In silico bioprospecting of receptors associated with the mechanism of action of Rondonin, an antifungal peptide from spider Acanthoscurria rondoniae haemolymph.

Multiple drug-resistant fungal species are associated with the development of diseases. Thus, more efficient drugs for the treatment of these aetiological agents are needed. Rondonin is a peptide isolated from the haemolymph of the spider Acanthoscurria rondoniae. Previous studies have shown that this peptide has antifungal activity against Candida sp. and Trichosporon sp. strains, acting on their genetic material. However, the molecular targets involved in its biological activity have not yet been described. Bioinformatics tools were used to determine the possible targets involved in the biological activity of Rondonin. The PharmMapper server was used to search for microorganismal targets of Rondonin. The PatchDock server was used to perform the molecular docking. UCSF Chimera software was used to evaluate these intermolecular interactions. In addition, the I-TASSER server was used to predict the target ligand sites. Then, these predictions were contrasted with the sites previously described in the literature. Molecular dynamics simulations were conducted for two promising complexes identified from the docking analysis. Rondonin demonstrated consistency with the ligand sites of the following targets: outer membrane proteins F (id: 1MPF) and A (id: 1QJP), which are responsible for facilitating the passage of small molecules through the plasma membrane; the subunit of the flavoprotein fumarate reductase (id: 1D4E), which is involved in the metabolism of nitrogenous bases; and the ATP-dependent Holliday DNA helicase junction (id: 1IN4), which is associated with histone proteins that package genetic material. Additionally, the molecular dynamics results indicated the stability of the interaction of Rondonin with 1MPF and 1IN4 during a 10 ns simulation. These interactions corroborate with previous in vitro studies on Rondonin, which acts on fungal genetic material without causing plasma membrane rupture. Therefore, the bioprospecting methods used in this research were considered satisfactory since they were consistent with previous results obtained via in vitro experimentation. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-024-00224-1.

In silico prospection of receptors associated with the biological activity of U1-SCTRX-lg1a: an antimicrobial peptide isolated from the venom of Loxosceles gaucho.

The emergence of antibiotic-resistant pathogens generates impairment to human health. U1-SCTRX-lg1a is a peptide isolated from a phospholipase D extracted from the spider venom of Loxosceles gaucho with antimicrobial activity against Gram-negative bacteria (between 1.15 and 4.6 µM). The aim of this study was to suggest potential receptors associated with the antimicrobial activity of U1-SCTRX-lg1a using in silico bioinformatics tools. The search for potential targets of U1-SCRTX-lg1a was performed using the PharmMapper server. Molecular docking between U1-SCRTX-lg1a and the receptor was performed using PatchDock software. The prediction of ligand sites for each receptor was conducted using the PDBSum server. Chimera 1.6 software was used to perform molecular dynamics simulations only for the best dock score receptor. In addition, U1-SCRTX-lg1a and native ligand interactions were compared using AutoDock Vina software. Finally, predicted interactions were compared with the ligand site previously described in the literature. The bioprospecting of U1-SCRTX-lg1a resulted in the identification of three hundred (300) diverse targets (Table S1), forty-nine (49) of which were intracellular proteins originating from Gram-negative microorganisms (Table S2). Docking results indicate Scores (10,702 to 6066), Areas (1498.70 to 728.40) and ACEs (417.90 to - 152.8) values. Among these, NAD + NH3-dependent synthetase (PDB ID: 1wxi) showed a dock score of 9742, area of 1223.6 and ACE of 38.38 in addition to presenting a Normalized Fit score of 8812 on PharmMapper server. Analysis of the interaction of ligands and receptors suggests that the peptide derived from brown spider venom can interact with residues SER48 and THR160. Furthermore, the C terminus (- 7.0 score) has greater affinity for the receptor than the N terminus (- 7.7 score). The molecular dynamics assay shown that free energy value for the protein complex of - 214,890.21 kJ/mol, whereas with rigid docking, this value was - 29.952.8 sugerindo that after the molecular dynamics simulation, the complex exhibits a more favorable energy value compared to the previous state. The in silico bioprospecting of receptors suggests that U1-SCRTX-lg1a may interfere with NAD + production in Escherichia coli, a Gram-negative bacterium, altering the homeostasis of the microorganism and impairing growth. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-024-00190-8.

