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Background: Mitochondrial acyl-CoA dehydrogenase family member 9 (ACAD9) is essential for the assembly of mitochondrial respiratory chain complex I. Disease causing biallelic variants in ACAD9 have been reported in individuals presenting with lactic acidosis and cardiomyopathy.
Results: We describe the genetic, clinical and biochemical findings in a cohort of 70 patients, of whom 29 previously unpublished. We found 34 known and 18 previously unreported variants in ACAD9. No patients harbored biallelic loss of function mutations, indicating that this combination is unlikely to be compatible with life. Causal pathogenic variants were distributed throughout the entire gene, and there was no obvious genotype-phenotype correlation.
Most of the patients presented in the first year of life. For this subgroup the survival was poor (50% not surviving the first 2 years) comparing to patients with a later presentation (more than 90% surviving 10 years). The most common clinical findings were cardiomyopathy (85%), muscular weakness (75%) and exercise intolerance (72%). Interestingly, severe intellectual deficits were only reported in one patient and severe developmental delays in four patients. More than 70% of the patients were able to perform the same activities of daily living when compared to peers.
Conclusions: Our data show that riboflavin treatment improves complex I activity in the majority of patient-derived fibroblasts tested. This effect was also reported for most of the treated patients and is mirrored in the survival data. In the patient group with disease-onset below 1 year of age, we observed a statistically-significant better survival for patients treated with riboflavin.
Mitochondrial derived reactive oxygen species (mtROS) are known for their signaling qualities in both physiology and pathology. To elucidate mitochondrial complex I-dependent ROS-signaling after lipopolysaccharide (LPS)-stimulation THP-1 macrophages with a knockdown of the transmembrane protein TMEM126B were generated. TMEM knockdown cells (sh126B) showed a reduced assembly of complex I and attenuated mtROS production. In these cells we identified protein oxidization by mtROS upon LPS-treatment using the BIAM switch assay coupled to liquid chromatography and mass spectrometry. One of the identified targets of mtROS was succinate dehydrogenase (SDH) flavoprotein subunit A (SDHA). Oxidation of SDHA decreased its enzymatic activity and pharmacological inhibition of SDH in turn stabilized hypoxia inducible factor (HIF)-1α and caused the subsequent, sustained expression of interleukin-1β (IL-1β). Oxidation of SDHA in sh126B cells was attenuated, while pharmacological inhibition of SDH by atpenin A5 restored IL-1β expression in sh126B cells upon LPS-treatment. Conclusively, oxidation of SDH by mtROS links an altered metabolism, i.e. succinate accumulation to HIF-1-driven, inflammatory changes in macrophages.
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).
Upregulations of neuronal nitric oxide synthase (nNOS) in the rodent brain have been associated with neuronal aging. To address underlying mechanisms we generated SH-SY5Y neuronal cells constitutively expressing nNOS at a level similar to mouse brain (nNOS+ versus MOCK). Initial experiments revealed S-nitrosylations (SNO) of key players of protein homeostasis: heat shock cognate HSC70/HSPA8 within its nucleotide-binding site, and UBE2D ubiquitin conjugating enzymes at the catalytic site cysteine. HSPA8 is involved in protein folding, organelle import/export and chaperone-mediated LAMP2a-dependent autophagy (CMA). A set of deep redox and full proteome analyses, plus analysis of autophagy, CMA and ubiquitination with rapamycin and starvation as stimuli confirmed the initial observations and revealed a substantial increase of SNO modifications in nNOS+ cells, in particular targeting protein networks involved in protein catabolism, ubiquitination, carbohydrate metabolism and cell cycle control. Importantly, NO-independent reversible oxidations similarly occurred in both cell lines. Functionally, nNOS caused an accumulation of proteins, including CMA substrates and loss of LAMP2a. UBE2D activity and proteasome activity were impaired, resulting in dysregulations of cell cycle checkpoint proteins. The observed changes of protein degradation pathways caused an expansion of the cytoplasm, large lysosomes, slowing of the cell cycle and suppression of proliferation suggesting a switch of the phenotype towards aging, supported by downregulations of neuronal progenitor markers but increase of senescence-associated proteins. Hence, upregulation of nNOS in neuronal cells imposes aging by SNOing of key players of ubiquitination, chaperones and of substrate proteins leading to interference with crucial steps of protein homeostasis.
