Henlea earthworm bioluminescence comprises violet-blue BRET from tryptophan 2-carboxylate to deazaflavin cofactor.

We recently identified the deazaflavin cofactor as a light emitter in novel bioluminescence (BL) system from Siberian earthworms Henlea sp. (Petushkov et al., 2023, Org. Biomol. Chem. 21:415-427). In the present communication we compared in vitro BL spectra in the absence and in the presence of the cofactor and found a wavelength shift from 420 to 476 nm. This violet-blue BRET to deazaflavin cofactor (acceptor of photonless transfer) masks the actual oxyluciferin as an emitter (BRET donor) in the novel BL system. The best candidate for that masked chromophore is tryptophan 2-carboxylate (T2C) found previously as a building block in some natural products isolated from Henlea sp. (Dubinnyi et al., 2020, ChemSelect 5:13155-13159). We synthesized T2C and acetyl-T2C, verified their presence in earthworms by nanoflow-HRMS, explored spectral properties of excitation and emission spectra and found a chain of excitation/emission maxima with a perfect potential for BRET: 300 nm (excitation of T2C) - 420 nm (emission of T2C) - 420 nm (excitation of deazaflavin) - 476 nm (emission of deazaflavin, BL). An array of natural products with T2C chromophore are present in BL earthworms as candidates for novel oxyluciferin. We demonstrated for the Henlea BL that the energy of the excited state of the T2C chromophore is transferred by the Förster mechanism and then emitted by deazaflavin (BRET), similarly to known examples: aequorin-GFP in Aequorea victoria and antenna proteins in bacterial BL systems (lumazine from Photobacterium and yellow fluorescent protein from Vibrio fischeri strain Y1).

A hybrid pathway for self-sustained luminescence.

The fungal bioluminescence pathway can be reconstituted in other organisms allowing luminescence imaging without exogenously supplied substrate. The pathway starts from hispidin biosynthesis-a step catalyzed by a large fungal polyketide synthase that requires a posttranslational modification for activity. Here, we report identification of alternative compact hispidin synthases encoded by a phylogenetically diverse group of plants. A hybrid bioluminescence pathway that combines plant and fungal genes is more compact, not dependent on availability of machinery for posttranslational modifications, and confers autonomous bioluminescence in yeast, mammalian, and plant hosts. The compact size of plant hispidin synthases enables additional modes of delivery of autoluminescence, such as delivery with viral vectors.

Structure elucidation of Keroplatus (Diptera:Keroplatidae) fungus gnat oxyluciferin.

Bioluminescence of insects is a well-known natural phenomenon in the focus of interest of scientific research. While the mechanisms of bioluminescence in Coleoptera have been extensively studied, there is a lack of information about the chemistry of light emission in Diptera species. Here we report the Keroplatus spp. oxyluciferin structure elucidation and identification as 3-hydroxykynurenic acid. Additionally, the present study provides the first direct evidence of the relationship between the bioluminescent systems of Orfelia and Keroplatus. However, the properties of the putative Orfelia oxyluciferin suggest that the light emission mechanisms are not identical.

Domain Truncation in Hispidin Synthase Orthologs from Non-Bioluminescent Fungi Does Not Lead to Hispidin Biosynthesis.

Hispidin is a polyketide found in plants and fungi. In bioluminescent fungi, hispidin serves as a precursor of luciferin and is produced by hispidin synthases. Previous studies revealed that hispidin synthases differ in orthologous polyketide synthases from non-bioluminescent fungi by the absence of two domains with predicted ketoreductase and dehydratase activities. Here, we investigated the hypothesis that the loss of these domains in evolution led to the production of hispidin and the emergence of bioluminescence. We cloned three orthologous polyketide synthases from non-bioluminescent fungi, as well as their truncated variants, and assessed their ability to produce hispidin in a bioluminescence assay in yeast. Interestingly, expression of the full-length enzyme hsPKS resulted in dim luminescence, indicating that small amounts of hispidin are likely being produced as side products of the main reaction. Deletion of the ketoreductase and dehydratase domains resulted in no luminescence. Thus, domain truncation by itself does not appear to be a sufficient step for the emergence of efficient hispidin synthases from orthologous polyketide synthases. At the same time, the production of small amounts of hispidin or related compounds by full-length enzymes suggests that ancestral fungal species were well-positioned for the evolution of bioluminescence.

