Biology subjects

Zgoda, V. G.

Publications and source records attributed to Zgoda, V. G..

13 recordsLinked to original sources

bioRxiv2026

A Joint Promoterome-Proteome Atlas Highlights the Molecular Diversity of Human Skeletal Muscles

More than 600 distinct skeletal muscles constitute up to 40% of the total mass of the human body. Human skeletal muscles differ in anatomical position, morphology, origin, and function, but the diversity of their molecular phenotypes, the gene expression and protein abundance profiles, remains poorly explored. Here, we report the large-scale CAGE-Seq promoterome profiling of 75 human skeletal muscles, complemented by 22 matched proteomes obtained with mass spectrometry. We identified 37001 transcribed regulatory elements and 1804 protein groups encompassing 1895 proteins, 80% of which demonstrated non-uniform expression across different muscles. The skeletal muscles of the eye, tongue, and diaphragm had the most distinctive molecular phenotypes, while the overall diversity was driven by hundreds of transcription factors with tissue-specific activity. By analyzing the allelic imbalance of CAGE-Seq reads, we discovered 6653 allele-specific single-nucleotide variants often coinciding with muscle-related GWAS SNPs, including muscle volume. Finally, we provide an interactive online atlas of transcriptomic and proteomic molecular phenotypes, facilitating further studies of gene regulation and heritable pathologies of skeletal muscles.

genomics↗

bioRxiv2025

Different effects of 3-week disuse on the phenotype and gene expression of the calf and thigh muscles

Disuse, like several other pathological conditions, has specific effects on various skeletal muscles; the mechanisms underlying these responses remain unclear. We aimed to compare the disuse-induced changes in the phenotype and proteome of the calf and thigh muscles, and to assess the extent to which these proteomic changes are regulated at the mRNA and other levels. Twelve healthy young males participated in 3-week bed rest. Disuse resulted in a greater decrease in lean mass, aerobic performance, and changes in the proteome and transcriptome of the calf muscles/m. soleus than the thigh muscles/m. vastus lateralis. A greater decrease in calf muscle mass was associated with a decrease in the expression/deactivation of translation regulators, but not to the expression of the main sarcomeric proteins. At the same time, a significant decrease in aerobic performance of the ankle plantar flexors occurred without changing the expression of oxidative enzymes - a marker of mitochondrial density. That decrease was associated with dysregulation of mitochondrial biogenesis. Most large-scale changes in the transcriptome did not translate into changes in the proteome, indicating post-transcription protein buffering. However, changes in the RNA levels were revealed to play a dominant role in regulating specific proteins, whereas for others, this factor played little or no role. In conclusion, our findings partially explain why calf muscles with a strong postural function are more sensitive to short-term disuse. This provides a foundation for developing targeted approaches to counteract the negative effects of disuse on different muscles.

physiology↗

bioRxiv2024

Multidirectional effect of low-intensity electrical myostimulation on gene expression and phenotype in thigh and calf muscles after one week of disuse

PurposeThis study investigated the effects of a one-week disuse, both with and without low-intensity neuromuscular electrical stimulation - a safe (non-traumatic) approach to prevent the loss of muscle mass, on the functional capacities and gene expression in thigh and calf muscles. MethodsThis study assessed the efficiency of low-intensity ([~]10% of maximal voluntary contraction) electrical stimulation in preventing the negative effects of 7-day disuse (dry immersion without and with daily stimulation) on the strength and aerobic performance of the ankle plantar flexors and knee extensors, mitochondrial function in permeabilized muscle fibers, and the proteomic (quantitative mass spectrometry-based analysis) and transcriptomic (RNA-sequencing) profiles of the soleus muscle and vastus lateralis muscle. ResultsApplication of electrical stimulation during dry immersion prevented a decrease in the maximal strength and a slight reduction in aerobic performance of the knee extensors, as well as a decrease in maximal (intrinsic) ADP-stimulated mitochondrial respiration and changes in the expression of genes encoding mitochondrial, extracellular matrix, and membrane proteins in the vastus lateralis muscle. In contrast, for the ankle plantar flexors/soleus muscle, electrical stimulation had a positive effect only on maximal mitochondrial respiration, but slightly accelerated the decline in the maximal strength and muscle fiber cross-sectional area, which appears to be related to the activation of inflammatory genes. ConclusionThe data obtained open up broad prospects for the use of low-intensity electrical stimulation to prevent the negative effects of disuse for "mixed" muscles, meanwhile, the optimization of the stimulation protocol is required for "slow" muscles. Practitioner PointsO_LILow-intensity electrical myostimulation is often used as an alternative to exercise and high-intensity electrical stimulation to prevent the loss of muscle mass and function in patients with severe chronic diseases and in spaceflight. However, its effect on muscles with different functional capacities remains uncertain. C_LIO_LIOne week of disuse (dry immersion) lead to a comparable decrease in the maximal strength and (intrinsic) mitochondrial respiration in both the ankle plantar flexors/soleus muscle and the knee extensors/vastus lateralis muscle. Meanwhile changes in gene expression (transcriptome) were three times more pronounced in the soleus muscle than in the vastus lateralis muscle. C_LIO_LIApplication of electrical stimulation during disuse prevented most of the negative effects of disuse in the knee extensors/vastus lateralis muscle, but accelerated the decline in the maximal strength/muscle fiber cross-sectional area in the ankle plantar flexors/soleus muscle, which may be related to the activation of genes regulating the inflammatory response. C_LI

