C M Gupta

Central Drug Research Institute, Lucknow, Uttar Pradesh, India

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Publications (59)217.56 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Actin related proteins are ubiquitous actin-like proteins that show high similarity with actin in terms of their amino acid sequence and three-dimensional structure. However, in lower eukaryotes, such as trypanosomatids, their functions have not yet been explored. Here, we show that a novel actin-related protein (ORF LmjF.13.0950) is localized mainly in the Leishmania mitochondrion. We further reveal that depletion of the intracellular levels of this protein leads to an appreciable decrease in the mitochondrial membrane potential as well as in the ATP production, which appears to be accompanied with impairment in the flagellum assembly and motility. Additionally, we report that the mutant so generated fail to survive inside the mouse peritoneal macrophages. These abnormalities are, however, reversed by the episomal gene complementation. Our results, for the first time indicate that apart from their classical roles in the cytoplasm and nucleus, actin-related proteins may also regulate the mitochondrial function, and in case of Leishmania donovani they may also serve as the essential factor for their survival in the host cells.
    Molecular Microbiology 12/2013; · 4.96 Impact Factor
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    ABSTRACT: Leishmania donovani coronin CRN12 is an actin-binding protein which consists of two domains: an N-terminal WD repeat domain and a C-terminal coiled-coil domain. The coiled-coil domain is 53 residues in length. Helix-helix interactions in general and coiled coils in particular are ubiquitous in the structure of proteins and play a significant role in the association among proteins, including supramolecular assemblies and transmembrane receptors that mediate cellular signalling, transport and actin dynamics. The L. donovani coronin CRN12 coiled-coil domain (5.8 kDa) was cloned, overexpressed, purified to homogeneity and the N-terminal 6×His tag was successfully removed by thrombin cleavage. Crystals of recombinant L. donovani coronin CRN12 coiled-coil domain were grown by vapour diffusion using a hanging-drop setup. Diffraction-quality crystals were obtained and data extending to 2.46 Å resolution were collected at 100 K on BM14, ESRF, Grenoble, France. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 118.0, b = 50.6, c = 46.0 Å, β = 111.0°. Matthews coefficient (VM) calculations suggested the presence of 4-6 molecules in the asymmetric unit, corresponding to a solvent content of ∼33-55%, and are consistent with self-rotation function calculations.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 05/2013; 69(Pt 5):535-9. · 0.55 Impact Factor
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    ABSTRACT: Leishmania, like other eukaryotes, contains large amounts of actin and a number of actin-related and actin binding proteins. Our earlier studies have shown that deletion of the gene corresponding to Leishmania actin-depolymerizing protein (ADF/cofilin) adversely affects flagellum assembly, intracellular trafficking, and cell division. To further analyze this, we have now created ADF/cofilin site-specific point mutants and then examined (i) the actin-depolymerizing, G-actin binding, and actin-bound nucleotide exchange activities of the mutant proteins and (ii) the effect of overexpression of these proteins in wild-type cells. Here we show that S4D mutant protein failed to depolymerize F-actin but weakly bound G-actin and inhibited the exchange of G-actin-bound nucleotide. We further observed that overexpression of this protein impaired flagellum assembly and consequently cell motility by severely impairing the assembly of the paraflagellar rod, without significantly affecting vesicular trafficking or cell growth. Taken together, these results indicate that dynamic actin is essentially required in assembly of the eukaryotic flagellum.
    Eukaryotic Cell 04/2012; 11(6):752-60. · 3.59 Impact Factor
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    ABSTRACT: Leishmania donovani ADF/cofilin (LdCof) is a novel member of ADF/cofilin family. LdCof depolymerizes, but does not co-sediment with, rabbit muscle actin filaments. Its F-actin depolymerizing activity is pH independent. Further, it possesses weak F-actin severing activity. In order to better understand its characteristic properties, we have determined the solution NMR structure of LdCof and have analyzed protein backbone dynamics from (15)N-relaxation measurements. The structure of LdCof possesses a conserved ADF/cofilin fold with a central mixed β-sheet consisting of six β-strands which is surrounded by five α-helices. LdCof structure has conserved G/F-actin binding site which includes the characteristic long kinked α-helix (α3). LdCof binds to rabbit muscle ADP-G-actin with 1:1 stoichiometry (K(d)∼0.2μM). The F-actin binding site is not well formed and analysis of (15)N-relaxation data shows that residues in the β4-β5 loop region and C-terminal are relatively flexible, which seems to be a determinant for the low F-actin severing activity of LdCof.