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.
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.
Colorectal cancer (CRC) is one of the most common cancers that is characterized by a high mortality due to the strong metastatic potential of the primary tumor and the high rate of therapy resistance. Hereby, evasion of apoptosis is the primary underlying cause of reduced sensitivity of tumor cells to chemo- and radiotherapy. Using RNA affinity chromatography, we identified the tripartite motif-containing protein 25 (TRIM25) as a bona fide caspase-2 mRNA-binding protein in colon carcinoma cells. Loss-of-function and gain-of-function approaches revealed that TRIM25 attenuates the protein levels of caspase-2 without significantly affecting caspase-2 mRNA levels. In addition, experiments with cycloheximide revealed that TRIM25 does not affect the protein stability of caspase-2. Furthermore, silencing of TRIM25 induced a significant redistribution of caspase-2 transcripts from RNP particles to translational active polysomes, indicating that TRIM25 negatively interferes with caspase-2 translation. Functionally, the elevation in caspase-2 upon TRIM25 depletion significantly increased the sensitivity of colorectal cells to drug-induced intrinsic apoptosis as implicated by increased caspase-3 cleavage and cytochrome c release. Importantly, the apoptosis-sensitizing effects by transient TRIM25 knockdown were rescued by concomitant silencing of caspase-2, demonstrating a critical role of caspase-2. Inhibition of caspase-2 by TRIM25 implies a survival mechanism that critically contributes to chemotherapeutic drug resistance in CRC.
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.
High-resolution cryo-EM structures of respiratory complex I: Mechanism, assembly, and disease
(2019)
Respiratory complex I is a redox-driven proton pump, accounting for a large part of the electrochemical gradient that powers mitochondrial adenosine triphosphate synthesis. Complex I dysfunction is associated with severe human diseases. Assembly of the one-megadalton complex I in the inner mitochondrial membrane requires assembly factors and chaperones. We have determined the structure of complex I from the aerobic yeast Yarrowia lipolytica by electron cryo-microscopy at 3.2-Å resolution. A ubiquinone molecule was identified in the access path to the active site. The electron cryo-microscopy structure indicated an unusual lipid-protein arrangement at the junction of membrane and matrix arms that was confirmed by molecular simulations. The structure of a complex I mutant and an assembly intermediate provide detailed molecular insights into the cause of a hereditary complex I-linked disease and complex I assembly in the inner mitochondrial membrane.
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.
Complexome profiling is an emerging ‘omics approach that systematically interrogates the composition of protein complexes (the complexome) of a sample, by combining biochemical separation of native protein complexes with mass-spectrometry based quantitation proteomics. The resulting fractionation profiles hold comprehensive information on the abundance and composition of the complexome, and have a high potential for reuse by experimental and computational researchers. However, the lack of a central resource that provides access to these data, reported with adequate descriptions and an analysis tool, has limited their reuse. Therefore, we established the ComplexomE profiling DAta Resource (CEDAR, www3.cmbi.umcn.nl/cedar/), an openly accessible database for depositing and exploring mass spectrometry data from complexome profiling studies. Compatibility and reusability of the data is ensured by a standardized data and reporting format containing the “minimum information required for a complexome profiling experiment” (MIACE). The data can be accessed through a user-friendly web interface, as well as programmatically using the REST API portal. Additionally, all complexome profiles available on CEDAR can be inspected directly on the website with the profile viewer tool that allows the detection of correlated profiles and inference of potential complexes. In conclusion, CEDAR is a unique, growing and invaluable resource for the study of protein complex composition and dynamics across biological systems.
Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.
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.
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.
