Biology subjects

Zubarev, R. A.

Publications and source records attributed to Zubarev, R. A..

16 recordsLinked to original sources

bioRxiv2025

Proteome integral solubility alteration assay for monitoring antibody-antigen cell interactions

The Proteome Integral Solubility Alteration (PISA) assay is commonly used to identify and characterize interactions between small molecules and the cellular proteome. In this study, a modified PISA approach identified tumor necrosis factor alpha (TNF) as the antigen of the monoclonal antibody infliximab in activated monocytes - uniquely among [~]8,000 cellular proteins. These results highlight the potential of PISA-based analysis not only for monitoring antibody-antigen interactions in therapeutic development, but also for identifying new antigens and characterizing the initial effector functions of antibodies within complex proteome environments. TeaserModified PISA pinpointed TNF- as Infliximabs antigen among 8000 proteins, highlighting broad potential for therapeutic antigen studies.

molecular biology↗

bioRxiv2025

Is it time to retire {triangleup}?

The paradigm postulating that tissue stable isotopic ratios ({delta}tissue) equal those of the diet ({delta}diet) plus a small, quasi-constant isotope discrimination factor {Delta} emerged in the late 1970s, establishing stable isotope analysis as a dietary proxy. Today, this framework is still widely used across multiple branches of science, despite growing contradicting evidence. Here, we reanalysed several well-controlled laboratory experiments and universally found that {Delta} not only varies strongly with{delta} diet, but also changes sign at a certain{delta} eq ={delta} diet ={delta} tissue value, which we term the {Delta}-equilibrium. The {Delta}-equilibrium phenomenon results from the sub-unity slope of the linear regression between{delta} diet and{delta} tissue and leads to converging of{delta} tissue values. The most frequently observed position of the {Delta}-equilibrium on the ({delta}13C,{delta} 15N) plane is (-21{+/-}1{per thousand}, 12{+/-}1{per thousand}). These findings firmly establish that stable isotopes are not neutral spectators but active participants in biochemical processes. If presented evidence holds in a much broader study, the paradigm{delta} tissue ={delta} food + {Delta} can finally be retired after half a century of service, being replaced by{delta} tissue = ax{delta}food + Const, where a is the newly defined isotope assimilation factor. The {Delta}-equilibrium position is then found as{delta} eq = Const/(1 - a). The reason for isotope convergence remains a subject for future research, but likely hypotheses include evolutionary adaptation and isotopic resonance.

biochemistry↗

bioRxiv2025

Beyond the known cuts: trypsin specificity in native proteins

Trypsin is a serine protease that plays a pivotal role in protein digestion, being extensively used in various proteomics workflows due to its cleavage profile and specificity. Understanding trypsins enzymatic behavior is thus critical. We have employed Above-Filter Digestion Proteomics (AFDIP) to investigate trypsins cleavage preferences in HeLa cell lysates, preserving the proteins native state. We quantified over 18,000 unique peptides and correlated their emergence rates with cleavage window sequence motifs and physicochemical properties. Contrary to previous studies performed on denatured proteomes, we found that in native proteins cleavages at lysine residues were more abundant and faster than at arginine residues, and that physicochemical properties of the peptides affected their emergence times. These findings may need to be taken into account when interpreting the results of limited proteolysis experiments as well as when designing a food protein with extremely fast digestion times.

biochemistry↗

bioRxiv2025

Towards a species search engine: KISSE offers a rigorous statistical framework for bone collagen tandem mass spectrometry data

DNA and bone collagen are two key sources of resilient molecular markers used to identify species from their remains. Collagen is more stable than DNA, and thus it is preferred for ancient and degraded samples. Current mass spectrometry-based collagen sequencing approaches are empirical and lack a rigorous statistical framework. Based on the well-developed approaches to protein identification in shotgun proteomics, we introduce a first approximation of the species search engine (SSE). Our SSE named KISSE is based on a species-specific library of collagenous peptides that uses both peptide sequences and their relative abundances. The developed statistical model can identify the species and the probability of correct identification, as well as determine the likelihood of the analyzed species not being in the library. We discuss the advantages and limitations of the proposed approach and the possibility of extending it to other tissues.

zoology↗

bioRxiv2024

One-Pot Time-Induced Proteome Integral Solubility Alteration (OPTI-PISA) assay for automated and sensitive drug target identification

