LipiDex 2 Integrates MSn Tree-Based Fragmentation Methods and Quality Control Modules to Improve Discovery Lipidomics.

As lipidomics experiments increase in scale and complexity, data processing tools must support workflows for new liquid chromatography-mass spectrometry (LC-MS) methods while simultaneously supporting quality controls to maximize the confidence in lipid identifications. LipiDex 2 improves lipidomics data processing algorithms from LipiDex 1 and introduces new tools for spectral matching and peak annotation functions, with improvements in speed and user-friendliness. In silico spectral library generation now supports tandem mass spectral (MSn) tree-based fragmentation methods, and the LipiDex 2 workflow fully integrates the fragmentation logic into the data processing steps to enable lipid identification at the appropriate level of structural resolution. Finally, LipiDex 2 features new modules for automated quality control checks that also allow users to visualize data quality in a data dashboard user interface.

Lysosomal storage disease proteo/lipidomic profiling using nMOST links ferritinophagy with mitochondrial iron deficiencies in cells lacking NPC2.

Lysosomal storage diseases (LSDs) comprised ~50 monogenic diseases characterized by the accumulation of cellular material in lysosomes and associated defects in lysosomal function, but systematic molecular phenotyping is lacking. Here, we develop a nanoflow-based multi-omic single-shot technology (nMOST) workflow allowing simultaneously quantify HeLa cell proteomes and lipidomes from more than two dozen LSD mutants, revealing diverse molecular phenotypes. Defects in delivery of ferritin and its autophagic receptor NCOA4 to lysosomes (ferritinophagy) were pronounced in NPC2-/- cells, which correlated with increased lyso-phosphatidylcholine species and multi-lamellar membrane structures visualized by cryo-electron-tomography. Ferritinophagy defects correlated with loss of mitochondrial cristae, MICOS-complex components, and electron transport chain complexes rich in iron-sulfur cluster proteins. Strikingly, mitochondrial defects were alleviated when iron was provided through the transferrin system. This resource reveals how defects in lysosomal function can impact mitochondrial homeostasis in trans and highlights nMOST as a discovery tool for illuminating molecular phenotypes across LSDs.

Scd1 and monounsaturated lipids are required for autophagy and survival of adipocytes.

OBJECTIVE: Exposure of adipocytes to 'cool' temperatures often found in the periphery of the body induces expression of Stearoyl-CoA Desaturase-1 (Scd1), an enzyme that converts saturated fatty acids to monounsaturated fatty acids. The goal of this study is to further investigate the roles of Scd in adipocytes. METHOD: In this study, we employed Scd1 knockout cells and mouse models, along with pharmacological Scd1 inhibition to dissect the enzyme's function in adipocyte physiology. RESULTS: Our study reveals that production of monounsaturated lipids by Scd1 is necessary for fusion of autophagosomes to lysosomes and that with a Scd1-deficiency, autophagosomes accumulate. In addition, Scd1-deficiency impairs lysosomal and autolysosomal acidification resulting in vacuole accumulation and eventual cell death. Blocking autophagosome formation or supplementation with monounsaturated fatty acids maintains vitality of Scd1-deficient adipocytes. CONCLUSION: This study demonstrates the indispensable role of Scd1 in adipocyte survival, with its inhibition in vivo triggering autophagy-dependent cell death and its depletion in vivo leading to the loss of bone marrow adipocytes.

High-content phenotypic analysis of a C. elegans recombinant inbred population identifies genetic and molecular regulators of lifespan.

Lifespan is influenced by complex interactions between genetic and environmental factors. Studying those factors in model organisms of a single genetic background limits their translational value for humans. Here, we mapped lifespan determinants in 85 genetically diverse C. elegans recombinant intercross advanced inbred lines (RIAILs). We assessed molecular profiles - transcriptome, proteome, and lipidome - and life-history traits, including lifespan, development, growth dynamics, and reproduction. RIAILs exhibited large variations in lifespan, which positively correlated with developmental time. Among the top candidates obtained from multi-omics data integration and QTL mapping, we validated known and novel longevity modulators, including rict-1, gfm-1 and mltn-1. We translated their relevance to humans using UK Biobank data and showed that variants in RICTOR and GFM1 are associated with an elevated risk of age-related heart disease, dementia, diabetes, kidney, and liver diseases. We organized our dataset as a resource (https://lisp-lms.shinyapps.io/RIAILs/) that allows interactive explorations for new longevity targets.

