Doklady Biochemistry and Biophysics最新文献

Alternative splicing is an important mechanism that provides genetic diversity of proteins. Unique loci have been identified in Drosophila melanogaster, where mRNA diversity arises as a result of trans-splicing—a process in which exons from different pre-mRNAs are joined together. The trans-splicing in the mod(mdg4) locus, which encodes more than 31 isoforms, has been studied in detail. Important elements for this process include previously described conserved sequences in the fourth intron. The aim of this study is to further characterize the conserved motifs of the fourth intron, specifically the element at the 5'-end of the intron. Using model transgenic lines, it has been shown that introduced changes in the sequence of the studied element lead to a disruption of trans-splicing. In contrast, similar changes in the endogenous locus did not result in a disruption of trans-splicing. Thus, the conserved element plays a role in trans-splicing but is not critical.

The CP190 protein binds to both housekeeping gene promoters and insulator/boundary elements and plays a critical role in their activity. The aim of this work was to study the effect of deletions in highly conserved regions of the CP190 protein on its functionality. It was shown that deletion of the sequence from 664 to 700 aa leads to a lethal phenotype. Thus, a new region was identified in CP190 that plays an important role in the interaction of the CP190 protein with as yet unidentified partner proteins, stabilization of protein complexes formed by CP190, and their recruitment to chromatin.

The autonomous functioning of regulatory domains in the Bithorax complex (BX-C) of Drosophila melanogaster is maintained by boundaries (insulators) that prevent inappropriate enhancer–promoter interactions. Polycomb response elements (PREs) maintain epigenetic memory within regulatory domains and are often located near insulators, enhancing their blocking activity. The bxd PRE is a well-characterized silencer that regulates the bxd/pbx domain of the Ubx gene in the first abdominal segment. We used a boundary replacement strategy to assess the insulator activity of the bxd PRE. Substituting the bxd PRE for the Fab-7 boundary, which separates the Abd-B regulatory domains in the BX-C, revealed that the bxd PRE has weak insulator activity.

The Bap170 subunit of the SWI/SNF chromatin remodeler exhibits activator functions when artificially recruited to the LacZ reporter promoter in enhancer-dependent transcription. In this study, the functional significance of Bap170 protein domains in reporter activation was analyzed. Deletion of the ARID domain does not reduce Bap170 activity. Increased expression of the LacZ reporter was observed in the form of Bap170 without a region that includes LXXLL motifs. Deletions of the central (RFX domain and IDRs) and C-terminal region (zinc fingers) of Bap170 lead to a significant decrease in transgene expression. Apparently, these regions of Bap170 are critical for the function of this protein in enhancer-dependent transcription.

The MLE helicase of D. melanogaster, like its ortholog DHX9 in mammals, is involved in a wide range of processes related to the regulation of gene expression. In the present study, we investigated the impact of the mle[9] mutation on its own mRNA expression level. It was shown that in addition to the previously described deletion in the catalytic domain of the protein, which impairs its helicase activity, the mle[9] mutation contains an additional small deletion in the C-terminal domain. In the mle[9] mutation background, there was a threefold increase in the expression of the main transcript of the mle gene encoding the full-length protein. Binding of MLE to chromatin at the coding region and promoters of the mle gene and nearby enhancers was analyzed. To exclude the influence of dosage compensation, experiments were performed on females. The data obtained indicate the role of MLE in specific regulation of its own mRNA expression level in vivo at the adult stage.

Splicing, a key step in mRNA maturation, plays a crucial role in the regulation of eukaryotic gene expression. The formation of chimeric mRNAs from two transcripts during splicing is typically a prohibited process; however, in insects, trans-splicing is a primary mechanism for increasing protein diversity for several loci. The aim of this study is to investigate the evolutionary conservativeness of sequences responsible for trans-splicing in the mod(mdg4) locus among species from the Drosophilidae family (order Diptera) and the silkworm (Bombyx mori), which belongs to the order Lepidoptera. Using model transgenic lines, it was shown that sequences from distant Drosophila species retain the ability to support trans-splicing in D. melanogaster. In contrast, analogous sequences in Bombyx mori do not support trans-splicing. Thus, the RNA motifs and their binding hypothetical protein factors, defining trans-splicing, remain conserved among the Drosophilidae group, but have functionally diverged between Diptera and Lepidoptera.

In the previously created modular nanotransporter (MNT) capable of delivering a monobody to Keap1 into the cytosol, the endosomolytic module, translocation domain of diphtheria toxin (DTox), was replaced by the endosomolytic peptide GALA3. It was found that this substitution more than doubles the lifetime of MNT in the blood. Using confocal microscopy, it was shown that MNT with GALA3 was internalized into AML12 cells mainly due to binding to the epidermal growth factor receptor, and is also able to exit from endosomes into the cytosol. Using cellular thermal shift assay, it was shown that MNT with GALA3 and MNT with DTox are equally effective in disrupting the formation of the Nrf2 complex with Keap1, which led to similar protection of AML12 cells from the action of hydrogen peroxide. The obtained results allow not only optimizing the systemic use of MNT, but can also serve as a basis for creating agents aimed at treating diseases associated with oxidative stress.

