Yakov E. Dunaevsky

In recent years, researchers have increasingly focused on a special class of proteins—pseudopeptidases—that have lost their catalytic activity while retaining the ability to bind targets with high specificity. The evolutionary transition from an active enzyme to a pseudopeptidase is often driven by point mutations in the active site that “switch off” hydrolysis, paradoxically increasing affinity for substrates or regulatory partners. This process of “functional exaptation” allows the cell to create complex regulatory networks by repurposing existing domains rather than synthesizing new structures. This review analyzes the mechanisms underlying the emergence of pseudopeptidases across all major peptidase families and their roles in signaling cascades as molecular scaffolds, allosteric modulators, or competitive inhibitors. Particular attention is paid to their contribution to pathophysiology: the loss of proteolytic function does not render these proteins “silent”; on the contrary, their dysfunction is critical in the development of cancer, as well as neurodegenerative and inflammatory diseases. The shift from catalytic inertness to active regulation opens new horizons in understanding the molecular mechanisms of pathologies, positioning pseudopeptidases as promising targets for therapeutic intervention.

In recent years, researchers have increasingly focused on a special class of proteins – pseudopeptidases, that have lost their catalytic activity while retaining the ability to bind targets with high specificity. The evolutionary transition from an active enzyme to a pseudopeptidase is often driven by point mutations in the active site that “switch off” hydrolysis, paradoxically increasing affinity for substrates or regulatory partners. This process of “functional exaptation” allows the cell to create complex regulatory networks by repurposing existing domains rather than synthesizing new structures. This review analyzes the mechanisms underlying the emergence of pseudopeptidases across all major peptidase families and their roles in signaling cascades as molecular scaffolds, allosteric modulators, or competitive inhibitors. Particular attention is paid to their contribution to pathophysiology: the loss of proteolytic function does not render these proteins “silent”; on the contrary, their dysfunction is critical in the development of cancer, as well as neurodegenerative and inflammatory diseases. The shift from catalytic inertness to active regulation opens new horizons in understanding the molecular mechanisms of pathologies, positioning pseudopeptidases as promising targets for therapeutic intervention.

Adaptive laboratory evolution (ALE) is aimed at elucidating the molecular basis of adaptation and is widely employed as a tool for gaining deeper insight into genetic and/or metabolic pathways underlying evolutionary processes. One of the primary goals of experimental evolution is to predict mutations representing the key driving forces of adaptation. The use of whole-genome resequencing enables easy identification of mutations that arise during ALE, and consequently, biochemical alterations that occur in the experimental lineages. ALE has also proven highly relevant in practical applications, as it provides an innovative approach to the construction of evolved microbial strains with desirable performance, such as rapid growth, stress resistance, efficient utilization of diverse substrates, and production of compounds with a high added value (amino acids, ethanol, aromatic compounds, lipids, etc.). In this review, we analyzed the results of studies focused on the demonstration and explanation of relationships between mutations and resulting phenotypic and biochemical changes, as well as discussed a potential of microorganisms as model systems for ALE experiments and testing of various evolutionary hypotheses. We also described achievements reached by using ALE strategies, as well as the still unresolved issues and methodological limitations of this approach.

Adaptive Laboratory Evolution (ALE) is a novel approach to generating microbial strains with desired characteristics and producing value-added products. Additionally, ALE is employed as a means of enhancing comprehension of the genetic and/or metabolic pathways of evolution. The objective of this review is to analyze the results of studies that elucidate and demonstrate the potential of microorganisms as model objects for laboratory evolutionary experiments. These experiments are becoming increasingly prevalent in the study of adaptation, the evaluation of evolutionary dynamics, and the testing of various evolutionary hypotheses. Concurrently, ALE has demonstrated itself to be a promising and efficacious methodology, which, when employed for biotechnological applications, has already resulted in the generation of novel and useful microbial strains. It is important to note that the current achievements not only demonstrate the power and versatility of this approach but also highlight a number of unanswered questions. The conclusions drawn on the basis of ALE require a cautious interpretation of the results obtained.

Transcriptomics is at the intersection of molecular biology and genetics, and studies the complete set of transcripts that are synthesized in a cell or organism under certain conditions

Serine peptidases (SPs) of the chymotrypsin S1A subfamily are an extensive group of enzymes found in all animal organisms, including insects. Here, we provide analysis of SPs in the yellow mealworm Tenebrio molitor transcriptomes and genomes datasets and profile their expression patterns at various stages of ontogeny. A total of 269 SPs were identified, including 137 with conserved catalytic triad residues, while 125 others lacking conservation were proposed as non-active serine peptidase homologs (SPHs). Seven deduced sequences exhibit a complex domain organization with two or three peptidase units (domains), predicted both as active or non-active. The largest group of 84 SPs and 102 SPHs had no regulatory domains in the propeptide, and the majority of them were expressed only in the feeding life stages, larvae and adults, presumably playing an important role in digestion. The remaining 53 SPs and 23 SPHs had different regulatory domains, showed constitutive or upregulated expression at eggs or/and pupae stages, participating in regulation of various physiological processes. The majority of polypeptidases were mainly expressed at the pupal and adult stages. The data obtained expand our knowledge on SPs/SPHs and provide the basis for further studies of the functions of proteins from the S1A subfamily in T. molitor.

