Biology subjects

Shevchenko, A.

Publications and source records attributed to Shevchenko, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Identification of lipid specificity in membrane organizing protein complexes

Organelle membrane identity is encoded by protein and lipid composition1. Human cells produce thousands of distinct lipid species for this purpose2,3. Yet, the precise molecular functions of most lipids remain unknown, owing to limited methods for in situ investigations of individual lipid species. Here, we use minimally modified bifunctional lipid probes to detect and functionally characterize lipid-protein interactions across time and subcellular compartments. We leverage this approach to map time-resolved protein interactomes of individual lipids representing major membrane lipid classes as they are transported through the organelle system of human cells. We identify hundreds of unique lipid-protein interaction candidates, measure lipid-induced protein abundance changes, and distinguish between directly and indirectly interacting subunits of associated protein complexes. Informed by this multimodal dataset, we interrogate the lipid specificity of membrane organizing complexes, in particular the selective co-association of phosphatidylethanolamine with the ancestral subunits of the nuclear pore (NDC1) and MICOS (MIC60) complexes. Using super-resolution microscopy and molecular dynamics simulations, we demonstrate that phosphatidylethanolamine stabilizes both NDC1 and MIC60 in highly curved membrane nanodomains, indicating that major membrane organizing complexes are positioned by selective lipid-protein interactions. Altogether, our experimental strategy provides a blueprint for discovering and characterizing cellular functions of individual lipids.

cell biology↗

bioRxiv2025

Ribosomal frameshifting is a carefully tuned modifier of proteins

Ribosomal frameshifting, occurring during protein translation, is one of the most consequential recoding events, as it leads to large changes to the protein sequence. However, beyond a few well-characterized cases, its prevalence and consequences across organisms remain poorly understood. To address this, we modeled and experimentally tested frameshifting and systematically characterized its evolutionary impact. We developed SLIPPERRS, a mechanistic model that identifies "slippery" frameshift sites achieving [~]70% accuracy. Building on SLIPPERS predictions and on our mass spectrometry and fluorescence-based assay, we characterized 165 short mRNA sequences. We identified dozens of novel sites with frameshift probabilities that often exceed those of known programmed frameshift cases, and found evidence for both translocation and decoding mechanisms. In Gammaproteobacteria, an average of [~]100 genes per species harbor frameshifting sites, which alter proteins by truncating, extending, and fusing canonical proteins. We detect selection for shorter and less disordered frameshifted proteins to mitigate their deleterious impact. We found negative correlation between GC-content and the occurrence of frameshifting sites. In GC-rich bacteria, where frameshift products are expected to be particularly long and disordered, there is selection for cryptic stop codons. While frameshifting is prevalent and expands protein diversity, it is carefully tuned to avoid its harmful consequences.

biochemistry↗

bioRxiv2025

Absolute Quantification of Fluorescent Protein Fusions by Proteomics

Fusions with fluorescence proteins (FPs) play a pivotal role in experimental biology because of their sensitive and spatially precise visualization by spectroscopy. However, observed fluorescence is not always proportional to their molar abundance. Only a fraction of the fusion protein containing the mature fluorescence chromophore is detectable by spectroscopy and there is no generic method for estimating its molar abundance. We developed a fluorescence-independent mass-spectrometry based approach for accurate absolute sub-femtomole quantification of FP-fusions that also estimates the protein faction having fully matured chromophore. The method exploits an isotopically labelled 68 kDa recombinant protein standard containing peptide proxies for 6 prototypical FPs (mCherry, mScarlet-I, mKate2, EGFP, mNeonGreen and Dendra2) and two self-labelling (Halo- and SNAP-) tags. It enables quantification of proteins fused to any of more than 70 FPs or self-labelling tags. It is versatile, robust, precise and can be used broadly for the absolute quantification of fluorescent fusions in in-vivo and in-vitro cellular systems. As case study we combined mass spectrometry with fluorescence spectroscopy to study expression kinetics of FP fusions in cell-free systems. Molar concentrations of the expressed fusion protein, its fraction containing the mature fluorescent chromophore and of the fluorescent protein were integrated into a mathematical model to obtain kinetic rates of translation, chromophore maturation and folding.