Complexome profiling is an emerging ‘omics’ approach that systematically interrogates the composition of protein complexes (the complexome) of a sample, by combining biochemical separation of native protein complexes with mass-spectrometry based quantitation proteomics. The resulting fractionation profiles hold comprehensive information on the abundance and composition of the complexome, and have a high potential for reuse by experimental and computational researchers. However, the lack of a central resource that provides access to these data, reported with adequate descriptions and an analysis tool, has limited their reuse. Therefore, we established the ComplexomE profiling DAta Resource (CEDAR, www3.cmbi.umcn.nl/cedar/), an openly accessible database for depositing and exploring mass spectrometry data from complexome profiling studies. Compatibility and reusability of the data is ensured by a standardized data and reporting format containing the “minimum information required for a complexome profiling experiment” (MIACE). The data can be accessed through a user-friendly web interface, as well as programmatically using the REST API portal. Additionally, all complexome profiles available on CEDAR can be inspected directly on the website with the profile viewer tool that allows the detection of correlated profiles and inference of potential complexes. In conclusion, CEDAR is a unique, growing and invaluable resource for the study of protein complex composition and dynamics across biological systems.
Mitochondrial dysfunction may activate innate immunity, e.g. upon abnormal handling of mitochondrial DNA in TFAM mutants or in altered mitophagy. Recent reports showed that also deletion of mitochondrial matrix peptidase ClpP in mice triggers transcriptional upregulation of inflammatory factors. Here, we studied ClpP-null mouse brain at two ages and mouse embryonal fibroblasts, to identify which signaling pathways are responsible, employing mass spectrometry, subcellular fractionation, immunoblots, and reverse transcriptase polymerase chain reaction. Several mitochondrial unfolded protein response factors showed accumulation and altered migration in blue-native gels, prominently the co-chaperone DNAJA3. Its mitochondrial dysregulation increased also its extra-mitochondrial abundance in the nucleus, a relevant observation given that DNAJA3 modulates innate immunity. Similar observations were made for STAT1, a putative DNAJA3 interactor. Elevated expression was observed not only for the transcription factors Stat1/2, but also for two interferon-stimulated genes (Ifi44, Gbp3). Inflammatory responses were strongest for the RLR pattern recognition receptors (Ddx58, Ifih1, Oasl2, Trim25) and several cytosolic nucleic acid sensors (Ifit1, Ifit3, Oas1b, Ifi204, Mnda). The consistent dysregulation of these factors from an early age might influence also human Perrault syndrome, where ClpP loss-of-function leads to early infertility and deafness, with subsequent widespread neurodegeneration.
Many proteins have been found to operate in a complex with various biomolecules such as proteins, nucleic acids, carbohydrates, or lipids. Protein complexes can be transient, stable or dynamic and their association is controlled under variable cellular conditions. Complexome profiling is a recently developed mass spectrometry-based method that combines mild separation techniques, native gel electrophoresis, and density gradient centrifugation with quantitative mass spectrometry to generate inventories of protein assemblies within a cell or subcellular fraction. This review summarizes applications of complexome profiling with respect to assembly ranging from single subunits to large macromolecular complexes, as well as their stability, and remodeling in health and disease.
The accumulation of functionally impaired mitochondria is a key event in aging. Previous works with the fungal aging model Podospora anserina demonstrated pronounced age-dependent changes of mitochondrial morphology and ultrastructure, as well as alterations of transcript and protein levels, including individual proteins of the oxidative phosphorylation (OXPHOS). The identified protein changes do not reflect the level of the whole protein complexes as they function in-vivo. In the present study, we investigated in detail the age-dependent changes of assembled mitochondrial protein complexes, using complexome profiling. We observed pronounced age-depen-dent alterations of the OXPHOS complexes, including the loss of mitochondrial respiratory supercomplexes (mtRSCs) and a reduction in the abundance of complex I and complex IV. Additionally, we identified a switch from the standard complex IV-dependent respiration to an alternative respiration during the aging of the P. anserina wild type. Interestingly, we identified proteasome components, as well as endoplasmic reticulum (ER) proteins, for which the recruitment to mitochondria appeared to be increased in the mitochondria of older cultures. Overall, our data demonstrate pronounced age-dependent alterations of the protein complexes involved in energy transduction and suggest the induction of different non-mitochondrial salvage pathways, to counteract the age-dependent mitochondrial impairments which occur during aging.