Serine peptidases (SPs) of the chymotrypsin S1A subfamily are an extensive group of enzymes found in all animal organisms, including insects. Here we provide analysis of SPs in the yellow mealworm Tenebrio molitor transcriptomes and genomes datasets and profile their expression pattern at various stages of ontogeny. A total of 269 SPs were identified, including 137 with con-served catalytic triad residues, while others 125 lacking conservation were proposed as non-active serine peptidase homologs (SPHs). Seven deduced sequences exhibit a complex do-main organization with two or three peptidase units (domains), predicted both as active or non-active. The largest group of 84 SP and 102 SPH had no regulatory domains in the propep-tide, and the majority of them were expressed only in the feeding life stages, larvae and adults, presumably playing an important role in digestion. The remaining 53 SP and 23 SPH had differ-ent regulatory domains, showed constitutive or upregulated expression at eggs or/and pupae stages, participating in regulation of various physiological processes. The majority of polypep-tidases was mainly expressed at the pupal and adult stages. The data obtained expand our knowledge on SPs/SPHs and provide a foundation for further research on the functions of this gene family in T. molitor.

The Podospora anserina long-term evolution experiment (PaLTEE) is the only running filamentous fungus study, which is still going on. The aim of our work is to trace the evolutionary dynamics of the accumulation of mutations in the genomes of eight haploid populations of P. anserina. The results of the genome-wide analysis of all of the lineages, performed 8 years after the start of the PaLTEE, are presented. Data analysis detected 312 single nucleotide polymorphisms (SNPs) and 39 short insertion-deletion mutations (indels) in total. There was a clear trend towards a linear increase in the number of SNPs depending on the experiment duration. Among 312 SNPs, 153 were fixed in the coding regions of P. anserina genome. Relatively few synonymous mutations were found, exactly 38; 42 were classified as nonsense mutations; 72 were assigned to missense mutations. In addition, 21 out of 39 indels identified were also localized in coding regions. Here, we also report the detection of parallel evolution at the paralog level in the P. anserina model system. Parallelism in evolution at the level of protein functions also occurs. The latter is especially true for various transcription factors, which may indicate selection leading to optimization of the wide range of cellular processes under experimental conditions.

A detailed analysis of the complexes of proline-specific peptidases (PSPs) in the midgut transcriptomes of the larvae of agricultural pests Tenebrio molitor and Tribolium castaneum and in the genome of T. castaneum is presented. Analysis of the T. castaneum genome revealed 13 PSP sequences from the clans of serine and metal-dependent peptidases, of which 11 sequences were also found in the gut transcriptomes of both tenebrionid species’ larvae. Studies of the localization of PSPs, evaluation of the expression level of their genes in gut transcriptomes, and prediction of the presence of signal peptides determining secretory pathways made it possible to propose a set of peptidases that can directly participate in the hydrolysis of food proteins in the larvae guts. The discovered digestive PSPs of tenebrionids in combination with the post-glutamine cleaving cysteine cathepsins of these insects effectively hydrolyzed gliadins, which are the natural food substrates of the studied pests. Based on the data obtained, a hypothetical scheme for the complete hydrolysis of immunogenic gliadin peptides by T. molitor and T. castaneum digestive peptidases was proposed. These results show promise regarding the development of a drug based on tenebrionid digestive enzymes for the enzymatic therapy of celiac disease and gluten intolerance.

Wheat gliadins contain a large amount of glutamine- and proline-rich peptides which are not hydrolyzed by human digestive peptidases and can cause autoimmune celiac disease and other forms of gluten intolerance in predisposed people. Peptidases that efficiently cleave such immunogenic peptides can be used in enzyme therapy. The stored product insect pest Tribolium castaneum efficiently hydrolyzes gliadins. The main digestive peptidase of T. castaneum is cathepsin L, which is from the papain C1 family with post-glutamine cleavage activity. We describe the isolation and characterization of T. castaneum recombinant procathepsin L (rpTcCathL1, NP_001164001), which was expressed in Pichia pastoris cells. The activation of the proenzyme was conducted by autocatalytic processing. The effects of pH and proenzyme concentration in the reaction mixture on the processing were studied. The mature enzyme retained high activity in the pH range from 5.0 to 9.0 and displayed high pH-stability from 4.0 to 8.0 at 20 °C. The enzyme was characterized according to electrophoretic mobility under native conditions, activity and stability at various pH values, a sensitivity to various inhibitors, and substrate specificity, and its hydrolytic effect on 8-, 10-, 26-, and 33-mer immunogenic gliadins peptides was demonstrated. Our results show that rTcCathL1 is an effective peptidase that can be used to develop a drug for the enzyme therapy of various types of gluten intolerance.