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Pawan Gupta - One of the best experts on this subject based on the ideXlab platform.

  • Stem Bromelain induced macrophage apoptosis and activation curtail mycobacterium tuberculosis persistence
    The Journal of Infectious Diseases, 2012
    Co-Authors: Sahil Mahajan, Vemika Chandra, Sandeep Dave, Ravikanth Nanduri, Pawan Gupta
    Abstract:

    BACKGROUND Mycobacterium tuberculosis, the causative agent of tuberculosis, has a remarkable ability to usurp its host's innate immune response, killing millions of infected people annually. One approach to manage infection is prevention through the use of natural agents. In this regard, Stem Bromelain (SBM), a pharmacologically active member of the sulfhydryl proteolytic enzyme family, obtained from Ananas comosus and possessing a remarkable ability to induce the innate and acquired immune syStems, is important. METHODS We evaluated SBM's ability to induce apoptosis and free-radical generation in macrophages. We also studied antimycobacterial properties of SBM and its effect on foamy macrophages. RESULTS SBM treatment of peritoneal macrophages resulted in the upregulation of proapoptotic proteins and downregulation of antiapoptotic proteins. Additionally, SBM treatment activated macrophages, curtailed the levels of free glutathione, and augmented the production of hydrogen peroxide, superoxide anion, peroxynitrite, and nitric oxide. SBM cleaves CD36 and reduced the formation of foam cells, the hallmark of M. tuberculosis infection. These conditions created an environment for the increased clearance of M. tuberculosis. CONCLUSIONS Together these data provide a mechanism for antimycobacterial activity of SBM and provide important insights for the use of cysteine proteases as immunomodulatory agents.

  • Stem Bromelain–Induced Macrophage Apoptosis and Activation Curtail Mycobacterium tuberculosis Persistence
    The Journal of infectious diseases, 2012
    Co-Authors: Sahil Mahajan, Vemika Chandra, Sandeep Dave, Ravikanth Nanduri, Pawan Gupta
    Abstract:

    BACKGROUND Mycobacterium tuberculosis, the causative agent of tuberculosis, has a remarkable ability to usurp its host's innate immune response, killing millions of infected people annually. One approach to manage infection is prevention through the use of natural agents. In this regard, Stem Bromelain (SBM), a pharmacologically active member of the sulfhydryl proteolytic enzyme family, obtained from Ananas comosus and possessing a remarkable ability to induce the innate and acquired immune syStems, is important. METHODS We evaluated SBM's ability to induce apoptosis and free-radical generation in macrophages. We also studied antimycobacterial properties of SBM and its effect on foamy macrophages. RESULTS SBM treatment of peritoneal macrophages resulted in the upregulation of proapoptotic proteins and downregulation of antiapoptotic proteins. Additionally, SBM treatment activated macrophages, curtailed the levels of free glutathione, and augmented the production of hydrogen peroxide, superoxide anion, peroxynitrite, and nitric oxide. SBM cleaves CD36 and reduced the formation of foam cells, the hallmark of M. tuberculosis infection. These conditions created an environment for the increased clearance of M. tuberculosis. CONCLUSIONS Together these data provide a mechanism for antimycobacterial activity of SBM and provide important insights for the use of cysteine proteases as immunomodulatory agents.

  • Inhibition of adipogenesis and induction of apoptosis and lipolysis by Stem Bromelain in 3T3-L1 adipocytes.
    PloS one, 2012
    Co-Authors: Sandeep Dave, H. Kitdorlang Dkhar, Ravikanth Nanduri, Naval Jit Kaur, Ashwani Kumar, Pawan Gupta
    Abstract:

    The phytotherapeutic protein Stem Bromelain (SBM) is used as an anti-obesity alternative medicine. We show at the cellular level that SBM irreversibly inhibits 3T3-L1 adipocyte differentiation by reducing adipogenic gene expression and induces apoptosis and lipolysis in mature adipocytes. At the molecular level, SBM suppressed adipogenesis by downregulating C/EBPα and PPARγ independent of C/EBPβ gene expression. Moreover, mRNA levels of adipocyte fatty acid-binding protein (ap2), fatty acid synthase (FAS), lipoprotein lipase (LPL), CD36, and acetyl-CoA carboxylase (ACC) were also downregulated by SBM. Additionally, SBM reduced adiponectin expression and secretion. SBM's ability to repress PPARγ expression seems to Stem from its ability to inhibit Akt and augment the TNFα pathway. The Akt–TSC2–mTORC1 pathway has recently been described for PPARγ expression in adipocytes. In our experiments, TNFα upregulation compromised cell viability of mature adipocytes (via apoptosis) and induced lipolysis. Lipolytic response was evident by downregulation of anti-lipolytic genes perilipin, phosphodiestersae-3B (PDE3B), and GTP binding protein Giα1, as well as sustained expression of hormone sensitive lipase (HSL). These data indicate that SBM, together with all-trans retinoic-acid (atRA), may be a potent modulator of obesity by repressing the PPARγ-regulated adipogenesis pathway at all stages and by augmenting TNFα-induced lipolysis and apoptosis in mature adipocytes.

