RESUMO
The ATM kinase is a central component of the DNA damage repair machinery and redox balance. ATM dysfunction results in the multisystem disease ataxia-telangiectasia (AT). A major cause of mortality in AT is respiratory bacterial infections. Whether ATM deficiency causes innate immune defects that might contribute to bacterial infections is not known. Here we have shown that loss of ATM impairs inflammasome-dependent anti-bacterial innate immunity. Cells from AT patients or Atm(-/-) mice exhibited diminished interleukin-1ß (IL-1ß) production in response to bacteria. In vivo, Atm(-/-) mice were more susceptible to pulmonary S. pneumoniae infection in a manner consistent with inflammasome defects. Our data indicate that such defects were due to oxidative inhibition of inflammasome complex assembly. This study reveals an unanticipated function of reactive oxygen species (ROS) in negative regulation of inflammasomes and proposes a theory for the notable susceptibility of AT patients to pulmonary bacterial infection.
Assuntos
Ataxia Telangiectasia/genética , Pulmão/imunologia , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Células Cultivadas , Dano ao DNA , Reparo do DNA , Humanos , Imunidade Inata , Inflamassomos/fisiologia , Interleucina-1beta , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Espécies Reativas de Oxigênio/metabolismoRESUMO
Dysfunction in Ataxia-telangiectasia mutated (ATM), a central component of the DNA repair machinery, results in Ataxia Telangiectasia (AT), a cancer-prone disease with a variety of inflammatory manifestations. By analyzing AT patient samples and Atm(-/-) mice, we found that unrepaired DNA lesions induce type I interferons (IFNs), resulting in enhanced anti-viral and anti-bacterial responses in Atm(-/-) mice. Priming of the type I interferon system by DNA damage involved release of DNA into the cytoplasm where it activated the cytosolic DNA sensing STING-mediated pathway, which in turn enhanced responses to innate stimuli by activating the expression of Toll-like receptors, RIG-I-like receptors, cytoplasmic DNA sensors, and their downstream signaling partners. This study provides a potential explanation for the inflammatory phenotype of AT patients and establishes damaged DNA as a cell intrinsic danger signal that primes the innate immune system for a rapid and amplified response to microbial and environmental threats.
Assuntos
Ataxia Telangiectasia/imunologia , Dano ao DNA , DNA/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Proteínas de Membrana/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Células da Medula Óssea/imunologia , Linhagem Celular , Citosol/imunologia , Citosol/microbiologia , Reparo do DNA/genética , Ativação Enzimática/imunologia , Células HEK293 , Humanos , Imunidade Inata , Interferon-alfa/biossíntese , Interferon beta/biossíntese , Interferon gama/biossíntese , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Liver resection (LR) is the primary treatment for hepatic tumors, yet posthepatectomy liver failure (PHLF) remains a significant concern. While the precise etiology of PHLF remains elusive, dysregulated inflammatory processes are pivotal. Therefore, we explored the theragnostic potential of extracellular high-mobility-group-box protein 1 (HMGB1), a key damage-associated molecular pattern (DAMP) released by hepatocytes, in liver recovery post LR in patients and animal models. Plasma from 96 LR patients and liver tissues from a subset of 24 LR patients were analyzed for HMGB1 levels, and associations with PHLF and liver injury markers were assessed. In a murine LR model, the HMGB1 inhibitor glycyrrhizin, was administered to assess its impact on liver regeneration. Furthermore, plasma levels of keratin-18 (K18) and cleaved cytokeratin-18 (ccK18) were quantified to assess suitability as predictive biomarkers for PHLF. Patients experiencing PHLF exhibited elevated levels of intrahepatic and circulating HMGB1, correlating with markers of liver injury. In a murine LR model, inhibition of HMGB1 improved liver function, reduced steatosis, enhanced regeneration and decreased hepatic cell death. Elevated levels of hepatic cell death markers K18 and ccK18 were detected in patients with PHLF and correlations with levels of circulating HMGB1 was observed. Our study underscores the therapeutic and predictive potential of HMGB1 in PHLF mitigation. Elevated HMGB1, K18, and ccK18 levels correlate with patient outcomes, highlighting their predictive significance. Targeting HMGB1 enhances liver regeneration in murine LR models, emphasizing its role in potential intervention and prediction strategies for liver surgery.
