Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.700
Filtrar
1.
Virulence ; 13(1): 684-697, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35400294

RESUMO

The vegetative insecticidal proteins (Vip3A) secreted by some Bacillus thuringiensis (Bt) strains during vegetative growth are regarded as a new generation of insecticidal toxins. Like insecticidal crystal proteins, they are also used in transgenic crops to control pests. However, their insecticidal mechanisms are far less defined than those of insecticidal crystal protein. Prohibitin 2 (PHB2) is a potential Vip3Aa binding receptor identified from the membrane of Sf9 cells in our previous work. In this paper, we demonstrated the interaction between Vip3Aa and PHB2 using pull-down, dot blotting, microscale thermophoresis, and co-immunoprecipitation assays. PHB2 is distributed on the cell membrane and in the cytoplasm, and the co-localization of PHB2 and Vip3Aa was observed in Sf9 cells using a confocal laser scanning microscope. Moreover, PHB2 could interact with scavenger receptor-C via its SPFH (stomatin, prohibitin, flotillin, and HflK/C) domain. Downregulation of phb2 expression reduced the degree of internalization of Vip3Aa, exacerbated Vip3Aa-mediated mitochondrial damage, and increased Vip3Aa toxicity to Sf9 cells. This suggested that PHB2 performs two different functions: Acting as an interacting partner to facilitate the internalization of Vip3Aa into Sf9 cells and maintaining the stability of mitochondria. The latter has a more important influence on the virulence of Vip3Aa.


Assuntos
Bacillus thuringiensis , Inseticidas , Animais , Proteínas de Bactérias/metabolismo , Inseticidas/metabolismo , Inseticidas/toxicidade , Mitocôndrias/metabolismo , Células Sf9 , Spodoptera , Virulência
2.
Nat Commun ; 13(1): 853, 2022 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-35165283

RESUMO

Neuropeptide Y (NPY) is highly abundant in the brain and involved in various physiological processes related to food intake and anxiety, as well as human diseases such as obesity and cancer. However, the molecular details of the interactions between NPY and its receptors are poorly understood. Here, we report a cryo-electron microscopy structure of the NPY-bound neuropeptide Y1 receptor (Y1R) in complex with Gi1 protein. The NPY C-terminal segment forming the extended conformation binds deep into the Y1R transmembrane core, where the amidated C-terminal residue Y36 of NPY is located at the base of the ligand-binding pocket. Furthermore, the helical region and two N-terminal residues of NPY interact with Y1R extracellular loops, contributing to the high affinity of NPY for Y1R. The structural analysis of NPY-bound Y1R and mutagenesis studies provide molecular insights into the activation mechanism of Y1R upon NPY binding.


Assuntos
Neuropeptídeo Y/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular , Microscopia Crioeletrônica , Ativação Enzimática/fisiologia , Humanos , Neuropeptídeo Y/genética , Ligação Proteica/fisiologia , Conformação Proteica , Receptores de Neuropeptídeo Y/genética , Células Sf9 , Transdução de Sinais
3.
Toxins (Basel) ; 14(2)2022 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-35202184

RESUMO

Fall armyworm (FAW), Spodoptera frugiperda, is a highly destructive and invasive global noctuid pest. Its control is based on insecticide applications and Bacillus thuringiensis (Bt) insecticidal Cry toxins expressed in transgenic crops, such as Cry1F in Bt corn. Continuous selection pressure has resulted in populations that are resistant to Bt corn, particularly in Brazil. FAW resistance to Cry1F was recently shown to be conferred by mutations of ATP-binding cassette transporter C2 (ABCC2), but several mutations, particularly indels in extracellular loop 4 (ECL4), are not yet functionally validated. We addressed this knowledge gap by baculovirus-free insect cell expression of ABCC2 variants (and ABCC3) by electroporation technology and tested their response to Cry1F, Cry1A.105 and Cry1Ab. We employed a SYTOXTM orange cell viability test measuring ABCC2-mediated Bt toxin pore formation. In total, we tested seven different FAW ABCC2 variants mutated in ECL4, two mutants modified in nucleotide binding domain (NBD) 2, including a deletion mutant lacking NBD2, and S. frugiperda ABCC3. All tested ECL4 mutations conferred high resistance to Cry1F, but much less to Cry1A.105 and Cry1Ab, whereas mutations in NBD2 hardly affected Bt toxin activity. Our study confirms the importance of indels in ECL4 for Cry1F resistance in S. frugiperda ABCC2.


