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1.
iScience ; 27(4): 109467, 2024 Apr 19.
Article in English | MEDLINE | ID: mdl-38558939

ABSTRACT

Alba domain proteins, owing to their functional plasticity, play a significant role in organisms. Here, we report an intrinsic DNase activity of PfAlba6 from Plasmodium falciparum, an etiological agent responsible for human malignant malaria. We identified that tyrosine28 plays a critical role in the Mg2+ driven 5'-3' DNase activity of PfAlba6. PfAlba6 cleaves both dsDNA as well as ssDNA. We also characterized PfAlba6-DNA interaction and observed concentration-dependent oligomerization in the presence of DNA, which is evident from size exclusion chromatography and single molecule AFM-imaging. PfAlba6 mRNA expression level is up-regulated several folds following heat stress and treatment with artemisinin, indicating a possible role in stress response. PfAlba6 has no human orthologs and is expressed in all intra-erythrocytic stages; thus, this protein can potentially be a new anti-malarial drug target.

2.
PLoS One ; 19(2): e0292479, 2024.
Article in English | MEDLINE | ID: mdl-38349923

ABSTRACT

Recombinase enzymes are extremely efficient at integrating very large DNA fragments into target genomes. However, intrinsic sequence specificities curtail their use to DNA sequences with sufficient homology to endogenous target motifs. Extensive engineering is therefore required to broaden applicability and robustness. Here, we describe the directed evolution of novel lambda integrase variants capable of editing exogenous target sequences identified in the diatom Phaeodactylum tricornutum and the algae Nannochloropsis oceanica. These microorganisms hold great promise as conduits for green biomanufacturing and carbon sequestration. The evolved enzyme variants show >1000-fold switch in specificity towards the non-natural target sites when assayed in vitro. A single-copy target motif in the human genome with homology to the Nannochloropsis oceanica site can also be efficiently targeted using an engineered integrase, both in vitro and in human cells. The developed integrase variants represent useful additions to the DNA editing toolbox, with particular application for targeted genomic insertion of large DNA cargos.


Subject(s)
Diatoms , Stramenopiles , Humans , Integrases/genetics , Genome, Human/genetics , DNA , Genomics , Diatoms/genetics , Stramenopiles/genetics , Gene Editing
3.
Front Bioeng Biotechnol ; 11: 1198465, 2023.
Article in English | MEDLINE | ID: mdl-37425360

ABSTRACT

Reliable cell-based platforms to test and/or produce biologics in a sustainable manner are important for the biotech industry. Utilizing enhanced λ integrase, a sequence-specific DNA recombinase, we developed a novel transgenesis platform involving a fully characterized single genomic locus as an artificial landing pad for transgene insertion in human Expi293F cells. Importantly, transgene instability and variation in expression were not observed in the absence of selection pressure, thus enabling reliable long-term biotherapeutics testing or production. The artificial landing pad for λ integrase can be targeted with multi-transgene constructs and offers future modularity involving additional genome manipulation tools to generate sequential or nearly seamless insertions. We demonstrated broad utility with expression constructs for anti PD-1 monoclonal antibodies and showed that the orientation of heavy and light chain transcription units profoundly affected antibody expression levels. In addition, we demonstrated encapsulation of our PD-1 platform cells into bio-compatible mini-bioreactors and the continued secretion of antibodies, thus providing a basis for future cell-based applications for more effective and affordable therapies.

4.
Cell Rep ; 42(4): 112292, 2023 04 25.
Article in English | MEDLINE | ID: mdl-36947546

ABSTRACT

Plasmodium falciparum Alba domain-containing protein Alba3 (PfAlba3) is ubiquitously expressed in intra-erythrocytic stages of Plasmodium falciparum, but the function of this protein is not yet established. Here, we report an apurinic/apyrimidinic site-driven intrinsic nuclease activity of PfAlba3 assisted by divalent metal ions. Surface plasmon resonance and atomic force microscopy confirm sequence non-specific DNA binding by PfAlba3. Upon binding, PfAlba3 cleaves double-stranded DNA (dsDNA) hydrolytically. Mutational studies coupled with mass spectrometric analysis indicate that K23 is the essential residue in modulating the binding to DNA through acetylation-deacetylation. We further demonstrate that PfSir2a interacts and deacetylates K23-acetylated PfAlba3 in favoring DNA binding. Hence, K23 serves as a putative molecular switch regulating the nuclease activity of PfAlba3. Thus, the nuclease activity of PfAlba3, along with its apurinic/apyrimidinic (AP) endonuclease feature identified in this study, indicates a role of PfAlba3 in DNA-damage response that may have a far-reaching consequence in Plasmodium pathogenicity.


