Institute of Protein Research

Abstract N⁶-methyladenosine (m⁶A) role in translation control and, particularly, in cap-independent initiation has attracted major attention. A common approach is to study the impact of m⁶A depending on its particular location in the transcripts, but the global impact of m⁶A along mRNAs remains unclear. Here, we combined ribosome profiling under conditions of mTOR inhibition and m⁶A mapping to identify distinct subsets of mRNAs that differ in their sensitivity to the suppression of cap-dependent initiation. The sensitivity was strongly correlated with the total m⁶A methylation of the transcripts. Further, we demonstrated that upon decreased mTOR activity, m⁶A methylation facilitated the enhanced association of mRNAs with components of the eIF4F complex. Thus, the efficiency of cap-independent translation initiation is primarily defined not by precise localization but by the total level of m⁶A methylation, and m⁶A has a compensatory role in maintaining translation when the canonical cap-dependent pathway is impaired. All in all, our findings underscore the significance of contemplating global m⁶A methylation status as a pivotal element in translational control, particularly under stress or signaling perturbations.

Objective: Laccases are known to be able to degrade phenolic compounds to simpler components. The main objective of our study was to analyze this property in transgenic aspen plants carrying the laccase gene Lac from Trametes hirsuta which can be potentially used in soil phytoremediation. Methods: We created transgenic aspen plants carrying the laccase gene Lac from Trametes hirsute using the agrobacterial transformation of stem explants with the pBI-Lac vector containing the Lac gene from the white rot fungus T. hirsuta 072 (NCBI GenBank accession number KP027478). Transgenic plants were micropropagated and cultivated in vitro in lines. The degradation of 2,4,6-trichlorophenol (2,4,6-TCP) by plant roots was analyzed by mass-spectrometry with electron ionization using a gas chromatograph. Results: Although plants have their own laccases, those of fungal origin are more effective. All transgenic plants that expressed the recombinant gene degraded 2,4,6-TCP more effectively than non-transformed plants in the control (the degradation efficiency ranged 92 to 98% versus 82% in non-transformed control). Line 47Lac8 demonstrated a 16% higher efficiency than the non-transformed plants in the control. There was also an inverse relationship between the viability of a transgenic line and its level of expression of the recombinant gene. Thus, line 47Lac4 was not viable under native conditions, probably due to lignin synthesis disruptions during the initiation of secondary tissues. This is confirmed by changes in the expression of native genes of lignin biosynthesis. The rest of the transgenic lines did not differ significantly from control in wood growth and biochemistry. The transgenic plant roots were shown to preserve the ability to express the Lac gene ex vitro. Conclusions: Three transgenic lines (47Lac5, 47Lac8, and 47Lac23) with the Lac gene can be recommended for use in soil phytoremediation.

The paper summarizes the main provisions of Alexander Spirin’s concept on the possible role of RNA, molecular machines, and molecular colonies in the emergence of life, including (1) the role of the capability of polyribonucleotides of spontaneous rearrangements and recombinations in the generation of RNA molecules and development of their structure, as well as in the early replication of RNA; (2) the principle of operation of molecular machines powered by the energy of Brownian motion and the need for their emergence to solve the problem of unwinding double-stranded molecules formed as a result of the complementary copying of RNA; (3) the role of molecular colonies formed during the replication of RNA in porous media as a mode of compartmentalization and as a prerequisite for the evolution in the precellular RNA world.

The pathogenic microorganism Staphylococcus aureus, one of the representatives of the staphylococcus family, is capable of developing resistance to antibiotics and antiseptics. The largest ribosomal protein, S1, from S. aureus contains four structural S1 domains, and short peptides synthesized based on its sequence have amyloidogenic and antimicrobial properties (antimicrobial peptides). In this work, in silico analysis of all available ribosomal protein S1 sequences from various S. aureus strains allowed us to identify residues that are characteristic of specific strains. Based on data from the I-Mutant service, changes in the stability of ribosomal protein S1 from various S. aureus strains were predicted. The results obtained will be used in the future for targeted mutations in the design of new antimicrobial peptides based on ribosomal protein S1.

Intermediate filaments (IFs) represented by a diverse range of proteins, are one of the three main cytoskeleton components in different types of animal cells. IFs provide mechanical strength to cells and help position the nucleus and organelles in the cell. Desmin is an IF protein typical of muscle cells, while vimentin, which has a similar structure, is expressed in many mesenchymal cells. Both proteins are synthesized during myogenesis and regeneration of damaged muscle tissue and form a mixed IF network. Both desmin and vimentin regulate mitochondrial activity, including mitochondrial localization and maintenance of mitochondrial membrane potential, in the corresponding cells, but the role of mixed IFs in the control of mitochondrial functions remains unclear. To investigate how a simultaneous presence of these proteins affects mitochondrial membrane potential, we used BHK21 cells expressing both vimentin and desmin IFs. Expression of vimentin or desmin individually or both proteins simultaneously was suppressed using gene knockout and/or RNA interference. It was found that disruption of biosynthesis of either vimentin or desmin did not affect the mitochondrial membrane potential, which remained unchanged compared to cells expressing both proteins. Simultaneous abolishment of both proteins resulted in a 20% reduction in the mitochondrial membrane potential, indicating that both vimentin and desmin play an equally important role in its maintenance.

Representatives of the colorless sulfur bacteria of the genus Beggiatoa use reduced sulfur compounds in the processes of lithotrophic growth, which is accompanied by the storage of intracellular sulfur. However, it is still unknown how the transformation of intracellular sulfur occurs in Beggiatoa representatives. Annotation of the genome of Beggiatoa leptomitoformis D-402 did not identify any genes for the oxidation or reduction of elemental sulfur. By searching BLASTP, two putative persulfide dioxygenase (PDO) homologs were found in the genome of B. leptomitoformis. In some heterotrophic prokaryotes, PDO is involved in the oxidation of sulfane sulfur. According to HPLC-MS/MS, the revealed protein was reliably detected in a culture sample grown only in the presence of endogenous sulfur and CO2. The recombinant protein from B. leptomitoformis was active in the presence of glutathione persulfide. The crystal structure of recombinant PDO exhibited consistency with known structures of type I PDO. Thus, it was shown that B. leptomitoformis uses PDO to oxidize endogenous sulfur. Additionally, on the basis of HPLC-MS/MS, RT-qPCR, and the study of PDO reaction products, we predicted the interrelation of PDO and Sox-system function in the oxidation of endogenous sulfur in B. leptomitoformis and the connection of this process with energy metabolism.

In recent years, the study of antimicrobial peptides (AMPs) has garnered considerable attention due to their potential in combating antibiotic-resistant pathogens. Mass spectrometry-based proteomics provides valuable information on microbial stress responses induced by AMPs. This work aims to unravel the proteomic alterations induced by the amyloidogenic antimicrobial peptide R23I, encompassing both inhibitory and non-inhibitory concentrations. This study investigates the effects of the R23I peptide on the protein abundance of Thermus thermophilus (T. thermophilus) at different concentrations (20, 50, and 100 μg/mL). We found 82 differentially expressed proteins, including 15 upregulated and 67 downregulated proteins. We also compared the protein identification results between the PEAKS and IdentiPy programs. Our proteomic analysis revealed distinct patterns of protein expression, suggesting compensatory mechanisms in response to the R23I peptide. Notably, the alterations predominantly affected membrane and cytoplasmic proteins that play a central role in critical cellular processes such as transcription, translation, and energy conversion. This study sheds light on the complex interactions between antimicrobial peptides and bacterial responses, offering insights into microbial adaptability and potential implications for antimicrobial strategies and the understanding of microbial physiology.