Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2.

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.

Osteocytes control myeloid cell proliferation and differentiation through Gsα-dependent and -independent mechanisms.

Osteocytes, the bone cells embedded in the mineralized matrix, control bone modeling, and remodeling through direct contact with adjacent cells and via paracrine and endocrine factors that affect cells in the bone marrow microenvironment or distant organs. Osteocytes express numerous G protein-coupled receptors (GPCRs) and thus mice lacking the stimulatory subunit of G-protein (Gsα) in osteocytes (Dmp1-GsαKO mice) have abnormal myelopoiesis, osteopenia, and reduced adipose tissue. We previously reported that the severe osteopenia and the changes in adipose tissue present in these mice were mediated by increased sclerostin, which suppress osteoblast functions and promote browning of white adipocytes. Inversely, the myeloproliferation was driven by granulocyte colony-stimulating factor (G-CSF) and administration of neutralizing antibodies against G-CSF only partially restored the myeloproliferation, suggesting that additional osteocyte-derived factors might be involved. We hypothesized that osteocytes secrete Gsα-dependent factor(s) which regulate the myeloid cells proliferation. To identify osteocyte-secreted proteins, we used the osteocytic cell line Ocy454 expressing or lacking Gsα expression (Ocy454-Gsαcont and Ocy454-GsαKO ) to delineate the osteocyte "secretome" and its regulation by Gsα. Here we reported that factors secreted by osteocytes increased the number of myeloid colonies and promoted macrophage proliferation. The proliferation of myeloid cells was further promoted by osteocytes lacking Gsα expression. Myeloid cells can differentiate into bone-resorbing osteoclasts, therefore, we hypothesized that osteocyte-secreted factors might also regulate osteoclastogenesis in a Gsα-dependent manner. Conditioned medium (CM) from Ocy454 (both Gsαcont and GsαKO ) significanlty increased the proliferation of bone marrow mononuclear cells (BMNC) and, at the same time, inhibited their differentiation into mature osteoclasts via a Gsα-dependent mechanism. Proteomics analysis of CM from Ocy454 Gsαcont and GsαKO cells identified neuropilin-1 (Nrp-1) and granulin (Grn) as osteocytic-secreted proteins upregulated in Ocy454-GsαKO cells compared to Ocy454-Gsαcont , whereas semaphorin3A was significantly suppressed. Treatment of Ocy454-Gsαcont cells with recombinant proteins or knockdown of Nrp-1 and Grn in Ocy454-GsαKO cells partially rescued the inhibition of osteoclasts, demonstrating that osteocytes control osteoclasts differentiation through Nrp-1 and Grn which are regulated by Gsα signaling.

Endothelial cell-specific redox gene modulation inhibits angiogenesis but promotes B16F0 tumor growth in mice.

Glutaredoxin-1 (Glrx) is a small cytosolic enzyme that removes S-glutathionylation, glutathione adducts of protein cysteine residues, thus modulating redox signaling and gene transcription. Although Glrx up-regulation prevented endothelial cell (EC) migration and global Glrx transgenic mice had impaired ischemic vascularization, the effects of cell-specific Glrx overexpression remained unknown. Here, we examined the role of EC-specific Glrx up-regulation in distinct models of angiogenesis; namely, hind limb ischemia and tumor angiogenesis. EC-specific Glrx transgenic (EC-Glrx TG) overexpression in mice significantly impaired EC migration in Matrigel implants and hind limb revascularization after femoral artery ligation. Additionally, ECs migrated less into subcutaneously implanted B16F0 melanoma tumors as assessed by decreased staining of EC markers. Despite reduced angiogenesis, EC-Glrx TG mice unexpectedly developed larger tumors compared with control mice. EC-Glrx TG mice showed higher levels of VEGF-A in the tumors, indicating hypoxia, which may stimulate tumor cells to form vascular channels without EC, referred to as vasculogenic mimicry. These data suggest that impaired ischemic vascularization does not necessarily associate with suppression of tumor growth, and that antiangiogenic therapies may be ineffective for melanoma tumors because of their ability to implement vasculogenic mimicry during hypoxia.-Yura, Y., Chong, B. S. H., Johnson, R. D., Watanabe, Y., Tsukahara, Y., Ferran, B., Murdoch, C. E., Behring, J. B., McComb, M. E., Costello, C. E., Janssen-Heininger, Y. M. W., Cohen, R. A., Bachschmid, M. M., Matsui, R. Endothelial cell-specific redox gene modulation inhibits angiogenesis but promotes B16F0 tumor growth in mice.