biochemistry↗

bioRxiv2025

Disruption of Plasma Membrane Lipid Asymmetry Alters Cellular Energetics

Biological membranes often feature an unequal and actively maintained concentration gradients of lipids between bilayer leaflets, termed lipid asymmetry.1-3 Lipid asymmetry has been linked to many cellular processes4,5, but the mechanisms that connect lipid trans-bilayer concentration gradients to cellular functions are poorly understood. Here we systematically map the cellular processes affected by dysregulation of lipid asymmetry by knocking out flippases, floppases, and scramblases in mammalian cells. We identify broad alterations to core metabolic pathways including nutrient uptake, neutral lipid turnover, pentose phosphate pathway and glycolysis. Lipidomics, respirometry, lipid and metabolic imaging, and live-cell calorimetry revealed elevated neutral lipid and ATP consumption rates which are coupled with increased heat loss per unit of biomass synthesized despite slower growth. This suggests that compensatory maintenance of lipid asymmetry strains the cellular energy budget, inducing a shift from a growth-promoting anabolic to a more energy-using catabolic state. Our data indicate that lipid asymmetry and its active maintenance is key for proper cell energetics, highlighting its putative role as a cellular store of potential energy akin to proton and ion transmembrane gradients.

cell biology↗

bioRxiv2024

A comparative analysis of planarian regeneration specificity reveals tissue polarity contributions of the axial cWnt signalling gradient.

Planarians exhibit remarkable whole-body regeneration abilities. The formation of heads at forward-facing wounds and tails at rearward-facing wounds suggests an intrinsic tissue polarity guiding regeneration. While the underlying mechanisms remain unclear, reports of double-headed regenerates from increasingly narrow tissue fragments have long been hypothesised to reflect gradient-based polarity specification. Here, we systematically re-examine this hypothesis in the modern model species Schmidtea mediterranea and a representative of the genus likely used in the original studies, Girardia sinensis. While we never observed double-heads in S. mediterranea, G. sinensis readily regenerated double-heads in a manner dependent on piece length, anatomical position and body size. We found that the reduced regeneration robustness of G. sinensis was partially explained by wound site-symmetric expression of the head determinant notum, which is highly anterior-specific in S. mediterranea. Exploring what else might mediate head/tail regeneration specificity in G. sinensis, we examined the role of the conserved tail-to-head cWnt signalling gradient. By developing a time-resolved pharmacological approach to reduce the cWnt gradient slope without affecting wound-induced cWnt signalling dynamics, we observed an increased incidence of double-headed regenerates. In addition, the body size-dependence of double-head formation correlated with the decreasing steepness of the cWnt gradient due to scaling. Taken together, our results indicate that the slope of the cWnt gradient may contribute to planarian head/tail regeneration specificity. Furthermore, they suggest that planarian tissue polarity is composed of multiple parallely-acting polarity cues, the differential reliance on which contributes to the observed interspecies variation in regeneration specificity.

developmental biology↗

bioRxiv2024

Snapshots of acetyl-CoA synthesis, the final step of CO2 fixation in the Wood-Ljungdahl pathway

In the ancient microbial Wood-Ljungdahl pathway, CO2 is fixed in a multi-step process ending with acetyl-CoA synthesis at the bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase complex (CODH/ACS). Here, we present catalytic snapshots of the CODH/ACS from the gas-converting acetogen Clostridium autoethanogenum, characterizing the molecular choreography of the overall reaction including electron transfer to the CODH for CO2 reduction, methyl transfer from the corrinoid iron-sulfur protein (CoFeSP) partner to the ACS active site and acetyl-CoA production. Unlike CODH, the multidomain ACS undergoes large conformational changes to form an internal connection to the CODH active site, accommodate the CoFeSP for methyl transfer and protect the reaction intermediates. Altogether, the structures allow us to draw a detailed reaction mechanism of this enzyme crucial for CO2 fixation in anaerobic organisms. One-Sentence Summary: Structural description of key states of CO2 fixation by the carbon monoxide dehydrogenase/acetyl-CoA synthase complex.

biophysics↗

bioRxiv2024

Carbon monoxide-driven bioethanol production operates via a tungsten-dependent catalyst