Assuntos
Toxinas de Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis/toxicidade , Bacillus thuringiensis/genética , Resistência a Inseticidas/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Proteínas Recombinantes/genética , Spodoptera/efeitos dos fármacos , Spodoptera/genética , Animais , Brasil , Variação Genética , Genótipo , Mutação , Células Sf9/efeitos dos fármacos
4.
Nat Commun ; 13(1): 731, 2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35136060

RESUMO

Lysophospholipids are bioactive lipids and can signal through G-protein-coupled receptors (GPCRs). The best studied lysophospholipids are lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). The mechanisms of lysophospholipid recognition by an active GPCR, and the activations of lysophospholipid GPCR-G-protein complexes remain unclear. Here we report single-particle cryo-EM structures of human S1P receptor 1 (S1P1) and heterotrimeric Gi complexes formed with bound S1P or the multiple sclerosis (MS) treatment drug Siponimod, as well as human LPA receptor 1 (LPA1) and Gi complexes in the presence of LPA. Our structural and functional data provide insights into how LPA and S1P adopt different conformations to interact with their cognate GPCRs, the selectivity of the homologous lipid GPCRs for S1P versus LPA, and the different activation mechanisms of these GPCRs by LPA and S1P. Our studies also reveal specific optimization strategies to improve the MS-treating S1P1-targeting drugs.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Animais , Azetidinas/farmacologia , Azetidinas/uso terapêutico , Compostos de Benzil/farmacologia , Compostos de Benzil/uso terapêutico , Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/isolamento & purificação , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Humanos , Lisofosfolipídeos/metabolismo , Conformação Molecular/efeitos dos fármacos , Simulação de Acoplamento Molecular , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/patologia , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/isolamento & purificação , Receptores de Ácidos Lisofosfatídicos/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Células Sf9 , Imagem Individual de Molécula , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/genética , Receptores de Esfingosina-1-Fosfato/isolamento & purificação , Receptores de Esfingosina-1-Fosfato/ultraestrutura , Spodoptera
5.
Nat Commun ; 13(1): 923, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35177668

RESUMO

N-methyl-D-aspartate receptors (NMDARs) are critically involved in basic brain functions and neurodegeneration as well as tumor invasiveness. Targeting specific subtypes of NMDARs with distinct activities has been considered an effective therapeutic strategy for neurological disorders and diseases. However, complete elimination of off-target effects of small chemical compounds has been challenging and thus, there is a need to explore alternative strategies for targeting NMDAR subtypes. Here we report identification of a functional antibody that specifically targets the GluN1-GluN2B NMDAR subtype and allosterically down-regulates ion channel activity as assessed by electrophysiology. Through biochemical analysis, x-ray crystallography, single-particle electron cryomicroscopy, and molecular dynamics simulations, we show that this inhibitory antibody recognizes the amino terminal domain of the GluN2B subunit and increases the population of the non-active conformational state. The current study demonstrates that antibodies may serve as specific reagents to regulate NMDAR functions for basic research and therapeutic objectives.


Assuntos
Anticorpos Monoclonais/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/ultraestrutura , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/farmacologia , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/isolamento & purificação , Região Variável de Imunoglobulina/farmacologia , Região Variável de Imunoglobulina/ultraestrutura , Simulação de Dinâmica Molecular , Oócitos , Ratos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/ultraestrutura , Células Sf9 , Spodoptera , Xenopus laevis
6.
Cell ; 185(4): 672-689.e23, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35114111

RESUMO

ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.