Subject(s)
DNA-(Apurinic or Apyrimidinic Site) Lyase , Plasmodium falciparum , Plasmodium falciparum/genetics , Protein Binding , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , DNA/metabolism , DNA Repair
5.
Br J Pharmacol ; 180(18): 2317-2340, 2023 09.
Article in English | MEDLINE | ID: mdl-36914615

ABSTRACT

BACKGROUND AND PURPOSE: Mitochondrial oxidative stress, inflammation and apoptosis primarily underlie gastric mucosal injury caused by the widely used non-steroidal anti-inflammatory drugs (NSAIDs). Alternative gastroprotective strategies are therefore needed. Sirtuin-3 pivotally maintains mitochondrial structural integrity and metabolism while preventing oxidative stress; however, its relevance to gastric injury was never explored. Here, we have investigated whether and how sirtuin-3 stimulation by the phytochemical, honokiol, could rescue NSAID-induced gastric injury. EXPERIMENTAL APPROACH: Gastric injury in rats induced by indomethacin was used to assess the effects of honokiol. Next-generation sequencing-based transcriptomics followed by functional validation identified the gastroprotective function of sirtuin-3. Flow cytometry, immunoblotting, qRT-PCR and immunohistochemistry were used measure effects on oxidative stress, mitochondrial dynamics, electron transport chain function, and markers of inflammation and apoptosis. Sirtuin-3 deacetylase activity was also estimated and gastric luminal pH was measured. KEY RESULTS: Indomethacin down-regulated sirtuin-3 to induce oxidative stress, mitochondrial hyperacetylation, 8-oxoguanine DNA glycosylase 1 depletion, mitochondrial DNA damage, respiratory chain defect and mitochondrial fragmentation leading to severe mucosal injury. Indomethacin dose-dependently inhibited sirtuin-3 deacetylase activity. Honokiol prevented mitochondrial oxidative damage and inflammatory tissue injury by attenuating indomethacin-induced depletion of both sirtuin-3 and its transcriptional regulators PGC1α and ERRα. Honokiol also accelerated gastric wound healing but did not alter gastric acid secretion, unlike lansoprazole. CONCLUSIONS AND IMPLICATIONS: Sirtuin-3 stimulation by honokiol prevented and reversed NSAID-induced gastric injury through maintaining mitochondrial integrity. Honokiol did not affect gastric acid secretion. Sirtuin-3 stimulation by honokiol may be utilized as a mitochondria-based, acid-independent novel gastroprotective strategy against NSAIDs.


Subject(s)
Sirtuin 3 , Rats , Animals , Sirtuin 3/metabolism , Rats, Sprague-Dawley , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Indomethacin/toxicity , Gastric Mucosa/metabolism , Apoptosis , Inflammation/metabolism
6.
Biochim Biophys Acta Gen Subj ; 1864(10): 129656, 2020 10.
Article in English | MEDLINE | ID: mdl-32512169