Assuntos
Proteína HMGB1 , Hepatectomia , Falência Hepática , Idoso , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Biomarcadores , Morte Celular , Modelos Animais de Doenças , Ácido Glicirrízico/farmacologia , Hepatectomia/efeitos adversos , Hepatócitos/metabolismo , Proteína HMGB1/metabolismo , Proteína HMGB1/sangue , Queratina-18/metabolismo , Queratina-18/sangue , Fígado/metabolismo , Fígado/patologia , Falência Hepática/etiologia , Falência Hepática/metabolismo , Falência Hepática/patologia , Regeneração Hepática , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: The IκB kinase (IKK) complex, comprising the two enzymes IKKα and IKKß, is the main activator of the inflammatory transcription factor NF-κB, which is constitutively active in many cancers. While several connections between NF-κB signaling and the oncogene c-Myc have been shown, functional links between the signaling molecules are still poorly studied. METHODS: Molecular interactions were shown by co-immunoprecipitation and FRET microscopy. Phosphorylation of c-Myc was shown by kinases assays and its activity by improved reporter gene systems. CRISPR/Cas9-mediated gene knockout and chemical inhibition were used to block IKK activity. The turnover of c-Myc variants was determined by degradation in presence of cycloheximide and by optical pulse-chase experiments.. Immunofluorescence of mouse prostate tissue and bioinformatics of human datasets were applied to correlate IKKα- and c-Myc levels. Cell proliferation was assessed by EdU incorporation and apoptosis by flow cytometry. RESULTS: We show that IKKα and IKKß bind to c-Myc and phosphorylate it at serines 67/71 within a sequence that is highly conserved. Knockout of IKKα decreased c-Myc-activity and increased its T58-phosphorylation, the target site for GSK3ß, triggering polyubiquitination and degradation. c-Myc-mutants mimicking IKK-mediated S67/S71-phosphorylation exhibited slower turnover, higher cell proliferation and lower apoptosis, while the opposite was observed for non-phosphorylatable A67/A71-mutants. A significant positive correlation of c-Myc and IKKα levels was noticed in the prostate epithelium of mice and in a variety of human cancers. CONCLUSIONS: Our data imply that IKKα phosphorylates c-Myc on serines-67/71, thereby stabilizing it, leading to increased transcriptional activity, higher proliferation and decreased apoptosis.
Assuntos
Quinase I-kappa B/metabolismo , Inflamação/enzimologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Células HEK293 , Humanos , Quinase I-kappa B/química , Inflamação/patologia , Masculino , Camundongos , Modelos Biológicos , Mutação/genética , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Próstata/metabolismo , Ligação Proteica , Estabilidade Proteica , Transcrição GênicaRESUMO
RATIONALE: Extracellular vesicles, including microvesicles, are increasingly recognized as important mediators in cardiovascular disease. The cargo and surface proteins they carry are considered to define their biological activity, including their inflammatory properties. Monocyte to endothelial cell signaling is a prerequisite for the propagation of inflammatory responses. However, the contribution of microvesicles in this process is poorly understood. OBJECTIVE: To elucidate the mechanisms by which microvesicles derived from activated monocytic cells exert inflammatory effects on endothelial cells. METHODS AND RESULTS: LPS (lipopolysaccharide)-stimulated monocytic cells release free mitochondria and microvesicles with mitochondrial content as demonstrated by flow cytometry, quantitative polymerase chain reaction, Western Blot, and transmission electron microscopy. Using RNAseq analysis and quantitative reverse transcription-polymerase chain reaction, we demonstrated that both mitochondria directly isolated from and microvesicles released by LPS-activated monocytic cells, as well as circulating microvesicles isolated from volunteers receiving low-dose LPS-injections, induce type I IFN (interferon), and TNF (tumor necrosis factor) responses in endothelial cells. Depletion of free mitochondria significantly reduced the ability of these microvesicles to induce type I IFN and TNF-dependent genes. We identified mitochondria-associated TNFα and RNA from stressed mitochondria as major inducers of these responses. Finally, we demonstrated that the proinflammatory potential of microvesicles and directly isolated mitochondria were drastically reduced when they were derived from monocytic cells with nonrespiring mitochondria or monocytic cells cultured in the presence of pyruvate or the mitochondrial reactive oxygen species scavenger MitoTEMPO. CONCLUSIONS: Mitochondria and mitochondria embedded in microvesicles constitute a major subset of extracellular vesicles released by activated monocytes, and their proinflammatory activity on endothelial cells is determined by the activation status of their parental cells. Thus, mitochondria may represent critical intercellular mediators in cardiovascular disease and other inflammatory settings associated with type I IFN and TNF signaling.
