RESUMO
Heat shock protein 90 (Hsp90) is known to mediate heme insertion and activation of heme-deficient neuronal nitric oxide (NO) synthase (apo-nNOS) in cells by a highly dynamic interaction that has been extremely difficult to study mechanistically with the use of subcellular systems. In that the heme content of many critical hemeproteins is regulated by Hsp90 and the heme chaperone GAPDH, the development of an in vitro system for the study of this chaperone-mediated heme regulation would be extremely useful. Here, we show that use of an antibody-immobilized apo-nNOS led not only to successful assembly of chaperone complexes but the ability to show a clear dependence on Hsp90 and GAPDH for heme-mediated activation of apo-nNOS. The kinetics of binding for Hsp70 and Hsp90, the ATP and K+ dependence, and the absolute requirement for Hsp70 in assembly of Hsp90â¢apo-nNOS heterocomplexes all point to a similar chaperone machinery to the well-established canonical machine regulating steroid hormone receptors. However, unlike steroid receptors, the use of a purified protein system containing Hsp90, Hsp70, Hsp40, Hop, and p23 is unable to activate apo-nNOS. Thus, heme insertion requires a unique Hsp90-chaperone complex. With this newly developed in vitro system, which recapitulates the cellular process requiring GAPDH as well as Hsp90, further mechanistic studies are now possible to better understand the components of the Hsp90-based chaperone system as well as how this heterocomplex works with GAPDH to regulate nNOS and possibly other hemeproteins.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases , Proteínas de Choque Térmico HSP70 , Proteínas de Choque Térmico HSP90 , Heme , Hemeproteínas , Chaperonas Moleculares , Óxido Nítrico Sintase , Heme/química , Hemeproteínas/química , Hemeproteínas/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Ligação Proteica , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/metabolismo , Enzimas Imobilizadas , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Ativação EnzimáticaRESUMO
BACKGROUND: Ebola virus disease (EVD) supportive care strategies are largely guided by retrospective observational research. This study investigated the effect of EVD supportive care algorithms on duration of survival in a controlled nonhuman primate (NHP) model. METHODS: Fourteen rhesus macaques were challenged intramuscularly with a target dose of Ebola virus (1000 plaque-forming units; Kikwit). NHPs were allocated to intensive care unit (ICU)-like algorithms (nâ =â 7), intravenous fluids plus levofloxacin (nâ =â 2), or a control group (nâ =â 5). The primary outcome measure was duration of survival, and secondary outcomes included changes in clinical laboratory values. RESULTS: Duration of survival was not significantly different between the pooled ICU-like algorithm and control groups (8.2 vs 6.9 days of survival; hazard ratio; 0.50; Pâ =â .25). Norepinephrine was effective in transiently maintaining baseline blood pressure. NHPs treated with ICU-like algorithms had delayed onset of liver and kidney injury. CONCLUSIONS: While an obvious survival difference was not observed with ICU-like care, clinical observations from this model may aid in EVD supportive care NHP model refinement.
Assuntos
Cuidados Críticos , Doença pelo Vírus Ebola , Unidades de Terapia Intensiva , Animais , Modelos Animais de Doenças , Ebolavirus , Doença pelo Vírus Ebola/terapia , Macaca mulatta , Primatas , Estudos RetrospectivosRESUMO
Crimean-Congo hemorrhagic fever (CCHF) is the most medically important tick-borne viral disease of humans and tuberculosis is the leading cause of death worldwide by a bacterial pathogen. These two diseases overlap geographically, however, concurrent infection of CCHF virus (CCHFV) with mycobacterial infection has not been assessed nor has the ability of virus to persist and cause long-term sequela in a primate model. In this study, we compared the disease progression of two diverse strains of CCHFV in the recently described cynomolgus macaque model. All animals demonstrated signs of clinical illness, viremia, significant changes in clinical chemistry and hematology values, and serum cytokine profiles consistent with CCHF in humans. The European and Asian CCHFV strains caused very similar disease profiles in monkeys, which demonstrates that medical countermeasures can be evaluated in this animal model against multiple CCHFV strains. We identified evidence of CCHFV persistence in the testes of three male monkeys that survived infection. Furthermore, the histopathology unexpectedly revealed that six additional animals had evidence of a latent mycobacterial infection with granulomatous lesions. Interestingly, CCHFV persisted within the granulomas of two animals. This study is the first to demonstrate the persistence of CCHFV in the testes and within the granulomas of non-human primates with concurrent latent tuberculosis. Our results have important public health implications in overlapping endemic regions for these emerging pathogens.