A Two-Step Mechanism for Creating Stable, Condensed Chromatin with the Polycomb Complex PRC1.

The Drosophila PRC1 complex regulates gene expression by modifying histone proteins and chromatin architecture. Two PRC1 subunits, PSC and Ph, are most implicated in chromatin architecture. In vitro, PRC1 compacts chromatin and inhibits transcription and nucleosome remodeling. The long disordered C-terminal region of PSC (PSC-CTR) is important for these activities, while Ph has little effect. In cells, Ph is important for condensate formation, long-range chromatin interactions, and gene regulation, and its polymerizing sterile alpha motif (SAM) is implicated in these activities. In vitro, truncated Ph containing the SAM and two other conserved domains (mini-Ph) undergoes phase separation with chromatin, suggesting a mechanism for SAM-dependent condensate formation in vivo. How the distinct activities of PSC and Ph on chromatin function together in PRC1 is not known. To address this question, we analyzed structures formed with large chromatin templates and PRC1 in vitro. PRC1 bridges chromatin into extensive fibrillar networks. Ph, its SAM, and SAM polymerization activity have little effect on these structures. Instead, the PSC-CTR controls their growth, and is sufficient for their formation. To understand how phase separation driven by Ph SAM intersects with the chromatin bridging activity of the PSC-CTR, we used mini-Ph to form condensates with chromatin and then challenged them with PRC1 lacking Ph (PRC1ΔPh). PRC1ΔPh converts mini-Ph chromatin condensates into clusters of small non-fusing condensates and bridged fibers. These condensates retain a high level of chromatin compaction and do not intermix. Thus, phase separation of chromatin by mini-Ph, followed by the action of the PSC-CTR, creates a unique chromatin organization with regions of high nucleosome density and extraordinary stability. We discuss how this coordinated sequential activity of two proteins found in the same complex may occur and the possible implications of stable chromatin architectures in maintaining transcription states.

In silico prospection of receptors associated with the biological activity of U1-SCTRX-lg1a: an antimicrobial peptide isolated from the venom of Loxosceles gaucho

The emergence of antibiotic-resistant pathogens generates impairment to human health. U1-SCTRX-lg1a is a peptide isolated from a phospholipase D extracted from the spider venom of Loxosceles gaucho with antimicrobial activity against Gram-negative bacteria (between 1.15 and 4.6 μM). The aim of this study was to suggest potential receptors associated with the antimicrobial activity of U1-SCTRX-lg1a using in silico bioinformatics tools. The search for potential targets of U1-SCRTX-lg1a was performed using the PharmMapper server. Molecular docking between U1-SCRTX-lg1a and the receptor was performed using PatchDock software. The prediction of ligand sites for each receptor was conducted using the PDBSum server. Chimera 1.6 software was used to perform molecular dynamics simulations only for the best dock score receptor. In addition, U1-SCRTX-lg1a and native ligand interactions were compared using AutoDock Vina software. Finally, predicted interactions were compared with the ligand site previously described in the literature. The bioprospecting of U1-SCRTX-lg1a resulted in the identification of three hundred (300) diverse targets (Table S1), forty-nine (49) of which were intracellular proteins originating from Gram-negative microorganisms (Table S2). Docking results indicate Scores (10,702 to 6066), Areas (1498.70 to 728.40) and ACEs (417.90 to – 152.8) values. Among these, NAD + NH3-dependent synthetase (PDB ID: 1wxi) showed a dock score of 9742, area of 1223.6 and ACE of 38.38 in addition to presenting a Normalized Fit score of 8812 on PharmMapper server. Analysis of the interaction of ligands and receptors suggests that the peptide derived from brown spider venom can interact with residues SER48 and THR160. Furthermore, the C terminus (– 7.0 score) has greater affinity for the receptor than the N terminus (– 7.7 score). The molecular dynamics assay shown that free energy value for the protein complex of – 214,890.21 kJ/mol, whereas with rigid docking, this value was – 29.952.8 sugerindo that after the molecular dynamics simulation, the complex exhibits a more favorable energy value compared to the previous state. The in silico bioprospecting of receptors suggests that U1-SCRTX-lg1a may interfere with NAD + production in Escherichia coli, a Gram-negative bacterium, altering the homeostasis of the microorganism and impairing growth.