  • Trifluoroethanol stabilizes the molten globule state and induces non-amyloidic turbidity in Stem Bromelain near its isoelectric point
    International journal of biological macromolecules, 2011
    Co-Authors: Sandeep Dave, Vemika Chandra, Sahil Mahajan, Pawan Gupta
    Abstract:

    Stem Bromelain (SBM) is a therapeutic protein that has been studied for alkaline denaturation in the intestines, the principal site of its absorption. In this study, we investigated fluorinated alcohol 2,2,2-trifluoroethanol (TFE)-induced conformational changes in the specific/pre-molten globule (SMG) state of SBM observed at pH 10 by spectroscopic methods. Far-UV circular dichroism (CD) spectra showed that the protein retained its native-like secondary structure at TFE concentrations of up to 30% with a pronounced minimum at 222 nm, characteristic of a helix. However, addition of slightly higher TFE concentrations (≥40%) resulted in an ∼2.5-fold induction of this helical feature and a time-dependent increase in non-amyloidic turbidity as evidenced by turbidometric, Congo red-binding, and Thioflavin T (ThT)-binding studies. Near-UV CD spectra suggested a gradual but significant loss of tertiary structure at 10-30% TFE. Tryptophan studies showed blue-shifted fluorescence, although the number of accessible tryptophans remained the same up to 30% TFE. The SMG showed enhanced binding of the fluorescent probe 1-anilino-8-naphthalene sulfonic acid (ANS) up to 30% TFE, beyond which binding plateaued. Thermal and guanidine hydrochloride (GdnHCl) transition studies in the near-UV range indicated a single cooperative transition for the SMG state in the presence of 30% TFE, similar to that observed for native SBM at pH 7.0 (although with different T(m)s), unlike the SMG state. TFE (30%) appeared to induce native-like stability to the original SMG. These observations suggest a transformation of the SMG to a characteristic molten globule (MG) conformation at 30% TFE, possibly due to TFE-induced rearrangement of hydrophobic interactions at the protein's isoelectric point.

  • hexafluoroisopropanol induced helix sheet transition of Stem Bromelain correlation to function
    The International Journal of Biochemistry & Cell Biology, 2010
    Co-Authors: Sandeep Dave, Manvendra Pratap Singh, Garima Gupta, Vemika Chandra, Sahil Mahajan, Kitdorlang H Dkhar, Pawan Gupta
    Abstract:

    Stem Bromelain is a proteolytic phytoprotein with a variety of therapeutic effects. Understanding its structural properties could provide insight into the mechanisms underlying its clinical utility. Stem Bromelain was evaluated for its conformational and folding properties at the pH conditions it encounters when administered orally. It exists as a partially folded intermediate at pH 2.0. The conformational changes to this intermediate state were evaluated using fluorinated alcohols known to induce changes similar to those seen in vivo. Studies using circular dichroism, fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid and mass spectrometry indicate that treatment with 10-30% hexafluoroisopropanol induces the partially folded intermediate to adopt much of the native protein's secondary structure, but only a rudimentary tertiary structure, characteristic of the molten globule state. Addition of slightly higher concentrations of hexafluoroisopropanol caused transformation from an alpha-helix to a beta-sheet and induced formation of a compact nonnative structure. This nonnative form was more inhibitory of cell survival than either the native or the partially folded intermediate forms, as measured by enhanced suppression of proliferative cues (e.g., extracellular-signal-regulated kinase) and initiation of apoptotic events. The nonnative form also showed better antitumorigenic properties, as evaluated using an induced two-stage mouse skin papilloma model. In contrast, the nonnative state showed only a fraction of the proteolytic activity of the native form. This study demonstrates that hexafluoroisopropanol can induce a conformational change in Stem Bromelain to a form with potentially useful therapeutic properties different from those of the native protein.

Rizwan Hasan Khan - One of the best experts on this subject based on the ideXlab platform.

  • Cysteine as a potential anti-amyloidogenic agent with protective ability against amyloid induced cytotoxicity.
    International journal of biological macromolecules, 2017
    Co-Authors: Masihuz Zaman, Saima Nusrat, Tajalli Ilm Chandel, Parvez Alam, Syed Mohammad Zakariya, Mohammad Ajmal, Syed M. Meeran, Wahiduzzaman, Rizwan Hasan Khan
    Abstract:

    Protein aggregation and misfolding have been allied with numerous human disorders and thus inhibition of such occurrence has been center for intense research efforts against these diseases. Here, we investigated anti-fibrillation activity of cysteine and its effect on kinetics of Stem Bromelain amyloid fibril formation. We established the anti-fibrillation and anti aggregation activities of cysteine by using multiple approaches like turbidity measurements, dye binding assays (ThT and ANS) and structural changes were monitored by circular dichroism (CD) followed by electron microscopy. Our experimental study inferred that cysteine inhibits temperature induced fibrillation of protein in a concentration dependent way. In addition, MDA-MB-231 cell viability of pre-formed amyloid was increased in presence of cysteine as compared to the fibrils alone. Furthermore, dynamic light scattering studies of native, aggregated as well as incubated (amyloids in presence of cysteine) samples indicates that cysteine restores native like structures of Stem Bromelain. Isothermal titration calorimetric results revealed that hydrogen bonding between cysteine and Stem Bromelain plays a significant role during inhibition of Stem Bromelain aggregation. However, thiophilic interaction between thiol group of cysteine and aromatic amino acid residue of Stem Bromelain may also have noteworthy role in inhibition of amyloid formation.