Assuntos
Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Interferon Tipo I/biossíntese , Mitocôndrias/metabolismo , Monócitos/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Adulto , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/imunologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lipopolissacarídeos/toxicidade , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/imunologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Adulto JovemRESUMO
Following genotoxic stress, cells activate a complex signalling network to arrest the cell cycle and initiate DNA repair or apoptosis. The tumour suppressor p53 lies at the heart of this DNA damage response. However, it remains incompletely understood, which signalling molecules dictate the choice between these different cellular outcomes. Here, we identify the transcriptional regulator apoptosis-antagonizing transcription factor (AATF)/Che-1 as a critical regulator of the cellular outcome of the p53 response. Upon genotoxic stress, AATF is phosphorylated by the checkpoint kinase MK2. Phosphorylation results in the release of AATF from cytoplasmic MRLC3 and subsequent nuclear translocation where AATF binds to the PUMA, BAX and BAK promoter regions to repress p53-driven expression of these pro-apoptotic genes. In xenograft experiments, mice exhibit a dramatically enhanced response of AATF-depleted tumours following genotoxic chemotherapy with adriamycin. The exogenous expression of a phospho-mimicking AATF point mutant results in marked adriamycin resistance in vivo. Nuclear AATF enrichment appears to be selected for in p53-proficient endometrial cancers. Furthermore, focal copy number gains at the AATF locus in neuroblastoma, which is known to be almost exclusively p53-proficient, correlate with an adverse prognosis and reduced overall survival. These data identify the p38/MK2/AATF signalling module as a critical repressor of p53-driven apoptosis and commend this pathway as a target for DNA damage-sensitizing therapeutic regimens.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose/fisiologia , Dano ao DNA/fisiologia , Proteínas Repressoras/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/genética , Pontos de Checagem do Ciclo Celular , Dano ao DNA/genética , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias do Endométrio/genética , Feminino , Amplificação de Genes , Dosagem de Genes , Células HEK293 , Humanos , Camundongos , Dados de Sequência Molecular , Complexos Multiproteicos , Cadeias Leves de Miosina/metabolismo , Neuroblastoma/genética , Neuroblastoma/mortalidade , Pressão Osmótica , Fosforilação , Prognóstico , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/genéticaRESUMO
Binding of TNF to its receptor (TNFR1) elicits the spatiotemporal assembly of two signaling complexes that coordinate the balance between cell survival and cell death. We have shown previously that, following TNF treatment, the mRNA decay protein tristetraprolin (TTP) is Lys-63-polyubiquitinated by TNF receptor-associated factor 2 (TRAF2), suggesting a regulatory role in TNFR signaling. Here we demonstrate that TTP interacts with TNFR1 in a TRAF2-dependent manner, thereby initiating the MEKK1/MKK4-dependent activation of JNK activities. This regulatory function toward JNK activation but not NF-κB activation depends on lysine 105 of TTP, which we identified as the corresponding TRAF2 ubiquitination site. Disabling TTP polyubiquitination results in enhanced TNF-induced apoptosis in cervical cancer cells. Together, we uncover a novel aspect of TNFR1 signaling where TTP, in alliance with TRAF2, acts as a balancer of JNK-mediated cell survival versus death.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Tristetraprolina/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lisina/metabolismo , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase Quinase 1/metabolismo , Camundongos Knockout , Mutação , NF-kappa B/metabolismo , Poliubiquitina/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Tristetraprolina/genética , Ubiquitinação/efeitos dos fármacosRESUMO