Proteome Integral Solubility Alteration (PISA) assay is widely used for identifying drug targets, but it is labour-intensive and time-consuming, and requires a substantial amount of biological sample. Aiming at enabling automation and greatly reducing the sample amount, we developed One-Pot Time-Induced (OPTI)-PISA. Here we demonstrate OPTI-PISA performance on identifying targets of multiple drugs in cell lysate and scaling down the sample amount to sub-microgram levels, making PISA method suitable for NanoProteomics.

biochemistry↗

bioRxiv2024

Gel-Assisted Proteome Position Integral Shift (GAPPIS) Assay Returns Molecular Weight to Shotgun Proteomics and Identifies Novel Caspase 3 Substrates

Here we present a high-throughput virtual top-down proteomics approach that restores the molecular weight (MW) information in shotgun proteomics, and demonstrate its utility in studying proteolytic events in programmed cell death. With Gel-Assisted Proteome Position Integral Shift (GAPPIS), we quantified over 7000 proteins in staurosporine-induced apoptotic HeLa cells and identified 84 proteins exhibiting in a statistically significant manner at least two of the following features: 1) a negative MW shift; 2) an elevated ratio in a pair of a semi-tryptic and tryptic peptide, 3) a negative shift in the standard deviation of MW estimated for different peptides, and 4) a negative shift in skewness of the same data. Of these proteins, 58 molecules were novel caspase 3 substrates. Further analysis identified the preferred cleavage sites consistent with the known caspase cleavages after the DXXD motif. As a powerful tool for high-throughput MW analysis simultaneously with the conventional expression analysis, GAPPIS assay can prove useful in studying a broad range of biological processes involving proteolytic events.

biochemistry↗

bioRxiv2023

Chemical proteomics reveals that the anticancer drug everolimus affects ubiquitin-proteasome system

Rapamycin is a natural antifungal, immunosuppressive, and antiproliferative compound allosterically inhibiting mTOR complex 1. The ubiquitin-proteasome system (UPS) responsible for protein turnover is usually not listed among the pathways affected by mTOR signaling; however, a number of reports indicated the interplay between UPS and mTOR. It is also reported that rapamycin and analogs can allosterically inhibit the proteasome itself. We studied the molecular effect of rapamycin and its analogs (rapalogs), everolimus, and temsirolimus by expression chemical proteomics on A549 cell line. The analysis revealed that the cellular response to everolimus differs dramatically from that of rapamycin and temsirolimus. In cluster analysis the effect of everolimus was similar to that of bortezomib, a well-established proteasome inhibitor. UPS-related pathways were enriched in the cluster of proteins specifically up-regulated upon everolimus and bortezomib treatments, suggesting that both compounds have similar proteasome inhibition effects. In particular, the total amount of ubiquitin was significantly elevated in the samples treated with everolimus and bortezomib, and the analysis of the polyubiquitination patterns revealed elevated intensities of the ubiquitin peptide with a GG modification at K48 residue, consistent with a bottleneck in the proteasomal protein degradation. Moreover, everolimus treatment resulted in both ubiquitin phosphorylation and a significant amount of semi-tryptic peptides illustrating the induction of protease activity. These observations suggest that everolimus affects the ubiquitin-proteasome system in a unique way and its mechanism of action is strikingly different from that of its close chemical analogs, rapamycin and temsirolimus. Data are available via ProteomeXchange with identifier PXD045774.

cancer biology↗

bioRxiv2023

Multifaceted proteome analysis at solubility, redox, and expression dimensions for target identification

Multifaceted interrogation of the proteome deepens the system-wide understanding of biological systems; however, mapping the redox changes in the proteome has so far been significantly more challenging than expression and solubility/stability analyses. Here, we devise the first high- throughput redox proteomics approach integrated with expression analysis (REX) and combine it with Proteome Integral Solubility Alteration (PISA) assay. The whole PISA-REX experiment with up to four biological replicates can be multiplexed into a single tandem mass tag TMTpro set. For benchmarking this compact tool, we analyzed HCT116 cells treated with auranofin, showing great improvement compared with previous such studies. Then we applied PISA-REX to study proteome remodeling upon stimulation of human monocytes by interferon . We also studied the proteome changes in plasmacytoid dendritic cells isolated from wild type vs. Ncf1- mutant mice treated with interferon , showing that NCF1 deficiency enhances the STAT1 pathway and modulates the expression, solubility and redox state of interferon-induced proteins. Providing comprehensive multifaceted information on the proteome, the compact PISA-REX has the potential to become an industry standard in proteomics and to open new windows into the biology of health and disease.