Glycomic and Proteomic Changes in Aging Brain Nigrostriatal Pathway.

Parkinson's disease (PD) is a neurological disorder characterized by the progressive loss of functional dopaminergic neurons in the nigrostriatal pathway in the brain. Although current treatments provide only symptomatic relief, gene therapy has the potential to slow or halt the degeneration of nigrostriatal dopamine neurons in PD patients. Adeno-associated viruses (AAV) are vectors of choice in gene therapy because of their well-characterized safety and efficacy profiles; however, although gene therapy has been successful in preclinical models of the disease, clinical trials in humans have failed to demonstrate efficacy. Significantly, all primary AAV receptors of the virus are glycans. We thus hypothesize that age related changes in glycan receptors of heparan sulfate (HS) proteoglycans (receptor for rAAV2), and/or N-glycans with terminal galactose (receptor for rAAV9) results in poor adeno-associated virus binding in either the striatum or substantia nigra, or both, affecting transduction and gene delivery. To test our hypothesis we analyzed the striatum and substantia nigra for changes in HS, N-glycans and proteomic signatures in young versus aged rat brain striatum and substantia nigra. We observed different brain region-specific HS disaccharide profiles in aged compared with young adult rats for brain region-specific profiles in striatum versus substantia nigra. We observed brain region- and age-specific N-glycan compositional profiles with respect to the terminal galactose units that serve as receptors for AAV9. We also observed brain region-specific changes in protein expression in the aging nigrostriatal pathway. These studies provide insight into age- and brain region-specific changes in glycan receptors and proteome that will inform design of improved viral vectors for Parkinson Disease (PD) gene therapy.

RuvbL1 and RuvbL2 enhance aggresome formation and disaggregate amyloid fibrils.

The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic aggregates. Similarly, downregulation of RuvbL orthologs in yeast suppressed the formation of an aggresome-like body and enhanced the aggregate toxicity. In contrast, their overproduction enhanced the resistance to proteotoxic stress independently of chaperone Hsp104. Mammalian RuvbL associated with the aggresome, and the aggresome substrate synphilin-1 interacted directly with the RuvbL1 barrel-like structure near the opening of the central channel. Importantly, polypeptides with unfolded structures and amyloid fibrils stimulated the ATPase activity of RuvbL. Finally, disassembly of protein aggregates was promoted by RuvbL. These data indicate that RuvbL complexes serve as chaperones in protein disaggregation.

Human Regulatory Protein Ki-1/57 Is a Target of SUMOylation and Affects PML Nuclear Body Formation.

Ki-1/57 is a nuclear and cytoplasmic regulatory protein first identified in malignant cells from Hodgkin's lymphoma. It is involved in gene expression regulation on both transcriptional and mRNA metabolism levels. Ki-1/57 belongs to the family of intrinsically unstructured proteins and undergoes phosphorylation by PKC and methylation by PRMT1. Previous characterization of its protein interaction profile by yeast two-hybrid screening showed that Ki-1/57 interacts with proteins of the SUMOylation machinery, the SUMO E2 conjugating enzyme UBC9 and the SUMO E3 ligase PIAS3, which suggested that Ki-1/57 could be involved with this process. Here we identified seven potential SUMO target sites (lysine residues) on Ki-1/57 sequence and observed that Ki-1/57 is modified by SUMO proteins in vitro and in vivo. We showed that SUMOylation of Ki-1/57 occurred on lysines 213, 276, and 336. In transfected cells expressing FLAG-Ki-1/57 wild-type, its paralog FLAG-CGI-55 wild-type, or their non-SUMOylated triple mutants, the number of PML-nuclear bodies (PML-NBs) is reduced compared with the control cells not expressing the constructs. More interestingly, after treating cells with arsenic trioxide (As2O3), the number of PML-NBs is no longer reduced when the non-SUMOylated triple mutant Ki-1/57 is expressed, suggesting that the SUMOylation of Ki-1/57 has a role in the control of As2O3-induced PML-NB formation. A proteome-wide analysis of Ki-1/57 partners in the presence of either SUMO-1 or SUMO-2 suggests that the involvement of Ki-1/57 with the regulation of gene expression is independent of the presence of either SUMO-1 or SUMO-2; however, the presence of SUMO-1 strongly influences the interaction of Ki-1/57 with proteins associated with cellular metabolism, maintenance, and cell cycle.