ABSTRACT

BACKGROUND: Intracellular protein trafficking is crucial for survival of cell and proper functioning of the organelles; however, these pathways are not well studied in the malaria parasite. Its unique cellular architecture and organellar composition raise an interesting question to investigate. METHODS: The interaction of Plasmodium falciparum Rab7 (PfRab7) with vacuolar protein sorting-associated protein 26 (PfVPS26) of retromer complex was shown by coimmunoprecipitation (co-IP). Confocal microscopy was used to show the localization of the complex in the parasite with respect to different organelles. Further chemical tools were employed to explore the role of digestive vacuole (DV) in retromer trafficking in parasite and GTPase activity of PfRab7 was examined. RESULTS: PfRab7 was found to be interacting with retromer complex that assembled mostly near DV and the Golgi in trophozoites. Chemical disruption of DV by chloroquine (CQ) led to its disassembly that was further validated by using compound 5f, a heme polymerization inhibitor in the DV. PfRab7 exhibited Mg2+ dependent weak GTPase activity that was inhibited by a specific Rab7 GTPase inhibitor, CID 1067700, which prevented the assembly of retromer complex in P. falciparum and inhibited its growth suggesting the role of GTPase activity of PfRab7 in retromer assembly. CONCLUSION: Retromer complex was found to be interacting with PfRab7 and the functional integrity of the DV was found to be important for retromer assembly in P. falciparum. GENERAL SIGNIFICANCE: This study explores the retromer trafficking in P. falciparum and describes amechanism to validate DV targeting antiplasmodial molecules.


Subject(s)
Plasmodium falciparum/metabolism , Vacuoles/metabolism , rab GTP-Binding Proteins/metabolism , Antimalarials/pharmacology , Chloroquine/pharmacology , Humans , Magnesium/metabolism , Malaria, Falciparum/drug therapy , Malaria, Falciparum/metabolism , Malaria, Falciparum/parasitology , Models, Molecular , Plasmodium falciparum/drug effects , Protein Interaction Maps/drug effects , Vacuoles/drug effects , rab7 GTP-Binding Proteins
7.
J Biol Chem ; 294(20): 8238-8258, 2019 05 17.
Article in English | MEDLINE | ID: mdl-30940726

ABSTRACT

The subcellular mechanism by which nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in gastric cancer and normal mucosal cells is elusive because of the diverse cyclooxygenase-independent effects of these drugs. Using human gastric carcinoma cells (AGSs) and a rat gastric injury model, here we report that the NSAID indomethacin activates the protein kinase Cζ (PKCζ)-p38 MAPK (p38)-dynamin-related protein 1 (DRP1) pathway and thereby disrupts the physiological balance of mitochondrial dynamics by promoting mitochondrial hyper-fission and dysfunction leading to apoptosis. Notably, DRP1 knockdown or SB203580-induced p38 inhibition reduced indomethacin-induced damage to AGSs. Indomethacin impaired mitochondrial dynamics by promoting fissogenic activation and mitochondrial recruitment of DRP1 and down-regulating fusogenic optic atrophy 1 (OPA1) and mitofusins in rat gastric mucosa. Consistent with OPA1 maintaining cristae architecture, its down-regulation resulted in EM-detectable cristae deformity. Deregulated mitochondrial dynamics resulting in defective mitochondria were evident from enhanced Parkin expression and mitochondrial proteome ubiquitination. Indomethacin ultimately induced mitochondrial metabolic and bioenergetic crises in the rat stomach, indicated by compromised fatty acid oxidation, reduced complex I- associated electron transport chain activity, and ATP depletion. Interestingly, Mdivi-1, a fission-preventing mito-protective drug, reversed indomethacin-induced DRP1 phosphorylation on Ser-616, mitochondrial proteome ubiquitination, and mitochondrial metabolic crisis. Mdivi-1 also prevented indomethacin-induced mitochondrial macromolecular damage, caspase activation, mucosal inflammation, and gastric mucosal injury. Our results identify mitochondrial hyper-fission as a critical and common subcellular event triggered by indomethacin that promotes apoptosis in both gastric cancer and normal mucosal cells, thereby contributing to mucosal injury.


Subject(s)
Apoptosis/drug effects , GTP Phosphohydrolases/metabolism , Gastric Mucosa/enzymology , Indomethacin/pharmacology , MAP Kinase Signaling System/drug effects , Microtubule-Associated Proteins/metabolism , Mitochondria/enzymology , Mitochondrial Dynamics/drug effects , Mitochondrial Proteins/metabolism , Neoplasm Proteins/metabolism , Protein Kinase C/metabolism , Stomach Neoplasms/enzymology , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Apoptosis/genetics , Cell Line, Tumor , Dynamins , GTP Phosphohydrolases/genetics , Gastric Mucosa/pathology , Humans , MAP Kinase Signaling System/genetics , Microtubule-Associated Proteins/genetics , Mitochondria/genetics , Mitochondrial Dynamics/genetics , Mitochondrial Proteins/genetics , Neoplasm Proteins/genetics , Protein Kinase C/genetics , Rats , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , p38 Mitogen-Activated Protein Kinases/genetics
8.
ACS Infect Dis ; 5(1): 63-73, 2019 01 11.
Article in English | MEDLINE | ID: mdl-30472841