Assuntos
Febre Hemorrágica da Crimeia/complicações , Tuberculose Latente/complicações , Testículo/patologia , Animais , Anticorpos Antivirais/sangue , Doenças Transmissíveis Emergentes/complicações , Doenças Transmissíveis Emergentes/patologia , Doenças Transmissíveis Emergentes/virologia , Citocinas/sangue , Modelos Animais de Doenças , Progressão da Doença , Granuloma/microbiologia , Granuloma/patologia , Granuloma/virologia , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Vírus da Febre Hemorrágica da Crimeia-Congo/patogenicidade , Febre Hemorrágica da Crimeia/patologia , Febre Hemorrágica da Crimeia/virologia , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Tuberculose Latente/microbiologia , Tuberculose Latente/patologia , Macaca fascicularis , Masculino , Testículo/microbiologia , Testículo/virologiaRESUMO
The polyglutamine (polyQ) diseases are a group of nine fatal, adult-onset neurodegenerative disorders characterized by the misfolding and aggregation of mutant proteins containing toxic expansions of CAG/polyQ tracts. The heat shock protein 90 and 70 (Hsp90/Hsp70) chaperone machinery is a key component of cellular protein quality control, playing a role in the regulation of folding, aggregation, and degradation of polyQ proteins. The ability of Hsp70 to facilitate disaggregation and degradation of misfolded proteins makes it an attractive therapeutic target in polyQ diseases. Genetic studies have demonstrated that manipulation of Hsp70 and related co-chaperones can enhance the disaggregation and/or degradation of misfolded proteins in models of polyQ disease. Therefore, the development of small molecules that enhance Hsp70 activity is of great interest. However, it is still unclear if currently available Hsp70 modulators can selectively enhance disaggregation or degradation of misfolded proteins without perturbing other Hsp70 functions essential for cellular homeostasis. This review discusses the multifaceted role of Hsp70 in protein quality control and the opportunities and challenges Hsp70 poses as a potential therapeutic target in polyQ disease.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Peptídeos/metabolismo , Agregação Patológica de Proteínas/metabolismo , Animais , Humanos , Doença de Huntington/tratamento farmacológico , Doença de Huntington/metabolismo , Terapia de Alvo Molecular , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/metabolismo , Agregação Patológica de Proteínas/tratamento farmacológico , Dobramento de Proteína/efeitos dos fármacos , Deficiências na Proteostase/tratamento farmacológico , Deficiências na Proteostase/metabolismo , Ataxias Espinocerebelares/tratamento farmacológico , Ataxias Espinocerebelares/metabolismoRESUMO
Background: For most classes of drugs, rapid development of therapeutics to treat emerging infections is challenged by the timelines needed to identify compounds with the desired efficacy, safety, and pharmacokinetic profiles. Fully human monoclonal antibodies (mAbs) provide an attractive method to overcome many of these hurdles to rapidly produce therapeutics for emerging diseases. Methods: In this study, we deployed a platform to generate, test, and develop fully human antibodies to Zaire ebolavirus. We obtained specific anti-Ebola virus (EBOV) antibodies by immunizing VelocImmune mice that use human immunoglobulin variable regions in their humoral responses. Results: Of the antibody clones isolated, 3 were selected as best at neutralizing EBOV and triggering FcγRIIIa. Binding studies and negative-stain electron microscopy revealed that the 3 selected antibodies bind to non-overlapping epitopes, including a potentially new protective epitope not targeted by other antibody-based treatments. When combined, a single dose of a cocktail of the 3 antibodies protected nonhuman primates (NHPs) from EBOV disease even after disease symptoms were apparent. Conclusions: This antibody cocktail provides complementary mechanisms of actions, incorporates novel specificities, and demonstrates high-level postexposure protection from lethal EBOV disease in NHPs. It is now undergoing testing in normal healthy volunteers in preparation for potential use in future Ebola epidemics.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Doença pelo Vírus Ebola/tratamento farmacológico , Animais , Anticorpos Monoclonais/isolamento & purificação , Glicoproteínas/imunologia , Cobaias , Células HEK293 , Humanos , Macaca mulatta , Masculino , CamundongosRESUMO
