Biology subjects

Hell, R.

Publications and source records attributed to Hell, R..

11 recordsLinked to original sources

bioRxiv2025

MetAlyzer to Perform Streamlined, Interactive, and Pathway-Mapping Analysis of Targeted Metabolomics Data from the biocrates Platform

Mass spectrometry (MS)-based metabolomics has emerged as a powerful tool to address multifaceted biological questions. Commercial solutions like the ones developed at biocrates allow reliable and quantitative targeted metabolic profiling, including the conversion of the raw MS spectra into absolute concentrations of metabolites. These results can be exported for further analysis under several formats with varying levels of human-vs. machine-readability. The default output format is an Excel spreadsheet that favours human readability and therefore requires extra preparation steps for downstream bioinformatic analysis and data exploration. To streamline this next step for users of this platform, we developed MetAlyzer, an R package (https://github.com/Lu-Group-UKHD/MetAlyzer) specifically designed to handle the spreadsheets generated by WebIDQ, the biocrates workflow manager software. MetAlyzer converts WebIDQ-generated spreadsheets into flexible SummarizedExperiment objects and provides functions for data preprocessing, statistical testing, and visualization of differential metabolites. To further support data exploration and hypothesis generation by users without coding experience, we also developed an interactive and intuitive Shiny app (https://metalyzer.shinyapps.io/MetAlyzer_ShinyApp/) that interfaces with MetAlyzers core functionality, enabling users to execute the complete analysis workflow without writing code. This combination can help scientists deepen their understanding of metabolomics results, supporting the broader adoption of metabolomics in the life sciences community.

bioinformatics↗

bioRxiv2025

Impaired methyl recycling induces substantial shifts in sulfur utilization in Arabidopsis

The tight coordination of sulfur metabolism and growth regulation is predicated upon nutrient availability. Central to this balancing act is the utilization of cysteine (Cys) for the formation of methionine (Met) and S-adenosylmethionine (SAM). Plants that are severely deficient in regenerating Met due to reduced methylthioadenosine (MTA) nucleosidase activity experience numerous developmental abnormalities. Here, we assess the developmental, metabolic, and regulatory effects associated with decreased MTA recycling. We show that MTA over-accumulation predominantly occurs in reproductive tissues and leads to reduced levels of Cys, Met, and SAM, as well as elevated S-adenosylhomocysteine. These disruptions of primary sulfur utilization also lead to the misregulation of energy metabolism and altered cell cycle progression. RNA-seq experiments show a general down-regulation of many developmental and reproductive genes. Targeted metabolite analyses demonstrate clear impacts on the methyl index which are reflected in the results of bisulfite-sequencing experiments including global alterations in CG gene-body methylation levels and decreases of CHG and CHH methylation in transposable elements. Our findings demonstrate the broad impacts of MTA metabolism on plant development, sulfur utilization and the maintenance of the methyl index.

plant biology↗

bioRxiv2023

A single-sample workflow for joint metabolomic and proteomic analysis of clinical specimens

Understanding the interplay of the proteome and the metabolome aids in understanding cellular phenotypes. To enable more robust inferences from such multi-omics analyses, combining proteomic and metabolomic datasets from the same sample provides major benefits by reducing technical variation between extracts during the pre-analytical phase, decreasing sample variation due to varying cellular content between aliquots, and limiting the required sample amount. We evaluated the advantages, practicality and feasibility of a single-sample workflow for combined proteome and metabolome analysis. In the workflow, termed MTBE-SP3, we combined a fully automated protein lysis and extraction protocol (autoSP3) with a semi-automated biphasic 75% EtOH/MTBE extraction for quantification of polar/non-polar metabolites. Additionally, we compared the resulting proteome of various biological matrices (FFPE tissue, fresh-frozen tissue, plasma, serum and cells) between autoSP3 and MTBE-SP3. Our analysis revealed that the single-sample workflow provided similar results to those obtained from autoSP3 alone, with an 85-98% overlap of proteins detected across the different biological matrices. Additionally, it provides distinct advantages by decreasing (tissue) heterogeneity by retrieving metabolomics and proteomic data from the identical biological material, and limiting the total amount of required material. Lastly, we applied MTBE-SP3 to a lung adenocarcinoma cohort of 10 patients. Integrating the metabolic and proteomic alterations between tumour and non-tumour adjacent tissue yielded consistent data independent of the method used. This revealed mitochondrial dysfunction in tumor tissue through deregulation of OGDH, SDH family enzymes and PKM. In summary, MTBE-SP3 enables the facile and confident parallel measurement of proteins and metabolites obtained from the same sample. This workflow is particularly applicable for studies with limited sample availability and offers the potential to enhance the integration of metabolomic and proteomic datasets.

