RESUMO
A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the global pandemic of COVID-19 in 2020. Through structural analysis, it was found that several amino acid residues in the human angiotensin-converting enzyme-2 (hACE2) receptor directly interact with those in the receptor binding domain (RBD) of the spike glycoprotein (S-protein). Various cell lines, including HEK293, HeLa cells, and the baculovirus expression vector system (BEVS) with the insect cell line Sf9, have been utilized to produce the RBD. In this study, we investigated the use of Bombyx mori nucleopolyhedrovirus (BmNPV) and BEVS. For efficient production of a highly pure recombinant RBD protein, we designed it with two tags (His tag and STREP tag) at the C-terminus and a solubilizing tag (SUMO) at the N-terminus. After expressing the protein using BmNPV and silkworm and purifying it with a HisTrap excel column, the eluted protein was digested with SUMO protease and further purified using a Strep-Tactin Superflow column. As a result, we obtained the RBD as a monomer with a yield of 2.6 mg/10 mL serum (equivalent to 30 silkworms). The RBD showed an affinity for the hACE2 receptor. Additionally, the RBDs from the Alpha, Beta, Gamma, Delta, and Omicron variants were expressed and purified using the same protocol. It was found that the RBD from the Alpha, Beta, Gamma, and Delta variants could be obtained with yields of 1.4-2.6 mg/10 mL serum and had an affinity to the hACE2 receptor.
Assuntos
Bombyx , COVID-19 , Nucleopoliedrovírus , Animais , Humanos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Bombyx/genética , Bombyx/metabolismo , Células HeLa , Células HEK293 , Proteínas Recombinantes , Ligação ProteicaRESUMO
The coronavirus disease 2019 (COVID-19) pandemic has necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 3C-like protease (3CLpro) is a promising target for COVID-19 treatment. Here, we report a new class of covalent inhibitors of 3CLpro that possess chlorofluoroacetamide (CFA) as a cysteine-reactive warhead. Based on an aza-peptide scaffold, we synthesized a series of CFA derivatives in enantiopure form and evaluated their biochemical efficiency. The data revealed that 8a (YH-6) with the R configuration at the CFA unit strongly blocks SARS-CoV-2 replication in infected cells, and its potency is comparable to that of nirmatrelvir. X-ray structural analysis showed that YH-6 formed a covalent bond with Cys145 at the catalytic center of 3CLpro. The strong antiviral activity and favorable pharmacokinetic properties of YH-6 suggest its potential as a lead compound for the treatment of COVID-19.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Humanos , Proteases 3C de Coronavírus , Peptídeo Hidrolases , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Inibidores de Proteases/química , Cisteína , Cisteína Endopeptidases/química , Antivirais/farmacologia , Antivirais/uso terapêutico , Antivirais/química , Peptídeos/químicaRESUMO
Diacylglycerol kinases (DGKs) are multi-domain lipid kinases that modulate the levels of lipid messengers, diacylglycerol, and phosphatidic acid. Recently, increasing attention has been paid to its α isozyme (DGKα) as a potential target for cancer immunotherapy. However, little progress has been made on the structural biology of DGKs, and a detailed understanding of the Ca2+ -triggered activation of DGKα, for which the N-terminal domains likely play a critical role, remains unclear. We have recently shown that Ca2+ binding to DGKα-EF induces conformational changes from a protease-susceptible "open" conformation in the apo state to a well-folded one in its holo state. Here, we further studied the structural properties of DGKα N-terminal (RVH and EF) domains using a series of biophysical techniques. We first revealed that the N-terminal RVH domain is a novel Ca2+ -binding domain, but the Ca2+ -induced conformational changes mainly occur in the EF domain. This was corroborated by NMR experiments showing that the EF domain adopts a molten-globule like structure in the apo state. Further analyses using SEC-SAXS and NMR indicate that the partially unfolded EF domain interacts with RVH domain, likely via hydrophobic interactions in the absence of Ca2+ , and this interaction is modified in the presence of Ca2+ . Taken together, these results present novel insights into the structural rearrangement of DGKα N-terminal domains upon binding to Ca2+ , which is essential for the activation of the enzyme.
