Biochemistry (Moscow)最新文献

Recombinant proteorhodopsin ESR of the gram-positive bacterium Exiguobacterium sibiricum isolated from permafrost deposits in northeastern Siberia binds retinal and acts as a light-dependent proton pump, but not much is known about its expression under natural conditions. In this work, expression of the esr gene in E. sibiricum cultures grown under various conditions was studied by quantitative PCR. It has been discovered that cultivation on poor media at low temperatures contributes to a significant increase in the content of the corresponding mRNA. The data obtained are confirmed by the results of the analysis of the membrane fraction of cells using label-free quantitative chromatography-mass spectrometry. Also, at 10°C, increased content of phytoene desaturases, which are involved in the biosynthesis of carotenoids, is observed. However, we were unable to detect the presence of a functional retinal-containing protein in the cells, presumably due to the lack of an enzymatic retinal synthesis system in E. sibiricum. The possible functions of ESR in E. sibiricum cells are discussed in connection with the characteristics of the extreme habitat of the bacterium. The results of this study contribute to expanding the understanding of the molecular mechanisms of microbial adaptation to environmental conditions and the potential role of microbial rhodopsins in these processes.

Photosystem II (PSII) is one of the most vulnerable components of photosynthetic apparatus of the thylakoid membrane to the action of inhibitory factors. The donor side of PSII exhibits high sensitivity to photoinhibition and thermal inactivation, which leads to the loss of O₂-evolving function of the water-oxidizing complex (WOC). The data obtained in this study demonstrated increased stability of WOC activity in the PSII membrane preparations from the wild-type (WT) Chlamydomonas reinhardtii compared to the PSII preparations from the cia3 mutant, which lack α-carbonic anhydrase (CA) CAH3, under conditions of moderate photoinhibition and thermal inactivation. This effect was completely eliminated by adding a CA inhibitor to the PSII preparations from WT. At the same time, addition of active recombinant CAH3 (rCAH3) protein to the preparations from cia3 restored increased resistance of PSII to these factors. Under the same conditions of photoinhibition and thermal inactivation, the PSII preparations from Arabidopsis thaliana demonstrated very low loss of O₂-evolving activity, regardless of the presence or absence of carbonic anhydrase α-CA4, which is similar to CAH3. More pronounced suppression of the O₂-evolving activity in the PSII from A. thaliana mutants lacking CA4 was observed only when they were incubated at elevated temperature, indicating the possibility of more significant conformational changes in the WOC proteins of PSII. Despite the clear binding of the rCAH3 to PSII membrane preparations from A. thaliana, the enzyme had little effect on the WOC activity in these preparations, suggesting absence of functional interaction between the rCAH3 and PSII from A. thaliana. The obtained results indicate different mechanisms of involvement of CAH3 and CA4, both of which are assumed to exist in close association with PSII in live systems, in the PSII functioning.

This paper reports the design of a thermostable temperature-activated mini-intein based on the full-length intein DnaE1 from Thermus thermophilus HB27 (TthDnaE1). We performed rational design of three mini-inteins TthDnaE1 Δ272, Δ280, and Δ287 through deletion mutations in the full-length intein sequence. Two mini-inteins (Δ272 and Δ280) were capable of efficient protein splicing at temperatures above 50°C. The most active mini-intein with the Δ280 deletion was selected as a platform for further design of a self-cleaving carrier of affinity tags through single-point mutagenesis. Three mutations – C1A, D405G, and the combined C1A/D405G – were introduced to inhibit N-terminal extein cleavage and extein ligation. As a result, the mini-intein Δ280 with double mutation C1A/D405G displayed the highest efficiency of C-terminal extein cleavage with temperature optimum around 60°C. Thus, we constructed thermostable temperature-activated mini-intein capable of efficient protein splicing or cleavage of the C-terminal extein. The engineered TthDnaE1 Δ280 C1A/D405G mini-intein can serve as a basis for the development of new expression system for intein-mediated production of pharmaceutically relevant recombinant proteins and peptides.

The untranslated regions (UTRs) of messenger RNAs (mRNAs) play a crucial role in regulating translational efficiency, stability, and tissue-specific expression. The review describes various applications and challenges of UTR design in the development of gene therapy and mRNA-based therapeutics. UTRs affect critical biological functions, such as mRNA stability, modulation of protein synthesis, and attenuation of immune response. Incorporating tissue-specific microRNA (miRNA)-binding sites into 3′ UTRs might improve precise targeting of transgene expression and minimize off-target effects. Nucleotide modifications (pseudouridine, N1-methyladenosine, and N4-acetylcytidine) in mRNA and UTRs in particular, improve mRNA stability and translational efficiency. At the same time, several challenges remain, such as lack of consensus on UTRs best suited for certain biomedical applications. Current efforts are focused on integrating high-throughput screening, computational modeling, and experimental validation to refine UTR-based therapeutic strategies. The review presents current information on the design of UTRs and their role in therapeutic applications, with special focus on the possibilities and limitations of existing approaches.

Quantitative analysis of gene transcription is widely used across various fields of biology and, in particular, in medicine, it serves as a tool for diagnostics and transcriptomic profiling of diseases. In recent years, transcriptome analysis methods based on large-scale next-generation sequencing have become widely adopted. Transcriptomic studies enable the identification of cellular processes that are active at specific time points, the investigation of transcriptome dynamics in different tissues or physiological states (such as during ontogenesis or adaptive responses) and the detection of differentially expressed genes in pathological conditions. A pronounced change in the transcription level of one or more genes under pathological conditions may be sufficient for diagnosis, serving as a transcriptional biomarker of disease. However, in some cases, altered transcription levels may indicate the presence of mutations, including those leading to disruption of splicing, activation of mobile elements, or pseudogenes. This review discusses cases in which transcriptional changes can provide insights into the genetic causes of disease, as well as the challenges that must be considered when using transcription as a diagnostic marker. In the future, specialized targeted panels based on transcriptome analysis are expected to be used not only as diagnostic and prognostic tools, but also as predictors of structural genomic abnormalities, thereby contributing to the development of novel strategies for effective disease treatment.

