Patent Grant
10968436
The instant application contains a Sequence Listing which has been submitted electronically in an ASCII text file named 7077-0004_SL.txt on Dec. 16, 2019. The Sequence Listing is hereby incorporated by reference in its entirety.
The present invention relates to novel NADPH oxidase proteins, the use thereof, the method of preparation thereof and the method for identifying them.
The NADPH oxidase proteins, or Nox, are tissue-specific modular proteins of very wide occurrence in eukaryotes. They share a catalytic core consisting of an FNR (flavin-containing ferredoxin NADPH oxidoreductase-like) domain and an FRD (ferric reductase domain), bound to the membrane, fixing 2 haems (Nox 1 to 4). They may also have, as Nox5, a calmodulin domain and in addition, as in the case of Duox1 and Duox2, an additional transmembrane segment and a domain of the peroxidase type at the N-terminal end (Lambeth J D, NOX enzymes and the biology of reactive oxygen, Nature Reviews, 2004, Immunology, 4:181-189). Although the two domains FNR and cytochrome b are found in prokaryotes, pure and simple sequence alignments have never allowed Nox or Fre proteins to be detected in the bacterial genomes.
In terms of function, in addition to Nox2, which is found both in phagocytes, in which it is involved in immune defence, and in the cardiac tissues, in which it plays a role in muscle contraction, the Nox enzymes are involved in multiple processes such as cellular proliferation, apoptosis, or intra- and intercellular communication. Moreover, the Duox enzymes are involved in the synthesis of the thyroid hormones (Prosser B L et al., X-ROS signaling: rapid mechano-chemo transduction in heart, 2011, Science, 333:1440-1445; De Deken X et al., Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family, The Journal of Biological Chemistry, 275:23227-23233). In yeast, the ferric reductases (Fre) for their part are involved in the reduction of ferric ions to ferrous ions during iron import.
In a controlled manner, the Nox proteins lead to the production of reactive oxygen species (ROS), such as the superoxide ions O2.−, hydrogen peroxide (H2O2), or the OH. hydroxyl radical. Recent accumulation of data indicates that the ROS have a much more subtle role than was initially attributed in the immune system. Their importance in molecular and cellular mechanisms such as signalling, chemical reactivity, or toxicity induced by the redox potential of the species produced, therefore places the Nox proteins henceforth in the rank of high-added-value therapeutic targets.
To identify new medicaments capable of specifically targeting this protein family, the pharmaceutical industry screens small molecules to determine their effects with respect to Nox proteins. These small molecules are then improved by organic synthesis in order to optimize their effects.
Now, taking into account the eukaryotic origin of the Nox proteins, their capacity as membrane proteins, their modes of regulation, and their specific post-translational modifications (Nox1-4, Nox5 and Duox1-2), current pharmaceutical research must tackle the difficulties inherent in the very nature of these Nox proteins. In other words, it is difficult to produce in sufficient quantities a highly regulated glycosylated membrane system, with multiple components.
For carrying out screening assays, it is however necessary on the one hand to obtain functional Nox proteins to evaluate the actual impact of the molecules tested, and on the other hand to produce sufficient quantities of Nox proteins so as to be able to test a large number of molecules. Now, the Nox proteins produced in large quantities are mainly inactive, whereas the functional Nox proteins themselves are obtained in insignificant quantities. This has the effect of making their production cost too high to envisage screening assays on a large scale.
Currently, human Nox proteins have already been produced in cells of the immune system (Taylor R M et al., Analysis of human phagocyte flavocytochrome b(558) by mass spectrometry, J Biol Chem 2006, Dec. 1, 281(48): 37045-56; Lord C I et al., Single-step immunoaffinity purification and functional reconstitution of human phagocyte flavocytochrome b, J Immunol Methods, 2008, Jan. 1, 329(1-2):201-7) and various attempts at heterologous expression have also been envisaged, but without success (de Mendez I, Leto T L, Functional reconstitution of the phagocyte NADPH oxidase by transfection of its multiple components in a heterologous system, Blood, 1995, Feb. 15, 85(4):1104-10; Price M O et al. Creation of a genetic system for analysis of the phagocyte respiratory burst: high-level reconstitution of the NADPH oxidase in a nonhematopoietic system. Blood, 2002, Apr. 15, 99(8):2653-61; Zhen L et al., Gene targeting of X chromosome-linked chronic granulomatous disease locus in a human myeloid leukemia cell line and rescue by expression of recombinant gp91phox. Proc Natl Acad Sci USA, 1993 Nov. 1, 90(21):9832-6).
Mammalian Nox proteins have in particular been expressed heterologously in various eukaryotic organisms, such as in insect cells (Rotrosen D et al., Production of recombinant cytochrome b558 allows reconstitution of the phagocyte NADPH oxidase solely from recombinant proteins, J Biol Chem, 1993, Jul. 5, 268(19):14256-60) or in yeast (Ostuni M A et al., Expression of functional mammal flavocytochrome b(558) in yeast: comparison with improved insect cell system. Biochim Biophys Acta, 2010 June, 1798(6): 1179-88).
However, none of these approaches has made it possible to produce functional Nox proteins in sufficient quantities to envisage high-throughput screening. Moreover, heterologous expression systems have also been tested in attempts to produce eukaryotic Nox proteins in prokaryotic organisms, in particular in the bacterium Escherichia coli, but only truncated forms of the eukaryotic Nox proteins have been produced (Han C H et al., Characterization of the flavoprotein domain of gp91phox which has NADPH diaphorase activity, J Biochem, 2001 April, 129(4):513-20; Nisimoto Y et al. Activation of the flavoprotein domain of gp91phox upon interaction with N-terminal p67phox (1-210) and the Rac complex, Biochemistry, 2004, Jul. 27, 43(29):9567-75).
In this context, the aim of the present invention is to propose novel Nox proteins, identified and characterized for the first time in prokaryotes. Use of these novel Nox proteins thus offers the advantage of allowing production of functional proteins in large quantity and at less cost compared to the eukaryotic Nox proteins.
An aspect of the invention is therefore to propose novel Nox proteins.
Another aspect of the invention is to propose a method for preparing these novel Nox proteins.
Another aspect of the invention is to propose a method for identifying these novel Nox proteins starting from bacterial protein sequences.
Another aspect of the invention finally relates to the applications of these novel Nox proteins.
In a first alternative, the invention relates to a Nox (or NADPH oxidase) protein having:
a domain comprising from 3 to 7 transmembrane helices, and having an amino acid sequence comprising:
at least 2 bis-histidyl motifs, each of the bis-histidyl motifs constituted by 2 histidine residues separated by 12, 13 or 14 amino acid residues, said 2 bis-histidyl motifs being located in the membrane part and separated by a transmembrane helix, and
at least one first motif constituted by the sequence:
at least one second motif constituted by the sequence:
at least one third motif constituted by the sequence:
at least one fourth motif constituted by the sequence:
In the invention, the term “Nox”, or “NADPH oxidase protein” refers to an enzyme catalysing the reaction of electron transfer from an electron donor, a homologue of NADPH, to a final acceptor, which is molecular oxygen. This results in the production of reactive oxygen species.
