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1.
Viruses ; 15(10)2023 10 19.
Artículo en Inglés | MEDLINE | ID: mdl-37896901

RESUMEN

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells by binding to the angiotensin-converting enzyme 2 (hACE2) receptor. This process is aided by the transmembrane protease serine 2 (TMPRSS2), which enhances entry efficiency and infectiousness by cleaving the SARS-CoV-2 surface glycoprotein (Spike). The cleavage primes the Spike protein, promoting membrane fusion instead of receptor-mediated endocytosis. Despite the pivotal role played by TMPRSS2, our understanding of its non-protease distinct domains remains limited. In this report, we present evidence indicating the potential phosphorylation of a minimum of six tyrosine residues within the cytosolic tail (CT) of TMPRSS2. Via the use of TMPRSS2 CT phospho-mimetic mutants, we observed a reduction in TMPRSS2 protease activity, accompanied by a decrease in SARS-CoV-2 pseudovirus transduction, which was found to occur mainly via the endosomal pathway. We expanded our investigation beyond TMPRSS2 CT and discovered the involvement of other non-protease domains in regulating infection. Our co-immunoprecipitation experiments demonstrated a strong interaction between TMPRSS2 and Spike. We revealed a 21 amino acid long TMPRSS2-Spike-binding region (TSBR) within the TMPRSS2 scavenger receptor cysteine-rich (SRCR) domain that contributes to this interaction. Our study sheds light on novel functionalities associated with TMPRSS2's cytosolic tail and SRCR region. Both of these regions have the capability to regulate SARS-CoV-2 entry pathways. These findings contribute to a deeper understanding of the complex interplay between viral entry and host factors, opening new avenues for potential therapeutic interventions.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Internalización del Virus , Péptido Hidrolasas , Serina , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo
2.
Viruses ; 15(5)2023 05 09.
Artículo en Inglés | MEDLINE | ID: mdl-37243215

RESUMEN

The COVID-19 pandemic resulted from the global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since its first appearance in 2019, new SARS-CoV-2 variants of concern (VOCs) have emerged frequently, changing the infection's dynamic. SARS-CoV-2 infects cells via two distinct entry routes; receptor-mediated endocytosis or membrane fusion, depending on the absence or presence of transmembrane serine protease 2 (TMPRSS2), respectively. In laboratory conditions, the Omicron SARS-CoV-2 strain inefficiently infects cells predominantly via endocytosis and is phenotypically characterized by decreased syncytia formation compared to the earlier Delta variant. Thus, it is important to characterize Omicron's unique mutations and their phenotypic manifestations. Here, by utilizing SARS-CoV-2 pseudovirions, we report that the specific Omicron Spike F375 residue decreases infectivity, and its conversion to the Delta S375 sequence significantly increases Omicron infectivity. Further, we identified that residue Y655 decreases Omicron's TMPRSS2 dependency and entry via membrane fusion. The Y655H, K764N, K856N and K969N Omicron revertant mutations, bearing the Delta variant sequence, increased the cytopathic effect of cell-cell fusion, suggesting these Omicron-specific residues reduced the severity of SARS-CoV-2. This study of the correlation of the mutational profile with the phenotypic outcome should sensitize our alertness towards emerging VOCs.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Pandemias , Mutación , Glicoproteína de la Espiga del Coronavirus/genética , Serina Endopeptidasas/genética
3.
Cell Rep Med ; 4(11): 101278, 2023 11 21.
Artículo en Inglés | MEDLINE | ID: mdl-37944529

RESUMEN

The choroid plexus (CP) plays a key role in remotely controlling brain function in health, aging, and disease. Here, we report that CP epithelial cells express the brain-specific cholesterol 24-hydroxylase (CYP46A1) and that its levels are decreased under different mouse and human brain conditions, including amyloidosis, aging, and SARS-CoV-2 infection. Using primary mouse CP cell cultures, we demonstrate that the enzymatic product of CYP46A1, 24(S)-hydroxycholesterol, downregulates inflammatory transcriptomic signatures within the CP, found here to be elevated across multiple neurological conditions. In vitro, the pro-inflammatory cytokine tumor necrosis factor α (TNF-α) downregulates CYP46A1 expression, while overexpression of CYP46A1 or its pharmacological activation in mouse CP organ cultures increases resilience to TNF-α. In vivo, overexpression of CYP46A1 in the CP in transgenic mice with amyloidosis is associated with better cognitive performance and decreased brain inflammation. Our findings suggest that CYP46A1 expression in the CP impacts the role of this niche as a guardian of brain immune homeostasis.