Tuberculosis is a leading cause of death from a bacterial infection agent. The development of new tuberculosis vaccines can reduce the number of new cases and tuberculosis-related deaths. One of the most promising areas in vaccination is development of mRNA vaccines, which have already proven their high effectiveness against COVID-19 and other viral infections. Using modern immunoinformatic methods, we developed four new antituberculosis multiepitope mRNA vaccines differing in the encoded adjuvants and codon composition and tested their immunogenicity and protectivity in mice. Most of the developed mRNA vaccines induced the formation of both cellular and humoral immunity. The adaptive response was stronger for the vaccines with the RpfE adjuvant; however, the best protective response was elicited by the mRNA-mEp21-FL-IDT vaccine with the FL adjuvant. This vaccine reduced the mycobacterial load in the lungs of mice infected with Mycobacterium tuberculosis and increased their survival rate. Altogether, our results indicate that the mRNA-mEp21-FL-IDT vaccine ensures effective protection against tuberculosis comparable to that provided by the BCG vaccine.

Telomere biology still remains a topic of interest in life sciences. Analysis of several thousand clinical samples from healthy individuals performed in recent years has shown that the telomere length (TL) in peripheral blood leukocytes correlates with the TL in cells of internal organ and reflects their condition. TL decreases under the influence of damaging factors and can serve as an indicator of health status. The telomere shortening leads to the cell proliferation arrest and is considered as a marker of replicative aging of proliferating cells. A decrease in the TL in peripheral blood leukocytes is viewed as an indicator of organism aging. Recent studies have allowed to formulate the concept on the role of the CST–polymerase α/primase in the C-strand fill in after completion of 3′G overhang synthesis by telomerase during telomere replication. The discovery of the telomeric RNA (TERRA) and its role in the regulation of telomerase activity (TA) and alternative lengthening of telomeres, as well as the possibility of TERRA translation, has provided evidence of the complex epigenetic regulation of the TL maintenance. Analysis of the published data indicates that telomeres are dynamic structures, whose length undergoes significant changes under the influence of damaging factors. TL is determined not only by the chronological age, but also by the exposure to the exogenous and endogenous deleterious factors during the lifetime. A decrease in the TL due to inherited mutations in the genes coding for proteins involved in the telomere structure formation and telomere replication (primarily, proteins of the shelterin and CST complexes and telomerase) has been found in a number of hereditary diseases – telomeropathies. The assessment of TL and TA is of great importance for the diagnostics of telomeropathies and can be useful in the diagnostics of cancer. Analysis of TL can be used for monitoring the health status (e.g., in the case of exposure to ionizing radiation and space flight factors), as well as predicting individual’s sensitivity to the action of various damaging agents. The application of modern advancement in genetic technologies in the analysis of TL and TA makes it available for the use in clinical and epidemiological studies, diagnostics of telomeropathies, and monitoring of astronauts’ health.

The worldwide number of deaths from complications caused by severe influenza and COVID-19 is about 1 million cases annually. Development of the effective antiviral therapy strategies for the disease treatment is one of the most important tasks. Use of the CRISPR/Cas13 system, which specifically degrades viral RNA and significantly reduces titer of the virus, could be a solution of this problem. Despite the fact that Cas13 nucleases have been discovered only recently, they already have shown high efficiency in suppressing viral transcripts in cell cultures. The recent advances in mRNA technology and improvements in non-viral delivery systems have made it possible to effectively use CRISPR/Cas13 in animal models as well. In this review, we analyzed experimental in vitro and in vivo studies on the use of CRISPR/Cas13 systems as an antiviral agent in cell cultures and animal models and discussed main directions for improving the CRISPR/Cas13 system. These data allow us to understand prospects and limitations of the further use of CRISPR/Cas13 in the treatment of viral diseases.

Gastric cancer (GC) is one of the most common malignant tumors worldwide and ranks fifth in the structure of cancer mortality. MicroRNAs are involved in the pathogenesis and progression of GC as epigenetic factors, and are considered as potential noninvasive markers. We selected microRNAs involved in the regulation of epigenetic mechanisms in GC (miR-1301-3p, miR-106a-5p, miR-129-5p, miR-3613-3p, miR-647) and analyzed their expression in plasma of GC patients. To assess their diagnostic and prognostic potential, we estimated correlations of differential expression with clinical and pathological characteristics of GC tumors. The study included 65 plasma samples from the GC patients and 48 plasma samples obtained from the individuals without tumor lesions, which were used as a control group. The expression was analyzed by using real-time polymerase chain reaction (RT-PCR) method. When comparing the expression levels of selected microRNAs in the plasma of GC patients and the control group, significant differences were found for miR-1301-3p (p = 0.040), miR-106a-5p (p = 0.029), miR-129-5p (p < 0.0001), miR-647 (p < 0.0001). MiR-129-5p expression was significantly associated with the prevalence of a primary tumor (p = 0.002), with the development of metastases to regional lymph nodes (p = 0.003), and distant metastases (p = 0.003), as well as with the late clinical stage (p = 0.003). There was a significant correlation between the miR-3613-3p expression and the clinical stage of GC (p = 0.049). ROC analysis revealed that combining miR-106a-5p, miR-129-5p, miR-1301-3p, and miR-647 improves diagnostic and prognostic properties of the potential panel of markers.