Mesenchymal stromal cell-derived extracellular vesicles afford neuroprotection by modulating PI3K/AKT pathway and calcium oscillations.

Mesenchymal stromal cells (MSC) are widely recognized as potential effectors in neuroprotective therapy. The protective properties of MSC were considered to be associated with the secretion of extracellular vesicles (MSC-EV). We explored the effects of MSC-EV in vivo on models of traumatic and hypoxia-ischemia (HI) brain injury. Neuroprotective mechanisms triggered by MSC-EV were also studied in vitro using a primary neuroglial culture. Intranasal administration of MSC-EV reduced the volume of traumatic brain damage, correlating with a recovery of sensorimotor functions. Neonatal HI-induced brain damage was mitigated by the MSC-EV administration. This therapy also promoted the recovery of sensorimotor functions, implying enhanced neuroplasticity, and MSC-EV-induced growth of neurites in vitro supports this. In the in vitro ischemic model, MSC-EV prevented cell calcium (Ca2+) overload and subsequent cell death. In mixed neuroglial culture, MSC-EV induced inositol trisphosphate (IP3) receptor-related Ca2+ oscillations in astrocytes were associated with resistance to calcium overload not only in astrocytes but also in co-cultured neurons, demonstrating intercellular positive crosstalk between neural cells. This implies that phosphatidylinositol 3-Kinase/AKT signaling is one of the main pathways in MSC-EV-mediated protection of neural cells exposed to ischemic challenge. Components of this pathway were identified among the most enriched categories in the MSC-EV proteome.

Molecular Basis of the Slow Growth of Mycoplasma hominis on Different Energy Sources.

Mycoplasma hominis is an opportunistic urogenital pathogen in vertebrates. It is a non-glycolytic species that produces energy via arginine degradation. Among genital mycoplasmas, M. hominis is the most commonly reported to play a role in systemic infections and can persist in the host for a long time. However, it is unclear how M. hominis proceeds under arginine limitation. The recent metabolic reconstruction of M. hominis has demonstrated its ability to catabolize deoxyribose phosphate to produce ATP. In this study, we cultivated M. hominis on two different energy sources (arginine and thymidine) and demonstrated the differences in growth rate, antibiotic sensitivity, and biofilm formation. Using label-free quantitative proteomics, we compared the proteome of M. hominis under these conditions. A total of 466 proteins were identified from M. hominis, representing approximately 85% of the predicted proteome, while the levels of 94 proteins changed significantly. As expected, we observed changes in the levels of metabolic enzymes. The energy source strongly affects the synthesis of enzymes related to RNA modifications and ribosome assembly. The translocation of lipoproteins and other membrane-associated proteins was also impaired. Our study, the first global characterization of the proteomic switching of M. hominis in arginine-deficiency media, illustrates energy source-dependent control of pathogenicity factors and can help to determine the mechanisms underlying the interaction between the growth rate and fitness of genome-reduced bacteria.

Gene Silencing through CRISPR Interference in Mycoplasmas.

Mycoplasmas are pathogenic, genome-reduced bacteria. The development of such fields of science as system and synthetic biology is closely associated with them. Despite intensive research of different representatives of this genus, genetic manipulations remain challenging in mycoplasmas. Here we demonstrate a single-plasmid transposon-based CRISPRi system for the repression of gene expression in mycoplasmas. We show that selected expression determinants provide a level of dCas9 that does not lead to a significant slow-down of mycoplasma growth. For the first time we describe the proteomic response of genome-reduced bacteria to the expression of exogenous dcas9. The functionality of the resulting vector is confirmed by targeting the three genes coding transcription factors-fur, essential spxA, whiA, and histone-like protein hup1 in Mycoplasma gallisepticum. As a result, the expression level of each gene was decreased tenfold and influenced the mRNA level of predicted targets of transcription factors. To illustrate the versatility of this vector, we performed a knockdown of metabolic genes in a representative member of another cluster of the Mycoplasma genus-Mycoplasma hominis. The developed CRISPRi system is a powerful tool to discover the functioning of genes that are essential, decipher regulatory networks and that can help to identify novel drug targets to control Mycoplasma infections.

Streptocinnamides A and B, Depsipeptides from Streptomyces sp. KMM 9044.

