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1.
Int J Mol Sci ; 22(13)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34281277

RESUMO

The aim of this research was to analyze the heterologous expression, purification, and immunoregulatory activity of recombinant YGP40 (rYGP40), the potential precursor of the yolkin peptide complex. The ygp40 coding sequence was codon optimized, successfully expressed in the E. coli system, and purified from inclusion bodies with a yield of about 1.1 mg/L of culture. This study showed that the protein exhibits immunomodulatory activity, expressed by the stimulation of TNF-α and IL-10 production and nitric oxide induction at a level comparable to that of the natural yolkin peptide complex obtained by other authors from hen egg yolk. At the highest dose of 100 µg/mL, rYGP40 also caused the up-regulation of iNOS expression in murine bone marrow-derived macrophages (BMDM). Moreover, no cytotoxic effects of rYGP40 on the BMDM cell line were observed.


Assuntos
Vitelogeninas/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Clonagem Molecular , Gema de Ovo/química , Feminino , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/genética , Fatores Imunológicos/farmacologia , Técnicas In Vitro , Interleucina-10/biossíntese , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Peso Molecular , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Vitelogeninas/genética , Vitelogeninas/farmacologia
2.
Int J Mol Sci ; 22(13)2021 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-34281258

RESUMO

Infection by Proteus mirabilis causes urinary stones and catheter incrustation due to ammonia formed by urease (PMU), one of its virulence factors. Non-enzymatic properties, such as pro-inflammatory and neurotoxic activities, were previously reported for distinct ureases, including that of the gastric pathogen Helicobacter pylori. Here, PMU was assayed on isolated cells to evaluate its non-enzymatic properties. Purified PMU (nanomolar range) was tested in human (platelets, HEK293 and SH-SY5Y) cells, and in murine microglia (BV-2). PMU promoted platelet aggregation. It did not affect cellular viability and no ammonia was detected in the cultures' supernatants. PMU-treated HEK293 cells acquired a pro-inflammatory phenotype, producing reactive oxygen species (ROS) and cytokines IL-1ß and TNF-α. SH-SY5Y cells stimulated with PMU showed high levels of intracellular Ca2+ and ROS production, but unlike BV-2 cells, SH-SY5Y did not synthesize TNF-α and IL-1ß. Texas Red-labeled PMU was found in the cytoplasm and in the nucleus of all cell types. Bioinformatic analysis revealed two bipartite nuclear localization sequences in PMU. We have shown that PMU, besides urinary stone formation, can potentially contribute in other ways to pathogenesis. Our data suggest that PMU triggers pro-inflammatory effects and may affect cells beyond the renal system, indicating a possible role in extra-urinary diseases.


Assuntos
Proteus mirabilis/enzimologia , Proteus mirabilis/patogenicidade , Urease/metabolismo , Urease/toxicidade , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/microbiologia , Modelos Moleculares , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/microbiologia , Neurotoxinas/química , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Sinais de Localização Nuclear , Agregação Plaquetária/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Urease/química , Virulência/fisiologia
3.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209932

RESUMO

Enzymatic transamidation of gliadins by microbial transglutaminase (mTG) inhibits interferon-γ (IFN-γ) secretion by intestinal T cell lines in patients with celiac disease (CD). To gain insight into the cellular mechanisms underlying the down-regulatory effects of transamidation, we tested a single recombinant α-gliadin (r-gliadin) harbouring two immunodominant peptides, p13 (aa. 120-139) and p23 (aa. 220-239), in HLA-DQ8 transgenic mice, a model of gluten sensitivity. Mice were intranasally immunised with r-gliadin or r-gliadin transamidated by mTG (K-r-gliadin) along with cholera toxin, and the response of mesenteric lymph node cells was analysed by cytokine multiplex assay. An in vitro challenge with r-gliadin was characterised by secretion of specific cytokines featuring both innate immunity and the Th1/Th2/Th17 pattern of the adaptive response. Notably, transamidation specifically down-regulated the Th1 response. Structural studies performed on K-r-gliadin confirmed that specific glutamine residues in p13 and p23, previously found to be deamidated by tissue transglutaminase, were also transamidated by mTG. In silico analysis, simulating p13 and p23 peptide binding to HLA-DQ8 showed that these glutamines, in the form of glutamate, could interact by means of salt bridges with peculiar amino acids of the alpha chain of HLA-DQ8, suggesting that their transamidation may influence the HLA-restricted recognition of these peptides. Thus, the structural findings provided a rationale to explain the down-regulation of the r-gliadin-specific Th1 response following transamidation.