This NADPH oxidase activity of the Nox proteins occurs naturally in vivo in the organisms that produce them, but it can also be determined and measured in vitro using specific assays, via reduction reactions showing the production of superoxide ion (by colorimetry tracking the reduction of cytochrome c, or the reduction of NBT (Nitro Blue Tetrazolium), or also by fluorescence using amplex Red, and by chemiluminescence using luminol).
The term “membrane part” refers to the amino acids that are arranged in the lipid bilayer of the cell, when the Nox protein is expressed functionally. This membrane part contains the 2 haems of the Nox protein and consists essentially of amino acids folded in the form of a helices.
The present invention is based on the Inventors' surprising finding that proteins of the Nox family are present in prokaryotes.
For the first time, the Inventors have identified motifs present on the amino acid sequence of these Nox proteins, and these motifs make it possible to identify and correctly ascribe said Nox proteins.
The Inventors' approach is based on the identification of (i) a consensus motif corresponding to the FAD-binding domain, (ii) a consensus motif corresponding to the NADPH-binding domain and (iii) a domain comprising from 3 to 7 transmembrane helices (identified from the amino acid sequence) containing 2 bis-histidyl motifs of 12 to 14 amino acids separated by at least 20 amino acids (i.e. the length of a transmembrane helix), which are found in association in Nox proteins.
Moreover, the Inventors also identified, surprisingly, additional motifs present in most of the prokaryotic Nox, allowing them to be distinguished from the eukaryotic Nox. The Nox proteins according to the invention may thus be identified from their amino acid sequence with the aid of the following 4 motifs:
this motif corresponds to the FAD-binding site on the Nox protein,
3rd motif: [K/R]-X-X-G-[D/G]-X-[T/S] (SEQ ID NO: 3),
4th motif: G-[I/S/V/L]-G-[V/I/A/F]-[T/A/S]-[P/Y/T/A] (SEQ ID NO: 4), this motif corresponds to the NADPH-binding site on the Nox protein.
The amino acids shown in square brackets correspond to alternative amino acids for the position considered on the consensus sequence.
In a second alternative, another aspect of the invention relates to a Nox protein having:
a domain comprising from 3 to 7 transmembrane helices, and having an amino acid sequence comprising:
at least 2 bis-histidyl motifs, each of the bis-histidyl motifs being constituted by 2 histidine residues separated by 12, 13 or 14 amino acid residues, said 2 bis-histidyl motifs being located in the membrane part and separated by a transmembrane helix, and
at least one motif A constituted by the sequence:
at least one motif B constituted by the sequence:
at least one motif C constituted by the sequence:
According to this aspect of the invention, the Nox proteins may be identified with the aid of the following 3 motifs:
motif B: H-[P/S/A]-F-[T/S]-[L/I/M/V] (SEQ ID NO: 6), this motif corresponds to the FAD-binding site on the Nox protein
motif C: G-[I/S/V/L]-G-[V/I/A/F]-[T/A/S]-[P/Y/T/A] (SEQ ID NO: 4), this motif corresponds to the NADPH-binding site on the Nox protein.
In a third alternative, another aspect of the invention relates to a Nox protein having:
a domain comprising from 3 to 7 transmembrane helices, and having an amino acid sequence comprising:
at least 2 bis-histidyl motifs, each of the bis-histidyl motifs being constituted by 2 histidine residues separated by 12, 13 or 14 amino acid residues, said 2 bis-histidyl motifs being located in the membrane part and separated by a transmembrane helix, and
at least one motif A′ constituted by the sequence:
at least one motif B′ constituted by the sequence:
at least one motif C′ constituted by the sequence:
According to this aspect of the invention, the Nox proteins may be identified with the aid of the following 3 motifs:
motif B′: [P/A/S/E/F]-H-[P/S/A]-F-[T/S]-[L/I/M/V] (SEQ ID NO: 2), this motif corresponds to the FAD-binding site on the Nox protein,
motif C′: G-[I/S/V/L]-G-[V/I/A/F]-[T/A/S]-[P/Y/T/A] (SEQ ID NO: 4), this motif corresponds to the NADPH-binding site on the Nox protein.
The three alternatives of the invention correspond to Nox proteins identified in prokaryotic organisms. However, it is not excluded that Nox proteins according to the invention might be identified in eukaryotic organisms.
In an embodiment of the first alternative, the invention relates to a Nox protein as defined above, in which said first motif is selected from the group constituted by the sequences SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.
The sequences SEQ ID NO: 8 to 10 correspond respectively to the motifs present on the Nox proteins of Streptococcus pneumoniae, Pseudomonas aeruginosa and Escherichia coli (SEQ ID NO: 8), Salmonella enterica and Bordetella pertussis (SEQ ID NO: 9) and Vibrio cholerae (SEQ ID NO: 10).
In an embodiment of the first alternative, the invention relates to a Nox protein as defined above, in which said second motif is selected from the group constituted by the sequences SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.
The sequences SEQ ID NO: 11 to 14 correspond respectively to the motifs present on the Nox proteins of Streptococcus pneumoniae (SEQ ID NO: 11), Pseudomonas aeruginosa, Salmonella enterica and Bordetella pertussis (SEQ ID NO: 12), Vibrio cholerae (SEQ ID NO: 13) and Escherichia coli (SEQ ID NO: 14).
In an embodiment of the first alternative, the invention relates to a Nox protein as defined above, in which said third motif is selected from the group constituted by the sequences SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.
The sequences SEQ ID NO: 15 to 20 correspond respectively to the motifs present on the Nox proteins of Streptococcus pneumoniae (SEQ ID NO: 15), Pseudomonas aeruginosa (SEQ ID NO: 16), Escherichia coli (SEQ ID NO: 17), Vibrio cholerae (SEQ ID NO: 18), Salmonella enterica (SEQ ID NO: 19) and Bordetella pertussis (SEQ ID NO: 20).
In an embodiment of the first alternative, the invention relates to a Nox protein as defined above, in which said fourth motif is selected from the group constituted by the sequences SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23.
The sequences SEQ ID NO: 21 to 23 correspond respectively to the motifs present on the Nox proteins of Streptococcus pneumoniae, Salmonella enterica, Escherichia coli and Bordetella pertussis (SEQ ID NO: 21), Pseudomonas aeruginosa (SEQ ID NO: 22) and Vibrio cholerae (SEQ ID NO: 23).
In a particular embodiment, the invention relates to a Nox protein as defined above, the amino acid sequence of which comprises, or consists of, a sequence having at least 90% identity, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 24.
The sequence SEQ ID NO: 24 corresponds to a Nox protein identified in Streptococcus pneumoniae.
The “percentage identity” between two amino acid sequences, within the meaning of the present invention, is determined by comparing the two optimally aligned sequences through a comparison window. The part of the amino acid sequence in the comparison window may thus comprise additions or deletions (for example “gaps”) relative to the reference sequence (which would not comprise these additions or deletions) so as to obtain optimum alignment between the two sequences.
The percentage identity is calculated by determining the number of positions for which an amino acid residue is identical in the two sequences being compared, then dividing the number of positions for which there is identity between the two amino acid residues, by the total number of positions in the comparison window, and then multiplying the result by one hundred to obtain the percentage amino acid identity between the two sequences.