    Journal of Structural Biology 12/2010; 172(3):219-24. · 3.36 Impact Factor
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    ABSTRACT: Actin-based myosin motors have a pivotal role in intracellular trafficking in eukaryotic cells. The parasitic protozoan organism Leishmania expresses a novel class of myosin, myosin XXI (Myo21), which is preferentially localized at the proximal region of the flagellum. However, its function in this organism remains largely unknown. Here, we show that Myo21 interacts with actin, and its expression is dependent of the growth stage. We further reveal that depletion of Myo21 levels results in impairment of the flagellar assembly and intracellular trafficking. These defects are, however, reversed by episomal complementation. Additionally, it is shown that deletion of the Myo21 gene leads to generation of ploidy, suggesting an essential role of Myo21 in survival of Leishmania cells. Together, these results indicate that actin-dependent trafficking activity of Myo21 is essentially required during assembly of the Leishmania flagellum.
    Journal of Cell Science 06/2010; 123(Pt 12):2035-44. · 5.88 Impact Factor
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    ABSTRACT: ADF/cofilin is an actin-dynamics-regulating protein that is required for several actin-based cellular processes such as cell motility and cytokinesis. A homologue of this protein has recently been identified in the protozoan parasite Leishmania, which has been shown to be essentially required in flagellum assembly and cell motility. However, the role of this protein in cytokinesis remains largely unknown. We show here that deletion of the gene encoding ADF/cofilin in these organisms results in several aberrations in the process of cell division. These aberrations include delay in basal body and kinetoplast separation, cleavage furrow progression and flagellar pocket division. In addition to these changes, the intracellular trafficking and actin dynamics are also adversely affected. All these abnormalities are, however, reversed by episomal complementation. Together, these results indicate that actin dynamics regulates early events in Leishmania cell division.
    Journal of Cell Science 06/2010; 123(Pt 11):1894-901. · 5.88 Impact Factor
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    ABSTRACT: Leishmania actin (LdACT) is an unconventional form of eukaryotic actin in that it markedly differs from other actins in terms of its filament forming as well as toxin and DNase-1-binding properties. Besides being present in the cytoplasm, cortical regions, flagellum and nucleus, it is also present in the kinetoplast where it appears to associate with the kinetoplast DNA (kDNA). However, nothing is known about its role in this organelle. Here, we show that LdACT is indeed associated with the kDNA disc in Leishmania kinetoplast, and under in vitro conditions, it specifically binds DNA primarily through electrostatic interactions involving its unique DNase-1-binding region and the DNA major groove. We further reveal that this protein exhibits DNA-nicking activity which requires its polymeric state as well as ATP hydrolysis and through this activity it converts catenated kDNA minicircles into open form. In addition, we show that LdACT specifically binds bacterial type II topoisomerase and inhibits its decatenation activity. Together, these results strongly indicate that LdACT could play a critical role in kDNA remodeling.
    Nucleic Acids Research 02/2010; 38(10):3308-17. · 8.81 Impact Factor
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    ABSTRACT: Compartmentalization of glycolytic enzymes in glycosomes is vital in trypanosomatid parasites. Retention of these enzymes in the cytosol induces sugar toxicity and accumulation of intermediate metabolites, notably the hexokinase product glucose-6-phosphate. However, the role of hexokinase in sugar mediated toxicity remains unexplored. We have generated Leishmania donovani transfectants expressing a catalytically active cytosolic mutant of hexokinase. In the presence of glucose, these transfectants exhibited toxicity during log and stationary phases of growth. These results suggest that targeting of hexokinase to the glycosome is required to prevent uncontrolled and cytotoxic glucose phosphorylation in L. donovani parasites.
    Molecular and Biochemical Parasitology 11/2009; 170(1):41-4. · 2.73 Impact Factor
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    ABSTRACT: In general, coronins play an important role in actin-based processes, and are expressed in a variety of eukaryotic cells, including Leishmania. Here, we show that Leishmania coronin preferentially distributes to the distal tip during cytokinesis, and interacts with microtubules through a microtubule-based motor, kinesin K39. We further show that reduction in coronin levels by 40-50% in heterozygous coronin mutants results in generation of bipolar cells (25-30%), specifically in the log phase, owing to unregulated growth of the corset microtubules. Further analysis of bipolar cells revealed that the main cause of generation of bipolar cell morphology is the intrusion of the persistently growing corset microtubules into the other daughter cell corset from the opposite direction. This defect in cytokinesis, however, disappears upon episomal gene complementation. Additionally, our attempts to prepare homozygous mutants were unsuccessful, as only the aneuploid cells survive the selection process. These results indicate that coronin regulates microtubule remodeling during Leishmania cytokinesis and is essentially required for survival of these parasites in culture.