Assuntos
Doença Celíaca/tratamento farmacológico , Toxina da Cólera/administração & dosagem , Citocinas/metabolismo , Gliadina/administração & dosagem , Antígenos HLA-DQ/genética , Transglutaminases/metabolismo , Administração Intranasal , Animais , Doença Celíaca/genética , Doença Celíaca/imunologia , Toxina da Cólera/imunologia , Citocinas/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo , Regulação da Expressão Gênica , Gliadina/química , Gliadina/genética , Gliadina/imunologia , Antígenos HLA-DQ/metabolismo , Imunização , Epitopos Imunodominantes/imunologia , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Células Th1/imunologia , Células Th17/imunologia , Células Th2/imunologia
4.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070642

RESUMO

Urate oxidase initiates the uric acid degradation pathways and is extensively used for protein drug development for gout therapy and serum uric acid diagnosis. We first present the biochemical and structural elucidation of a urate oxidase from the extremophile microorganism Deinococcus radiodurans (DrUox). From enzyme characterization, DrUox showed optimal catalytic ability at 30 °C and pH 9.0 with high stability under physiological conditions. Only the Mg2+ ion moderately elevated its activity, which indicates the characteristic of the cofactor-free urate oxidase family. Of note, DrUox is thermostable in mesophilic conditions. It retains almost 100% activity when incubated at 25 °C and 37 °C for 24 h. In this study, we characterized a thermostable urate oxidase, DrUox with high catalytic efficiency and thermal stability, which strengthens its potential for medical applications.


Assuntos
Proteínas de Bactérias , Deinococcus , Gota/tratamento farmacológico , Hiperuricemia/tratamento farmacológico , Urato Oxidase , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/uso terapêutico , Deinococcus/enzimologia , Deinococcus/genética , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , Urato Oxidase/química , Urato Oxidase/genética , Urato Oxidase/uso terapêutico
5.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073266

RESUMO

The monocot chimeric jacalin OsJAC1 from Oryza sativa consists of a dirigent and a jacalin-related lectin domain. The corresponding gene is expressed in response to different abiotic and biotic stimuli. However, there is a lack of knowledge about the basic function of the individual domains and their contribution to the physiological role of the entire protein. In this study, we have established a heterologous expression in Escherichia coli with high yields for the full-length protein OsJAC1 as well as its individual domains. Our findings showed that the secondary structure of both domains is dominated by ß-strand elements. Under reducing conditions, the native protein displayed clearly visible transition points of thermal unfolding at 59 and 85 °C, which could be attributed to the lectin and the dirigent domain, respectively. Our study identified a single carbohydrate-binding site for each domain with different specificities towards mannose and glucose (jacalin domain), and galactose moieties (dirigent domain), respectively. The recognition of different carbohydrates might explain the ability of OsJAC1 to respond to different abiotic and biotic factors. This is the first report of specific carbohydrate-binding activity of a DIR domain, shedding new light on its function in the context of this monocot chimeric jacalin.


Assuntos
Oryza/química , Lectinas de Plantas/química , Proteínas de Plantas/química , Oryza/genética , Lectinas de Plantas/genética , Proteínas de Plantas/genética , Conformação Proteica em Folha beta , Domínios Proteicos , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
6.
Sci Rep ; 11(1): 12410, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34127732