SCD1 and monounsaturated lipids are required for autophagy and survival of adipocytes.

Exposure of adipocytes to 'cool' temperatures often found in the periphery of the body induces expression of Stearoyl-CoA Desaturase-1 (SCD1), an enzyme that converts saturated fatty acids to monounsaturated fatty acids. In this study, we employed Scd1 knockout cells and mouse models, along with pharmacological SCD1 inhibition, to investigate further the roles of SCD1 in adipocytes. Our study reveals that production of monounsaturated lipids by SCD1 is necessary for fusion of autophagosomes to lysosomes and that with a SCD1-deficiency, autophagosomes accumulate. In addition, SCD1-deficiency impairs lysosomal and autolysosomal acidification resulting in vacuole accumulation and eventual cell death. Blocking autophagosome formation or supplementation with monounsaturated fatty acids maintains vitality of SCD1-deficient adipocytes. Taken together, our results demonstrate that in vitro inhibition of SCD1 in adipocytes leads to autophagy-dependent cell death, and in vivo depletion leads to loss of bone marrow adipocytes.

A transcriptional network governing ceramide homeostasis establishes a cytokine-dependent developmental process.

Transcriptional mechanisms controlling developmental processes establish and maintain proteomic networks, which can govern the levels of intracellular small molecules. Although dynamic changes in bioactive small molecules can link transcription factor and genome activity with cell state transitions, many mechanistic questions are unresolved. Using quantitative lipidomics and multiomics, we discover that the hematopoietic transcription factor GATA1 establishes ceramide homeostasis during erythroid differentiation by regulating genes encoding sphingolipid metabolic enzymes. Inhibiting a GATA1-induced sphingolipid biosynthetic enzyme, delta(4)-desaturase, or disrupting ceramide homeostasis with cell-permeable dihydroceramide or ceramide is detrimental to erythroid, but not myeloid, progenitor activity. Coupled with genetic editing-based rewiring of the regulatory circuitry, we demonstrate that ceramide homeostasis commissions vital stem cell factor and erythropoietin signaling by opposing an inhibitory protein phosphatase 2A-dependent, dual-component mechanism. Integrating bioactive lipids as essential components of GATA factor mechanisms to control cell state transitions has implications for diverse cell and tissue types.

Hem25p is required for mitochondrial IPP transport in fungi.

Coenzyme Q (CoQ, ubiquinone) is an essential cellular cofactor composed of a redox-active quinone head group and a long hydrophobic polyisoprene tail. How mitochondria access cytosolic isoprenoids for CoQ biosynthesis is a longstanding mystery. Here, via a combination of genetic screening, metabolic tracing and targeted uptake assays, we reveal that Hem25p-a mitochondrial glycine transporter required for haem biosynthesis-doubles as an isopentenyl pyrophosphate (IPP) transporter in Saccharomyces cerevisiae. Mitochondria lacking Hem25p failed to efficiently incorporate IPP into early CoQ precursors, leading to loss of CoQ and turnover of CoQ biosynthetic proteins. Expression of Hem25p in Escherichia coli enabled robust IPP uptake and incorporation into the CoQ biosynthetic pathway. HEM25 orthologues from diverse fungi, but not from metazoans, were able to rescue hem25∆ CoQ deficiency. Collectively, our work reveals that Hem25p drives the bulk of mitochondrial isoprenoid transport for CoQ biosynthesis in fungi.

Plasma metabolomics supports non-fasted sampling for metabolic profiling across a spectrum of glucose tolerance in the Nile rat model for type 2 diabetes.

