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Human RNF213, which encodes the protein mysterin, is a known susceptibility gene for moyamoya disease (MMD), a cerebrovascular condition with occlusive lesions and compensatory angiogenesis. Mysterin mutations, together with exposure to environmental trigger factors, lead to an elevated stroke risk since childhood. Mysterin is induced during cell stress, to function as cytosolic AAA+ ATPase and ubiquitylation enzyme. Little knowledge exists, in which context mysterin is needed. Here, we found that genetic ablation of several mitochondrial matrix factors, such as the peptidase ClpP, the transcription factor Tfam, as well as the peptidase and AAA+ ATPase Lonp1, potently induces Rnf213 transcript expression in various organs, in parallel with other components of the innate immune system. Mostly in mouse fibroblasts and human endothelial cells, the Rnf213 levels showed prominent upregulation upon Poly(I:C)-triggered TLR3-mediated responses to dsRNA toxicity, as well as upon interferon gamma treatment. Only partial suppression of Rnf213 induction was achieved by C16 as an antagonist of PKR (dsRNA-dependent protein kinase). Since dysfunctional mitochondria were recently reported to release immune-stimulatory dsRNA into the cytosol, our results suggest that mysterin becomes relevant when mitochondrial dysfunction or infections have triggered RNA-dependent inflammation. Thus, MMD has similarities with vasculopathies that involve altered nucleotide processing, such as Aicardi-Goutières syndrome or systemic lupus erythematosus. Furthermore, in MMD, the low penetrance of RNF213 mutations might be modified by dysfunctions in mitochondria or the TLR3 pathway.
Diabetes mellitus is a major risk factor for cardiovascular disease. Platelets from diabetic patients are hyperreactive and release microparticles that carry activated cysteine proteases or calpains. Whether platelet-derived calpains contribute to the development of vascular complications in diabetes is unknown. Here we report that platelet-derived calpain1 (CAPN1) cleaves the protease-activated receptor 1 (PAR-1) on the surface of endothelial cells, which then initiates a signaling cascade that includes the activation of the tumor necrosis factor (TNF)-α converting enzyme (TACE). The latter elicits the shedding of the endothelial protein C receptor and the generation of TNF-α, which in turn, induces intracellular adhesion molecule (ICAM)-1 expression to promote monocyte adhesion. All of the effects of CAPN1 were mimicked by platelet-derived microparticles from diabetic patients or from wild-type mice but not from CAPN1−/− mice, and were not observed in PAR-1-deficient endothelial cells. Importantly, aortae from diabetic mice expressed less PAR-1 but more ICAM-1 than non-diabetic mice, effects that were prevented by treating diabetic mice with a calpain inhibitor as well as by the platelet specific deletion of CAPN1. Thus, platelet-derived CAPN1 contributes to the initiation of the sterile vascular inflammation associated with diabetes via the cleavage of PAR-1 and the release of TNF-α from the endothelial cell surface.
Aims: Parkinson's disease (PD) is frequently associated with a prodromal sensory neuropathy manifesting with sensory loss and chronic pain. We have recently shown that PD-associated sensory neuropathy in patients is associated with high levels of glucosylceramides. Here, we assessed the underlying pathology and mechanisms in Pink1−/−SNCAA53T double mutant mice. Methods: We studied nociceptive and olfactory behaviour and the neuropathology of dorsal root ganglia (DRGs), including ultrastructure, mitochondrial respiration, transcriptomes, outgrowth and calcium currents of primary neurons, and tissue ceramides and sphingolipids before the onset of a PD-like disease that spontaneously develops in Pink1−/−SNCAA53T double mutant mice beyond 15 months of age. Results: Similar to PD patients, Pink1−/−SNCAA53T mice developed a progressive prodromal sensory neuropathy with a loss of thermal sensitivity starting as early as 4 months of age. In analogy to human plasma, lipid analyses revealed an accumulation of glucosylceramides (GlcCer) in the DRGs and sciatic nerves, which was associated with pathological mitochondria, impairment of mitochondrial respiration, and deregulation of transient receptor potential channels (TRPV and TRPA) at mRNA, protein and functional levels in DRGs. Direct exposure of DRG neurons to GlcCer caused transient hyperexcitability, followed by a premature decline of the viability of sensory neurons cultures upon repeated GlcCer application. Conclusions: The results suggest that pathological GlcCer contribute to prodromal sensory disease in PD mice via mitochondrial damage and calcium channel hyperexcitability. GlcCer-associated sensory neuron pathology might be amenable to GlcCer lowering therapeutic strategies.
