VapC-15 components assemble into one heterotetramer (VapB2C2) and two heterotrimers (VapBC2) in each asymmetric unit of the crystal. The active site of VapC-15 toxin consists of a cluster of acidic amino acid residues and two divalent metal ions, forming a well organised ribonuclease active site. The distribution of the catalytic-site residues of the VapC-15 toxin is similar to that of T4 RNase H and of Methanococcus jannaschii FEN-1, providing strong evidence that these three proteins share a similar mechanism of activity. The presence of both VapB2C2 and VapBC2 emphasizes the fact that the same antitoxin can bind the toxin in 1:1 and 1:2 ratios. The crystal structure determination of the VapBC-15 complex reveals for the first time a PIN-domain ribonuclease protein that shows two metal ions at the active site and a variable mode of toxin-antitoxin assembly. The structure further shows that VapB-15 antitoxin binds to the same groove meant for the binding of putative substrate (RNA), resulting in the inhibition of VapC-15's toxicity.

Addition of magnesium chloride to enhance mono-dispersity of a coiled-coil recombinant mouse macrophage protein.

X-ray crystallography for the determination of three-dimensional structures of protein macromolecules represents an important tool in function assignment of uncharacterized proteins. However, crystallisation is often difficult to achieve. A protein sample fully characterized in terms of dispersity may increase the likelihood of successful crystallisation by improving the predictability of the crystallisation process. To maximize the probability of crystallisation of a novel mouse macrophage protein (rMMP), target molecule was characterized and refined to improve monodispersity. Addition of MgCl2 at low concentrations resolves the rMMP into a monodisperse solution, and finally successful crystallization of rMMP was achieved. The effect of MgCl2 was studied using gel filtration chromatography and dynamic light scattering.

MALDI-TOF mass spectrometry proteomic based identification of clinical bacterial isolates.

BACKGROUND & OBJECTIVES: Pathogenic bacteria often cause life threatening infections especially in immunocompromised individuals. Therefore, rapid and reliable species identification is essential for a successful treatment and disease management. We evaluated a rapid, proteomic based technique for identification of clinical bacterial isolates by protein profiling using matrix-assisted laser desorption-ionization time - of - flight mass spectrometry (MALDI-TOF MS). METHODS: Freshly grown bacterial isolates were selected from culture plates. Ethanol/formic acid extraction procedure was carried out, followed by charging of MALDI target plate with the extract and overlaying with α-cyano-4 hydroxy-cinnamic acid matrix solution. Identification was performed using the MALDI BioTyper 1.1, software for microbial identification (Bruker Daltonik GmbH, Bremen, Germany). RESULTS: A comparative analysis of 82 clinical bacterial isolates using MALDI -TOF MS and conventional techniques was carried out. Amongst the clinical isolates, the accuracy at the species level for clinical isolates was 98.78%. One out of 82 isolates was not in accordance with the conventional assays because MALDI-TOF MS established it as Streptococcus pneumoniae and conventional methods as Streptococcus viridans. INTERPRETATION & CONCLUSIONS: MALDI - TOF MS was found to be an accurate, rapid, cost-effective and robust system for identification of clinical bacterial isolates. This innovative approach holds promise for earlier therapeutic intervention leading to better patient care.

Delineation of the Structural Elements of Oriental Liver Fluke PLA2 Isoforms for Potent Drug Designing.

Clonorchis sinensis or the Chinese liver fluke is one of the most prevalent parasites affecting a major population in the oriental countries. The parasite lacks lipid generating mechanisms but is exposed to fatty acid rich bile in the liver. A secretory phospholipase A2, an enzyme that breaks down complex lipids, is important for the growth of the parasite. The enzyme is also implicated in the pathogenesis leading up to the hepatic fibrosis and its complications including cancer. The five isoforms of this particular enzyme from the parasite therefore qualify as potential drug targets. In this study, a detailed structural and ligand binding analysis of the isoforms has been done by modeling. The overall three dimensional structures of the isoforms are well conserved with three helices and a ß-wing stabilized by four disulfide bonds. There are characteristic differences at the calcium binding loop, hydrophobic channel and the C-terminal domain that can potentially be exploited for drug binding. But the most significant feature pertains to the catalytic site where the isoforms exhibit three variations of either a histidine-aspartate-tyrosine or histidine-glutamate-tyrosine or histidine-aspartate-phenylalanine. Molecular docking studies show that isoform specific residues and their conformations in the substrate binding hydrophobic channel make unique interactions with certain inhibitor molecules resulting in a perfect tight fit. The proposed ligand molecules have a predicted affinity in micro-molar to nano-molar range. Interestingly, few of the ligand binding interaction patterns is in accordance to the phylogenetic studies to thereby establish the usefulness of evolutionary mechanisms in aiding ligand design. The molecular diversity of the parasitic PLA2 described in this study provides a platform for personalized medicine in the therapeutics of clonorchiasis.

Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk.

Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk-secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI-TOF/TOF MS and 1D-Gel-LC-MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT-PCR and Western blotting. The 1D-Gel-LC-MS/MS and 2DE-MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC-specific proteins that will help the researchers to understand the molecular events taking place during lactation.

Apolipoprotein A1 as a potential biomarker in the ascitic fluid for the differentiation of advanced ovarian cancers.

CONTEXT: Primary ovarian cancer and ovarian metastasis from non-ovarian cancers in advanced stage are closely mimicking conditions whose therapeutics and prognosis are different. OBJECTIVE: To identify biomarkers that can differentiate the two variants of advanced ovarian cancers. METHODS: Gel-based proteomics and antibody-based assays were used to study the differentially expressed proteins in the ascitic fluid of fourteen patients with advanced ovarian cancers. RESULTS: Programmed Cell Death 1-Ligand 2, apolipoprotein A1, apolipoprotein A4 and anti-human fas antibody are differentially expressed proteins. CONCLUSIONS: Apolipoprotein A1 with a 61.8 ng/ml cut-off is a potential biomarker with the best differentiating statistical parameters.

Preliminary crystallographic analysis of recombinant VapBC-15 toxin-antitoxin complex from Mycobacterium tuberculosis.

The Mycobacterium tuberculosis vapBC15 locus encodes a toxin-antitoxin complex. VapC-15 is a toxin and possesses ribonuclease activity and VapB-15 is an antitoxin which both binds and inhibits the VapC-15 toxin. In this study, vapBC15 genes were cloned and co-expressed in Escherichia coli. The complex was purified to homogeneity by affinity and size-exclusion chromatography. The VapBC-15 complex was crystallized using the sitting-drop vapour-diffusion technique. The crystals diffracted to 2.6 Šresolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 85.63, b = 139.09, c = 148.86 Å. The self-rotation function combined with Matthews coefficient and solvent-content calculations suggests the presence of either six or eight molecules of the complex in the asymmetric unit.

Experimental inhibition of peptide fibrillogenesis by synthetic peptides, carbohydrates and drugs.

Peptide fibrillogenesis generally begins by the transformation of normally soluble proteins into elongated aggregates which are called as amyloid. These fibrils mainly consist of ß-sheets. They share certain common characteristics such as a cross-ß x-ray diffraction pattern, association with other common proteins and typical staining by the dye Congo Red. The individual form of the deposit consists of a disease-specific peptide/protein. The disease-specific protein serves as the basis for the classification of the amyloids. The association of fibril-forming peptides/proteins with diseases makes them primary disease-targets. Understanding the molecular interactions involved in the fibril formation becomes the foremost requirement to characterize the target. Interference with these interactions of ß-sheets in vitro prevents and sometimes reverses the fibril assembly. A small molecule capable of interfering with the formation of fibril could have therapeutic applications in these diseases. This anti-aggregation approach appears to be a viable treatment option. A search for such a molecule is pursued actively world over. All types of compounds and approaches to slow down or prevent the aggregation process have been described in the literature. These efforts are reviewed in this chapter.

Physicochemical characteristics and antioxidant efficacy of chitosan from the internal shell of spineless cuttlefish Sepiella inermis.