biochemistry↗

bioRxiv2023

SpotLight proteomics identifies variable sequences of blood antibodies specific against deamidated human serum albumin

Spontaneous deamidation of asparaginyl residues in proteins, if not repaired or cleared, can set in motion a cascade that leads to deteriorated health. Previously, we have discovered that deamidated human serum albumin (HSA) is elevated in blood of patients with Alzheimers disease and other neurodegenerative diseases, while the level of endogenous antibodies against deamidated HSA is significantly diminished, creating an imbalance between the risk factor and the defense against it. Endogenous antibodies against deamidated proteins are still unexplored. In the current study, we employed the SpotLight proteomics approach to identify novel amino acid sequences in antibodies specific to deamidated HSA. The results provide new insights into the clearance mechanism of deamidated proteins, a possible avenue for prevention of neurodegeneration.

immunology↗

bioRxiv2023

DOPA residues endow collagen with radical scavenging capacity

Here we uncover collagen, the main structural protein of all connective tissues, as a redox-active material. We identify dihydroxyphenylalanine (DOPA) residues, post-translational oxidation products of tyrosine residues, to be common in collagen derived from different connective tissues. We observe that these DOPA residues endow collagen with substantial radical scavenging capacity. When reducing radicals, DOPA residues work as redox relay: they convert to the quinone and generate hydrogen peroxide. In this dual function, DOPA outcompetes its amino acid precursors and ascorbic acid. Our results establish DOPA residues as redox-active side chains of collagens, probably protecting connective tissues against radicals formed under mechanical stress and/or inflammation.

biophysics↗

bioRxiv2023

Massive solubility changes of neuronal proteins upon simulated traumatic brain injury reveal the role of shockwave in irreversible damage

The immediate molecular consequences of traumatic brain injuries or TBI are poorly understood. Here, we simulated TBI using an innovative laboratory apparatus that employs a 5.1 kg dummy head holding neuronal cells and generating a [≤]4,000 g-force acceleration upon impact. Dynamic impact led to both reduction in neuron viability and massive solubility changes in the proteome profiled using Proteome Integral Solubility Alteration (PISA) assay. The affected proteins mapped not only to the expected pathways like cell adhesion, collagen and laminin structures, as well as response to stress, but also to other dense protein networks, such as immune response, complement and coagulation cascades. The cellular effects are found to be mainly due to the shockwave rather than the g-force acceleration. Soft materials could reduce the impact severity only until being fully compressed. This study shows way to develop a proteome-based meter for measuring irreversible shockwave-induced cell damage and provides a resource for identifying TBI protein biomarkers and potential drug targets for developing products aiming at primary prevention and intervention.

neuroscience↗

bioRxiv2022

Mapping the GALNT1 substrate landscape with versatile proteomics tools

O-GalNAc type glycosylation is a common post-translational modification (PTM) of proteins catalyzed by polypeptide GalNAc transferases, but the substrate specificity of these transferases is poorly understood. Here we develop a strategy based on integral thermal proteome solubility profiling to identify and prioritize the protein substrates of polypeptide N-acetylgalactosaminyltransferase 1 (GALNT1). Combined with glycoprotein enrichment followed by HCD and soft EThcD gas-phase fragmentation technique, we uncover hundreds of novel GALNT1 substrates in two model human cell lines. GALNT1-mediated O-glycosylation is more common on Thr than Ser residues, with a strong preference for Pro at positions +3 and +4 in respect to O-glycosylation. These results implicate GALNT1 in potentially regulating proteins in several diverse pathways, including some unexpected processes, such as TCA cycle and DNA transcription. This study depicts a roadmap for identification of functional substrates for glycosyltransferases, facilitating fundamental insight into the role of glycosylation in homeostasis and disease.