Several hundred proteins cycle into heterocomplexes with a dimer of the chaperone heat shock protein 90 (Hsp90), regulating their activity and turnover. There are two isoforms of Hsp90, Hsp90α and Hsp90ß, and their relative chaperone activities and composition in these client proteinâ¢Hsp90 heterocomplexes has not been determined. Here, we examined the activity of human Hsp90α and Hsp90ß in a purified five-protein chaperone machinery that assembles glucocorticoid receptor (GR)â¢Hsp90 heterocomplexes to generate high-affinity steroid-binding activity. We found that human Hsp90α and Hsp90ß have equivalent chaperone activities, and when mixed together in this assay, they formed only GRâ¢Hsp90αα and GRâ¢Hsp90ßß homodimers and no GRâ¢Hsp90αß heterodimers. In contrast, GRâ¢Hsp90 heterocomplexes formed in human embryonic kidney (HEK) cells also contain GRâ¢Hsp90αß heterodimers. The formation of GRâ¢Hsp90αß heterodimers in HEK cells probably reflects the longer time permitted for exchange to form Hsp90αß heterodimers in the cell versus in the cell-free assembly conditions. This purified GR-activating chaperone machinery can be used to determine how modifications of Hsp90 affect its chaperone activity. To that effect, we have tested whether the unique phosphorylation of Hsp90α at threonines 5 and 7 that occurs during DNA damage repair affects its chaperone activity. We showed that the phosphomimetic mutant Hsp90α T5/7D has the same intrinsic chaperone activity as wild-type human Hsp90α in activation of GR steroid-binding activity by the five-protein machinery, supporting the conclusion that T5/7 phosphorylation does not affect Hsp90α chaperone activity.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Multimerização Proteica/fisiologia , Receptores de Glucocorticoides/metabolismo , Animais , Células HEK293 , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP90/química , Humanos , Camundongos , Chaperonas Moleculares/química , Ligação Proteica/fisiologia , Receptores de Glucocorticoides/químicaRESUMO
Currently available therapies for adult onset neurodegenerative diseases provide symptomatic relief but do not modify disease progression. Here we explore a new neuroprotective approach based on drugs targeting chaperone-directed protein quality control. Critical target proteins that unfold and aggregate in these diseases, such as the polyglutamine androgen receptor in spinal and bulbar muscular atrophy, huntingtin in Huntington's disease, α-synuclein in Parkinson's disease, and tau in Alzheimer's disease, are client proteins of heat shock protein 90 (Hsp90), and their turnover is regulated by the protein quality control function of the Hsp90/Hsp70-based chaperone machinery. Hsp90 and Hsp70 have opposing effects on client protein stability in protein quality control; Hsp90 stabilizes the clients and inhibits their ubiquitination, whereas Hsp70 promotes ubiquitination dependent on CHIP (C terminus of Hsc70-interacting protein) and proteasomal degradation. We discuss how drugs that modulate proteostasis by inhibiting Hsp90 function or promoting Hsp70 function enhance the degradation of the critical aggregating proteins and ameliorate toxic symptoms in cell and animal disease models.
Assuntos
Proteínas de Choque Térmico HSP70/agonistas , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Sistema Nervoso/efeitos dos fármacos , Doenças Neurodegenerativas/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Adulto , Idade de Início , Animais , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Sistema Nervoso/metabolismo , Sistema Nervoso/fisiopatologia , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Estabilidade Proteica , Transdução de Sinais/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Unprotected sexual intercourse between persons residing in or traveling from regions with Zika virus transmission is a risk factor for infection. To model risk for infection after sexual intercourse, we inoculated rhesus and cynomolgus macaques with Zika virus by intravaginal or intrarectal routes. In macaques inoculated intravaginally, we detected viremia and virus RNA in 50% of macaques, followed by seroconversion. In macaques inoculated intrarectally, we detected viremia, virus RNA, or both, in 100% of both species, followed by seroconversion. The magnitude and duration of infectious virus in the blood of macaques suggest humans infected with Zika virus through sexual transmission will likely generate viremias sufficient to infect competent mosquito vectors. Our results indicate that transmission of Zika virus by sexual intercourse might serve as a virus maintenance mechanism in the absence of mosquito-to-human transmission and could increase the probability of establishment and spread of Zika virus in regions where this virus is not present.