  • Surfactant-mediated amyloidogenesis behavior of Stem Bromelain; a biophysical insight.
    Journal of biomolecular structure & dynamics, 2016
    Co-Authors: Masihuz Zaman, Saima Nusrat, Atiyatul Qadeer, Syed Mohammad Zakariya, Mohsin Vahid Khan, Mohammad Ajmal, Rizwan Hasan Khan
    Abstract:

    Neurodegenerative disorders are mainly associated with amyloid fibril formation of different proteins. Stem Bromelain (SB), a cysteine protease, is known to exist as a molten globule state at pH 10.0. It passes through the identical surrounding (pH 10.0) in the gut epithelium of intestine upon oral administration. Protein-surfactant complexes are widely employed as drug carriers, so the nature of surfactant toward protein is of great interest. The present work describes the effect of cationic surfactants (CTAB & DTAB) and their hydrophobic behavior toward amyloidogenesis behavior of SB at pH 10.0. Multiple approaches including light scattering, far UV-CD, turbidity measurements, and dye binding assay (ThT, Congo red and ANS) were performed to measure the aggregation propensity of SB. Further, we monitored the hydrodynamic radii of aggregates formed using dynamic light scattering technique. Structure of fibrils was also visualized through fluorescence microscopy as well as TEM. At pH 10.0, low concentration of CTAB (0-200 μM) induced amyloid formation in SB as evident from a prominent increase in turbidity and light scattering, gain in β-sheet content, and enhanced ThT fluorescence intensity. However, further increase in CTAB concentration suppressed the fibrillation phenomenon. In contrast, DTAB did not induce fibril formation at any concentration used (0-500 μM) due to lower hydrophobicity. Net negative charge developed on protein at high pH (10.0) might have facilitated amyloid formation at low concentration of cationic surfactant (CTAB) due to electrostatic and hydrophobic interactions.

  • Amyloidogenic behavior of different intermediate state of Stem Bromelain: A biophysical insight
    International journal of biological macromolecules, 2016
    Co-Authors: Masihuz Zaman, Aquib Ehtram, Sumit Kumar Chaturvedi, Saima Nusrat, Rizwan Hasan Khan
    Abstract:

    Stem Bromelain, a cysteine proteases from Ananas comosus is a widely accepted therapeutic drug with broad medicinal application. It exists as intermediate states at pH 2.0 and 10.0, where it encountered in gastrointestinal tract during adsorption (acidic pH) and in gut epithelium (alkaline pH), respectively. In this study, we monitored the thermal aggregation/amyloid formation of SB at different pH intermediate states. Thermal treatment of Stem Bromelain at pH 10.0 favors the fibrillation in which the extent of aggregation increases with increase in protein concentration. However, no fibril formation in Stem Bromelain at pH 2.0 was found at all the concentration used at pH 10.0. The fibril formation was confirmed by various techniques such as turbidity measurements, Rayleigh light scattering, dye binding assays and far UV circular dichroism. The Dynamic light scattering confirmed the formation of aggregates by measuring the hydrodynamic radii pattern. Moreover, microscopic techniques were performed to analyze the morphology of fibrils. The aggregation behavior may be due to variation in number of charged amino acid residues. The less negative charge developed at pH 10.0 may be responsible for aggregation. This work helps to overcome the aggregation related problems of Stem Bromelain during formulations in pharmaceutical industry.

  • Biophysical Insight of DNA Induced Aggregation of Stem Bromelain
    Biophysical Journal, 2016
    Co-Authors: Masihuz Zaman, Rizwan Hasan Khan
    Abstract:

    Stem Bromelain, a widely accepted phytotherapeutical drug, exists as a partially folded intermediate at pH 2.0, condition also encountered in the gastrointestinal tract (its site of adsorption). Numerous studies have demonstrated that various environmental and intracellular factors affect the fibrillation property of Stem Bromelain, by accelerating the process of assembly. In this study, the effect of calf thymus DNA (CT-DNA) on Stem Bromelain is investigated using multiple approaches such as turbidity measurements, ANS binding assays, Rayleigh scattering, ThT binding, far-UV circular dichroism, dynamic light scattering fluorescence microscopy and transmission electron microscopy. CT-DNA induced large sized β-sheet aggregates of SB at pH 2.0, and propensity of aggregation concomitantly increases with increasing concentration of CT-DNA (0-100 μM) followed by saturation phase at higher CT-DNA concentration. Furthermore, isothermal titration calorimetric measurements suggested that electrostatic interaction between positively charged SB at pH 2.0 and negatively charged phosphate group of CT-DNA is the probable mechanism that leads to aggregate formation. However, the hydrophobic interaction between CT-DNA and SB cannot be neglected.