The bacterium Streptomyces sp. KMM 9044 from a sample of marine sediment collected in the northwestern part of the Sea of Japan produces highly chlorinated depsiheptapeptides streptocinnamides A (1) and B (2), representatives of a new structural group of antibiotics. The structures of 1 and 2 were determined using nuclear magnetic resonance and mass spectrometry studies and confirmed by a series of chemical transformations. Streptocinnamide A potently inhibits Micrococcus sp. KMM 1467, Arthrobacter sp. ATCC 21022, and Mycobacterium smegmatis MC2 155.

Thymidine utilisation pathway is a novel phenotypic switch of Mycoplasma hominis.

Introduction. Mycoplasma hominis is a bacterium belonging to the class Mollicutes. It causes acute and chronic infections of the urogenital tract. The main features of this bacterium are an absence of cell wall and a reduced genome size (517-622 protein-encoding genes). Previously, we have isolated morphologically unknown M. hominis colonies called micro-colonies (MCs) from the serum of patients with inflammatory urogenital tract infection.Hypothesis. MCs are functionally different from the typical colonies (TCs) in terms of metabolism and cell division.Aim. To determine the physiological differences between MCs and TCs of M. hominis and elucidate the pathways of formation and growth of MCs by a comparative proteomic analysis of these two morphological forms.Methodology. LC-MS proteomic analysis of TCs and MCs using an Ultimate 3000 RSLC nanoHPLC system connected to a QExactive Plus mass spectrometer.Results. The study of the proteomic profiles of M. hominis colonies allowed us to reconstruct their energy metabolism pathways. In addition to the already known pentose phosphate and arginine deamination pathways, M. hominis can utilise ribose phosphate and deoxyribose phosphate formed by nucleoside catabolism as energy sources. Comparative proteomic HPLC-MS analysis revealed that the proteomic profiles of TCs and MCs were different. We assume that MC cells preferably utilised deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Utilisation of deoxyribonucleosides is less efficient as compared with that of ribonucleosides and arginine in terms of energy production. Thymidine phosphorylase DeoA is one of the key enzymes of deoxyribonucleosides utilisation. We obtained a DeoA overexpressing mutant that exhibited a phenotype similar to that of MCs, which confirmed our hypothesis.Conclusion. In addition to the two known pathways for energy production (arginine deamination and the pentose phosphate pathway) M. hominis can use deoxyribonucleosides and ribonucleosides. MC cells demonstrate a reorganisation of energy metabolism: unlike TC cells, they preferably utilise deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Thus MC cells enter a state of energy starvation, which helps them to survive under stress, and in particular, to be resistant to antibiotics.

Simple In-House Ultra-High Performance Capillary Column Manufacturing with the FlashPack Approach.

Capillary ultra-high performance liquid chromatography (UHPLC) is currently a method of choice for the sample separation step in LC-MS-based proteomics. However, capillary columns are much less robust in comparison to their higher flow countertypes. Because of easy contamination and blocking, they often need replacement. That makes them a markedly expensive part of the total LC-MS analysis cost. In-house packing of UHPLC capillary columns saves a lot of money and allows customization. However, the standard packing procedure in the 100-bar pressure bomb works well only for HPLC columns but is too slow for UHPLC sorbents. Here we provide a description of an optimized FlashPack protocol applied to the same 100-bar pressure bomb setup. The method is based on packing from ultra-high sorbent concentration slurry and is developed for in-house manufacturing of UHPLC capillary columns of unlimited length in reasonable time.

The Dynamics of Mycoplasma gallisepticum Nucleoid Structure at the Exponential and Stationary Growth Phases.

The structure and dynamics of bacterial nucleoids play important roles in regulating gene expression. Bacteria of class Mollicutes and, in particular, mycoplasmas feature extremely reduced genomes. They lack multiple structural proteins of the nucleoid, as well as regulators of gene expression. We studied the organization of Mycoplasma gallisepticum nucleoids in the stationary and exponential growth phases at the structural and protein levels. The growth phase transition results in the structural reorganization of M. gallisepticum nucleoid. In particular, it undergoes condensation and changes in the protein content. The observed changes corroborate with the previously identified global rearrangement of the transcriptional landscape in this bacterium during the growth phase transition. In addition, we identified that the glycolytic enzyme enolase functions as a nucleoid structural protein in this bacterium. It is capable of non-specific DNA binding and can form fibril-like complexes with DNA.

Unexpected Coelenterazine Degradation Products of Beroe abyssicola Photoprotein Photoinactivation.