Assuntos
Channelrhodopsins/química , Channelrhodopsins/metabolismo , Ativação do Canal Iônico , Animais , Channelrhodopsins/ultraestrutura , Microscopia Crioeletrônica , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Modelos Moleculares , Optogenética , Filogenia , Ratos Sprague-Dawley , Bases de Schiff/química , Células Sf9 , Relação Estrutura-Atividade
7.
Nat Commun ; 13(1): 876, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35169143

RESUMO

The membrane receptor kinases HAESA and HSL2 recognize a family of IDA/IDL signaling peptides to control cell separation processes in different plant organs. The homologous HSL1 has been reported to regulate epidermal cell patterning by interacting with a different class of signaling peptides from the CLE family. Here we demonstrate that HSL1 binds IDA/IDL peptides with high, and CLE peptides with lower affinity, respectively. Ligand sensing capability and receptor activation of HSL1 require a SERK co-receptor kinase. Crystal structures with IDA/IDLs or with CLE9 reveal that HSL1-SERK1 complex recognizes the entire IDA/IDL signaling peptide, while only parts of CLE9 are bound to the receptor. In contrast, the receptor kinase BAM1 interacts with the entire CLE9 peptide with high affinity and specificity. Furthermore, the receptor tandem BAM1/BAM2 regulates epidermal cell division homeostasis. Consequently, HSL1-IDLs and BAM1/BAM2-CLEs independently regulate cell patterning in the leaf epidermal tissue.


Assuntos
Proteínas de Arabidopsis/metabolismo , Células Epidérmicas/citologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Folhas de Planta/embriologia , Proteínas Quinases/metabolismo , Proteínas Repressoras/metabolismo , Animais , Arabidopsis , Proteínas de Arabidopsis/genética , Linhagem Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Repressoras/genética , Células Sf9 , Tabaco
8.
Viruses ; 14(1)2022 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-35062344

RESUMO

The baculovirus display system (BDS), an excellent eukaryotic surface display technology that offers the advantages of safety, efficiency, and economy, is widely used in biomedicine. A previous study using rBacmid-Δgp64-ires-gp64 expressed in low copy numbers of the gp64 gene achieved high-efficiency expression and co-display of three fluorescent proteins (GFP, YFP, and mCherry). However, low expression of GP64 in recombinant baculoviruses also reduces the efficiency of recombinant baculovirus transduction into mammalian cells. In addition, the baculovirus promoter has no expression activity in mammalian cells and thus cannot meet the application requirements of baculoviral vectors for the BDS. Based on previous research, this study first determined the expression activity of promoters in insect Spodoptera frugiperda 9 cells and mammalian cells and successfully screened the very early promoter pie1 to mediate the co-expression of multiple genes. Second, utilizing the envelope display effect of the INVASIN and VSVG proteins, the efficiency of transduction of recombinant baculovirus particles into non-host cells was significantly improved. Finally, based on the above improvement, a recombinant baculovirus vector displaying four antigen proteins with high efficiency was constructed. Compared with traditional BDSs, the rBacmid-Δgp64 system exhibited increased display efficiency of the target protein by approximately 3-fold and induced an approximately 4-fold increase in the titer of serum antibodies to target antigens in Bal B/c mice. This study systematically explored the application of a new multi-gene co-display technology applicable to multi-vaccine research, and the results provide a foundation for the development of novel BDS technologies.


Assuntos
Baculoviridae/genética , Vetores Genéticos/imunologia , Vacinas/genética , Vacinas/imunologia , Animais , Anticorpos Antivirais/sangue , Escherichia coli , Regulação Viral da Expressão Gênica , Vetores Genéticos/genética , Células HEK293 , Humanos , Imunogenicidade da Vacina , Camundongos , Regiões Promotoras Genéticas , Células Sf9
9.
Elife ; 112022 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-34986091

RESUMO

Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A2A receptor (A2AR) in reconstituted phospholipid nanodiscs. This model system conveniently excludes possible effects arising from cholesterol-induced phase separation or receptor oligomerization and focuses on the question of allostery. GTP hydrolysis assays show that cholesterol weakly enhances the basal signaling of A2AR while decreasing the agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy shows that this enhancement arises from an increase in the receptor's active state population and a G-protein-bound precoupled state. 19F NMR of fluorinated cholesterol analogs reveals transient interactions with A2AR, indicating a lack of high-affinity binding or direct allosteric modulation. The combined results suggest that the observed allosteric effects are largely indirect and originate from cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Colesterol/metabolismo , Receptor A2A de Adenosina/química , Animais , Escherichia coli , Espectroscopia de Ressonância Magnética , Saccharomycetales , Células Sf9 , Spodoptera
10.
Nat Commun ; 13(1): 251, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-35017564

