RÉSUMÉ
Once thought of in terms of bioenergetics, mitochondria are now widely accepted as both the orchestrator of cellular health and the gatekeeper of cell death. The pulmonary disease field has performed extensive efforts to explore the role of mitochondria in regulating inflammation, cellular metabolism, apoptosis, and oxidative stress. However, a critical component of these processes needs to be more studied: mitochondrial network dynamics. Mitochondria morphologically change in response to their environment to regulate these processes through fusion, fission, and mitophagy. This allows mitochondria to adapt their function to respond to cellular requirements, a critical component in maintaining cellular homeostasis. For that reason, mitochondrial network dynamics can be considered a bridge that brings multiple cellular processes together, revealing a potential pathway for therapeutic intervention. In this review, we discuss the critical modulators of mitochondrial dynamics and how they are affected in pulmonary diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), acute lung injury (ALI), and pulmonary arterial hypertension (PAH). A dysregulated mitochondrial network plays a crucial role in lung disease pathobiology, and aberrant fission/fusion/mitophagy pathways are druggable processes that warrant further exploration. Thus, we also discuss the candidates for lung disease therapeutics that regulate mitochondrial network dynamics.
Sujet(s)
Dynamique mitochondriale , Broncho-pneumopathie chronique obstructive , Humains , Stress oxydatif , Inflammation , Métabolisme énergétiqueRÉSUMÉ
Mechanical ventilation (MV) is a life-supporting strategy employed in the Intensive Care Unit (ICU). However, MV-associated mechanical stress exacerbates existing lung inflammation in ICU patients, resulting in limited improvement in mortality and a condition known as Ventilator-Induced Lung Injury (VILI). Sphingosine-1-phosphate (S1P) is a circulating bioactive lipid that maintains endothelial integrity primarily through S1P receptor 1 (S1PR1). During VILI, mechanical stress upregulates endothelial S1PR3 levels. Unlike S1PR1, S1PR3 mediates endothelial barrier disruption through Rho-dependent pathways. However, the specific impact of elevated S1PR3 on lung endothelial function, apart from Rho activation, remains poorly understood. In this study, we investigated the effects of S1PR3 in endothelial pathobiology during VILI using an S1PR3 overexpression adenovirus. S1PR3 overexpression caused cytoskeleton rearrangement, formation of paracellular gaps, and a modified endothelial response towards S1P. It resulted in a shift from S1PR1-dependent barrier enhancement to S1PR3-dependent barrier disruption. Moreover, S1PR3 overexpression induced an ADAM10-dependent cleavage of Vascular Endothelial (VE)-cadherin, which hindered endothelial barrier recovery. S1PR3-induced cleavage of VE-cadherin was at least partially regulated by S1PR3-mediated NFκB activation. Additionally, we employed an S1PR3 inhibitor TY-52156 in a murine model of VILI. TY-52156 effectively attenuated VILI-induced increases in bronchoalveolar lavage cell counts and protein concentration, suppressed the release of pro-inflammatory cytokines, and inhibited lung inflammation as assessed via a histological evaluation. These findings confirm that mechanical stress associated with VILI increases S1PR3 levels, thereby altering the pulmonary endothelial response towards S1P and impairing barrier recovery. Inhibiting S1PR3 is validated as an effective therapeutic strategy for VILI.
Sujet(s)
Pneumopathie infectieuse , Lésion pulmonaire induite par la ventilation mécanique , Humains , Souris , Animaux , Récepteurs de la sphingosine-1-phosphate , Cadhérines , Sphingosine/pharmacologie , Lésion pulmonaire induite par la ventilation mécanique/métabolisme , Lysophospholipides/pharmacologie , Récepteurs aux lysosphingolipides/métabolisme , Protéine ADAM10 , Protéines membranaires , Amyloid precursor protein secretasesRÉSUMÉ
Pulmonary arterial hypertension (PAH) is a progressive disease in which pulmonary arterial (PA) endothelial cell (EC) dysfunction is associated with unrepaired DNA damage. BMPR2 is the most common genetic cause of PAH. We report that human PAEC with reduced BMPR2 have persistent DNA damage in room air after hypoxia (reoxygenation), as do mice with EC-specific deletion of Bmpr2 (EC-Bmpr2-/-) and persistent pulmonary hypertension. Similar findings are observed in PAEC with loss of the DNA damage sensor ATM, and in mice with Atm deleted in EC (EC-Atm-/-). Gene expression analysis of EC-Atm-/- and EC-Bmpr2-/- lung EC reveals reduced Foxf1, a transcription factor with selectivity for lung EC. Reducing FOXF1 in control PAEC induces DNA damage and impaired angiogenesis whereas transfection of FOXF1 in PAH PAEC repairs DNA damage and restores angiogenesis. Lung EC targeted delivery of Foxf1 to reoxygenated EC-Bmpr2-/- mice repairs DNA damage, induces angiogenesis and reverses pulmonary hypertension.
