RESUMO
Inflammasomes are innate immune sensing and signaling complexes critical for defense against pathogens and response to cellular stresses. A core component of inflammasomes is the sensor protein, which, upon sensing pathogen- or danger-associated molecular patterns (PAMPs or DAMPs), converts from inactive to active signaling platform for initiation of inflammatory signaling. A reliable source for the production and purification of recombinant inflammasome sensors is therefore invaluable for biochemical and structural characterizations, as well as drug screening for the development of therapeutics. Here, we describe an expression and purification protocol using the baculovirus-insect cell expression system to generate recombinant NLRP1, an important member of the NOD-like receptor (NLR) family of inflammasome sensors.
Assuntos
Inflamassomos , Proteínas NLR , Inflamassomos/metabolismo , Proteínas NLR/genética , Transdução de Sinais , Baculoviridae/genética , Baculoviridae/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLRRESUMO
Metallocarboxypeptidases play critical roles in the development of mosquitoes and influence pathogen/parasite infection of the mosquito midgut. Here, we report the crystal structure of Aedes aegypti procarboxypeptidase B1 (PCPBAe1), characterized its substrate specificity and mechanism of binding to and inhibiting Dengue virus (DENV). We show that the activated PCPBAe1 (CPBAe1) hydrolyzes both Arg- and Lys-substrates, which is modulated by residues Asp251 and Ser239 Notably, these residues are conserved in CPBs across mosquito species, possibly required for efficient digestion of basic dietary residues that are necessary for mosquito reproduction and development. Importantly, we characterized the interaction between PCPBAe1 and DENV envelope (E) protein, virus-like particles, and infectious virions. We identified residues Asp18A, Glu19A, Glu85, Arg87, and Arg89 of PCPBAe1 are essential for interaction with DENV. PCPBAe1 maps to the dimeric interface of the E protein domains I/II (Lys64-Glu84, Val238-Val252, and Leu278-Leu287). Overall, our studies provide general insights into how the substrate-binding property of mosquito carboxypeptidases could be targeted to potentially control mosquito populations or proposes a mechanism by which PCPBAe1 binds to and inhibits DENV.
Assuntos
Aedes/enzimologia , Aedes/virologia , Carboxipeptidase B/metabolismo , Vírus da Dengue , Dengue/transmissão , Interações entre Hospedeiro e Microrganismos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Carboxipeptidase B/química , Carboxipeptidase B/genética , Domínio Catalítico , Dengue/prevenção & controle , Dengue/virologia , Vírus da Dengue/fisiologia , Controle de Infecções , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Análise de Sequência de DNA , Relação Estrutura-Atividade , Especificidade por Substrato , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismoRESUMO
Over the past two decades, deadly coronaviruses, with the most recent being the severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) 2019 pandemic, have majorly challenged public health. The path for virus invasion into humans and other hosts is mediated by host-pathogen interactions, specifically virus-receptor binding. An in-depth understanding of the virus-receptor binding mechanism is a prerequisite for the discovery of vaccines, antibodies, and small-molecule inhibitors that can interrupt this interaction and prevent or cure infection. In this review, we discuss the viral entry mechanism, the known structural aspects of virus-receptor interactions (SARS-CoV-2 S/humanACE2, SARS-CoV S/humanACE2, and MERS-CoV S/humanDPP4), the key protein domains and amino acid residues involved in binding, and the small-molecule inhibitors and other drugs that have (as of June 2020) exhibited therapeutic potential. Specifically, we review the potential clinical utility of two transmembrane serine protease 2 (TMPRSS2)-targeting protease inhibitors, nafamostat mesylate and camostat mesylate, as well as two novel potent fusion inhibitors and the repurposed Ebola drug, remdesivir, which is specific to RNA-dependent RNA polymerase, against human coronaviruses, including SARS-CoV-2.
Assuntos
Enzima de Conversão de Angiotensina 2/efeitos dos fármacos , Antivirais/farmacologia , Antivirais/uso terapêutico , Tratamento Farmacológico da COVID-19 , Receptores Virais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Humanos , Inibidores de Proteases/uso terapêuticoRESUMO
The 2019 coronavirus pandemic remains a major public health concern. Neutralizing antibodies (nAbs) represent a cutting-edge antiviral strategy. We focus here on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-CoV, and discuss current progress in antibody research against rampant SARS-CoV-2 infections. We provide a perspective on the mechanisms of SARS-CoV-2-derived nAbs, comparing these with existing SARS-CoV-derived antibodies. We offer insight into how these antibodies cross-react and cross-neutralize by analyzing available structures of spike (S) glycoprotein-antibody complexes. We also propose ways of adopting antibody-based strategies - such as cocktail antibody therapeutics against SARS-CoV-2 - to overcome the possible resistance of currently identified mutants and mitigate possible antibody-dependent enhancement (ADE) pathologies. This review provides a platform for the progression of antibody and vaccine design against SARS-CoV-2, and possibly against future coronavirus pandemics.