In a particular embodiment, the invention relates to a Nox protein as defined above, the amino acid sequence of which comprises, or consists of, a sequence having at least 90% identity, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 25.
The sequence SEQ ID NO: 25 corresponds to a Nox protein identified in Pseudomonas aeruginosa.
In a particular embodiment, the invention relates to a Nox protein as defined above, the amino acid sequence of which comprises, or consists of, a sequence having at least 90% identity, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 26.
The sequence SEQ ID NO: 26 corresponds to a Nox protein identified in Escherichia coli.
In a particular embodiment, the invention relates to a Nox protein as defined above, the amino acid sequence of which comprises, or consists of, a sequence having at least 90% identity, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 27.
The sequence SEQ ID NO: 27 corresponds to a Nox protein identified in Vibrio cholerae.
In a particular embodiment, the invention relates to a Nox protein as defined above, the amino acid sequence of which comprises, or consists of, a sequence having at least 90% identity, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 28.
The sequence SEQ ID NO: 28 corresponds to a Nox protein identified in Salmonella enterica.
In a particular embodiment, the invention relates to a Nox protein as defined above, the amino acid sequence of which comprises, or consists of, a sequence having at least 90% identity, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 29.
The sequence SEQ ID NO: 29 corresponds to a Nox protein identified in Bordetella pertussis.
In an embodiment, the invention relates to a Nox protein as defined above, the amino acid sequence of which comprises, or consists of, a sequence selected from the group constituted by the sequences SEQ ID NO: 24 to 351.
The sequences SEQ ID NO: 24 to 351 correspond to 328 sequences of Nox proteins identified among the sequences accessible in the databases at 22 Jan. 2014.
In an embodiment, the invention relates to a Nox protein as defined above the amino acid sequence of which comprises, or consists of, a sequence selected from the group constituted by the sequences SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29.
In a particular embodiment, the invention relates to a Nox protein as defined above, said Nox protein being a protein of a prokaryotic organism, said prokaryotic organism preferably being selected from the group constituted by Streptococcus pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Vibrio cholerae, Salmonella enterica and Bordetella pertussis.
In another particular embodiment, the invention relates to a Nox protein as defined above, said Nox protein being an isolated.
In another particular embodiment, the invention relates to a Nox protein as defined above, said Nox protein being a recombinant protein.
In the invention, the term “recombinant protein” refers to a protein produced in a cell in which the gene coding for this protein has been inserted, for example using a plasmid vector. A recombinant protein can be produced heterologously in a cell of an organism different from that in which it was identified originally.
In another particular embodiment, the invention relates to a Nox protein as defined above, said Nox protein having a spectral signature in spectrophotometry from 250 to 700 nm that is similar, preferably identical, to one of the spectral signatures from 250 to 700 nm presented in
The presence of the haems in the Nox protein can in fact be visualized by carrying out a measurement by the spectrophotometry technique. The presence of the haems induces a characteristic spectral signature from 250 to 700 nm, with a peak at 410 nm when the protein is in its oxidized state. Another characteristic spectrum of the Nox is the difference spectrum (reduced spectrum minus oxidized spectrum) having a major peak at 425 nm and another peak at 558 nm. Thus, analysis of the spectral signature makes it possible to visualize the presence of the haems and consequently verify the good folding and stability of the Nox protein analysed.
In another particular embodiment, the invention relates to a Nox protein as defined above, said Nox protein having NADPH oxidase activity leading to production of ROS in the presence of an electron donor, said NADPH oxidase activity capable of being detected in a sample comprising said Nox protein via a reaction showing production of superoxide ion, preferably by reduction of cytochrome c or by reduction of NBT (Nitro Blue Tetrazolium), and preferably by measuring absorbance by spectrophotometry, by chemiluminescence or by a colorimetric assay, carried out on the sample.
In another aspect, the invention also relates to a nucleic acid coding for a Nox protein defined above.
In an embodiment, the invention relates to a nucleic acid as defined above comprising, or consisting of, a sequence selected from the group constituted by the sequences SEQ ID NO: 352, SEQ ID NO: 353, SEQ ID NO: 354, SEQ ID NO: 355, SEQ ID NO: 356, SEQ ID NO: 357, SEQ ID NO: 358, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 361, SEQ ID NO: 362 and SEQ ID NO: 363.
The sequences SEQ ID NO: 352 to 357 correspond to nucleic acids coding for Nox proteins identified in S. pneumoniae (SEQ ID NO: 352), P. aeruginosa (SEQ ID NO: 353), E. coli (SEQ ID NO: 354), V. cholerae (SEQ ID NO: 355), S. enterica (SEQ ID NO: 356) and B. pertussis (SEQ ID NO: 357).
The sequences SEQ ID NO: 358 to 363 correspond to nucleic acids, the codons of which have been optimized to allow expression of Nox proteins of S. pneumoniae (SEQ ID NO: 358), P. aeruginosa (SEQ ID NO: 359), E. coli (SEQ ID NO: 360), V. cholerae (SEQ ID NO: 361), S. enterica (SEQ ID NO: 362) and B. pertussis (SEQ ID NO: 363), having a polyhistidine tail and a thrombin cleavage site.
In another aspect, the invention relates to an expression vector comprising a nucleic acid as defined above.
In an embodiment, the invention relates to an expression vector as defined above comprising, or consisting of, a sequence selected from the group constituted by the sequences SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 366, SEQ ID NO: 367, SEQ ID NO: 368 and SEQ ID NO: 369.
In another aspect, the invention relates to a cell containing at least one expression vector as defined above.
In another embodiment, the invention relates to a cell as defined above, said cell being a bacterium, preferably selected from the group constituted by Escherichia coli, Streptococcus pneumoniae, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella enterica and Bordetella pertussis.
Another aspect of the invention also relates to a method for preparing a Nox protein as defined above, starting from a cellular extract from cells producing said protein, contained in a suitable solution, comprising a step of solubilization of the cell membranes of said cellular extract in order to put said protein in suspension in said suitable solution.
Taking into account their complex structure and their membrane nature, the Nox proteins are difficult to produce and purify as it is difficult to dissolve the membrane to release the Nox protein without destabilizing its structure. Use of an unsuitable detergent leads, moreover, to degradation of the Nox protein.
However, the Inventors found, surprisingly, that the use of detergents of the maltoside type makes it possible to dissolve the Nox protein.
In an embodiment, the invention relates to a method as defined above, in which the step of solubilization of said cell membrane is carried out using at least one detergent from the maltoside family, preferably selected from the group of dodecyl-maltosides.
The detergent will thus coat the membrane part, replacing the membrane lipids. The detergent will then maintain the three-dimensional structure of the Nox protein, which will keep the haems in the membrane part during purification.
In an embodiment, the invention relates to a method as defined above, in which said at least one detergent is selected from the group constituted by:
In an embodiment, the invention relates to a method as defined above, in which said at least one detergent is Lauryl Maltose Neopentyl Glycol (MNG3).
In a particular embodiment, the invention relates to a method as defined above, in which said at least one detergent is a mixture of detergents, in particular of maltosides, preferably a CYMAL-7/DDM or DDM/MNG3 mixture.