Assuntos
Macaca fascicularis , Macaca mulatta , Infecção por Zika virus/virologia , Zika virus/fisiologia , Animais , Feminino , Masculino , Vagina , Replicação Viral , Eliminação de Partículas Virais , Infecção por Zika virus/transmissãoRESUMO
We sought new strategies to reduce amounts of the polyglutamine androgen receptor (polyQ AR) and achieve benefits in models of spinobulbar muscular atrophy, a protein aggregation neurodegenerative disorder. Proteostasis of the polyQ AR is controlled by the heat shock protein 90 (Hsp90)- and Hsp70-based chaperone machinery, but mechanisms regulating the protein's turnover are incompletely understood. We demonstrate that overexpression of Hsp70 interacting protein (Hip), a co-chaperone that enhances binding of Hsp70 to its substrates, promotes client protein ubiquitination and polyQ AR clearance. Furthermore, we identify a small molecule that acts similarly to Hip by allosterically promoting Hsp70 binding to unfolded substrates. Like Hip, this synthetic co-chaperone enhances client protein ubiquitination and polyQ AR degradation. Both genetic and pharmacologic approaches targeting Hsp70 alleviate toxicity in a Drosophila model of spinobulbar muscular atrophy. These findings highlight the therapeutic potential of allosteric regulators of Hsp70 and provide new insights into the role of the chaperone machinery in protein quality control.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Peptídeos/química , Animais , Relação Dose-Resposta a Droga , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacologia , Drosophila , Feminino , Células HEK293 , Células HeLa , Humanos , Concentração Inibidora 50 , Modelos Químicos , Chaperonas Moleculares/química , Transtornos Musculares Atróficos/metabolismo , Neurotoxinas/química , Células PC12 , Estrutura Terciária de Proteína , Proteínas/química , Piridinas/farmacologia , Ratos , Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Tiazóis/farmacologia , UbiquitinaçãoRESUMO
Antibody therapies to prevent or limit filovirus infections have received modest interest in recent years, in part because of early negative experimental evidence. We have overcome the limitations of this approach, leveraging the use of antibody from nonhuman primates (NHPs) that survived challenge to filoviruses under controlled conditions. By using concentrated, polyclonal IgG antibody from these survivors, we treated filovirus-infected NHPs with multiple doses administered over the clinical phase of disease. In the first study, Marburg virus (MARV)-infected NHPs were treated 15 to 30 min postexposure with virus-specific IgG, with additional treatments on days 4 and 8 postexposure. The postexposure IgG treatment was completely protective, with no signs of disease or detectable viremia. MARV-specific IgM antibody responses were generated, and all macaques survived rechallenge with MARV, suggesting that they generated an immune response to virus replication. In the next set of studies, NHPs were infected with MARV or Ebola virus (EBOV), and treatments were delayed 48 h, with additional treatments on days 4 and 8 postexposure. The delayed treatments protected both MARV- and EBOV-challenged NHPs. In both studies, two of the three IgG-treated NHPs had no clinical signs of illness, with the third NHP developing mild and delayed signs of disease followed by full recovery. These studies clearly demonstrate that postexposure antibody treatments can protect NHPs and open avenues for filovirus therapies for human use using established Food and Drug Administration-approved polyclonal or monoclonal antibody technologies.