Ca2+-regulated photoproteins of ctenophores lose bioluminescence activity when exposed to visible light. Little is known about the chemical nature of chromophore photoinactivation. Using a total synthesis strategy, we have established the structures of two unusual coelenterazine products, isolated from recombinant berovin of the ctenophore Beroe abyssicola, which are Z/E isomers. We propose that during light irradiation, these derivatives are formed from 2-hydroperoxycoelenterazine via the intermediate 8a-peroxide by a mechanism reminiscent of that previously described for the auto-oxidation of green-fluorescent-protein-like chromophores.

Yeast Ppz1 protein phosphatase toxicity involves the alteration of multiple cellular targets.

Control of the protein phosphorylation status is a major mechanism for regulation of cellular processes, and its alteration often lead to functional disorders. Ppz1, a protein phosphatase only found in fungi, is the most toxic protein when overexpressed in Saccharomyces cerevisiae. To investigate the molecular basis of this phenomenon, we carried out combined genome-wide transcriptomic and phosphoproteomic analyses. We have found that Ppz1 overexpression causes major changes in gene expression, affecting ~ 20% of the genome, together with oxidative stress and increase in total adenylate pools. Concurrently, we observe changes in the phosphorylation pattern of near 400 proteins (mainly dephosphorylated), including many proteins involved in mitotic cell cycle and bud emergence, rapid dephosphorylation of Snf1 and its downstream transcription factor Mig1, and phosphorylation of Hog1 and its downstream transcription factor Sko1. Deletion of HOG1 attenuates the growth defect of Ppz1-overexpressing cells, while that of SKO1 aggravates it. Our results demonstrate that Ppz1 overexpression has a widespread impact in the yeast cells and reveals new aspects of the regulation of the cell cycle.

Unusually efficient CUG initiation of an overlapping reading frame in POLG mRNA yields novel protein POLGARF.

While near-cognate codons are frequently used for translation initiation in eukaryotes, their efficiencies are usually low (<10% compared to an AUG in optimal context). Here, we describe a rare case of highly efficient near-cognate initiation. A CUG triplet located in the 5' leader of POLG messenger RNA (mRNA) initiates almost as efficiently (∼60 to 70%) as an AUG in optimal context. This CUG directs translation of a conserved 260-triplet-long overlapping open reading frame (ORF), which we call POLGARF (POLG Alternative Reading Frame). Translation of a short upstream ORF 5' of this CUG governs the ratio between POLG (the catalytic subunit of mitochondrial DNA polymerase) and POLGARF synthesized from a single POLG mRNA. Functional investigation of POLGARF suggests a role in extracellular signaling. While unprocessed POLGARF localizes to the nucleoli together with its interacting partner C1QBP, serum stimulation results in rapid cleavage and secretion of a POLGARF C-terminal fragment. Phylogenetic analysis shows that POLGARF evolved ∼160 million y ago due to a mammalian-wide interspersed repeat (MIR) transposition into the 5' leader sequence of the mammalian POLG gene, which became fixed in placental mammals. This discovery of POLGARF unveils a previously undescribed mechanism of de novo protein-coding gene evolution.

Data on proteome of Mycoplasma hominis cultivated with arginine or thymidine as a carbon source.

Mycoplasma hominis is an opportunistic bacterium that can cause acute and chronic infections of the urogenital tract. This bacterium, like all other Mycoplasma species, is characterized by the reduced genome size, and, consequently, reduction of the main metabolic pathways. M. hominis cells cannot effectively use glucose as a carbon and energy source. Therefore, the main pathway of energy metabolism is the arginine dihydrolase pathway. However, several bacteria can use nucleosides as the sole energy source. Biochemical studies using Salmonella typhimurium have shown that three enzymes (thymidine phosphorylase, phosphopentose mutase and deoxyribose-phosphate aldolase) are involved in the thymidine catabolic pathway. All these enzymes are present in M. hominis. For understanding changes in the energy metabolism of M. hominis we performed shotgun proteome analysis of M. hominis cells in liquid medium with arginine or thymidine as a carbon source. LC-MS analysis was performed with an Ultimate 3000 Nano LC System (Thermo Fisher Scientific) coupled to a Q Exactive HF benchtop Orbitrap mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific). Data are available via ProteomeXchange with identifier PXD018714 (https://www.ebi.ac.uk/pride/archive/projects/PXD018714).

Data on nucleoid-associated proteins isolated from Mycoplasma Gallisepticum in different growth phases.