cancer biology↗

bioRxiv2023

Altered iron-sulfur cluster transfer in Arabidopsis mitochondria reveals lipoyl synthase as a Janus-faced enzyme that generates toxic sulfide

Iron-sulfur (Fe-S) cluster are vital cofactors in all domains of life. Mitochondrial Fe-S cluster assembly occurs in two major steps to first build [2Fe-2S] clusters and subsequently assemble these into [4Fe-4S] clusters. The two assembly machineries are interconnected by glutaredoxin S15 (GRXS15) that transfers [2Fe-2S] clusters to the second machinery. Diminished cluster transfer activity of GRXS15 in Arabidopsis mitochondria causes specific defects associated with lipoyl synthase (LIP1) activity. Conversely, overexpression of LIP1 in wild-type plants causes the release of toxic amounts of sulfide that can be detoxified by increasing the capacity for sulfide fixation through overexpression of O-acetylserine-(thiol)-lyase. The release of sulfide by lipoyl synthase causes a disturbance of mitochondrial sulfide homeostasis resulting in distinct and readily observable macroscopic phenotypes. These phenotypes enable a direct readout of consequences resulting from defects in Fe-S cluster assembly or targeted modulation of Fe-S cluster flux in mitochondria.

plant biology↗

bioRxiv2022

Pre-analytical processing of plasma and serum samples for combined proteome and metabolome analysis

Metabolomic and proteomic analyses of human plasma and serum samples harbour the power to advance our understanding of disease biology. Pre-analytical factors may contribute to variability and bias in the detection of analytes, especially when multiple labs are involved, caused by sample handling, processing time, and differing operating procedures. To better understand the impact of pre-analytical factors that are relevant to implement a unified proteomic and metabolomic approach in a clinical setting, we assessed the influence of temperature, sitting times, and centrifugation speed on the plasma and serum metabolomes and proteomes from six healthy volunteers. We used targeted metabolic profiling (497 metabolites) and data-independent acquisition (DIA) proteomics (572 proteins) on the same samples generated with well-defined pre-analytical conditions to evaluate criteria for pre-analytical SOPs for plasma and serum samples. Time and temperature showed the strongest influence on the integrity of plasma and serum proteome and metabolome. While rapid handling and low temperatures (4{degrees}C) are imperative for metabolic profiling, the analysed proteome showed variability when exposed to temperatures of 4{degrees}C for more than 2 hours, highlighting the need for compromises in a combined analysis. We formalised a quality control scoring system to objectively rate sample stability and tested this score using external data sets from other pre-analytical studies. Stringent and harmonised standard operating procedures (SOPs) are required for pre-analytical sample handling when combining proteomics and metabolomics of clinical samples to yield robust and interpretable data on a longitudinal scale and across different clinics. To ensure an adequate level of practicability in a clinical routine for metabolomics and proteomics studies we suggest to keep blood samples up to 2 hours on ice (4{degrees}C) prior to snap-freezing as a compromise between stability and operability. Finally, we provide the methodology as an open source R package allowing the systematic scoring of proteomics and metabolomics datasets to assess the stability of plasma and serum samples.

biochemistry↗

bioRxiv2021

Deep metabolic profiling assessment of tissue extraction protocols for three model organisms