Sujet(s)
Hypertension pulmonaire , Hypertension artérielle pulmonaire , Souris , Humains , Animaux , Hypertension artérielle pulmonaire/génétique , Hypertension pulmonaire/génétique , Hypertension pulmonaire/métabolisme , Hypertension artérielle pulmonaire primitive familiale/métabolisme , Artère pulmonaire/métabolisme , Altération de l'ADN , Récepteurs de la protéine morphogénique osseuse de type II/génétique , Facteurs de transcription Forkhead/génétique , Facteurs de transcription Forkhead/métabolismeRÉSUMÉ
Mitochondria are highly dynamic organelles essential for cell metabolism, growth, and function. It is becoming increasingly clear that endothelial cell dysfunction significantly contributes to the pathogenesis and vascular remodeling of various lung diseases, including pulmonary arterial hypertension (PAH), and that mitochondria are at the center of this dysfunction. The more we uncover the role mitochondria play in pulmonary vascular disease, the more apparent it becomes that multiple pathways are involved. To achieve effective treatments, we must understand how these pathways are dysregulated to be able to intervene therapeutically. We know that nitric oxide signaling, glucose metabolism, fatty acid oxidation, and the TCA cycle are abnormal in PAH, along with alterations in the mitochondrial membrane potential, proliferation, and apoptosis. However, these pathways are incompletely characterized in PAH, especially in endothelial cells, highlighting the urgent need for further research. This review summarizes what is currently known about how mitochondrial metabolism facilitates a metabolic shift in endothelial cells that induces vascular remodeling during PAH.
Sujet(s)
Hypertension pulmonaire , Maladies vasculaires , Humains , Hypertension pulmonaire/métabolisme , Remodelage vasculaire , Cellules endothéliales/métabolisme , Poumon/métabolisme , Stress oxydatif , Maladies vasculaires/anatomopathologie , Artère pulmonaire/métabolisme , Artère pulmonaire/anatomopathologie , Prolifération cellulaireRÉSUMÉ
OBJECTIVE: Mutations in BMPR2 (bone morphogenetic protein receptor 2) are associated with familial and sporadic pulmonary arterial hypertension (PAH). The functional and molecular link between loss of BMPR2 in pulmonary artery smooth muscle cells (PASMC) and PAH pathogenesis warrants further investigation, as most investigations focus on BMPR2 in pulmonary artery endothelial cells. Our goal was to determine whether and how decreased BMPR2 is related to the abnormal phenotype of PASMC in PAH. METHODS: SMC-specific Bmpr2-/- mice (BKOSMC) were created and compared to controls in room air, after 3 weeks of hypoxia as a second hit, and following 4 weeks of normoxic recovery. Echocardiography, right ventricular systolic pressure, and right ventricular hypertrophy were assessed as indices of pulmonary hypertension. Proliferation, contractility, gene and protein expression of PASMC from BKOSMC mice, human PASMC with BMPR2 reduced by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation were compared to controls, to investigate the phenotype and underlying mechanism. RESULTS: BKOSMC mice showed reduced hypoxia-induced vasoconstriction and persistent pulmonary hypertension following recovery from hypoxia, associated with sustained muscularization of distal pulmonary arteries. PASMC from mutant compared to control mice displayed reduced contractility at baseline and in response to angiotensin II, increased proliferation and apoptosis resistance. Human PASMC with reduced BMPR2 by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation showed a similar phenotype related to upregulation of pERK1/2 (phosphorylated extracellular signal related kinase 1/2)-pP38-pSMAD2/3 mediating elevation in ARRB2 (ß-arrestin2), pAKT (phosphorylated protein kinase B) inactivation of GSK3-beta, CTNNB1 (ß-catenin) nuclear translocation and reduction in RHOA (Ras homolog family member A) and RAC1 (Ras-related C3 botulinum toxin substrate 1). Decreasing ARRB2 in PASMC with reduced BMPR2 restored normal signaling, reversed impaired contractility and attenuated heightened proliferation and in mice with inducible loss of BMPR2 in SMC, decreasing ARRB2 prevented persistent pulmonary hypertension. CONCLUSIONS: Agents that neutralize the elevated ARRB2 resulting from loss of BMPR2 in PASMC could prevent or reverse the aberrant hypocontractile and hyperproliferative phenotype of these cells in PAH.