Biallelic pathogenic variants in CLPP, encoding mitochondrial matrix peptidase ClpP, cause a rare autosomal recessive condition, Perrault syndrome type 3 (PRLTS3). It is characterized by primary ovarian insufficiency and early sensorineural hearing loss, often associated with progressive neurological deficits. Mouse models showed that accumulations of (i) its main protein interactor, the substrate-selecting AAA+ ATPase ClpX, (ii) mitoribosomes, and (iii) mtDNA nucleoids are the main cellular consequences of ClpP absence. However, the sequence of these events and their validity in human remain unclear. Here, we studied global proteome profiles to define ClpP substrates among mitochondrial ClpX interactors, which accumulated consistently in ClpP-null mouse embryonal fibroblasts and brains. Validation work included novel ClpP-mutant patient fibroblast proteomics. ClpX co-accumulated in mitochondria with the nucleoid component POLDIP2, the mitochondrial poly(A) mRNA granule element LRPPRC, and tRNA processing factor GFM1 (in mouse, also GRSF1). Only in mouse did accumulated ClpX, GFM1, and GRSF1 appear in nuclear fractions. Mitoribosomal accumulation was minor. Consistent accumulations in murine and human fibroblasts also affected multimerizing factors not known as ClpX interactors, namely, OAT, ASS1, ACADVL, STOM, PRDX3, PC, MUT, ALDH2, PMPCB, UQCRC2, and ACADSB, but the impact on downstream metabolites was marginal. Our data demonstrate the primary impact of ClpXP on the assembly of proteins with nucleic acids and show nucleoid enlargement in human as a key consequence.
Aims: We investigated N471D WASH complex subunit strumpellin (Washc5) knock-in and Washc5 knock-out mice as models for hereditary spastic paraplegia type 8 (SPG8). Methods: We generated heterozygous and homozygous N471D Washc5 knock-in mice and subjected them to a comprehensive clinical, morphological and laboratory parameter screen, and gait analyses. Brain tissue was used for proteomic analysis. Furthermore, we generated heterozygous Washc5 knock-out mice. WASH complex subunit strumpellin expression was determined by qPCR and immunoblotting. Results: Homozygous N471D Washc5 knock-in mice showed mild dilated cardiomyopathy, decreased acoustic startle reactivity, thinner eye lenses, increased alkaline phosphatase and potassium levels and increased white blood cell counts. Gait analyses revealed multiple aberrations indicative of locomotor instability. Similarly, the clinical chemistry, haematology and gait parameters of heterozygous mice also deviated from the values expected for healthy animals, albeit to a lesser extent. Proteomic analysis of brain tissue depicted consistent upregulation of BPTF and downregulation of KLHL11 in heterozygous and homozygous knock-in mice. WASHC5-related protein interaction partners and complexes showed no change in abundancies. Heterozygous Washc5 knock-out mice showing normal WASHC5 levels could not be bred to homozygosity. Conclusions: While biallelic ablation of Washc5 was prenatally lethal, expression of N471D mutated WASHC5 led to several mild clinical and laboratory parameter abnormalities, but not to a typical SPG8 phenotype. The consistent upregulation of BPTF and downregulation of KLHL11 suggest mechanistic links between the expression of N471D mutated WASHC5 and the roles of both proteins in neurodegeneration and protein quality control, respectively.
Long non-coding RNA aerrie controls DNA damage repair via YBX1 to maintain endothelial cell function
(2021)
Aging is accompanied by many physiological changes. These changes can progressively lead to many types of cardiovascular diseases. During this process blood vessels lose their ability to maintain vascular homeostasis, ultimately resulting in hypertension, stroke, or myocardial infarction. Increase in DNA damage is one of the hallmarks of aging and can be repaired by the DNA signaling and repair system. In our study we show that long non-coding RNA Aerrie (linc01013) contributes to the DNA signaling and repair mechanism. Silencing of Aerrie in endothelial cells impairs angiogenesis, migration, and barrier function. Aerrie associates with YBX1 and together they act as important factors in DNA damage signaling and repair. This study identifies Aerrie as a novel factor in genomic stability and as a binding partner of YBX1 in responding to DNA damage.