Assuntos
Anticorpos Antivirais/imunologia , Infecções por Filoviridae/imunologia , Infecções por Filoviridae/prevenção & controle , Filoviridae/imunologia , Macaca mulatta/imunologia , Macaca mulatta/virologia , Animais , Fracionamento Químico , Ebolavirus/imunologia , Infecções por Filoviridae/virologia , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/imunologia , Marburgvirus/imunologia , Testes de Neutralização , Especificidade da Espécie , Análise de SobrevidaRESUMO
An eight-amino acid segment is known to be responsible for the marked difference in the rates of degradation of the EGF receptor (ErbB1) and ErbB2 upon treatment of cells with the Hsp90 inhibitor geldanamycin. We have scrambled the first six amino acids of this segment of the EGF receptor (EGFR), which lies in close association with the ATP binding cleft and the dimerization face. Scrambling these six amino acids markedly reduces EGFR stability, EGF-stimulated receptor dimerization, and autophosphorylation activity. Two peptides were synthesized as follows: one containing the wild-type sequence of the eight-amino acid segment, which we call Disruptin; and one with the scrambled sequence. Disruptin inhibits Hsp90 binding to the EGFR and causes slow degradation of the EGFR in two EGFR-dependent cancer cell lines, whereas the scrambled peptide is inactive. This effect is specific for EGFR versus other Hsp90 client proteins. In the presence of EGF, Disruptin, but not the scrambled peptide, inhibits EGFR dimerization and causes rapid degradation of the EGFR. In contrast to the Hsp90 inhibitor geldanamycin, Disruptin inhibits cancer cell growth by a nonapoptotic mechanism. Disruptin provides proof of concept for the development of a new class of anti-tumor drugs that specifically cause EGFR degradation.
Assuntos
Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , Animais , Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Células CHO , Linhagem Celular Tumoral , Cricetulus , Dimerização , Desenho de Fármacos , Receptores ErbB/farmacologia , Humanos , Lactamas Macrocíclicas/farmacologia , Mutagênese Sítio-Dirigida , Mutação , Fosforilação , Ligação ProteicaRESUMO
There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine.
Assuntos
Ebolavirus/imunologia , Vírus da Encefalite Equina Venezuelana/genética , Doença pelo Vírus Ebola/prevenção & controle , Replicon , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Ebolavirus/genética , Vírus da Encefalite Equina Venezuelana/fisiologia , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Macaca fascicularis , Vacinação , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
Ebola hemorrhagic fever is an acute and often deadly disease caused by Ebola virus (EBOV). The possible intentional use of this virus against human populations has led to design of vaccines that could be incorporated into a national stockpile for biological threat reduction. We have evaluated the immunogenicity and efficacy of an EBOV vaccine candidate in which the viral surface glycoprotein is biomanufactured as a fusion to a monoclonal antibody that recognizes an epitope in glycoprotein, resulting in the production of Ebola immune complexes (EICs). Although antigen-antibody immune complexes are known to be efficiently processed and presented to immune effector cells, we found that codelivery of the EIC with Toll-like receptor agonists elicited a more robust antibody response in mice than did EIC alone. Among the compounds tested, polyinosinic:polycytidylic acid (PIC, a Toll-like receptor 3 agonist) was highly effective as an adjuvant agent. After vaccinating mice with EIC plus PIC, 80% of the animals were protected against a lethal challenge with live EBOV (30,000 LD(50) of mouse adapted virus). Surviving animals showed a mixed Th1/Th2 response to the antigen, suggesting this may be important for protection. Survival after vaccination with EIC plus PIC was statistically equivalent to that achieved with an alternative viral vector vaccine candidate reported in the literature. Because nonreplicating subunit vaccines offer the possibility of formulation for cost-effective, long-term storage in biothreat reduction repositories, EIC is an attractive option for public health defense measures.
