scholarly journals Comparative characterisation of Plasmodium falciparum Hsp70-1 relative to E. coli DnaK reveals functional specificity of the parasite chaperone

2020 ◽  
Author(s):  
Charity Mekgwa Lebepe ◽  
Pearl Rutendo Matambanadzo ◽  
Xolani Henry Makhoba ◽  
Ikechukwu Achilonu ◽  
Tawanda Zininga ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Molecular Chaperone ◽  
Chimeric Protein ◽  
Cellular Functions ◽  
E Coli ◽  
Adenosylmethionine Decarboxylase ◽  
Comparative Structure ◽  
Functional Features ◽  
Self Association

ABSTRACTHsp70 is one of the most prominent molecular chaperones. Although Hsp70s from various organisms are generally conserved, they exhibit specialised cellular functions. It remains to be fully understood how these highly conserved molecules exhibit specialised functional features. Plasmodium falciparum Hsp70-1 (PfHsp70-1) is a cytosol localised molecular chaperone that is implicated in the cyto-protection and pathogenicity of the malaria parasite. In the current study, we investigated the comparative structure-function features of PfHsp70-1 relative to its homologue, E. coli Hsp70 (DnaK) and a chimeric protein, KPf, that was constituted by the ATPase domain of DnaK and the substrate binding domain (SBD) of PfHsp70-1. Recombinant forms of all the three Hsp70s exhibited similar secondary and tertiary structural fold. We further established that compared to DnaK, both KPf and PfHsp70-1 were more stable to heat stress and exhibited higher basal ATPase activity. A recombinant P. falciparum Hsp40 (PfHsp40) stimulated the ATPase activities of all the three Hsp70s. In addition, both PfHsp70-1 and KPf exhibited preference for asparagine rich peptides as opposed to DnaK. Furthermore, all the three proteins exhibited self-association capabilities in vitro. Recombinant P. falciparum adenosylmethionine decarboxylase (PfAdoMetDC) co-expressed in E. coli with either KPf or PfHsp70-1 was produced as a fully folded product. On the other hand, co-expression of PfAdoMetDC with heterologous DnaK in E. coli did not promote folding of the former. These findings demonstrated that the SBD of PfHsp70-1 regulates several functional features of the protein and that this molecular chaperone is tailored to facilitate folding of plasmodial proteins.

scholarly journals Comparative Characterization of Plasmodium falciparum Hsp70-1 Relative to E. coli DnaK Reveals the Functional Specificity of the Parasite Chaperone

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 856
Author(s):  
Charity Mekgwa Lebepe ◽  
Pearl Rutendo Matambanadzo ◽  
Xolani Henry Makhoba ◽  
Ikechukwu Achilonu ◽  
Tawanda Zininga ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Molecular Chaperone ◽  
Chimeric Protein ◽  
Peptide Substrates ◽  
E Coli ◽  
Adenosylmethionine Decarboxylase ◽  
Comparative Structure ◽  
Functional Features ◽  
Substrate Binding Domain

Hsp70 is a conserved molecular chaperone. How Hsp70 exhibits specialized functions across species remains to be understood. Plasmodium falciparum Hsp70-1 (PfHsp70-1) and Escherichia coli DnaK are cytosol localized molecular chaperones that are important for the survival of these two organisms. In the current study, we investigated comparative structure-function features of PfHsp70-1 relative to DnaK and a chimeric protein, KPf, constituted by the ATPase domain of DnaK and the substrate binding domain (SBD) of PfHsp70-1. Recombinant forms of the three Hsp70s exhibited similar secondary and tertiary structural folds. However, compared to DnaK, both KPf and PfHsp70-1 were more stable to heat stress and exhibited higher basal ATPase activity. In addition, PfHsp70-1 preferentially bound to asparagine rich peptide substrates, as opposed to DnaK. Recombinant P. falciparum adenosylmethionine decarboxylase (PfAdoMetDC) co-expressed in E. coli with either KPf or PfHsp70-1 was produced as a fully folded product. Co-expression of PfAdoMetDC with heterologous DnaK in E. coli did not promote folding of the former. However, a combination of supplementary GroEL plus DnaK improved folding of PfAdoMetDC. These findings demonstrated that the SBD of PfHsp70-1 regulates several functional features of the protein and that this molecular chaperone is tailored to facilitate folding of plasmodial proteins.