Nucleoredoxin is a thioredoxin-like redoxin that has been recognized as redox modulator of WNT signaling. Using a Yeast-2-Hybrid screen, we identified calcium calmodulin kinase 2a, Camk2a, as a prominent prey in a brain library. Camk2a is crucial for nitric oxide dependent processes of neuronal plasticity of learning and memory. Therefore, the present study assessed functions of NXN in neuronal Nestin-NXN-/- deficient mice. The NXN-Camk2a interaction was confirmed by coimmunoprecipitation, and by colocalization in neuropil and dendritic spines. Functionally, Camk2a activity was reduced in NXN deficient neurons and restored with recombinant NXN. Proteomics revealed reduced oxidation in the hippocampus of Nestin-NXN-/- deficient mice, including Camk2a, further synaptic and mitochondrial proteins, and was associated with a reduction of mitochondrial respiration. Nestin-NXN-/- mice were healthy and behaved normally in behavioral tests of anxiety, activity and sociability. They had no cognitive deficits in touchscreen based learning & memory tasks, but omitted more trials showing a lower interest in the reward. They also engaged less in rewarding voluntary wheel running, and in exploratory behavior in IntelliCages. Accuracy was enhanced owing to the loss of exploration. The data suggested that NXN maintained the oxidative state of Camk2a and thereby its activity. In addition, it supported oxidation of other synaptic and mitochondrial proteins, and mitochondrial respiration. The loss of NXN-dependent pro-oxidative functions manifested in a loss of exploratory drive and reduced interest in reward in behaving mice.
Epoxides and diols of polyunsaturated fatty acids (PUFAs) are bioactive and can influence processes such as tumor cell proliferation and angiogenesis. Studies with inhibitors of the soluble epoxide hydrolase (sEH) in animals overexpressing cytochrome P450 enzymes or following the systemic administration of specific epoxides revealed a markedly increased incidence of tumor metastases. To determine whether PUFA epoxides increased metastases in a model of spontaneous breast cancer, sEH-/- mice were crossed onto the polyoma middle T oncogene (PyMT) background. We found that the deletion of the sEH accelerated the growth of primary tumors and increased both the tumor macrophage count and angiogenesis. There were small differences in the epoxide/diol content of tumors, particularly in epoxyoctadecamonoenic acid versus dihydroxyoctadecenoic acid, and marked changes in the expression of proteins linked with cell proliferation and metabolism. However, there was no consequence of sEH inhibition on the formation of metastases in the lymph node or lung. Taken together, our results confirm previous reports of increased tumor growth in animals lacking sEH but fail to substantiate reports of enhanced lymph node or pulmonary metastases.
Besides transcription, RNA decay accounts for a large proportion of regulated gene expression and is paramount for cellular functions. Classical RNA surveillance pathways, like nonsense-mediated decay (NMD), are also implicated in the turnover of non-mutant transcripts. Whereas numerous protein factors have been assigned to distinct RNA decay pathways, the contribution of long non-coding RNAs (lncRNAs) to RNA turnover remains unknown. Here we identify the lncRNA CALA as a potent regulator of RNA turnover in endothelial cells. We demonstrate that CALA forms cytoplasmic ribonucleoprotein complexes with G3BP1 and regulates endothelial cell functions. A detailed characterization of these G3BP1-positive complexes by mass spectrometry identifies UPF1 and numerous other NMD factors having cytoplasmic G3BP1-association that is CALA-dependent. Importantly, CALA silencing impairs degradation of NMD target transcripts, establishing CALA as a non-coding regulator of RNA steady-state levels in the endothelium.
Pathologies associated with tissue ischemia/reperfusion (I/R) in highly metabolizing organs such as the brain and heart are leading causes of death and disability in humans. Molecular mechanisms underlying mitochondrial dysfunction during acute injury in I/R are tissue-specific, but their details are not completely understood. A metabolic shift and accumulation of substrates of reverse electron transfer (RET) such as succinate are observed in tissue ischemia, making mitochondrial complex I of the respiratory chain (NADH:ubiquinone oxidoreductase) the most vulnerable enzyme to the following reperfusion. It has been shown that brain complex I is predisposed to losing its flavin mononucleotide (FMN) cofactor when maintained in the reduced state in conditions of RET both in vitro and in vivo. Here we investigated the process of redox-dependent dissociation of FMN from mitochondrial complex I in brain and heart mitochondria. In contrast to the brain enzyme, cardiac complex I does not lose FMN when reduced in RET conditions. We proposed that the different kinetics of FMN loss during RET is due to the presence of brain-specific long 50 kDa isoform of the NDUFV3 subunit of complex I, which is absent in the heart where only the canonical 10 kDa short isoform is found. Our simulation studies suggest that the long NDUFV3 isoform can reach toward the FMN binding pocket and affect the nucleotide affinity to the apoenzyme. For the first time, we demonstrated a potential functional role of tissue-specific isoforms of complex I, providing the distinct molecular mechanism of I/R-induced mitochondrial impairment in cardiac and cerebral tissues. By combining functional studies of intact complex I and molecular structure simulations, we defined the critical difference between the brain and heart enzyme and suggested insights into the redox-dependent inactivation mechanisms of complex I during I/R injury in both tissues.