RESUMO

Influenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral RNA-dependent RNA polymerase which transcribes and replicates the viral RNA genome. The polymerase undergoes conformational rearrangements and interacts with viral and host proteins to perform these functions. Here we determine the structure of the 1918 influenza virus polymerase in transcriptase and replicase conformations using cryo-electron microscopy (cryo-EM). We then structurally and functionally characterise the binding of single-domain nanobodies to the polymerase of the 1918 pandemic influenza virus. Combining these functional and structural data we identify five sites on the polymerase which are sensitive to inhibition by nanobodies. We propose that the binding of nanobodies at these sites either prevents the polymerase from assuming particular functional conformations or interactions with viral or host factors. The polymerase is highly conserved across the influenza A subtypes, suggesting these sites as effective targets for potential influenza antiviral development.


Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Orthomyxoviridae/genética , Pandemias , Anticorpos de Domínio Único/química , Animais , Microscopia Crioeletrônica , Genoma Viral , Células HEK293 , Humanos , Vírus da Influenza A/genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA , Células Sf9 , Anticorpos de Domínio Único/genética , Proteínas Virais/química , Proteínas Virais/genética
11.
Biochem Biophys Res Commun ; 594: 69-73, 2022 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-35074588

RESUMO

BacMam system utilizes baculovirus to deliver exogenous genes into mammalian cells and is extensively used for recombinant production of eukaryotic proteins. Here, we described the development of a BacMam vector (pBMCL1), which allows convenient tracing of virus production, provides higher infection efficiency towards mammalian cells, minimizes unwanted transcription of toxic genes in insect cells, and provides the capability for co-expression of multiple proteins via a single virus. We demonstrate the successful application of the pBMCL1 vector for the expression of homo-tetrameric human TRPC3 channel and hetero-octameric KATP channel.


Assuntos
Baculoviridae/metabolismo , Regulação da Expressão Gênica , Animais , DNA Complementar/metabolismo , Eletrofisiologia , Expressão Gênica , Genes Reporter , Vetores Genéticos , Células HEK293 , Humanos , Insetos , Mesocricetus , Camundongos , Proteínas Recombinantes/química , Células Sf9 , Canais de Cátion TRPC/química , Transdução Genética
12.
Sci Rep ; 12(1): 1086, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-35058492

RESUMO

The antibiotic resistance crisis has prompted research into alternative candidates such as antimicrobial peptides (AMPs). However, the demand for such molecules can only be met by continuous production processes, which achieve high product yields and offer compatibility with the Quality-by-Design initiative by implementing process analytical technologies such as turbidimetry and dielectric spectroscopy. We developed batch and perfusion processes at the 2-L scale for the production of BR033, a cecropin-like AMP from Lucilia sericata, in stably-transformed polyclonal Sf-9 cells. This is the first time that BR033 has been expressed as a recombinant peptide. Process analytical technology facilitated the online monitoring and control of cell growth, viability and concentration. The perfusion process increased productivity by ~ 180% compared to the batch process and achieved a viable cell concentration of 1.1 × 107 cells/mL. Acoustic separation enabled the consistent retention of 98.5-100% of the cells, viability was > 90.5%. The recombinant AMP was recovered from the culture broth by immobilized metal affinity chromatography and gel filtration and was able to inhibit the growth of Escherichia coli K12. These results demonstrate a successful, integrated approach for the development and intensification of a process from cloning to activity testing for the production of new biopharmaceutical candidates.


Assuntos
/biossíntese , Técnicas de Cultura de Células/métodos , Animais , Reatores Biológicos , Biotecnologia/métodos , Insetos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Células Sf9/metabolismo
13.
Elife ; 112022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-35049497

RESUMO

Integrin adhesion complexes (IACs) are integrin-based plasma-membrane-associated compartments where cells sense environmental cues. The physical mechanisms and molecular interactions that mediate initial IAC formation are unclear. We found that both p130Cas ('Cas') and Focal adhesion kinase ('FAK') undergo liquid-liquid phase separation in vitro under physiologic conditions. Cas- and FAK- driven phase separation is sufficient to reconstitute kindlin-dependent integrin clustering in vitro with recombinant mammalian proteins. In vitro condensates and IACs in mouse embryonic fibroblasts (MEFs) exhibit similar sensitivities to environmental perturbations including changes in temperature and pH. Furthermore, mutations that inhibit or enhance phase separation in vitro reduce or increase the number of IACs in MEFs, respectively. Finally, we find that the Cas and FAK pathways act synergistically to promote phase separation, integrin clustering, IAC formation and partitioning of key components in vitro and in cells. We propose that Cas- and FAK-driven phase separation provides an intracellular trigger for integrin clustering and nascent IAC formation.