Type 2 diabetes is a challenge in modern healthcare, and animal models are necessary to identify underlying mechanisms. The Nile rat (Arvicanthis niloticus) develops diet-induced diabetes rapidly on a conventional rodent chow diet without genetic or chemical manipulation. Unlike common laboratory models, the outbred Nile rat model is diurnal and has a wide range of overt diabetes onset and diabetes progression patterns in both sexes, better mimicking the heterogeneous diabetic phenotype in humans. While fasted blood glucose has historically been used to monitor diabetic progression, postprandial blood glucose is more sensitive to the initial stages of diabetes. However, there is a long-held assumption that ad libitum feeding in rodent models leads to increased variance, thus masking diabetes-related metabolic changes in the plasma. Here we compared repeatability within triplicates of non-fasted or fasted plasma samples and assessed metabolic changes relevant to glucose tolerance in fasted and non-fasted plasma of 8-10-week-old male Nile rats. We used liquid chromatography-mass spectrometry lipidomics and polar metabolomics to measure relative metabolite abundances in the plasma samples. We found that, compared to fasted metabolites, non-fasted plasma metabolites are not only more strongly associated with glucose tolerance on the basis of unsupervised clustering and elastic net regression model, but also have a lower replicate variance. Between the two sampling groups, we detected 66 non-fasted metabolites and 32 fasted metabolites that were associated with glucose tolerance using a combined approach with multivariable elastic net and individual metabolite linear models. Further, to test if metabolite replicate variance is affected by age and sex, we measured non-fasted replicate variance in a cohort of mature 30-week-old male and female Nile rats. Our results support using non-fasted plasma metabolomics to study glucose tolerance in Nile rats across the progression of diabetes.

Decreased plasma cartilage acidic protein 1 in COVID-19.

Cartilage acidic protein-1 (CRTAC1) is produced by several cell types, including Type 2 alveolar epithelial (T2AE) cells that are targeted by SARS-CoV2. Plasma CRTAC1 is known based on proteomic surveys to be low in patients with severe COVID-19. Using an ELISA, we found that patients treated for COVID-19 in an ICU almost uniformly had plasma concentrations of CRTAC1 below those of healthy controls. Magnitude of decrease in CRTAC1 distinguished COVID-19 from other causes of acute respiratory decompensation and correlated with established metrics of COVID-19 severity. CRTAC1 concentrations below those of controls were found in some patients a year after hospitalization with COVID-19, long COVID after less severe COVID-19, or chronic obstructive pulmonary disease. Decreases in CRTAC1 in severe COVID-19 correlated (r = 0.37, p = 0.0001) with decreases in CFP (properdin), which interacts with CRTAC1. Thus, decreases of CRTAC1 associated with severe COVID-19 may result from loss of production by T2AE cells or co-depletion with CFP. Determination of significance of and reasons behind decreased CRTAC1 concentration in a subset of patients with long COVID will require analysis of roles of preexisting lung disease, impact of prior acute COVID-19, age, and other confounding variables in a larger number of patients.

The citrate transporters SLC13A5 and SLC25A1 elicit different metabolic responses and phenotypes in the mouse.

Cytosolic citrate is imported from the mitochondria by SLC25A1, and from the extracellular milieu by SLC13A5. In the cytosol, citrate is used by ACLY to generate acetyl-CoA, which can then be exported to the endoplasmic reticulum (ER) by SLC33A1. Here, we report the generation of mice with systemic overexpression (sTg) of SLC25A1 or SLC13A5. Both animals displayed increased cytosolic levels of citrate and acetyl-CoA; however, SLC13A5 sTg mice developed a progeria-like phenotype with premature death, while SLC25A1 sTg mice did not. Analysis of the metabolic profile revealed widespread differences. Furthermore, SLC13A5 sTg mice displayed increased engagement of the ER acetylation machinery through SLC33A1, while SLC25A1 sTg mice did not. In conclusion, our findings point to different biological responses to SLC13A5- or SLC25A1-mediated import of citrate and suggest that the directionality of the citrate/acetyl-CoA pathway can transduce different signals.

Mass Spectrometry-Based Multi-omics Integration with a Single Set of C. elegans Samples.