  • DNA induced aggregation of Stem Bromelain; a mechanistic insight
    RSC Advances, 2016
    Co-Authors: Masihuz Zaman, Sumit Kumar Chaturvedi, Saima Nusrat, Nida Zaidi, Atiyatul Qadeer, Tajalli Ilm Chandel, Parvez Alam, Rizwan Hasan Khan
    Abstract:

    Negatively charged species such as nucleic acids have commonly been found to be associated with the proteinaceous deposits in the tissues of patients with amyloid diseases. Numerous studies have demonstrated that various environmental and intracellular factors affect the fibrillation property of proteins, by accelerating the process of assembly. Thus in the present study, the effect of calf thymus DNA (CT-DNA) on Stem Bromelain, a proteolytic phytoprotein, is investigated at pH 2.0, using multiple approaches that include turbidity measurements, Rayleigh light scattering, dye binding assay (ThT and ANS), far-UV circular dichroism, dynamic light scattering fluorescence microscopy and transmission electron microscopy. Large sized β-sheet aggregates of SB are found in the presence of CT-DNA at pH 2.0. The propensity for aggregation concomitantly increases with increasing concentration of CT-DNA (0–100 μM) and levels off at higher concentration of CT-DNA (beyond 100 μM). Isothermal titration calorimetric results confirmed that an electrostatic interaction between positively charged SB at pH 2.0 and the negatively charged phosphate group of CT-DNA is the probable mechanism behind aggregate formation. However, the hydrophobic interaction between CT-DNA and SB cannot be neglected. The survival of aggregates even after treatment with DNase indicates that intact CT-DNA is not necessarily required for SB aggregation.

Pannuru Venkatesu - One of the best experts on this subject based on the ideXlab platform.

  • can Stem Bromelain a pineapple waste product be used as a drug alternative a mechanistic insight into protein protein interactions
    New Journal of Chemistry, 2020
    Co-Authors: Samima Khatun, Anamika Sindhu, Pannuru Venkatesu
    Abstract:

    Stem Bromelain (SB) is a mixture of cysteine protease and non-protease components that is extracted from the pineapple plant (Ananas comosus). It has several therapeutic applications, such as antithrombotic, anti-inflammatory and anticancer. It also relieves osteoarthritis, diarrhea and various cardiovascular disorders. Bovine serum albumin (BSA), a blood plasma protein, is used as a model transport protein for studying protein folding and ligand binding mechanisms. We investigated the interactions between SB and BSA using UV-visible, fluorescence, isothermal titration calorimetry (ITC), circular dichroism (CD) and Fourier transform infrared (FTIR) techniques. The fluorescence results indicate that the fluorescence intensity of native BSA was quenched with increasing concentration of SB, with a blue shift in its wavelength maxima. The binding constant (Kb) was found to be on the order of 106, which is greater than that of protein–drug interactions; this reflects that SB can be used as an alternative to drugs in treating various diseases. The thermodynamic parameters obtained from isothermal titration calorimetry suggest that hydrophobic interactions play a major role in the association process, and it is an entropically driven process. The CD and FTIR spectroscopic results confirmed conformational alteration in BSA in the presence of SB, which was further substantiated by dynamic light scattering (DLS). The DLS results showed that the hydrodynamic diameter (dH) of BSA increases due to interaction with SB, resulting in unfolding of the native structure of BSA. This study is expected to provide important insight into the binding mechanism of SB with BSA, which may be useful in the pharmacology and clinical medicinal fields.

  • Excellency of pyrimidinyl moieties containing α-aminophosphonates over benzthiazolyl moieties for thermal and structural stability of Stem Bromelain.
    International journal of biological macromolecules, 2020
    Co-Authors: Gundluru Mohan, Pannuru Venkatesu, Sumit Kuma, Murali Sudileti, C. Sridevi, Cirandur Suresh Reddy
    Abstract:

    Abstract An efficient approach has been made for the synthesis of a series of novel di α-aminophosphonates by the reaction of terephthalaldehyde with various pyrimidine/benzthiazole amines and diethyl phosphite using sulfonated graphitic carbon nitride – SA@g-C3N4 as catalyst under room temperature and solvent free conditions. Later, the different effects of these newly synthesized α-aminophosphonates as a function of concentration gradient has been scrutinized on the thermal and structural stability of Stem Bromelain (SBM) through combining the results of various spectroscopic techniques like UV–vis, steady state fluorescence and circular dichroism (CD). Lastly the competitive and distinctive behaviour of α-aminophosphonates towards the stability of SBM has been envisaged using molecular docking simulations which suggest that nature of α-aminophosphonates plays a crucial role for their interactions with SBM. Molecular docking results clearly show that α-aminophosphonates with pyrimidine ring are having more number of hydrogen bonding interaction with amino acid residues of SBM than α-aminophosphonates with benzthiazolyl ring. Sequentially for thermal and structure stability of SBM, concentration of α-aminophosphonates plays an inexorable role and through these results it must be concluded that most of the α-aminophosphonates are stabilizing the SBM upto the 0. 1 mM concentration.

  • Experimental and molecular docking studies in understanding the biomolecular interactions between Stem Bromelain and imidazolium-based ionic liquids
    Journal of Molecular Liquids, 2020
    Co-Authors: Pannuru Kiran Kumar, Pannuru Venkatesu, Indrani Jha, Indra Bahadur, Anamika Sindhu, Eno E. Ebenso
    Abstract:

    Abstract The present study reports the influence of imidazolium-based ILs such as 1-butyl-3-methylimidazolium hydrogen sulphate [C4mi]+[HSO4]-, 1-butyl-3-methylimidazolium acetate [C4mi]+[CH3COO]- and 1-butyl-3-methylimidazolium nitrate [C4mi]+[NO3]- on the structural and thermal stability of cysteine proteinase enzyme-Stem Bromelain (BMN) by employing several biophysical techniques. This work is intended to study the action of various anions and concentration of ILs on the structural stability of BMN. The results reveal that all the ILs acted as stabilizers at lower concentrations such as 0.01 and 0.05 M. Moreover, we have also tried to unveil the type and different existing interactions between BMN and the cation and anions of ILs using molecular docking studies. The results show that the stability of BMN is dependent of the nature of ions of ILs and their concentration, the ILs are supposed to alter the properties of the protein by the mechanism of their interaction with the functional groups present on the surface of protein.