RESUMO

In situ generation of antibacterial and antiviral agents by harnessing the catalytic activity of enzymes on surfaces provides an effective eco-friendly approach for disinfection. The perhydrolase (AcT) from Mycobacterium smegmatis catalyzes the perhydrolysis of acetate esters to generate the potent disinfectant, peracetic acid (PAA). In the presence of AcT and its two substrates, propylene glycol diacetate and H2O2, sufficient and continuous PAA is generated over an extended time to kill a wide range of bacteria with the enzyme dissolved in aqueous buffer. For extended self-disinfection, however, active and stable AcT bound onto or incorporated into a surface coating is necessary. In the current study, an active, stable and reusable AcT-based coating was developed by incorporating AcT into a polydopamine (PDA) matrix in a single step, thereby forming a biocatalytic composite onto a variety of surfaces. The resulting AcT-PDA composite coatings on glass, metal and epoxy surfaces yielded up to 7-log reduction of Gram-positive and Gram-negative bacteria when in contact with the biocatalytic coating. This composite coating also possessed potent antiviral activity, and dramatically reduced the infectivity of a SARS-CoV-2 pseudovirus within minutes. The single-step approach enables rapid and facile fabrication of enzyme-based disinfectant composite coatings with high activity and stability, which enables reuse following surface washing. As a result, this enzyme-polymer composite technique may serve as a general strategy for preparing antibacterial and antiviral surfaces for applications in health care and common infrastructure safety, such as in schools, the workplace, transportation, etc.


Assuntos
Antibacterianos/química , Antivirais/química , Proteínas de Bactérias/química , Hidrolases/química , Indóis/química , Polímeros/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antivirais/metabolismo , Antivirais/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , COVID-19/patologia , COVID-19/virologia , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/metabolismo , Materiais Revestidos Biocompatíveis/farmacologia , Estabilidade de Medicamentos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Cinética , Mycobacterium smegmatis/enzimologia , Ácido Peracético/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , SARS-CoV-2/efeitos dos fármacos
7.
Biochem Biophys Res Commun ; 563: 92-97, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34062392

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has the characteristic accessory protein ORF8. Although clinical reports indicate that ORF8 variant strains (Δ382 and L84S variants) are less likely to cause severe illness, functional differences between wild-type and variant ORF8 are unknown. Furthermore, the physicochemical properties of the ORF8 protein have not been analyzed. In this study, the physicochemical properties of the wild-type ORF8 and its L84S variant were analyzed and compared. Using the tobacco BY-2 cell production system, which has been successfully used to produce the wild-type ORF8 protein with a single conformation, was used to successfully produce the ORF8 L84S variant protein at the same level as wild-type ORF8. The produced proteins were purified, and their temperature and pH dependencies were examined using nuclear magnetic resonance spectra. Our data suggested that the wild-type and L84S variant ORF8 structures are highly stable over a wide temperature range. Both proteins displayed an aggregated conformation at higher temperature that reverted when the temperature was decreased to room temperature. Moreover, ORF8 precipitated at acidic pH and this precipitation was reversed when the solution pH was shifted to neutral. Interestingly, the L84S variant exhibited greater solubility than wild-type ORF8 under acidic conditions. Thus, the finding indicated that conformational stability and reversibility of ORF8 are key properties related to function in oppressive environments.


Assuntos
COVID-19/virologia , SARS-CoV-2/química , Proteínas Virais/química , COVID-19/metabolismo , COVID-19/patologia , Humanos , Conformação Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Relação Estrutura-Atividade , Proteínas Virais/genética , Proteínas Virais/metabolismo
8.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070875

RESUMO

TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation. In this study, we have investigated the conformational dynamics of TRAF2-C through circular dichroism, fluorescence, and dynamic light scattering, performing temperature-dependent measurements. The data indicate that the protein retains its oligomeric state and most of its secondary structure, while displaying a significative increase in the heterogeneity of the tyrosines signal, increasing the temperature from ≈15 to ≈35 °C. The peculiar crowding of tyrosine residues (12 out of 18) at the three subunit interfaces and the strong dependence on the trimer concentration indicate that such conformational changes mainly involve the contact areas between each pair of monomers, affecting the oligomeric state. Molecular dynamic simulations in this temperature range suggest that the interfaces heterogeneity is an intrinsic property of the trimer that arises from the continuous, asymmetric approaching and distancing of its subunits. Such dynamics affect the results of molecular docking on the external protein surface using receptor peptides, indicating that the TRAF2-receptor interaction in the solution might not involve three subunits at the same time, as suggested by the static analysis obtainable from the crystal structure. These findings shed new light on the role that the TRAF2 oligomeric state might have in regulating the protein binding activity in vivo.