Francisella tularensis is a highly infectious intracellular pathogen that has evolved an efficient strategy to subvert host defense response to survive inside the host. The molecular mechanisms regulating these host-pathogen interactions and especially those that are initiated at the time of the bacterial entry via its attachment to the host plasma membrane likely predetermine the intracellular fate of pathogen. Here, we provide the evidence that infection of macrophages with F. tularensis leads to changes in protein composition of macrophage-derived lipid rafts, isolated as detergent-resistant membranes (DRMs). Using SILAC-based quantitative proteomic approach, we observed the accumulation of autophagic adaptor protein p62 at the early stages of microbe-host cell interaction. We confirmed the colocalization of the p62 with ubiquitinated and LC3-decorated intracellular F. tularensis microbes with its maximum at 1 h postinfection. Furthermore, the infection of p62-knockdown host cells led to the transient increase in the intracellular number of microbes up to 4 h after in vitro infection. Together, these data suggest that the activation of the autophagy pathway in F. tularensis infected macrophages, which impacts the early phase of microbial proliferation, is subsequently circumvented by ongoing infection.
Assuntos
Autofagia , Microdomínios da Membrana/metabolismo , Proteômica , Tularemia/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Microdomínios da Membrana/química , Camundongos , Dados de Sequência MolecularRESUMO
The proteome of two newborn meningitis Escherichia coli K1 (NMEC) morphotypes was examined via a label-free proteomics approach. Besides shared NMEC virulence factors, the two strains have different evolutionary strategies-strain IHE3034 tends to perform anaerobic respiration continuously, while strain RS218 maintains its filamentous morphotype due to active SOS response.
RESUMO
Ubiquitin modifications alter protein function and stability, thereby regulating cell homeostasis and viability, particularly under stress. Ischemic stroke induces protein ubiquitination at the ischemic periphery, wherein cells remain viable, however the identity of ubiquitinated proteins is unknown. Here, we employed a proteomics approach to identify these proteins in mice undergoing ischemic stroke. The data are available in a searchable web interface ( https://hochrainerlab.shinyapps.io/StrokeUbiOmics/ ). We detected increased ubiquitination of 198 proteins, many of which localize to the postsynaptic density (PSD) of glutamatergic neurons. Among these were proteins essential for maintaining PSD architecture, such as PSD95, as well as NMDA and AMPA receptor subunits. The largest enzymatic group at the PSD with elevated post-ischemic ubiquitination were kinases, such as CaMKII, PKC, Cdk5, and Pyk2, whose aberrant activities are well-known to contribute to post-ischemic neuronal death. Concurrent phospho-proteomics revealed altered PSD-associated phosphorylation patterns, indicative of modified kinase activities following stroke. PSD-located CaMKII, PKC, and Cdk5 activities were decreased while Pyk2 activity was increased after stroke. Removal of ubiquitin restored kinase activities to pre-stroke levels, identifying ubiquitination as the responsible molecular mechanism for post-ischemic kinase regulation. These findings unveil a previously unrecognized role of ubiquitination in the regulation of essential kinases involved in ischemic injury.
Assuntos
AVC Isquêmico , Acidente Vascular Cerebral , Camundongos , Animais , Proteína 4 Homóloga a Disks-Large , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Quinase 2 de Adesão Focal , Densidade Pós-Sináptica , Fosfotransferases , Ubiquitinação , Isquemia , UbiquitinaRESUMO
The transcription factor Sox18 plays a role in angiogenesis, including lymphangiogenesis, where it is upregulated by growth factors and directs the expression of genes encoding, e.g., guidance molecules and a matrix metalloproteinase. Conversely, we found that in human umbilical vein endothelial cells (HUVEC) Sox18 is repressed by the pro-inflammatory mediator TNFα (as well as IL-1 and LPS). Since a common feature of these mediators is the activation of the NF-κB signaling pathway, we investigated whether Sox18 downregulation is dependent on this transcription factor. Transduction of HUVEC with an adenoviral vector directing the expression of the NF-κB inhibitor IκBα prevented the downregulation of Sox18. Transient transfections of Sox18 promoter reporter genes revealed that the downregulation takes place on the level of transcription, and that the p65/RelA subunit of NF-κB was operative. Furthermore, the responsible promoter region of Sox18 is located within -1.0kb from the transcriptional start site. The repression of Sox18 and its target genes may lead to altered formation of vessels in inflamed settings.