  • Can Stem Bromelain, a pineapple waste product, be used as a drug alternative? A mechanistic insight into protein–protein interactions
    New Journal of Chemistry, 2020
    Co-Authors: Samima Khatun, Anamika Sindhu, Pannuru Venkatesu
    Abstract:

    Stem Bromelain (SB) is a mixture of cysteine protease and non-protease components that is extracted from the pineapple plant (Ananas comosus). It has several therapeutic applications, such as antithrombotic, anti-inflammatory and anticancer. It also relieves osteoarthritis, diarrhea and various cardiovascular disorders. Bovine serum albumin (BSA), a blood plasma protein, is used as a model transport protein for studying protein folding and ligand binding mechanisms. We investigated the interactions between SB and BSA using UV-visible, fluorescence, isothermal titration calorimetry (ITC), circular dichroism (CD) and Fourier transform infrared (FTIR) techniques. The fluorescence results indicate that the fluorescence intensity of native BSA was quenched with increasing concentration of SB, with a blue shift in its wavelength maxima. The binding constant (Kb) was found to be on the order of 106, which is greater than that of protein–drug interactions; this reflects that SB can be used as an alternative to drugs in treating various diseases. The thermodynamic parameters obtained from isothermal titration calorimetry suggest that hydrophobic interactions play a major role in the association process, and it is an entropically driven process. The CD and FTIR spectroscopic results confirmed conformational alteration in BSA in the presence of SB, which was further substantiated by dynamic light scattering (DLS). The DLS results showed that the hydrodynamic diameter (dH) of BSA increases due to interaction with SB, resulting in unfolding of the native structure of BSA. This study is expected to provide important insight into the binding mechanism of SB with BSA, which may be useful in the pharmacology and clinical medicinal fields.

  • A biophysical strategy to examine the impact of newly synthesized polymerizable ammonium-based ionic liquids on the structural stability and proteolytic activity of Stem Bromelain
    International journal of biological macromolecules, 2019
    Co-Authors: Navin Kumar Mogha, Anamika Sindhu, Pannuru Venkatesu
    Abstract:

    Abstract The present manuscript reports the synthesis and characterization of polymerizable ammonium-based ionic liquids (ILs) and explores their influence on structural stability of Stem Bromelain (BMN) by using various biophysical techniques. Thermal fluorescence results showed that N-ethyl-2-(methylacryloyloxy)-N,N-dimethylethan-1-ammonium bromide (IL2C) (at lower concentration (0.1 mg/mL)) is found to be increasing the thermal stability of BMN which can be evident from the transition temperature (Tm) for BMN in IL2C (68.51 °C) is higher than BMN in buffer (66.24 °C). Whereas, N-(2-(methacryloyloxy)ethyl)-N,N-dimethylpropan-1-ammonium bromide (IL3C) and N-(2-(methacryloyloxy)ethyl)-N,N-dimethylpentan-1-ammonium bromide (IL5C) are maintaining the Tm values very near to Tm of pure BMN. Though, N-(2-(methacryloyloxy)ethyl)-N,N-dimethylhexan-1-ammonium bromide (IL6C) is found to be destabilizing IL as it significantly decreased the Tm value of BMN at all concentrations. Additionally, consequence of ILs on the proteolytic activity of BMN has also performed for IL2C up to 5 mg/mL while IL3C and IL5C at 0.1 mg/mL and 0.5 mg/mL are enhancing the proteolytic activity of BMN. Later, molecular docking studies are also performed with AutoDock vina and results showed that all ILs have different binding sites on BMN however IL6C is observed to be binding to the catalytic site of the BMN, it turns out to be the most destabilizing IL.

Sandeep Dave - One of the best experts on this subject based on the ideXlab platform.

  • Stem Bromelain induced macrophage apoptosis and activation curtail mycobacterium tuberculosis persistence
    The Journal of Infectious Diseases, 2012
    Co-Authors: Sahil Mahajan, Vemika Chandra, Sandeep Dave, Ravikanth Nanduri, Pawan Gupta
    Abstract:

    BACKGROUND Mycobacterium tuberculosis, the causative agent of tuberculosis, has a remarkable ability to usurp its host's innate immune response, killing millions of infected people annually. One approach to manage infection is prevention through the use of natural agents. In this regard, Stem Bromelain (SBM), a pharmacologically active member of the sulfhydryl proteolytic enzyme family, obtained from Ananas comosus and possessing a remarkable ability to induce the innate and acquired immune syStems, is important. METHODS We evaluated SBM's ability to induce apoptosis and free-radical generation in macrophages. We also studied antimycobacterial properties of SBM and its effect on foamy macrophages. RESULTS SBM treatment of peritoneal macrophages resulted in the upregulation of proapoptotic proteins and downregulation of antiapoptotic proteins. Additionally, SBM treatment activated macrophages, curtailed the levels of free glutathione, and augmented the production of hydrogen peroxide, superoxide anion, peroxynitrite, and nitric oxide. SBM cleaves CD36 and reduced the formation of foam cells, the hallmark of M. tuberculosis infection. These conditions created an environment for the increased clearance of M. tuberculosis. CONCLUSIONS Together these data provide a mechanism for antimycobacterial activity of SBM and provide important insights for the use of cysteine proteases as immunomodulatory agents.