Assuntos
Diacilglicerol Quinase , Diglicerídeos , Diacilglicerol Quinase/genética , Endopeptidases , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The introduction of intermolecular disulfide bonds by amino acid mutations is an effective method for stabilizing dimeric proteins. X-ray crystal structure of Fab of a therapeutic antibody, adalimumab, revealed the first loop of the CH1 domain to be partially unsolved at position 135-141. To find new sites for the introduction of intermolecular disulfide bonds in adalimumab Fab, Fab mutants targeting the unsolved region were predicted using molecular simulation software. Four Fab mutants, H:K137C-L:I117C, H:K137C-L:F209C, H:S138C-L:F116C and H:S140C-L:S114C, were expressed in the methylotrophic yeast Pichia pastoris. SDS-PAGE analysis of these mutants indicated that H:K137C-L:F209C, H:S138C-L:F116C and H:S140C-L:S114C mutants mostly formed intermolecular disulfide bonds, whereas some H:K137C-L:I117C mutants formed intermolecular disulfide bonds and some did not. Differential scanning calorimetry measurements showed increased thermal stability in all Fab mutants with engineered disulfide bonds. The bio-layer interferometry measurements, for binding of the antigen tumor necrotic factor α, indicated that Fab mutants had less antigen-binding activity than wild-type Fab. In particular, the KD value of H:K137C-L:F209C was ~17 times higher than that of wild-type Fab. Thus, we successfully introduced intermolecular disulfide bonds between the first loop region of the CH1 and CL domains and observed that it increases the thermostability of Fab and affects the antigen-binding activity.
Assuntos
Dissulfetos , Fragmentos Fab das Imunoglobulinas , Adalimumab/genética , Dissulfetos/química , Fragmentos Fab das Imunoglobulinas/genéticaRESUMO
AL amyloidosis is a life-threatening disease characterized by the deposition of amyloidogenic immunoglobulin light chain secreted from clonal plasma cells. Here we established an in-vitro screening system of amyloid inhibition of a variable domain in λ6 light chain mutant (Vλ6), Wil, and screened a food-additive compound library to identify compounds inhibiting the fibril formation. We found gossypetin and isoquercitrin as novel inhibitors. NMR analysis showed that both compounds directly interacted with natively-folded Wil, and proteolysis experiments demonstrated that these compounds conferred proteolytic resistance, suggesting that the compounds enhance the kinetic stability of Wil. Since gossypetin and isoquercitrin specifically interacted with the protein at micromolar concentrations, these compounds could be used as lead to further develop inhibitors against AL amyloidosis.
Assuntos
Amiloide/antagonistas & inibidores , Flavonoides/farmacologia , Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Cadeias lambda de Imunoglobulina/metabolismo , Quercetina/análogos & derivados , Amiloide/genética , Amiloide/metabolismo , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Catequina/análogos & derivados , Catequina/metabolismo , Catequina/farmacologia , Relação Dose-Resposta a Droga , Flavonoides/química , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/química , Cadeias lambda de Imunoglobulina/genética , Cinética , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Mutação , Ligação Proteica , Estabilidade Proteica/efeitos dos fármacos , Quercetina/química , Quercetina/farmacologia , Fatores de TempoRESUMO
We have recently developed a mouse monoclonal antibody (12-10H) binding to the head domain region in rat P2X4 receptor (rP2X4R, which is crucial for the pathogenesis of neuropathic pain) expressed on the cell with the highest binding affinity (KD = 20 nM). However, the 12-10H antibody failed to detect endogenously expressed P2X4Rs in microglia isolated from the spinal cord of rats whose spinal nerves were injured. Then, we prepared R5 mutant, in which five arginine residues were introduced into variable regions except for the "hot spot" in the 12-10H antibody to increase electrostatic interactions with the head domain, an anionic region, in rP2X4R. The mutation resulted in an increase of 50-fold in the affinity of the R5 mutant for the head domain with respect to the intact 12-10H antibody. As a result, detection of P2X4Rs endogenously expressed on primary cultured microglial cells originated from the neonatal rat brain and spinal cord microglia isolated from a rat model of neuropathic pain was achieved. These findings suggest a strategy to improve the affinity of a monoclonal antibody for an anionic antigen by the introduction of several arginine residues into variable regions other than the "hot spot" in the paratope.