Sujet(s)
Hypertension pulmonaire , Hypertension artérielle pulmonaire , Animaux , Humains , Souris , bêta-Arrestine 2/métabolisme , Récepteurs de la protéine morphogénique osseuse de type II/génétique , Récepteurs de la protéine morphogénique osseuse de type II/métabolisme , Prolifération cellulaire , Cellules cultivées , Cellules endothéliales/métabolisme , Glycogen Synthase Kinase 3/métabolisme , Hypertension pulmonaire/métabolisme , Hypoxie/complications , Hypoxie/génétique , Hypoxie/métabolisme , Myocytes du muscle lisse/métabolisme , Hypertension artérielle pulmonaire/génétique , Artère pulmonaire/métabolisme , ARN/métabolismeRÉSUMÉ
Rationale: The role of neutrophils and their extracellular vesicles (EVs) in the pathogenesis of pulmonary arterial hypertension is unclear. Objectives: To relate functional abnormalities in pulmonary arterial hypertension neutrophils and their EVs to mechanisms uncovered by proteomic and transcriptomic profiling. Methods: Production of elastase, release of extracellular traps, adhesion, and migration were assessed in neutrophils from patients with pulmonary arterial hypertension and control subjects. Proteomic analyses were applied to explain functional perturbations, and transcriptomic data were used to find underlying mechanisms. CD66b-specific neutrophil EVs were isolated from plasma of patients with pulmonary arterial hypertension, and we determined whether they produce pulmonary hypertension in mice. Measurements and Main Results: Neutrophils from patients with pulmonary arterial hypertension produce and release increased neutrophil elastase, associated with enhanced extracellular traps. They exhibit reduced migration and increased adhesion attributed to elevated ß1-integrin and vinculin identified by proteomic analysis and previously linked to an antiviral response. This was substantiated by a transcriptomic IFN signature that we related to an increase in human endogenous retrovirus K envelope protein. Transfection of human endogenous retrovirus K envelope in a neutrophil cell line (HL-60) increases neutrophil elastase and IFN genes, whereas vinculin is increased by human endogenous retrovirus K deoxyuridine triphosphate diphosphatase that is elevated in patient plasma. Neutrophil EVs from patient plasma contain increased neutrophil elastase and human endogenous retrovirus K envelope and induce pulmonary hypertension in mice, mitigated by elafin, an elastase inhibitor. Conclusions: Elevated human endogenous retroviral elements and elastase link a neutrophil innate immune response to pulmonary arterial hypertension.
Sujet(s)
Rétrovirus endogènes , Hypertension pulmonaire , Hypertension artérielle pulmonaire , Animaux , Antiviraux , Élafine/génétique , Élafine/métabolisme , Élafine/pharmacologie , Rétrovirus endogènes/métabolisme , Hypertension artérielle pulmonaire primitive familiale/génétique , Humains , Hypertension pulmonaire/génétique , Intégrines/génétique , Intégrines/métabolisme , Leukocyte elastase/métabolisme , Souris , Granulocytes neutrophiles/métabolisme , Protéomique , Vinculine/génétique , Vinculine/métabolismeRÉSUMÉ
Since antibiotic development lags, we search for potential drug targets through directed evolution experiments. A challenge is that many resistance genes hide in a noisy mutational background as mutator clones emerge in the adaptive population. Here, to overcome this noise, we quantify the impact of mutations through evolutionary action (EA). After sequencing ciprofloxacin or colistin resistance strains grown under different mutational regimes, we find that an elevated sum of the evolutionary action of mutations in a gene identifies known resistance drivers. This EA integration approach also suggests new antibiotic resistance genes which are then shown to provide a fitness advantage in competition experiments. Moreover, EA integration analysis of clinical and environmental isolates of antibiotic resistant of E. coli identifies gene drivers of resistance where a standard approach fails. Together these results inform the genetic basis of de novo colistin resistance and support the robust discovery of phenotype-driving genes via the evolutionary action of genetic perturbations in fitness landscapes.