Asunto(s)
Amiloidosis , Plexo Coroideo , Humanos , Ratones , Animales , Colesterol 24-Hidroxilasa/metabolismo , Plexo Coroideo/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Encéfalo/patología , Homeostasis/fisiología , Ratones Transgénicos , Amiloidosis/metabolismo , Amiloidosis/patología
4.
Sci Rep ; 12(1): 5758, 2022 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-35388061

RESUMEN

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal agent of the COVID-19 pandemic. More than 274 million individuals have suffered from COVID-19 and over five million people have died from this disease so far. Therefore, there is an urgent need for therapeutic drugs. Repurposing FDA approved drugs should be favored since evaluation of safety and efficacy of de-novo drug design are both costly and time consuming. We report that imatinib, an Abl tyrosine kinase inhibitor, robustly decreases SARS-CoV-2 infection and uncover a mechanism of action. We show that imatinib inhibits the infection of SARS-CoV-2 and its surrogate lentivector pseudotype. In latter, imatinib inhibited both routes of viral entry, endocytosis and membrane-fusion. We utilized a system to quantify in real-time cell-cell membrane fusion mediated by the SARS-CoV-2 surface protein, Spike, and its receptor, hACE2, to demonstrate that imatinib inhibits this process in an Abl1 and Abl2 independent manner. Furthermore, cellular thermal shift assay revealed a direct imatinib-Spike interaction that affects Spike susceptibility to trypsin digest. Collectively, our data suggest that imatinib inhibits Spike mediated viral entry by an off-target mechanism. These findings mark imatinib as a promising therapeutic drug in inhibiting the early steps of SARS-CoV-2 infection.


Asunto(s)
Tratamiento Farmacológico de COVID-19 , Humanos , Mesilato de Imatinib/farmacología , Pandemias , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus
5.
iScience ; 25(10): 105193, 2022 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-36188189

RESUMEN

Blocking the interaction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with its angiotensin-converting enzyme 2 (ACE2) receptor was proved to be an effective therapeutic option. Various protein binders as well as monoclonal antibodies that effectively target the receptor-binding domain (RBD) of SARS-CoV-2 to prevent interaction with ACE2 were developed. The emergence of SARS-CoV-2 variants that accumulate alterations in the RBD can severely affect the efficacy of such immunotherapeutic agents, as is indeed the case with Omicron that resists many of the previously isolated monoclonal antibodies. Here, we evaluate an ACE2-based immunoadhesin that we have developed early in the pandemic against some of the recent variants of concern (VoCs), including the Delta and the Omicron variants. We show that our ACE2-immunoadhesin remains effective in neutralizing these variants, suggesting that immunoadhesin-based immunotherapy is less prone to escape by the virus and has a potential to remain effective against future VoCs.

6.
Front Pharmacol ; 12: 671929, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34234670

RESUMEN

Silent information regulator 2-related enzyme 1 (SIRT1) is an NAD+-dependent class III deacetylase and a key component of the cellular metabolic sensing pathway. The requirement of NAD+ for SIRT1 activity led us to assume that NQO1, an NADH oxidoreductase producing NAD+, regulates SIRT1 activity. We show here that SIRT1 is capable of increasing NQO1 (NAD(P)H Dehydrogenase Quinone 1) transcription and protein levels. NQO1 physically interacts with SIRT1 but not with an enzymatically dead SIRT1 H363Y mutant. The interaction of NQO1 with SIRT1 is markedly increased under mitochondrial inhibition. Interestingly, under this condition the nuclear pool of NQO1 is elevated. Depletion of NQO1 compromises the role of SIRT1 in inducing transcription of several target genes and eliminates the protective role of SIRT1 following mitochondrial inhibition. Our results suggest that SIRT1 and NQO1 form a regulatory loop where SIRT1 regulates NQO1 expression and NQO1 binds and mediates the protective role of SIRT1 during mitochondrial stress. The interplay between an NADH oxidoreductase enzyme and an NAD+ dependent deacetylase may act as a rheostat in sensing mitochondrial stress.