In a particular embodiment, the invention relates to a method as defined above, in which the step of solubilization of said cell membrane is carried out using a solution comprising 50 mM of Tris HCl at pH 7, 300 mM of NaCl and 5 mM of MNG3.
In an embodiment, the invention relates to a method as defined above, comprising, at the end of the solubilization step, a step of purification of said Nox protein in suspension in the suitable solution.
In an embodiment, the invention relates to a method as defined above, in which said at least one detergent is used at a concentration from 0.01 mM to 0.2 mM, preferably at 0.03 mM during said purification step.
In an embodiment, the invention relates to a method as defined above, in which the step of purification of said Nox protein is carried out by chromatography, preferably using a resin having a strong affinity for an amino acid motif present on said protein, said amino acid motif preferably being a polyhistidine tail.
In a particular embodiment, the invention relates to a method as defined above, in which said resin is sepharose.
Preferably, said resin is equilibrated with the same solution as that used for solubilization of the membrane, preferably with a solution comprising 50 mM of Tris HCl at pH 7, 300 mM of NaCl and 5 mM of MNG3.
In an embodiment, the invention relates to a method as defined above, in which said cells are bacteria, preferably selected from the group consisting of Streptococcus pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Vibrio cholerae, Salmonella enterica and Bordetella pertussis.
In an embodiment, the invention relates to a method as defined above, comprising the steps of:
production of said Nox protein in bacteria,
lysis of the bacteria to obtain a cellular extract in a suitable solution,
bringing the cellular extract into contact with a detergent to dissolve the cell membranes and obtain said Nox protein in suspension in the suitable solution,
purification of said Nox protein in suspension in the suitable solution.
In an embodiment, the invention relates to a method as defined above, comprising the steps of:
production of said Nox protein in E. coli bacteria,
mechanical lysis of the bacteria by a microfluidizer to obtain a cellular extract in a suitable solution,
bringing the cellular extract into contact with a solution comprising 50 mM of Tris HCl at pH 7, 300 mM of NaCl and 5 mM of MNG3, to dissolve the cell membranes and obtain said Nox protein in suspension in the suitable solution,
purification of said Nox protein in suspension in the suitable solution, by affinity chromatography, with a buffer of 50 mM Tris pH7, 300 mM NaCl, 0.03 mM MNG3.
In an embodiment, the invention relates to a method as defined above, comprising, after said purification step, a step of size exclusion chromatography, in particular in a column, preferably using a buffer of 50 mM Tris HCl pH 7, 300 mM NaCl and 20 to 100 μM MNG3.
Preferably, the buffer used for the purification step contains 30 μM of MNG3.
In an embodiment, the invention relates to a method as defined above, comprising, after said step of size exclusion chromatography, a step of depletion of the detergent.
The step of depletion of the detergent makes it possible to remove the excess detergent from the Nox protein in an affinity column (or ion exchange column). Preferably, 10 column volumes are used for washing the surplus of MNG3, and elution is carried out with a buffer of 50 mM Tris HCl pH 7, 300 mM NaCl and 20 μM MNG3.
The Nox proteins of the invention can be expressed effectively in bacteria, with a high yield of the order of a milligram of pure, functional protein per litre of culture, which allows large-scale production at very low cost.
Another aspect of the invention relates to the use in vitro, ex vivo or in vivo of the Nox protein as defined above as a research tool for screening molecules of therapeutic interest.
Besides their production in large quantity and their low production cost, the Nox proteins of the invention offer many advantages for screening molecules of therapeutic interest. Non-limitatively, the latter can for example be activated with NADPH alone, or an analogue of NADPH, and are practical for implementation, provided a one-component enzyme system is concerned.
In an embodiment, the invention relates to the above use in which said Nox protein is used in vitro.
In an embodiment, the invention relates to the above use in which said Nox protein is used ex vivo.
In an embodiment, the invention relates to the above use in which said Nox protein is used in vivo.
In an embodiment, the invention relates to the above use in which said Nox protein is used ex vivo or in vivo in humans.
In an embodiment, the invention relates to the above use in which said Nox protein is used ex vivo or in vivo in a non-human animal.
In an embodiment, the invention relates to the above use in which said Nox protein is not used ex vivo or in vivo in humans.
In an embodiment, the invention relates to the above use in which said Nox protein is not used ex vivo or in vivo in a non-human animal.
In a particular embodiment, the invention relates to the use of the Nox protein defined above, in which said Nox protein constitutes a model therapeutic target.
The Nox proteins of the invention constitute particularly interesting model proteins, all the more so as they are homologues of eukaryotic Nox proteins, and in particular of human Nox proteins. In fact, the similarity between SpNox and Nox2 is identical to the similarity between Nox2 and Nox5. The use of the Nox proteins of the invention for pre-screening or screening of molecules of therapeutic interest would therefore make it possible to increase the probability of identifying new drugs that are active for the eukaryotic Nox.
In a particular embodiment, the invention relates to the use of the Nox protein defined above, in which said protein is brought into contact with at least one molecule of therapeutic interest.
In an embodiment, the invention relates to the use of the Nox protein defined above, in which said Nox protein is adsorbed on a support, preferably from a polyhistidine tag present on said Nox protein.
In an embodiment, the invention relates to the use of the Nox protein defined above, in which said support is a plate, preferably a 96-well plate.
In a particular embodiment, the invention relates to the use of the Nox protein defined above, in which the NADPH oxidase activity of said Nox protein and/or the production of ROS is measured, in the presence or absence of said at least one molecule of therapeutic interest.
In an embodiment, the invention relates to the use of the Nox protein defined above for identifying molecules capable of modulating its NADPH oxidase activity.
In an embodiment, the invention relates to the use of the Nox protein defined above for identifying molecules capable of modulating its NADPH oxidase activity, said Nox protein being a prokaryotic protein.
In an embodiment, the invention relates to the use of the Nox protein defined above for identifying molecules capable of modulating the NADPH oxidase activity of another Nox protein.
In a particular embodiment, the invention relates to the use of a prokaryotic Nox protein as defined above, for identifying molecules capable of modulating the NADPH oxidase activity of a eukaryotic Nox protein.
In a particular embodiment, the invention relates to the use of the Nox protein defined above for screening molecules capable of modulating the production of ROS by said Nox protein or by another Nox protein.
In a particular embodiment, the invention relates to the use of the Nox protein defined above for screening molecules capable of reducing or inhibiting the production of ROS by said Nox protein or by another Nox protein.
In a particular embodiment, the invention relates to the use of the Nox protein defined above for screening molecules capable of stimulating the production of ROS by said Nox protein or by another Nox protein.
In a particular embodiment, the invention relates to the use of the Nox protein defined above for screening molecules capable of having antibiotic properties. Identification of molecules capable of stimulating the production of ROS by a bacterial Nox may also allow potentiation of known antibiotics. In fact, the endogenous production of ROS in the bacterium may have the consequence of making resistant bacteria sensitive to antibiotics.
Such molecules that increase the production of ROS by the NOX may thus be used in combination with an antibiotic for treating certain bacterial infections.
