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1.
J Am Chem Soc ; 144(36): 16604-16611, 2022 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-36049228

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the infectious agent of the COVID-19 pandemic, remains a global medical problem. Angiotensin-converting enzyme 2 (ACE2) was identified as the primary viral entry receptor, and transmembrane serine protease 2 primes the spike protein for membrane fusion. However, ACE2 expression is generally low and variable across tissues, suggesting that auxiliary receptors facilitate viral entry. Identifying these factors is critical for understanding SARS-Cov-2 pathophysiology and developing new countermeasures. However, profiling host-virus interactomes involves extensive genetic screening or complex computational predictions. Here, we leverage the photocatalytic proximity labeling platform µMap to rapidly profile the spike interactome in human cells and identify eight novel candidate receptors. We systemically validate their functionality in SARS-CoV-2 pseudoviral uptake assays with both Wuhan and Delta spike variants and show that dual expression of ACE2 with either neuropilin-2, ephrin receptor A7, solute carrier family 6 member 15, or myelin and lymphocyte protein 2 significantly enhances viral uptake. Collectively, our data show that SARS-CoV-2 synergistically engages several host factors for cell entry and establishes µMap as a powerful tool for rapidly interrogating host-virus interactomes.


Assuntos
COVID-19 , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Humanos , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus
2.
N Engl J Med ; 375(3): 220-8, 2016 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-27468058

RESUMO

BACKROUND: In December 2013, a multicomponent meningococcal serogroup B (4CMenB) vaccine was used before licensure on the basis of special consideration by the Food and Drug Administration to respond to an outbreak of Neisseria meningitidis B at a U.S. university. Data suggested that vaccination would control the outbreak because isolates expressed antigens that were closely related to the vaccine antigens (factor H-binding protein [fHbp] and neisserial heparin-binding antigen). We quantified the immune responses induced by 4CMenB during the outbreak. METHODS: We conducted a seroprevalence survey among students to assess vaccination status and collect serum specimens to quantify titers of serum bactericidal antibodies (SBA) with an assay that included human complement (hSBA). We compared the proportion of vaccinated and unvaccinated participants who were seropositive for the outbreak strain and for one closely related reference strain (44/76-SL, which included fHbp) and one mismatched reference strain (5/99, which included neisserial adhesin A), both of which were used in vaccine development. Seropositivity was defined as an hSBA titer of 4 or higher. RESULTS: Among the 499 participants who received two doses of the 4CMenB vaccine 10 weeks apart, 66.1% (95% confidence interval [CI], 61.8 to 70.3) were seropositive for the outbreak strain, although the geometric mean titer was low at 7.6 (95% CI, 6.7 to 8.5). Among a random subgroup of 61 vaccinees who also received two doses but did not have a detectable protective response to the outbreak strain, 86.9% (95% CI, 75.8 to 94.2) were seropositive for the 44/76-SL strain, for which there was a geometric mean titer of 17.4 (95% CI, 13.0 to 23.2), whereas 100% of these vaccinees (95% CI, 94.1 to 100) were seropositive for the 5/99 strain and had a higher geometric mean titer (256.3; 95% CI, 187.3 to 350.7). The response to the outbreak strain was moderately correlated with the response to the 44/76-SL strain (Pearson's correlation,0.64; P<0.001) but not with the response to the 5/99 strain (Pearson's correlation,-0.06; P=0.43). CONCLUSIONS: Eight weeks after the second dose of the 4CMenB vaccine was administered, there was no evidence of an hSBA response against the outbreak strain in 33.9% of vaccinees, although no cases of meningococcal disease caused by N. meningitidis B were reported among vaccinated students. (Funded by Princeton University and others.).