7.
Prion ; 13(1): 160-172, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-31476957

RESUMEN

After the discovery of two atypical bovine spongiform encephalopathy (BSE) forms in France and Italy designated H- and L-BSE, the question arose whether these new forms differed from classical BSE (C-BSE) in their pathogenesis. Samples collected from cattle in the clinical stage of BSE during an intracranial challenge study with L- and H-BSE were analysed using biochemical and histological methods as well as in a transgenic mouse bioassay. Our results generally confirmed what had been described for C-BSE to be true also for both atypical BSE forms, namely the restriction of the pathological prion protein (PrPSc) and BSE infectivity to the nervous system. However, analysis of samples collected under identical conditions from both atypical H- and L-BSE forms allowed us a more precise assessment of the grade of involvement of different tissues during the clinical end stage of disease as compared to C-BSE. One important feature is the involvement of the peripheral nervous and musculoskeletal tissues in both L-BSE and H-BSE affected cattle. We were, however, able to show that in H-BSE cases, the PrPSc depositions in the central and peripheral nervous system are dominated by a glial pattern, whereas a neuronal deposition pattern dominates in L-BSE cases, indicating differences in the cellular and topical tropism of both atypical BSE forms. As a consequence of this cell tropism, H-BSE seems to spread more rapidly from the CNS into the periphery via the glial cell system such as Schwann cells, as opposed to L-BSE which is mostly propagated via neuronal cells.


Asunto(s)
Bovinos , Encefalopatía Espongiforme Bovina/diagnóstico , Proteínas PrPSc/análisis , Animales , Bovinos/fisiología , Sistema Nervioso Central/patología , Encefalopatía Espongiforme Bovina/patología , Femenino , Ratones , Neuroglía/patología , Neuronas/patología , Médula Espinal/patología , Ganglio del Trigémino/patología
8.
Toxicol Lett ; 293: 51-61, 2018 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-29183814

RESUMEN

BACKGROUND: Sulfur mustard (SM) is a potent blistering chemical warfare agent, which was first used in 1917. Despite the Chemical Weapons Convention, a use was recently reported in Syria in 2015. This emphasizes the importance to develop countermeasures against chemical warfare agents. Despite intensive research, there is still no antidote or prophylaxis available against SM. METHODS: The newly developed SM-resistant keratinocyte cell line HaCaT/SM was used to identify new target structures for drug development, particularly the adaptations in protective measures against oxidative stress. For this purpose, glutathione (GSH) and NAD(P)H levels, the effect of glutathione S-transferase (GST) inhibition as well as activation and expression of Nrf2, GST, glutamate cysteine ligase (GCL) and glutathione-disulfide reductase (GSR) as well as multi-drug resistance (MDR) proteins 1, 3 and 5 were investigated. RESULTS: The HaCaT/SM cells showed not only a better survival after treatment with SM or cytostatic drugs, but also hydrogen peroxide (H2O2). They exhibit more GSH even after SM treatment. Nrf2 levels were significantly lower. Inhibition of GST led to significantly decreased, activation to slightly higher IC50 values after SM treatment and a lower expression of GST was observed. The cells also expressed less GCLC and GSR. Expression of MDR1, MDR3 and MDR5 was higher under control conditions, but less stimulated by SM treatment. An increased NADP+/NADPH ratio as well as higher NAD+ levels were shown. CONCLUSION: In summary, an improved response of the resistant cell line to oxidative stress was observed. The underlying mechanisms are elevated GSH levels as well as lower expression of Nrf2 and its targets GCLC and GST as well as GSR and MDR1, MDR3 and MDR5. GST is an especially interesting target because its inhibition already induced a significant SM sensitivity. SM resistance also caused redox equivalent level differences. Taken together, these findings provide further insight into the mechanism of SM resistance and may open a window for novel therapeutic targets in SM therapy.


Asunto(s)
Antioxidantes/metabolismo , Sustancias para la Guerra Química/farmacología , Glutatión/metabolismo , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Gas Mostaza/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Resistencia a Múltiples Medicamentos , Humanos , NAD/metabolismo , NADP/metabolismo , Factor 2 Relacionado con NF-E2/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos
9.
Toxicol Lett ; 244: 49-55, 2016 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-26456177

RESUMEN

BACKGROUND: The cell line HaCaT/SM was derived from the human keratinocyte cell line HaCaT. HaCaT/SM cells display a high resistance against sulfur mustard (SM). Intention of the presented study was to determine the cellular and molecular differences between HaCaT/SM and HaCaT so as to evaluate which changes might be responsible for being resistant against SM. METHODS: Both cell lines HaCaT and HaCaT/SM were analyzed with respect to their cell growth, nuclei perimeter, clonogenicity and secretion profile. Moreover DNA alkylation pattern under presence of SM was investigated. RESULTS: In comparison to HaCaT, the HaCaT/SM showed a significant smaller nuclei perimeter. For DNA alkylation a significant difference was observed over time. The clonogenicity of HaCaT/SM was increased to 150%. The secretion profile of these cells demonstrated a strong increase of ANG, PDGF-AA, TIMP1, TIMP2, and a decrease of AREG, CCL5, CXC1, CXC2/3, CXCL6, CXCL7, CXCL8, CXCL10, MIF, Trappin-1. CONCLUSION: The sulfur mustard (SM) resistant cell line HaCaT/SM demonstrates a wide range of significant differences to their origin cell line HaCaT. These differences might be responsible to provide resistance against SM and might also be useful to establish treatment concepts for humans after SM exposure.