Assuntos
Subunidades Proteicas/química , Fator 2 Associado a Receptor de TNF/química , Tirosina/química , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Pró-Proteína Convertases/química , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteína de Domínio de Morte Associada a Receptor de TNF/química , Proteína de Domínio de Morte Associada a Receptor de TNF/genética , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Termodinâmica , Tirosina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
9.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069064

RESUMO

Extracellular vesicles (EVs) are valued candidates for the development of new tools for medical applications. Vesicles carrying melanoma-associated antigen A (MAGEA) proteins, a subfamily of cancer-testis antigens, are particularly promising tools in the fight against cancer. Here, we have studied the biophysical and chemical properties of MAGEA4-EVs and show that they are stable under common storage conditions such as keeping at +4 °C and -80 °C for at least 3 weeks after purification. The MAGEA4-EVs can be freeze-thawed two times without losing MAGEA4 in detectable quantities. The attachment of MAGEA4 to the surface of EVs cannot be disrupted by high salt concentrations or chelators, but the vesicles are sensitive to high pH. The MAGEA4 protein can bind to the surface of EVs in vitro, using robust passive incubation. In addition, EVs can be loaded with recombinant proteins fused to the MAGEA4 open reading frame within the cells and also in vitro. The high stability of MAGEA4-EVs ensures their potential for the development of EV-based anti-cancer applications.


Assuntos
Antígenos de Neoplasias/química , Vesículas Extracelulares/química , Proteínas de Neoplasias/química , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/isolamento & purificação , Antígenos de Neoplasias/metabolismo , Armazenamento de Medicamentos , Vesículas Extracelulares/metabolismo , Congelamento , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Concentração de Íons de Hidrogênio , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/isolamento & purificação , Proteínas de Neoplasias/metabolismo , Octoxinol/química , Proteínas Recombinantes/química , Sais/química
10.
Structure ; 29(7): 655-663.e4, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34111408

RESUMO

Emerging SARS-CoV-2 strains, B.1.1.7 and B.1.351, from the UK and South Africa, respectively, show decreased neutralization by monoclonal antibodies and convalescent or vaccinee sera raised against the original wild-type virus, and are thus of clinical concern. However, the neutralization potency of two antibodies, 1-57 and 2-7, which target the receptor-binding domain (RBD) of the spike, was unaffected by these emerging strains. Here, we report cryo-EM structures of 1-57 and 2-7 in complex with spike, revealing each of these antibodies to utilize a distinct mechanism to bypass or accommodate RBD mutations. Notably, each antibody represented an immune response with recognition distinct from those of frequent antibody classes. Moreover, many epitope residues recognized by 1-57 and 2-7 were outside hotspots of evolutionary pressure for ACE2 binding and neutralizing antibody escape. We suggest the therapeutic use of antibodies, such as 1-57 and 2-7, which target less prevalent epitopes, could ameliorate issues of monoclonal antibody escape.


Assuntos
Enzima de Conversão de Angiotensina 2/química , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Receptores Virais/química , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Epitopos/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/genética , Receptores Virais/imunologia , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
11.
Sci Signal ; 14(689)2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34131072

RESUMO

Capping of viral messenger RNAs is essential for efficient translation, for virus replication, and for preventing detection by the host cell innate response system. The SARS-CoV-2 genome encodes the 2'-O-methyltransferase nsp16, which, when bound to the coactivator nsp10, uses S-adenosylmethionine (SAM) as a donor to transfer a methyl group to the first ribonucleotide of the mRNA in the final step of viral mRNA capping. Here, we provide biochemical and structural evidence that this reaction requires divalent cations, preferably Mn2+, and a coronavirus-specific four-residue insert. We determined the x-ray structures of the SARS-CoV-2 2'-O-methyltransferase (the nsp16-nsp10 heterodimer) in complex with its reaction substrates, products, and divalent metal cations. These structural snapshots revealed that metal ions and the insert stabilize interactions between the capped RNA and nsp16, resulting in the precise alignment of the ribonucleotides in the active site. Comparison of available structures of 2'-O-methyltransferases with capped RNAs from different organisms revealed that the four-residue insert unique to coronavirus nsp16 alters the backbone conformation of the capped RNA in the binding groove, thereby promoting catalysis. This insert is highly conserved across coronaviruses, and its absence in mammalian methyltransferases makes this region a promising site for structure-guided drug design of selective coronavirus inhibitors.


