scholarly journals Mutation of GGMP Repeat Segments of Plasmodium falciparum Hsp70-1 Compromises Chaperone Function and Hop Co-Chaperone Binding

2021 ◽  
Vol 22 (4) ◽  
pp. 2226
Author(s):  
Stanley Makumire ◽  
Tendamudzimu Harmfree Dongola ◽  
Graham Chakafana ◽  
Lufuno Tshikonwane ◽  
Cecilia Tshikani Chauke ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Chimeric Protein ◽  
Catalytic Efficiency ◽  
Peptide Substrate ◽  
E Coli ◽  
Chaperone Function ◽  
Hsp70 Chaperone ◽  
Chaperone Binding ◽  
Substrate Binding Domain

Parasitic organisms especially those of the Apicomplexan phylum, harbour a cytosol localised canonical Hsp70 chaperone. One of the defining features of this protein is the presence of GGMP repeat residues sandwiched between α-helical lid and C-terminal EEVD motif. The role of the GGMP repeats of Hsp70s remains unknown. In the current study, we introduced GGMP mutations in the cytosol localised Hsp70-1 of Plasmodium falciparum (PfHsp70-1) and a chimeric protein (KPf), constituted by the ATPase domain of E. coli DnaK fused to the C-terminal substrate binding domain of PfHsp70-1. A complementation assay conducted using E. coli dnaK756 cells demonstrated that the GGMP motif was essential for chaperone function of the chimeric protein, KPf. Interestingly, insertion of GGMP motif of PfHsp70-1 into DnaK led to a lethal phenotype in E. coli dnaK756 cells exposed to elevated growth temperature. Using biochemical and biophysical assays, we established that the GGMP motif accounts for the elevated basal ATPase activity of PfHsp70-1. Furthermore, we demonstrated that this motif is important for interaction of the chaperone with peptide substrate and a co-chaperone, PfHop. Our findings suggest that the GGMP may account for both the specialised chaperone function and reportedly high catalytic efficiency of PfHsp70-1.

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 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 Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1

2020 ◽  
Vol 75 (9) ◽  
pp. 2554-2563 ◽  
Author(s):  
Christopher Fröhlich ◽  
Vidar Sørum ◽  
Sandra Huber ◽  
Ørjan Samuelsen ◽  
Fanny Berglund ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Homology Modelling ◽  
Catalytic Efficiency ◽  
Hydrolytic Activity ◽  
Human Pathogens ◽  
E Coli ◽  
X Ray Crystallography ◽  
Environmental Reservoir

Abstract Background MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to β-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure–activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure. Objectives To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1. Methods The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different β-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1). Results Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8°C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity. Conclusions These results show the importance of environmental species acting as reservoirs for MBL-encoding genes.

scholarly journals Phenotypic and Biochemical Comparison of the Carbapenem-Hydrolyzing Activities of Five Plasmid-Borne AmpC β-Lactamases

2010 ◽  
Vol 54 (11) ◽  
pp. 4556-4560 ◽  
Author(s):  
Hedi Mammeri ◽  
Hélène Guillon ◽  
François Eb ◽  
Patrice Nordmann
Keyword(s):  
Biochemical Analysis ◽  
Carbapenem Resistance ◽  
Catalytic Efficiency ◽  
Wild Type ◽  
E Coli ◽  
Genes Encoding ◽  
High Level ◽  
Biochemical Comparison ◽  
Outer Membrane Permeability