  • Stem Bromelain–Induced Macrophage Apoptosis and Activation Curtail Mycobacterium tuberculosis Persistence
    The Journal of infectious diseases, 2012
    Co-Authors: Sahil Mahajan, Vemika Chandra, Sandeep Dave, Ravikanth Nanduri, Pawan Gupta
    Abstract:

    BACKGROUND Mycobacterium tuberculosis, the causative agent of tuberculosis, has a remarkable ability to usurp its host's innate immune response, killing millions of infected people annually. One approach to manage infection is prevention through the use of natural agents. In this regard, Stem Bromelain (SBM), a pharmacologically active member of the sulfhydryl proteolytic enzyme family, obtained from Ananas comosus and possessing a remarkable ability to induce the innate and acquired immune syStems, is important. METHODS We evaluated SBM's ability to induce apoptosis and free-radical generation in macrophages. We also studied antimycobacterial properties of SBM and its effect on foamy macrophages. RESULTS SBM treatment of peritoneal macrophages resulted in the upregulation of proapoptotic proteins and downregulation of antiapoptotic proteins. Additionally, SBM treatment activated macrophages, curtailed the levels of free glutathione, and augmented the production of hydrogen peroxide, superoxide anion, peroxynitrite, and nitric oxide. SBM cleaves CD36 and reduced the formation of foam cells, the hallmark of M. tuberculosis infection. These conditions created an environment for the increased clearance of M. tuberculosis. CONCLUSIONS Together these data provide a mechanism for antimycobacterial activity of SBM and provide important insights for the use of cysteine proteases as immunomodulatory agents.

  • Inhibition of adipogenesis and induction of apoptosis and lipolysis by Stem Bromelain in 3T3-L1 adipocytes.
    PloS one, 2012
    Co-Authors: Sandeep Dave, H. Kitdorlang Dkhar, Ravikanth Nanduri, Naval Jit Kaur, Ashwani Kumar, Pawan Gupta
    Abstract:

    The phytotherapeutic protein Stem Bromelain (SBM) is used as an anti-obesity alternative medicine. We show at the cellular level that SBM irreversibly inhibits 3T3-L1 adipocyte differentiation by reducing adipogenic gene expression and induces apoptosis and lipolysis in mature adipocytes. At the molecular level, SBM suppressed adipogenesis by downregulating C/EBPα and PPARγ independent of C/EBPβ gene expression. Moreover, mRNA levels of adipocyte fatty acid-binding protein (ap2), fatty acid synthase (FAS), lipoprotein lipase (LPL), CD36, and acetyl-CoA carboxylase (ACC) were also downregulated by SBM. Additionally, SBM reduced adiponectin expression and secretion. SBM's ability to repress PPARγ expression seems to Stem from its ability to inhibit Akt and augment the TNFα pathway. The Akt–TSC2–mTORC1 pathway has recently been described for PPARγ expression in adipocytes. In our experiments, TNFα upregulation compromised cell viability of mature adipocytes (via apoptosis) and induced lipolysis. Lipolytic response was evident by downregulation of anti-lipolytic genes perilipin, phosphodiestersae-3B (PDE3B), and GTP binding protein Giα1, as well as sustained expression of hormone sensitive lipase (HSL). These data indicate that SBM, together with all-trans retinoic-acid (atRA), may be a potent modulator of obesity by repressing the PPARγ-regulated adipogenesis pathway at all stages and by augmenting TNFα-induced lipolysis and apoptosis in mature adipocytes.

  • Trifluoroethanol stabilizes the molten globule state and induces non-amyloidic turbidity in Stem Bromelain near its isoelectric point
    International journal of biological macromolecules, 2011
    Co-Authors: Sandeep Dave, Vemika Chandra, Sahil Mahajan, Pawan Gupta
    Abstract:

    Stem Bromelain (SBM) is a therapeutic protein that has been studied for alkaline denaturation in the intestines, the principal site of its absorption. In this study, we investigated fluorinated alcohol 2,2,2-trifluoroethanol (TFE)-induced conformational changes in the specific/pre-molten globule (SMG) state of SBM observed at pH 10 by spectroscopic methods. Far-UV circular dichroism (CD) spectra showed that the protein retained its native-like secondary structure at TFE concentrations of up to 30% with a pronounced minimum at 222 nm, characteristic of a helix. However, addition of slightly higher TFE concentrations (≥40%) resulted in an ∼2.5-fold induction of this helical feature and a time-dependent increase in non-amyloidic turbidity as evidenced by turbidometric, Congo red-binding, and Thioflavin T (ThT)-binding studies. Near-UV CD spectra suggested a gradual but significant loss of tertiary structure at 10-30% TFE. Tryptophan studies showed blue-shifted fluorescence, although the number of accessible tryptophans remained the same up to 30% TFE. The SMG showed enhanced binding of the fluorescent probe 1-anilino-8-naphthalene sulfonic acid (ANS) up to 30% TFE, beyond which binding plateaued. Thermal and guanidine hydrochloride (GdnHCl) transition studies in the near-UV range indicated a single cooperative transition for the SMG state in the presence of 30% TFE, similar to that observed for native SBM at pH 7.0 (although with different T(m)s), unlike the SMG state. TFE (30%) appeared to induce native-like stability to the original SMG. These observations suggest a transformation of the SMG to a characteristic molten globule (MG) conformation at 30% TFE, possibly due to TFE-induced rearrangement of hydrophobic interactions at the protein's isoelectric point.