Assuntos
Moléculas de Adesão Celular/metabolismo , Adesão Celular , Fibroblastos/fisiologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Integrinas/metabolismo , Animais , Linhagem Celular , Proteína-Tirosina Quinases de Adesão Focal/genética , Integrinas/genética , Camundongos , Fosforilação , Células Sf9 , Transdução de Sinais
14.
Biochem J ; 479(3): 289-304, 2022 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-35037691

RESUMO

Ubiquitination and ADP-ribosylation are post-translational modifications that play major roles in pathways including the DNA damage response and viral infection. The enzymes responsible for these modifications are therefore potential targets for therapeutic intervention. DTX3L is an E3 Ubiquitin ligase that forms a heterodimer with PARP9. In addition to its ubiquitin ligase activity, DTX3L-PARP9 also acts as an ADP-ribosyl transferase for Gly76 on the C-terminus of ubiquitin. NAD+-dependent ADP-ribosylation of ubiquitin by DTX3L-PARP9 prevents ubiquitin from conjugating to protein substrates. To gain insight into how DTX3L-PARP9 generates these post-translational modifications, we produced recombinant forms of DTX3L and PARP9 and studied their physical interactions. We show the DTX3L D3 domain (230-510) mediates the interaction with PARP9 with nanomolar affinity and an apparent 1 : 1 stoichiometry. We also show that DTX3L and PARP9 assemble into a higher molecular weight oligomer, and that this is mediated by the DTX3L N-terminal region (1-200). Lastly, we show that ADP-ribosylation of ubiquitin at Gly76 is reversible in vitro by several Macrodomain-type hydrolases. Our study provides a framework to understand how DTX3L-PARP9 mediates ADP-ribosylation and ubiquitination through both intra- and inter-subunit interactions.


Assuntos
Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Multimerização Proteica/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , ADP-Ribosilação/genética , Adenosina Difosfato Ribose/metabolismo , Animais , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas de Neoplasias/genética , Poli(ADP-Ribose) Polimerases/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Transfecção , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
15.
Cell ; 185(1): 158-168.e11, 2022 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-34995514

RESUMO

Small molecule chaperones have been exploited as therapeutics for the hundreds of diseases caused by protein misfolding. The most successful examples are the CFTR correctors, which transformed cystic fibrosis therapy. These molecules revert folding defects of the ΔF508 mutant and are widely used to treat patients. To investigate the molecular mechanism of their action, we determined cryo-electron microscopy structures of CFTR in complex with the FDA-approved correctors lumacaftor or tezacaftor. Both drugs insert into a hydrophobic pocket in the first transmembrane domain (TMD1), linking together four helices that are thermodynamically unstable. Mutating residues at the binding site rendered ΔF508-CFTR insensitive to lumacaftor and tezacaftor, underscoring the functional significance of the structural discovery. These results support a mechanism in which the correctors stabilize TMD1 at an early stage of biogenesis, prevent its premature degradation, and thereby allosterically rescuing many disease-causing mutations.


Assuntos
Aminopiridinas/metabolismo , Benzodioxóis/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Indóis/metabolismo , Dobramento de Proteína , Aminopiridinas/química , Aminopiridinas/uso terapêutico , Animais , Benzodioxóis/química , Benzodioxóis/uso terapêutico , Sítios de Ligação , Células CHO , Membrana Celular/química , Membrana Celular/metabolismo , Cricetulus , Microscopia Crioeletrônica , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Indóis/química , Indóis/uso terapêutico , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/uso terapêutico , Mutação , Domínios Proteicos/genética , Células Sf9 , Transfecção
16.
Int J Biol Macromol ; 194: 9-16, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34861271