Sujet(s)
Antibactériens , Résistance bactérienne aux médicaments , Protéines Escherichia coli , Escherichia coli , Antibactériens/pharmacologie , Ciprofloxacine/pharmacologie , Colistine/pharmacologie , Résistance bactérienne aux médicaments/génétique , Escherichia coli/effets des médicaments et des substances chimiques , Escherichia coli/génétique , Protéines Escherichia coli/génétique , Tests de sensibilité microbienne , MutationRÉSUMÉ
RNA polymerase III (Pol III) includes two alternate isoforms, defined by mutually exclusive incorporation of subunit POLR3G (RPC7α) or POLR3GL (RPC7ß), in mammals. The contributions of POLR3G and POLR3GL to transcription potential has remained poorly defined. Here, we discover that loss of subunit POLR3G is accompanied by a restricted repertoire of genes transcribed by Pol III. Particularly sensitive is snaR-A, a small noncoding RNA implicated in cancer proliferation and metastasis. Analysis of Pol III isoform biases and downstream chromatin features identifies loss of POLR3G and snaR-A during differentiation, and conversely, re-establishment of POLR3G gene expression and SNAR-A gene features in cancer contexts. Our results support a model in which Pol III identity functions as an important transcriptional regulatory mechanism. Upregulation of POLR3G, which is driven by MYC, identifies a subgroup of patients with unfavorable survival outcomes in specific cancers, further implicating the POLR3G-enhanced transcription repertoire as a potential disease factor.
Sujet(s)
Tumeurs , Petit ARN non traduit , Animaux , Chromatine , Humains , Mammifères/génétique , Tumeurs/génétique , Isoformes de protéines/génétique , RNA polymerase III/génétique , RNA polymerase III/métabolismeRÉSUMÉ
We previously reported heightened expression of the human endogenous retroviral protein HERV-K deoxyuridine triphosphate nucleotidohydrolase (dUTPase) in circulating monocytes and pulmonary arterial (PA) adventitial macrophages of patients with PA hypertension (PAH). Furthermore, recombinant HERV-K dUTPase increased IL-6 in PA endothelial cells (PAECs) and caused pulmonary hypertension in rats. Here we show that monocytes overexpressing HERV-K dUTPase, as opposed to GFP, can release HERV-K dUTPase in extracellular vesicles (EVs) that cause pulmonary hypertension in mice in association with endothelial mesenchymal transition (EndMT) related to induction of SNAIL/SLUG and proinflammatory molecules IL-6 as well as VCAM1. In PAECs, HERV-K dUTPase requires TLR4-myeloid differentiation primary response-88 to increase IL-6 and SNAIL/SLUG, and HERV-K dUTPase interaction with melanoma cell adhesion molecule (MCAM) is necessary to upregulate VCAM1. TLR4 engagement induces p-p38 activation of NF-κB in addition to p-pSMAD3 required for SNAIL and pSTAT1 for IL-6. HERV-K dUTPase interaction with MCAM also induces p-p38 activation of NF-κB in addition to pERK1/2-activating transcription factor-2 (ATF2) to increase VCAM1. Thus in PAH, monocytes or macrophages can release HERV-K dUTPase in EVs, and HERV-K dUTPase can engage dual receptors and signaling pathways to subvert PAEC transcriptional machinery to induce EndMT and associated proinflammatory molecules.
Sujet(s)
Rétrovirus endogènes , Transition épithélio-mésenchymateuse/immunologie , Hypertension pulmonaire , Macrophages/immunologie , Monocytes/immunologie , Artère pulmonaire , Pyrophosphatases/métabolisme , Animaux , Antigènes CD146/métabolisme , Rétrovirus endogènes/métabolisme , Rétrovirus endogènes/pathogénicité , Cellules endothéliales/métabolisme , Hypertension pulmonaire/immunologie , Hypertension pulmonaire/métabolisme , Hypertension pulmonaire/virologie , Inflammation/métabolisme , Inflammation/virologie , Souris , Artère pulmonaire/immunologie , Artère pulmonaire/anatomopathologie , Transduction du signal , Facteurs de transcription de la famille Snail/métabolismeRÉSUMÉ
MOTIVATION: Since the first recognized case of COVID-19, more than 100 million people have been infected worldwide. Global efforts in drug and vaccine development to fight the disease have yielded vaccines and drug candidates to cure COVID-19. However, the spread of SARS-CoV-2 variants threatens the continued efficacy of these treatments. In order to address this, we interrogate the evolutionary history of the entire SARS-CoV-2 proteome to identify evolutionarily conserved functional sites that can inform the search for treatments with broader coverage across the coronavirus family. RESULTS: Combining coronavirus family sequence information with the mutations observed in the current COVID-19 outbreak, we systematically and comprehensively define evolutionarily stable sites that may provide useful drug and vaccine targets and which are less likely to be compromised by the emergence of new virus strains. Several experimentally validated effective drugs interact with these proposed target sites. In addition, the same evolutionary information can prioritize cross reactive antigens that are useful in directing multi-epitope vaccine strategies to illicit broadly neutralizing immune responses to the betacoronavirus family. Although the results are focused on SARS-CoV-2, these approaches stem from evolutionary principles that are agnostic to the organism or infective agent. AVAILABILITY AND IMPLEMENTATION: The results of this work are made interactively available at http://cov.lichtargelab.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Sujet(s)