In silico bioprospecting of receptors for Doderlin: an antimicrobial peptide isolated from Lactobacillus acidophilus.

The emergence of resistant bacteria strains against traditional antibiotics and treatments increases each year. Doderlin is a cationic and amphiphilic peptide active against gram-positive, negative and yeast stains. The aim of the present work was prospect potentials receptors associated of antimicrobial activity of Doderlin using in silico bioinformatics tools. To search for potential targets of Doderlin, PharmMapper software was used. Molecular docking between Doderlin and the receptor was performed by PatchDock. Additional interaction and ligand site prediction for each receptor was performed by I-TASSER software. Those PDB Id, 1XDJ (score: 11,746), 1JMH (score: 11,046), 1YR3 (score: 10,578), 1NG3 (score: 10,082) showed highest dock score. Doderlin was found to predicted/real sites co-localize with 1XDJ and 1JMH, enzymes accountable for nitrogenic bases synthesis. The resulting receptor bioprospecting is highly correlated and suggests that Doderlin might act by interfering with DNA metabolism/production of bacteria, altering microorganism homeostasis and growth impairment. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00149-1.

In silico bioprospecting of receptors for Doderlin: an antimicrobial peptide isolated from Lactobacillus acidophilus

The emergence of resistant bacteria strains against traditional antibiotics and treatments increases each year. Doderlin is a cationic and amphiphilic peptide active against gram-positive, negative and yeast stains. The aim of the present work was prospect potentials receptors associated of antimicrobial activity of Doderlin using in silico bioinformatics tools. To search for potential targets of Doderlin, PharmMapper software was used. Molecular docking between Doderlin and the receptor was performed by PatchDock. Additional interaction and ligand site prediction for each receptor was performed by I-TASSER software. Those PDB Id, 1XDJ (score: 11,746), 1JMH (score: 11,046), 1YR3 (score: 10,578), 1NG3 (score: 10,082) showed highest dock score. Doderlin was found to predicted/real sites co-localize with 1XDJ and 1JMH, enzymes accountable for nitrogenic bases synthesis. The resulting receptor bioprospecting is highly correlated and suggests that Doderlin might act by interfering with DNA metabolism/production of bacteria, altering microorganism homeostasis and growth impairment.

Phase separation by the polyhomeotic sterile alpha motif compartmentalizes Polycomb Group proteins and enhances their activity.

Polycomb Group (PcG) proteins organize chromatin at multiple scales to regulate gene expression. A conserved Sterile Alpha Motif (SAM) in the Polycomb Repressive Complex 1 (PRC1) subunit Polyhomeotic (Ph) has been shown to play an important role in chromatin compaction and large-scale chromatin organization. Ph SAM forms helical head to tail polymers, and SAM-SAM interactions between chromatin-bound Ph/PRC1 are believed to compact chromatin and mediate long-range interactions. To understand the underlying mechanism, here we analyze the effects of Ph SAM on chromatin in vitro. We find that incubation of chromatin or DNA with a truncated Ph protein containing the SAM results in formation of concentrated, phase-separated condensates. Ph SAM-dependent condensates can recruit PRC1 from extracts and enhance PRC1 ubiquitin ligase activity towards histone H2A. We show that overexpression of Ph with an intact SAM increases ubiquitylated H2A in cells. Thus, SAM-induced phase separation, in the context of Ph, can mediate large-scale compaction of chromatin into biochemical compartments that facilitate histone modification.

Export of Precursor tRNAIle from the Nucleus to the Cytoplasm in Human Cells.