ABSTRACT The CMY-2, ACT-1, DHA-1, ACC-1, and FOX-1 enzymes are representative of five plasmid-mediated AmpC (pAmpC) β-lactamase clusters. Resistance to imipenem has been reported in Enterobacteriaceae as a result of pAmpC expression combined with decreased outer membrane permeability. The aim of this study was to determine the role of different pAmpCs in carbapenem resistance and to define the structure/activity relationship supporting carbapenemase activity. The ampC genes encoding the five pAmpCs and the chromosomal AmpC of Escherichia coli EC6, which was used as a reference cephalosporinase, were cloned and introduced into wild-type E. coli TOP10 and OmpC/OmpF porin-deficient E. coli HB4 strains. The MICs of β-lactams for the recombinant strains revealed that CMY-2, ACT-1, and DHA-1 β-lactamases conferred a high level of resistance to ceftazidime and cefotaxime once expressed in E. coli TOP10 and reduced significantly the susceptibility to imipenem once expressed in E. coli HB4. In contrast, FOX-1 and ACC-1 enzymes did not confer resistance to imipenem. Biochemical analysis showed that CMY-2 β-lactamase and, to a lesser extent, ACT-1 exhibited the highest catalytic efficiency toward imipenem and showed low Km values. A modeling study revealed that the large R2 binding site of these two enzymes may support the carbapenemase activity. Therefore, CMY-2-type, ACT-1-type, and DHA-1-type β-lactamases may promote the emergence of carbapenem resistance in porin-deficient clinical isolates.

scholarly journals Insights into Enzymic Catalysis from Studies on Dihydrofolate Reductases

Pteridines ◽  
1989 ◽  
Vol 1 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Stephen J. Benkovic ◽  
Joseph A. Adams ◽  
Carol A. Fierke ◽  
Adel M. Naylor
Keyword(s):  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Structural Similarity ◽  
E Coli ◽  
Energy Profiles ◽  
Dihydrofolate Reductases ◽  
Cellular Dna ◽  
High Degree ◽  
Conserved Amino Acids

Summary The role of DHFR in the maintenance of cellular DNA has sparked wide interest in the structure and dynamics of this enzyme. Kinetic studies of specific amino acid replacements on the enzyme isolated from E. coli has proved useful in the detailing of hydrophobic and ionic interactions both proximal and distal to the site of chemical transformation (e. g. Phe-31, Leu-54 and Arg-44). Despite the low sequence homology shared by the E. coli and L. easei enzymes, the free energy profiles are surprisingly comparable. This probably is the result of the high degree of structural similarity of the active site surfaces, but the deleterious effects of subtle replacements (e. g. Leu-54-Ile) at strictly conserved amino acids underscore the latters unique role in attaining the required catalytic efficiency for the enzyme.

scholarly journals Substrate Specificity of the Escherichia coli Outer Membrane Protease OmpP

2006 ◽  
Vol 189 (2) ◽  
pp. 522-530 ◽  
Author(s):  
Bum-Yeol Hwang ◽  
Navin Varadarajan ◽  
Haixin Li ◽  
Sarah Rodriguez ◽  
Brent L. Iverson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Outer Membrane ◽  
Amino Acid Sequence Identity ◽  
Catalytic Efficiency ◽  
Wild Type ◽  
Peptide Substrate ◽  
Peptide Substrates ◽  
E Coli ◽  
Sequence Identity

ABSTRACT Escherichia coli OmpP is an F episome-encoded outer membrane protease that exhibits 71% amino acid sequence identity with OmpT. These two enzymes cleave substrate polypeptides primarily between pairs of basic amino acids. We found that, like OmpT, purified OmpP is active only in the presence of lipopolysaccharide. With optimal peptide substrates, OmpP exhibits high catalytic efficiency (k cat/Km = 3.0 × 106 M−1s−1). Analysis of the extended amino acid specificity of OmpP by substrate phage revealed that both Arg and Lys are strongly preferred at the P1 and P1′ sites of the enzyme. In addition, Thr, Arg, or Ala is preferred at P2; Leu, Ala, or Glu is preferred at P4; and Arg is preferred at P3′. Notable differences in OmpP and OmpT specificities include the greater ability of OmpP to accept Lys at the P1 or P1′, site as well as the prominence of Ser at P3 in OmpP substrates. Likewise, the OmpP P1 site could better accommodate Ser; as a result, OmpP was able to cleave a peptide substrate between Ser-Arg about 120 times more efficiently than was OmpT. Interestingly, OmpP and OmpT cleave peptides with three consecutive Arg residues at different sites, a difference in specificity that might be important in the inactivation of cationic antimicrobial peptides. Accordingly, we show that the presence of an F′ episome results in increased resistance to the antimicrobial peptide protamine both in ompT mutants and in wild-type E. coli cells.