Assuntos
Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/fisiologia , NF-kappa B/metabolismo , Neovascularização Fisiológica/genética , Fatores de Transcrição SOXF/metabolismo , Regulação para Baixo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Regiões Promotoras Genéticas , Fatores de Transcrição SOXF/genética , Transcrição Gênica , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
There is little doubt that final victories over pandemics, such as COVID-19, are attributed to herd immunity, either through post-disease convalescence or active immunization of a high percentage of the world's population with vaccines, which demonstrate protection from infection and transmission and are available in large quantities at reasonable prices. However, it is assumable that humans with immune defects or immune suppression, e.g., as a consequence of allograft transplantation, cannot be immunized actively nor produce sufficient immune responses to prevent SARS-CoV-2 infections. These subjects desperately need other strategies, such as sophisticated protection measures and passive immunization. Hypertonic salt solutions attack vulnerable core areas of viruses; i.e., salt denatures surface proteins and thus prohibits virus penetration of somatic cells. It has to be ensured that somatic proteins are not affected by denaturation regarding this unspecific virus protection. Impregnating filtering facepieces with hypertonic salt solutions is a straightforward way to inactivate viruses and other potential pathogens. As a result of the contact of salt crystals on the filtering facepiece, these pathogens become denatured and inactivated almost quantitatively. Such a strategy could be easily applied to fight against the COVID-19 pandemic and other ones that may occur in the future. Another possible tool to fight the COVID-19 pandemic is passive immunization with antibodies against SARS-CoV-2, preferably from human origin. Such antibodies can be harvested from human patients' sera who have successfully survived their SARS-CoV-2 infection. The disadvantage of a rapid decrease in the immunoglobulin titer after the infection ends can be overcome by immortalizing antibody-producing B cells via fusion with, e.g., mouse myeloma cells. The resulting monoclonal antibodies are then of human origin and available in, at least theoretically, unlimited amounts. Finally, dry blood spots are a valuable tool for surveilling a population's immunity. The add-on strategies were selected as examples for immediate, medium and long-term assistance and therefore did not raise any claim to completeness.
Assuntos
COVID-19 , Animais , Camundongos , Humanos , COVID-19/prevenção & controle , SARS-CoV-2 , Pandemias/prevenção & controle , Vacinação , Anticorpos Antivirais , Anticorpos NeutralizantesRESUMO
Proteomics is an indispensable analytical technique to study the dynamic functioning of biological systems via different proteins and their proteoforms. In recent years, bottom-up shotgun has become more popular than gel-based top-down proteomics. The current study examined the qualitative and quantitative performance of these two fundamentally different methodologies by the parallel measurement of six technical and three biological replicates of the human prostate carcinoma cell line DU145 using its two most common standard techniques, label-free shotgun and two-dimensional differential gel electrophoresis (2D-DIGE). The analytical strengths and limitations were explored, finally focusing on the unbiased detection of proteoforms, exemplified by discovering a prostate cancer-related cleavage product of pyruvate kinase M2. Label-free shotgun proteomics quickly yields an annotated proteome but with reduced robustness, as determined by three times higher technical variation compared to 2D-DIGE. At a glance, only 2D-DIGE top-down analysis provided valuable, direct stoichiometric qualitative and quantitative information from proteins to their proteoforms, even with unexpected post-translational modifications, such as proteolytic cleavage and phosphorylation. However, the 2D-DIGE technology required almost 20 times as much time per protein/proteoform characterization with more manual work. Ultimately, this work should expose both techniques' orthogonality with their different contents of data output to elucidate biological questions.
