RESUMO
Derailed cytokine and immune cell networks account for the organ damage and the clinical severity of COVID-19 (refs. 1-4). Here we show that SARS-CoV-2, like other viruses, evokes cellular senescence as a primary stress response in infected cells. Virus-induced senescence (VIS) is indistinguishable from other forms of cellular senescence and is accompanied by a senescence-associated secretory phenotype (SASP), which comprises pro-inflammatory cytokines, extracellular-matrix-active factors and pro-coagulatory mediators5-7. Patients with COVID-19 displayed markers of senescence in their airway mucosa in situ and increased serum levels of SASP factors. In vitro assays demonstrated macrophage activation with SASP-reminiscent secretion, complement lysis and SASP-amplifying secondary senescence of endothelial cells, which mirrored hallmark features of COVID-19 such as macrophage and neutrophil infiltration, endothelial damage and widespread thrombosis in affected lung tissue1,8,9. Moreover, supernatant from VIS cells, including SARS-CoV-2-induced senescence, induced neutrophil extracellular trap formation and activation of platelets and the clotting cascade. Senolytics such as navitoclax and a combination of dasatinib plus quercetin selectively eliminated VIS cells, mitigated COVID-19-reminiscent lung disease and reduced inflammation in SARS-CoV-2-infected hamsters and mice. Our findings mark VIS as a pathogenic trigger of COVID-19-related cytokine escalation and organ damage, and suggest that senolytic targeting of virus-infected cells is a treatment option against SARS-CoV-2 and perhaps other viral infections.
Assuntos
Tratamento Farmacológico da COVID-19 , COVID-19/patologia , COVID-19/virologia , Senescência Celular/efeitos dos fármacos , Terapia de Alvo Molecular , SARS-CoV-2/patogenicidade , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Animais , COVID-19/complicações , Linhagem Celular , Cricetinae , Dasatinibe/farmacologia , Dasatinibe/uso terapêutico , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Quercetina/farmacologia , Quercetina/uso terapêutico , SARS-CoV-2/efeitos dos fármacos , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Trombose/complicações , Trombose/imunologia , Trombose/metabolismoRESUMO
Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) has developed substantial antigenic variability. As the majority of the population now has pre-existing immunity due to infection or vaccination, the use of experimentally generated animal immune sera can be valuable for measuring antigenic differences between virus variants. Here, we immunized Syrian hamsters by two successive infections with one of nine SARS-CoV-2 variants. Their sera were titrated against 16 SARS-CoV-2 variants, and the resulting titers were visualized using antigenic cartography. The antigenic map shows a condensed cluster containing all pre-Omicron variants (D614G, Alpha, Delta, Beta, Mu, and an engineered B.1+E484K variant) and considerably more diversity among a selected panel of Omicron subvariants (BA.1, BA.2, BA.4/BA.5, the BA.5 descendants BF.7 and BQ.1.18, the BA.2.75 descendant BN.1.3.1, the BA.2-derived recombinants XBB.2 and EG.5.1, and the BA.2.86 descendant JN.1). Some Omicron subvariants were as antigenically distinct from each other as the wildtype is from the Omicron BA.1 variant. Compared to titers measured in human sera, titers in hamster sera are of higher magnitude, show less fold change, and result in a more compact antigenic map topology. The results highlight the potential of sera from hamsters for the continued antigenic characterization of SARS-CoV-2.