In other words, a molecule that activates a prokaryotic Nox protein is capable of leading to the production of ROS when it is administered to a bacterium and, consequently, is capable of constituting an effective antibacterial molecule.
Another aspect of the invention relates to the use in vitro, ex vivo or in vivo:
of a nucleic acid coding for a Nox protein as defined above,
of an expression vector containing said nucleic acid, or
of a cell containing said expression vector,
as a research tool for screening molecules of therapeutic interest.
In an embodiment, the invention relates to the above use in which said nucleic acid, said expression vector or said cell is used in vitro.
In an embodiment, the invention relates to the above use in which said nucleic acid, said expression vector or said cell is used ex vivo.
In an embodiment, the invention relates to the above use in which said nucleic acid, said expression vector or said cell is used in vivo.
In an embodiment, the invention relates to the above use in which said nucleic acid, said expression vector or said cell is used ex vivo or in vivo in humans.
In an embodiment, the invention relates to the above use in which said nucleic acid, said expression vector or said cell is used ex vivo or in vivo in a non-human animal.
In an embodiment, the invention relates to the above use in which said nucleic acid, said expression vector or said cell is not used ex vivo or in vivo in humans.
In an embodiment, the invention relates to the above use in which said nucleic acid, said expression vector or said cell is not used ex vivo or in vivo in a non-human animal.
In another aspect, the invention relates to a method for identifying, from a bank of molecules to be tested, at least one molecule capable of modifying the production of ROS by a eukaryotic Nox protein, comprising:
a step a) in which said molecules to be tested are screened, by being brought into contact with a prokaryotic Nox protein, for selecting the molecules capable of modifying the production of ROS by the prokaryotic Nox protein, then
a step b) in which said molecules capable of modifying the production of ROS by the prokaryotic Nox protein are brought into contact with the eukaryotic Nox protein, for identifying at least one molecule capable of modifying the production of ROS by the eukaryotic Nox protein.
As the prokaryotic Nox proteins can be produced on a large scale and at low cost, it is beneficial to use them for testing and pre-selecting molecules that are capable of acting on the eukaryotic Nox proteins, the latter being more difficult to produce in sufficient quantities.
In an embodiment, the production of ROS by said prokaryotic Nox protein in step a), and/or by said eukaryotic Nox protein in step b), is determined by measuring absorbance by spectrophotometry, and in particular by a colorimetric assay, by chemiluminescence or by fluorescence.
In an embodiment, steps a) and b) are carried out in plates, preferably 96-well plates.
In an embodiment, step a) is carried out in plates, preferably 96-well plates, and step b) is carried out in a spectrophotometry cell.
In an embodiment, step a) is carried out in a spectrophotometry cell and step b) is carried out in plates, preferably 96-well plates.
In another aspect, the invention relates to the use of the motif:
for identifying an amino acid sequence capable of encoding a Nox protein.
In another aspect, the invention relates to the use of the motif:
for identifying an amino acid sequence capable of encoding a Nox protein.
In another aspect, the invention relates to the use of the motif:
and the motif:
for identifying an amino acid sequence capable of encoding a Nox protein.
Another aspect of the invention also relates to a method for identifying a Nox protein comprising:
a step of detecting, in an amino acid sequence, an NADPH motif comprising the following sequence:
a step of detecting, in said amino acid sequence, an FAD motif comprising the following sequence:
In an embodiment, the invention also relates to a method for identifying in silico a Nox protein comprising:
a step of detecting, in an amino acid sequence, an NADPH motif comprising the following sequence:
a step of detecting, in said amino acid sequence, an FAD motif comprising the following sequence:
In a particular embodiment, the invention relates to a method of identification as defined above, further comprising:
a step of detecting, in said amino acid sequence, 3 to 7 regions capable of forming transmembrane helices, in particular with the aid of the TMHMM software, and
a step of detecting, in said amino acid sequence, at least 2 bis-histidyl motifs separated by at least 20 amino acids, each of the bis-histidyl motifs being constituted by 2 histidine residues separated by 12, 13 or 14 amino acid residues, and in which, if said amino acid sequence comprises said 3 to 7 regions capable of forming transmembrane helices and said at least 2 bis-histidyl motifs, then said amino acid sequence is capable of encoding a Nox protein.
In a particular embodiment, the invention relates to a method of identification as defined above, in which said FAD motif from step b) comprises the sequence:
In a particular embodiment, the invention relates to a method of identification as defined above, comprising a step of detecting, in said amino acid sequence, an additional motif S1:
In a particular embodiment, the invention relates to a method of identification as defined above, comprising a step of detecting, in said amino acid sequence, an additional motif S2:
In a particular embodiment, the invention relates to a method of identification as defined above, in which said amino acid sequence is encoded by a nucleotide sequence present on a bacterial genome.
In a particular embodiment, the invention relates to a method as defined above comprising:
a step a) of detecting, in an amino acid sequence, an NADPH motif comprising the following sequence:
a step b) of detecting, in said amino acid sequence, an FAD motif comprising the following sequence:
a step c) of detecting, in said amino acid sequence, 3 to 7 regions capable of forming transmembrane helices, in particular with the aid of the TMHMM software, and
a step d) of detecting, in said amino acid sequence, at least 2 bis-histidyl motifs, each of the bis-histidyl motifs constituted by 2 histidine residues separated by 12, 13 or 14 amino acid residues, said bis-histidyl motifs being separated by 20 amino acids, in which, if said amino acid sequence comprises said NADPH motif from step a), said FAD motif from step b), said 3 to 7 regions capable of forming transmembrane helices from step c) and said at least 2 bis-histidyl motifs from step d), then said amino acid sequence is capable of encoding an NADPH oxidase protein.
In another aspect, the invention relates to a method for identifying a prokaryotic Nox protein comprising:
a step a) of detecting, in an amino acid sequence, an NADPH motif comprising the following sequence:
a step b) of detecting, in said amino acid sequence, an FAD motif comprising the following sequence:
a step c) of detecting, in said amino acid sequence, 3 to 7 regions capable of forming transmembrane helices, in particular with the aid of the TMHMM software, and
a step d) of detecting, in said amino acid sequence, at least 2 bis-histidyl motifs, each of the bis-histidyl motifs being constituted by 2 histidine residues separated by 12, 13 or 14 amino acid residues, said bis-histidyl motifs being separated by 20 amino acids, and
a step e) of detecting, in said amino acid sequence, an additional motif S1:
a step f) of detecting, in said amino acid sequence, a second additional motif S2:
in which, if said amino acid sequence comprises said NADPH motif from step a), said FAD motif from step b), said 3 to 7 regions capable of forming transmembrane helices from step c), said at least 2 bis-histidyl motifs from step d), the additional motif S1 from step e) and the additional motif S2 from step f), then said amino acid sequence is capable of encoding a prokaryotic Nox protein.