Assuntos
Surtos de Doenças/prevenção & controle , Meningite Meningocócica/imunologia , Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo B/imunologia , Anticorpos Antibacterianos/sangue , Feminino , Humanos , Masculino , Meningite Meningocócica/epidemiologia , Meningite Meningocócica/prevenção & controle , New Jersey/epidemiologia , Estudos Soroepidemiológicos , Estados Unidos/epidemiologia , Universidades , Adulto Jovem
3.
Proc Natl Acad Sci U S A ; 110(43): 17368-73, 2013 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-24101513

RESUMO

Large tumor suppressor (LATS)1/2 protein kinases transmit Hippo signaling in response to intercellular contacts and serum levels to limit cell growth via the inhibition of Yes-associated protein (YAP). Here low serum and high LATS1 activity are found to enhance the levels of the 130-kDa isoform of angiomotin (Amot130) through phosphorylation by LATS1/2 at serine 175, which then forms a binding site for 14-3-3. Such phosphorylation, in turn, enables the ubiquitin ligase atrophin-1 interacting protein (AIP)4 to bind, ubiquitinate, and stabilize Amot130. Consistently, the Amot130 (S175A) mutant, which lacks LATS phosphorylation, bound AIP4 poorly under all conditions and showed reduced stability. Amot130 and AIP4 also promoted the ubiquitination and degradation of YAP in response to serum starvation, unlike Amot130 (S175A). Moreover, silencing Amot130 expression blocked LATS1 from inhibiting the expression of connective tissue growth factor, a YAP-regulated gene. Concordant with phosphorylated Amot130 specifically mediating these effects, wild-type Amot130 selectively induced YAP phosphorylation and reduced transcription of connective tissue growth factor in an AIP4-dependent manner versus Amot130 (S175A). Further, Amot130 but not Amot130 (S175A) strongly inhibited the growth of MDA-MB-468 breast cancer cells. The dominant-negative effects of Amot130 (S175A) on YAP signaling also support that phosphorylated Amot130 transduces Hippo signaling. Likewise, Amot130 expression provoked premature growth arrest during mammary cell acini formation, whereas Amot130 (S175A)-expressing cells formed enlarged and poorly differentiated acini. Taken together, the phosphorylation of Amot130 by LATS is found to be a key feature that enables it to inhibit YAP-dependent signaling and cell growth.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proliferação de Células/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Angiomotinas , Animais , Sítios de Ligação/genética , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Células MCF-7 , Proteínas de Membrana/genética , Proteínas dos Microfilamentos , Microscopia Confocal , Mutação , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina/genética , Serina/metabolismo , Fatores de Transcrição , Transcrição Gênica/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Sinalização YAP
4.
J Biol Chem ; 288(21): 15181-93, 2013 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-23564455

RESUMO

The adaptor protein Amot130 scaffolds components of the Hippo pathway to promote the inhibition of cell growth. This study describes how Amot130 through binding and activating the ubiquitin ligase AIP4/Itch achieves these effects. AIP4 is found to bind and ubiquitinate Amot130 at residue Lys-481. This both stabilizes Amot130 and promotes its residence at the plasma membrane. Furthermore, Amot130 is shown to scaffold a complex containing overexpressed AIP4 and the transcriptional co-activator Yes-associated protein (YAP). Consequently, Amot130 promotes the ubiquitination of YAP by AIP4 and prevents AIP4 from binding to large tumor suppressor 1. Amot130 is found to reduce YAP stability. Importantly, Amot130 inhibition of YAP dependent transcription is reversed by AIP4 silencing, whereas Amot130 and AIP4 expression interdependently suppress cell growth. Thus, Amot130 repurposes AIP4 from its previously described role in degrading large tumor suppressor 1 to the inhibition of YAP and cell growth.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proliferação de Células , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Angiomotinas , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Proteínas dos Microfilamentos , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade Proteica , Proteólise , Proteínas Repressoras/genética , Fatores de Transcrição , Transcrição Gênica/fisiologia , Ubiquitina-Proteína Ligases/genética , Proteínas de Sinalização YAP
5.
Sci Signal ; 16(806): eadf5494, 2023 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-37816090

RESUMO

Interferons (IFNs) play crucial roles in antiviral defenses. Despite using the same Janus-activated kinase (JAK)-signal transducer and activator of transcription (STAT) signaling cascade, type I and III IFN receptors differ in the magnitude and dynamics of their signaling in terms of STAT phosphorylation, gene transcription, and antiviral responses. These differences are not due to ligand-binding affinity and receptor abundance. Here, we investigated the ability of the intracellular domains (ICDs) of IFN receptors to differentiate between type I and III IFN signaling. We engineered synthetic, heterodimeric type I and III IFN receptors that were stably expressed at similar amounts in human cells and responded to a common ligand. We found that our synthetic type I IFN receptors stimulated STAT phosphorylation and gene expression to greater extents than did the corresponding type III IFN receptors. Furthermore, we identified short "box motifs" within ICDs that bind to JAK1 that were sufficient to encode differences between the type I and III IFN receptors. Together, our results indicate that specific regions within the ICDs of IFN receptor subunits encode different downstream signaling strengths that enable type I and III IFN receptors to produce distinct signaling outcomes.