Cuttlefish chitosan was extracted from the cuttlebone of Sepiella inermis by demineralization and deproteinization and produced by deacetylation, and its physical and chemical parameters were also compared with that of commercial chitosan. Ash, moisture, and mineral and metal content of the chitosan was estimated by adopting standard methodologies. The rate of deacetylation was calculated as 79.64% by potentiometric titration. Through viscometry and gel permeation chromatography, the molecular weight of chitosan was found to be significantly lower than that of the commercial chitosan. Optical activity was found to be levorotatory. The structure of the chitosan was elucidated with spectral techniques such as Fourier-transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy. Cuttlefish chitosan showed a melting endothermic peak at 117.32 °C. The x-ray diffraction (XRD) pattern of chitosan and standard chitosan exhibited the same crystalline peaks. Through scanning electron microscopy (SEM) the fine structure of chitosan was studied. The binding capacity (water and fat) of cuttlefish chitosan was found to be significantly higher than that of the commercial chitosan. The antioxidant efficacy of chitosan was determined through the conjugated diene method, scavenging ability on DPPH radicals, reducing power, and chelating ability on ferrous ions. This study has brought out the importance of shell as a potential source for obtaining another natural antioxidant.

Balsamin, a novel ribosome-inactivating protein from the seeds of Balsam apple Momordica balsamina.

Plant seeds, a rich source of proteins, are considered important for their application as functional ingredients in a food system. A novel ribosome-inactivating protein (RIP), balsamin was purified from the seeds of Balsam apple, Momordica balsamina. Balsamin was purified by ion exchange chromatography on CM Sepharose and gel filtration on superdex-75. It has a molecular weight of 28 kDa as shown by SDS-PAGE analysis. Balsamin inhibits protein synthesis in a rabbit reticulocyte lysate-based cell free translation assay with an IC(50) of 90.6 ng ml(-1). It has RNA N-glycosidase activity and releases a 400-base long fragment termed the Endo fragment from 28S rRNA in the same manner as does saporin-6 from Saponaria officinalis. The N-terminal sequence analysis of the first 12 amino acids of balsamin revealed that it shares 83% similarity with type I RIP α-MMC from Momordica charantia and 50% similarity with ß-MMC (from Momordica charantia), bryodin I (from Bryonia dioica) and luffin a (from Luffa cylindrica). Balsamin was further characterized by mass spectrometry. CD spectroscopic studies indicate that secondary structure of balsamin contains helix (23.5%), ß-strand (24.6%), turn (20%) and random coil (31.9%). Thus RIPs activity expressed in vegetables like Momordica sp. advocates its usage in diet.

Biomineralization process in hard tissues: The interaction complexity within protein and inorganic counterparts.

Biomineralization can be considered as nature's strategy to produce and sustain biominerals, primarily via creation of hard tissues for protection and support. This review examines the biomineralization process within the hard tissues of the human body with special emphasis on the mechanisms and principles of bone and teeth mineralization. We describe the detailed role of proteins and inorganic ions in mediating the mineralization process. Furthermore, we highlight the various available models for studying bone physiology and mineralization starting from the historical static cell line-based methods to the most advanced 3D culture systems, elucidating the pros and cons of each one of these methods. With respect to the mineralization process in teeth, enamel and dentin mineralization is discussed in detail. The key role of intrinsically disordered proteins in modulating the process of mineralization in enamel and dentine is given attention. Finally, nanotechnological interventions in the area of bone and teeth mineralization, diseases and tissue regeneration is also discussed. STATEMENT OF SIGNIFICANCE: This article provides an overview of the biomineralization process within hard tissues of the human body, which encompasses the detailed mechanism innvolved in the formation of structures like teeth and bone. Moreover, we have discussed various available models used for studying biomineralization and also explored the nanotechnological applications in the field of bone regeneration and dentistry.

Structural and binding studies of C-terminal half (C-lobe) of lactoferrin protein with COX-2-specific non-steroidal anti-inflammatory drugs (NSAIDs).