Assuntos
Variação Antigênica , COVID-19 , Mesocricetus , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , SARS-CoV-2/imunologia , SARS-CoV-2/genética , COVID-19/imunologia , COVID-19/virologia , Cricetinae , Variação Antigênica/imunologia , Variação Antigênica/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Antígenos Virais/imunologia , Antígenos Virais/genética , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Humanos , Soros Imunes/imunologiaRESUMO
Live attenuated vaccines (LAVs) administered via the mucosal route may offer better control of the COVID-19 pandemic than non-replicating vaccines injected intramuscularly. Conceptionally, LAVs have several advantages, including presentation of the entire antigenic repertoire of the virus, and the induction of strong mucosal immunity. Thus, immunity induced by LAV could offer superior protection against future surges of COVID-19 cases caused by emerging SARS-CoV-2 variants. However, LAVs carry the risk of unintentional transmission. To address this issue, we investigated whether transmission of a SARS-CoV-2 LAV candidate can be blocked by removing the furin cleavage site (FCS) from the spike protein. The level of protection and immunity induced by the attenuated virus with the intact FCS was virtually identical to the one induced by the attenuated virus lacking the FCS. Most importantly, removal of the FCS completely abolished horizontal transmission of vaccine virus between cohoused hamsters. Furthermore, the vaccine was safe in immunosuppressed animals and showed no tendency to recombine in vitro or in vivo with a SARS-CoV-2 field strain. These results indicate that removal of the FCS from SARS-CoV-2 LAV is a promising strategy to increase vaccine safety and prevent vaccine transmission without compromising vaccine efficacy.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Animais , Cricetinae , Humanos , COVID-19/prevenção & controle , Pandemias , SARS-CoV-2 , Vacinas Atenuadas , Anticorpos Antivirais , Anticorpos NeutralizantesRESUMO
As virus outbreaks continue to pose a challenge, a nonspecific viral inhibitor can provide significant benefits, especially against respiratory viruses. Polyglycerol sulfates recently emerge as promising agents that mediate interactions between cells and viruses through electrostatics, leading to virus inhibition. Similarly, hydrophobic C60 fullerene can prevent virus infection via interactions with hydrophobic cavities of surface proteins. Here, two strategies are combined to inhibit infection of SARS-CoV-2 variants in vitro. Effective inhibitory concentrations in the millimolar range highlight the significance of bare fullerene's hydrophobic moiety and electrostatic interactions of polysulfates with surface proteins of SARS-CoV-2. Furthermore, microscale thermophoresis measurements support that fullerene linear polyglycerol sulfates interact with the SARS-CoV-2 virus via its spike protein, and highlight importance of electrostatic interactions within it. All-atom molecular dynamics simulations reveal that the fullerene binding site is situated close to the receptor binding domain, within 4 nm of polyglycerol sulfate binding sites, feasibly allowing both portions of the material to interact simultaneously.
Assuntos
COVID-19 , Fulerenos , Humanos , SARS-CoV-2 , Fulerenos/farmacologia , Ligação ProteicaRESUMO
For coronavirus disease 2019 (COVID-19), effective and well-understood treatment options are still scarce. Since vaccine efficacy is challenged by novel variants, short-lasting immunity, and vaccine hesitancy, understanding and optimizing therapeutic options remains essential. We aimed at better understanding the effects of two standard-of-care drugs, dexamethasone and anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies, on infection and host responses. By using two COVID-19 hamster models, pulmonary immune responses were analyzed to characterize effects of single or combinatorial treatments. Pulmonary viral burden was reduced by anti-SARS-CoV-2 antibody treatment and unaltered or increased by dexamethasone alone. Dexamethasone exhibited strong anti-inflammatory effects and prevented fulminant disease in a severe disease model. Combination therapy showed additive benefits with both anti-viral and anti-inflammatory potency. Bulk and single-cell transcriptomic analyses confirmed dampened inflammatory cell recruitment into lungs upon dexamethasone treatment and identified a specifically responsive subpopulation of neutrophils, thereby indicating a potential mechanism of action. Our analyses confirm the anti-inflammatory properties of dexamethasone and suggest possible mechanisms, validate anti-viral effects of anti-SARS-CoV-2 antibody treatment, and reveal synergistic effects of a combination therapy, thus informing more effective COVID-19 therapies.