COVID-19 , Vaccins antiviraux , Humains , SARS-CoV-2/génétique , Protéome , Vaccins contre la COVID-19 , Vaccins antiviraux/génétiqueRÉSUMÉ
BACKGROUND: Metabolic alterations provide substrates that influence chromatin structure to regulate gene expression that determines cell function in health and disease. Heightened proliferation of smooth muscle cells (SMC) leading to the formation of a neointima is a feature of pulmonary arterial hypertension (PAH) and systemic vascular disease. Increased glycolysis is linked to the proliferative phenotype of these SMC. METHODS: RNA sequencing was applied to pulmonary arterial SMC (PASMC) from PAH patients with and without a BMPR2 (bone morphogenetic receptor 2) mutation versus control PASMC to uncover genes required for their heightened proliferation and glycolytic metabolism. Assessment of differentially expressed genes established metabolism as a major pathway, and the most highly upregulated metabolic gene in PAH PASMC was aldehyde dehydrogenase family 1 member 3 (ALDH1A3), an enzyme previously linked to glycolysis and proliferation in cancer cells and systemic vascular SMC. We determined if these functions are ALDH1A3-dependent in PAH PASMC, and if ALDH1A3 is required for the development of pulmonary hypertension in a transgenic mouse. Nuclear localization of ALDH1A3 in PAH PASMC led us to determine whether and how this enzyme coordinately regulates gene expression and metabolism in PAH PASMC. RESULTS: ALDH1A3 mRNA and protein were increased in PAH versus control PASMC, and ALDH1A3 was required for their highly proliferative and glycolytic properties. Mice with Aldh1a3 deleted in SMC did not develop hypoxia-induced pulmonary arterial muscularization or pulmonary hypertension. Nuclear ALDH1A3 converted acetaldehyde to acetate to produce acetyl coenzyme A to acetylate H3K27, marking active enhancers. This allowed for chromatin modification at NFYA (nuclear transcription factor Y subunit α) binding sites via the acetyltransferase KAT2B (lysine acetyltransferase 2B) and permitted NFY-mediated transcription of cell cycle and metabolic genes that is required for ALDH1A3-dependent proliferation and glycolysis. Loss of BMPR2 in PAH SMC with or without a mutation upregulated ALDH1A3, and transcription of NFYA and ALDH1A3 in PAH PASMC was ß-catenin dependent. CONCLUSIONS: Our studies have uncovered a metabolic-transcriptional axis explaining how dividing cells use ALDH1A3 to coordinate their energy needs with the epigenetic and transcriptional regulation of genes required for SMC proliferation. They suggest that selectively disrupting the pivotal role of ALDH1A3 in PAH SMC, but not endothelial cells, is an important therapeutic consideration.
Sujet(s)
Aldehyde oxidoreductases/génétique , Régulation de l'expression des gènes , Hypertension artérielle pulmonaire/génétique , Aldehyde oxidoreductases/métabolisme , Animaux , Modèles animaux de maladie humaine , Humains , Mâle , Souris , Souris de lignée C57BL , Muscles lisses/métabolisme , Muscles lisses/anatomopathologie , Hypertension artérielle pulmonaire/métabolisme , Hypertension artérielle pulmonaire/anatomopathologie , ARN messager/génétique , ARN messager/métabolisme , Transduction du signalRÉSUMÉ
RATIONALE: In pulmonary arterial hypertension (PAH), endothelial dysfunction and obliterative vascular disease are associated with DNA damage and impaired signaling of BMPR2 (bone morphogenetic protein type 2 receptor) via two downstream transcription factors, PPARγ (peroxisome proliferator-activated receptor gamma), and p53. OBJECTIVE: We investigated the vasculoprotective and regenerative potential of a newly identified PPARγ-p53 transcription factor complex in the pulmonary endothelium. METHODS AND RESULTS: In this study, we identified a pharmacologically inducible vasculoprotective mechanism in pulmonary arterial and lung MV (microvascular) endothelial cells in response to DNA damage and oxidant stress regulated in part by a BMPR2 dependent transcription factor complex between PPARγ and p53. Chromatin immunoprecipitation sequencing and RNA-sequencing established an inducible PPARγ-p53 mediated regenerative program regulating 19 genes