Respiratory chain signalling is essential for adaptive remodelling following cardiac ischaemia
(2020)
Cardiac ischaemia‐reperfusion (I/R) injury has been attributed to stress signals arising from an impaired mitochondrial electron transport chain (ETC), which include redox imbalance, metabolic stalling and excessive production of reactive oxygen species (ROS). The alternative oxidase (AOX) is a respiratory enzyme, absent in mammals, that accepts electrons from a reduced quinone pool to reduce oxygen to water, thereby restoring electron flux when impaired and, in the process, blunting ROS production. Hence, AOX represents a natural rescue mechanism from respiratory stress. This study aimed to determine how respiratory restoration through xenotopically expressed AOX affects the re‐perfused post‐ischaemic mouse heart. As expected, AOX supports ETC function and attenuates the ROS load in post‐anoxic heart mitochondria. However, post‐ischaemic cardiac remodelling over 3 and 9 weeks was not improved. AOX blunted transcript levels of factors known to be up‐regulated upon I/R such as the atrial natriuretic peptide (Anp) whilst expression of pro‐fibrotic and pro‐apoptotic transcripts were increased. Ex vivo analysis revealed contractile failure at nine but not 3 weeks after ischaemia whilst label‐free quantitative proteomics identified an increase in proteins promoting adverse extracellular matrix remodelling. Together, this indicates an essential role for ETC‐derived signals during cardiac adaptive remodelling and identified ROS as a possible effector.
Progranulin deficiency is associated with neurodegeneration in humans and in mice. The mechanisms likely involve progranulin-promoted removal of protein waste via autophagy. We performed a deep proteomic screen of the pre-frontal cortex in aged (13–15 months) female progranulin-deficient mice (GRN−/−) and mice with inducible neuron-specific overexpression of progranulin (SLICK-GRN-OE) versus the respective control mice. Proteins were extracted and analyzed per liquid chromatography/mass spectrometry (LC/MS) on a Thermo Scientific™ Q Exactive Plus equipped with an ultra-high performance liquid chromatography unit and a Nanospray Flex Ion-Source. Full Scan MS-data were acquired using Xcalibur and raw files were analyzed using the proteomics software Max Quant. The mouse reference proteome set from uniprot (June 2015) was used to identify peptides and proteins. The DiB data file is a reduced MaxQuant output and includes peptide and protein identification, accession numbers, protein and gene names, sequence coverage and label free quantification (LFQ) values of each sample. Differences in protein expression in genotypes are presented in "Progranulin overexpression in sensory neurons attenuates neuropathic pain in mice: Role of autophagy" (C. Altmann, S. Hardt, C. Fischer, J. Heidler, H.Y. Lim, A. Haussler, B. Albuquerque, B. Zimmer, C. Moser, C. Behrends, F. Koentgen, I. Wittig, M.H. Schmidt, A.M. Clement, T. Deller, I. Tegeder, 2016) [1].
Leigh syndrome is one of the most common neurological phenotypes observed in pediatric mitochondrial disease presentations. It is characterized by symmetrical lesions found on neuroimaging in the basal ganglia, thalamus, and brainstem and by a loss of motor skills and delayed developmental milestones. Genetic diagnosis of Leigh syndrome is complicated on account of the vast genetic heterogeneity with >75 candidate disease-associated genes having been reported to date. Candidate genes are still emerging, being identified when “omics” tools (genomics, proteomics, and transcriptomics) are applied to manipulated cell lines and cohorts of clinically characterized individuals who lack a genetic diagnosis. NDUFAF8 is one such protein; it has been found to interact with the well-characterized complex I (CI) assembly factor NDUFAF5 in a large-scale protein-protein interaction screen. Diagnostic next-generation sequencing has identified three unrelated pediatric subjects, each with a clinical diagnosis of Leigh syndrome, who harbor bi-allelic pathogenic variants in NDUFAF8. These variants include a recurrent splicing variant that was initially overlooked due to its deep-intronic location. Subject fibroblasts were found to express a complex I deficiency, and lentiviral transduction with wild-type NDUFAF8-cDNA ameliorated both the assembly defect and the biochemical deficiency. Complexome profiling of subject fibroblasts demonstrated a complex I assembly defect, and the stalled assembly intermediates corroborate the role of NDUFAF8 in early complex I assembly. This report serves to expand the genetic heterogeneity associated with Leigh syndrome and to validate the clinical utility of orphan protein characterization. We also highlight the importance of evaluating intronic sequence when a single, definitively pathogenic variant is identified during diagnostic testing.
Myeloid-specific deletion of the AMPK2 subunit alters monocyte protein expression and atherogenesis
(2019)
The AMP-activated protein kinase (AMPK) is an energy sensing kinase that is activated by a drop in cellular ATP levels. Although several studies have addressed the role of the AMPKα1 subunit in monocytes and macrophages, little is known about the α2 subunit. The aim of this study was to assess the consequences of AMPKα2 deletion on protein expression in monocytes/macrophages, as well as on atherogenesis. A proteomics approach was applied to bone marrow derived monocytes from wild-type mice versus mice specifically lacking AMPKα2 in myeloid cells (AMPKα2∆MC mice). This revealed differentially expressed proteins, including methyltransferases. Indeed, AMPKα2 deletion in macrophages increased the ratio of S-adenosyl methionine to S-adenosyl homocysteine and increased global DNA cytosine methylation. Also, methylation of the vascular endothelial growth factor and matrix metalloproteinase-9 (MMP9) genes was increased in macrophages from AMPKα2∆MC mice, and correlated with their decreased expression. To link these findings with an in vivo phenotype, AMPKα2∆MC mice were crossed onto the ApoE-/- background and fed a western diet. ApoExAMPKα2∆MC mice developed smaller atherosclerotic plaques than their ApoExα2fl/fl littermates, that contained fewer macrophages and less MMP9 than plaques from ApoExα2fl/fl littermates. These results indicate that the AMPKα2 subunit in myeloid cells influences DNA methylation and thus protein expression and contributes to the development of atherosclerotic plaques.