ABSTRACT

The rapid emergence of resistance against frontline antimalarial drugs essentially warrants the identification of new-generation antimalarials. Here, we describe the synthesis of ( E)-2-isopropyl-5-methyl-4-((2-(pyridin-4-yl)hydrazono)methyl)phenol (18), which binds ferriprotoporphyrin-IX (FeIII-PPIX) ( Kd = 33 nM) and offers antimalarial activity against chloroquine-resistant and sensitive strains of Plasmodium falciparum in vitro. Structure-function analysis reveals that compound 18 binds FeIII-PPIX through the -C═N-NH- moiety and 2-pyridyl substitution at the hydrazine counterpart plays a critical role in antimalarial efficacy. Live cell confocal imaging using a fluorophore-tagged compound confirms its accumulation inside the acidic food vacuole (FV) of P. falciparum. Furthermore, this compound concentration-dependently elevates the pH in FV, implicating a plausible interference with FeIII-PPIX crystallization (hemozoin formation) by a dual function: increasing the pH and binding free FeIII-PPIX. Different off-target bioassays reduce the possibility of the promiscuous nature of compound 18. Compound 18 also exhibits potent in vivo antimalarial activity against chloroquine-resistant P. yoelii and P. berghei ANKA (causing cerebral malaria) in mice with negligible toxicity.


Subject(s)
Antimalarials/chemical synthesis , Antimalarials/pharmacology , Hemin/metabolism , Hydrazones/pharmacology , Malaria, Falciparum/prevention & control , Phenols/chemistry , Phenols/pharmacology , Vacuoles/drug effects , Animals , Biological Assay , Drug Resistance , Hemeproteins/antagonists & inhibitors , Hemeproteins/biosynthesis , Hydrazones/chemical synthesis , Hydrogen-Ion Concentration , Mice , Microscopy, Confocal , Plasmodium berghei/drug effects , Plasmodium falciparum/drug effects , Plasmodium yoelii/drug effects , Protein Binding , Vacuoles/chemistry
9.
J Biol Chem ; 293(51): 19740-19760, 2018 12 21.
Article in English | MEDLINE | ID: mdl-30366984

ABSTRACT

The indispensable role of macrophage migration inhibitory factor (MIF) in cancer cell proliferation is unambiguous, although which specific roles the cytokine plays to block apoptosis by preserving cell growth is still obscure. Using different cancer cell lines (AGS, HepG2, HCT116, and HeLa), here we report that the silencing of MIF severely deregulated mitochondrial structural dynamics by shifting the balance toward excess fission, besides inducing apoptosis with increasing sub-G0 cells. Furthermore, enhanced mitochondrial Bax translocation along with cytochrome c release, down-regulation of Bcl-xL, and Bcl-2 as well as up-regulation of Bad, Bax, and p53 indicated the activation of a mitochondrial pathway of apoptosis upon MIF silencing. The data also indicate a concerted down-regulation of Opa1 and Mfn1 along with a significant elevation of Drp1, cumulatively causing mitochondrial fragmentation upon MIF silencing. Up-regulation of Drp1 was found to be further coupled with fissogenic serine 616 phosphorylation and serine 637 dephosphorylation, thus ensuring enhanced mitochondrial translocation. Interestingly, MIF silencing was found to be associated with decreased NF-κB activation. In fact, NF-κB knockdown in turn increased mitochondrial fission and cell death. In addition, the silencing of CD74, the cognate receptor of MIF, remarkably increased mitochondrial fragmentation in addition to preventing cell proliferation, inducing mitochondrial depolarization, and increasing apoptotic cell death. This indicates the active operation of a MIF-regulated CD74-NF-κB signaling axis for maintaining mitochondrial stability and cell growth. Thus, we propose that MIF, through CD74, constitutively activates NF-κB to control mitochondrial dynamics and stability for promoting carcinogenesis via averting apoptosis.