Assuntos
COVID-19/virologia , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Humanos , Manganês/metabolismo , Metilação , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Capuzes de RNA/química , Capuzes de RNA/genética , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/genética , Transdução de Sinais , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
12.
Nat Commun ; 12(1): 3902, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162863

RESUMO

Self-assembly of proteins holds great promise for the bottom-up design and production of synthetic biomaterials. In conventional approaches, designer proteins are pre-programmed with specific recognition sites that drive the association process towards a desired organized state. Although proven effective, this approach poses restrictions on the complexity and material properties of the end-state. An alternative, hierarchical approach that has found wide adoption for inorganic systems, relies on the production of crystalline nanoparticles that become the building blocks of a next-level assembly process driven by oriented attachment (OA). As it stands, OA has not yet been observed for protein systems. Here we employ cryo-transmission electron microscopy (cryoEM) in the high nucleation rate limit of protein crystals and map the self-assembly route at molecular resolution. We observe the initial formation of facetted nanocrystals that merge lattices by means of OA alignment well before contact is made, satisfying non-trivial symmetry rules in the process. As these nanocrystalline assemblies grow larger we witness imperfect docking events leading to oriented aggregation into mesocrystalline assemblies. These observations highlight the underappreciated role of the interaction between crystalline nuclei, and the impact of OA on the crystallization process of proteins.


Assuntos
Aldose-Cetose Isomerases/química , Nanoestruturas/química , Proteínas Recombinantes/química , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Microscopia Crioeletrônica , Cristalização , Cristalografia por Raios X , Cinética , Modelos Moleculares , Nanoestruturas/ultraestrutura , Tamanho da Partícula , Mutação Puntual , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
13.
Molecules ; 26(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071665

RESUMO

Halogens have been reported to play a major role in the inhibition of monoamine oxidase (MAO), relating to diverse cognitive functions of the central nervous system. Pyrazoline/halogenated pyrazolines were investigated for their inhibitory activities against human monoamine oxidase-A and -B. Halogen substitutions on the phenyl ring located at the fifth position of pyrazoline showed potent MAO-B inhibition. Compound 3-(4-ethoxyphenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazole (EH7) showed the highest potency against MAO-B with an IC50 value of 0.063 µM. The potencies against MAO-B were increased in the order of -F (in EH7) > -Cl (EH6) > -Br (EH8) > -H (EH1). The residual activities of most compounds for MAO-A were > 50% at 10 µM, except for EH7 and EH8 (IC50 = 8.38 and 4.31 µM, respectively). EH7 showed the highest selectivity index (SI) value of 133.0 for MAO-B, followed by EH6 at > 55.8. EH7 was a reversible and competitive inhibitor of MAO-B in kinetic and reversibility experiments with a Ki value of 0.034 ± 0.0067 µM. The molecular dynamics study documented that EH7 had a good binding affinity and motional movement within the active site with high stability. It was observed by MM-PBSA that the chirality had little effect on the overall binding of EH7 to MAO-B. Thus, EH7 can be employed for the development of lead molecules for the treatment of various neurodegenerative disorders.


Assuntos
Simulação de Dinâmica Molecular , Inibidores da Monoaminoxidase/química , Pirazóis/química , Barreira Hematoencefálica/efeitos dos fármacos , Domínio Catalítico , Química Farmacêutica/métodos , Cognição/efeitos dos fármacos , Desenho de Fármacos , Halogênios/química , Humanos , Concentração Inibidora 50 , Cinética , Modelos Químicos , Simulação de Acoplamento Molecular , Estrutura Molecular , Monoaminoxidase/metabolismo , Movimento (Física) , Análise de Componente Principal , Ligação Proteica , Proteínas Recombinantes/química , Estereoisomerismo , Relação Estrutura-Atividade
14.
Methods Mol Biol ; 2268: 43-60, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085260

RESUMO

Large-scale recombinant expression of G protein-coupled receptors (GPCRs) is required for structure and function studies where there is a need for milligram amounts of protein in pure form. Here we describe a procedure for the construction of human embryonic kidney 293S (HEK293S) stable cell lines for inducible expression of the gene encoding bovine rhodopsin. The HEK293S cell line is particularly suitable for this application because of several favorable properties as a recombinant host including: its ease of transfection, its capacity for handling large amounts of protein cargo, and its ability to perform the necessary co- and post-translational modifications required for correct folding and processing of complex membrane proteins such as GPCRs. The procedures described here will focus on the HEK293S GnTI- cell line, an HEK293S derivative that is widely used for the production of glycoproteins modified homogeneously with truncated N-glycans.