Assuntos
Tratamento Farmacológico da COVID-19 , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Anticorpos Antivirais , Antivirais , Cricetinae , Dexametasona/farmacologia , SARS-CoV-2 , TranscriptomaRESUMO
Mucins are the key component of the defensive mucus barrier. They are extended fibers of very high molecular weight with diverse biological functions depending strongly on their specific structural parameters. Here, we present a mucin-inspired nanostructure, produced via a synthetic methodology to prepare methacrylate-based dendronized polysulfates (MIP-1) on a multi gram-scale with high molecular weight (MW=450â kDa) and thiol end-functionalized mucin-inspired polymer (MIP) via RAFT polymerization. Cryo-electron tomography (Cryo-ET) analysis of MIP-1 confirmed a mucin-mimetic wormlike single-chain fiber structure (length=144±59â nm) in aqueous solution. This biocompatible fiber showed promising activity against SARS-CoV-2 and its mutant strain, with a remarkable low half maximal (IC50 ) inhibitory concentration (IC50 =10.0â nM). Additionally, we investigate the impact of fiber length on SARS-CoV-2 inhibition by testing other functional polymers (MIPs) of varying fiber lengths.
Assuntos
COVID-19 , Impressão Molecular , Humanos , Mucinas , SARS-CoV-2 , Polímeros/farmacologia , Polímeros/química , Impressão Molecular/métodosRESUMO
Hypertension is a complex and multifactorial disorder caused by lifestyle and environmental factors, inflammation and disease-related genetic factors and is a risk factor for stroke, ischemic heart disease and renal failure. Although circulating monocytes and tissue macrophages contribute to the pathogenesis of hypertension, the underlying mechanisms are poorly understood. Cysteine rich protein 1 (CRIP1) is highly expressed in immune cells, and CRIP1 mRNA expression in monocytes associates with blood pressure (BP) and is up-regulated by proinflammatory modulation suggesting a link between CRIP1 and BP regulation through the immune system. To address this functional link, we studied CRIP1 expression in immune cells in relation to BP using a human cohort study and hypertensive mouse models. CRIP1 expression in splenic monocytes/macrophages and in circulating monocytes was significantly affected by angiotensin II (Ang II) in a BP-elevating dose (2 mg/kg/day). In the human cohort study, monocytic CRIP1 expression levels were associated with elevated BP, whereas upon differentiation of monocytes to macrophages this association along with the CRIP1 expression level was diminished. In conclusion, CRIP1-positive circulating and splenic monocytes seem to play an important role in hypertension related inflammatory processes through endogenous hormones such as Ang II. These findings suggest that CRIP1 may affect the interaction between the immune system, in particular monocytes, and the pathogenesis of hypertension.
Assuntos
Proteínas de Transporte/metabolismo , Hipertensão/fisiopatologia , Monócitos/metabolismo , Angiotensina II/administração & dosagem , Animais , Pressão Sanguínea , Proteínas de Transporte/genética , Diferenciação Celular , Estudos de Coortes , Modelos Animais de Doenças , Humanos , Hipertensão/induzido quimicamente , Hipertensão/metabolismo , Macrófagos , Masculino , NG-Nitroarginina Metil Éster/administração & dosagem , Baço , TranscriptomaRESUMO
The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 has revolutionized genome editing by providing a simple and robust means to cleave specific genomic sequences. However, introducing templated changes at the targeted site usually requires homology-directed repair (HDR), active in only a small subset of cells in culture. To enrich for HDR-dependent edited cells, we employed a co-editing strategy, editing a gene of interest (GOI) concomitantly with rescuing an endogenous pre-made temperature-sensitive (ts) mutation. By using the repair of the ts mutation as a selectable marker, the selection is "scarless" since editing restores the wild-type (wt) sequence. As proof of principle, we used HEK293 and HeLa cells with a ts mutation in the essential TAF1 gene. CRISPR co-editing