Annotation of the results obtained in 2018
As a result of the work carried out in 2018, our research team was able to come closer to understanding the spatial structure of a protein from a fundamentally new family that does not have the homologs described, the fungal luciferase. Unfortunately, due to the low predictive power of algorithms that calculate three-dimensional spatial structures for proteins whose homolog structure has not been described experimentally, we were unable to obtain a reliable model of the three-dimensional structure of luciferase, but we have quite successfully determined the conservative parts necessary for the functioning of luciferase, predicted elements of the secondary structure (6 alpha helices and 5 to 6 beta sites for luciferases of various fungi). Using experimental approaches of directional and random mutagenesis, we determined the role of cysteine ​​residues in the N. nambi luciferase protein: in particular, the point substitution for serine residues or residues typical for the other fungi luciferases, a decrease in luminescence was observed to various degrees; during random mutagenesis, a tendency towards disappearance of luminescent activity was revealed with the replacement of amino acid residues Asp127, Arg136, Ser144, Asp173, Arg176, Glu237, Glu238, that provide a base for further research by site-directed mutagenesis of these particular amino acids. A method of isolation of functionally-active luciferase was developed through expression of gene nnLuz-CHis in P. pastoris cells; analysis of the circular dichroism spectrum of this protein sample indicates that luciferase NNLuz-CHis isolated after expression in P.pastoris cells has a well defined secondary structure and can be used for further establishment of its structure using methods of X-ray crystallography; and NMR. In addition, we have created a specific quantitative test-system for determination of caffeic acid, operating within substrate concentrations range from 10 mkM to 12.5 mM, and shown the detection limit up to 50 pmol.

 