Assuntos
Anticorpos Monoclonais/farmacologia , Microglia/efeitos dos fármacos , Neuralgia/tratamento farmacológico , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X4/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Células CHO , Linhagem Celular Tumoral , Cricetulus , Modelos Animais de Doenças , Humanos , Masculino , Microglia/imunologia , Microglia/metabolismo , Mutação , Neuralgia/imunologia , Neuralgia/metabolismo , Ligação Proteica , Conformação Proteica , Antagonistas do Receptor Purinérgico P2X/imunologia , Ratos Wistar , Receptores Purinérgicos P2X4/genética , Receptores Purinérgicos P2X4/imunologia , Receptores Purinérgicos P2X4/metabolismo , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
Glycosylation is one of the major post-translational modifications in eukaryotic cells and has been reported to affect the amyloid fibril formation in several amyloidogenic proteins and peptides. In this study, we expressed a Vλ6 light chain mutant, Wil, which is an amyloidogenic mutant in AL amyloidosis, by the yeast Pichia pastoris. After separation by cation exchange chromatography, we obtained the O-glycosylated and non-glycosylated Wil mutants in high yield. The structures of these Wil mutants were identical except with respect to glycosylation, and the stabilities were also identical. On the other hand, the O-glycosylation retarded the amyloid fibril formation in a sugar size-dependent manner. From these results, we discussed the role of covalently attached glycan in the retardation of amyloid fibril formation.
Assuntos
Amiloide/química , Região Variável de Imunoglobulina/química , Cadeias lambda de Imunoglobulina/química , Mutação , Processamento de Proteína Pós-Traducional , Amiloide/genética , Amiloide/metabolismo , Glicosilação , Humanos , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Cadeias lambda de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/metabolismo , Multimerização Proteica , Estabilidade Proteica , SaccharomycetalesRESUMO
In Escherichia coli, DNA replication is restarted following DNA repair by the PriA-dependent pathway, in which the binding and dissociation of proteins such as PriA, PriB, and DnaT on ssDNA lead to the formation of a protein-DNA complex for recruiting the DnaB-DnaC replication protein complex. However, the structure of the PriB-DnaT complex, which is an essential step in the PriA-dependent pathway, remains elusive. In this study, the importance of His26 in PriB for replication restart was reconfirmed using plasmid complementation. Furthermore, we used NMR to examine the DnaT interaction sites on PriB. We also evaluated the PriB-DnaT peptide complex model, which was prepared by in silico docking, using molecular dynamic simulation. From these data, we propose a structural model that provides insight into the PriB-DnaT interaction.
Assuntos
DNA Bacteriano/química , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Sítios de Ligação , Replicação do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Teste de Complementação Genética , Simulação de Dinâmica Molecular , Mutação , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , TermodinâmicaRESUMO
Conjugation with polyethylene glycol (PEG) is performed to increase serum half-life of the Fab for clinical applications. However, current designs for recombinant Fab only allow PEGylation at the interchain SS bond (disulfide bond) at the C-terminal end of the heavy chain and light chain of the Fab, which the decrease of thermostability occurred by partial reduction of the interchain SS bond. An adalimumab Fab mutant with a novel interchain SS bond (CH1 : C177-CL : C160) and one cysteine at the C-terminal end (mutSS FabSH) was designed to maintain Fab thermostability and for site-specific PEGylation. MutSS FabSH was expressed in Pichia pastoris and purified mutSS FabSH was conjugated with 20-kDa PEG targeted at the free cysteine. Based on enzyme-linked immunosorbent assay (ELISA), PEGylation did not affect the binding capacity of the mutSS FabSH. To confirm the influence of PEGylation on the pharmacokinetic behavior of the Fab, PEGylated mutSS FabSH was administered to rats via tail vein injection. Analysis of the mean serum concentration of the PEGylated mutSS FabSH versus time through ELISA indicated an increase in half-life compared to that of non-PEGylated wild-type Fab. Consequently, we have successfully demonstrated that a Fab mutant with a novel interchain SS bond and one free cysteine at the C-terminal end can be PEGylated without changes in functionality. This design can potentially be used as a platform for modification of other recombinant Fabs.