physiology↗

bioRxiv2023

Age-related changes in human skeletal muscle transcriptome and proteome are moreaffected by chronic inflammation and physical inactivity than primary aging

Evaluation of the influence of primary and secondary aging on the manifestation of molecular and cellular hallmarks of aging is a challenging and currently unresolved issue. Our study represents the first demonstration of the distinct role of primary aging and chronic inflammation/physical inactivity - the most important drivers of secondary aging, in the regulation of transcriptomic and proteomic profiles in human skeletal muscle. To achieve this purpose, young healthy people (n=15), young (n=8) and older (n=37) patients with knee/hip osteoarthritis, a model to study the effect of long-term inactivity and chronic inflammation on the vastus lateralis muscle, were included in the study. It was revealed that widespread and substantial age-related changes in gene expression in older patients relative to young healthy people ([~]4,000 genes regulating mitochondrial function, proteostasis, cell membrane, secretory and immune response) were related to the long-term physical inactivity and chronic inflammation rather than primary aging. Primary aging contributed mainly to the regulation of genes ([~]200) encoding nuclear proteins (regulators of DNA repair, RNA processing, and transcription), mitochondrial proteins (genes encoding respiratory enzymes, mitochondrial complex assembly factors, regulators of cristae formation and mitochondrial reactive oxygen species production), as well as regulators of proteostasis. It was found that proteins associated with aging were regulated mainly at the post-transcriptional level. The set of putative primary aging genes and their potential transcriptional regulators can be used as a resource for further targeted studies investigating the role of individual genes and related transcription factors in the emergence of a senescent cell phenotype.

physiology↗

bioRxiv2022

Massive proteogenomic reanalysis of publicly available proteomic datasets of human tissues in search for protein recoding via adenosine-to-inosine RNA editing

The proteogenomic search pipeline developed in this work has been applied for re-analysis of 40 publicly available shotgun proteomic datasets from various human tissues comprising more than 8,000 individual LC-MS/MS runs, of which 5442 .raw data files were processed in total. The scope of this re-analysis was focused on searching for ADAR-mediated RNA editing events, their clustering across samples of different origin, and classification. In total, 33 recoded protein sites were identified in 21 datasets. Of those, 18 sites were detected in at least two datasets representing the core human protein editome. In agreement with prior art works, neural and cancer tissues were found being enriched with recoded proteins. Quantitative analysis indicated that recoding of specific sites did not directly depend on the levels of ADAR enzymes or targeted proteins themselves, rather it was provided by differential and yet undescribed regulation of interaction of enzymes with mRNA. Nine recoding sites conservative between human and rodents were validated by targeted proteomics using stable isotope standards in murine brain cortex and cerebellum, and an additional one was validated in human cerebrospinal fluid. In addition to previous data of the same type from cancer proteomes, we provide a comprehensive catalog of recoding events caused by ADAR RNA editing in the human proteome.

bioinformatics↗

bioRxiv2021

Number of detected proteins as the function of the sensitivity of proteomic technology in human liver cells