O-Linked N-Acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Interact with Mi2ß Protein at the Aγ-Globin Promoter.

One mode of γ-globin gene silencing involves a GATA-1·FOG-1·Mi2ß repressor complex that binds to the -566 GATA site relative to the (A)γ-globin gene cap site. However, the mechanism of how this repressor complex is assembled at the -566 GATA site is unknown. In this study, we demonstrate that the O-linked N-acetylglucosamine (O-GlcNAc) processing enzymes, O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA), interact with the (A)γ-globin promoter at the -566 GATA repressor site; however, mutation of the GATA site to GAGA significantly reduces OGT and OGA promoter interactions in ß-globin locus yeast artificial chromosome (ß-YAC) bone marrow cells. When WT ß-YAC bone marrow cells are treated with the OGA inhibitor Thiamet-G, the occupancy of OGT, OGA, and Mi2ß at the (A)γ-globin promoter is increased. In addition, OGT and Mi2ß recruitment is increased at the (A)γ-globin promoter when γ-globin becomes repressed in postconception day E18 human ß-YAC transgenic mouse fetal liver. Furthermore, we show that Mi2ß is modified with O-GlcNAc, and both OGT and OGA interact with Mi2ß, GATA-1, and FOG-1. Taken together, our data suggest that O-GlcNAcylation is a novel mechanism of γ-globin gene regulation mediated by modulating the assembly of the GATA-1·FOG-1·Mi2ß repressor complex at the -566 GATA motif within the promoter.

Epithelial Mesenchymal Transition Induces Aberrant Glycosylation through Hexosamine Biosynthetic Pathway Activation.

Deregulated cellular metabolism is a hallmark of tumors. Cancer cells increase glucose and glutamine flux to provide energy needs and macromolecular synthesis demands. Several studies have been focused on the importance of glycolysis and pentose phosphate pathway. However, a neglected but very important branch of glucose metabolism is the hexosamine biosynthesis pathway (HBP). The HBP is a branch of the glucose metabolic pathway that consumes ∼2-5% of the total glucose, generating UDP-GlcNAc as the end product. UDP-GlcNAc is the donor substrate used in multiple glycosylation reactions. Thus, HBP links the altered metabolism with aberrant glycosylation providing a mechanism for cancer cells to sense and respond to microenvironment changes. Here, we investigate the changes of glucose metabolism during epithelial mesenchymal transition (EMT) and the role of O-GlcNAcylation in this process. We show that A549 cells increase glucose uptake during EMT, but instead of increasing the glycolysis and pentose phosphate pathway, the glucose is shunted through the HBP. The activation of HBP induces an aberrant cell surface glycosylation and O-GlcNAcylation. The cell surface glycans display an increase of sialylation α2-6, poly-LacNAc, and fucosylation, all known epitopes found in different tumor models. In addition, modulation of O-GlcNAc levels was demonstrated to be important during the EMT process. Taken together, our results indicate that EMT is an applicable model to study metabolic and glycophenotype changes during carcinogenesis, suggesting that cell glycosylation senses metabolic changes and modulates cell plasticity.

Microfluidic Capillary Electrophoresis-Mass Spectrometry for Analysis of Monosaccharides, Oligosaccharides, and Glycopeptides.