In an embodiment, the invention relates to a method for identifying in silico a prokaryotic Nox protein comprising:
a step a) of detecting, in an amino acid sequence, an NADPH motif comprising the following sequence:
a step b) of detecting, in said amino acid sequence, an FAD motif comprising the following sequence:
a step c) of detecting, in said amino acid sequence, 3 to 7 regions capable of forming transmembrane helices, in particular with the aid of the TMHMM software, and
a step d) of detecting, in said amino acid sequence, at least 2 bis-histidyl motifs, each of the bis-histidyl motifs being constituted by 2 histidine residues separated by 12, 13 or 14 amino acid residues, said bis-histidyl motifs being separated by 20 amino acids, and
a step e) of detecting, in said amino acid sequence, an additional motif S1:
a step f) of detecting, in said amino acid sequence, a second additional motif S2:
in which, if said amino acid sequence comprises said NADPH motif from step a), said FAD motif from step b), said 3 to 7 regions capable of forming transmembrane helices from step c), said at least 2 bis-histidyl motifs from step d), the additional motif S1 from step e) and the additional motif S2 from step f), then said amino acid sequence is capable of encoding a prokaryotic Nox protein.
Another aspect of the invention finally relates to the use of a detergent from the maltoside family for solubilization of Nox proteins.
In another embodiment, the invention relates to the use of a detergent from the maltoside family for solubilization of Nox proteins, said detergent preferably being selected from the group of dodecyl-maltosides.
In another embodiment, the invention relates to the use of a detergent from the maltoside family for solubilization of Nox proteins, said detergent preferably being selected from the group constituted by:
The program used for identifying the Nox sequences from the bacterial genomes was written in Python 3.0. This program is based on the use of 3 complementary filters.
The first 2 filters select the sequences containing the motifs G[I/S/V/L]G[V/I/A/F][T/A/S][P/Y/T/A] (SEQ ID NO: 4) (NADPH-binding domain) and H[P/S/A]F[T/S][L/I/M/V] (SEQ ID NO: 6) (FAD-binding domain), defined on the basis of the sequences of the 48 most similar proteins relative to human Nox2 protein (Uniprot code: P04839). The sequences of the 48 proteins were identified by performing a BlastP search on the public databases.
The third filter selects the sequences that contain the 4 histidine residues responsible for coordinating the 2 haem parts of the cytochrome b motif. Thus, a search is carried out for 2 bis-histidyl motifs separated by at least 20 amino acids (i.e. the size of a transmembrane helix), each of the bis-histidyl motifs being constituted by 2 histidine residues separated by 12, 13 or 14 amino acid residues. The TMHMM software was used for locating the transmembrane α helices. Sequences with more than 7 predicted α helices (i.e. the number of helices found in Duox1-2) were not retained, whereas the sequences with at least 3 predicted α helices were selected to take account of possible errors of prediction.
The multiple alignments of the sequences of Nox proteins identified were carried out using the Clustal Omega software, performing 2 iterations. A phylogenetic tree was constructed using the PhyML 3.0 software based on the sequences of 150 putative Nox proteins (
The gene coding for the protein SpNox (Uniprot code #Q8CZ28) from Streptococcus pneumoniae R6 (taxonomic identifier #171101) was synthesized and optimized for heterologous expression in Escherichia coli. Bases coding for a polyhistidine tag as well as a thrombin digestion site were added on the 5′ end of the complementary DNA encoding SpNox.
Translation of the gene introduces 18 amino acids on the amino-terminal end of the protein before digestion by thrombin and only 4 amino acids (GSRS) afterwards. The gene thus modified was cloned into the pUC57 vector.
In order to produce the protein in large quantities, the modified gene was inserted in the pQlinkN expression vector between the NotI and BamHI restriction sites under the control of a tac promoter, inducible with IPTG (isopropyl β-D-1-thiogalactopyranoside), and this vector also carries an ampicillin resistance gene. The vector was introduced by transformation by thermal shock into a chloramphenicol-resistant strain of Escherichia coli BL21 (DE3) pLysS (phenotype: F− ompT hsdSB (rB−mB−) gal dcm (DE3) pLysS (CamR)). The bacteria were then immediately cultured for 1 h at 37° C. in LB (Luria Bertani) medium without selection pressure. A fraction of this culture was then spread on a Petri dish and incubated overnight at 37° C.
Starting from a single colony, a culture was started in liquid medium in the presence of chloramphenicol (34 μg/ml) and ampicillin (100 μg/ml) at 37° C., with stirring overnight. The culture was inoculated from the pre-culture at dilution to 1/20th of the final volume. The culture was placed at 37° C. in an orbital stirrer with a speed of 180 rpm. The optical density of the medium was monitored at 600 nm until a value of 1.5 was reached. Overexpression of the SpNOX protein was induced by adding a concentration of 0.1 mM of IPTG to the medium for 4 h at 210 rpm. At the end of culture, the bacteria were collected by centrifugation at low speed (7000 g, 30 mM, 4° C.) and were stored at −80° C. The expression level of the SpNOX protein was then monitored by SDS-PAGE analysis and confirmed by Western blot (
The bacterial pellet was taken up in a buffer of 50 mM Tris HCl at pH 7, 250 mM NaCl and 10% glycerol, to which a cocktail of anti-proteases (complete ULTRA Tablets, EDTA-free, Roche Applied Science) was added, as well as DNase at a concentration of 10 μg/ml (Sigma-Aldrich). The cells were lysed mechanically using a microfluidizer (M-1105, Microfluidics, USA) at 1400 Psi in 5 successive passes. Visual monitoring of sample viscosity is important in this step. The unlysed bacteria, the cellular debris and the inclusion bodies were separated by centrifugation at low speed (8000 g, 20 min., 4° C.). The supernatant was centrifuged at very high speed for 1 h at 250000 g, at 4° C. The pellet was taken up in a buffer of 50 mM Tris pH7 and the total protein concentration was determined by the BCA (bicinchoninic acid assay) technique and the absorbance was measured at 562 nm according to the protocol provided by the supplier (Sigma-Aldrich). The sample was stored at −80° C. at a concentration of 20 mg/ml of total protein.
The SpNox activity was tested in this step in order to check its integrity after the lysis step (
The membranes were dissolved in a buffer of 50 mM Tris HCl at pH 7, 300 mM NaCl and 5 mM of MNG3 (lauryl Maltose NeoPentyl Glycol—NG310—MNG-Affymetrix to a final concentration of 0.5 mg/ml of total protein. Ni-NTA Sepharose High performance resin (GE Healthcare Life Sciences), initially equilibrated in the same solubilization buffer, was added to the sample to a concentration of 1 ml of resin to 15 mg of protein. The whole was incubated with stirring at 4° C. overnight.
The resin loaded with the protein was deposited on a gravity column until the filtrate had flowed away completely. Several washing operations were carried out with a buffer of 50 mM Tris HCl pH7, 300 mM NaCl, 50 μM MNG3 and 50 mM imidazole until the absorbance at 280 nm had stabilized. A plateau at 300 mM of imidazole makes it possible to elute the protein by spreading the retentate at 3 to 5 ml.