The Local Sanarelli-Shwartzman Phenomenon in Rats Induced by Human Leukaemic Cells

1973 ◽  
Vol 29 (02) ◽  
pp. 353-362
Author(s):  
J Lisiewicz ◽  
A Pituch ◽  
J. A Litwin
Keyword(s):  
Chronic Lymphocytic Leukaemia ◽  
Intravenous Injection ◽  
Peripheral Blood ◽  
Lymphocytic Leukaemia ◽  
Acute Myeloblastic Leukaemia ◽  
Demarcation Line ◽  
E Coli ◽  
Leukaemic Cells ◽  
Shwartzman Phenomenon

SummaryThe local Sanarelli-Shwartzman phenomenon (SSP-L) in the skin of 30 rats was induced by an intr a cutaneous sensitizing injection of leukaemic leucocytes isolated from the peripheral blood of patients with chronic lymphocytic leukaemia (CLL), acute myeloblastic leukaemia (AL) and chronic granulocytic leukaemia (CGL) and challenged by an intravenous injection of 100(μ of E. coli endotoxin. SSP-L was observed in 7 rats after injection of CLL lymphocytes and in 6 and 2 rats after AL myeloblasts and the CGL granulocytes, respectively. The lesions in the skin after AL myeloblasts appeared in a shorter time and were of longer duration compared with those observed after CLL lymphocytes and CGL granulocytes. Histologically, the lesions consisted of areas of destruction in the superficial layers of the skin ; the demarcation line showed the presence of neutrophils, macrophages and erythrocytes. Haemorrhages and fibrin deposits near the demarcation line were larger after injection of CLL lymphocytes and AL myeloblasts than after CGL granulocytes. The possible role of leucocyte procoagulative substances in the differences observed have been discussed.

Bioenhancing Effect of Cow Urine Distillate and Pepper Extract on Antibacterial Activity of Azadirachta Indica Leaves

1970 ◽  
Vol 9 (2) ◽  
pp. 3212-3214
Author(s):  
Pramod Dhakal ◽  
Ankit a Achary ◽  
Vedamurthy Joshi
Keyword(s):  
Antibacterial Activity ◽  
Azadirachta Indica ◽  
Pathogenic Bacteria ◽  
Ethanol Extract ◽  
Watery Diarrhea ◽  
Diffusion Method ◽  
E Coli ◽  
Drug Molecules ◽  
Cow Urine

Bioenhancers are drug facilitator which do not show the typical drug activity but in combination to enhance the activity of other molecule in several way including increase the bioavailability of drug across the membrane, potentiating the drug molecules by conformational interaction, acting as receptor for drug molecules and making target cell more receptive to drugs and promote and increase the bioactivity or bioavailability or the uptake of drugs in combination therapy. The objective of the present study was to evaluate the antibacterial and activity of combination in Azadirachta indica extract with cow urine distillate and pepper extract against common pathogenic bacteria, a causative agent of watery diarrhea. It has been found that Indian indigenous cow urine and its distillate also possess bioenhancing ability. Bioenhancing role of cow urine distillate (CUD) and pepper extract was investigated on antibacterial activity of ethanol extract of Azadirachta indica. Antibacterial activity of ethanol extract neem alone and in combination with CUD and pepper extract were determined the ATCC strains against Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and E-coli by cup plate diffusion method. Ethanol extract of neem has showed more effect on P. aeruginosa, E-coli than S. aureus and K. pneumonia with combination of CUD and pepper extract. CUD and pepper did not show any inhibition of test bacteria in low concentration. The antibacterial effect of combination of extract and CUD was higher than the inhibition caused by extract alone and is suggestive of the bioenhancing role of cow urine distillate and pepper. Moreover, inhibition of test bacteria was observed with less concentration of extract on combining with CUD

scholarly journals E. coli Enterotoxin LtB Enhances Vaccine-Induced Anti-H. pylori Protection by Promoting Leukocyte Migration into Gastric Mucus via Inflammatory Lesions