scholarly journals In vitro reconstitution of the GTPase-associated centre of the archaebacterial ribosome: the functional features observed in a hybrid form with Escherichia coli 50S subunits

2006 ◽  
Vol 396 (3) ◽  
pp. 565-571 ◽  
Author(s):  
Takaomi Nomura ◽  
Kohji Nakano ◽  
Yasushi Maki ◽  
Takao Naganuma ◽  
Takashi Nakashima ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Proteins ◽  
Synthetic Activity ◽  
23S Rrna ◽  
Elongation Factors ◽  
Hybrid Form ◽  
Pyrococcus Horikoshii ◽  
E Coli ◽  
Functional Features

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0·Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0·Ph-L12 complex and Ph-L11 could replace L10·L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.

scholarly journals MazG, a Nucleoside Triphosphate Pyrophosphohydrolase, Interacts with Era, an Essential GTPase in Escherichia coli

2002 ◽  
Vol 184 (19) ◽  
pp. 5323-5329 ◽  
Author(s):  
Junjie Zhang ◽  
Masayori Inouye
Keyword(s):  
Escherichia Coli ◽  
Direct Interaction ◽  
Kanamycin Resistance ◽  
Nucleoside Triphosphate ◽  
Cellular Functions ◽  
Genomic Libraries ◽  
E Coli ◽  
Yeast Two Hybrid System ◽  
Nucleoside Triphosphate Pyrophosphohydrolase

ABSTRACT Era is an essential GTPase in Escherichia coli, and Era has been implicated in a number of cellular functions. Homologues of Era have been identified in various bacteria and some eukaryotes. Using the era gene as bait in the yeast two-hybrid system to screen E. coli genomic libraries, we discovered that Era interacts with MazG, a protein of unknown function which is highly conserved among bacteria. The direct interaction between Era and MazG was also confirmed in vitro, being stronger in the presence of GDP than in the presence of GTPγS. MazG was characterized as a nucleoside triphosphate pyrophosphohydrolase which can hydrolyze all eight of the canonical ribo- and deoxynucleoside triphosphates to their respective monophosphates and PPi, with a preference for deoxynucleotides. A mazG deletion strain of E. coli was constructed by replacing the mazG gene with a kanamycin resistance gene. Unlike mutT, a gene for another conserved nucleotide triphosphate pyrophosphohydrolase that functions as a mutator gene, the mazG deletion did not result in a mutator phenotype in E. coli.

Irreversible inhibition of S-adenosylmethionine decarboxylase in Plasmodium falciparum-infected erythrocytes: Growth inhibition in vitro

1991 ◽  
Vol 41 (11) ◽  
pp. 1713-1718 ◽  
Author(s):  
Paul S. Wright ◽  
Timothy L. Byers ◽  
Doreen E. Cross-Doersen ◽  
Peter P. McCann ◽  
Alan J. Bitonti
Keyword(s):  
Plasmodium Falciparum ◽  
Growth Inhibition ◽  
Adenosylmethionine Decarboxylase ◽  
Irreversible Inhibition

scholarly journals Plasmodium falciparum Merozoite Surface Protein 1 (MSP-1)-MSP-3 Chimeric Protein: Immunogenicity Determined with Human-Compatible Adjuvants and Induction of Protective Immune Response

2009 ◽  
Vol 78 (2) ◽  
pp. 872-883 ◽  
Author(s):  
Suman Mazumdar ◽  
Paushali Mukherjee ◽  
Syed Shams Yazdani ◽  
S. K. Jain ◽  
Asif Mohmmed ◽  
...  
Keyword(s):  
Immune Response ◽  
Plasmodium Falciparum ◽  
Fusion Protein ◽  
Malaria Vaccine ◽  
Chimeric Protein ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Protective Immune Response ◽  
Merozoite Surface Protein 1