    Journal of Cell Science 05/2009; 122(Pt 10):1691-9. · 5.88 Impact Factor
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    ABSTRACT: Leishmania major genome analysis revealed the presence of putative genes corresponding to two myosins, which have been designated to class IB and a novel class, class XXI, specifically present in kinetoplastids. To characterize these myosin homologs in Leishmania, we have cloned and over-expressed the full-length myosin XXI gene and variable region of myosin IB gene in bacteria, purified the corresponding proteins, and then used the affinity purified anti-sera to analyze the expression and intracellular distribution of these proteins. Whereas myosin XXI was expressed in both the promastigote and amastigote stages, no expression of myosin IB could be detected in any of the two stages of these parasites. Further, myosin XXI expression was more predominant in the promastigote stage where it was preferentially localized in the proximal region of the flagellum. The observed flagellar localization was not dependent on the myosin head region or actin but was exclusively determined by the myosin tail region, as judged by over-expressing GFP conjugates of full-length myosin XXI, its head domain and its tail domain separately in Leishmania. Furthermore, immunofluorescence and immuno-gold electron microscopy analyses revealed that this protein was partly associated with paraflagellar rod proteins but not with tubulins in the flagellar axoneme. Our results, for the first time, report the expression and detailed analysis of cellular localization of a novel class of myosin, myosin XXI in trypanosomatids.
    Molecular and Biochemical Parasitology 01/2009; 164(2):105-10. · 2.73 Impact Factor
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    ABSTRACT: ADF/cofilins are ubiquitous actin dynamics-regulating proteins that have been mainly implicated in actin-based cell motility. Trypanosomatids, e.g. Leishmania and Trypanosoma, which mediate their motility through flagellum, also contain a putative ADF/cofilin homologue, but its role in flagellar motility remains largely unexplored. We have investigated the role of this protein in assembly and motility of the Leishmania flagellum after knocking out the ADF/cofilin gene by targeted gene replacement. The resultant mutants were completely immotile, short and stumpy, and had reduced flagellar length and severely impaired beat. In addition, the assembly of the paraflagellar rod was lost, vesicle-like structures were seen throughout the length of the flagellum and the state and distribution of actin were altered. However, episomal complementation of the gene restored normal morphology and flagellar function. These results for the first time indicate that the actin dynamics-regulating protein ADF/cofilin plays a critical role in assembly and motility of the eukaryotic flagellum.
    Molecular Microbiology 10/2008; 70(4):837-52. · 4.96 Impact Factor
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    ABSTRACT: Leishmania actin was cloned, overexpressed in baculovirus-insect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg2+ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg2+ alone, (ii) it polymerized in a restricted range of pH 7.0-8.5, (iii) its critical concentration for polymerization was found to be 3-4-fold lower than of muscle actin, (iv) it predominantly formed bundles rather than single filaments at pH 8.0, (v) it displayed considerably higher ATPase activity during polymerization, (vi) it did not inhibit DNase-I activity, and (vii) it did not bind the F-actin-binding toxin phalloidin or the actin polymerization disrupting agent Latrunculin B. Computational and molecular modeling studies revealed that the observed unconventional behavior of Leishmania actin is related to the diverged amino acid stretches in its sequence, which may lead to changes in the overall charge distribution on its solvent-exposed surface, ATP binding cleft, Mg2+ binding sites, and the hydrophobic loop that is involved in monomer-monomer interactions. Phylogenetically, it is related to ciliate actins, but to the best of our knowledge, no other actin with such unconventional properties has been reported to date. It is therefore suggested that actin in Leishmania may serve as a novel target for design of new antileishmanial drugs.
