Browsing by Author "Vikash K. Dubey"

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A novel serine protease cryptolepain from Cryptolepis buchanani: Purification and biochemical characterization
(2006) Monu Pande; Vikash K. Dubey; Subhash C. Yadav; Medicherla V. Jagannadham
A novel protease is purified to homogeneity from the latex of a medicinally important plant Cryptolepis buchanani of family Apocynaceae (formerly Asclepiadaceae). The enzyme named cryptolepain has a molecular mass of 50.5 kDa. The isoelectric point and extinction coefficient (∈280nm1%) are 6.0 and 26.4, respectively. Cryptolepain contains 15 tryptophans, 41 tyrosines, and eight cysteine residues forming four disulfide bridges. The detectable carbohydrate moiety in the enzyme was found to be 6-7%. Cryptolepain hydrolyzes denatured natural substrates like casein, azocasein, and azoalbumin with high specific activity. The protease is exclusively inhibited by serine protease inhibitors phenylmethansulfonyl fluoride and diisopropyl fluorophosphate. Hydrolysis of azoalbumin by the cryptolepain is optimal in the pH range of 8-10 and temperatures of 65-75°C. The enzyme shows high stability against pH (2.5-11.5), temperature (up to 80°C), and chemical denaturants. The K m value of the enzyme was found to be 10 μM with azocasein as the substrate. The N-terminal sequence of cryptolepain is unique and shows only little homology to other known serine proteases, which makes this enzyme an ideal candidate for our ongoing biochemical and structure-function investigations of proteases. Easy availability of the latex and simple purification procedures make the enzyme a good system for exploring the biophysical chemistry of serine proteases as well as applications in the food industry. © 2006 American Chemical Society.
A squalene oil emulsified MPL-A and anti-CD200/CD300a antibodies adjuvanted whole-killed Leishmania vaccine provides durable immunity against L. donovani parasites
(Elsevier Ltd, 2024) Baishakhi Mahapatra; Abhishek Singh; Arpita Banerjee; Shruti Sirohi; Samer Singh; Vikash K. Dubey; Rakesh K. Singh
Antigenic inefficacy to induce robust immune responses and durable memory are major causes of constantly failing prophylactic approaches in leishmaniasis. Here, we determine the potential of a standardized whole-killed Leishmania vaccine (Leishvacc) adjuvanted with anti-CD200 and anti-CD300a antibodies, either alone or with monophosphoryl lipid A (MPL-SE) emulsified in squalene oil, in restoring the compromised antigen presenting abilities of dendritic cells (DCs), effector properties of CD4+T cells and providing protection against Leishmania donovani parasites. In animals vaccinated with antibodies adjuvanted vaccines, either alone or with MPL-SE, the antigen presenting abilities of CD11c+ DCs against Leishmania antigens, measured in terms of CD80, CD86, MHC-I, and MHC-II surface receptors and intracellular IL-12 were found enhanced than non-adjuvanted vaccine. We observed more proliferative and pro-inflammatory cytokines i.e. IL-2, IFN-γ, IL-23, and IL-12 producing CD4+T cells in antibodies/MPL-SE adjuvanted vaccinated animals further suggesting that this approach helps antigen activated CD4+T cells to acquire pro-inflammatory cytokines producing abilities. In antibodies, either alone or with MPL-SE, vaccinated animals, the number of CD4+ central memory T cells and their longevity were found significantly enhanced that further evidenced the impact of this vaccination approach in inducing long term protective immunity. The animals, receiving antibodies adjuvanted vaccines, either alone or with MPL-SE, exhibited excellent protection against virulent parasites by restricting their growth, which correlated with the significantly reduced parasitemia, splenomegaly, and hepatomegaly, along with fewer numbers of liver granulomas. Our findings provide an insight to a new immunoprophylactic approach against visceral leishmaniasis, which not only satisfies the safety criteria, but also provides a robust immunogenic response with remarkable potential for parasites control. However, further in-depth investigations are needed to ascertain its ability in inducing long-lasting immunity. © 2024
Accumulation of partly folded states in the equilibrium unfolding of ervatamin A: Spectroscopic description of the native, intermediate, and unfolded states
(2007) Sreedevi Nallamsetty; Vikash K. Dubey; Monu Pande; P.K. Ambasht; M.V. Jagannadham
Ervatamin A, a cysteine proteases from Ervatamia coronaria, has been used as model system to examine structure-function relationship by equilibrium unfolding methods. Ervatamin A belongs to α+β class of proteins and exhibit stability towards temperature and chemical denaturants. Acid induced unfolding of ervatamin A was incomplete with respect to the structural content of the enzyme. Between pH 0.5 and 2.0, the enzyme is predominantly in β-sheet conformation and shows a strong ANS binding suggesting the existence of a partially unfolded intermediate state (IA state). Surprisingly, high concentrations of GuHCl required to unfold this state and the transition mid points GuHCl induced unfolding curves are significantly higher. GuHCl induced unfolding of ervatamin A at pH 3.0 as well as at pH 4.0 is complex and cannot be satisfactorily fit to a two-state model for unfolding. Besides, a strong ANS binding to the protein is observed at low concentration of GuHCl, indicating the presence of intermediate in the unfolding pathway. On the other hand, even in the presence of urea (8 M) the enzyme retains all the activity as well as structural parameters at neutral pH. However, the protein is susceptible to urea unfolding at pH 3.0 and below. Urea induced unfolding of ervatamin A at pH 3.0 is cooperative and the transitions curves obtained by different probes are and non-coincidental. Temperature denaturation of ervatamin A in IA state is non-cooperative, contrary to the cooperativity seen with native protein, suggesting the presence of two parts in the molecular structure of ervatamin A may be domains, with different stability that unfolds in steps. Careful inspection of biophysical properties of intermediate states populated in urea and GuHCl (IUG state) induced unfolding suggests all these three intermediates are identical and populated in different conditions. However, the properties of the intermediate (IA state) identified at pH ∼1.5 are different from those of the IUG state. © 2007 Elsevier Masson SAS. All rights reserved.