RESUMO

Spodoptera litura is a serious polyphagous pest in the whole world, which has developed resistance to most conventional insecticides and even some Bacillus thuringiensis (Bt) toxins. Cry1Ca has excellent insecticide activity against S. litura with potential application to control S. litura and delay the development of insect resistance. However, the mode of action of Cry1Ca in S. litura is poorly understood. Here, Cry1Ca-binding proteins were identified from S. litura by using pull down assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that aminopeptidase-N (APN), ATP binding cassette subfamily C member 2 (ABCC2), polycalin, actin and V-type proton ATPase subunit A may bind with Cry1Ca. Further study confirmed that ABCC2 fragment expressed in vitro can bind to Cry1Ca as demonstrated by Ligand blot and homologous competition experiments. The over-expression of endogenous SlABCC2 in Sf9 cells increased Cry1Ca cytotoxicity. Correspondingly, the vivo loss of function analyses by SlABCC2 small interfering RNAs (siRNAs) in S. litura larvae decreased the toxicity of Cry1Ca to larvae. Altogether, these results show that ABCC2 of S. litura is a functional receptor that is involved in the action mode of Cry1Ca.


Assuntos
Bacillus thuringiensis/fisiologia , Interações Hospedeiro-Patógeno , Spodoptera/metabolismo , Spodoptera/microbiologia , Animais , Toxinas de Bacillus thuringiensis/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Células Sf9 , Spodoptera/genética
17.
Int J Biol Macromol ; 195: 609-619, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34871658

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) is a coronavirus which can cause severe human respiratory diseases with a fatality rate of almost 36%. In this study, we report the generation, characterization and epitope mapping of several monoclonal antibodies against the spike receptor-binding domain (RBD) of MERS-CoV. Two monoclonal antibodies (4C7 and 6E8) that can react with linearized RBD have been selected for subsequent identification of RBD mAb-binding epitopes. Two distinct novel linear epitopes, 423FTCSQIS429 and 546SPLEGGGWL554,were precisely located at the outermost surface of RBD by dot-blot hybridization and ELISAs. Multiple sequence alignment analysis showed that these two peptides were highly conserved. Alanine (A)-scanning mutagenesis demonstrated that residues 423F, 428I, and 429S are the crucial residues for the linear epitope 423FTCSQIS429 while residues 548L, 550G, 553W, 554L for epitope 546SPLEGGGWL554. These findings may be helpful for further understanding of the function of RBD protein and the development of subsequent diagnosis and detection methods.


Assuntos
Anticorpos Monoclonais Murinos/imunologia , Anticorpos Antivirais/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais Murinos/genética , Anticorpos Antivirais/genética , Epitopos/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Domínios Proteicos , Células Sf9 , Glicoproteína da Espícula de Coronavírus/genética , Spodoptera
18.
J Cell Biochem ; 123(1): 65-76, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34741485

RESUMO

Pancreatic ductal adenoma carcinoma (PDAC) is considered one of the deadliest solid cancers as it is usually diagnosed in advanced stages and has a poor response to treatment. The enormous effort made in the last 2 decades in the oncology field has not led to significant progress in improving early diagnosis or therapy for PDAC. The stroma of PDAC plays an active role in tumour initiation and progression and includes immune cells and stromal cells. We previously reported that Bcl2-associated athanogene (BAG3) secreted by PDAC cells activates tumour-associated macrophages to promote tumour growth. The disruption of this tumour-stroma axis by the anti-BAG3 H2L4 therapeutic antibody is sufficient to delay tumour growth and limit metastatic spreading in different PDAC preclinical models. In the present study, we examined the role of BAG3 to activate human fibroblasts (HF) in releasing cytokines capable of supporting tumour progression. Treatment of fibroblasts with recombinant BAG3 induced important changes in the organisation of the cytoskeleton of these cells and stimulated the production of interleukin-6, monocyte chemoattractant protein-1/C-C motif chemokine ligand 2, and hepatocyte growth factor. Specifically, we observed that BAG3 triggered a depolymerisation of microtubules at the periphery of the cell while they were conserved in the perinuclear area. Conversely, the vimentin-based intermediate filaments increased and spread to the edges of the cells. Finally, the conditioned medium (CM) collected from BAG3-treated HF promoted the survival, proliferation, and migration of the PDAC cells. Blocking of the PDAC-fibroblast axis by the H2L4 therapeutic anti-BAG3 antibody, resulted in inhibition of cytokine release and, consequently, the inhibition of the migratory phenotype conferred by the CM to PDAC cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/farmacologia , Proteínas Reguladoras de Apoptose/farmacologia , Carcinoma Ductal Pancreático/metabolismo , Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Citocinas/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Humanos , Neoplasias Pancreáticas/patologia , Proteínas Recombinantes/farmacologia , Células Sf9 , Spodoptera
19.
Biomed Pharmacother ; 145: 112434, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34801853