The spectral signature of the various fractions was evaluated between 200 and 700 nm and purification was monitored by SDS-PAGE. The purest fractions were combined and were concentrated by membrane filtration with a size limit of 30 kDa (Amicon® Ultra-4—Millipore). The sample thus prepared was centrifuged at high speed, 150000 g, for 20 minutes at 4° C. The yield and the quality of purification were evaluated by spectroscopy (
In order to perfect the purification of SpNox, a step of size exclusion chromatography is necessary. For this, a Superdex 200 10/300 GL column (GE Healthcare Life Sciences) was used with a mobile phase constituted by a buffer of 50 mM Tris HCl pH 7, 300 mM NaCl and 50 μM MNG3. Elution is monitored by absorbance at 280 nm using a system of the ÄKTA FPLC type (GE Healthcare Life Sciences). The purity of the protein was analysed by SDS-PAGE and mass spectrometry (
Its activity was assayed (
A CM Sepharose Fast Flow affinity column (GE Healthcare Life Sciences) was used in order to deplete the excess detergent from the SpNox protein. The sample was diluted in order to bring the concentration of NaCl below the threshold of 15 mM with the aid of a buffer of 50 mM Tris HCl pH7 and 20 μm MNG3. The quantity of resin used depends on the quantity of protein available with a ratio of 0.5 ml of resin for 3 mg of SpNox. Ten column volumes were used for washing the surplus MNG3. 65 to 70% of the protein was recovered.
The sample was checked by FT-IR spectroscopy and analytical ultracentrifugation (
The sample obtained may be concentrated and/or stored.
The oxidase activity is determined by measuring the kinetics of reduction of cytochrome c or Nitro blue tetrazolium chloride, by O2.−, while monitoring the increase in absorbance at 550 nm (molar extinction coefficient of cytochrome c: ε550=21.1 mM−1·cm−1).
This assay is carried out on 10 μg of membrane proteins of BL21 (DE3) pLys overexpressing SpNox in a total volume of 1 mL. The spectrophotometer is used for kinetics of 5 min and at a wavelength of 550 nm.
The buffer for the reaction is the buffer for resuspending the membranes (50 mM Tris pH7).
The blank test is carried out on 10 μg of membrane of BL21 (DE3) pLys overexpressing SpNox and 100 μm cytochrome c. The reaction is then started by adding 100 μM FAD and 200 μM NADPH.
The NADPH oxidase activity is then quantified by calculating the slope of the curve thus obtained (
Starting from 10 μg of membrane proteins of BL21 (DE3) pLys overexpressing SpNox, the d(OD)/D(t) ratio obtained is between 0.1 and 0.5.
Measurement of the NADPH oxidase activity of the membrane proteins of BL21 (DE3) pLysS not overexpressing SpNox (transformed by the empty vector) on the reduction of cytochrome c makes it possible to confirm that the reduction observed beforehand is indeed due to the presence of SpNox in the membranes.
This assay is carried out on 1 μg SpNox in a total volume of 1 mL using the spectrophotometer for kinetics of 5 mM and at a wavelength of 550 nm. The reaction buffer is the protein storage buffer (50 mM Tris pH7, 300 mM NaCl, 0.03±0.02 mM MNG3).
The blank test is carried out on 1 μg SpNox and 100 μm cytochrome c. The reaction is then started by adding 100 μM FAD and 200 μM NADPH.
The NADPH oxidase activity is then measured using the slope of the curve thus obtained. The result obtained is 24 nanomoles of cytochrome c reduced per minute per microgram of SpNox (or more generally, between 12 and 60 nanomoles of cytochrome c reduced per minute per microgram of SpNox).
This activity can be measured at different concentrations of NaCl (0-1M), KCl and MNG3.
The following protocol, described for PLB985 cells, can be applied to other cell lines such as HEK 293 (Human Embryonic Kidney) and PMN (Polymorphonuclear leukocytes).
This assay is carried out on 30 to 100 μg of PLB985 membranes in a total volume of 1000 μL.
The reaction buffer is PBS 1×.
Evaluation of the effect of a molecule A on the NADPH oxidase activity of eukaryotic Nox
30-100 μg of PLB985 membrane is incubated with increasing concentrations of a molecule A for 5-20 min at 25° C. in a volume of 100 μl. 0.5-4 mM of arachidonic acid, 10-30 μM of FAD and 100-500 nM of trimer (unprenylated trimer, rac 1Q61L) are added to the reaction mixture and incubated with the membranes for 5-20 min at 25° C. according to the protocol described in Mizrahi et al., A prenylated p47phox-p67phox-Rac1 chimera is a Quintessential NADPH oxidase activator: membrane association and functional capacity. J Biol Chem, 2010. 285(33): p. 25485-99.
The reaction is then started by adding 900 μL of PBS containing the substrate of NADPH oxidase, 100-400 μm NADPH (final) and 100 μm final of cytochrome c (or of NBT), which will allow the reaction to be monitored.
After homogenization, the reaction kinetics at 550 nm is monitored for 5 min.
This technique is based on the principle of dissipation of energy by emission of light by a compound when it returns from an excited state to a ground state. The chemiluminescence reaction applied is based on an oxidation reaction of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) dependent on the peroxidase HRP (horseradish peroxidase). The return of excited luminol to its ground state is accompanied by emission of light, which is quantified by a luminometer. The emission of light is directly proportional to the NADPH oxidase activity.
This assay is carried out on 0.1-1 μg of SpNox in a total volume of 100-200 μL. The plate reader is used for kinetics of 5 min-20 min
The reaction buffer is 50 mM Tris pH7, 300 mM NaCl, 0.03±0.02 mM MNG3.
Evaluation of the effect of a molecule A on the NADPH oxidase activity of SpNox
0.1-1 μg SpNox is incubated with increasing concentrations of a molecule A for 5-20 min at 25° C. 10-100 μm luminol and 10-50 U/ml HRP are added to the reaction mixture and incubated with the SpNox protein and molecule A at 25° C. away from the light, for 5 min.
The reaction is then started by adding 20-50 μm FAD and 20-150 μM NADPH.
For each molecule A tested, 2 checks should be done:
By replacing the SpNox protein with 50-500 μM pyrogallol (without adding FAD or NADPH), which will undergo autoxidation in the presence of oxygen, releasing superoxide ions in solution.
In the absence of SpNox in the reaction mixture, in order to verify that the chemiluminescence measured can indeed be attributed to SpNox and not to molecule A. Incubation of SpNox with molecule A and with luminol and HRP may be done simultaneously.
The oxidase activity is determined by measuring the kinetics of reduction of cytochrome c (or Nitro blue tetrazolium chloride) by O2.−, by monitoring the increase in absorbance at 550 nm (molar extinction coefficient of cytochrome c: ε550=21.1 mM−1.cm−1).
This assay is carried out on 0.1-1 μg SpNox in a total volume of 100-200 μL. The plate reader is used for kinetics of 5-20 min and at a wavelength of 550 nm. The reaction buffer is the SpNox storage buffer (50 mM Tris pH7, 300 mM NaCl, 0.03 mM±0.02 mM MNG3)
Evaluation of the effect of a molecule A on the NADPH oxidase activity of SpNox
0.1-1 μg SpNox is incubated with increasing concentrations of a molecule A for 5-20 mM at 25° C. 10-30 μM cytochrome c is added to the reaction mixture and the reaction is then started by adding 20-50 μm FAD and 20-150 μM NADPH.
The NADPH oxidase activity is then quantified by calculating the slope of the curve obtained following reduction of cytochrome c (or of NBT).