Mass spectrometry-based large-scale multi-omics research has proven to be powerful in answering biological questions; nonetheless, it faces many challenges from sample preparation to downstream data integration. To efficiently extract biomolecules of different physicochemical properties, preparation of various sample type needs specific tailoring, especially of difficult ones, such as Caenorhabditis elegans. In this study, we sought to develop a multi-omics sample preparation method starting with a single set ofC. elegans samples to save time, minimize variability, expand biomolecule coverage, and promote multi-omics integration. We investigated tissue disruption methods to effectively release biomolecules and optimized extraction strategies to achieve broader and more reproducible biomolecule coverage in proteomics, lipidomics, and metabolomics workflows. In our assessment, we also considered speediness and usability of the approaches. The developed method was validated through a study of 16C. elegans samples designed to shine light on mitochondrial unfolded protein response (UPRmt), induced by three unique stressors─knocking down electron transfer chain element cco-1, mitochondrial ribosome protein S5 mrps-5, and antibiotic treatment Doxycycline. Our findings suggested that the method achieved great coverage of proteome, lipidome, and metabolome with high reproducibility and validated that all stressors triggered UPRmt in C. elegans, although generating unique molecular signatures. Innate immune response was activated, and triglycerides were decreased under all three stressor conditions. Additionally, Doxycycline treatment elicited more distinct proteomic, lipidomic, and metabolomic response than the other two treatments. This method has been successfully used to process Saccharomyces cerevisiae (data not shown) and can likely be applied to other organisms for multi-omics research.

PKA regulatory subunit Bcy1 couples growth, lipid metabolism, and fermentation during anaerobic xylose growth in Saccharomyces cerevisiae.

Organisms have evolved elaborate physiological pathways that regulate growth, proliferation, metabolism, and stress response. These pathways must be properly coordinated to elicit the appropriate response to an ever-changing environment. While individual pathways have been well studied in a variety of model systems, there remains much to uncover about how pathways are integrated to produce systemic changes in a cell, especially in dynamic conditions. We previously showed that deletion of Protein Kinase A (PKA) regulatory subunit BCY1 can decouple growth and metabolism in Saccharomyces cerevisiae engineered for anaerobic xylose fermentation, allowing for robust fermentation in the absence of division. This provides an opportunity to understand how PKA signaling normally coordinates these processes. Here, we integrated transcriptomic, lipidomic, and phospho-proteomic responses upon a glucose to xylose shift across a series of strains with different genetic mutations promoting either coupled or decoupled xylose-dependent growth and metabolism. Together, results suggested that defects in lipid homeostasis limit growth in the bcy1Δ strain despite robust metabolism. To further understand this mechanism, we performed adaptive laboratory evolutions to re-evolve coupled growth and metabolism in the bcy1Δ parental strain. The evolved strain harbored mutations in PKA subunit TPK1 and lipid regulator OPI1, among other genes, and evolved changes in lipid profiles and gene expression. Deletion of the evolved opi1 gene partially reverted the strain's phenotype to the bcy1Δ parent, with reduced growth and robust xylose fermentation. We suggest several models for how cells coordinate growth, metabolism, and other responses in budding yeast and how restructuring these processes enables anaerobic xylose utilization.

Improved Structural Characterization of Glycerophospholipids and Sphingomyelins with Real-Time Library Searching.