BIAM switch assay coupled to mass spectrometry identifies novel redox targets of NADPH oxidase 4
(2019)
Aim: NADPH oxidase (Nox) -derived reactive oxygen species have been implicated in redox signaling via cysteine oxidation in target proteins. Although the importance of oxidation of target proteins is well known, the specificity of such events is often debated. Only a limited number of Nox-oxidized proteins have been identified thus far; especially little is known concerning redox-targets of the constitutively active NADPH oxidase Nox4.
In this study, HEK293 cells with tetracycline-inducible Nox4 overexpression (HEK-tet-Nox4), as well as podocytes of WT and Nox4-/- mice, were utilized to identify Nox4-dependent redox-modified proteins.
Results: TGFβ1 induced an elevation in Nox4 expression in podocytes from WT but not Nox4-/- mice. Using BIAM based redox switch assay in combination with mass spectrometry and western blot analysis, 142 proteins were identified as differentially oxidized in podocytes from wild type vs. Nox4-/- mice and 131 proteins were differentially oxidized in HEK-tet-Nox4 cells upon Nox4 overexpression. A predominant overlap was found for peroxiredoxins and thioredoxins, as expected. More interestingly, the GRB2-associated-binding protein 1 (Gab1) was identified as being differentially oxidized in both approaches. Further analysis using mass spectrometry-coupled BIAM switch assay and site directed mutagenesis, revealed Cys374 and Cys405 as the major Nox4 targeted oxidation sites in Gab1.
Innovation & conclusion: BIAM switch assay coupled to mass spectrometry is a powerful and versatile tool to identify differentially oxidized proteins in a global untargeted way. Nox4, as a source of hydrogen peroxide, changes the redox-state of numerous proteins. Of those, we identified Gab1 as a novel redox target of Nox4.
Upregulations of neuronal nitric oxide synthase (nNOS/NOS1) in the mouse brain upon aging suggest a role in age-associated changes of protein homeostasis. We generated a cell model, in which constitutive expression of nNOS in SH-SY5Y cells at a level comparable to mouse brain replicates the aging phenotype i.e. slowing of cell proliferation, cell enlargement and expression of senescence markers. nNOS+ and MOCK cells were exposed to proteostasis stress by treatment with rapamycin or serum-free starvation. The proteomes were analyzed per SILAC or label-free using hybrid liquid chromatography/mass spectrometry (LC/MS). Full scan MS-data were acquired using Xcalibur, and raw mass spectra were analyzed using the proteomics software MaxQuant. The human reference proteome from uniprot was used as template to identify peptides and proteins and quantify protein expression. The DiB data file contains essential MaxQuant output tables and includes peptide and protein identification, accession numbers, protein and gene names, sequence coverage and quantification values of each sample. Differences in protein expression in MOCK versus nNOS+ SH-SY5Y cells and interpretation of results are presented in Valek et al. (2018). Raw mass spectra and MaxQuant output files have been deposited to the ProteomeXchange Consortium (Vizcaino et al., 2014) via the PRIDE partner repository with the dataset identifier PRIDE: PXD010538.
Upregulations of neuronal nitric oxide synthase (nNOS/NOS1) in the mouse brain upon aging and stress suggest a role of NO-dependent redox protein modifications for age-associated protein imbalances or dysfunctions. We generated a cell model, in which constitutive expression of nNOS in SH-SY5Y cells at a level comparable with mouse brain replicates the aging phenotype, that is, slowing of cell proliferation, cell enlargement, and expression of senescence markers. nNOS+ and MOCK cells were exposed to proteostasis stress by the treatment with rapamycin or serum-free starvation versus control conditions. To analyze NO-mediated S-nitrosylations (SNO) and other reversible protein modifications including disulfides and sulfoxides, we used complimentary proteomic approaches encompassing 2D-SNO-DIGE (differential gel electrophoresis), SNO-site identification (SNOSID), SNO Super-SILAC, SNO BIAM-Switch, and Redox-BIAM switch. The redox proteomes were analyzed using hybrid liquid chromatography/mass spectrometry (LC/MS). Full scan MS-data were acquired using Xcalibur, and raw mass spectra were analyzed using the proteomics software MaxQuant. The human reference proteome sets from uniprot were used as templates to identify peptides and proteins and quantify protein expression. The DiB data file contains MaxQuant output tables of the redox-modified proteins.The tables include peptide and protein identification, accession numbers, protein, and gene names, sequence coverage and quantification values of each sample. Differences in protein redox modifications in MOCK versus nNOS+ SH-SY5Y cells and interpretation of results are presented in (Valek et al., 2018).