Subject(s)
Antigens, Differentiation, B-Lymphocyte/metabolism , Histocompatibility Antigens Class II/metabolism , Macrophage Migration-Inhibitory Factors/metabolism , Mitochondrial Dynamics , NF-kappa B/metabolism , Signal Transduction , Apoptosis , Cell Line, Tumor , Cell Proliferation , Cell Survival , Down-Regulation , Dynamins , GTP Phosphohydrolases/metabolism , Gene Silencing , Humans , Macrophage Migration-Inhibitory Factors/deficiency , Macrophage Migration-Inhibitory Factors/genetics , Microtubule-Associated Proteins/metabolism , Mitochondrial Proteins/metabolism , Protein Transport , Up-Regulation
10.
Biochim Biophys Acta Proteins Proteom ; 1866(5-6): 722-730, 2018.
Article in English | MEDLINE | ID: mdl-29654975

ABSTRACT

Retromer complex plays a crucial role in intracellular protein trafficking and is conserved throughout the eukaryotes including malaria parasite, Plasmodium falciparum, where it is partially conserved. The assembly of retromer complex in RBC stages of malarial parasite is extremely difficult to explore because of its complicated physiology, small size, and intra-erythrocytic location. Nonetheless, understanding of retromer assembly may pave new ways for the development of novel antimalarials targeting parasite-specific protein trafficking pathways. Here, we investigated the assembly of retromer complex in P. falciparum, by an immunosensing method through highly sensitive Surface Plasmon Resonance (SPR) technique. After taking leads from the bioinformatics search and literature, different interacting proteins were identified and specific antibodies were raised against them. The sensor chip was prepared by covalently linking antibody specific to one component and the whole cell lysate was passed through it in order to trap the interacting complex. Antibodies raised against other interacting components were used to detect them in the trapped complex on the SPR chip. We were able to detect three different components in the retromer complex trapped by the immobilized antibody specific against a different component on a sensor chip. The assay was reproduced and validated in a different two-component CD74-MIF system in mammalian cells. We, thus, illustrate the assembly of retromer complex in P. falciparum through a bio-sensing approach that combines SPR with immunosensing requiring a very small amount of sample from the native source.


Subject(s)
Biosensing Techniques , Multiprotein Complexes/metabolism , Plasmodium falciparum/metabolism , Protozoan Proteins/metabolism , Surface Plasmon Resonance , Vesicular Transport Proteins/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Blotting, Western , Computational Biology , Hep G2 Cells , Humans , Immunoprecipitation , Kinetics , Mice , Multiprotein Complexes/genetics , Multiprotein Complexes/immunology , NIH 3T3 Cells , Plasmodium falciparum/genetics , Plasmodium falciparum/immunology , Protein Binding , Protein Transport , Protozoan Proteins/genetics , Protozoan Proteins/immunology , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/immunology
11.
Free Radic Biol Med ; 113: 424-438, 2017 12.
Article in English | MEDLINE | ID: mdl-28993273

ABSTRACT

Psychological stress, depression and anxiety lead to multiple organ dysfunctions wherein stress-related mucosal disease (SRMD) is common to people experiencing stress and also occur as a side effect in patients admitted to intensive care units; however the underlying molecular aetiology is still obscure. We report that in rat-SRMD model, cold restraint-stress severely damaged gut mitochondrial functions to generate superoxide anion (O2•-), depleted ATP and shifted mitochondrial fission-fusion dynamics towards enhanced fission to induce mucosal injury. Activation of mitophagy to clear damaged and fragmented mitochondria was evident from mitochondrial translocation of Parkin and PINK1 along with enhanced mitochondrial proteome ubiquitination, depletion of mitochondrial DNA copy number and TOM 20. However, excess and sustained accumulation of O2•--generating defective mitochondria overpowered the mitophagic machinery, ultimately triggering Bax-dependent apoptosis and NF-κB-intervened pro-inflammatory mucosal injury. We further observed that stress-induced enhanced serum corticosterone stimulated mitochondrial recruitment of glucocorticoid receptor (GR), which contributed to gut mitochondrial dysfunctions as documented from reduced ETC complex 1 activity, mitochondrial O2•- accumulation, depolarization and hyper-fission. GR-antagonism by RU486 or specific scavenging of mitochondrial O2•- by a mitochondrially targeted antioxidant mitoTEMPO ameliorated stress-induced mucosal damage. Gut mitopathology and mucosal injury were also averted when the perception of mental stress was blocked by pre-treatment with a sedative or antipsychotic. Altogether, we suggest the role of mitochondrial GR-O2•--fission cohort in brain-mitochondria cross-talk during acute mental stress and advocate the utilization of this pathway as a potential target to prevent mitochondrial unrest and gastropathy bypassing central nervous system.