Three COX-2-specific non-steroidal anti-inflammatory drugs (NSAIDs), etoricoxib, parecoxib, and nimesulide are widely prescribed against inflammatory conditions. However, their long term administration leads to severe conditions of cardiovascular complications and gastric ulceration. In order to minimize these side effects, C-terminal half (C-lobe) of colostrum protein lactoferrin has been indicated to be useful if co-administered with NSAIDs. Lactoferrin is an 80kDa glycoprotein with two similar halves designated as N- and C-lobes. Since NSAID-binding site is located in the C-terminal half of lactoferrin, C-lobe was prepared from lactoferrin by limited proteolysis using proteinase K. The incubation of lactoferrin with serine proteases for extended periods showed that N-lobe was completely digested but C-lobe was resistant for more than 72h indicating its long half life in the animal gut. The solution studies have shown that COX-2-specific NSAIDs bind to C-lobe with binding constants ranging from 10(-4) to 10(-5)M showing significant affinities for sequestering these compounds. In order to understand the mode of binding and sequestering properties, the complexes of C-lobe with all these three compounds, etoricoxib, parecoxib, and nimesulide were prepared and the structures of their complexes with C-lobe were determined at 2.2, 2.9, and 2.7A resolutions, respectively. The analysis of the structures of complexes of C-lobe with NSAIDs clearly show that all the three compounds bind firmly at the same ligand-binding site in the C-lobe revealing the details of the interactions between C-lobe and NSAIDs. The mode of binding of COX-2-specific NSAIDs to C-lobe is similar to that of the binding of COX-2 non-specific NSAIDs to C-lobe.

Mapping the Inorganic and Proteomic Differences among Different Types of Human Teeth: A Preliminary Compositional Insight.

In recent years, studies on mineralized tissues are becoming increasingly popular not only due to the diverse mechanophysical properties of such materials but also because of the growing need to understand the intricate mechanism involved in their assembly and formation. The biochemical mechanism that results in the formation of such hierarchical structures through a well-coordinated accumulation of inorganic and organic components is termed biomineralization. Some prime examples of such tissues in the human body are teeth and bones. Our current study is an attempt to dissect the compositional details of the inorganic and organic components in four major types of human teeth using mass spectrometry-based approaches. We quantified inorganic materials using inductively coupled plasma resonance mass spectrometry (ICP-MS). Differential level of ten different elements, Iron (Fe), Cadmium (Cd), Potassium (K), Sulphur (S), Cobalt (Co), Magnesium (Mg), Manganese (Mn), Zinc (Zn), Aluminum (Al), and Copper (Cu) were quantified across different teeth types. The qualitative and quantitative details of their respective proteomic milieu revealed compositional differences. We found 152 proteins in total tooth protein extract. Differential abundance of proteins in different teeth types were also noted. Further, we were able to find out some significant protein-protein interaction (PPI) backbone through the STRING database. Since this is the first study analyzing the differential details of inorganic and organic counterparts within teeth, this report will pave new directions to the compositional understanding and development of novel in-vitro repair strategies for such biological materials.

Accelerated identification of serine racemase inhibitor from Centella asiatica.

Serine racemase (SR) converts the free form of L-serine into D-serine (DS) in the mammalian brain. The DS functions as a co-agonist of N-methyl D-aspartate (NMDA) receptor. The over- activation of NMDA receptor leads to many neurological disorders like stroke, amyotrophic lateral sclerosis, Alzheimer's disease and an effective inhibitor of SR could be a corrective method for the receptor over-activation. We report for the first time here a rapid way of purifying and identifying an inhibitor from medicinal plants known to have the neuro-protective effect. We have purified SR inhibitor from the methanolic extract of Centella asiatica by affinity method. High resolution mass spectrometry and infrared spectroscopy were used to identify the ligand to be madecassoside. We have shown the madecassoside binding in silico and its inhibition of recombinant human serine racemase in vitro and ex vivo.

Disorder-to-order transition in PE-PPE proteins of Mycobacterium tuberculosis augments the pro-pathogen immune response.