In this work, two new compounds, N⁶-(4,5-dimethoxyphenyl)adenine and N⁶-(3,5-di-trifluoromethylphenyl)adenine, with a broad range of antiviral activity against RNA viruses were identified. We showed that these compounds exhibit pronounced antiviral activity against human poliovirus types 1, 2, and 3, belonging to enterovirus C species. Both compounds also demonstrated pronounced antiviral activity against Coxsackie viruses B3, B5, and B6, belonging to enterovirus B species. In addition, the compounds demonstrated antiviral activity against Newcastle disease virus, which belongs to the paramyxovirus genus. The compounds discovered in this work can subsequently serve as prototypes for the development of new antiviral drugs against epidemiologically significant human RNA viruses.

Graves’ disease is caused by overactivation of the thyroid-stimulating hormone receptor (TSHR). One approach for its treatment may be the use of negative allosteric modulators (NAM) of TSHR, which normalize TSHR activity and do not cause thyroid hormone (TH) deficiency. The aim of the work was to study the effect of a new compound 5-amino-4-(4-bromophenyl)-2-(methylthio)thieno[2,3-d]pyrimidine-6-carboxylic acid N-tert-butylamide (TPY4) on the basal and TSH-stimulated TH production in cultured FRTL-5 thyrocytes and on basal and thyrotropin-releasing hormone (TRH)-stimulated TH levels in the blood of rats. TPY4 stimulated TH production by thyrocytes and increased TH levels when administered intraperitoneally and orally in rats. It also decreased the TSH-stimulated TH production in thyrocytes and the TRH-stimulated TH levels in rats. Thus, TPY4 is the first known allosteric regulator of TSHR, combining the properties of NAM and a partial agonist, and can be considered as a prototype of drugs for the treatment of Graves’ disease.

Introduction. Breast cancer resistance protein (BCRP) is an efflux membrane transporter that controls the pharmacokinetics of a large number of drugs. Its activity may change when taking some endo- and exogenous substances, thus making it a link in drug interactions. Aim. The aim of the study was to develop a methodology for testing drugs for belonging to BCRP substrates and inhibitors in vitro. Materials and methods. The work was performed on Caco-2 cells overexpressing BCRP, the cultivation was performed in a transwell system consisting of the apical and basolateral chambers. Cells were seeded at the bottom of the apical chamber, which is a semi-permeable membrane. Primarily, the transport of BCRP substrates—methotrexate, mitoxantrone, and quercetin—was evaluated in the concentration range of 1, 5, 10, and 50 μM in the direction from the basal chamber to the apical one (Papp b-a) and in the opposite direction (Papp a-b). The Papp b-a/Papp a-b ratio more than 2 characterizes the involvement of transporter proteins in the transcellular transport of substances. To confirm the involvement of BCRP in their transport, an experiment was carried out with the addition of a transporter inhibitor, reserpine, at a concentration of 50 μM to the transport medium. The concentration of substrates in the chambers was analyzed by HPLC–MS/MS. Results. After the addition of methotrexate (1 μM), mitoxantrone (1 μM), and quercetin (1–10 μM) to the apical or basolateral chambers of the transwell system, their content in the recipient chamber was not detected. At methotrexate concentration of 5 μM, the Papp b-a/Papp a-b ratio was 3.38 ± 0.08, which indicates the involvement of transporters in its transfer. When methotrexate was added to the donor chamber at concentrations of 10 and 50 μM, the Papp b-a/Papp a-b ratio decreased to values below 2. At mitoxantrone concentration of 5 μM, the Papp b-a/Papp a-b ratio was 2.72 ± 0.16. An increase in the concentration to 10 μM led to an increase in the Papp b-a/Papp a-b ratio to 6.18 ± 0.08. At the substance concentration of 50 μM, the index decreased but remained above 2. At the quercetin concentration of 50 µM, the Papp b-a/Papp ratio was below 2. Reserpine reduced the Papp b-a/Papp a-b ratio of methotrexate 3.31 times (p = 0.0002), which indicates the elimination of asymmetry in the transport of the substance. At a mitoxantrone concentration of 10 µM, reserpine reduced its Papp b-a/Papp a-b ratio 3.36 times (p < 0.0001). These results indicate the involvement of BCRP in the control of the transfer of both substances through the cellular monolayer. Conclusions. A method of testing drugs for affiliation to BCRP substrates and inhibitors using methotrexate (5 μM) and mitoxantrone (10 μM) as marker substrates and reserpine (50 μM) as inhibitor was developed and tested on Caco-2 cells.