involved in lung endothelial cell survival, angiogenesis and DNA repair including, EPHA2 (ephrin type-A receptor 2), FHL2 (four and a half LIM domains protein 2), JAG1 (jagged 1), SULF2 (extracellular sulfatase Sulf-2), and TIGAR (TP53-inducible glycolysis and apoptosis regulator). Expression of these genes was partially impaired when the PPARγ-p53 complex was pharmacologically disrupted or when BMPR2 was reduced in pulmonary artery endothelial cells (PAECs) subjected to oxidative stress. In endothelial cell-specific Bmpr2-knockout mice unable to stabilize p53 in endothelial cells under oxidative stress, Nutlin-3 rescued endothelial p53 and PPARγ-p53 complex formation and induced target genes, such as APLN (apelin) and JAG1, to regenerate pulmonary microvessels and reverse pulmonary hypertension. In PAECs from BMPR2 mutant PAH patients, pharmacological induction of p53 and PPARγ-p53 genes repaired damaged DNA utilizing genes from the nucleotide excision repair pathway without provoking PAEC apoptosis. CONCLUSIONS: We identified a novel therapeutic strategy that activates a vasculoprotective gene regulation program in PAECs downstream of dysfunctional BMPR2 to rehabilitate PAH PAECs, regenerate pulmonary microvessels, and reverse disease. Our studies pave the way for p53-based vasculoregenerative therapies for PAH by extending the therapeutic focus to PAEC dysfunction and to DNA damage associated with PAH progression.
Sujet(s)
Agents angiogéniques/pharmacologie , Cellules endothéliales/effets des médicaments et des substances chimiques , Imidazoles/pharmacologie , Néovascularisation physiologique/effets des médicaments et des substances chimiques , Récepteur PPAR gamma/métabolisme , Pipérazines/pharmacologie , Hypertension artérielle pulmonaire/traitement médicamenteux , Artère pulmonaire/effets des médicaments et des substances chimiques , Régénération/effets des médicaments et des substances chimiques , Protéine p53 suppresseur de tumeur/métabolisme , Animaux , Récepteurs de la protéine morphogénique osseuse de type II/génétique , Récepteurs de la protéine morphogénique osseuse de type II/métabolisme , Cellules cultivées , Cellules endothéliales/métabolisme , Cellules endothéliales/anatomopathologie , Femelle , Régulation de l'expression des gènes , Humains , Mâle , Souris , Souris knockout , Stress oxydatif , Récepteur PPAR gamma/génétique , Hypertension artérielle pulmonaire/génétique , Hypertension artérielle pulmonaire/métabolisme , Hypertension artérielle pulmonaire/physiopathologie , Artère pulmonaire/métabolisme , Artère pulmonaire/anatomopathologie , Artère pulmonaire/physiopathologie , Transduction du signal , Protéine p53 suppresseur de tumeur/génétiqueRÉSUMÉ
Since the first recognized case of COVID-19, more than 100 million people have been infected worldwide. Global efforts in drug and vaccine development to fight the disease have yielded vaccines and drug candidates to cure COVID-19. However, the spread of SARS-CoV-2 variants threatens the continued efficacy of these treatments. In order to address this, we interrogate the evolutionary history of the entire SARS-CoV-2 proteome to identify evolutionarily conserved functional sites that can inform the search for treatments with broader coverage across the coronavirus family. Combining this information with the mutations observed in the current COVID-19 outbreak, we systematically and comprehensively define evolutionarily stable sites that may provide useful drug and vaccine targets and which are less likely to be compromised by the emergence of new virus strains. Several experimentally-validated effective drugs interact with these proposed target sites. In addition, the same evolutionary information can prioritize cross reactive antigens that are useful in directing multi-epitope vaccine strategies to illicit broadly neutralizing immune responses to the betacoronavirus family. Although the results are focused on SARS-CoV-2, these approaches stem from evolutionary principles that are agnostic to the organism or infective agent.
RÉSUMÉ
The human metabolome is the cumulative product of ingested metabolites and those produced by the body and its microbiota. Together these metabolites can dynamically report on the health and disease state of an individual, as well as their response to drug treatments and other external perturbations. Profiling metabolites in human body fluids provides an opportunity to identify biomarkers and stratify patients for personalized treatments but requires the development of high-throughput approaches compatible with large cohort and longitudinal studies. Here we review in detail sample preparation and analytical liquid chromatography-mass spectrometry (LC-MS) methods to measure the broad chemical diversity of metabolites found in human plasma and urine.