Microbial alcohol production from waste gasses is a game changer for sustainable carbon cycling and remediation. While the biotechnological process employing Clostridium autoethanogenum to transform syngas (H2/CO2/CO) is blooming, the reactions involved in ethanol biosynthesis remain to be demonstrated. Here, we experimentally showed that ethanol production initiates via a tungsten-dependent aldehyde:ferredoxin oxidoreductase (AFOR), which reduces acetate to acetaldehyde. Such an unfavourable reaction has often been considered unsuitable for a biological process. To answer this riddle, we demonstrated that the thermodynamic pull of CO-oxidation and ethanol synthesis is crucial for triggering acetate reduction. The experimental setup performed with native CO-dehydrogenase and AFOR highlighted the key role of ferredoxin in stimulating the activity of both metalloenzymes and electron shuttling. The crystal structure of holo AFOR refined to 1.59-[A] resolution, together with its biochemical characterisation, provides new insights into the reaction mechanism and the specificities of this enzyme fundamental to sustainable biofuel production.

biochemistry↗

bioRxiv2024

A multi-year longitudinal harmonization of quality controls in mass spectrometry proteomics core facilities

Quality control procedures play a pivotal role in ensuring the reliability and consistency of data generated in mass spectrometry-based proteomics laboratories. However, the lack of standardized quality control practices across laboratories poses challenges for data comparability and reproducibility. In response, we conducted a harmonization study within proteomics laboratories of the Core for Life alliance with the aim to establish a common quality control framework, which facilitates comprehensive quality assessment and identification of potential sources of performance drift. Through collaborative efforts, we developed a consensus quality control standard for longitudinal assessment and we adopted a common processing software. We generated a 4-year longitudinal dataset from multiple instruments and laboratories, which enabled us to assess intra- and inter-laboratory variability, to identify causes of performance drift, and to establish community reference values for several quality control parameters. Our study enhances data comparability and reliability and fosters a culture of collaboration and continuous improvement within the proteomics community to ensure the integrity of proteomics data.

biochemistry↗

bioRxiv2024

Quantitative imaging of species-specific lipid transport in mammalian cells

Eukaryotic cells produce over 1000 different lipid species which tune organelle membrane properties, control signalling and store energy1,2. How lipid species are selectively sorted between organelles to maintain specific membrane identities is largely unknown due to the difficulty to image lipid transport in cells3. Here, we measured transport and metabolism of individual lipid species in mammalian cells using time-resolved fluorescence imaging of bifunctional lipid probes in combination with ultra-high resolution mass spectrometry and mathematical modelling. Quantification of lipid flux between organelles revealed that directional, non-vesicular lipid transport is responsible for fast, species-selective lipid sorting compared to slow, unspecific vesicular membrane trafficking. Using genetic perturbations, we found that coupling between active lipid flipping and passive non-vesicular transport is a mechanism for directional lipid transport. Comparison of metabolic conversion and transport rates showed that non-vesicular transport dominates the organelle distribution of lipids while species-specific phospholipid metabolism controls neutral lipid accumulation. Our results provide the first quantitative map of retrograde lipid flux in cells4. We anticipate that our pipeline for quantitative mapping of lipid flux through physical and chemical space in cells will boost our understanding of lipids in cell biology and disease.

cell biology↗

bioRxiv2024

Hepatocyte differentiation requires anisotropic expansion of bile canaliculi

During liver development, bipotential progenitor cells called hepatoblasts differentiate into hepatocytes or cholangiocytes. Hepatocyte differentiation is uniquely associated with multi-axial polarity, enabling the anisotropic expansion of apical lumina between adjacent cells and formation of a three-dimensional network of bile canaliculi (BC). Cholangiocytes, the cells forming the bile ducts, exhibit the vectorial polarity common to epithelial cells. Whether and how cell polarization feeds back on the gene regulatory pathways governing hepatoblast differentiation is unknown. Here, we used primary hepatoblasts to investigate the contribution of anisotropic apical expansion to hepatocyte differentiation. Silencing of the small GTPase Rab35 caused isotropic lumen expansion and formation of multicellular cysts with the vectorial polarity of cholangiocytes. Gene expression profiling revealed that these cells express reduced levels of hepatocyte markers and upregulate genes associated with cholangiocyte identity. Time-course RNA sequencing demonstrated that loss of lumen anisotropy precedes these transcriptional changes. Independent alterations in apical lumen morphology induced either by modulation of the subapical actomyosin cortex or increased intraluminal pressure caused similar transcriptional changes. These findings suggest that cell polarity and lumen morphogenesis feedback to hepatoblast-to-hepatocyte differentiation. Summary statementDifferentiation of liver progenitors to functional hepatocytes requires anisotropic elongation of their nascent apical surfaces into tubular bile canaliculi.