In mass spectrometry-based lipidomics, complex lipid mixtures undergo chromatographic separation, are ionized, and are detected using tandem MS (MSn) to simultaneously quantify and structurally characterize eluting species. The reported structural granularity of these identified lipids is strongly reliant on the analytical techniques leveraged in a study. For example, lipid identifications from traditional collisionally activated data-dependent acquisition experiments are often reported at either species level or molecular species level. Structural resolution of reported lipid identifications is routinely enhanced by integrating both positive and negative mode analyses, requiring two separate runs or polarity switching during a single analysis. MS3+ can further elucidate lipid structure, but the lengthened MS duty cycle can negatively impact analysis depth. Recently, functionality has been introduced on several Orbitrap Tribrid mass spectrometry platforms to identify eluting molecular species on-the-fly. These real-time identifications can be leveraged to trigger downstream MSn to improve structural characterization with lessened impacts on analysis depth. Here, we describe a novel lipidomics real-time library search (RTLS) approach, which utilizes the lipid class of real-time identifications to trigger class-targeted MSn and to improve the structural characterization of phosphotidylcholines, phosphotidylethanolamines, phosphotidylinositols, phosphotidylglycerols, phosphotidylserine, and sphingomyelins in the positive ion mode. Our class-based RTLS method demonstrates improved selectivity compared to the current methodology of triggering MSn in the presence of characteristic ions or neutral losses.

A role for cytoglobin in regulating intracellular hydrogen peroxide and redox signals in the vasculature.

The oxidant hydrogen peroxide serves as a signaling molecule that alters many aspects of cardiovascular functions. Recent studies suggest that cytoglobin - a hemoglobin expressed in the vasculature - may promote electron transfer reactions with proposed functions in hydrogen peroxide decomposition. Here, we determined the extent to which cytoglobin regulates intracellular hydrogen peroxide and established mechanisms. We found that cytoglobin decreased the hyperoxidation of peroxiredoxins and maintained the activity of peroxiredoxin 2 following challenge with exogenous hydrogen peroxide. Cytoglobin promoted a reduced intracellular environment and facilitated the reduction of the thiol-based hydrogen peroxide sensor Hyper7 after bolus addition of hydrogen peroxide. Cytoglobin also limited the inhibitory effect of hydrogen peroxide on glycolysis and reversed the oxidative inactivation of the glycolytic enzyme GAPDH. Our results indicate that cytoglobin in cells exists primarily as oxyferrous cytoglobin (CygbFe 2+ -O 2 ) with its cysteine residues in the reduced form. We found that the specific substitution of one of two cysteine residues on cytoglobin (C83A) inhibited the reductive activity of cytoglobin on Hyper7 and GAPDH. Carotid arteries from cytoglobin knockout mice were more sensitive to glycolytic inhibition by hydrogen peroxide than arteries from wildtype mice. Together, these results support a role for cytoglobin in regulating intracellular redox signals associated with hydrogen peroxide through oxidation of its cysteine residues, independent of hydrogen peroxide reaction at its heme center.

Hem25p is a mitochondrial IPP transporter.

Coenzyme Q (CoQ, ubiquinone) is an essential cellular cofactor comprised of a redox-active quinone head group and a long hydrophobic polyisoprene tail. How mitochondria access cytosolic isoprenoids for CoQ biosynthesis is a longstanding mystery. Here, via a combination of genetic screening, metabolic tracing, and targeted uptake assays, we reveal that Hem25p-a mitochondrial glycine transporter required for heme biosynthesis-doubles as an isopentenyl pyrophosphate (IPP) transporter in Saccharomyces cerevisiae. Mitochondria lacking Hem25p fail to efficiently incorporate IPP into early CoQ precursors, leading to loss of CoQ and turnover of CoQ biosynthetic proteins. Expression of Hem25p in Escherichia coli enables robust IPP uptake demonstrating that Hem25p is sufficient for IPP transport. Collectively, our work reveals that Hem25p drives the bulk of mitochondrial isoprenoid transport for CoQ biosynthesis in yeast.

Genetic mapping of microbial and host traits reveals production of immunomodulatory lipids by Akkermansia muciniphila in the murine gut.

The molecular bases of how host genetic variation impacts the gut microbiome remain largely unknown. Here we used a genetically diverse mouse population and applied systems genetics strategies to identify interactions between host and microbe phenotypes including microbial functions, using faecal metagenomics, small intestinal transcripts and caecal lipids that influence microbe-host dynamics. Quantitative trait locus (QTL) mapping identified murine genomic regions associated with variations in bacterial taxa; bacterial functions including motility, sporulation and lipopolysaccharide production and levels of bacterial- and host-derived lipids. We found overlapping QTL for the abundance of Akkermansia muciniphila and caecal levels of ornithine lipids. Follow-up in vitro and in vivo studies revealed that A. muciniphila is a major source of these lipids in the gut, provided evidence that ornithine lipids have immunomodulatory effects and identified intestinal transcripts co-regulated with these traits including Atf3, which encodes for a transcription factor that plays vital roles in modulating metabolism and immunity. Collectively, these results suggest that ornithine lipids are potentially important for A. muciniphila-host interactions and support the role of host genetics as a determinant of responses to gut microbes.