of TAF1ts and a GOI resulted in up to 90% of the temperature-resistant cells bearing the desired mutation in the GOI. We used this system to insert large cassettes encoded by plasmid donors and smaller changes encoded by single-stranded oligonucleotide donors (ssODN). Of note, among the genes we edited was the introduction of a T35A mutation in the proteasome subunit PSMB6, which eliminates its caspase-like activity. The edited cells showed a specific reduction in this activity, demonstrating this system's utility in generating cell lines with biologically relevant mutations in endogenous genes. This approach offers a rapid, efficient, and scarless method for selecting genome-edited cells requiring HDR.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Histona Acetiltransferases/genética , Mutagênese , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Células HEK293 , Células HeLa , HumanosRESUMO
The cysteine-rich LIM-only protein 4 (CRP4), a LIM-domain and zinc finger containing adapter protein, has been implicated as a downstream effector of the second messenger 3',5'-cyclic guanosine monophosphate (cGMP) pathway in multiple cell types, including vascular smooth muscle cells (VSMCs). VSMCs and nitric oxide (NO)-induced cGMP signaling through cGMP-dependent protein kinase type I (cGKI) play fundamental roles in the physiological regulation of vascular tone and arterial blood pressure (BP). However, it remains unclear whether the vasorelaxant actions attributed to the NO/cGMP axis require CRP4. This study uses mice with a targeted deletion of the CRP4 gene (CRP4 KO) to elucidate whether cGMP-elevating agents, which are well known for their vasorelaxant properties, affect vessel tone, and thus, BP through CRP4. Cinaciguat, a NO- and heme-independent activator of the NO-sensitive (soluble) guanylyl cyclase (NO-GC) and NO-releasing agents, relaxed both CRP4-proficient and -deficient aortic ring segments pre-contracted with prostaglandin F2α. However, the magnitude of relaxation was slightly, but significantly, increased in vessels lacking CRP4. Accordingly, CRP4 KO mice presented with hypotonia at baseline, as well as a greater drop in systolic BP in response to the acute administration of cinaciguat, sodium nitroprusside, and carbachol. Mechanistically, loss of CRP4 in VSMCs reduced the Ca2+-sensitivity of the contractile apparatus, possibly involving regulatory proteins, such as myosin phosphatase targeting subunit 1 (MYPT1) and the regulatory light chain of myosin (RLC). In conclusion, the present findings confirm that the adapter protein CRP4 interacts with the NO-GC/cGMP/cGKI pathway in the vasculature. CRP4 seems to be part of a negative feedback loop that eventually fine-tunes the NO-GC/cGMP axis in VSMCs to increase myofilament Ca2+ desensitization and thereby the maximal vasorelaxant effects attained by (selected) cGMP-elevating agents.
Assuntos
Pressão Sanguínea , Vasos Sanguíneos/fisiologia , GMP Cíclico/metabolismo , Proteínas com Domínio LIM/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Vasos Sanguíneos/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Feminino , Masculino , Camundongos Knockout , Modelos Biológicos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Norepinefrina/farmacologia , Transdução de Sinais , Guanilil Ciclase Solúvel/metabolismo , Vasodilatadores/farmacologiaRESUMO
The 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type I (cGKI aka PKGI) is a major cardiac effector acting downstream of nitric oxide (NO)-sensitive soluble guanylyl cyclase and natriuretic peptides (NPs), which signal through transmembrane guanylyl cyclases. Consistent with the wide distribution of the cGMP-generating guanylyl cyclases, cGKI, which usually elicits its cellular effects by direct phosphorylation of its targets, is present in multiple cardiac cell types including cardiomyocytes (CMs). Although numerous targets of cGMP/cGKI in heart were identified in the past, neither their exact patho-/physiological functions nor cell-type specific roles are clear. Herein, we inform about the current knowledge on the signal transduction