Sujet(s)
Chromatographie en phase liquide , Métabolomique/méthodes , Médecine de précision/méthodes , Spectrométrie de masse en tandem , Marqueurs biologiques/sang , Liquides biologiques/métabolisme , Fèces/composition chimique , Humains , Marquage isotopique , Métabolome/génétiqueRÉSUMÉ
Using proteomic approaches, we uncovered a DNA damage response (DDR) function for peroxisome proliferator activated receptor γ (PPARγ) through its interaction with the DNA damage sensor MRE11-RAD50-NBS1 (MRN) and the E3 ubiquitin ligase UBR5. We show that PPARγ promotes ATM signaling and is essential for UBR5 activity targeting ATM interactor (ATMIN). PPARγ depletion increases ATMIN protein independent of transcription and suppresses DDR-induced ATM signaling. Blocking ATMIN in this context restores ATM activation and DNA repair. We illustrate the physiological relevance of PPARγ DDR functions by using pulmonary arterial hypertension (PAH) as a model that has impaired PPARγ signaling related to endothelial cell (EC) dysfunction and unresolved DNA damage. In pulmonary arterial ECs (PAECs) from PAH patients, we observed disrupted PPARγ-UBR5 interaction, heightened ATMIN expression, and DNA lesions. Blocking ATMIN in PAH PAEC restores ATM activation. Thus, impaired PPARγ DDR functions may explain the genomic instability and loss of endothelial homeostasis in PAH.
Sujet(s)
Réparation de l'ADN , Cellules endothéliales/métabolisme , Homéostasie , Récepteur PPAR gamma/métabolisme , Facteurs de transcription/métabolisme , Ubiquitin-protein ligases/métabolisme , Protéines mutées dans l'ataxie-télangiectasie/métabolisme , Altération de l'ADN , Instabilité du génome , Cellules HEK293 , Humains , Modèles biologiques , Liaison aux protéines , Artère pulmonaire/anatomopathologie , Transduction du signal , UbiquitinationRÉSUMÉ
Nuclear receptor-related 1 protein (Nurr1/NR4A2) is an orphan nuclear receptor (NR) that is considered to function without a canonical ligand-binding pocket (LBP). A crystal structure of the Nurr1 ligand-binding domain (LBD) revealed no physical space in the conserved region where other NRs with solvent accessible apo-protein LBPs bind synthetic and natural ligands. Using solution nuclear magnetic resonance spectroscopy, hydrogen/deuterium exchange mass spectrometry, and molecular dynamics simulations, we show that the putative canonical Nurr1 LBP is dynamic with high solvent accessibility, exchanges between two or more conformations on the microsecond-to-millisecond timescale, and can expand from the collapsed crystallized conformation to allow binding of unsaturated fatty acids. These findings should stimulate future studies to probe the ligandability and druggability of Nurr1 for both endogenous and synthetic ligands, which could lead to new therapeutics for Nurr1-related diseases, including Parkinson's disease and schizophrenia.
Sujet(s)
Simulation de docking moléculaire , Membre-2 du groupe A de la sous-famille-4 de récepteurs nucléaires/composition chimique , Sites de fixation , Acides gras insaturés/composition chimique , Humains , Ligands , Simulation de dynamique moléculaire , Membre-2 du groupe A de la sous-famille-4 de récepteurs nucléaires/métabolisme , Liaison aux protéinesRÉSUMÉ
CRISPR advances genome engineering by directing endonuclease sequence specificity with a guide RNA molecule (gRNA). For precisely targeting a gene for modification, each genetic construct requires a unique gRNA. By generating a gRNA against the flippase recognition target (FRT) site, a common genetic element shared by multiple genetic collections, CRISPR-FRT circumvents this design constraint to provide a broad platform for fast, scarless, off-the-shelf genome engineering.
Sujet(s)
Systèmes CRISPR-Cas , DNA nucleotidyltransferases/métabolisme , Édition de gène/méthodes , /métabolisme , Sites de fixation/génétique , DNA nucleotidyltransferases/génétique , ADN bactérien/génétique , ADN bactérien/métabolisme , Escherichia coli/génétique , Escherichia coli/métabolisme , Techniques de knock-out de gènes , Génome bactérien/génétique , Modèles génétiques , Mutation , /génétiqueRÉSUMÉ
Nuclear receptor (NR) transcription factors bind various coreceptors, small-molecule ligands, DNA response element sequences, and transcriptional coregulator proteins to affect gene transcription. Small-molecule ligands and DNA are known to influence receptor structure, coregulator protein interaction, and function; however, little is known on the mechanism of synergy between ligand and DNA. Using quantitative biochemical, biophysical, and solution structural methods, including 13C-detected nuclear magnetic resonance and hydrogen/deuterium exchange (HDX) mass spectrometry, we show that ligand and DNA cooperatively recruit the intrinsically disordered steroid receptor coactivator-2 (SRC-2/TIF2/GRIP1/NCoA-2) receptor interaction domain to peroxisome proliferator-activated receptor gamma-retinoid X receptor alpha (PPARγ-RXRα) heterodimer and reveal the binding determinants of the complex. Our data reveal a thermodynamic mechanism by which DNA binding propagates a conformational change in PPARγ-RXRα, stabilizes the receptor ligand binding domain dimer interface, and impacts ligand potency and cooperativity in NR coactivator recruitment.