Subject(s)
Adenosine Triphosphate/metabolism , Gastric Mucosa/metabolism , Immobilization/psychology , Mitochondria/metabolism , Stress, Psychological/metabolism , Animals , Antipsychotic Agents/pharmacology , Cold Temperature , Corticosterone/blood , Electron Transport Complex I/genetics , Electron Transport Complex I/metabolism , Gastric Mucosa/drug effects , Gastric Mucosa/pathology , Gene Expression Regulation , Immobilization/methods , Inflammation , Membrane Transport Proteins , Mifepristone/pharmacology , Mitochondria/drug effects , Mitochondria/pathology , Mitochondrial Dynamics/drug effects , Mitochondrial Dynamics/genetics , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Mitophagy/drug effects , Mitophagy/genetics , NF-kappa B/genetics , NF-kappa B/metabolism , Organophosphorus Compounds/pharmacology , Oxidative Stress , Piperidines/pharmacology , Protein Kinases/genetics , Protein Kinases/metabolism , Rats, Sprague-Dawley , Receptors, Cell Surface , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Glucocorticoid/antagonists & inhibitors , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Stomach , Stress, Psychological/genetics , Stress, Psychological/pathology , Superoxides/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism
12.
Biochem Pharmacol ; 121: 33-51, 2016 Dec 01.
Article in English | MEDLINE | ID: mdl-27693316

ABSTRACT

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat multiple inflammatory diseases and pain but severe gastric mucosal damage is the worst outcome of NSAID-therapy. Here we report that mitoTEMPO, a mitochondrially targeted superoxide (O2-) scavenger protected as well as healed gastric injury induced by diclofenac (DCF), the most commonly used NSAID. Common existing therapy against gastric injury involves suppression of gastric acid secretion by proton pump inhibitors and histamine H2 receptor antagonists; however, dyspepsia, vitamin B12 deficiency and gastric microfloral dysbalance are the major drawbacks of acid suppression. Interestingly, mitoTEMPO did not inhibit gastric acid secretion but offered gastroprotection by preventing DCF-induced generation of O2- due to mitochondrial respiratory chain failure and by preventing mitochondrial oxidative stress (MOS)-mediated mitopathology. MitoTEMPO even restored DCF-stimulated reduced fatty acid oxidation, mitochondrial depolarization and bioenergetic crisis in gastric mucosa. MitoTEMPO also prevented the activation of mitochondrial pathway of apoptosis and MOS-mediated proinflammatory signaling through NF-κB by DCF. Furthermore, mitoTEMPO when administered in rats with preformed gastric lesions expedited the healing of gastric injury and the healed stomach exhibited its normal physiology as evident from gastric acid and pepsin secretions under basal or stimulated conditions. Thus, in contrast to the existing antiulcer drugs, mitochondrially targeted O2- scavengers like mitoTEMPO may represent a novel class of gastroprotective molecules that does not affect gastric acid secretion and may be used in combination with DCF, keeping its anti-inflammatory action intact, while reducing its gastrodamaging effects.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Diclofenac/adverse effects , Gastric Mucosa/drug effects , Gastritis/prevention & control , Mitochondria/metabolism , Organophosphorus Compounds/therapeutic use , Piperidines/therapeutic use , Superoxides/antagonists & inhibitors , Animals , Apoptosis/drug effects , Cell Line , Chemotaxis, Leukocyte/drug effects , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/pathology , Gastric Acid/metabolism , Gastric Mucosa/injuries , Gastric Mucosa/metabolism , Gastritis/metabolism , Gastritis/pathology , Humans , Microscopy, Fluorescence , Neutrophil Infiltration/drug effects , Organophosphorus Compounds/administration & dosage , Oxidative Stress/drug effects , Piperidines/administration & dosage , Rats, Sprague-Dawley
13.
Protein Expr Purif ; 120: 7-15, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26690372