Vascular integrity is essential for organ homeostasis to prevent edema formation and infiltration of inflammatory cells. Long non-coding RNAs (lncRNAs) are important regulators of gene expression and often expressed in a cell type-specific manner. By screening for endothelial-enriched lncRNAs, we identified the undescribed lncRNA NTRAS to control endothelial cell functions. Silencing of NTRAS induces endothelial cell dysfunction in vitro and increases vascular permeability and lethality in mice. Biochemical analysis revealed that NTRAS, through its CA-dinucleotide repeat motif, sequesters the splicing regulator hnRNPL to control alternative splicing of tight junction protein 1 (TJP1; also named zona occludens 1, ZO-1) pre-mRNA. Deletion of the hnRNPL binding motif in mice (Ntras∆CA/∆CA) significantly repressed TJP1 exon 20 usage, favoring expression of the TJP1α- isoform, which augments permeability of the endothelial monolayer. Ntras∆CA/∆CA mice further showed reduced retinal vessel growth and increased vascular permeability and myocarditis. In summary, this study demonstrates that NTRAS is an essential gatekeeper of vascular integrity.
The MICOS complex subunit MIC13 is essential for mitochondrial cristae organization. Mutations in MIC13 cause severe mitochondrial hepato-encephalopathy displaying defective cristae morphology and loss of the MIC10-subcomplex. Here we identified SLP2 as a novel interacting partner of MIC13 and decipher a critical role of SLP2 for MICOS assembly at distinct steps. SLP2 provides a large interaction hub for MICOS subunits and loss of SLP2 imparted YME1L-mediated proteolysis of MIC26 and drastic alterations in cristae morphology. We further identified a MIC13-specific role in stabilizing the MIC10-subcomplex via a MIC13-YME1L axis. SLP2 together with the stabilized MIC10-subcomplex promotes efficient assembly of the MIC60-subcomplex forming the MICOS-MIB complex. Consistently, super-resolution nanoscopy showed a dispersed distribution of the MIC60 in cells lacking SLP2 and MIC13. Our study reveals converging and interdependent assembly pathways for the MIC10- and MIC60-subcomplexes which are controlled in two ways, the MIC13-YME1L and the SLP2-YME1L axes, revealing mechanistic insights of these factors in cristae morphogenesis. These results will be helpful in understanding the human pathophysiology linked to mutations in MIC13 or its interaction partners.
The interaction of Eph receptor tyrosine kinases with their transmembrane ligands; the ephrins, is important for the regulation of cell-cell communication. Ephrin-Eph signaling is probably best known for the discrimination of arterial and venous territories by repulsion of venous endothelial cells away from those with an arterial fate. Ultimately, cell repulsion is mediated by initiating the collapse of the actin cytoskeleton in membrane protrusions. Here, we investigated the role of the Ena/VASP family of actin binding proteins in endothelial cell repulsion initiated by ephrin ligands. Human endothelial cells dynamically extended sheet-like lamellipodia over ephrin-B2 coated surfaces. While lamellipodia of control siRNA transfected cells rapidly collapsed, resulting in a pronounced cell repulsion from the ephrin-B2 surfaces, the knockdown of Ena/VASP proteins impaired the cytoskeletal collapse of membrane protrusions and the cells no longer avoided the repulsive surfaces. Mechanistically, ephrin-B2 stimulation elicited the EphB-mediated tyrosine phosphorylation of VASP, which abrogated its interaction with the focal adhesion protein Zyxin. Nck2 was identified as a novel VASP binding protein, which only interacted with the tyrosine phosphorylated VASP protein. Nck links Eph-receptors to the actin cytoskeleton. Therefore, we hypothesize that Nck-Ena/VASP complex formation is required for actin reorganization and/or Eph receptor internalization downstream of ephrin-Eph interaction in endothelial cells, with implications for endothelial navigation and pathfinding.