Sujet(s)
ADN/métabolisme , Complexes multiprotéiques/composition chimique , Coactivateur-2 de récepteur nucléaire/composition chimique , Coactivateur-2 de récepteur nucléaire/métabolisme , Sites de fixation , Spectroscopie par résonance magnétique du carbone-13 , Mesure d'échange de deutérium , Régulation de l'expression des gènes , Humains , Ligands , Récepteur PPAR gamma/composition chimique , Récepteur PPAR gamma/métabolisme , Liaison aux protéines , Récepteur des rétinoïdes X type alpha/composition chimique , Récepteur des rétinoïdes X type alpha/métabolismeRÉSUMÉ
The nuclear pore complex subunit TPR is found in at least five different oncogenic fusion kinases, including TPR-MET, yet how TPR fusions promote activation of kinases and their oncogenic activities remains poorly understood. Here we report the crystal structure of TPR(2-142), the MET fusion partner of oncogenic TPR-MET. TPR(2-142) contains a continuous 124-residue α helix that forms an antiparallel tetramer from two leucine zipper-containing parallel coiled coils. Remarkably, single mutations cause strikingly different conformations of the coiled coil, indicating its highly dynamic nature. We further show that fusion of TPR(2-142) to the MET intracellular domain strongly and selectively stabilizes the αG helix of the MET kinase domain, and mutations of only the TPR leucine zipper residues at the junction to MET, but not other leucine zipper residues, abolish kinase activation. Together, these results provide critical insight into the TPR structure and its ability to induce dimerization and activation of fusion kinases.
Sujet(s)
Protéine oncogène tpr-met/composition chimique , Protéine oncogène tpr-met/métabolisme , Cristallographie aux rayons X , Activation enzymatique , Humains , Glissières à leucine , Modèles moléculaires , Mutation , Protéine oncogène tpr-met/génétique , Conformation des protéines , Domaines protéiques , Pliage des protéines , Multimérisation de protéines , Stabilité protéiqueRÉSUMÉ
BACKGROUND AND PURPOSE: Data about non-invasive follow-up of aneurysm after stent-assisted coiling is scarce. We aimed to compare time-of-flight (TOF) magnetic resonance angiography (MRA) (3D-TOF-MRA) and contrast-enhanced MRA (CE-MRA) at 3-Tesla, with digital subtraction angiography (DSA) for evaluating aneurysm occlusion and parent artery patency after stent-assisted coiling. MATERIALS AND METHODS: In this retrospective single-center study, patients were included if they had an intracranial aneurysm treated by stent-assisted coiling between March 2008 and June 2015, followed with both MRA sequences (3D-TOF-MRA and CE-MRA) at 3-Tesla and DSA, performed in an interval<48hours. RESULTS: Thirty-five aneurysms were included. Regarding aneurysm occlusion evaluation, agreement with DSA was better for CE-MRA (K=0.53) than 3D-TOF-MRA (K=0.28). Diagnostic accuracies for aneurysm remnant depiction were similar for 3D-TOF-MRA and CE-MRA (P=1). Both 3D-TOF-MRA (K=0.05) and CE-MRA (K=-0.04) were unable to detect pathological vessel compared to DSA, without difference in accuracy (P=0.68). For parent artery occlusion detection, agreement with DSA was substantial for 3D-TOF-MRA (K=0.64) and moderate for CE-MRA (K=0.45), with similar good diagnostic accuracies (P=1). CONCLUSION: After stent-assisted coiling treatment, 3D-TOF-MRA and CE-MRA demonstrated good accuracy to detect aneurysm remnant (but tended to overestimation). Although CE-MRA agreement with DSA was better, there was no statistical difference between 3D-TOF-MRA and CE-MRA accuracies. Both MRAs were unable to provide a precise evaluation of in-stent status but could detect parent vessel occlusion.