ABSTRACT

Translocation of various proteins to the subcellular organelles is an essential mechanism to regulate the metabolic pathways and often vacuolar protein sorting (VPS) proteins are involved in this transportation. Plasmodium falciparum VPS29 (PfVPS29) is predicted to be a functional component in the assembly of the retromer complex; however, so far detailed characterization of PfVPS29 in its native form is not yet done. We report the successful expression and purification of tag-free recombinant PfVPS29 with a yield of 5.6 mg from 1 L of Escherichia coli culture. PfVPS29 was purified by combined anion-exchange and size exclusion chromatography. The protein showed a single band in SDS-PAGE and it exhibited molecular mass of 21.7 kDa as measured by MALDI-TOF mass spectrometry. Secondary structure was elucidated by circular dichroism spectroscopy. It was found to be a monomeric protein in solution as evident from dynamic light scattering studies, chemical cross-linking experiments and size exclusion chromatography. Subsequently, polyclonal anti-PfVPS29 antibody was generated and used for evaluating protein expression by western blot and following subcellular localization in P. falciparum by confocal immunofluoroscence microscopy. PfVPS29 was found to be located in cytoplasm and expressed from early trophozoite to schizont stages with maximum expression in trophozoite stage. This study provides purification, biophysical characterization and subcellular localization of PfVPS29 in different asexual stages of P. falciparum.


Subject(s)
Plasmodium falciparum/metabolism , Protozoan Proteins/metabolism , Vesicular Transport Proteins/metabolism , Amino Acid Sequence , Blotting, Western , Circular Dichroism , Cloning, Molecular , Cytoplasm/metabolism , Electrophoresis, Polyacrylamide Gel , Gene Expression , Life Cycle Stages , Mass Spectrometry , Molecular Sequence Data , Molecular Weight , Plasmodium falciparum/physiology , Protein Structure, Secondary , Protozoan Proteins/genetics , Protozoan Proteins/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Alignment , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/isolation & purification
14.
Infect Immun ; 82(8): 3113-26, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24818663

ABSTRACT

The liver efficiently restores function after damage induced during malarial infection once the parasites are cleared from the blood. However, the molecular events leading to the restoration of liver function after malaria are still obscure. To study this, we developed a suitable model wherein mice infected with Plasmodium yoelii (45% parasitemia) were treated with the antimalarial α/ß-arteether to clear parasites from the blood and, subsequently, restoration of liver function was monitored. Liver function tests clearly indicated that complete recovery of liver function occurred after 25 days of parasite clearance. Analyses of proinflammatory gene expression and neutrophil infiltration further indicated that hepatic inflammation, which was induced immediately after parasite clearance from the blood, was gradually reduced. Moreover, the inflammation in the liver after parasite clearance was found to be correlated positively with oxidative stress and hepatocyte apoptosis. We investigated the role of heme oxygenase 1 (HO-1) in the restoration of liver function after malaria because HO-1 normally renders protection against inflammation, oxidative stress, and apoptosis under various pathological conditions. The expression and activity of HO-1 were found to be increased significantly after parasite clearance. We even found that chemical silencing of HO-1 by use of zinc protoporphyrin enhanced inflammation, oxidative stress, hepatocyte apoptosis, and liver injury. In contrast, stimulation of HO-1 by cobalt protoporphyrin alleviated liver inflammation and reduced oxidative stress, hepatocyte apoptosis, and associated tissue injury. Therefore, we propose that selective induction of HO-1 in the liver would be beneficial for the restoration of liver function after parasite clearance.


Subject(s)
Antimalarials/therapeutic use , Heme Oxygenase-1/metabolism , Liver/pathology , Malaria/drug therapy , Malaria/pathology , Plasmodium yoelii/growth & development , Animals , Liver Function Tests , Malaria/parasitology , Male , Mice , Mice, Inbred BALB C
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