    Journal of Biological Chemistry 07/2008; 283(33):22760-73. · 4.65 Impact Factor
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    Molecular and Biochemical Parasitology 07/2007; 153(2):216-9. · 2.73 Impact Factor
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    ABSTRACT: The presence of actin in Leishmania has recently been demonstrated, but the functional form of this protein (filamentous actin) has not yet been identified. We report here that the putative coronin homologue identified in the Leishmania genome is invariably associated with the filament-like structures of actin in Leishmania promastigotes. The occurrence of filamentous structures is significantly increased upon overexpression of Leishmania coronin as its GFP fusion product in Leishmania cells. However, expression of Leishmania actin or coronin alone in mammalian cells does not result in formation of any filament-like structures of Leishmania actin or association of Leishmania coronin with mammalian filamentous actin, but coexpression of both the proteins in these cells leads to formation of filamentous structures containing Leishmania actin and coronin. The high specificity of Leishmania coronin for Leishmania actin could be attributed to its unique structure as it differs from other coronins not only in the unique region but also in the actin-binding site and leucine zipper motif. These results taken together indicate that Leishmania contains a novel form of coronin which colocalizes with actin in filament-like structures in these cells.
    Molecular and Biochemical Parasitology 11/2005; 143(2):152-64. · 2.73 Impact Factor
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    M Owais, C M Gupta
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    ABSTRACT: Successful homing of drugs to the desired biological compartment of the host usually depends on the intrinsic properties of the drug molecules. However, it can always be manipulated by appropriate designing of the carrier/delivery system, as little can be done to influence the target and its surroundings. Various carrier systems have emerged to deliver drugs to macrophages, albeit the efficacy, reliability and selectivity of these carriers are still in question. To date, the most extensively studied carriers are liposomes and microspheres. In fact, physicochemical properties of these carriers can alter their efficacy and specificity to a great extent. These properties include hydrophilicity, surface charge, composition, concentration, and presence of various target specific ligands on their surface. Incidentally, the particulate nature of these vehicles may facilitate passive homing of the entrapped drug molecules to the macrophages, which may harbour many of the important pathogens in their intracellular compartments, such as Mycobacterium sps, Leishmania and dengue virus etc., belonging to three different major classes of microbes. Moreover, macrophages upon interaction with particulate drug delivery vehicles may act as secondary drug depot, thus helping in localized delivery of the drug at the infected site. In the present article, a comprehensive review of literature is presented on the suitability of some lipid-based and polymeric materials as vehicles in delivery of drugs to macrophages in parasitic infections.
    Current Drug Delivery 11/2005; 2(4):311-8.
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    C M Gupta, W Haq
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    ABSTRACT: Tuftsin is a tetrapeptide (Thr-Lys-Pro-Arg) that specifically binds monocytes, macrophages, and polymorphonuclear leukocytes and potentiates their natural killer activity against tumors and pathogens. The antimicrobial activity of this peptide is significantly increased by attaching at the C-terminus a fatty acyl residue through the ethylenediamine spacer arm. This activity is further augmented by incorporating the modified tuftsin in the liposomes. The tuftsin-bearing liposomes not only enhance the host's resistance against a variety of infections but also serve as useful vehicles for the site-specific delivery of drugs in a variety of macrophage-based infections, such as tuberculosis and leishmaniasis.
    Methods in Enzymology 02/2005; 391:291-304. · 2.00 Impact Factor
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    ABSTRACT: To study the occurrence and subcellular distribution of actin in trypanosomatid parasites, we have cloned and overexpressed Leishmania donovani actin gene in bacteria, purified the protein, and employed the affinity purified rabbit polyclonal anti-recombinant actin antibodies as a probe to study the organisation and subcellular distribution of actin in Leishmania cells. The Leishmania actin did not cross react with antimammalian actin antibodies but was readily recognized by the anti-Leishmania actin antibodies in both the promastigote and amastigote forms of the parasite. About 10(6) copies per cell of this protein (M(r) 42.05 kDa) were present in the Leishmania promastigote. Unlike other eukaryotic actins, the oligomeric forms of Leishmania actin were not stained by phalloidin nor were dissociated by actin filament-disrupting agents, like Latrunculin B and Cytochalasin D. Analysis of the primary structure of this protein revealed that these unusual characteristics may be related to the presence of highly diverged amino acids in the DNase I-binding loop (amino acids 40-50) and the hydrophobic plug (amino acids 262-272) regions of Leishmania actin. The subcellular distribution of actin was studied in the Leishmania promastigotes by employing immunoelectron and immunofluorescence microscopies. This protein was present not only in the flagella, flagellar pocket, nucleus and the kinetoplast but it was also localized on the nuclear, vacuolar and cytoplasmic face of the plasma membranes. Further, the plasma membrane-associated actin was colocalised with subpellicular microtubules, while most of the actin present in the kinetoplast colocalised with the k-DNA network. These results clearly indicate that Leishmania contains a novel form of actin which may structurally and functionally differ from other eukaryotic actins. The functional significance of these observations is discussed.