Conformational plasticity of cryptolepain: Accumulation of partially unfolded states in denaturants induced equilibrium unfolding
(2007) Monu Pande; Vikash K. Dubey; Vishal Sahu; Medicherla V. Jagannadham
pH and chemical denaturant dependent conformational changes of a serine protease cryptolepain from Cryptolepis buchanani are presented in this paper. Activity measurements, near UV, far UV CD, fluorescence emission spectroscopy, and ANS binding studies have been carried out to understand the folding mechanism of the protein in the presence of denaturants. pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions. The preliminary biophysical study on cryptolepain shows that major elements of secondary structure are β-sheets. Under neutral conditions the enzyme was stable in urea while GuHCl-induced equilibrium unfolding was cooperative. Cryptolepain shows little ANS binding even under neutral conditions due to more hydrophobicity of β-sheets. Multiple intermediates were populated during the pH-induced unfolding of cryptolepain. Temperature-induced denaturation of cryptolepain in the molten globule like state is non-cooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two parts, possibly domains, in the molecular structure of cryptolepain, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of A state (molten globule state) of cryptolepain is unique, as lower concentration of denaturant, not only induces structure but also facilitate transition from one molten globule like state (MG1) into another (MG2). The increase of pH drives the protein into alkaline denatured state characterized by the absence of any ANS binding. GuHCl- and urea-induced unfolding transition curves at pH 12.0 were non-coincidental indicating the presence of an intermediate in the unfolding pathway. © 2007 Elsevier B.V. All rights reserved.
Crystallization and preliminary X-ray analysis of cryptolepain, a novel glycosylated serine protease from Cryptolepis buchanani
(2007) Monu Pande; Vikash K. Dubey; Medicherla V. Jagannadham
Cryptolepain is a stable glycosylated novel serine protease purified from the latex of the medicinally important plant Cryptolepis buchanani. The molecular weight of the enzyme is 50.5 kDa, as determined by mass spectrometry. The sequence of the first 15 N-terminal resides of the protease showed little homology with those of other plant serine proteases, suggesting it to be structurally unique. Thus, it is of interest to solve the structure of the enzyme in order to better understand its structure-function relationship. X-ray diffraction data were collected from a crystal of cryptolepain and processed to 2.25 Å with acceptable statistics. The crystals belong to the orthorhombic space group C2221, with unit-cell parameters a = 81.78, b = 108.15, c = 119.86 Å. The Matthews coefficient was 2.62 Å3 Da-1 with one molecule in the asymmetric unit. The solvent content was found to be 53%. Structure determination of the enzyme is under way. © International Union of Crystallography 2007.