Assuntos
Adalimumab/química , Fragmentos Fab das Imunoglobulinas/química , Animais , Cisteína/química , Masculino , Mutação , Polietilenoglicóis/química , Ratos , Ratos Sprague-DawleyRESUMO
P2X purinergic receptors are ATP-driven ionic channels expressed as trimers and showing various functions. A subtype, the P2X4 receptor present on microglial cells is highly involved in neuropathic pain. In this study, in order to prepare antibodies recognizing the native structure of rat P2X4 (rP2X4) receptor, we immunized mice with rP2X4's head domain (rHD, Gln111-Val167), which possesses an intact structure stabilized by S-S bond formation (Igawa and Abe et al. FEBS Lett. 2015), as an antigen. We generated five monoclonal antibodies with the ability to recognize the native structure of its head domain, stabilized by S-S bond formation. Site-directed mutagenesis revealed that Asn127 and Asp131 of the rHD, in which combination of these amino acid residues is only conserved in P2X4 receptor among P2X family, were closely involved in the interaction between rHD and these antibodies. We also demonstrated the antibodies obtained here could detect rP2X4 receptor expressed in 1321N1 human astrocytoma cells.
Assuntos
Anticorpos Monoclonais , Receptores Purinérgicos P2X4 , Animais , Humanos , Camundongos , Domínios Proteicos , Ratos , Receptores Purinérgicos P2X4/análise , Receptores Purinérgicos P2X4/químicaRESUMO
Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1-10 µM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton's tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI.
Assuntos
Acetamidas/síntese química , Cisteína/metabolismo , Quinazolinas/síntese química , Acetamidas/química , Acetamidas/farmacologia , Animais , Antineoplásicos , Linhagem Celular , Receptores ErbB , Humanos , Camundongos , Camundongos Nus , Neoplasias , Fosfotransferases/fisiologia , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/antagonistas & inibidores , Quinazolinas/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Light chains are abnormally overexpressed from disordered monoclonal B-cells and form amyloid fibrils, which are then deposited on the affected organ, leading to a form of systemic amyloidosis known as AL (Amyloid Light chain) amyloidosis. A green tea catechin, epigallocatechin-3-O-gallate (EGCG), which is thought to inhibit various amyloidoses, is a potent inhibitor of amyloid fibril formation in AL amyloidosis. METHODS: An amyloidogenic variable domain in λ6 light chain mutant, Wil was incubated in the presence of EGCG. The incubation products were analyzed by SDS-PAGE and reverse-phase HPLC. The interaction between Wil and EGCG was observed by using NMR and tryptophan fluorescence. RESULTS: EGCG inhibited the amyloid fibril formation of Wil at pHâ¯7.5 and 42⯰C. Under these conditions, most Wil populations were in the unfolded state and several chemical reactions, i.e., oxidation and/or covalent bond oligomerization could be induced by auto-oxidated EGCG. Moreover, we found that EGCG bound to the unfolded state of Wil with higher affinity (Kdâ¯=â¯7⯵M). CONCLUSIONS: Inhibition of amyloid fibril formation of Wil was caused by 1) EGCG binding to unfolded state rather than folded state and 2) chemical modifications of Wil by auto oxidation of EGCG. GENERAL SIGNIFICANCE: In the competitive formation of amyloid fibrils and off-pathway oligomers, EGCG produces the latter immediately after it preferentially binds to the unfolded state. It may be general mechanism of EGCG inhibition for amyloidosis.