In the current concept, tRNA maturation in vertebrate cells, including splicing of introns, trimming of 5' leader and 3' trailer, and adding of CCA, is thought to occur exclusively in the nucleus. Here we provide evidence to challenge this concept. Unspliced intron-containing precursor tRNAIle was identified in Human Immunodeficiency Virus type 1 (HIV-1) virions, which are synthesized in the cytoplasm. Northern blot, confocal microscopy and quantitative RT-PCR further verified enrichment of this unspliced tRNAIle within the cytoplasm in human cells. In addition to containing an intron, the cytoplasmic precursor tRNAIle also contains a short incompletely processed 5´ leader and a 3´ trailer, which abundance is around 1000 fold higher than the nuclear precursor tRNAIle with long 5' leader and long 3' trailer. In vitro data also suggest that the cytoplasmic unspliced end-immature precursor tRNAIle could be processed by short isoform of RNase Z, but not long isoform of RNase Z. These data suggest that precursor tRNAs could export from the nucleus to the cytoplasm in human cells, instead of be processed only in the nucleus.

In virio SHAPE analysis of tRNA(Lys3) annealing to HIV-1 genomic RNA in wild type and protease-deficient virus.

BACKGROUND: tRNA(Lys3) annealing to the viral RNA of human immunodeficiency virus type-1 (HIV-1) is an essential step in the virus life cycle, because this tRNA serves as the primer for initiating reverse transcription. tRNA(Lys3) annealing to viral RNA occurs in two steps. First, Gag promotes annealing of tRNA(Lys3) to the viral RNA during cytoplasmic HIV-1 assembly. Second, mature nucleocapsid (NCp7), produced from the processing of Gag by viral protease during viral budding from the cell, remodels the annealed complex to form a more stable interaction between the viral RNA and tRNA(Lys3), resulting in a more tightly bound and efficient primer for reverse transcription. RESULTS: In this report, we have used in virio SHAPE analysis of both the 5´-untranslated region in HIV-1 RNA and the annealed tRNA(Lys3) to determine structural differences of the annealed complex that occur between protease-negative (Pr-) and wild type viruses. Our results indicate that the weaker binding of tRNA(Lys3) annealed by Gag in Pr- virions reflects both missing interactions of tRNA(Lys3) with viral RNA regions in the upper PBS stem, and a weaker interaction with the internal stem-loop found within the unannealed primer binding site in viral RNA. CONCLUSIONS: We propose secondary structure models for the tRNA(Lys3)/viral RNA annealed complexes in PR- and wild type viruses that support the two-step annealing model by showing that Gag promotes a partial annealing of tRNA(Lys3) to HIV-1 viral RNA, followed by a more complete annealing by NCp7.

Annealing to sequences within the primer binding site loop promotes an HIV-1 RNA conformation favoring RNA dimerization and packaging.

The 5' untranslated region (5' UTR) of HIV-1 genomic RNA (gRNA) includes structural elements that regulate reverse transcription, transcription, translation, tRNA(Lys3) annealing to the gRNA, and gRNA dimerization and packaging into viruses. It has been reported that gRNA dimerization and packaging are regulated by changes in the conformation of the 5'-UTR RNA. In this study, we show that annealing of tRNA(Lys3) or a DNA oligomer complementary to sequences within the primer binding site (PBS) loop of the 5' UTR enhances its dimerization in vitro. Structural analysis of the 5'-UTR RNA using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) shows that the annealing promotes a conformational change of the 5' UTR that has been previously reported to favor gRNA dimerization and packaging into virus. The model predicted by SHAPE analysis is supported by antisense experiments designed to test which annealed sequences will promote or inhibit gRNA dimerization. Based on reports showing that the gRNA dimerization favors its incorporation into viruses, we tested the ability of a mutant gRNA unable to anneal to tRNA(Lys3) to be incorporated into virions. We found a ∼60% decrease in mutant gRNA packaging compared with wild-type gRNA. Together, these data further support a model for viral assembly in which the initial annealing of tRNA(Lys3) to gRNA is cytoplasmic, which in turn aids in the promotion of gRNA dimerization and its incorporation into virions.