Assuntos
Processamento de Proteína Pós-Traducional , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/metabolismo , Rodopsina/metabolismo , Animais , Bovinos , Glicosilação , Células HEK293 , Humanos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Rodopsina/genética , Rodopsina/isolamento & purificação , Transfecção
15.
Methods Mol Biol ; 2268: 159-178, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085268

RESUMO

A wealth of assays for screening GPCR activity have been developed. Biosensors that employ Förster Resonance Energy transfer (FRET) are specific and enable dynamic measurements. Moreover, FRET biosensors are ideally suited for the analysis of single living cells. The FRET biosensors described in this manuscript are entirely genetically encoded by plasmids. Here, protocols for employing FRET-based biosensors to detect G protein activity upon GPCR activation are reported. The protocols include details on the isolation of plasmids, transfection, generation of stable cell lines with the FRET biosensors, FRET ratio imaging, and data analysis.


Assuntos
Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas de Bactérias/química , Proteínas de Fluorescência Verde/química , Células HEK293 , Humanos , Proteínas Luminescentes/química , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transdução de Sinais
16.
Artigo em Inglês | MEDLINE | ID: mdl-34098178

RESUMO

Commercially approved conventional antibody-drug conjugates (ADCs) are produced as heterogeneous mixtures containing a stochastic distribution of payloads decorating the antibody molecules resulting in decreased efficacy and thus lowering their therapeutic index. Control of the DAR and conjugation site in the development of next-generation ADCs is believed to assist in increasing the therapeutic index of these targeted biologics leading to overall enhanced clinical efficacy and reduced toxicity. A chemical site-specific conjugation technology termed AJICAP® allows ADC developers to control both the location and quantity of the payload conjugation to an antibody. Furthermore, this simplified ADC composition enables a streamlined chemical analysis. Here we report the chromatographic separation of site-specific ADCs produced by AJICAP® technology using an analytical affinity chromatography HPLC column containing a recombinant FcγIIIa receptor-ligand immobilized on a non-porous polymer resin (NPR). These HPLC analyses provided visually clear chromatogram results reflecting the heterogeneity of each ADC. The affinity strength was also measured by biolayer interferometry (BLI) and predicted by molecular structure analysis. The results indicate that AJICAP® technology is a promising solution to link hydrophobic payloads to antibodies without compromising antibody receptor function. This study also shows that FcγIIIa-NPR column can be used to characterize site-specific conjugated ADCs compared to ADCs synthesized using conventional methods.


Assuntos
Cromatografia de Afinidade/métodos , Imunoconjugados , Receptores de IgG , Proteínas Recombinantes , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Imunoconjugados/análise , Imunoconjugados/química , Imunoconjugados/metabolismo , Modelos Moleculares , Porosidade , Receptores de IgG/análise , Receptores de IgG/química , Receptores de IgG/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
17.
Int J Mol Sci ; 22(10)2021 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-34065754

RESUMO

Cyanobacteriochromes (CBCRs) are promising optogenetic tools for their diverse absorption properties with a single compact cofactor-binding domain. We previously uncovered the ultrafast reversible photoswitching dynamics of a red/green photoreceptor AnPixJg2, which binds phycocyanobilin (PCB) that is unavailable in mammalian cells. Biliverdin (BV) is a mammalian cofactor with a similar structure to PCB but exhibits redder absorption. To improve the AnPixJg2 feasibility in mammalian applications, AnPixJg2_BV4 with only four mutations has been engineered to incorporate BV. Herein, we implemented femtosecond transient absorption (fs-TA) and ground state femtosecond stimulated Raman spectroscopy (GS-FSRS) to uncover transient electronic dynamics on molecular time scales and key structural motions responsible for the photoconversion of AnPixJg2_BV4 with PCB (Bpcb) and BV (Bbv) cofactors in comparison with the parent AnPixJg2 (Apcb). Bpcb adopts the same photoconversion scheme as Apcb, while BV4 mutations create a less bulky environment around the cofactor D ring that promotes a faster twist. The engineered Bbv employs a reversible clockwise/counterclockwise photoswitching that requires a two-step twist on ~5 and 35 picosecond (ps) time scales. The primary forward Pfr → Po transition displays equal amplitude weights between the two processes before reaching a conical intersection. In contrast, the primary reverse Po → Pfr transition shows a 2:1 weight ratio of the ~35 ps over 5 ps component, implying notable changes to the D-ring-twisting pathway. Moreover, we performed pre-resonance GS-FSRS and quantum calculations to identify the Bbv vibrational marker bands at ~659,797, and 1225 cm-1. These modes reveal a stronger H-bonding network around the BV cofactor A ring with BV4 mutations, corroborating the D-ring-dominant reversible photoswitching pathway in the excited state. Implementation of BV4 mutations in other PCB-binding GAF domains like AnPixJg4, AM1_1870g3, and NpF2164g5 could promote similar efficient reversible photoswitching for more directional bioimaging and optogenetic applications, and inspire other bioengineering advances.