Metabolic profiling harbors the potential to better understand various disease entities such as cancer, diabetes, Alzheimers, Parkinsons disease or COVID-19. Deciphering these intricate pathways in human studies requires large sample sizes as a means of reducing variability. While such broad human studies have discovered new associations between a given disease and certain affected metabolites, i.e. biomarkers, they often provide limited functional insights. To design more standardized experiments, reduce variability in the measurements and better resolve the functional component of such dynamic metabolic profiles, model organisms are frequently used. Standardized rearing conditions and uniform sampling strategies are prerequisites towards a successful metabolomic study. However, further aspects such as the choice of extraction protocol and analytical technique can influence the outcome drastically. Here, we employed a highly standardized metabolic profiling assay analyzing 630 metabolites across three commonly used model organisms (Drosophila, mouse and Zebrafish) to find the optimal extraction protocols for various matrices. Focusing on parameters such as metabolite coverage, metabolite yield and variance between replicates we compared seven extraction protocols. We found that the application of a combination of 75% ethanol and methyl tertiary-butyl ether (MTBE), while not producing the broadest coverage and highest yields, was the most reproducible extraction protocol. We were able to determine up to 530 metabolites in mouse kidney samples, 509 in mouse liver, 422 in Zebrafish and 388 in Drosophila and discovered a core overlap of 261 metabolites in these four matrices. To enable other scientists to search for the most suitable extraction protocol in their experimental context and interact with this comprehensive data, we have integrated our data set in the open-source shiny app "MetaboExtract". This will enable scientists to search for their metabolite or metabolite class of interest, compare it across the different tested extraction protocols and sample types as well as find reference concentrations.

biochemistry↗

bioRxiv2020

Branched-chain amino acid catabolism depends on GRXS15 through mitochondrial lipoyl cofactor homeostasis

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors in all life and are used in a wide array of diverse biological processes, including electron transfer chains and several metabolic pathways. Biosynthesis machineries for Fe-S clusters exist in plastids, the cytosol and mitochondria. A single monothiol glutaredoxin (GRX) has been shown to be involved in Fe-S cluster assembly in mitochondria of yeast and mammals. In plants, the role of the mitochondrial homologue GRXS15 has only partially been characterized. Arabidopsis grxs15 null mutants are not viable, but mutants complemented with the variant GRXS15 K83A develop with a dwarf phenotype. In an in-depth metabolic analysis, we show that most Fe-S cluster-dependent processes are not affected, including biotin biosynthesis, molybdenum cofactor biosynthesis and the electron transport chain. Instead, we observed an increase in most TCA cycle intermediates and amino acids, especially pyruvate, 2-oxoglutarate, glycine and branched-chain amino acids (BCAAs). The most pronounced accumulation occurred in branched-chain -keto acids (BCKAs), the first degradation products resulting from deamination of BCAAs. In wild-type plants, pyruvate, 2-oxoglutarate, glycine and BCKAs are all metabolized through decarboxylation by four mitochondrial lipoyl cofactor-dependent dehydrogenase complexes. Because these enzyme complexes are very abundant and the biosynthesis of the lipoyl cofactor depends on continuous Fe-S cluster supply to lipoyl synthase, this could explain why lipoyl cofactor-dependent processes are most sensitive to restricted Fe-S supply in GRXS15 K83A mutants. One-sentence summaryDeficiency in GRXS15 restricts protein lipoylation and causes metabolic defects in lipoyl cofactor-dependent dehydrogenase complexes, with branched-chain amino acid catabolism as dominant bottleneck.

plant biology↗

bioRxiv2019

Redox-mediated Kick-Start of Mitochondrial Energy Metabolism drives Resource-efficient Seed Germination

Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is re-activated to drive germination when the external conditions are favorable. Since the switchover from quiescence to re-activation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism of Arabidopsis seeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag-based (iodoTMT) thiol redox proteomics. The redox state across all Cys-peptides was shifted towards reduction from 27.1 % to 13.0 %. A large number of Cys-peptides (412) were redox-switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid cycle (TCA). Active site Cys-peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.

plant biology↗

bioRxiv2019

Staphylococcus aureus uses the bacilliredoxin (BrxAB)/bacillithiol disulfide reductase (YpdA) redox pathway to defend against oxidative stress under infections