RNA-protein mutually induced fit: structure of Escherichia coli isopentenyl-tRNA transferase in complex with tRNA(Phe).

tRNAs that read codons starting with U are usually modified at their A37 by isopentenyl-tRNA transferases to minimize peptidyl-tRNA slippage in translation. The consensus substrate requirements of the isopentenyl-tRNA transferase of Escherichia coli, MiaA, have been the focus of extensive study. However, the molecular basis of tRNA-MiaA recognition remains unknown. Here we describe the 2.5A crystal structure of MiaA in complex with substrate tRNA(Phe). Comparative structural analysis reveals that the enzymatic reaction involves an RNA-protein mutually induced fit mechanism in which large domain movements in MiaA provoke the partial unfolding of the substrate tRNA anticodon loop. In addition, we show how substrate tRNAs are recognized by MiaA through a combination of direct and indirect sequence readouts.

RNase P cleaves the adenine riboswitch and stabilizes pbuE mRNA in Bacillus subtilis.

RNase P from Bacillus subtilis cleaves in vitro the adenine riboswitch upstream of pbuE, which codes for an adenine efflux pump. The guanine riboswitch, encoded upstream of xpt-pbuX operon, is not cleaved. The cleavage sites do not occur at any predicted structures that should be recognized by RNase P in the theoretical model of the adenine riboswitch. However, it is possible to draw alternative secondary structure models that match the apparent requirements for RNase P substrates at these cleavage sites. Support for these models is provided by appropriate mutagenesis experiments. Adenine showed no effect on the cleavage in vitro of the pbuE adenine riboswitch by RNase P holoenzyme from B. subtilis. The results of genetic experiments performed in B. subtilis support the cleavage of adenine riboswitch by RNase P in vivo and suggest that it induces the stabilization of pbuE mRNA under normal conditions.

Hybrid E. coli--Mitochondrial ribonuclease P RNAs are catalytically active.

RNase P is a ribonucleoprotein that cleaves tRNA precursors at their 5'-end. Mitochondrion-encoded RNA subunits of mitochondrial RNase P (mtP-RNA) have been identified in jakobid flagellates such as Reclinomonas americana, in the prasinophyte alga Nephroselmis olivacea, and in several ascomycete and zygomycete fungi. While the structures of ascomycete mtP-RNAs are highly reduced, those of jakobids, prasinophytes, and zygomycetes retain most conserved features of their bacterial counterparts. Therefore, these mtP-RNAs might be active in vitro in the absence of a protein subunit, as are bacterial P-RNAs. Here we present a comparative structural analysis including seven newly characterized jakobid mtP-RNAs. We investigate ribozyme activities of mtP-RNAs and find that even the most bacteria-like molecules of jakobids are inactive in vitro. However, when certain domains of jakobid and N. olivacea mtP-RNAs are replaced with those from Escherichia coli, these hybrid RNAs show catalytic activity. In vitro mutagenesis of these hybrid mtP-RNAs shows that various structural elements play a critical role in ribozyme catalysis and provide further support for the presence of these elements in mtP-RNAs. These include GNRA tetraloops in helix P14 and P18 of Jakoba libera, and a remnant P3 pairing in Seculamonas ecuadoriensis. Finally, we will discuss reasons for the failure of mtP-RNAs to show catalytic activity in the absence of P-proteins based on our mutagenesis analysis.

Comparative mitochondrial genomics in zygomycetes: bacteria-like RNase P RNAs, mobile elements and a close source of the group I intron invasion in angiosperms.