biochemistry↗

bioRxiv2022

Testing the link between isoaspartate and Alzheimer's disease etiology

Isoaspartate (isoAsp) is a damaging amino acid residue formed in proteins as a result of spontaneous deamidation. IsoAsp disrupts the secondary and higher order structures of proteins, damaging their functions and making them prone to aggregation. An association has been suggested between isoAsp and Alzheimers Disease (AD). Here we strengthened the link between isoAsp and AD by novel approaches to isoAsp analysis in blood human serum albumin (HSA), the most abundant blood protein, a major carrier of amyloid beta (A{beta}) peptide and phosphorylated tau (pTau) protein in blood and a key participant in their clearance pathway. We discovered a reduced amount of anti-isoAsp antibodies (P < .0001), an elevated isoAsp level in HSA (P < .001), more HSA aggregates (P < .0001) and increased levels of free A{beta} (P < .01) in AD blood compared to healthy controls. We also found that deamidation significantly reduces HSA capacity to bind with A{beta} and pTau (P < .05). These findings support the presence in AD of a bottleneck in clearance of A{beta} and pTau leading to their increased concentrations in brain and facilitating their aggregations there. RESEARCH IN CONTEXTO_LISystematic review: We reviewed the evidence that associates isoaspartate (isoAsp) residue in blood proteins with the etiology of Alzheimers disease (AD). However, the link between isoAsp in blood and aggregation of amyloid beta (A{beta}) peptide and phosphorylated tau (pTau) protein in brain remained unclear. C_LIO_LIInterpretation: For the first time we demonstrate that isoAsp-containing human serum albumin (HSA) forms aggregates with reduced binding capacity toward A{beta} peptide and pTau protein. Using a novel ELISA, we discovered in AD blood elevated levels of isoAsp in HSA, together with reduced endogenous anti-isoAsp antibody levels, suggesting hampered A{beta} and pTau clearance in AD. C_LIO_LIFuture directions: As degradation of the innate anti-isoAsp defenses may take years to develop, investigation of the isoAsp role in early stages of AD is warranted. And enrollment of different neurodegenerative disease cohorts will illustrate if isoAsp is AD-specific or universal to diseases related to aging. C_LI O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

neuroscience↗

bioRxiv2021

Redox regulation of PTPN22 affects the severity of T cell dependent autoimmune inflammation

Chronic autoimmune diseases are associated with mutations in PTPN22, a modifier of T cell receptor signaling. As with all protein tyrosine phosphatases the activity of PTPN22 is redox regulated, but if or how such regulation can modulate inflammatory pathways in vivo is not known. To determine this, we created a mouse with a cysteine-to-serine mutation at position 129 in PTPN22 (C129S), a residue proposed to alter the redox regulatory properties of PTPN22 by forming a disulfide with the catalytic C227 residue. The C129S mutant mouse showed a stronger T cell-dependent inflammatory response and development of T cell dependent autoimmune arthritis due to enhanced TCR signaling and activation of T cells, an effect neutralized by a mutation in Ncf1, a component of the NOX2 complex. Activity assays with purified proteins suggest that the functional results can be explained by an increased sensitivity to oxidation of the C129S mutated PTPN22 protein. We also observed that the disulfide of native PTPN22 can be directly reduced by the thioredoxin system, while the C129S mutant lacking this disulfide was less amenable to reductive reactivation. In conclusion, we show that PTPN22 functionally interacts with Ncf1 and is regulated by oxidation via the non-catalytic C129 residue and oxidation-prone PTPN22 leads to increased severity in the development of T cell-dependent autoimmunity. Significance statementA hitherto unstudied aspect of PTPN22 biology is its regulation by cell redox states. Here we created a mouse model where PTPN22 was mutated to respond differentially to redox levels in vivo and found that PTPN22 function is regulated by reactive oxygen species and that redox regulation of PTPN22 impacts T-cell-dependent autoimmune inflammation.

immunology↗

bioRxiv2018

Proteome Integral Stability Alteration assay dramatically increases throughput and sensitivity in profiling factor-induced proteome changes

Various factors, including drugs as well as non-molecular influences, induce alterations in the stability of proteins in cell lysates, living cells and organisms. These alterations can be probed by applying a stability-modifying agent, such as elevated temperature, to a varying degree. As a second dimension of variation, drug concentration or factor intensity can be used. However, the corresponding analysis scheme has a low throughput and high cost. Additionally, since traditional data analysis employs curve fitting, proteins with unusual behavior are frequently ignored. The novel Proteome Integral Stability Alteration (PISA) assay avoids these issues altogether, increasing the analysis throughput by one to two orders of magnitude for unlimited number of parameter variation points. The consumption of the compound and biological material decreases by the same factor. We envision widespread use of the PISA approach in chemical biology and drug development.

cancer 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↗