Staphylococcus aureus is a major human pathogen and has to cope with reactive oxygen and chlorine species (ROS, RCS) during infections. The low molecular weight thiol bacillithiol (BSH) is an important defense mechanism of S. aureus for detoxification of ROS and HOCl stress to maintain the reduced state of the cytoplasm. Under HOCl stress, BSH forms mixed disulfides with proteins, termed as S-bacillithiolations, which are reduced by bacilliredoxins (BrxA and BrxB). The NADPH-dependent flavin disulfide reductase YpdA is phylogenetically associated with the BSH synthesis and BrxA/B enzymes and was proposed to function as BSSB reductase. Here, we investigated the role of the bacilliredoxin BrxAB/BSH/YpdA pathway in S. aureus COL under oxidative stress and macrophage infection conditions in vivo and in biochemical assays in vitro. Using HPLC thiol metabolomics, a strongly enhanced BSSB level and a decreased BSH/BSSB ratio were measured in the S. aureus COL ypdA deletion mutant under control and NaOCl stress. Monitoring the BSH redox potential (EBSH) using the Brx-roGFP2 biosensor revealed that YpdA is required for regeneration of the reduced EBSH upon recovery from oxidative stress. In addition, the ypdA mutant was impaired in H2O2 detoxification as measured with the novel H2O2-specific Tpx-roGFP2 biosensor. Phenotype analyses further showed that BrxA and YpdA are required for survival under NaOCl and H2O2 stress in vitro and inside murine J-774A.1 macrophages in infection assays in vivo. Finally, NADPH-coupled electron transfer assays provide evidence for the function of YpdA in BSSB reduction, which depends on the conserved Cys14 residue. YpdA acts together with BrxA and BSH in de-bacillithiolation of S-bacilithiolated GapDH. In conclusion, our results point to a major role of the BrxA/BSH/YpdA pathway in BSH redox homeostasis in S. aureus during recovery from oxidative stress and under infections.

microbiology↗

bioRxiv2019

Arabidopsis glutathione reductase 2 is indispensable in plastids, while mitochondrial glutathione is safeguarded by additional reduction and transport systems

O_LIA highly negative glutathione redox potential (EGSH) is maintained in the cytosol, plastids and mitochondria of plant cells to support fundamental processes, including antioxidant defence, redox regulation and iron-sulfur cluster biogenesis. Out of two glutathione reductase (GR) proteins in Arabidopsis, GR2 is predicted to be dual-targeted to plastids and mitochondria, but its differential roles in these organelles remain unclear.\nC_LIO_LIWe dissected the role of GR2 in organelle glutathione redox homeostasis and plant development using a combination of genetic complementation and stacked mutants, biochemical activity studies, immunogold labelling and in vivo biosensing.\nC_LIO_LIOur data demonstrate that GR2 is dual-targeted to plastids and mitochondria, but embryo lethality of gr2 null mutants is caused specifically in plastids. Whereas lack of mitochondrial GR2 leads to a partially oxidised glutathione pool in the matrix, the ABC transporter ATM3 and the mitochondrial thioredoxin system provide functional backup and maintain plant viability.\nC_LIO_LIWe identify GR2 as essential in the plastid stroma, where it counters GSSG accumulation and developmental arrest. By contrast a functional triad of GR2, ATM3 and the thioredoxin system in the mitochondria provides resilience to excessive glutathione oxidation.\nC_LI

plant biology↗

bioRxiv2018

Pyrophosphate modulates stress responses via SUMOylation

Pyrophosphate (PPi), a byproduct of macromolecule biosynthesis is maintained at low levels by soluble inorganic pyrophosphatases (sPPase) found in all eukaryotes. In plants, H+-pumping pyrophosphatases (H+-PPase) convert the substantial energy present in PPi into an electrochemical gradient. We show here, that both cold- and heat stress sensitivity of fugu5 mutants lacking the major H+-PPase isoform AVP1 is caused by reduced SUMOylation. In addition, we show that increased PPi concentrations interfere with SUMOylation in yeast and we provide evidence that SUMO activating E1-enzymes are inhibited by micromolar concentrations of PPi in a non-competitive manner. Taken together, our results do not only provide a mechanistic explanation for the beneficial effects of AVP1 overexpression in plants but they also highlight PPi as an important integrator of metabolism and stress tolerance.

biochemistry↗