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 982
Author(s):  
Xiaoyan Peng ◽  
Rongguang Zhang ◽  
Chen Wang ◽  
Feiyan Yu ◽  
Mingyang Yu ◽  
...  
Keyword(s):  
Cellular Immunity ◽  
Protective Efficacy ◽  
Gastric Injury ◽  
Oral Vaccines ◽  
Gastric Mucus ◽  
E Coli ◽  
Non Invasive ◽  
Considerable Impact ◽  
H Pylori

Current studies indicate that the anti-H. pylori protective efficacy of oral vaccines to a large extent depends on using mucosal adjuvants like E. coli heat-lable enterotoxin B unit (LtB). However, the mechanism by which Th17/Th1-driven cellular immunity kills H. pylori and the role of LtB remains unclear. Here, two L. lactis strains, expressing H. pylori NapA and LtB, respectively, were orally administrated to mice. As observed, the administration of LtB significantly enhanced the fecal SIgA level and decreased gastric H. pylori colonization, but also markedly aggravated gastric inflammatory injury. Both NapA group and NapA+LtB group had elevated splenocyte production of IL-8, IL-10, IL-12, IL-17, IL-23 and INF-γ. Notably, gastric leukocytes’ migration or leakage into the mucus was observed more frequently in NapA+LtB group than in NapA group. This report is the first that discusses how LtB enhances vaccine-induced anti-H. pylori efficacy by aggravating gastric injury and leukocytes’ movement into the mucus layer. Significantly, it brings up a novel explanation for the mechanism underlying mucosal cellular immunity destroying the non-invasive pathogens. More importantly, the findings suggest the necessity to further evaluate LtB’s potential hazards to humans before extending its applications. Thus, this report can provide considerable impact on the fields of mucosal immunology and vaccinology.

scholarly journals Reconstitution in Proteoliposomes of the Recombinant Human Riboflavin Transporter 2 (SLC52A2) Overexpressed in E. coli

2019 ◽  
Vol 20 (18) ◽  
pp. 4416 ◽  
Author(s):  
Lara Console ◽  
Maria Tolomeo ◽  
Matilde Colella ◽  
Maria Barile ◽  
Cesare Indiveri
Keyword(s):  
Cardiovascular Disease ◽  
Amino Acids ◽  
Risk Factor ◽  
Biologically Active ◽  
Suitable Model ◽  
Native Protein ◽  
E Coli ◽  
Few Data ◽  
Riboflavin Transport

Background: the SLC52A2 gene encodes for the riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed. It mediates the transport of Riboflavin across cell membranes. Riboflavin plays a crucial role in cells since its biologically active forms, FMN and FAD, are essential for the metabolism of carbohydrates, amino acids, and lipids. Mutation of the Riboflavin transporters is a risk factor for anemia, cancer, cardiovascular disease, neurodegeneration. Inborn mutations of SLC52A2 are associated with Brown-Vialetto-van Laere syndrome, a rare neurological disorder characterized by infancy onset. In spite of the important metabolic and physio/pathological role of this transporter few data are available on its function and regulation. Methods: the human recombinant RFVT2 has been overexpressed in E. coli, purified and reconstituted into proteoliposomes in order to characterize its activity following the [3H]Riboflavin transport. Results: the recombinant hRFVT2 showed a Km of 0.26 ± 0.07 µM and was inhibited by lumiflavin, FMN and Mg2+. The Riboflavin uptake was also regulated by Ca2+. The native protein extracted from fibroblast and reconstituted in proteoliposomes also showed inhibition by FMN and lumiflavin. Conclusions: proteoliposomes represent a suitable model to assay the RFVT2 function. It will be useful for screening the mutation of RFVT2.

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