A growing body of evidence supports the hypothesis that intrinsically disordered proteins often mediate host-pathogen interactions and modulate host functions for pathogen survival and virulence. Mycobacterium tuberculosis (M.tb) has evolved largely through reductive evolution, with a few exceptions such as the glycine-alanine-rich PE-PPE/PGRS protein family, which has been expanding in pathogenic mycobacteria. Here, our analyses of the M.tb proteome and secretome revealed that the PE-PGRS subfamily is enriched for disordered regions and disordered binding sites, pointing to their importance in host-pathogen interactions. As a case study, the secondary structure of PE35-PPE68 and PE32-PPE65 of the pathogenesis-related RD1 and RD8 regions was analyzed through Fourier-transform infrared spectroscopy. These disordered proteins displayed a considerable structural shift from disordered to ordered while engaged in the formation of complexes. While these proteins are immunogenic individually and enhance the pro-pathogen response, their corresponding complexes enhanced the responses manifold as displayed here by PE35 and PPE68. It is likely that M.tb exploits such disorder-order structural dynamics as a strategy to mount a pro-pathogen response and subvert host defense for productive infection. This functional gain also serves as a means to compensate genomic content loss due to reductive evolution.

The structural basis for the prevention of nonsteroidal antiinflammatory drug-induced gastrointestinal tract damage by the C-lobe of bovine colostrum lactoferrin.

Nonsteroidal antiinflammatory drugs (NSAIDs), due to their good efficacy in the treatment of pain, inflammation, and fever, are among the most prescribed class of medicines in the world. The main drawback of NSAIDs is that they induce gastric complications such as peptic ulceration and injury to the intestine. Four NSAIDs, indomethacin, diclofenac, aspirin, and ibuprofen were selected to induce gastropathy in mouse models. It was found that the addition of C-terminal half of bovine lactoferrin (C-lobe) reversed the NSAID-induced injuries to the extent of 47-70% whereas the coadministration of C-lobe prevented it significantly. The C-lobe was prepared proteolytically using serine proteases. The binding studies of C-lobe with NSAIDs showed that these compounds bind to C-lobe with affinities ranging from 2.6 to 4.8 x 10(-4) M. The complexes of C-lobe were prepared with the above four NSAIDs. All four complexes were crystallized and their detailed three-dimensional structures were determined using x-ray crystallographic method. The structures showed that all the four NSAID molecules bound to C-lobe at the newly identified ligand binding site in C-lobe that is formed involving two alpha-helices, alpha10 and alpha11. The ligand binding site is separated from the well known iron binding site by the longest and the most stable beta-strand, betaj, in the structure. Similar results were also obtained with the full length lactoferrin molecule. This novel, to our knowledge, binding site in C-lobe of lactoferrin shows a good complementarity for the acidic and lipophilic compounds such as NSAIDs. We believe this indicates that C-lobe of lactoferrin can be exploited for the prevention of NSAID-induced gastropathy.

Simultaneous inhibition of anti-coagulation and inflammation: crystal structure of phospholipase A2 complexed with indomethacin at 1.4 A resolution reveals the presence of the new common ligand-binding site.

A novel ligand-binding site with functional implications has been identified in phospholipase A(2) (PLA(2)). The binding of non-steroidal anti-inflammatory agent indomethacin at this site blocks both catalytic and anti-coagulant actions of PLA(2). A group IIA PLA(2) has been isolated from Daboia russelli pulchella (Russell's viper) which is enzymatically active as well as induces a strong anti-coagulant action. The binding studies have shown that indomethacin reduces the effects of both anti-coagulant and pro-inflammatory actions of PLA(2). A group IIA PLA(2) was co-crystallized with indomethacin and the structure of the complex has been determined at 1.4 A resolution. The structure determination has revealed the presence of an indomethacin molecule in the structure of PLA(2) at a site which is distinct from the conventional substrate-binding site. One of the carboxylic group oxygen atoms of indomethacin interacts with Asp 49 and His 48 through the catalytically important water molecule OW 18 while the second carboxylic oxygen atom forms an ionic interaction with the side chain of Lys 69. It is well known that the residues, His 48 and Asp 49 are essential for catalysis while Lys 69 is a part of the anti-coagulant loop (residues, 54-77). Indomethacin binds in such a manner that it blocks the access to both, it works as a dual inhibitor for catalytic and anti-coagulant actions of PLA(2). This new binding site in PLA(2) has been observed for the first time and indomethacin is the first compound that has been shown to bind at this novel site resulting in the prevention of anti-coagulation and inflammation.