The main goal of the Russian part of C-HPP is to detect and functionally annotate missing proteins (PE2-PE4) encoded by human chromosome 18. However, identifying such proteins in a complex biological mixture using mass spectrometry (MS)-based methods is difficult due to the insufficient sensitivity of proteomic analysis methods. In this study, we determined the proteomic technology sensitivity using a standard set of UPS1 proteins as an example. The results revealed that 100% of proteins in a mixture could only be identified at a concentration of at least 10-9 M. The decrease in concentration leads to protein losses associated with technology sensitivity, and no UPS1 protein is detected at a concentration of 10-13 M. Therefore, two-dimensional fractionation of samples was applied to improve sensitivity. The human liver tissue was examined by selected reaction monitoring and shotgun methods of MS analysis using one-dimensional and two-dimensional fractionation to identify the proteins encoded by human chromosome 18. A total of 134 proteins were identified. The overlap between proteomic and transcriptomic data in human liver tissue was ~50%. This weak convergence is due to the low sensitivity of proteomic technology compared to transcriptomic approaches. Data is available via ProteomeXchange with identifier PXD026997.

systems biology↗

bioRxiv2021

Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Cross-Linking Mass Spectrometry

Aiming to elucidate the grounds of system-wide effects of the alcohol-induced increase in the content of cytochrome P450 2E1 (CYP2E1) on drug metabolism, we explored the array of its protein-protein interactions (proteome) in human liver microsomes (HLM) with chemical crosslinking mass spectrometry (CXMS). Our strategy employs membrane incorporation of purified CYP2E1 modified with photoreactive crosslinkers benzophenone-4-maleimide (BPM) and 4-(N-succinimidylcarboxy)benzophenone (BPS). Exposure of bait-incorporated HLM samples to light was followed by isolating the His-tagged bait protein and its crosslinked aggregates on Ni-NTA agarose. Analyzing the individual bands of SDS-PAGE slabs of thereby isolated protein with the toolset of untargeted proteomics, we detected the crosslinked dimeric and trimeric complexes of CYP2E1 with other drug-metabolizing enzymes. Among the most extensively crosslinked partners of CYP2E1 are the cytochromes P450 2A6, 2C8, 3A4, 4A11, and 4F2. Of particular interest are the interactions of CYP2E1 with the latter two enzymes, which are involved in the synthesis and disposal of vasoactive and pro-inflammatory eicosanoids. We also detected the conjugates of CYP2E1 with UDP-glucuronosyltransferases (UGTs) 1A and 2B, fatty aldehyde dehydrogenase (ALDH3A2), epoxide hydrolase 1 (EPHX1), disulfide oxidase 1 (ERO1L), and ribophorin II (RPN2). These results demonstrate the exploratory power of the proposed CXMS strategy and corroborate the concept of tight functional integration in the human drug-metabolizing ensemble through protein-protein interactions of the constituting enzymes.

biochemistry↗

bioRxiv2020

Human Chr18: "Stakhanovite" Genes, Missing and uPE1 Proteins in Liver Tissue and HepG2 Cells

Missing (MP) and functionally uncharacterized proteins (uPE1) comprise less than 5% of the total number of human Chr18 genes. Within half a year, since the January 2020 version of NextProt, the number of entries in the MP+uPE1 datasets has changed, mainly due to the achievements of antibody-based proteomics. Assuming that the proteome is closely related to the transcriptome scaffold, quantitative PCR, Illumina HiSeq, and Oxford Nanopore Technology were applied to characterize the liver samples of three male donors compared with the HepG2 cell line. The data mining of Expression Atlas (EMBL-EBI) and the profiling of our biospecimens using orthogonal methods of transcriptome analysis have shown that in HepG2 cells and the liver, the genes encoding functionally uncharacterized proteins (uPE1) are expressed as low as for the missing proteins (less than 1 copy per cell), except for selected cases of HSBP1L1, TMEM241, C18orf21, and KLHL14. The initial expectation that uPE1 genes might be expressed at higher levels than MP genes, was compromised by severe discrepancies in our semi-quantitative gene expression data and in public databanks. Such discrepancy forced us to revisit the transcriptome of Chr18, the target of Russian C-HPP Consortia. Tanglegram of highly expressed genes and further correlation analysis have shown the severe dependencies on the mRNA extraction method and analytical platform. Targeted gene expression analysis by quantitative PCR (qPCR) and high-throughput transcriptome profiling (Illumina HiSeq and ONT MinION) for the same set of samples from normal liver tissue and HepG2 cells revealed the detectable expression of 250+ (92%) protein-coding genes of Chr18 (at least one method). The expression of slightly more than 50% protein-coding genes was detected simultaneously by all three methods. Correlation analysis of the gene expression profiles showed that the grouping of the datasets depended almost equally on both the type of biological material and the experimental method, particularly cDNA/mRNA isolation and library preparation. The dependence on the choice of bioinformatics analysis pipeline was also noticeable but significantly less. Furthermore, the combination of Illumina HiSeq and ONT MinION sequencing to validate proteotypic peptides of missing and uPE1 proteins was performed for the heat-shock factor binding protein HSBP1L1 (missing protein, recently transferred to PE1 category) and uncharacterized protein C18orf21 (uPE1). We observed that a nonsynonymous SNP led to the loss of the site of trypsinolysis in HSBP1L1. The modified version of HSBP1L1 was included in the sequence database and searched against the MS/MS dataset from Kulak, Geyer & Mann (2017), but delivered no significant identification. Thus, HSBP1L1 is still missing for the MS-pillar of C-HPP, although its existence at the protein level has been confirmed.