downstream of CM cGKI. We believe that better insights into the specific actions of cGMP and cGKI in these cells will help to guide future studies in the search for predictive biomarkers for the response to pharmacological cGMP pathway modulation. In addition, targets downstream of cGMP/cGKI may be exploited for refined and optimized diagnostic and therapeutic strategies in different types of heart disease and their causes. Importantly, key functions of these proteins and particularly sites of regulatory phosphorylation by cGKI should, at least in principle, remain intact, although upstream signaling through the second messenger cGMP is impaired or dysregulated in a stressed or diseased heart state.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , GMP Cíclico/metabolismo , Cardiopatias/enzimologia , Miócitos Cardíacos/enzimologia , Sistemas do Segundo Mensageiro , Animais , Fármacos Cardiovasculares/uso terapêutico , Cardiopatias/tratamento farmacológico , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Fosforilação , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Especificidade por SubstratoRESUMO
The polyomavirus middle T antigen (PyMT) oncogene activates the cellular nonreceptor tyrosine kinase c-Src and recruits the Hippo pathway effectors, Yap (yes-associated protein) and Taz (transcriptional coactivator with PDZ-binding motif), as key steps in oncogenesis. Yap and Taz are transcription coactivators shuttling from the cytoplasm to the nucleus. The Hippo pathway kinase Lats1/2 (large tumor suppressor homolog) reduces Yap/Taz nuclear localization and minimizes their cytoplasmic levels by facilitating their ubiquitination by the E3 ligase SCF(ß-TrCP). In contrast, PyMT increases the cytoplasmic Taz level. Here we show that this unique PyMT behavior is mediated by Src. We demonstrate that PyMT-induced Src activation inhibits degradation of both wild-type and tyrosine-less Taz, ruling out Taz modification as a mechanism of escaping degradation. Instead, we found that Src attenuates the SCF(ß-TrCP) E3-ligase activity in blunting Taz proteasomal degradation. The role of Src in rescuing Taz from TrCP-mediated degradation gives rise to higher cell proliferation under dense cell culture. Finally, IkB (NF-kappa-B inhibitor), a known substrate of ß-TrCP, was rescued by Src, suggesting a wider effect of Src on ß-TrCP substrates. These findings introduce the Src tyrosine kinase as a regulator of SCF(ß-TrCP).
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Proteína Tirosina Quinase CSK , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Células HCT116 , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Células NIH 3T3 , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Sinalização YAP , Proteínas Contendo Repetições de beta-Transducina/genética , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Background: Many patients with complex regional pain syndrome (CRPS) report some foreignness of the affected limb, which is referred to as "neglect-like symptoms" (NLS). Despite similarities of the NLS reports to symptoms of body image disturbances in mental disorders, no study has been conducted to examine such associations. Methods: We investigated 50 patients with CRPS and 45 pain control patients (N = 27, chronic limb pain; N = 18, migraine headache). NLS, anxiety, depression, depersonalization, and somatization were assessed using validated questionnaires. Results: Seventy-two percent of the CRPS patients reported at least one NLS vs 29.6% and 33.3% in the two patient control groups. In limb pain controls, NLS correlated with pain intensity. In CRPS patients, NLS correlated with anxiety (rho = 0.658, P < 0.001), somatization (rho = 0.616, P < 0.001), depersonalization (rho = 0.634, P < 0.001), and pain catastrophizing (rho = 0.456, P < 0.01), but not with intensity of pain, duration of pain, or pain disability. Conclusions: In CRPS patients, NLS could be a result of somatization, depression, anxiety, and depersonalization, but probably not of pain. Whether these associations are causative must be clarified in longitudinal psychological studies.