    Molecular and Biochemical Parasitology 04/2004; 134(1):105-14. · 2.73 Impact Factor
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    ABSTRACT: In the present study, we evaluated the potential of an immunomodulator tuftsin in increasing the efficacy of liposomised diethylcarbamazine (DEC) against experimental filarial infection of Brugia malayi. The liposomised form of DEC, when used at sub-optimal dose of 25 mg/kg body weight, successfully eliminated filarial parasite from systemic circulation in animals inflicted with B. malayi infection. However, the formulation was effective upto 60 days post infection only, followed by recurrence of the infection. In contrast, the co-administration of liposomal formulation of DEC along with an immunomodulator tuftsin was found to be competent enough to suppress microfilarial stage of parasite till 90 days post treatment. Interestingly, tuftsin bearing DEC liposomes were found to be effective against adult parasite as well.
    Journal of Drug Targeting 05/2003; 11(4):247-51. · 2.77 Impact Factor
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    ABSTRACT: Chemotherapeutic efficacy of the amphotericin B (Amp B), which is the drug of choice for treatment of the leishmanial infections (kala-azar) that become resistant to the conventional chemotherapy using antimonials, has been examined in the Leishmania donovani infected hamsters after encapsulating the drug in tuftsin-free as well as tuftsin-bearing liposomes. The activity was significantly increased (p < 0.05) by delivering Amp B in tuftsin-free liposomes. This antileishmanial effect of the liposomized Amp B was further increased (p < 0.05) by grafting the natural macrophage-activator tetrapeptide, tuftsin (Thr-Lys-Pro-Arg), on the liposome's surface. This could possibly be attributed to both the enhanced drug tolerance after liposomization as well as to the increased uptake of tuftsin-bearing Amp B-laden liposomes by the macrophages. In addition to the increased efficacy, encapsulation of Amp B in the tuftsin-bearing liposomes also enhanced the drug accessibility to areas (e.g. bone marrow) that are otherwise inaccessible to the free drug. These results further demonstrate the usefulness of tuftsin-bearing liposomes as drug vehicles in treatment of the macrophage-based infections that have been reviewed recently (Agrawal, A.K. and Gupta, C.M. (2000). Tuftsin-bearing liposomes in treatment of macrophage-based infections, Adv. Drug Deliv. Rev., 41, 135-146).
    Journal of Drug Targeting 02/2002; 10(1):41-5. · 2.77 Impact Factor
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    A K Agrawal, A Puri, W Haq, C M Gupta
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    ABSTRACT: The immunostimulant activity of non-pyrogenic, sugar-free immunomodulator lipopeptide, Ala-D-Glu(Gly-Lys-CO.C11H23)-NH2 (comp. no 84/201), and its liposomized formulation has been studied. Liposomization of this lipopeptide significantly enhanced its antigen specific as well as nonspecific immune responses, as compared to the free lipopeptide. The liposomized formulation of lipopeptide significantly stimulated both the antibody and delayed-type hypersensitivity responses in Balb/c mice, and also enhanced nonspecifically the macrophage migration index, phagocytic activity and incorporation of 14C glucosamine in peritoneal macrophages of the mice that received pretreatment with this preparation. Further, the mice that received pretreatment with the liposomized preparation strongly resisted lethal P. berghei infection and consequently survived for longer period of times. These results indicate that liposomization of the compound no 84/201 significantly improves its ability to enhance not only antigen-specific immune response but also the nonspecific host's resistance against infections.
    Journal of Drug Targeting 02/2001; 9(4):231-9. · 2.77 Impact Factor

Publication Stats

742 Citations
217.56 Total Impact Points

Institutions

  • 1981–2012
    • Central Drug Research Institute
      • • Molecular and Structural Biology Division (CDRI)
      • • Biochemistry Division (CDRI)
      • • Parasitology Division (CDRI)
      Lucknow, Uttar Pradesh, India
  • 2000–2005
    • Aligarh Muslim University
      • Interdisciplinary Biotechnology Unit
      Alīgarh, Uttar Pradesh, India
    • GW Pharmaceuticals plc
      New Sarum, England, United Kingdom
  • 1993–1995
    • Institute of Microbial Technology
      Chandigarh, Chandīgarh, India
  • 1990
    • King George's Medical University
      Lakhnau, Uttar Pradesh, India
  • 1979–1981
    • Massachusetts Institute of Technology
      • Department of Biology
      Cambridge, Massachusetts, United States