Mycoplasma gallisepticum (MG) is one of the smallest free-living and self-replicating organisms, it is characterized by lack of cell wall and reduced genome size. As a result of genome reduction, MG has a limited variety of DNA-binding proteins and transcription factors. To investigate the dynamic changes of the proteomic profile of MG nucleoid, that may assist in revealing its mechanisms of functioning, regulation of chromosome organization and stress adaptation, a quantitative proteomic study was performed on MG nucleoids obtained from the cell culture in logarithmic and stationary phases of synchronous growth. MG cells were grown on a liquid medium with a 9 h starvation period. Nucleoids were obtained from the cell culture at the 26th and the 50th hour (logarithmic and stationary growth phases respectively) by sucrose density gradient centrifugation. LC-MS analysis was carried out on an Ultimate 3000 RSLCnano HPLC system connected to a Fusion Lumos mass spectrometer, controlled by XCalibur software (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific). For comprehensive peptide library generation one sample from each biological replicate was run in DDA mode. Then, all the samples were run in a single LC-MS DIA run. Identification of DDA files and DIA quantitation was performed with MaxQuant and Skyline software, correspondingly. All raw data generated from IDA and DDA acquisitions are presented in the PRIDE database with identifier PXD019077.

Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context.

Eukaryotic translation initiation involves preinitiation ribosomal complex 5'-to-3' directional probing of mRNA for codons suitable for starting protein synthesis. The recognition of codons as starts depends on the codon identity and on its immediate nucleotide context known as Kozak context. When the context is weak (i.e., nonoptimal), leaky scanning takes place during which a fraction of ribosomes continues the mRNA probing. We explored the relationship between the context of AUG codons annotated as starts of protein-coding sequences and the next AUG codon occurrence. We found that AUG codons downstream from weak starts occur in the same frame more frequently than downstream from strong starts. We suggest that evolutionary selection on in-frame AUGs downstream from weak start codons is driven by the advantage of the reduction of wasteful out-of-frame product synthesis and also by the advantage of producing multiple proteoforms from certain mRNAs. We confirmed translation initiation downstream from weak start codons using ribosome profiling data. We also tested translation of alternative start codons in 10 specific human genes using reporter constructs. In all tested cases, initiation at downstream start codons was more productive than at the annotated ones. In most cases, optimization of Kozak context did not completely abolish downstream initiation, and in the specific example of CMPK1 mRNA, the optimized start remained unproductive. Collectively, our work reveals previously uncharacterized forces shaping the evolution of protein-coding genes and points to the plurality of translation initiation and the existence of sequence features influencing start codon selection, other than Kozak context.

Correction to: Draft genome sequences of Hirudo medicinalis and salivary transcriptome of three closely related medicinal leeches.

An amendment to this paper has been published and can be accessed via the original article.

Draft genome sequences of Hirudo medicinalis and salivary transcriptome of three closely related medicinal leeches.

BACKGROUND: Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today. RESULTS: We annotated the Hirudo medicinalis genome and performed RNA-seq on salivary cells isolated from three closely related leech species, H. medicinalis, Hirudo orientalis, and Hirudo verbana. Differential expression analysis verified by proteomics identified salivary cell-specific gene expression, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants have been found to be expressed not only in salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis. CONCLUSIONS: Here we report a genome draft of Hirudo medicinalis and describe identification of novel salivary proteins and new homologs of genes encoding known anticoagulants in transcriptomes of three medicinal leech species. Our data provide new insights in genetics of blood-feeding lifestyle in leeches.

Middle-Down Proteomic Analyses with Ion Mobility Separations of Endogenous Isomeric Proteoforms.

Biological functions of many proteins are governed by post-translational modifications (PTMs). In particular, the rich PTM complement in histones controls the gene expression and chromatin structure with major health implications via a combinatoric language. Deciphering that "histone code" is the great challenge for proteomics given an astounding number of possible proteoforms, including isomers with different PTM positions. These must be disentangled on the top- or middle-down level to preserve the key PTM connectivity, which condensed-phase separations failed to achieve. We reported the capability of ion mobility spectrometry (IMS) methods to resolve such isomers for model histone tails. Here, we advance to biological samples, showing middle-down analyses of histones from mouse embryonic stem cells via online chromatography to fractionate proteoforms with distinct PTM sets, differential or field asymmetric waveform IMS (FAIMS) to resolve the isomers, and Orbitrap mass spectrometry with electron transfer dissociation to identify the resolved species.