Assuntos
Ansiedade/epidemiologia , Transtornos Dismórficos Corporais/epidemiologia , Síndromes da Dor Regional Complexa/epidemiologia , Despersonalização/epidemiologia , Depressão/epidemiologia , Transtornos da Percepção/epidemiologia , Transtornos Somatoformes/epidemiologia , Ansiedade/diagnóstico , Ansiedade/psicologia , Transtornos Dismórficos Corporais/diagnóstico , Transtornos Dismórficos Corporais/psicologia , Catastrofização , Causalidade , Comorbidade , Síndromes da Dor Regional Complexa/diagnóstico , Síndromes da Dor Regional Complexa/psicologia , Despersonalização/diagnóstico , Despersonalização/psicologia , Depressão/diagnóstico , Depressão/psicologia , Feminino , Alemanha/epidemiologia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Transtornos da Percepção/diagnóstico por imagem , Transtornos da Percepção/psicologia , Fatores de Risco , Transtornos Somatoformes/diagnóstico , Transtornos Somatoformes/psicologiaRESUMO
Adipocyte differentiation, or adipogenesis, is a complex and highly regulated process. A recent proteomic analysis has predicted that the nonreceptor tyrosine kinase Abelson murine leukemia viral oncogene (c-Abl) is a putative key regulator of adipogenesis, but the underlying mechanism remained obscure. We found that c-Abl was activated during the early phase of mouse 3T3-L1 preadipocyte differentiation. Moreover, c-Abl activity was essential and its inhibition blocked differentiation to mature adipocytes. c-Abl directly controlled the expression and activity of the master adipogenic regulator peroxisome proliferator-activator receptor gamma 2 (PPARγ2). PPARγ2 physically associated with c-Abl and underwent phosphorylation on two tyrosine residues within its regulatory activation function 1 (AF1) domain. We demonstrated that this process positively regulates PPARγ2 stability and adipogenesis. Remarkably, c-Abl binding to PPARγ2 required the Pro12 residue that has a phenotypically well-studied common human genetic proline 12 alanine substitution (Pro12Ala) polymorphism. Our findings establish a critical role for c-Abl in adipocyte differentiation and explain the behavior of the known Pro12Ala polymorphism.
Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , PPAR gama/fisiologia , Proteínas Proto-Oncogênicas c-abl/fisiologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Animais , Benzamidas/farmacologia , Células HEK293 , Humanos , Mesilato de Imatinib , Camundongos , Mutação de Sentido Incorreto , Células NIH 3T3 , PPAR gama/química , PPAR gama/genética , Fosforilação , Fosfotirosina/química , Piperazinas/farmacologia , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Prolina/química , Ligação Proteica , Mapeamento de Interação de Proteínas , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/química , Pirimidinas/farmacologia , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Transcrição GênicaRESUMO
The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser(46) in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser(46), and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate.
Assuntos
Proteínas de Transporte/metabolismo , Dano ao DNA , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Apoptose/efeitos da radiação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Dano ao DNA/efeitos da radiação , Dimerização , Ativação Enzimática/efeitos da radiação , Raios gama , Humanos , Fosforilação/efeitos da radiação , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-abl/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Raios UltravioletaRESUMO
BACKGROUND: Depersonalization-derealization syndrome (DDS) is an underdiagnosed and underresearched clinical phenomenon. In Germany, its administrative prevalence is far below the threshold for orphan diseases, although according to epidemiological surveys the diagnosis should be comparable frequent as anorexia nervosa for instance. Against this background, we carried out a large comprehensive survey of a DDS series in a tertiary mental health center with a specialized depersonalization-derealization clinic. To reveal differential characteristics, we compared the DDS patients, who consulted the specialized depersonalization-derealization clinic, with a group of patients with depressive disorders without comorbid DDS from the regular outpatient clinic of the mental health center. METHODS: The sample comprised 223 patients with a diagnosis of depersonalization-derealization-syndrome and 1129 patients with a depressive disorder but without a comorbid diagnosis of DDS. DDS patients were described and compared with depressive outpatients in terms of sociodemographic characteristics, treatment history, treatment wishes, clinical symptomatology, prevailing psychosocial stressors, family history of common mental disorders and history of childhood trauma. RESULTS: Despite the high comorbidity of DDS patients with depressive disorders and comparable burden with symptoms of depression and anxiety, the clinical picture and course of both patient groups differed strongly. DDS patients were younger, had a significant preponderance of male sex, longer disease duration and an earlier age of onset, a higher education but were more often unemployed. They tended to show more severe functional impairment. They had higher rates of previous or current mental health care utilization. Nearly all DDS patients endorsed the wish for a symptom specific counseling and 70.7 % were interested in the internet-based treatment of their problems. DDS patients had lower levels of self-rated traumatic childhood experiences and current psychosocial stressors. However, they reported a family history of anxiety disorders more often. CONCLUSION: In consideration of the selection bias of this study, this case series supports the view that the course of the DDS tends to be long-lasting. DDS patients are severely impaired, utilizing mental health care to a high degree, which nevertheless might not meet their treatment needs, as patients strongly opt for obtaining disorder specific counseling. In view of the size of the problem, more research on the disorder, its course and its optimal treatment is urgently required.