molecular biology↗

bioRxiv2020

Proteome-Wide Analysis of ADAR-mediated Messenger RNA Editing During Fruit Fly Ontogeny

Adenosine-to-inosine RNA editing is an enzymatic post-transcriptional modification which modulates immunity and neural transmission in multicellular organisms. Some of its functions are enforced through editing of mRNA codons with the resulting amino acid substitutions. We identified these sites originated from the RNA editing for developmental proteomes of Drosophila melanogaster at the protein level using available proteomic data for fifteen stages of fruit fly development from egg to imago and fourteen time points of embryogenesis. In total, 42 sites each belonging to a unique protein were found including four sites related to embryogenesis. The interactome analysis has revealed that most of the edited proteins are associated with synaptic vesicle trafficking and actomyosin organization. Quantitation data analysis suggested the existence of phase-specific RNA editing regulation by yet unknown mechanisms. These results support transcriptome analyses showing that a burst in RNA editing occurs during insect metamorphosis from pupa to imago. Further, targeted proteomics was employed to quantify edited and genomically encoded versions of five proteins in brains of larvae, pupae, and imago insects showing a clear trend towards an increase in editing rate for all of them. Our results may help to reveal the protein functions in physiological effects of RNA editing. SignificanceAdenosine-to-inosine RNA editing has multiple effects on body functions in many multicellular organisms from insects and molluscs to humans. Recent studies show that at least some of these effects are mediated by changes in protein sequences due to editing of codons in mRNA. However, it is not known how exactly the edited proteins can participate in RNA editing-mediated pathways. Moreover, most studies of edited proteins are based on the deduction of protein sequence changes from analysis of transcriptome without measurements of proteins themselves. Earlier, we explored for the first time the edited proteins of Drosophila melanogaster proteome. In this work, we continued the proteome-wide analysis of RNA editome using shotgun proteomic data of ontogeny phases of this model insect. It was found that non-synonymous RNA editing, which led to translation of changed proteins, is specific to the life cycle phase. Identification of tryptic peptides containing edited protein sites provides a basis for further direct and quantitative analysis of their editing rate by targeted proteomics. The latter was demonstrated in this study by multiple reaction monitoring experiments which were used to observe the dynamics of editing in selected brain proteins during developmental phases of fruit fly. HighlightsO_LIProteogenomic approach was applied to shotgun proteomics data of fruit fly ontogeny for identification of proteoforms originating from adenosine-to-inosine RNA editing. C_LIO_LIEdited proteins identified at all life cycle stages are enriched in annotated protein-protein interactions at statistically significant level with many of them associated with actomyosin and synaptic vesicle functions. C_LIO_LIProteome-wide RNA editing event profiles were found specific to life cycle phase and independent of the protein abundances. C_LIO_LIA majority of RNA editing events at the protein level was observed after metamorphosis in late pupae to adult insects, which was consistent with transcriptome data. C_LIO_LITargeted proteomic analysis of five selected edited sites and their genomic counterparts in brains for three phases of the fruit fly life cycle have demonstrated a clear increase in editing rate of up to 80% for the endophilin A protein in adult flies. C_LI

systems biology↗

bioRxiv2020

Multifold Activation of CYP3A4 Caused by Alcohol-Induced Increase in the Content of CYP2E1 in Human Liver Microsomes