developmental biology↗

bioRxiv2023

Multi-species benchmark analysis for LC-MS/MS validation and performance evaluation in bottom-up proteomics

We present an instrument-independent benchmarking procedure and software (LFQ_bout) for validation and comparative evaluation of the performance of LC-MS/MS and data processing workflows in bottom-up proteomics. It enables back-to-back comparison of common and emerging workflows, e.g. diaPASEF or ScanningSWATH, and evaluates the impact of arbitrary, inadequately documented settings or black-box data processing algorithms. The procedure enhances the overall performance and quantitative accuracy while enabling the detection of major error types.

biochemistry↗

bioRxiv2023

Lipidome unsaturation affects the morphology and proteome of the Drosophila eye.

While the proteome of an organism is largely determined by the genome, the lipidome is shaped by a poorly understood interplay of environmental factors and metabolic processes. To gain insights into the underlying mechanisms, we analyzed the impacts of dietary lipid manipulations on the ocular proteome of Drosophila melanogaster. We manipulated the lipidome with synthetic food media that differed in the supplementation of an equal amount of saturated or polyunsaturated triacylglycerols. This allowed us to generate flies whose eyes had a highly contrasting length and unsaturation of glycerophospholipids, the major lipid class of biological membranes, while the abundance of other membrane lipid classes remained unchanged. By bioinformatically comparing the resulting ocular proteomic trends and contrasting them with the impacts of vitamin A deficiency, we identified ocular proteins whose abundances are differentially affected by lipid saturation and unsaturation. For instance, we unexpectedly identified a group of proteins that have muscle-related functions and increase their abundances in the eye upon lipidome unsaturation but are unaffected by lipidome saturation. Moreover, we identified two differentially lipid-responsive proteins involved in stress responses, Turandot A and Smg5, whose abundances decrease with lipid unsaturation. Lastly, we discovered that the ocular lipid class composition is robust to dietary changes and propose that this may be a general homeostatic feature of the organization of eukaryotic tissues, while the length and unsaturation of fatty acid moieties is more variable to compensate environmental challenges. We anticipate that these insights into the molecular responses of the Drosophila eye proteome to specific lipid manipulations will guide the genetic dissection of the mechanisms that maintain visual function when the eye is exposed to dietary challenges.

neuroscience↗

bioRxiv2023

Eye proteome of Drosophila melanogaster

The Drosophila melanogaster eye is a popular model to elucidate the molecular mechanisms that underlie the structure and function of the eye as well as the causes of retinopathies. For instance, the Drosophila eye has been used to investigate the impacts of ageing and environmental stresses such as light-induced damage or dietary deficiencies. Moreover, large-scale screens have isolated genes whose mutation causes morphological and functional ocular defects, which includes key components of the phototransduction cascade. However, the proteome of the Drosophila eye is poorly characterized. Here, we used GeLC-MS/MS to quantify 3516 proteins he adult Drosophila melanogaster eye and provide a generic and expandable resource for further genetic, pharmacological, and dietary studies.

neuroscience↗

bioRxiv2023

Extracellular vesicles of Euryarchaeida: precursor to eukaryotic membrane trafficking

Since their discovery, extracellular vesicles (EVs) have changed our view on how organisms interact with their extracellular world. EVs are able to traffic a diverse array of molecules across different species and even domains, facilitating numerous functions. In this study, we investigate EV production in Haloferax volcanii, as representative for Euryarchaeida. We uncover that EVs enclose RNA, with specific transcripts preferentially enriched, including those with regulatory potential, and conclude that EVs can act as an RNA communication system between haloarchaea. We demonstrate the key role of an EV-associated Ras superfamily GTPase for EV formation in H. volcanii that is also present across other diverse evolutionary branches of Archaea. Ras superfamily GTPases are key players in eukaryotic intracellular vesicle formation and trafficking mechanisms that have been crucial for the emergence of Eukaryotes. Therefore, we propose that archaeal EV formation could reveal insights into the origin of the compartmentalized eukaryotic cell.