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Annotation of the results obtained in 2016
To address the challenge of detailed description of a new unique bioluminescent system of fungi, for the first year of the Project we planned obtaining a sample of purified fungal luciferase – the enzyme responsible of light emission. This work was carried out using the biomass of the tropical fungus Neonothopanus nambi cultivated in our laboratory. Our preliminary results have shown that soaking live mycelium of N, nambi in distilled water leads to multifold enhancement of its bioluminescence. We have also found that luciferase activity is not secreted into the cultural liquid, and is localized solely within mycelium cells. Based on these observations we developed a method for preparation of homogenous luciferase which includes 4 main stages: preparation of N.nambi mycelium lysate, concentration of target protein, chromatographic separation and native gel electrophoresis. At the first stage, mycelium cells were destroyed by freezing, mechanical homogenization of frozen mass by grinding in a mortar and ultrasonication. Extraction of the target enzyme utilized microsomal fraction of the homogenate which contained nearly all luminescence activity and was prepared by three-stage centrifugation of the extract. Maximal concentration of the luciferase was achieved by its solubilization into the micelles of dodecylmaltoside detergent. Purification of the luciferase was performed in several stages utilizing anion exchange chromatography at DEAE-Sepharose and gel filtration using Superdex 200 sorbent. At the next stage we applied gel electrophoresis in standard denaturing as well as in native (non-denaturing) and conditions to find a band corresponding to luminescence activity. The required solid result was obtained only using the latter approach. Thus, we designed a procedure for 2D non-denaturing electrophoresis with subsequent bioluminescence detection by incubation of gel in a buffer containing synthetic fungal luciferin. This approach allowed identification of a luminescent spot on 2D gel. Its subsequent staining with silver allowed reliable identification of the target spot and its precise cutting from the gel. A series of described experiments provided purified protein in the amounts required for Edman degradation sequencing and mass-spectrometric sequencing. In order to address the task concerning identification of structural fragments of fungal luciferin molecule responsible of luminescent reaction and to obtain fungal luciferin analogs with modified spectral properties, we synthesized six derivatives of fungal luciferin containing different electron donating substituents in the benzene ring: (E)-3,4-dihydroxy-6-(4-hydroxystyryl)-2H-pyran-2-one; (E)-3,4-dihydroxy -6-(2-(6-hydroxynaphthalen-2-yl)vinyl)-2H-pyran-2-one; (E)-3,4-dihydroxy -6-(2-(thien-2-yl)vinyl)-2H-pyran-2-one; (E)-6-(2-(1H-indol-3-yl)vinyl)-3,4-dihydroxy-2H-pyran-2-one; (E)-6-(4-(diethylamino)styryl)-3,4-dihydroxy-2H-pyran-2-one; (E)-6-(2-(1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinolin-9-yl)vinyl)-3,4- dihydroxy-2H-pyran-2-one. All of them except the thiophene derivative showed bioluminescent activities. We studied spectral properties of five active compounds. In case of naphthalene derivative its bioluminescence maximum is red-shifted compared to natural luciferin, while the rest 4 analogs demonstrated blue-shifted spectra. Our data indicate that aminated analogs (E)-6-(4-(diethylamino)styryl)-3,4-dihydroxy-2H-pyran-2-one and (E)-6-(2-(1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinolin-9-yl)vinyl)-3,4- dihydroxy-2H-pyran-2-one demonstrate higher activity compared to natural luciferin 2- and 4-fold respectively. The derivatives of naphthalene and indol are less active. The closest luciferin analog containing phenol group instead of catechol demonstrated activity at 75% level compared natural luciferin. The above data clearly indicate that the key role in the bioluminescence reaction belongs to a pyranone fragment of luciferin molecule, whereas a catechol fragment plays a role of auxochromous substituent. The possibility of wide variation of luciferin structure at catechol fragment with retaining the activity of these synthetic analogs provides clear evidence that the luminescent reaction involves only a pyranone moiety of fungal luciferin. In order to establish the structure of the luminescent reaction product – fungal oxyluciferin – we performed incubation of the reaction substrate (luciferin) with the protein extract of N. nambi, containing active luciferase. In the course of incubation we monitored the chemical composition of the reaction mixture by HPLC. We found that the primarily formed oxyluciferin undergoes further transformations to form 4 products. We isolated these products and determined their structures. Based on those structures we proposed the structure of oxyluciferin itself to be (2Z,5E)-6-(3,4-dihydroxyphenyl)-2-hydroxy-4-oxohexa-2,5-dienoic acid. We also confirmed this structure by total synthesis. The synthetic oxyluciferin showed identical chromatographic retention time and UV absorbance spectrum to the enzymatically obtained oxyluciferin. Based on the above data we proposed the scheme describing the 2-stage mechanism of bioluminescent reaction in fungi.

 