Assuntos
Proteoma , Proteômica , Masculino , Humanos , Proteômica/métodos , Proteoma/análise , Processamento de Proteína Pós-Traducional , Eletroforese em Gel Bidimensional , FosforilaçãoRESUMO
Age represents the major risk factor for fatal disease outcome in coronavirus disease (COVID-19) due to age-related changes in immune responses. On the one hand lymphocyte counts continuously decline with advancing age, on the other hand somatic hyper-mutations of B-lymphocytes and levels of class-switched antibodies diminish, resulting in lower neutralizing antibody titers. To date the impact of age on immunoglobulin G (IgG) production in response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is unknown. Therefore, we investigated the impact of age on the onset of IgG production and its association with outcome, viral persistence, inflammatory and thrombotic markers in consecutive, hospitalized COVID-19 patients admitted to the Clinic Favoriten (Vienna, Austria) between April and October 2020 that fulfilled predefined inclusion criteria. Three different IgGs against SARS-CoV-2 (spike protein S1, nucleocapsid (NC), and the spike protein receptor binding domain (RBD)) were monitored in plasma of 97 patients upon admission and three times within the first week followed by weekly assessment during their entire hospital stay. We analyzed the association of clinical parameters including C-reactive protein (CRP), D-dimer levels and platelet count as well as viral persistence with the onset and concentration of different anti-SARS-CoV-2 specific IgGs. Our data demonstrate that in older individuals anti-SARS-CoV-2 IgG production increases earlier after symptom onset and that deceased patients have the highest amount of antibodies against SARS-CoV-2 whereas intensive care unit (ICU) survivors have the lowest titers. In addition, anti-SARS-CoV-2 IgG concentrations are not associated with curtailed viral infectivity, inflammatory or thrombotic markers, suggesting that not only serological memory but also other adaptive immune responses are involved in successful viral killing and protection against a severe COVID-19 infection.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Idoso , Imunoglobulina G , Glicoproteína da Espícula de Coronavírus , Inflamação , Anticorpos AntiviraisRESUMO
Ubiquitin modifications alter protein function and stability, thereby regulating cell homeostasis and viability, particularly under stress. Ischemic stroke induces protein ubiquitination at the ischemic periphery, wherein cells remain viable, however the identity of ubiquitinated proteins is unknown. Here, we employed a proteomics approach to identify these proteins in mice undergoing ischemic stroke. The data are available in a searchable web interface ( https://hochrainerlab.shinyapps.io/StrokeUbiOmics/ ). We detected increased ubiquitination of 198 proteins, many of which localize to the postsynaptic density (PSD) of glutamatergic neurons. Among these were proteins essential for maintaining PSD architecture, such as PSD95, as well as NMDA and AMPA receptor subunits. The largest enzymatic group at the PSD with elevated post-ischemic ubiquitination were kinases, such as CaMKII, PKC, Cdk5, and Pyk2, whose aberrant activities are well-known to contribute to post-ischemic neuronal death. Concurrent phospho-proteomics revealed altered PSD-associated phosphorylation patterns, indicative of modified kinase activities following stroke. PSD-located CaMKII, PKC, and Cdk5 activities were decreased while Pyk2 activity was increased after stroke. Removal of ubiquitin restored kinase activities to pre-stroke levels, identifying ubiquitination as the responsible molecular mechanism for post-ischemic kinase regulation. These findings unveil a previously unrecognized role of ubiquitination in the regulation of essential kinases involved in ischemic injury.
