In this study, we investigate the ability of ethanol-inducible CYP2E1 to interact with other cytochrome P450 species and affect the metabolism of their substrates. As a model system, we used CYP2E1-enriched human liver microsomes (HLM) obtained by the incorporation of purified CYP2E1. Using a technique based on homo-FRET in oligomers of CYP2E1 labeled with BODIPY 577/618 maleimide we demonstrated that the interactions of CYP2E1 with HLM result in the formation of its mixed oligomers with other P450 species present in the microsomal membrane. Incorporation of CYP2E1 results in a multifold increase in the rate of metabolism of CYP2E1-specific substrates p-Nitrophenol and Chlorzaxozone. The rate of their oxidation remains proportional to the amount of incorporated CYP2E1 up to the content of 0.3–0.4 nmol/mg protein (or ∼50% CYP2E1 in the P450 pool). The incorporated CYP2E1 becomes a fully functional member of the P450 ensemble and do not exhibit any detectable functional differences with the endogenous CYP2E1. Enrichment of HLM with CYP2E1 results in pronounced changes in the metabolism of 7-ethoxy-4-cyanocoumarin (CEC), the substrate of CYP2C19 and CYP1A2 suggesting an increase in the involvement of the latter in its metabolism. This effect goes together with an augmentation of the rate of dealkylation of CYP1A2-specific substrate 7-ethoxyresorufin. Furthermore, probing the interactions of CYP2E1 with model microsomes containing individual P450 enzymes we found that CYP2E1 efficiently interacts with CYP1A2, but lacks any ability to form complexes with CYP2C19. This finding goes inline with CYP2E1-induced redirection of the main route of CEC metabolism from CYP2C19 to CYP1A2.

No abstract

The cutoff level applied in sequencing analysis varies according to the sequencing technology, sample type, and study purpose, which can largely affect the coverage and reliability of the data obtained. In this study, we aimed to determine the optimal combination of parameters for reliable RNA transcriptome data analysis. Toward this end, we compared the results obtained from different transcriptome analysis platforms (quantitative polymerase chain reaction, Illumina RNASeq, and Oxford Nanopore Technologies MinION) for the transcriptome encoded by human chromosome 18 (Chr 18) using the same sample types (HepG2 cells and liver tissue). A total of 275 protein-coding genes encoded by Chr 18 was taken as the gene set for evaluation. The combination of Illumina RNASeq and MinION nanopore technologies enabled the detection of at least one transcript for each protein-coding gene encoded by Chr 18. This combination also reduced the probability of false-positive detection of low-copy transcripts due to the simultaneous confirmation of the presence of a transcript by the two fundamentally different technologies: short reads essential for reliable detection (Illumina RNASeq) and long-read sequencing data (MinION). The combination of these technologies achieved complete coverage of all 275 protein-coding genes on Chr 18, identifying transcripts with non-zero expression levels. This approach can improve distinguishing the biological and technical reasons for the absence of mRNA detection for a given gene in transcriptomics.

One of the main goals of the Chromosome-Centric Human Proteome Project (C-HPP) is detection of “missing proteins” (PE2-PE4). Using the UPS2 (Universal proteomics standard 2) set as a model to simulate the range of protein concentrations in the cell, we have previously shown that 2D fractionation enables the detection of more than 95% of UPS2 proteins in a complex biological mixture. In this study, we propose a novel experimental workflow for protein detection during the analysis of biological samples. This approach is extremely important in the context of the C-HPP and the neXt-MP50 Challenge, which can be solved by increasing the sensitivity and the coverage of the proteome encoded by a particular human chromosome. In this study, we used 2D fractionation for in-depth analysis of the proteins encoded by human chromosome 18 (Chr 18) in the HepG2 cell line. Use of 2D fractionation increased the sensitivity of the SRM SIS method by 1.3-fold (68 and 88 proteins were identified by 1D fractionation and 2D fractionation, respectively) and the shotgun MS/MS method by 2.5-fold (21 and 53 proteins encoded by Chr 18 were detected by 1D fractionation and 2D fractionation, respectively). The results of all experiments indicate that 111 proteins encoded by human Chr 18 have been identified; this list includes 42% of the Chr 18 protein-coding genes and 67% of the Chr 18 transcriptome species (Illumina RNaseq) in the HepG2 cell line obtained using a single sample. Corresponding mRNAs were not registered for 13 of the detected proteins. The combination of 2D fractionation technology with SRM SIS and shotgun mass spectrometric analysis did not achieve full coverage, i.e., identification of at least one protein product for each of the 265 protein-coding genes of the selected chromosome. To further increase the sensitivity of the method, we plan to use 5–10 crude synthetic peptides for each protein to identify the proteins and select one of the peptides based on the obtained mass spectra for the synthesis of an isotopically labeled standard for subsequent quantitative analysis. Data are available via ProteomeXchange with the identifier PXD019263.

We explore the principles of pressure tolerance in enzymes of deep-sea fishes using lactate dehydrogenases (LDH) as a case study. We compared the effects of pressure on the activities of LDH from hadal snailfishes Notoliparis kermadecensis and Pseudoliparis swirei with those from a shallowadapted Liparis florae and an abyssal grenadier Coryphaenoides armatus. We then quantified the LDH content in muscle homogenates using mass-spectrometric determination of the LDH-specific conserved peptide LNLVQR. Existing theory suggests that adaptation to high pressure requires a decrease in volume changes in enzymatic catalysis. Accordingly, evolved pressure tolerance must be accompanied with an important reduction in the volume change associated with pressure-promoted alteration of enzymatic activity (DV PP). Our results suggest an important revision to this paradigm. Here, we describe an opposite effect of pressure adaptation-a substantial increase in the absolute value of DV PP in deep-living species compared to shallow-water counterparts. With this change, the enzyme activities in abyssal and hadal species do not substantially decrease their activity with pressure increasing up to 1-2 kbar, well beyond full-ocean depth pressures. In contrast, the activity of the enzyme from the tidepool snailfish, L. florae, decreases nearly linearly from 1 to 2500 bar. The increased tolerance of LDH activity to pressure comes at the expense of decreased catalytic efficiency, which is compensated with increased enzyme contents in high-pressure-adapted species. The newly discovered strategy is presumably used when the enzyme mechanism involves the formation of potentially unstable excited transient states associated with substantial changes in enzyme-solvent interactions.