ABSTRACT A chimeric gene, MSP-Fu24 , was constructed by genetically coupling immunodominant, conserved regions of the two leading malaria vaccine candidates, Plasmodium falciparum merozoite surface protein 1 (C-terminal 19-kDa region [PfMSP-119]) and merozoite surface protein 3 (11-kDa conserved region [PfMSP-311]). The recombinant MSP-Fu24 protein was produced in Escherichia coli cells and purified to homogeneity by a two-step purification process with a yield of ∼30 mg/liter. Analyses of conformational properties of MSP-Fu24 using PfMSP-119-specific monoclonal antibody showed that the conformational epitopes of PfMSP-119 that may be critical for the generation of the antiparasitic immune response remained intact in the fusion protein. Recombinant MSP-Fu24 was highly immunogenic in mice and in rabbits when formulated with two different human-compatible adjuvants and induced an immune response against both PfMSP-119 and PfMSP-311. Purified anti-MSP-Fu24 antibodies showed invasion inhibition of P. falciparum 3D7 and FCR parasites, and this effect was found to be dependent on antibodies specific for the PfMSP-119 component. The protective potential of MSP-Fu24 was demonstrated by in vitro parasite growth inhibition using an antibody-dependent cell inhibition (ADCI) assay with anti-MSP-Fu24 antibodies. Overall, the antiparasitic activity was mediated by a combination of growth-inhibitory antibodies generated by both the PfMSP-119 and PfMSP-311 components of the MSP-Fu24 protein. The antiparasitic activities elicited by anti-MSP-Fu24 antibodies were comparable to those elicited by antibodies generated with immunization with a physical mixture of two component antigens, PfMSP-119 and PfMSP-311. The fusion protein induces a protective immune response with human-compatible adjuvants and may form a part of a multicomponent malaria vaccine.

scholarly journals Functional Analysis of PvdS, an Iron Starvation Sigma Factor of Pseudomonas aeruginosa

2000 ◽  
Vol 182 (6) ◽  
pp. 1481-1491 ◽  
Author(s):  
Livia Leoni ◽  
Nicola Orsi ◽  
Victor de Lorenzo ◽  
Paolo Visca
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sigma Factor ◽  
Sigma Factors ◽  
Gene Copy ◽  
Wild Type ◽  
Iron Starvation ◽  
E Coli ◽  
Positive Regulator ◽  
Functional Features

ABSTRACT In Pseudomonas aeruginosa, iron modulates gene expression through a cascade of negative and positive regulatory proteins. The master regulator Fur is involved in iron-dependent repression of several genes. One of these genes, pvdS, was predicted to encode a putative sigma factor responsible for the transcription of a subset of genes of the Fur regulon. PvdS appears to belong to a structurally and functionally distinct subgroup of the extracytoplasmic function family of alternative sigma factors. Members of this subgroup, also including PbrA from Pseudomonas fluorescens, PfrI and PupI from Pseudomonas putida, and FecI from Escherichia coli, are controlled by the Fur repressor, and they activate transcription of genes for the biosynthesis or the uptake of siderophores. Evidence is provided that the PvdS protein of P. aeruginosa is endowed with biochemical properties of eubacterial sigma factors, as it spontaneously forms 1:1 complexes with the core fraction of RNA polymerase (RNAP, α2ββ′ subunits), thereby promoting in vitro binding of the PvdS-RNAP holoenzyme to the promoter region of the pvdA gene. These functional features of PvdS are consistent with the presence of structural domains predicted to be involved in core RNAP binding, promoter recognition, and open complex formation. The activity of pyoverdin biosynthetic (pvd) promoters was significantly lower in E. coli overexpressing the multicopy pvdS gene than in wild-type P. aeruginosa PAO1 carrying the single gene copy, andpvd::lacZ transcriptional fusions were silent in both pfrI (the pvdS homologue) and pfrA (a positive regulator of pseudobactin biosynthetic genes) mutants of P. putida WCS358, while they are expressed at PAO1 levels in wild-type WCS358. Moreover, the PvdS-RNAP holoenzyme purified from E. coli lacked the ability to generate in vitro transcripts from the pvdA promoter. These observations suggest that at least one additional positive regulator could be required for full activity of the PvdS-dependent transcription complex both in vivo and in vitro. This is consistent with the presence of a putative activator binding site (the iron starvation box) at variable distance from the transcription initiation sites of promoters controlled by the iron starvation sigma factors PvdS, PfrI, and PbrA of fluorescent pseudomonads.