To generate data for comparative analyses of zygomycete mitochondrial gene expression, we sequenced mtDNAs of three distantly related zygomycetes, Rhizopus oryzae, Mortierella verticillata and Smittium culisetae. They all contain the standard fungal mitochondrial gene set, plus rnpB, the gene encoding the RNA subunit of the mitochondrial RNase P (mtP-RNA) and rps3, encoding ribosomal protein S3 (the latter lacking in R.oryzae). The mtP-RNAs of R.oryzae and of additional zygomycete relatives have the most eubacteria-like RNA structures among fungi. Precise mapping of the 5' and 3' termini of the R.oryzae and M.verticillata mtP-RNAs confirms their expression and processing at the exact sites predicted by secondary structure modeling. The 3' RNA processing of zygomycete mitochondrial mRNAs, SSU-rRNA and mtP-RNA occurs at the C-rich sequence motifs similar to those identified in fission yeast and basidiomycete mtDNAs. The C-rich motifs are included in the mature transcripts, and are likely generated by exonucleolytic trimming of RNA 3' termini. Zygomycete mtDNAs feature a variety of insertion elements: (i) mtDNAs of R.oryzae and M.verticillata were subject to invasions by double hairpin elements; (ii) genes of all three species contain numerous mobile group I introns, including one that is closest to an intron that invaded angiosperm mtDNAs; and (iii) at least one additional case of a mobile element, characterized by a homing endonuclease insertion between partially duplicated genes [Paquin,B., Laforest,M.J., Forget,L., Roewer,I., Wang,Z., Longcore,J. and Lang,B.F. (1997) Curr. Genet., 31, 380-395]. The combined mtDNA-encoded proteins contain insufficient phylogenetic signal to demonstrate monophyly of zygomycetes.

Loss of the mRNA-like region in mitochondrial tmRNAs of jakobids.

It has been postulated that a highly reduced form of transfer messenger RNA (tmRNA), a bacterial molecule involved in the rescue of stalled ribosomes during translation, is expressed in the mitochondrion of the jakobid Reclinomonas americana. Here we show that genes encoding both one-piece and two-piece tmRNAs are present in six different jakobid mitochondrial DNAs. Mitochondrial tmRNAs have retained the highly conserved tRNA(Ala)-like domain, but they apparently lack the mRNA-like region present in all bacterial tmRNAs. Comparative analysis of jakobid mitochondrial genomes shows that a potential mRNA-like region in R. americana (orf64) is located at distant genomic positions in other jakobids. Our results strongly suggest that orf64 is a tatA homolog. Through Northern hybridization we confirm the postulated reduced size of both a one-piece tmRNA in Jakoba libera and a two-piece tmRNA in Seculamonas ecuadoriensis. The J. libera tmRNA is post-transcriptionally modified by addition of a 3' CCA tail, processed in vitro by RNase P RNA, and specifically charged with alanine in vitro by alanyl-tRNA synthetase. Our results strongly support the functionality of these reduced mitochondrial tmRNAs.

Mitochondrial RNase P RNAs in ascomycete fungi: lineage-specific variations in RNA secondary structure.

The RNA subunit of mitochondrial RNase P (mtP-RNA) is encoded by a mitochondrial gene (rnpB) in several ascomycete fungi and in the protists Reclinomonas americana and Nephroselmis olivacea. By searching for universally conserved structural elements, we have identified previously unknown rnpB genes in the mitochondrial DNAs (mtDNAs) of two fission yeasts, Schizosaccharomyces pombe and Schizosaccharomyces octosporus; in the budding yeast Pichia canadensis; and in the archiascomycete Taphrina deformans. The expression of mtP-RNAs of the predicted size was experimentally confirmed in the two fission yeasts, and their precise 5' and 3' ends were determined by sequencing of cDNAs generated from circularized mtP-RNAs. Comparative RNA secondary structure modeling shows that in contrast to mtP-RNAs of the two protists R. americana and N. olivacea, those of ascomycete fungi all have highly reduced secondary structures. In certain budding yeasts, such as Saccharomycopsis fibuligera, we find only the two most conserved pairings, P1 and P4. A P18 pairing is conserved in Saccharomyces cerevisiae and its close relatives, whereas nearly half of the minimum bacterial consensus structure is retained in the RNAs of fission yeasts, Aspergillus nidulans and Taphrina deformans. The evolutionary implications of the reduction of mtP-RNA structures in ascomycetes will be discussed.