The MICOS complex subunit MIC13 is essential for mitochondrial cristae organization. Mutations in MIC13 cause severe mitochondrial hepato-encephalopathy displaying defective cristae morphology and loss of the MIC10-subcomplex. Here we identified stomatin-like protein 2 (SLP2) as an interacting partner of MIC13 and decipher a critical role of SLP2 as an auxiliary MICOS subunit, modulating cristae morphology. SLP2 provides a large interaction hub for MICOS subunits and loss of SLP2 leads to drastic alterations in cristae morphology. Double deletion of SLP2 and MIC13 showed reduced assembly of core MICOS subunit, MIC60 into MICOS and dispersion of MIC60-specific puncta, demonstrating a critical role of SLP2-MIC13 in MICOS assembly and crista junction (CJ) formation. We further identified that the mitochondrial i-AAA protease YME1L in coordination either with MIC13 or SLP2 differentially regulates MICOS assembly pathways thereby interlinking MIC13-specific or scaffolding-specific role of SLP2 with quality control and assembly of the MICOS complex. YME1L- depletion in MIC13 KO could restore MIC10-subcomplex and reform the nascent CJ. Taken together, we propose ‘seeder’ model for MICOS assembly and CJ formation, where SLP2- MIC13 seed the assembly of MIC60 into MICOS complex and promote the formation of CJ by regulating the quality and stability of MIC10-subcomplex.
Long non-coding RNAs (lncRNAs) orchestrate various biological processes and regulate the development of cardiovascular diseases. Their potential therapeutic benefit to tackle disease progression has recently been extensively explored. Our study investigates the role of lncRNA Nudix Hydrolase 6 (NUDT6) and its antisense target fibroblast growth factor 2 (FGF2) in two vascular pathologies: abdominal aortic aneurysms (AAA) and carotid artery disease. Using tissue samples from both diseases, we detected a substantial increase of NUDT6, whereas FGF2 was downregulated. Targeting Nudt6 in vivo with antisense oligonucleotides in three murine and one porcine animal model of carotid artery disease and AAA limited disease progression. Restoration of FGF2 upon Nudt6 knockdown improved vessel wall morphology and fibrous cap stability. Overexpression of NUDT6 in vitro impaired smooth muscle cell (SMC) migration, while limiting their proliferation and augmenting apoptosis. By employing RNA pulldown followed by mass spectrometry as well as RNA immunoprecipitation, we identified Cysteine and Glycine Rich Protein 1 (CSRP1) as another direct NUDT6 interaction partner, regulating cell motility and SMC differentiation. Overall, the present study identifies NUDT6 as a well-conserved antisense transcript of FGF2. NUDT6 silencing triggers SMC survival and migration and could serve as a novel RNA-based therapeutic strategy in vascular diseases.
Tight control over transcription factor activity is necessary for a sensible balance between cellular proliferation and differentiation in the embryo and during tissue homeostasis by adult stem cells, but mechanistic details have remained incomplete. The homeodomain transcription factor MEIS2 is an important regulator of neurogenesis in the ventricular–subventricular zone (V-SVZ) adult stem cell niche in mice. We here identify MEIS2 as direct target of the intracellular protease calpain-2 (composed of the catalytic subunit CAPN2 and the regulatory subunit CAPNS1). Phosphorylation at conserved serine and/or threonine residues, or dimerization with PBX1, reduced the sensitivity of MEIS2 towards cleavage by calpain-2. In the adult V-SVZ, calpain-2 activity is high in stem and progenitor cells, but rapidly declines during neuronal differentiation, which is accompanied by increased stability of MEIS2 full-length protein. In accordance with this, blocking calpain-2 activity in stem and progenitor cells, or overexpression of a cleavage-insensitive form of MEIS2, increased the production of neurons, whereas overexpression of a catalytically active CAPN2 reduced it. Collectively, our results support a key role for calpain-2 in controlling the output of adult V-SVZ neural stem and progenitor cells through cleavage of the neuronal fate determinant MEIS2.