scholarly journals Excision of the doubly methylated base N 4 ,5-dimethylcytosine from DNA by Escherichia coli Nei and Fpg proteins

2018 ◽  
Vol 373 (1748) ◽  
pp. 20170337 ◽  
Author(s):  
Marina Alexeeva ◽  
Prashanna Guragain ◽  
Almaz N. Tesfahun ◽  
Miglė Tomkuvienė ◽  
Aysha Arshad ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Alkylating Agents ◽  
High Sensitivity ◽  
Cellular Functions ◽  
Repair Enzyme ◽  
E Coli ◽  
Chemical Evidence ◽  
Adenine Thymine

Cytosine (C) in DNA is often modified to 5-methylcytosine (m 5 C) to execute important cellular functions. Despite the significance of m 5 C for epigenetic regulation in mammals, damage to m 5 C has received little attention. For instance, almost no studies exist on erroneous methylation of m 5 C by alkylating agents to doubly or triply methylated bases. Owing to chemical evidence, and because many prokaryotes express methyltransferases able to convert m 5 C into N 4 ,5-dimethylcytosine (m N 4,5 C) in DNA, m N 4,5 C is probably present in vivo . We screened a series of glycosylases from prokaryotic to human and found significant DNA incision activity of the Escherichia coli Nei and Fpg proteins at m N 4,5 C residues in vitro . The activity of Nei was highest opposite cognate guanine followed by adenine, thymine (T) and C. Fpg-complemented Nei by exhibiting the highest activity opposite C followed by lower activity opposite T. To our knowledge, this is the first description of a repair enzyme activity at a further methylated m 5 C in DNA, as well as the first alkylated base allocated as a Nei or Fpg substrate. Based on our observed high sensitivity to nuclease S1 digestion, we suggest that m N 4,5 C occurs as a disturbing lesion in DNA and that Nei may serve as a major DNA glycosylase in E. coli to initiate its repair. This article is part of a discussion meeting issue ‘Frontiers in epigenetic chemical biology’.

scholarly journals Novel Strategy for Biofilm Inhibition by Using Small Molecules Targeting Molecular Chaperone DnaK

2014 ◽  
Vol 59 (1) ◽  
pp. 633-641 ◽  
Author(s):  
Ken-ichi Arita-Morioka ◽  
Kunitoshi Yamanaka ◽  
Yoshimitsu Mizunoe ◽  
Teru Ogura ◽  
Shinya Sugimoto
Keyword(s):  
Molecular Chaperone ◽  
Biofilm Formation ◽  
Antimicrobial Agents ◽  
Dependent Manner ◽  
Cellular Functions ◽  
Biofilm Inhibition ◽  
Content Type ◽  
E Coli ◽  
Chaperone Dnak ◽  
Biofilm Development

ABSTRACTBiofilms are complex communities of microorganisms that attach to surfaces and are embedded in a self-produced extracellular matrix. Since these cells acquire increased tolerance against antimicrobial agents and host immune systems, biofilm-associated infectious diseases tend to become chronic. We show here that the molecular chaperone DnaK is important for biofilm formation and that chemical inhibition of DnaK cellular functions is effective in preventing biofilm development. Genetic, microbial, and microscopic analyses revealed that deletion of thednaKgene markedly reduced the production of the extracellular functional amyloid curli, which contributes to the robustness ofEscherichia colibiofilms. We tested the ability of DnaK inhibitors myricetin (Myr), telmisartan, pancuronium bromide, and zafirlukast to prevent biofilm formation ofE. coli. Only Myr, a flavonol widely distributed in plants, inhibited biofilm formation in a concentration-dependent manner (50% inhibitory concentration [IC50] = 46.2 μM); however, it did not affect growth. Transmission electron microscopy demonstrated that Myr inhibited the production of curli. Phenotypic analyses of thermosensitivity, cell division, intracellular level of RNA polymerase sigma factor RpoH, and vulnerability to vancomycin revealed that Myr altered the phenotype ofE. coliwild-type cells to make them resemble those of the isogenicdnaKdeletion mutant, indicating that Myr inhibits cellular functions of DnaK. These findings provide insights into the significance of DnaK in curli-dependent biofilm formation and indicate that DnaK is an ideal target for antibiofilm drugs.