Assuntos
Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Vacinas de Subunidades Antigênicas/química , Animais , Anticorpos/química , Anticorpos Monoclonais/química , Vacinas contra Ebola/imunologia , Ebolavirus/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Imunoglobulina G/química , Glicoproteínas de Membrana/química , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Poli I-C/química , Receptor 3 Toll-Like/agonistasRESUMO
Background: Hepatitis C virus (HCV) infection can now be cured with well-tolerated direct-acting antiviral (DAA) therapy. However, a potential barrier to HCV elimination is the emergence of resistance-associated substitutions (RASs) that reduce the efficacy of antiviral drugs, but real-world studies assessing the clinical impact of RASs are limited. Here, an analysis of the impact of RASs on retreatment outcomes for different salvage regimens in patients nationally who failed first-line DAA therapy is reported. Methods: We collected data from 363 Australian patients who failed first-line DAA therapy, including: age, sex, fibrosis stage, HCV genotype, NS3/NS5A/NS5B RASs, details of failed first-line regimen, subsequent salvage regimens, and treatment outcome. Results: Of 240 patients who were initially retreated as per protocol, 210 (87.5%) achieved sustained virologic response (SVR) and 30 (12.5%) relapsed or did not respond. The SVR rate for salvage regimens that included sofosbuvir/velpatasvir/voxilaprevir was 94.3% (n = 140), sofosbuvir/velpatasvir 75.0% (n = 52), elbasvir/grazoprevir 81.6% (n = 38), and glecaprevir/pibrentasvir 84.6% (n = 13). NS5A RASs were present in 71.0% (n = 210) of patients who achieved SVR and in 66.7% (n = 30) of patients who subsequently relapsed. NS3 RASs were detected in 20 patients (20%) in the SVR group and 1 patient in the relapse group. NS5B RASs were observed in only 3 patients. Cirrhosis was a predictor of relapse after retreatment, as was previous treatment with sofosbuvir/velpatasvir. Conclusions: In our cohort, the SVR rate for sofosbuvir/velpatasvir/voxilaprevir was higher than with other salvage regimens. The presence of NS5A, NS5B, or NS3 RASs did not appear to negatively influence retreatment outcomes.
RESUMO
Like other nitric-oxide synthase (NOS) enzymes, neuronal NOS (nNOS) turnover and activity are regulated by the Hsp90/Hsp70-based chaperone machinery, which regulates signaling proteins by modulating ligand binding clefts (Pratt, W. B., Morishima, Y., and Osawa, Y. (2008) J. Biol. Chem. 283, 22885-22889). We have previously shown that nNOS turnover is due to Hsp70/CHIP-dependent ubiquitination and proteasomal degradation. In this work, we use an intracellular cross-linking approach to study both chaperone binding and nNOS ubiquitination in intact HEK293 cells. Treatment of cells with N(G)-nitro-L-arginine, a slowly reversible competitive inhibitor that stabilizes nNOS, decreases both nNOS ubiquitination and binding of Hsp90, Hsp70, and CHIP. Treatment with the calcium ionophore A23187, which increases Ca(2+)-calmodulin binding to nNOS, increases nNOS ubiquitination and binding of Hsp90, Hsp70, and CHIP in a manner that is specific for changes in the heme/substrate binding cleft. Both Hsp90 and Hsp70 are bound to the expressed nNOS oxygenase domain, which contains the heme/substrate binding cleft, but not to the reductase domain, and binding is increased to an expressed fragment containing both the oxygenase domain and the calmodulin binding site. Overexpression of Hsp70 promotes nNOS ubiquitination and decreases nNOS protein, and overexpression of Hsp90 inhibits nNOS ubiquitination and increases nNOS protein, showing the opposing effects of the two chaperones as they participate in nNOS quality control in the cell. These observations support the notion that changes in the state of the heme/substrate binding cleft affect chaperone binding and thus nNOS ubiquitination.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Proteólise , Ubiquitinação/fisiologia , Animais , Calcimicina/farmacologia , Ionóforos de Cálcio/farmacologia , Domínio Catalítico/fisiologia , Células HEK293 , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP90/genética , Heme/genética , Heme/metabolismo , Humanos , Óxido Nítrico Sintase Tipo I/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
We have reported that heme-dependent activation of apo-neuronal nitric oxide synthase (apo-nNOS) to the active holo-enzyme dimer is dependent upon factors present in reticulocyte lysate and other cytosols. Here, we find that both Hsp70 and thioredoxin are components of the activation system. The apo-nNOS activating activity of reticulocyte lysate is retained in a pool of fractions containing Hsp70 that elute from DE52 prior to Hsp90. All of the activating activity and 20-30% of the Hsp70 elute in the flow-through fraction upon subsequent ATP-agarose chromatography. Apo-nNOS activation by this flow-through fraction is inhibited by pifithrin-µ, a small molecule inhibitor of Hsp70, suggesting that a non-ATP-binding form of Hsp70 is involved in heme-dependent apo-nNOS activation. Previous work has shown that apo-nNOS can be activated by thiol-disulfide exchange, and we show substantial activation with a small molecule dithiol modeled on the active motifs of thioredoxin and protein disulfide isomerase. Further fractionation of the ATP-agarose flow-through on Sephacryl S-300 separates free thioredoxin from apo-nNOS activating activity, Hsp70, and a small amount of thioredoxin, all of which are eluted throughout the macromolecular peak. Incubation of apo-nNOS with the macromolecular fraction in combination either with the thioredoxin-containing fraction or with purified recombinant human thioredoxin restores full heme-dependent activating activity. This supports a model in which Hsp70 binding to apo-nNOS stabilizes an open state of the heme/substrate binding cleft to facilitate thioredoxin access to the active site cysteine that coordinates with heme iron, permitting heme binding and dimerization to the active enzyme.