Assuntos
Despersonalização/diagnóstico , Transtorno Depressivo/diagnóstico , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Fatores de Risco , Inquéritos e Questionários , Síndrome , Adulto JovemRESUMO
The 26S proteasome is the end point of the ubiquitin- and ATP-dependent degradation pathway. The 26S proteasome complex (26S PC) integrity and function has been shown to be highly dependent on ATP and its homolog nucleotides. We report here that the redox molecule NADH binds the 26S PC and is sufficient in maintaining 26S PC integrity even in the absence of ATP. Five of the 19S proteasome complex subunits contain a putative NADH binding motif (GxGxxG) including the AAA-ATPase subunit, Psmc1 (Rpt2). We demonstrate that recombinant Psmc1 binds NADH via the GxGxxG motif. Introducing the ΔGxGxxG Psmc1 mutant into cells results in reduced NADH-stabilized 26S proteasomes and decreased viability following redox stress induced by the mitochondrial inhibitor rotenone. The newly identified NADH binding of 26S proteasomes advances our understanding of the molecular mechanisms of protein degradation and highlights a new link between protein homeostasis and the cellular metabolic/redox state.
Assuntos
NADP/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Motivos de Aminoácidos , Animais , Estabilidade Enzimática/fisiologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , NADP/genética , Células NIH 3T3 , Oxirredução , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica/fisiologiaRESUMO
BACKGROUND: Depersonalization (DP) involves unpleasant experiences of detachment from one's sense of self or unreality in the environment. DP may occur in a broad range of conditions, among healthy persons due to sleep loss, drug induced, secondary to anxiety disorders or primary in depersonalization disorder. Although DP has an early age of onset, little is known about the prevalence and correlates of DP among adolescents. METHODS: Between January and June 2011, we conducted a questionnaire-based representative survey of pupils aged 12-18 years in the federal state Rhineland-Palatinate of Germany. The final sample comprised 3,809 pupils. We analyzed the prevalence of depersonalization and its correlates regarding sociodemographic characteristics, substance abuse, global mental distress and resilience factors. RESULTS: One-third of the sample showed severe global mental distress, and 11.9 % were in the range of clinically significant depersonalization. Depersonalized students were less often living with both parents (67.3 vs. 75.7 %), came more often from an disadvantaged socioeconomic background, had a very severe level of global mental distress (comparable to psychiatric inpatients), were more often smoking and abusing cannabis and they suffered from specific impairments regarding social insecurity, global self-efficacy and active coping abilities. CONCLUSIONS: Experiences of depersonalization were very common among adolescents and may indicate an increased risk for poor academic achievement and mental health in the long term. Prospective studies are needed to investigate the course and clinical relevance of depersonalization for the development of the adolescents.