Asunto(s)
Sustancias para la Guerra Química/toxicidad , Resistencia a Medicamentos , Queratinocitos/efectos de los fármacos , Gas Mostaza/toxicidad , Biomarcadores/metabolismo , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/patología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Aductos de ADN/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Queratinocitos/metabolismo , Queratinocitos/patología , Fenotipo , Factores de Tiempo
10.
Toxicol Lett ; 244: 56-71, 2016 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-26383629

RESUMEN

Mustard agents are potent DNA alkylating agents with mutagenic, cytotoxic and vesicant properties. They include bi-functional agents, such as sulfur mustard (SM) or nitrogen mustard (mustine, HN2), as well as mono-functional agents, such as "half mustard" (CEES). Whereas SM has been used as a chemical warfare agent, several nitrogen mustard derivatives, such as chlorambucil and cyclophosphamide, are being used as established chemotherapeutics. Upon induction of specific forms of genotoxic stimuli, several poly(ADP-ribose) polymerases (PARPs) synthesize the nucleic acid-like biopolymer poly(ADP-ribose) (PAR) by using NAD(+) as a substrate. Previously, it was shown that SM triggers cellular poly(ADP-ribosyl) ation (PARylation), but so far this phenomenon is poorly characterized. In view of the protective effects of PARP inhibitors, the latter have been proposed as a treatment option of SM-exposed victims. In an accompanying article (Debiak et al., 2016), we have provided an optimized protocol for the analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to further analyze mustard-induced PARylation and its functional consequences, in general. Thus, in the present study, we performed a comprehensive characterization of the PARylation response in HaCaT cells after treatment with four different mustard agents, i.e., SM, CEES, HN2, and chlorambucil, on a qualitative, quantitative and functional level. In particular, we recorded substance-specific as well as dose- and time-dependent PARylation responses using independent bioanalytical methods based on single-cell immuno-fluorescence microscopy and quantitative isotope dilution mass spectrometry. Furthermore, we analyzed if and how PARylation contributes to mustard-induced toxicity by treating HaCaT cells with CEES, SM, and HN2 in combination with the clinically relevant PARP inhibitor ABT888. As evaluated by a novel immunofluorescence-based protocol for the detection of N7-ETE-guanine DNA adducts, the excision rate of CEES-induced DNA adducts was not affected by PARP inhibition. Furthermore, while CEES induced moderate changes in cellular NAD(+) levels, annexin V/PI flow cytometry analysis revealed that these changes did not affect CEES-induced short-term cytotoxicity 24h after treatment. In contrast, PARP inhibition impaired cell proliferation and clonogenic survival, and potentiated micronuclei formation of HaCaT cells upon CEES treatment. Similarly, PARP inhibition affected clonogenic survival of cells treated with bi-functional mustards such as SM and HN2. In conclusion, we demonstrate that PARylation plays a functional role in mustard-induced cellular stress response with substance-specific differences. Since PARP inhibitors exhibit therapeutic potential to treat SM-related pathologies and to sensitize cancer cells for mustard-based chemotherapy, potential long-term effects of PARP inhibition on genomic stability and carcinogenesis should be carefully considered when pursuing such a strategy.


Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Antineoplásicos Alquilantes/toxicidad , Sustancias para la Guerra Química/toxicidad , Queratinocitos/efectos de los fármacos , Gas Mostaza/toxicidad , Compuestos de Mostaza Nitrogenada/toxicidad , Poli(ADP-Ribosa) Polimerasas/metabolismo , Antídotos/toxicidad , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Clorambucilo/toxicidad , Aductos de ADN/metabolismo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Inestabilidad Genómica/efectos de los fármacos , Humanos , Queratinocitos/enzimología , Queratinocitos/patología , Mecloretamina/toxicidad , Micronúcleos con Defecto Cromosómico/inducido químicamente , Gas Mostaza/análogos & derivados , Inhibidores de Poli(ADP-Ribosa) Polimerasas/toxicidad , Transducción de Señal/efectos de los fármacos , Factores de Tiempo
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