microbiology↗

bioRxiv2023

ENVIRONMENT MODULATES PROTEIN HETEROGENEITY THROUGH TRANSCRIPTIONAL AND TRANSLATIONAL STOP CODON MISCODING

Stop codon miscoding events give rise to longer proteins, which may alter the proteins function and thereby generate short-lasting phenotypic variability from a single gene. In order to systematically assess the frequency and origin of stop codon miscoding events, we designed a library of reporters. We introduced premature stop codons into mScarlet that enabled high-throughput quantification of protein synthesis termination errors in E.coli using fluorescent microscopy. We found that under stress conditions, stop codon miscoding may occur with a rate as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon miscoding events. The analysis of selected reporters by mass spectrometry and RNA-seq showed that not only translation but also transcription errors contribute to stop codon miscoding. The RNA polymerase is more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon miscoding by mass spectrometry revealed that temperature regulates the expression of cryptic sequences generated by stop codon miscoding in E.coli. Overall, our findings suggest that the environment influences the accuracy of protein production, which increases protein heterogeneity when the organisms need to adapt to new conditions.

systems biology↗

bioRxiv2023

Absolute quantification of photoreceptor outer segment proteins

Photoreceptor cells generate neuronal signals in response to capturing light. This process, called phototransduction, takes place in a highly specialized outer segment organelle. There are significant discrepancies in the reported amounts of many proteins supporting this process, particularly those of low abundance, which limits our understanding of their molecular organization and function. In this study, we used quantitative mass spectrometry to simultaneously determine the abundances of twenty key structural and functional proteins residing in mouse rod outer segments. We computed the absolute number of molecules of each protein residing within an individual outer segment and the molar ratio amongst all twenty proteins. The molar ratios of proteins comprising three well-characterized constitutive complexes in outer segments differed from the established subunit stoichiometries of these complexes by less than 7%, highlighting the exceptional precision of our quantification. Overall, this study resolves multiple existing discrepancies regarding the outer segment abundances of these proteins, thereby advancing our understanding of how the phototransduction pathway functions as a single, well-coordinated molecular ensemble.

biochemistry↗

bioRxiv2022

Activation of mTOR by release of extracellular cholesterol stores controls the transition from quiescence to growth in C. elegans

Recovery from the quiescent developmental stage called dauer is an essential process in C. elegans and provides an excellent model to understand how metabolic transitions contribute to developmental plasticity. We here show that the depletion of sterol-binding proteins SCL-12 and SCL-13 is the key change in the C. elegans proteome in early dauer recovery. This process releases a cholesterol store that is sequestered in the gut lumen during the dauer state to facilitate the transition into reproductive development. First, the stored cholesterol undergoes endocytosis into the lysosomes of the intestinal cells, where it activates mTOR to promote protein synthesis and growth. Second, it is used for the production of dafachronic acids that switch metabolic programs at the transcriptional level. These processes are essential for population fitness and survival, as loss of SCL-12 and SCL-13, depletion of sterols, and loss of mTOR precludes quiescence exit, ultimately leading to the expiration of the entire population.

cell biology↗

bioRxiv2022

Distinct glycerophospholipids potentiate Gsα-activated adenylyl cyclase activity

Nine mammalian adenylyl cyclases (AC) are pseudoheterodimers with two hexahelical membrane domains which are isoform-specifically conserved. Previously we proposed that these membrane domains are orphan receptors (10.7554/eLife.13098; 10.1016/j.cellsig.2020.109538). Lipids extracted from fetal bovine serum at pH 1 inhibited several mAC activities. Guided by a lipidomic analysis we tested glycerophospholipids as potential ligands. Contrary to expectations we surprisingly discovered that 1-stearoyl-2-docosahexaenoyl-phosphatidic acid (SDPA) potentiated Gs-activated activity of human AC isoform 3 seven-fold. The specificity of fatty acyl esters at glycerol positions 1 and 2 was rather stringent. 1-Stearoyl-2-docosahexaenoyl-phosphatidylserine and 1-stearoyl-2-docosahexaenoyl-phosphatidylethanolamine significantly potentiated several Gs-activated mAC isoforms to different extents. SDPA appears not interact with forskolin activation of AC isoform 3. SDPA enhanced Gs-activated AC activities in membranes from mouse brain cortex. The action of SDPA was reversible. Unexpectedly, SDPA did not affect cAMP generation in HEK293 cells stimulated by isoproterenol, PGE2 and adenosine, virtually excluding a role as an extracellular ligand and, instead, suggesting an intracellular role. In summary, we discovered a new dimension of intracellular AC regulation by chemically defined glycerophospholipids.

biochemistry↗