Glycomics and glycoproteomics analyses by mass spectrometry require efficient front-end separation methods to enable deep characterization of heterogeneous glycoform populations. Chromatography methods are generally limited in their ability to resolve glycoforms using mobile phases that are compatible with online liquid chromatography-mass spectrometry (LC-MS). The adoption of capillary electrophoresis-mass spectrometry methods (CE-MS) for glycomics and glycoproteomics is limited by the lack of convenient interfaces for coupling the CE devices to mass spectrometers. Here, we describe the application of a microfluidics-based CE-MS system for analysis of released glycans, glycopeptides and monosaccharides. We demonstrate a single CE method for three different modalities, thus contributing to comprehensive glycoproteomics analyses. In addition, we explored compatible sample derivatization methods. We used glycan TMT-labeling to improve electrophoretic migration and enable multiplexed quantitation by tandem MS. We used sialic acid linkage-specific derivatization methods to improve separation and the level of information obtained from a single analytical step. Capillary electrophoresis greatly improved glycoform separation for both released glycans and glycopeptides over that reported for chromatography modes more frequently employed for such analyses. Overall, the CE-MS method described here enables rapid setup and analysis of glycans and glycopeptides using mass spectrometry.

Comparative Proteomics Reveals Dysregulated Mitochondrial O-GlcNAcylation in Diabetic Hearts.

O-linked ß-N-acetylglucosamine (O-GlcNAc), a post-translational modification on serine and threonine residues of many proteins, plays crucial regulatory roles in diverse biological events. As a nutrient sensor, O-GlcNAc modification (O-GlcNAcylation) on nuclear and cytoplasmic proteins underlies the pathology of diabetic complications including cardiomyopathy. However, mitochondrial O-GlcNAcylation, especially in response to chronic hyperglycemia in diabetes, has been poorly explored. We performed a comparative O-GlcNAc profiling of mitochondria from control and streptozotocin (STZ)-induced diabetic rat hearts by using an improved ß-elimination/Michael addition with isotopic DTT reagents (BEMAD) followed by tandem mass spectrometric analysis. In total, 86 mitochondrial proteins, involved in diverse pathways, were O-GlcNAcylated. Among them, many proteins have site-specific alterations in O-GlcNAcylation in response to diabetes, which suggests that protein O-GlcNAcylation is a novel layer of regulation mediating adaptive changes in mitochondrial metabolism during the progression of diabetic cardiomyopathy.

The aryl hydrocarbon receptor directs hematopoietic progenitor cell expansion and differentiation.

The evolutionarily conserved aryl hydrocarbon receptor (AhR) has been studied for its role in environmental chemical-induced toxicity. However, recent studies have demonstrated that the AhR may regulate the hematopoietic and immune systems during development in a cell-specific manner. These results, together with the absence of an in vitro model system enabling production of large numbers of primary human hematopoietic progenitor cells (HPs) capable of differentiating into megakaryocyte- and erythroid-lineage cells, motivated us to determine if AhR modulation could facilitate both progenitor cell expansion and megakaryocyte and erythroid cell differentiation. Using a novel, pluripotent stem cell-based, chemically-defined, serum and feeder cell-free culture system, we show that the AhR is expressed in HPs and that, remarkably, AhR activation drives an unprecedented expansion of HPs, megakaryocyte-lineage cells, and erythroid-lineage cells. Further AhR modulation within rapidly expanding progenitor cell populations directs cell fate, with chronic AhR agonism permissive to erythroid differentiation and acute antagonism favoring megakaryocyte specification. These results highlight the development of a new Good Manufacturing Practice-compliant platform for generating virtually unlimited numbers of human HPs with which to scrutinize red blood cell and platelet development, including the assessment of the role of the AhR critical cell fate decisions during hematopoiesis.

Does reversible cysteine oxidation link the Western diet to cardiac dysfunction?