For each molecule A tested, 2 checks should be done:
By replacing the SpNox protein with 50-500 μM pyrogallol (without adding FAD or NADPH), which will undergo autoxidation in the presence of oxygen, releasing superoxide ions in solution. This assay will make it possible to verify that the effect of molecule A is exerted on SpNox and not on the superoxide radicals that it produces (scavenger effect).
In the absence of SpNox in the reaction mixture, in order to verify that the chemiluminescence measured can indeed be attributed to SpNox and not to molecule A. This activity can be measured at various concentrations of NaCl (0-1M) and of MNG3.
General protocol for measuring the kinetics of reduction of NBT
The oxidase activity is determined by measuring the kinetics of reduction of NBT by the O2.− ions, by monitoring the increase in absorbance at 560 nm (molar extinction coefficient of NBT: ε560=30 000 mM−1.cm−1). It is also possible to use a wavelength of 550 nm.
SpNox is incubated with a reaction mixture containing the molecule to be tested, NBT, FAD and the protein storage buffer for 5 to 20 min at 25° C. The reaction is then started by adding NADPH.
The NADPH oxidase activity is then quantified at the end point by determining the intensity of the coloration compared to a positive control. The positive control is carried out with the SpNox protein alone, without the molecule to be tested.
The compounds present in each well of the plate are, as final concentration:
All the experiments were carried out in triplicate and each molecule was tested at 5 different concentrations. The concentration range is deliberately wide so as to be able to include the possible IC50 values of each molecule. The concentrations selected are: 0.01-0.1-1-10-100 μM.
Experimental validation of the high-throughput screening assay:
Two preliminary assays are carried out in the presence of:
a bioactive molecule (inhibitory effect) on SpNox, diphenyleneiodonium chloride (DPI);
a bio-inactive molecule (without inhibitory effect) on SpNox, DMSO. The suitability, quality and feasibility of the high-throughput approach are evaluated by establishing a “Z′ score” (Zhang et al., J Biomol Screen, 4(2): 67-73, 1999), namely:
in which:
σc+/σc−: standard deviations of the results obtained for a positive/negative control;
μc+/μc−: mean values of the results obtained for a positive/negative control.
If Z′=1, the suitability, quality and feasibility of the screening assay are ideal.
If 0.5<Z′<1, the suitability, quality and feasibility of the screening assay are excellent.
If 0<Z′<0.5, the suitability, quality and feasibility of the screening assay are marginal.
If Z′<0, the suitability, quality and feasibility of the screening assay cannot be interpreted.
Each preliminary assay must be carried out a minimum of 20 times in order to yield significant values to allow calculation of the “Z′ score”.
In the present case, the “Z′ score” is between 0.7 and 0.8 according to the experiments.
Consequently, an assay in a 96-well plate with SpNox with detection by colorimetry (NBT) constitutes a sufficiently robust and reliable assay to be applied with high throughput.
Screening against commercial molecules targeting the eukaryotic NOX:
A total of 8 molecules was selected and tested, 7 inhibitors and 1 activator of the NADPH oxidase activity of the eukaryotic NOX (Table 1).
The NADPH oxidase activity of the enzyme may be monitored by colorimetry (or chemiluminescence) (
The experimental results thus demonstrate that ebselen, celastrol, Thr101, VAS2870, catechin, epicatechin gallate and DPI are inhibitors of SpNox (
These results therefore confirm that it is possible to screen molecules modulating the activity of the eukaryotic Nox by using a prokaryotic protein, such as SpNox.
Moreover, these results also indicate that the prokaryotic Nox proteins can also be used for identifying antibacterial molecules, whether or not said molecules have an effect on the eukaryotic Nox proteins.
In fact, the molecules that have an activating effect on the prokaryotic Nox proteins are capable of having a bactericidal or bacteriostatic effect on bacteria, by increasing the production of ROS in the bacteria.
This technique is based on the principle of dissipation of energy by emission of light by a compound when it returns from an excited state to its initial state. The chemiluminescence reaction applied is based on an oxidation reaction of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) dependent on horseradish peroxidase (HRP) and the intrinsic peroxidase MPO. The return of excited luminol to its ground state is accompanied by emission of light, which is quantified by a luminometer. The emission of light is directly proportional to the NADPH oxidase activity according to the protocol described in Dahlgren, C. and A. Karlsson, Respiratory burst in human neutrophils. J Immunol Methods, 1999. 232(1-2): p. 3-14.
This assay is carried out on 5×105 cells in a total volume of 250 μL. The plate reader is used for kinetics of 15-45 min
The reaction buffer is PBS.
Evaluation of the effect of a molecule A on the NADPH oxidase activity of eukaryotic Nox
5×105 PLB985 cells are incubated with increasing concentrations of a molecule A for 5-20 min at 25° C. 20 mM glucose, 20 μM luminol and 10 U/ml HRP are added to the reaction mixture and incubated with the cells and molecule A at 25° C., away from the light for 5 min.
The reaction is then started by adding a soluble activator of NADPH oxidase:
After homogenization, the reaction kinetics is monitored for 15-45 min.
Select the programme depending on the activator used in the experiment:
Zymosan programme: reading for 30 min with one measurement every 15 seconds after stimulation with OPZ (OPsonized Zymosan)
PMA programme: reading for 30 min with one measurement every 15 seconds after stimulation with PMA
fMLP programme: reading for 15 min with one measurement every 5 seconds after stimulation with fMLP.
Rates are obtained from 0.00015 to 0.0005 mM of cytochrome c reduced per minute for 5 micrograms of total membrane proteins.
This assay is carried out on 4-20 μg of PLB985 membrane in a total volume of 200 μL. The plate reader is used for kinetics of 5 min
The reaction buffer is PBS 1×.
Evaluation of the effect of a molecule A on the NADPH oxidase activity of eukaryotic Nox
4-20 μg of PLB985 membrane is incubated with increasing concentrations of a molecule A for 5 to 20 min at 25° C. in a volume of 100 μl. 0.10-1 mM of arachidonic acid, 10-50 μm of FAD and the trimer are added to the reaction mixture and incubated with the membranes for 5-20 min at 25° C. according to the protocol described in Mizrahi, A., et al., A prenylated p47phox-p67phox-Rac1 chimera is a Quintessential NADPH oxidase activator: membrane association and functional capacity. J Biol Chem, 2010. 285(33): p. 25485-99.
The reaction is then started by adding 100 μL of PBS containing the substrate of NADPH oxidase, 100-400 μm NADPH and 100 μm of cytochrome c (or NBT), which will allow monitoring of the reaction.
After homogenization, the reaction kinetics at 550 nm is monitored for 5 min.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1453616 | Apr 2014 | FR | national |
This application is a continuation application of U.S. patent application Ser. No. 15/306,137 filed on Oct. 24, 2016, which is the U.S. national phase under 35 U.S.C. 371 of international patent application number PCT/FR2015/051101 filed on Apr. 22, 2015, which designated the U.S. and claims priority to French patent application number 1453616 filed on Apr. 22, 2014.
| Number | Date | Country | |
|---|---|---|---|
| 20200181583 A1 | Jun 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15306137 | US | |
| Child | 16716202 | US |