Here we investigate the effect of alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. In these studies we used a model that implements enrichment of HLM samples with CYP2E1 through membrane incorporation of the purified protein. Enrichment of HLM with CYP2E1 considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. Incorporation of CYP2E1 also eliminates the activating effect of -Naphthoflavone (ANF) on BQ metabolism seen in some untreated HLM samples. To probe the physiological relevance of these effects we compared three pooled preparations of HLM from normal donors (HLM-N) with a preparation obtained from heavy alcohol consumers (HLM-A). The composition of the P450 pool in all four samples was characterized with mass-spectrometric determination of 11 cytochrome P450 species. The molar content of CYP2E1 in HLM-A was from 2.5 to 3.3 times higher than that found in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all four HLM samples. Despite of that, HLM-A exhibited much higher rate of metabolism and lower degree of homotropic cooperativity with BQ, similar to that observed in CYP2E1-enriched HLM-N. In order to substantiate the hypothesis on the involvement of physical interactions between CYP2E1 and CYP3A4 in the observed effects we probed hetero-association of these proteins in Supersomes containing recombinant CYP3A4 with a technique based on homo-FRET and employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments demonstrated high affinity interactions between the two enzymes and revealed an inhibitory effect of ANF on their hetero-association. Our results demonstrate that the catalytic activity and allosteric properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and imply a profound impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4.

biochemistry↗

bioRxiv2019

Non-additive effects of changing the cytochrome P450 ensemble: incorporation of CYP2E1 into human liver microsomes and its impact on CYP1A2

The aim of this study is to 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 microsomes obtained by incorporation of purified CYP2E1 protein into HLM. Using the method based on homo-FRET in homo-oligomers of CYP2E1 labeled with BODIPY 577/618 maleimide we demonstrated that the interactions of CYP2E1 with microsomes result in dissociation of the protein homo-oligomers. The finding that this effect is much better pronounced in HLM as compared to the microsomes containing no P450 proteins indicates the formation of mixed oligomers of CYP2E1 with other P450 species that takes place in expense of dissociation of the homo-oligomers.\n\nIncorporation of CYP2E1 into HLM results in a multifold increase in the rate of metabolism of CYP2E1-specific substrates p-Nitrophenol (pNP) and Chlorzaxozone (CLZ). 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 about 50% CYP2E1 in the P450 pool). These results demonstrate that the incorporated CYP2E1 becomes a fully-functional member of the P450 ensemble and do not exhibit any detectable functional differences with the endogenous CYP2E1 in HLM.\n\nEnrichment of HLM with CYP2E1 results in a pronounced alteration of the metabolism of 7-etoxy-4-cyanocoumarin (CEC), the substrate of CYP2C19 and CYP1A2, that suggests an important augmentation of the involvement of CYP1A2 in its metabolism. This effect goes together with a remarkable increase in the rate of dealkylation of CYP1A2-specific substrate 7-ethoxyresorufin by CYP2E1-enriched HLM. Furthermore, probing the interactions of CYP2E1 with model microsomes (Supersomes) 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.

biochemistry↗

bioRxiv2018

Far beyond common leeching: insights into an ancient medical device through integrated omics data

Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today. We annotated the Hirudo medicinalis genome and performed RNA-seq on salivary cells isolated from three closely related leech species, H. medicinalis, Hirudo orientalis, and Hirudo verbana. Differential expression analysis verified by proteomics identified salivary cell-specific genes, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants were not differentially expressed in the salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis. Thus, our study provides one of the most comprehensive knowledge of the genetic fundamentals of the blood-sucking lifestyle in leeches.

systems biology↗

bioRxiv2017

Proteogenomics of adenosine-to-inosine RNA editing in fruit fly

Adenosine-to-inosine RNA editing is one of the most common types of RNA editing, a posttranscriptional modification made by special enzymes. We present a proteomic study on this phenomenon for Drosophila melanogaster. Three proteome data sets were used in the study: two taken from public repository and the third one obtained here. A customized protein sequence database was generated using results of genome-wide adenosine-to-inosine RNA studies and applied for identifying the edited proteins. The total number of 68 edited peptides belonging to 59 proteins was identified in all data sets. Eight of them being shared between the whole insect, head and brain proteomes. Seven edited sites belonging to synaptic vesicle and membrane trafficking proteins were selected for validation by orthogonal analysis by Multiple Reaction Monitoring. Five editing events in cpx, Syx1A, Cadps, CG4587 and EndoA were validated in fruit fly brain tissue at the proteome level using isotopically labeled standards. Ratios of unedited-to-edited proteoforms varied from 35:1 (Syx1A) to 1:2 (EndoA). Lys-137 to Glu editing of endophilin A may have functional consequences for its interaction to membrane. The work demonstrates the feasibility to identify the RNA editing event at the proteome level using shotgun proteomics and customized edited protein database.

biochemistry↗