Assuntos
Amiloide/antagonistas & inibidores , Amiloide/química , Catequina/análogos & derivados , Cadeias Leves de Imunoglobulina/química , Sequência de Aminoácidos , Amiloide/metabolismo , Ácido Ascórbico/química , Catequina/metabolismo , Catequina/farmacologia , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Metionina/metabolismo , Mutação , Oxirredução , Mapeamento de Peptídeos , Ligação Proteica , Dobramento de Proteína , Desdobramento de Proteína , Análise Espectral/métodos , TermodinâmicaRESUMO
Protein tyrosine sulfation (PTS) is a post-translational modification regulating numerous biological events. PTS generally occurs at flexible regions of proteins, enhancing intermolecular interactions between proteins. Because of the high flexibility associated with the regions where PTS is generally encountered, an atomic-level understanding has been difficult to achieve by X-ray crystallography or nuclear magnetic resonance techniques. In this study, we focused on the conformational behavior of a flexible sulfated peptide and its interaction with an antibody. Molecular dynamics simulations and thermodynamic analysis indicated that PTS reduced the main-chain fluctuations upon the appearance of sulfate-mediated intramolecular H-bonds. Collectively, our data suggested that one of the mechanisms by which PTS may enhance protein-protein interactions consists of the limitation of conformational dynamics in the unbound state, thus reducing the loss of entropy upon binding and boosting the affinity for its partner.
Assuntos
Anticorpos/metabolismo , Peptídeos/metabolismo , Tirosina/análogos & derivados , Anticorpos/química , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Peptídeos/química , Ligação Proteica , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Termodinâmica , Tirosina/química , Tirosina/metabolismoRESUMO
Rheumatoid factors (RFs) are autoantibodies against the fragment-crystallizable (Fc) region of IgG. In individuals with hematological diseases such as cryoglobulinemia and certain B cell lymphoma forms, the RFs derived from specific heavy- and light-chain germline pairs, so-called "stereotypic RFs," are frequently produced in copious amounts and form immune complexes with IgG in serum. Of note, many structural details of the antigen recognition mechanisms in RFs are unclear. Here we report the crystal structure of the RF YES8c derived from the IGHV1-69/IGKV3-20 germline pair, the most common of the stereotypic RFs, in complex with human IgG1-Fc at 2.8 Å resolution. We observed that YES8c binds to the CH2-CH3 elbow in the canonical antigen-binding manner involving a large antigen-antibody interface. On the basis of this observation, combined with mutational analyses, we propose a recognition mechanism common to IGHV1-69/IGKV3-20 RFs: (1) the interaction of the Leu432-His435 region of Fc enables the highly variable complementarity-determining region (CDR)-H3 to participate in the binding, (2) the hydrophobic tip in the CDR-H2 typical of IGHV1-69 antibodies recognizes the hydrophobic patch on Fc, and (3) the interaction of the highly conserved RF light chain with Fc is important for RF activity. These features may determine the putative epitope common to the IGHV1-69/IGKV3-20 RFs. We also showed that some mutations in the binding site of RF increase the affinity to Fc, which may aggravate hematological diseases. Our findings unravel the structural basis for germline-encoded antibody autoreactivity.
Assuntos
Autoanticorpos/química , Autoanticorpos/imunologia , Células Germinativas , Fator Reumatoide/metabolismo , Substituição de Aminoácidos , Afinidade de Anticorpos , Sítios de Ligação , Regiões Determinantes de Complementaridade , Cristalografia por Raios X , Epitopos/imunologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoglobulina G/química , Imunoglobulina G/imunologia , Mutagênese , Conformação Proteica , Receptores Fc , Fator Reumatoide/química , Fator Reumatoide/imunologia , Relação Estrutura-AtividadeRESUMO
We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH1 and CL domains was deleted by substitution of Cys with Ala (FabΔSS). DSC measurements showed that the Tm values of FabΔSS were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of FabΔSS. The resulting Fab (mutSS FabΔSS) had the mutations H:V177C and L:Q160C in FabΔSS, confirming the formation of the disulfide bond between CH1 and CL. The thermostability of mutSS FabΔSS was approximately 5 °C higher than that of FabΔSS. Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of FabΔSS and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation.