Using a novel cysteine thiol labeling strategy coupled with mass spectrometric analysis, we identified and quantified the changes in global reversible cysteine oxidation of proteins in the left ventricle of hearts from mice with metabolic syndrome-associated diastolic dysfunction. This phenotype was induced by feeding a high-fat, high-sucrose, type-2 diabetogenic diet to C57BL/6J mice for 8 mo. The extent of reversible thiol oxidation in relationship to the total available (free and reducible) level of each cysteine could be confidently determined for 173 proteins, of which 98 contained cysteines differentially modified ≥1.5-fold by the diet. Our findings suggest that the metabolic syndrome leads to potentially deleterious changes in the oxidative modification of metabolically active proteins. These alterations may adversely regulate energy substrate flux through glycolysis, ß-oxidation, citric acid (TCA) cycle, and oxidative phosphorylation (oxphos), thereby contributing to maladaptive tissue remodeling that is associated with, and possibly contributing to, diastolic left ventricular dysfunction.

Protein Expression by Human Pulmonary Artery Smooth Muscle Cells Containing a BMPR2 Mutation and the Action of ET-1 as Determined by Proteomic Mass Spectrometry.

Pulmonary arterial hypertension (PAH) is a disease characterized by increased pulmonary vascular resistance and remodeling. Increase in the population of vascular smooth muscle cells is among the key events contributing to the remodeling. Endothelin-1 (ET-1), a potent vasoconstrictor, is linked to the etiology and progression of PAH. Here we analyze changes in protein expressions in response to ET-1 in pulmonary arterial smooth muscle cells (PASMC) from a healthy Control (non-PAH) and a PAH subject presenting a bone morphogenetic protein type II receptor (BMPR2) mutation with exon 1-8 deletion. Protein expressions were analyzed by proteomic mass spectrometry using label-free quantitation and the correlations were subjected to Ingenuity™ Pathway Analysis. The results point to eIF2/mTOR/p70S6K, RhoA/actin cytoskeleton/integrin and protein unbiquitination as canonical pathways whose protein expressions increase with the development of PAH. These pathways have an intimal function in the PAH-related physiology of smooth muscle proliferation, apoptosis, contraction and cellular stress. Exposure of the cells to ET-1 further increases protein expression within these pathways. Thus our results show changes in signaling pathways as a consequence of PAH and the effect of ET-1 interference on Control and PAH-affected cells.

Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry: the ABRF glycoprotein research multi-institutional study 2012.

One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods.

Characterization of the human NEK7 interactome suggests catalytic and regulatory properties distinct from those of NEK6.

Human NEK7 is a regulator of cell division and plays an important role in growth and survival of mammalian cells. Human NEK6 and NEK7 are closely related, consisting of a conserved C-terminal catalytic domain and a nonconserved and disordered N-terminal regulatory domain, crucial to mediate the interactions with their respective proteins. Here, in order to better understand NEK7 cellular functions, we characterize the NEK7 interactome by two screening approaches: one using a yeast two-hybrid system and the other based on immunoprecipitation followed by mass spectrometry analysis. These approaches led to the identification of 61 NEK7 interactors that contribute to a variety of biological processes, including cell division. Combining additional interaction and phosphorylation assays from yeast two-hybrid screens, we validated CC2D1A, TUBB2B, MNAT1, and NEK9 proteins as potential NEK7 interactors and substrates. Notably, endogenous RGS2, TUBB, MNAT1, NEK9, and PLEKHA8 localized with NEK7 at key sites throughout the cell cycle, especially during mitosis and cytokinesis. Furthermore, we obtained evidence that the closely related kinases NEK6 and NEK7 do not share common interactors, with the exception of NEK9, and display different modes of protein interaction, depending on their N- and C-terminal regions, in distinct fashions. In summary, our work shows for the first time a comprehensive NEK7 interactome that, combined with functional in vitro and in vivo assays, suggests that NEK7 is a multifunctional kinase acting in different cellular processes in concert with cell division signaling and independently of NEK6.

Epithelial wounds induce differential phosphorylation changes in response to purinergic and EGF receptor activation.