Leishmania antigens activated CD4+ T cells expressing CD200R receptors are the prime IL-10 producing phenotype and an important determinant of visceral leishmaniasis pathogenesis
(Academic Press, 2024) Abhishek Singh; Baishakhi Mahapatra; Arpita Banerjee; Samer Singh; Sangram Singh; Vikash K. Dubey; Pradeep Das; Rakesh K. Singh
The excessive production of IL-10, an anti-inflammatory cytokine, by Leishmania antigen-activated T cells is supposed to be a key player in the onset and progression of visceral leishmaniasis (VL). The IL-10-producing sources in VL remain unidentified and uncharacterized. In this study, we reveal that antigen-activated CD4+ T cells, i.e., CD44+CD4+ T cells expressing CD200R receptors, are the prime IL-10-producing phenotypes in Leishmania donovani infection-induced pathogenesis. These phenotypes are separate from CD25+Foxp3+CD4+ T regulatory cells, which are classical IL-10-producing phenotypes. In order to ascertain the role of CD200R and CD25 receptors in IL-10 overexpression-associated VL pathogenesis, we abrogated CD200R and CD25 receptor-mediated signaling in the infected mice. The splenic load of parasites and the size of the liver and spleen were significantly reduced in CD200-blocked mice as compared to CD25-blocked mice. Further, the CD200 blocking polarized CD4+ T cells to pro-inflammatory cytokines-producing phenotypes, as we observed a higher frequency of IFN-γ, TNF-α, and IL-12 positive cells as compared to controls including the CD25 blocking. Our findings suggest that in L. donovani infection-induced pathogenesis the expression of CD200R on antigen-activated T cells helps them to acquire IL-10-producing abilities as part of its one of the survival strategies. However, more studies would be warranted to better understand CD200R receptors role in VL pathogenesis and to develop the next generation of therapeutic and prophylactic control measures. © 2023 Elsevier Ltd
Miltefosine-unresponsive Leishmania donovani has a greater ability than miltefosine-responsive L. donovani to resist reactive oxygen species
(2013) Mousumi Das; Prakash Saudagar; Shyam Sundar; Vikash K. Dubey
Resistance of Leishmania parasites to miltefosine, which is only available oral drug, is a great concern. We have analyzed global gene expression profiles of miltefosine-unresponsive and miltefosine-responsive Leishmania donovani in order to understand the various metabolic processes involved in miltefosine drug resistance. The microarray data clearly indicated a role of oxidative metabolism in miltefosine resistance. Furthermore, fluorescence microscopy experiments suggested that miltefosine-unresponsive L. donovani resists the accumulation of reactive oxygen species and subsequent mitochondrial membrane damage leading to apoptotic death. In contrast, in miltefosine-responsive L. donovani, the accumulation of reactive oxygen species causes apoptotic death. Overall, this study provides fundamental insights into miltefosine resistance in L. donovani. Database The microarray data have been deposited in the Gene Expression Omnibus database under the accession number GSE45496 We have analyzed global gene expression profiles of miltefosine-unresponsive and miltefosine-responsive Leishmania donovani in order to understand various metabolic processes involved in miltefosine drug resistance. The microarray data clearly indicated a role of oxidative metabolism in miltefosine resistance. The experimental data supports that the miltefosine unresponsive Leishmania donovani can resists formation of reactive oxygen species more efficiently. © 2013 FEBS.
Modeled structure of trypanothione reductase of Leishmania infantum
(The Biochemical Society of the Republic of Korea, 2008) Bishal K. Singh; Nandini Sarkar; M.V. Jagannadham; Vikash K. Dubey
Trypanothione reductase is an important target enzyme for structure-based drug design against Leishmania. We used homology modeling to construct a three-dimensional structure of the trypanothione reductase (TR) of Leishmania infantum. The structure shows acceptable Ramachandran statistics and a remarkably different active site from glutathione reductase(GR). Thus, a specific inhibitor against TR can be designed without interfering with host (human) GR activity.
Papain-like proteases: Applications of their inhibitors
(2007) Vikash K. Dubey; Monu Pande; Bishal Kumar Singh; Medicherla V. Jagannadham
Proteases are one of the most important classes of enzyme and expressed throughout the animal and plant kingdoms as well as in viruses and bacteria. The protease family has drawn special attention for drug target for cure of several diseases such as osteoporosis, arthritis and cancer. Many proteases from various sources are being studied extensively with respect to activity, inhibition and structure. In this review, we hope to bring together the information available about the proteases with particular emphasis on papain-like plant cysteine proteases. Besides, protease inhibitors and their potential utilities are also discussed. © 2007 Academic Journals.
Roles for cavities in protein structure: New insights
(2008) Vikash K. Dubey; M.V. Jagannadham
"Packing defects" which result in the presence of a cavity in the protein continue to be a subject of great interest. Naturally occurring or engineered cavities in the protein structure are empty or hydrated may depend on several factors (size, hydrophobicity, etc.) and is currently the subject of active debate. The role of protein cavities is considerable controversy and still needs further investigation. The presence of internal cavities appears to be crucial for conferring the conformational flexibility needed for their biological function. In several studies, stabilities of the protein could be improved by cavity filling mutations but concomitant loss of functionality, indicating their role in stability-function tradeoff. In this review, we hope to bring together the experimental studies available on protein cavities. ©2008 Bentham Science Publishers Ltd.