  • hexafluoroisopropanol induced helix sheet transition of Stem Bromelain correlation to function
    The International Journal of Biochemistry & Cell Biology, 2010
    Co-Authors: Sandeep Dave, Manvendra Pratap Singh, Garima Gupta, Vemika Chandra, Sahil Mahajan, Kitdorlang H Dkhar, Pawan Gupta
    Abstract:

    Stem Bromelain is a proteolytic phytoprotein with a variety of therapeutic effects. Understanding its structural properties could provide insight into the mechanisms underlying its clinical utility. Stem Bromelain was evaluated for its conformational and folding properties at the pH conditions it encounters when administered orally. It exists as a partially folded intermediate at pH 2.0. The conformational changes to this intermediate state were evaluated using fluorinated alcohols known to induce changes similar to those seen in vivo. Studies using circular dichroism, fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid and mass spectrometry indicate that treatment with 10-30% hexafluoroisopropanol induces the partially folded intermediate to adopt much of the native protein's secondary structure, but only a rudimentary tertiary structure, characteristic of the molten globule state. Addition of slightly higher concentrations of hexafluoroisopropanol caused transformation from an alpha-helix to a beta-sheet and induced formation of a compact nonnative structure. This nonnative form was more inhibitory of cell survival than either the native or the partially folded intermediate forms, as measured by enhanced suppression of proliferative cues (e.g., extracellular-signal-regulated kinase) and initiation of apoptotic events. The nonnative form also showed better antitumorigenic properties, as evaluated using an induced two-stage mouse skin papilloma model. In contrast, the nonnative state showed only a fraction of the proteolytic activity of the native form. This study demonstrates that hexafluoroisopropanol can induce a conformational change in Stem Bromelain to a form with potentially useful therapeutic properties different from those of the native protein.

Sahil Mahajan - One of the best experts on this subject based on the ideXlab platform.

  • Stem Bromelain induced macrophage apoptosis and activation curtail mycobacterium tuberculosis persistence
    The Journal of Infectious Diseases, 2012
    Co-Authors: Sahil Mahajan, Vemika Chandra, Sandeep Dave, Ravikanth Nanduri, Pawan Gupta
    Abstract:

    BACKGROUND Mycobacterium tuberculosis, the causative agent of tuberculosis, has a remarkable ability to usurp its host's innate immune response, killing millions of infected people annually. One approach to manage infection is prevention through the use of natural agents. In this regard, Stem Bromelain (SBM), a pharmacologically active member of the sulfhydryl proteolytic enzyme family, obtained from Ananas comosus and possessing a remarkable ability to induce the innate and acquired immune syStems, is important. METHODS We evaluated SBM's ability to induce apoptosis and free-radical generation in macrophages. We also studied antimycobacterial properties of SBM and its effect on foamy macrophages. RESULTS SBM treatment of peritoneal macrophages resulted in the upregulation of proapoptotic proteins and downregulation of antiapoptotic proteins. Additionally, SBM treatment activated macrophages, curtailed the levels of free glutathione, and augmented the production of hydrogen peroxide, superoxide anion, peroxynitrite, and nitric oxide. SBM cleaves CD36 and reduced the formation of foam cells, the hallmark of M. tuberculosis infection. These conditions created an environment for the increased clearance of M. tuberculosis. CONCLUSIONS Together these data provide a mechanism for antimycobacterial activity of SBM and provide important insights for the use of cysteine proteases as immunomodulatory agents.

  • Stem Bromelain–Induced Macrophage Apoptosis and Activation Curtail Mycobacterium tuberculosis Persistence
    The Journal of infectious diseases, 2012
    Co-Authors: Sahil Mahajan, Vemika Chandra, Sandeep Dave, Ravikanth Nanduri, Pawan Gupta
    Abstract:

    BACKGROUND Mycobacterium tuberculosis, the causative agent of tuberculosis, has a remarkable ability to usurp its host's innate immune response, killing millions of infected people annually. One approach to manage infection is prevention through the use of natural agents. In this regard, Stem Bromelain (SBM), a pharmacologically active member of the sulfhydryl proteolytic enzyme family, obtained from Ananas comosus and possessing a remarkable ability to induce the innate and acquired immune syStems, is important. METHODS We evaluated SBM's ability to induce apoptosis and free-radical generation in macrophages. We also studied antimycobacterial properties of SBM and its effect on foamy macrophages. RESULTS SBM treatment of peritoneal macrophages resulted in the upregulation of proapoptotic proteins and downregulation of antiapoptotic proteins. Additionally, SBM treatment activated macrophages, curtailed the levels of free glutathione, and augmented the production of hydrogen peroxide, superoxide anion, peroxynitrite, and nitric oxide. SBM cleaves CD36 and reduced the formation of foam cells, the hallmark of M. tuberculosis infection. These conditions created an environment for the increased clearance of M. tuberculosis. CONCLUSIONS Together these data provide a mechanism for antimycobacterial activity of SBM and provide important insights for the use of cysteine proteases as immunomodulatory agents.