Assuntos
Interferon Tipo I , Receptores de Interferon , Humanos , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Ligantes , Interferons/metabolismo , Transdução de Sinais , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Janus Quinases/metabolismo , Fosforilação , Antivirais/farmacologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo
6.
Emerg Microbes Infect ; 11(1): 227-239, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34931940

RESUMO

Dengue is caused by four genetically distinct viral serotypes, dengue virus (DENV) 1-4. Following transmission by Aedes mosquitoes, DENV can cause a broad spectrum of clinically apparent disease ranging from febrile illness to dengue hemorrhagic fever and dengue shock syndrome. Progress in the understanding of different dengue serotypes and their impacts on specific host-virus interactions has been hampered by the scarcity of tools that adequately reflect their antigenic and genetic diversity. To bridge this gap, we created and characterized infectious clones of DENV1-4 originating from South America, Africa, and Southeast Asia. Analysis of whole viral genome sequences of five DENV isolates from each of the four serotypes confirmed their broad genetic and antigenic diversity. Using a modified circular polymerase extension reaction (CPER), we generated de novo viruses from these isolates. The resultant clones replicated robustly in human and insect cells at levels similar to those of the parental strains. To investigate in vivo properties of these genetically diverse isolates, representative viruses from each DENV serotype were administered to NOD Rag1-/-, IL2rgnull Flk2-/- (NRGF) mice, engrafted with components of a human immune system. All DENV strains tested resulted in viremia in humanized mice and induced cellular and IgM immune responses. Collectively, we describe here a workflow for rapidly generating de novo infectious clones of DENV - and conceivably other RNA viruses. The infectious clones described here are a valuable resource for reverse genetic studies and for characterizing host responses to DENV in vitro and in vivo.


Assuntos
Vírus da Dengue/genética , Vírus da Dengue/imunologia , Dengue/virologia , Aedes/fisiologia , Aedes/virologia , Animais , Anticorpos Antivirais/imunologia , Variação Antigênica , Dengue/genética , Dengue/imunologia , Dengue/transmissão , Vírus da Dengue/classificação , Vírus da Dengue/isolamento & purificação , Variação Genética , Humanos , Imunoglobulina M/imunologia , Camundongos , Camundongos Knockout , Genética Reversa , Sorogrupo
7.
Cell Rep ; 39(3): 110714, 2022 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-35421379

RESUMO

The human immunological mechanisms defining the clinical outcome of SARS-CoV-2 infection remain elusive. This knowledge gap is mostly driven by the lack of appropriate experimental platforms recapitulating human immune responses in a controlled human lung environment. Here, we report a mouse model (i.e., HNFL mice) co-engrafted with human fetal lung xenografts (fLX) and a myeloid-enhanced human immune system to identify cellular and molecular correlates of lung protection during SARS-CoV-2 infection. Unlike mice solely engrafted with human fLX, HNFL mice are protected against infection, severe inflammation, and histopathological phenotypes. Lung tissue protection from infection and severe histopathology associates with macrophage infiltration and differentiation and the upregulation of a macrophage-enriched signature composed of 11 specific genes mainly associated with the type I interferon signaling pathway. Our work highlights the HNFL model as a transformative platform to investigate, in controlled experimental settings, human myeloid immune mechanisms governing lung tissue protection during SARS-CoV-2 infection.


Assuntos
COVID-19 , Animais , COVID-19/genética , Modelos Animais de Doenças , Humanos , Imunidade Inata , Pulmão/patologia , Macrófagos , Camundongos , SARS-CoV-2
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