scholarly journals Intramolecular Regulation of the Opposing (p)ppGpp Catalytic Activities of RelSeq, the Rel/Spo Enzyme from Streptococcus equisimilis

2002 ◽  
Vol 184 (11) ◽  
pp. 2878-2888 ◽  
Author(s):  
Undine Mechold ◽  
Helen Murphy ◽  
Larissa Brown ◽  
Michael Cashel
Keyword(s):  
Specific Activity ◽  
Chimeric Protein ◽  
Stringent Response ◽  
In Vitro Assays ◽  
Synthetic Activity ◽  
E Coli ◽  
Regulatory Domains ◽  
Degradation Activity ◽  
Regulatory Effects

ABSTRACT Catalytic and regulatory domains of the Rel/Spo homolog of Streptococcus equisimilis affecting (p)ppGpp synthesis and degradation activities have been defined, and opposing activities of the purified protein and its fragments have been compared. Two major domains of the 739-residue Rel Seq protein are defined by limited proteolytic digestion. In vitro assays of the purified N-terminal half-protein reveal synthesis of (p)ppGpp by an ATP-GTP 3′-pyrophosphotransferase as well as an ability to degrade (p)ppGpp by a Mn2+-dependent 3′-pyrophosphohydrolase. Removal of the C-terminal half-protein has reciprocal regulatory effects on the activities of the N-terminal half-protein. Compared to the full-length protein, deletion activates (p)ppGpp synthesis specific activity about 12-fold and simultaneously inhibits (p)ppGpp degradation specific activity about 150-fold to shift the balance of the two activities in favor of synthesis. Cellular (p)ppGpp accumulation behavior is consistent with these changes. The bifunctional N-terminal half-protein can be further dissected into overlapping monofunctional subdomains, since purified peptides display either degradation activity (residues 1 to 224) or synthetic activity (residues 79 to 385) in vitro. These assignments can also apply to RelA and SpoT. The ability of Rel Seq to mediate (p)ppGpp accumulation during amino acid starvation in S. equisimilis is absent when the protein is expressed ectopically in Escherichia coli. Fusing the N-terminal half of Rel Seq with the C-terminal domain of RelA creates a chimeric protein that restores the stringent response in E. coli by inhibiting unregulated degradation and restoring regulated synthetic activity. Reciprocal intramolecular regulation of the dual activities may be a general intrinsic feature of Rel/Spo homolog proteins.

scholarly journals Biochemical and Immunological Characterization of Bacterially Expressed and Refolded Plasmodium falciparum 42-Kilodalton C-Terminal Merozoite Surface Protein 1

2003 ◽  
Vol 71 (12) ◽  
pp. 6766-6774 ◽  
Author(s):  
Sanjay Singh ◽  
Michael C. Kennedy ◽  
Carole A. Long ◽  
Allan J. Saul ◽  
Louis H. Miller ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Native Form ◽  
Immunological Characterization ◽  
E Coli ◽  
Merozoite Surface Protein 1 ◽  
Vaccine Trials

ABSTRACT Protection against Plasmodium falciparum can be induced by vaccination in animal models with merozoite surface protein 1 (MSP1), which makes this protein an attractive vaccine candidate for malaria. In an attempt to produce a product that is easily scaleable and inexpensive, we expressed the C-terminal 42 kDa of MSP1 (MSP142) in Escherichia coli, refolded the protein to its native form from insoluble inclusion bodies, and tested its ability to elicit antibodies with in vitro and in vivo activities. Biochemical, biophysical, and immunological characterization confirmed that refolded E. coli MSP142 was homogeneous and highly immunogenic. In a formulation suitable for human use, rabbit antibodies were raised against refolded E. coli MSP142 and tested in vitro in a P. falciparum growth invasion assay. The antibodies inhibited the growth of parasites expressing either homologous or heterologous forms of P. falciparum MSP142. However, the inhibitory activity was primarily a consequence of antibodies directed against the C- terminal 19 kDa of MSP1 (MSP119). Vaccination of nonhuman primates with E. coli MSP142 in Freund's adjuvant protected six of seven Aotus monkeys from virulent infection with P. falciparum. The protection correlated with antibody-dependent mechanisms. Thus, this new construct, E. coli MSP142, is a viable candidate for human vaccine trials.

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