  • Trifluoroethanol stabilizes the molten globule state and induces non-amyloidic turbidity in Stem Bromelain near its isoelectric point
    International journal of biological macromolecules, 2011
    Co-Authors: Sandeep Dave, Vemika Chandra, Sahil Mahajan, Pawan Gupta
    Abstract:

    Stem Bromelain (SBM) is a therapeutic protein that has been studied for alkaline denaturation in the intestines, the principal site of its absorption. In this study, we investigated fluorinated alcohol 2,2,2-trifluoroethanol (TFE)-induced conformational changes in the specific/pre-molten globule (SMG) state of SBM observed at pH 10 by spectroscopic methods. Far-UV circular dichroism (CD) spectra showed that the protein retained its native-like secondary structure at TFE concentrations of up to 30% with a pronounced minimum at 222 nm, characteristic of a helix. However, addition of slightly higher TFE concentrations (≥40%) resulted in an ∼2.5-fold induction of this helical feature and a time-dependent increase in non-amyloidic turbidity as evidenced by turbidometric, Congo red-binding, and Thioflavin T (ThT)-binding studies. Near-UV CD spectra suggested a gradual but significant loss of tertiary structure at 10-30% TFE. Tryptophan studies showed blue-shifted fluorescence, although the number of accessible tryptophans remained the same up to 30% TFE. The SMG showed enhanced binding of the fluorescent probe 1-anilino-8-naphthalene sulfonic acid (ANS) up to 30% TFE, beyond which binding plateaued. Thermal and guanidine hydrochloride (GdnHCl) transition studies in the near-UV range indicated a single cooperative transition for the SMG state in the presence of 30% TFE, similar to that observed for native SBM at pH 7.0 (although with different T(m)s), unlike the SMG state. TFE (30%) appeared to induce native-like stability to the original SMG. These observations suggest a transformation of the SMG to a characteristic molten globule (MG) conformation at 30% TFE, possibly due to TFE-induced rearrangement of hydrophobic interactions at the protein's isoelectric point.

  • hexafluoroisopropanol induced helix sheet transition of Stem Bromelain correlation to function
    The International Journal of Biochemistry & Cell Biology, 2010
    Co-Authors: Sandeep Dave, Manvendra Pratap Singh, Garima Gupta, Vemika Chandra, Sahil Mahajan, Kitdorlang H Dkhar, Pawan Gupta
    Abstract:

    Stem Bromelain is a proteolytic phytoprotein with a variety of therapeutic effects. Understanding its structural properties could provide insight into the mechanisms underlying its clinical utility. Stem Bromelain was evaluated for its conformational and folding properties at the pH conditions it encounters when administered orally. It exists as a partially folded intermediate at pH 2.0. The conformational changes to this intermediate state were evaluated using fluorinated alcohols known to induce changes similar to those seen in vivo. Studies using circular dichroism, fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid and mass spectrometry indicate that treatment with 10-30% hexafluoroisopropanol induces the partially folded intermediate to adopt much of the native protein's secondary structure, but only a rudimentary tertiary structure, characteristic of the molten globule state. Addition of slightly higher concentrations of hexafluoroisopropanol caused transformation from an alpha-helix to a beta-sheet and induced formation of a compact nonnative structure. This nonnative form was more inhibitory of cell survival than either the native or the partially folded intermediate forms, as measured by enhanced suppression of proliferative cues (e.g., extracellular-signal-regulated kinase) and initiation of apoptotic events. The nonnative form also showed better antitumorigenic properties, as evaluated using an induced two-stage mouse skin papilloma model. In contrast, the nonnative state showed only a fraction of the proteolytic activity of the native form. This study demonstrates that hexafluoroisopropanol can induce a conformational change in Stem Bromelain to a form with potentially useful therapeutic properties different from those of the native protein.

  • Specific molten globule conformation of Stem Bromelain at alkaline pH.
    Archives of biochemistry and biophysics, 2010
    Co-Authors: Sandeep Dave, H. Kitdorlang Dkhar, Vemika Chandra, Sahil Mahajan, Sambhavi, Pawan Gupta
    Abstract:

    Stem Bromelain (SBM), a therapeutic protein, is rapidly absorbed across the gut epithelium. Because SBM encounters an alkaline pH at its principal site of absorption, we investigated the alkaline-induced denaturation of SBM. From pH 7 to 10, the protein's secondary structure remained the same, although a slight loss of tertiary structure was observed. Above pH 10, there was a significant and irreversible loss of secondary and tertiary structure. At pH 10, SBM showed enhanced tryptophan fluorescence, however, the number of accessible tryptophans remained the same. The thermodynamics of temperature transition at pH 7 and 10 were strikingly different, with the former showing a two-phase transition endotherm, and the latter a broad non-two-state transition. At pH 10, SBM showed a significant increase in 8-anilino-1-naphthalene-sulfonate binding relative to the native state, suggestive of a specific molten globule (SMG) state. These studies suggest a distinct conformational rearrangement in SBM, at the protein's isoelectric point.