Assuntos
Adalimumab/química , Adalimumab/genética , Adalimumab/metabolismo , Dissulfetos/química , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Modelos Moleculares , Pichia/genética , Conformação Proteica , Engenharia de Proteínas , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
AIM: To determine the possibility that diabetes mellitus promotes pancreatic ductal adenocarcinoma via glyceraldehyde (GA)-derived advanced glycation-end products (GA-AGEs). METHODS: PANC-1, a human pancreatic cancer cell line, was treated with 1-4 mmol/L GA for 24 h. The cell viability and intracellular GA-AGEs were measured by WST-8 assay and slot blotting. Moreover, immunostaining of PANC-1 cells with an anti-GA-AGE antibody was performed. Western blotting (WB) was used to analyze the molecular weight of GA-AGEs. Heat shock proteins 90α, 90ß, 70, 27 and cleaved caspase-3 were analyzed by WB. In addition, PANC-1 cells were treated with GA-AGEs-bovine serum albumin (GA-AGEs-BSA), as a model of extracellular GA-AGEs, and proliferation of PANC-1 cells was measured. RESULTS: In PANC-1 cells, GA induced the production of GA-AGEs and cell death in a dose-dependent manner. PANC-1 cell viability was approximately 40% with a 2 mmol/L GA treatment and decreased to almost 0% with a 4 mmol/L GA treatment (each significant difference was P < 0.01). Cells treated with 2 and 4 mmol/L GA produced 6.4 and 21.2 µg/mg protein of GA-AGEs, respectively (P < 0.05 and P < 0.01). The dose-dependent production of some high-molecular-weight (HMW) complexes of HSP90ß, HSP70, and HSP27 was observed following administration of GA. We considered HMW complexes to be dimers and trimers with GA-AGEs-mediated aggregation. Cleaved caspase-3 could not be detected with WB. Furthermore, 10 and 20 µg/mL GA-AGEs-BSA was 27% and 34% greater than that of control cells, respectively (P < 0.05 and P < 0.01). CONCLUSION: Although intracellular GA-AGEs induce pancreatic cancer cell death, their secretion and release may promote the proliferation of other pancreatic cancer cells.
Assuntos
Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/patologia , Diabetes Mellitus Tipo 2/patologia , Produtos Finais de Glicação Avançada/metabolismo , Gliceraldeído/metabolismo , Neoplasias Pancreáticas/patologia , Adenocarcinoma/etiologia , Apoptose/efeitos dos fármacos , Western Blotting , Carcinoma Ductal Pancreático/etiologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 2/complicações , Proteínas de Choque Térmico/metabolismo , Humanos , Pâncreas/patologia , Neoplasias Pancreáticas/etiologia , Sais de Tetrazólio/farmacologia , Regulação para CimaRESUMO
The P2X receptor is an ATP-gated cation channel expressed on the plasma membrane. The head domain (Gln111-Val167 in the rat P2X4 receptor) regulates ATP-induced cation influx. In this study, we prepared a head domain with three disulfide bonds, such as the intact rat P2X4 receptor contains. NMR analysis showed that the head domain possessed the same fold as in the zebrafish P2X4 receptor previously determined by crystallography. Furthermore, the inhibitory, divalent, metal ion binding sites were determined by NMR techniques. These findings will be useful for the design of specific inhibitors for the P2X receptor family.