Assuntos
Citosol/metabolismo , Dissulfetos/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Heme/metabolismo , Óxido Nítrico Sintase/metabolismo , Compostos de Sulfidrila/metabolismo , Tiorredoxinas/metabolismo , Animais , Apoenzimas , Fatores de Coagulação Sanguínea/metabolismo , Dimerização , Ativação Enzimática , Humanos , Immunoblotting , Óxido Nítrico Sintase Tipo I , Ligação Proteica , Proteínas de Ligação a RNA , Coelhos , Ratos , Reticulócitos , Proteínas Ribossômicas , Sefarose/análogos & derivadosRESUMO
It is established that suicide inactivation of neuronal nitric-oxide synthase (nNOS) by drugs and other xenobiotics leads to ubiquitination and proteasomal degradation of the enzyme. The exact mechanism is not known, although it is widely thought that the covalent alteration of the active site during inactivation triggers the degradation. A mechanism that involves recognition of the altered nNOS by Hsp70 and its cochaperone CHIP, an E3-ubiquitin ligase, has been proposed. To further address how alterations of the active site trigger ubiquitination of nNOS, we examined a C331A nNOS mutant, which was reported to have impaired ability to bind L-arginine and tetrahydrobiopterin. We show here that C331A nNOS is highly susceptible to ubiquitination by a purified system containing ubiquitinating enzymes and chaperones, by the endogenous ubiquitinating system in reticulocyte lysate fraction II, and by intact HEK293 cells. The involvement of the altered heme cleft in regulating ubiquitination is confirmed by the finding that the slowly reversible inhibitor of nNOS, N(G)-nitro-L-arginine, but not its inactive D-isomer, protects the C331A nNOS from ubiquitination in all these experimental systems. We also show that both Hsp70 and CHIP play a major role in the ubiquitination of C331A nNOS, although Hsp90 protects from ubiquitination. Thus, these studies further strengthen the link between the mobility of the substrate-binding cleft and chaperone-dependent ubiquitination of nNOS. These results support a general model of chaperone-mediated protein quality control and lead to a novel mechanism for substrate stabilization based on nNOS interaction with the chaperone machinery.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Proteínas de Choque Térmico HSP70/metabolismo , Mutação , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina/química , Domínio Catalítico , Linhagem Celular , Imunoprecipitação da Cromatina , Proteínas de Choque Térmico/química , Humanos , Ligantes , Chaperonas Moleculares/química , Estrutura Terciária de Proteína , Frações Subcelulares , Xenobióticos/químicaRESUMO
The Hsp90/Hsp70-based chaperone machinery regulates the activity and degradation of many signaling proteins. Cycling with Hsp90 stabilizes client proteins, whereas Hsp70 interacts with chaperone-dependent E3 ubiquitin ligases to promote protein degradation. To probe these actions, small molecule inhibitors of Hsp70 would be extremely useful; however, few have been identified. Here we test the effects of methylene blue, a recently described inhibitor of Hsp70 ATPase activity, in three well established systems of increasing complexity. First, we demonstrate that methylene blue inhibits the ability of the purified Hsp90/Hsp70-based chaperone machinery to enable ligand binding by the glucocorticoid receptor and show that this effect is due to specific inhibition of Hsp70. Next, we establish that ubiquitination of neuronal nitric-oxide synthase by the native ubiquitinating system of reticulocyte lysate is dependent upon both Hsp70 and the E3 ubiquitin ligase CHIP and is blocked by methylene blue. Finally, we demonstrate that methylene blue impairs degradation of the polyglutamine expanded androgen receptor, an Hsp90 client mutated in spinal and bulbar muscular atrophy. In contrast, degradation of an amino-terminal fragment of the receptor, which lacks the ligand binding domain and, therefore, is not a client of the Hsp90/Hsp70-based chaperone machinery, is enhanced through homeostatic induction of autophagy that occurs when Hsp70-dependent proteasomal degradation is inhibited by methylene blue. Our data demonstrate the utility of methylene blue in defining Hsp70-dependent functions and reveal divergent effects on polyglutamine protein degradation depending on whether the substrate is an Hsp90 client.
Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico HSP70/metabolismo , Azul de Metileno/farmacologia , Peptídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos , Animais , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Camundongos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , RatosRESUMO
BACKGROUND: Undetected clinical deterioration is a major cause of high mortality events in Emergency Department (ED) patients. Yet, there is no known model to guide the recognition and response to clinical deterioration in the ED, integrating internal and external resources. METHODS: An integrative review was firstly conducted to identify the critical components of recognising and responding to clinical deterioration in the ED. Components identified from the review were analysed by clinical experts and informed the development of an ED Clinical Emergency Response System (EDCERS). RESULTS: Twenty four eligible studies were included in the review. Eight core components were identified: 1) vital sign monitoring; 2) track and trigger system; 3) communication plan; 4) response time; 5) emergency nurse response; 6) emergency physician response; 7) critical care team response; and 8) specialty team response. These components informed the development of the EDCERS protocol, integrating responses from staff internal and external to the ED. CONCLUSIONS: EDCERS was based on the best available evidence and considered the cultural context of care. Future research is needed to determine the useability and impact of EDCERS on patient and health outcomes.
Assuntos
Deterioração Clínica , Serviço Hospitalar de Emergência , Humanos , PolíticasRESUMO
Background and Objectives: Early warning of bacterial and viral infection, prior to the development of overt clinical symptoms, allows not only for improved patient care and outcomes but also enables faster implementation of public health measures (patient isolation and contact tracing). Our primary objectives in this effort are 3-fold. First, we seek to determine the upper limits of early warning detection through physiological measurements. Second, we investigate whether the detected physiological response is specific to the pathogen. Third, we explore the feasibility of extending early warning detection with wearable devices. Research Methods: For the first objective, we developed a supervised random forest algorithm to detect pathogen exposure in the asymptomatic period prior to overt symptoms (fever). We used high-resolution physiological telemetry data (aortic blood pressure, intrathoracic pressure, electrocardiograms, and core temperature) from non-human primate animal models exposed to two viral pathogens: Ebola and Marburg (N = 20). Second, to determine reusability across different pathogens, we evaluated our algorithm against three independent physiological datasets from non-human primate models (N = 13) exposed to three different pathogens: Lassa and Nipah viruses and Y. pestis. For the third objective, we evaluated performance degradation when the algorithm was restricted to features derived from electrocardiogram (ECG) waveforms to emulate data from a non-invasive wearable device. Results: First, our cross-validated random forest classifier provides a mean early warning of 51 ± 12 h, with an area under the receiver-operating characteristic curve (AUC) of 0.93 ± 0.01. Second, our algorithm achieved comparable performance when applied to datasets from different pathogen exposures - a mean early warning of 51 ± 14 h and AUC of 0.95 ± 0.01. Last, with a degraded feature set derived solely from ECG, we observed minimal degradation - a mean early warning of 46 ± 14 h and AUC of 0.91 ± 0.001. Conclusion: Under controlled experimental conditions, physiological measurements can provide over 2 days of early warning with high AUC. Deviations in physiological signals following exposure to a pathogen are due to the underlying host's immunological response and are not specific to the pathogen. Pre-symptomatic detection is strong even when features are limited to ECG-derivatives, suggesting that this approach may translate to non-invasive wearable devices.