Protein phosphorylation is a dynamic post-translational modification. Mass spectrometry-based quantitation was performed to determine the phosphoproteome profile of epithelial cells in response to injury, nucleotide, or epidermal growth factor. Phosphotyrosine enrichment used immunoprecipitation and immobilized metal affinity chromatography. Nucleotides released after scratch wounding activate purinergic receptors, leading to a distinct phosphorylation profile on epidermal growth factor receptor (EGFR) compared with its natural ligand. ATP induced a 2- to 15-fold phosphorylation increase over control on EGFR Y974, Y1086, and Y1148, with minimal phosphorylation intensity on EGFR Y1173 compared with the level measured in response to epidermal growth factor. Differential phosphorylation induced by epidermal growth factor or ATP was site specific on Src, Shc, phospholipase Cγ, protein kinase C, focal adhesion kinase, paxillin, and mitogen-activated protein kinases 1, 12, and 13. After wounding, the P2Y2 receptor mRNA expression increased, and after knockdown, migration and Ca(2+) mobilization were impaired. To examine phosphorylation mediated by P2Y2, cells were cultured in media containing stable isotope-labeled amino acids, the receptor was knocked down, and the cells were stimulated. Mass spectrometry-based comparison of the phosphorylation profiles of control versus transfected cells revealed a 50-fold decrease in phosphorylation of EGFR Y974 and 1086, with no decrease in Y1173 phosphorylation. A similarfold decrease in Src Y421 and Y446 and paxillin Y118 was detected, indicating the far-reaching importance of the P2Y2 receptor in mediating migration.

PANAMA-enabled high-sensitivity dual nanoflow LC-MS metabolomics and proteomics analysis.

High-sensitivity nanoflow liquid chromatography (nLC) is seldom employed in untargeted metabolomics because current sample preparation techniques are inefficient at preventing nanocapillary column performance degradation. Here, we describe an nLC-based tandem mass spectrometry workflow that enables seamless joint analysis and integration of metabolomics (including lipidomics) and proteomics from the same samples without instrument duplication. This workflow is based on a robust solid-phase micro-extraction step for routine sample cleanup and bioactive molecule enrichment. Our method, termed proteomic and nanoflow metabolomic analysis (PANAMA), improves compound resolution and detection sensitivity without compromising the depth of coverage as compared with existing widely used analytical procedures. Notably, PANAMA can be applied to a broad array of specimens, including biofluids, cell lines, and tissue samples. It generates high-quality, information-rich metabolite-protein datasets while bypassing the need for specialized instrumentation.

Corrigendum: Integrated metabolomics and proteomics reveal biomarkers associated with hemodialysis in end-stage kidney disease.

[This corrects the article DOI: 10.3389/fphar.2023.1243505.].

Oxidation of HRas cysteine thiols by metabolic stress prevents palmitoylation in vivo and contributes to endothelial cell apoptosis.

Here we demonstrate a new paradigm in redox signaling, whereby oxidants resulting from metabolic stress directly alter protein palmitoylation by oxidizing reactive cysteine thiolates. In mice fed a high-fat, high-sucrose diet and in cultured endothelial cells (ECs) treated with high palmitate and high glucose (HPHG), there was decreased HRas palmitoylation on Cys181/184 (61±24% decrease for cardiac tissue and 38±7.0% in ECs). This was due to oxidation of Cys181/184, detected using matrix-assisted laser desorption/ionization time of flight (MALDI TOF)-TOF. Decrease in HRas palmitoylation affected its compartmentalization and Ras binding domain binding activity, with a shift from plasma membrane tethering to Golgi localization. Loss of plasma membrane-bound HRas decreased growth factor-stimulated ERK phosphorylation (84±8.6% decrease) and increased apoptotic signaling (24±6.5-fold increase) after HPHG treatment that was prevented by overexpressing wild-type but not C181/184S HRas. The essential role of HRas in metabolic stress was made evident by the similar effects of expressing an inactive dominant negative N17-HRas or a MEK inhibitor. Furthermore, the relevance of thiol oxidation was demonstrated by overexpressing manganese superoxide dismutase, which improved HRas palmitoylation and ERK phosphorylation, while lessening apoptosis in HPHG treated ECs.