RESUMO
Pre-clinical studies from the recent past have indicated that senescent cells can negatively affect health and contribute to premature aging. Targeted eradication of these cells has been shown to improve the health of aged experimental animals, leading to a clinical interest in finding compounds that selectively eliminate senescent cells while sparing non-senescent ones. In our study, we identified a senolytic capacity of statins, which are lipid-lowering drugs prescribed to patients at high risk of cardiovascular events. Using two different models of senescence in human vascular endothelial cells (HUVECs), we found that statins preferentially eliminated senescent cells, while leaving non-senescent cells unharmed. We observed that the senolytic effect of statins could be negated with the co-administration of mevalonic acid and that statins induced cell detachment leading to anoikis-like apoptosis, as evidenced by real-time visualization of caspase-3/7 activation. Our findings suggest that statins possess a senolytic property, possibly also contributing to their described beneficial cardiovascular effects. Further studies are needed to explore the potential of short-term, high-dose statin treatment as a candidate senolytic therapy.
Assuntos
Senescência Celular , Inibidores de Hidroximetilglutaril-CoA Redutases , Animais , Humanos , Idoso , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Células Endoteliais , Anoikis , SenoterapiaRESUMO
Acute versus chronic inflammation is controlled by the accurate activation and regulation of interdependent signaling cascades. TNF-receptor 1 engagement concomitantly activates NF-κB and JNK signaling. The correctly timed activation of these pathways is the key to account for the balance between NF-κB-mediated cell survival and cell death, the latter fostered by prolonged JNK activation. Tristetraprolin (TTP), initially described as an mRNA destabilizing protein, acts as negative feedback regulator of the inflammatory response: it destabilizes cytokine-mRNAs but also acts as an NF-κB inhibitor by interfering with the p65/RelA nuclear import pathway. Our biochemical studies provide evidence that TTP contributes to the NF-κB/JNK balance. We find that the MAP 3-kinase MEKK1 acts as a novel TTP kinase that, together with the TNF receptor-associated factor 2 (TRAF2), constitutes not only a main determinate of the NF-κB-JNK cross-talk but also facilitates "TTP hypermodification": MEKK1 triggers TTP phosphorylation as prerequisite for its Lys-63-linked, TRAF2-mediated ubiquitination. Consequently, TTP no longer affects NF-κB activity but promotes the activation of JNK. Based on our data, we suggest a model where upon TNFα induction, TTP transits a hypo- to hypermodified state, thereby contributing to the molecular regulation of NF-κB versus JNK signaling cascades.
Assuntos
MAP Quinase Quinase Quinase 1/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Tristetraprolina/química , Ubiquitina/química , Adenoviridae/metabolismo , Sobrevivência Celular , Células HEK293 , Células HeLa , Humanos , Quinase I-kappa B/metabolismo , Inflamação , Sistema de Sinalização das MAP Quinases , Mutação , NF-kappa B/metabolismo , Fosforilação , Receptores Tipo I de Fatores de Necrose Tumoral/química , Transdução de SinaisRESUMO
Myeloperoxidase is abundantly present in inflammatory diseases where activation of monocytes/macrophages and T-cell-mediated immune response occurs. The potent oxidant hypochlorous acid (HOCl), generated by the myeloperoxidase-H(2)O(2)-chloride system of activated phagocytes, converts low-density lipoprotein (LDL) into a proinflammatory lipoprotein particle. Here, we investigated the apoptotic effect of HOCl-LDL, an in vivo occurring LDL modification, on human T-cell lymphoblast-like Jurkat cells. Experiments revealed that HOCl-LDL, depending on the oxidant:lipoprotein molar ratio, induces apoptosis via activation of caspase-3, PARP cleavage and accumulation of reactive oxygen species. The absence of Fas-associated protein with death domain or caspase-8 in mutant cells did not prevent HOCl-LDL induced apoptosis. In contrast, overexpression of the anti-apoptotic Bcl-2 protein protects Jurkat cells against HOCl-LDL-induced apoptosis and prevents accumulation of reactive oxygen species. We conclude that HOCl-LDL-mediated apoptosis in Jurkat cells follows predominantly the intrinsic, mitochondrial pathway. Insitu experiments revealed that an antibody raised against HOCl-LDL recognized epitopes that colocalize both with myeloperoxidase and CD3-positive T-cells in human decidual tissue where local stimulation of the immune system occurs. We provide convincing evidence that formation of HOCl-modified (lipo)proteins generated by the myeloperoxidase-H(2)O(2)-chloride system contributes to apoptosis in T-cells.