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Annotation of the results obtained in 2017
To solve the problem of detailed study of the new unique fungal bioluminescent system, it was planned to obtain a cDNA library of the fungus Neonothopanus nambi during the second year of the project. For this purpose total RNA was isolated from the biomass of the bioluminescent mycelium of this fungus, and then total RNA was enriched with mRNA fraction with magnetic particles bearing oligo-dT oligonucleotides. In the next step, a DNA-RNA hybrid was synthesized on the matrix of this enriched RNA fraction and the cDNA of Neonothopanus nambi fungus was amplified to form a double-stranded DNA library. This library was prepared for high-performance sequencing on the Illumina platform by ligation of TruSeq (Illumina) adapters and specific amplification with primers from the manufacturer was performed to enrich the molecules successfully ligated. The resulting PCR product was purified and, by excision from the agarose electrophoresis gel, the corresponding band was enriched with fragments of 300-500 b.p. length. After this, the DNA specimen was denatured in 0.1 M NaOH and dissolved in HT1 buffer (Illumina), to a concentration of 10 pM. Clusters were then generated in cBot (Illumina) using the TruSeq PE Cluster Kit v3-cBot-HS reagent kit and sequencing was performed using a paired reading (2x100bp) protocol using the Hiseq200 and TruSeq SBS chemistry kits. Demultiplexing was performed using CASAVA-1.8.2 (Illumina). Paired readings received on the Illumina platform were analyzed and fragments of adapter sequences were removed from them, as well as nucleotide sequences having low Phred values. Then short readings less than 75 nucleotides long were removed from the data. Both phases were performed by the Trimmomatic program using the following parameters: ILLUMINACLIP: ../ TruSeq3-PE-2.fa: 2: 30: 10 LEADING: 10 TRAILING: 10 SLIDINGWINDOW: 5: 15 MINLEN: 70 HEADCROP: 10. The resulting processed sequences were further filtered: purification from potential contaminants was carried out using the Bowtie2 program and the GenBank RefSeq database. Finally, from the remaining readings, the assembly of the transcriptome Neonothopanus nambi de novo was carried out using the Trinity program with default program parameters. To obtain the amino acid sequence of luciferase, hydrolysis of the protein in the polyacrylamide gel was carried out. A polyacrylamide gel fragment corresponding to the protein band or spot was excised and prepared for mass spectroscopy to obtain a peptide mixture. For this peptide mixture, a mass spectrometric study was performed using the LC-MS method. Among the obtained mass-spectrometric data processed by the operator, candidates were searched for the role of luciferase in the transcriptome of N. nambi. As a result, several possible candidate genes were identified, which were subsequently cloned into E. coli bacteria. However, the results of the activity test have produced questionable results that do not allow the identification of luciferase with confidence, which, as it later turned out, was associated with suboptimal protein expression conditions, as well as the fact that luciferase of N. nambi fungus is a membrane protein, which makes it difficult to fold properly in prokaryotic cells. In connection with the obtained data, we decided to create a library of cDNA of N. nambi fungus and clone it into yeast for subsequent identification of a clone expressing luciferase. To establish the nucleotide sequence of the gene for the novel luciferase of N. nambi, a cDNA library was created. For this purpose total RNA was isolated from the fresh mycelium of N. nambi fungus, enriched with mRNA fraction and used as a template for the synthesis of the cDNA library. This library was inclined to the vector GAP-pPic9K for expression in Pichia pastoris yeast. A linearized library of cDNA plasmids was used to transform the Pichia pastoris GS115 strain by electroporation. The transformed cells were spread onto Petri dishes with a solid growth medium and grown to the condition of individual distinct colonies. The final library of N. nambi cDNA contained about 1 million yeast colonies. Each Petri dish with transformants was sprayed with a solution of luciferin fungus and analyzed by IVIS Spectrum CT (PerkinElmer). Genomic DNA was isolated from the luminescent clones and amplification with specific primers to the inserted sequence was performed. All sequenced clones contained the same sequence corresponding to the sequence of the nnLuz gene. Finally, our plans included the cloning of the gene for the novel N. nambi luciferase, the production of the bacterial and/or eukaryotic strain of the recombinant luciferase producer strain. We selected bacterial vector pET23b, yeast vector pGAP-Kan, and the C1 vector for expression in mammalian cell cultures. Based on these vectors, plasmids encoding the nnLuz gene were created, which were named, respectively, nnLuz/pGAP_Kan, nnLuz/pET23, nnLuz/C1. The work of these DNA constructs was tested in the cells of the corresponding organisms: nnLuz/pGAP_Kan in the yeast of P. pastoris GS115, nnLuz/pET23 in the E. coli BL21 CodonPlus (DE3) bacteria  and nnLuz/C1 in human cell cultures HEK293T, HeLa and mouse cell culture CT26. In each case in response to the addition of fungal luciferin to the cells, bioluminescence appeared, which could be detected with a Glomax 20/20 luminometer (Promega). With the vectors nnLuz/pGAP_Kan, nnLuz/pET23, producer strains of recombinant luciferase were created: yeast strain P. pastoris GS115 and bacterial strain E. coli BL21 CodonPlus (DE3), respectively.

 

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