Rapid Multi-Omics Sample Preparation for Mass Spectrometry.

Multi-omics analysis is a powerful and increasingly utilized approach to gain insight into complex biological systems. One major hindrance with multi-omics, however, is the lengthy and wasteful sample preparation process. Preparing samples for mass spectrometry (MS)-based multi-omics involves extraction of metabolites and lipids with organic solvents, precipitation of proteins, and overnight digestion of proteins. These existing workflows are disparate and laborious. Here, we present a simple, efficient, and unified approach to prepare lipids, metabolites, and proteins for MS analysis. Our approach, termed the Bead-enabled Accelerated Monophasic Multi-omics (BAMM) method, combines an n-butanol-based monophasic extraction with unmodified magnetic beads and accelerated protein digestion. We demonstrate that the BAMM method affords comparable depth, quantitative reproducibility, and recovery of biomolecules as state-of-the-art multi-omics methods (e.g., Matyash extraction and overnight protein digestion). However, the BAMM method only requires about 3 h to perform, which saves 11 steps and 19 h on average compared to published multi-omics methods. Furthermore, we validate the BAMM method for multiple sample types and formats (biofluid, culture plate, and pellet) and show that in all cases, it produces high biomolecular coverage and data quality.

Spatial snapshots of amyloid precursor protein intramembrane processing via early endosome proteomics.

Degradation and recycling of plasma membrane proteins occurs via the endolysosomal system, wherein endosomes bud into the cytosol from the plasma membrane and subsequently mature into degradative lysosomal compartments. While methods have been developed for rapid selective capture of lysosomes (Lyso-IP), analogous methods for isolation of early endosome intermediates are lacking. Here, we develop an approach for rapid isolation of early/sorting endosomes through affinity capture of the early endosome-associated protein EEA1 (Endo-IP) and provide proteomic and lipidomic snapshots of EEA1-positive endosomes in action. We identify recycling, regulatory and membrane fusion complexes, as well as candidate cargo, providing a proteomic landscape of early/sorting endosomes. To demonstrate the utility of the method, we combined Endo- and Lyso-IP with multiplexed targeted proteomics to provide a spatial digital snapshot of amyloid precursor protein (APP) processing by ß and γ-Secretases, which produce amyloidogenic Aß species, and quantify small molecule modulation of Secretase action on endosomes. We anticipate that the Endo-IP approach will facilitate systematic interrogation of processes that are coordinated on EEA1-positive endosomes.

Structure and functionality of a multimeric human COQ7:COQ9 complex.

Coenzyme Q (CoQ) is a redox-active lipid essential for core metabolic pathways and antioxidant defense. CoQ is synthesized upon the mitochondrial inner membrane by an ill-defined "complex Q" metabolon. Here, we present structure-function analyses of a lipid-, substrate-, and NADH-bound complex comprising two complex Q subunits: the hydroxylase COQ7 and the lipid-binding protein COQ9. We reveal that COQ7 adopts a ferritin-like fold with a hydrophobic channel whose substrate-binding capacity is enhanced by COQ9. Using molecular dynamics, we further show that two COQ7:COQ9 heterodimers form a curved tetramer that deforms the membrane, potentially opening a pathway for the CoQ intermediates to translocate from the bilayer to the proteins' lipid-binding sites. Two such tetramers assemble into a soluble octamer with a pseudo-bilayer of lipids captured within. Together, these observations indicate that COQ7 and COQ9 cooperate to access hydrophobic precursors within the membrane and coordinate subsequent synthesis steps toward producing CoQ.