Assuntos
Biliverdina/química , Cianobactérias/genética , Fotorreceptores Microbianos/química , Fitocromo/química , Substituição de Aminoácidos , Biliverdina/genética , Sítios de Ligação , Cianobactérias/metabolismo , Eletrônica , Cinética , Processos Fotoquímicos , Fotorreceptores Microbianos/genética , Fitocromo/genética , Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Análise Espectral , Análise Espectral Raman , Tempo , Fatores de Tempo
18.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065885

RESUMO

Genetic engineering of plants has turned out to be an attractive approach to produce various secondary metabolites. Here, we attempted to produce kynurenine, a health-promoting metabolite, in plants of Nicotiana tabacum (tobacco) transformed by Agrobacterium tumefaciens with the gene, coding for human indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme responsible for the kynurenine production because of tryptophan degradation. The presence of IDO1 gene in transgenic plants was confirmed by PCR, but the protein failed to be detected. To confer higher stability to the heterologous human IDO1 protein and to provide a more sensitive method to detect the protein of interest, we cloned a gene construct coding for IDO1-GFP. Analysis of transiently transfected tobacco protoplasts demonstrated that the IDO1-GFP gene led to the expression of a detectable protein and to the production of kynurenine in the protoplast medium. Interestingly, the intracellular localisation of human IDO1 in plant cells is similar to that found in mammal cells, mainly in cytosol, but in early endosomes as well. To the best of our knowledge, this is the first report on the expression of human IDO1 enzyme capable of secreting kynurenines in plant cells.


Assuntos
Agrobacterium tumefaciens/fisiologia , Proteínas de Fluorescência Verde/genética , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Cinurenina/metabolismo , Tabaco/microbiologia , Agrobacterium tumefaciens/genética , Clonagem Molecular , Proteínas de Fluorescência Verde/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Plasmídeos/genética , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tabaco/genética , Tabaco/metabolismo , Transformação Bacteriana
19.
Nat Commun ; 12(1): 3287, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078893

RESUMO

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.


Assuntos
Magnésio/química , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Magnésio/metabolismo , Metilação , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Capuzes de RNA/química , Capuzes de RNA/genética , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética
20.
Nat Commun ; 12(1): 3305, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083522

RESUMO

Dopamine D1 receptor (D1R) is an important drug target implicated in many psychiatric and neurological disorders. Selective agonism of D1R are sought to be the therapeutic strategy for these disorders. Most selective D1R agonists share a dopamine-like catechol moiety in their molecular structure, and their therapeutic potential is therefore limited by poor pharmacological properties in vivo. Recently, a class of non-catechol D1R selective agonists with a distinct scaffold and pharmacological properties were reported. Here, we report the crystal structure of D1R in complex with stimulatory G protein (Gs) and a non-catechol agonist Compound 1 at 3.8 Å resolution. The structure reveals the ligand bound to D1R in an extended conformation, spanning from the orthosteric site to extracellular loop 2 (ECL2). Structural analysis reveals that the unique features of D1R ligand binding pocket explains the remarkable selectivity of this scaffold for D1R over other aminergic receptors, and sheds light on the mechanism for D1R activation by the non-catechol agonist.


Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Técnicas In Vitro , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química
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