Assuntos
Despersonalização/epidemiologia , Fumar/epidemiologia , Estresse Psicológico/epidemiologia , Adolescente , Criança , Comorbidade , Feminino , Alemanha/epidemiologia , Inquéritos Epidemiológicos , Humanos , Masculino , Fumar Maconha , Saúde Mental , Prevalência , Estudos Prospectivos , Fatores de Risco , Fatores Socioeconômicos , EstudantesRESUMO
We present novel research on the cortical dynamics of atypical perceptual and emotional processing in people with symptoms of depersonalization-derealization disorder (DP-DR). We used electroencephalography (EEG)/event-related potentials (ERPs) to delineate the early perceptual mechanisms underlying emotional face recognition and mirror touch in adults with low and high levels of DP-DR symptoms (low-DP and high-DP groups). Face-sensitive visual N170 showed markedly less differentiation for emotional versus neutral face-voice stimuli in the high- than in the low-DP group. This effect was related to self-reported bodily symptoms like disembodiment. Emotional face-voice primes altered mirror touch at somatosensory cortical components P45 and P100 differently in the two groups. In the high-DP group, mirror touch occurred only when seeing touch after being confronted with angry face-voice primes. Mirror touch in the low-DP group, however, was unaffected by preceding emotions. Modulation of mirror touch following angry others was related to symptoms of self-other confusion. Results suggest that others' negative emotions affect somatosensory processes in those with an altered sense of bodily self. Our findings are in line with the idea that disconnecting from one's body and self (core symptom of DP-DR) may be a defence mechanism to protect from the threat of negative feelings, which may be exacerbated through self-other confusion. This article is part of the theme issue 'Sensing and feeling: an integrative approach to sensory processing and emotional experience'.
Assuntos
Despersonalização , Eletroencefalografia , Emoções , Potenciais Evocados , Humanos , Emoções/fisiologia , Masculino , Feminino , Adulto , Despersonalização/psicologia , Despersonalização/fisiopatologia , Adulto Jovem , Reconhecimento Facial/fisiologia , Percepção do Tato/fisiologiaRESUMO
During the COVID-19 pandemic, the early emergence of viral variants repeatedly undermined the effects of vaccination. Our aim here is to explore strategies for improving spike vaccine gene antigenicity by merging mutations from different variants of concern (VOCs) in a single vaccine gene. To this end, newly developed recombinant vaccine genes were designed, cloned into adenoviral vectors, and applied to C57BL/6 mice; then, serum-neutralizing antibodies against the wildtype SARS-CoV-2 strains were determined in neutralization assays. The merger of mutations from different variants of concern (alpha, beta, gamma, and delta) in a single recombinant spike-based vaccine gene provided a substantial improvement in neutralizing immunity to all variants of concern, including the omicron strains. To date, only unmodified spike genes of the original SARS-CoV-2 Wuhan strain (B.1) or dominant variants (BA.1, BA.5, and XBB.1.5) have been used as vaccine genes. The employment of unmodified vaccine genes is afflicted by limited cross-protection among variant strains. In contrast, recombinant vaccine genes that combine mutations from different strains in a single gene hold the potential to broaden and improve immune protection and might help to reduce the need for frequent vaccine adaptations in the future.
RESUMO
Mucus is a dynamic biological hydrogel, composed primarily of the glycoprotein mucin, exhibits unique biophysical properties and forms a barrier protecting cells against a broad-spectrum of viruses. Here, this work develops a polyglycerol sulfate-based dendronized mucin-inspired copolymer (MICP-1) with ≈10% repeating units of activated disulfide as cross-linking sites. Cryo-electron microscopy (Cryo-EM) analysis of MICP-1 reveals an elongated single-chain fiber morphology. MICP-1 shows potential inhibitory activity against many viruses such as herpes simplex virus 1 (HSV-1) and SARS-CoV-2 (including variants such as Delta and Omicron). MICP-1 produces hydrogels with viscoelastic properties similar to healthy human sputum and with tuneable microstructures using linear and branched polyethylene glycol-thiol (PEG-thiol) as cross-linkers. Single particle tracking microrheology, electron paramagnetic resonance (EPR) and cryo-scanning electron microscopy (Cryo-SEM) are used to characterize the network structures. The synthesized hydrogels exhibit self-healing properties, along with viscoelastic properties that are tuneable through reduction. A transwell assay is used to investigate the hydrogel's protective properties against viral infection against HSV-1. Live-cell microscopy confirms that these hydrogels can protect underlying cells from infection by trapping the virus, due to both network morphology and anionic multivalent effects. Overall, this novel mucin-inspired copolymer generates mucus-mimetic hydrogels on a multi-gram scale. These hydrogels can be used as models for disulfide-rich airway mucus research, and as biomaterials.