Assuntos
Receptores Purinérgicos P2X4/química , Animais , Sítios de Ligação , Cobre/química , Cistina/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Soluções , Zinco/químicaRESUMO
Taurine is one of the osmolytes that maintain the structure of proteins in cells exposed to denaturing environmental stressors. Recently, cryoelectron tomographic analysis of eukaryotic cells has revealed that their cytoplasms are crowded with proteins. Such crowding conditions would be expected to hinder the efficient folding of nascent polypeptide chains. Therefore, we examined the role of taurine on the folding of denatured and reduced lysozyme, as a model protein, under a crowding condition. The results confirmed that taurine had a better effect on protein folding than did ß-alanine, which has a similar chemical structure, when the protein to be folded was present at submillimolar concentration. NMR analyses further revealed that under the crowding condition, taurine had more interactions than did ß-alanine with the lysozyme molecule in both the folded and denatured states. We concluded that taurine improves the folding of the reduced lysozyme at submillimolar concentration to allow it to interact more favorably with the lysozyme molecule. Thus, the role of taurine, as an osmolyte in vivo, may be to assist in the efficient folding of proteins.
Assuntos
Muramidase/química , Taurina/química , beta-Alanina/química , Animais , Galinhas , Clara de Ovo/química , Feminino , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Muramidase/genética , Ressonância Magnética Nuclear Biomolecular , Concentração Osmolar , Oxirredução , Pichia/genética , Pichia/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Non-alcoholic fatty liver disease (NAFLD) is a major cause of liver disease around the world. It includes a spectrum of conditions from simple steatosis to non-alcoholic steatohepatitis (NASH) and can lead to fibrosis, cirrhosis, liver failure, and/or hepatocellular carcinoma. NAFLD is also associated with other medical conditions such as obesity, diabetes mellitus (DM), metabolic syndrome, hypertension, insulin resistance, hyperlipidemia, and cardiovascular disease (CVD). In diabetes, chronic hyperglycemia contributes to the development of both macro- and microvascular conditions through a variety of metabolic pathways. Thus, it can cause a variety of metabolic and hemodynamic conditions, including upregulated advanced glycation end-products (AGEs) synthesis. In our previous study, the most abundant type of toxic AGEs (TAGE); i.e., glyceraldehyde-derived AGEs, were found to make a significant contribution to the pathogenesis of DM-induced angiopathy. Furthermore, accumulating evidence suggests that the binding of TAGE with their receptor (RAGE) induces oxidative damage, promotes inflammation, and causes changes in intracellular signaling and the expression levels of certain genes in various cell populations including hepatocytes and hepatic stellate cells. All of these effects could facilitate the pathogenesis of hypertension, cancer, diabetic vascular complications, CVD, dementia, and NASH. Thus, inhibiting TAGE synthesis, preventing TAGE from binding to RAGE, and downregulating RAGE expression and/or the expression of associated effector molecules all have potential as therapeutic strategies against NASH. Here, we examine the contributions of RAGE and TAGE to various conditions and novel treatments that target them in order to prevent the development and/or progression of NASH.
RESUMO
UNLABELLED: The ß-N-acetylglucosaminidase from the hyperthermophilic bacteria Thermotoga maritima (NagA) hydrolyzes chitooligomers into monomer ß-N-acetylglucosamine. Although NagA contains a highly conserved sequence motif found in glycoside hydrolase (GH) family 3, it can be distinguished from other GH family 3 ß-N-acetylglucosaminidases by its substrate specificity and biological assembly. To investigate its unique structure around the active site, we determined the crystal structure of NagA at a resolution of 2.43 Å. The NagA forms a dimer structure in which the monomer structure consists of an N- and a C-terminal domain. The dimer structure exhibits high solvation free energy for dimer formation. From mutagenesis analyses, the catalytic nucleophile and general acid-base residues were supposed to be Asp245 and His173, respectively. The most striking characteristic of NagA was that it forms the active site cleft from the N-terminal domain and the C-terminal domain of the next polypeptide chain, whereas the other two-domain GH family 3 enzymes form the site within the same molecule. Another striking feature is that the loops located around the active site show high flexibility. One of the flexible loops contains the general acid-base His173 and was thought to be involved in substrate distortion during catalysis. In addition, a loop in close contact with the active site, which comes from the C-terminal domain of the next polypeptide chain, contains a region of high B-factor values, indicating the possibility that the C-terminal domain is involved in catalysis. These results suggest that the dimer structure of NagA is important for its activity and thermostability. DATABASE: Structural data are available in the Protein Data Bank under accession number 3WO8.