RESUMO

Lipoxygenases (ALOX-isoforms) are lipid peroxidizing enzymes, which have been implicated in cell differentiation and maturation but also in the biosynthesis of lipid mediators playing important roles in the pathogenesis of inflammatory, hyperproliferative and neurological diseases. In mammals these enzymes are widely distributed and the human genome involves six functional genes encoding for six distinct human ALOX paralogs. In mice, there is an orthologous enzyme for each human ALOX paralog but the catalytic properties of human and mouse ALOX orthologs show remarkable differences. ALOX inhibitors are frequently employed for deciphering the biological role of these enzymes in mouse models of human diseases but owing to the functional differences between mouse and human ALOX orthologs the uncritical use of such inhibitors is sometimes misleading. In this study we evaluated the paralog- and ortholog-specificity of 13 frequently employed ALOX-inhibitors against four recombinant human and mouse ALOX paralogs (ALOX15, ALOX15B, ALOX12, ALOX5) under different experimental conditions. Our results indicated that except for zileuton, which exhibits a remarkable paralog-specificity for mouse and human ALOX5, no other inhibitor was strictly paralog specific but some compounds exhibit an interesting ortholog-specificity. Because of the variable isoform specificities of the currently available ALOX inhibitors care must be taken when the biological effects of these compounds observed in complex in vitro and in vivo systems are interpreted.


Assuntos
Araquidonato 15-Lipoxigenase/efeitos dos fármacos , Inibidores de Lipoxigenase/farmacologia , Animais , Araquidonato 15-Lipoxigenase/genética , Linhagem Celular , Humanos , Isoenzimas , Camundongos , Células Sf9 , Especificidade da Espécie
20.
Insect Biochem Mol Biol ; 140: 103696, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34800643

RESUMO

Insect CYP2 and mitochondrial clan P450s are relatively conserved genes encoding enzymes generally thought to be involved in biosynthesis or metabolism of endobiotics. However, emerging evidence argues they have potential roles in chemical defense as well, but their actual detoxification functions remain largely unknown. Here, we focused on the full complement of 8 CYP2 and 10 mitochondrial P450s in the generalist herbivore, Helicoverpa armigera. Their varied spatiotemporal expression profiles were analyzed and reflected their specific functions. For functional study of the mitochondrial clan P450s, the redox partners, adrenodoxin reductase (AdR) and adrenodoxin (Adx), were identified from genomes of eight insects and an efficient in vitro electron transfer system of mitochondrial P450 was established by co-expression with Adx and AdR of H. armigera. All CYP2 clan P450s and 8 mitochondrial P450s were successfully expressed in Sf9 cells and compared functionally. In vitro metabolism assays showed that two CYP2 clan P450s (CYP305B1 and CYP18A1) and CYP333B3 (mito clan) could epoxidize aldrin to dieldrin, while CYP305B1 and CYP339A1 (mito clan) have limited but significant hydroxylation capacities to esfenvalerate. CYP303A1 of the CYP2 clan exhibits high metabolic efficiency to 2-tridecanone. Screening the xenobiotic metabolism competence of CYP2 and mitochondrial clan P450s not only provides new insights on insect chemical defense but also can give indications on their physiological functions in H. armigera and other insects.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450/metabolismo , Inativação Metabólica , Mariposas , Xenobióticos/metabolismo , Aldrina/metabolismo , Animais , Inativação Metabólica/genética , Inativação Metabólica/fisiologia , Proteínas de Insetos/metabolismo , Mariposas/genética , Mariposas/metabolismo , Mariposas/fisiologia , Células Sf9
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...