RESUMO
Reovirus (Reo) has shown promising potential in specifically killing tumor cells, and offering new possibilities for ovarian cancer (OC) treatment. However, neutralizing antibodies in the ascites from OC patients greatly limit the further application of Reo. In this study, we employed cationic liposomes (Lipo) to deliver Reo, significantly enhancing its ability to enter OC cells and its effectiveness in killing these cells under ascitic conditions. Pre-treatment with the MßCD inhibitor notably decreased Reo-mediated tumor cell death, indicating that Lipo primarily enables Reo's cellular uptake through caveolin-mediated endocytosis. Our results demonstrate that Lipo effectively facilitates the entry of Reo into the cytoplasm and triggers cell apoptosis. The above findings provide a new strategy to overcome the obstacle of neutralizing antibodies in the clinical application of Reo.
Assuntos
Anticorpos Neutralizantes , Lipossomos , Neoplasias Ovarianas , Reoviridae , Feminino , Humanos , Neoplasias Ovarianas/imunologia , Anticorpos Neutralizantes/imunologia , Reoviridae/imunologia , Reoviridae/fisiologia , Linhagem Celular Tumoral , Terapia Viral Oncolítica/métodos , Apoptose , Animais , Cátions , Vírus Oncolíticos/imunologia , CamundongosRESUMO
In addition to chemotherapy, oncolytic viruses are an efficient treatment for acute myeloid leukemia (AML). Like other oncolytic viruses, the anti-tumor efficacy of reovirus when administered intravenously is reduced due to the presence of neutralizing antibodies. In this study, we evaluated the role of exosomes in human umbilical cord-derived mesenchymal stem cells (UC-MSCs) to deliver reovirus to AML cells. We show that UC-MSCs loaded with reovirus can deliver reovirus to tumor cells without cellular contact. We further demonstrate that the exosome inhibitor, GW4869, inhibits the release of exosomes as well as inhibited the transfer of reovirus from UC-MSCs to tumor cells. Mechanistically, we show that exosomes derived from reovirus-infected UC-MSCs (MSCREO-EXOs) have a tumor lysis effect and transmit reovirus to tumor cells mainly through clathrin-mediated endocytosis (CME) and macropinocytosis. In addition, we demonstrate the feasibility of using MSC-derived exosomes (MSC-EXOs) as a reovirus carrier to exert an anti-tumor effect on AML cells. Collectively, our data indicate that UC-MSCs transfer reovirus to AML cells via exosome release and prompt further study of MSC-EXOs as a potential reovirus carrier to treat AML.
Assuntos
Exossomos , Leucemia Mieloide Aguda , Células-Tronco Mesenquimais , Terapia Viral Oncolítica , Vírus Oncolíticos , Cordão Umbilical , Humanos , Exossomos/metabolismo , Células-Tronco Mesenquimais/virologia , Células-Tronco Mesenquimais/metabolismo , Leucemia Mieloide Aguda/terapia , Cordão Umbilical/citologia , Vírus Oncolíticos/fisiologia , Terapia Viral Oncolítica/métodos , Linhagem Celular Tumoral , Reoviridae/fisiologia , Compostos de Anilina/farmacologia , Endocitose , Compostos de BenzilidenoRESUMO
Efficient identification of epitopes is crucial for drug discovery and design as it enables the selection of optimal epitopes, expansion of lead antibody diversity, and verification of binding interface. Although high-resolution low throughput methods like x-ray crystallography can determine epitopes or protein-protein interactions accurately, they are time-consuming and can only be applied to a limited number of complexes. To overcome these limitations, we have developed a rapid computational method that incorporates N-linked glycans to mask epitopes or protein interaction surfaces, thereby providing a mapping of these regions. Using human coagulation factor IXa (fIXa) as a model system, we computationally screened 158 positions and expressed 98 variants to test experimentally for epitope mapping. We were able to delineate epitopes rapidly and reliably through the insertion of N-linked glycans that efficiently disrupted binding in a site-selective manner. To validate the efficacy of our method, we conducted ELISA experiments and high-throughput yeast surface display assays. Furthermore, x-ray crystallography was employed to verify the results, thereby recapitulating through the method of N-linked glycans a coarse-grained mapping of the epitope.
Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Humanos , Epitopos/química , Mapeamento de Epitopos/métodos , Ensaios de Triagem em Larga Escala/métodosRESUMO
Exosomes are lipid bilayer-enclosed vesicles, actively secreted by cells, containing proteins, lipids, nucleic acids, and other substances with multiple biological functions after entering target cells. Exosomes derived from NK cells have been shown to have certain anti-tumor effects and potential applications as chemotherapy drug carriers. These developments have resulted in high demand for exosomes. Although there has been large-scale industrial preparation of exosomes, they are only for generally engineered cells such as HEK 293T. The large-scale preparation of specific cellular exosomes is still a major problem in laboratory studies. Therefore, in this study, we used tangential flow filtration (TFF) to concentrate the culture supernatants isolated from NK cells and isolated NK cell-derived exosomes (NK-Exo) by ultracentrifugation. Through a series of characterization and functional verification of NK-Exo, the characterization, phenotype, and anti-tumor activity of NK-Exo were verified. Our study provides a considerably time- and labor-saving protocol for the isolation of NK-Exo.
RESUMO
Hypoxia in water environment has become increasingly frequent and serious due to global warming and environmental pollution. Revealing the molecular mechanism of fish hypoxia adaptation will help to develop markers of environmental pollution caused by hypoxia. Here, we used a multi-omics method to identify the hypoxia-associated mRNA, miRNA, protein, and metabolite involved in various biological processes in Pelteobagrus vachelli brain. The results showed that hypoxia stress caused brain dysfunction by inhibiting energy metabolism. Specifically, the biological processes involved in energy synthesis and energy consumption are inhibited in P. vachelli brain under hypoxia, such as oxidative phosphorylation, carbohydrate metabolism and protein metabolism. Brain dysfunction is mainly manifested as blood-brain barrier injury accompanied by neurodegenerative diseases and autoimmune diseases. In addition, compared with previous studies, we found that P. vachelli has tissue specificity in response to hypoxia stress and the muscle suffers more damage than the brain. This is the first report to the integrated analysis of the transcriptome, miRNAome, proteome, and metabolome in fish brain. Our findings could provide insights into the molecular mechanisms of hypoxia, and the approach could also be applied to other fish species. DATA AVAILABILITY: The raw data of transcriptome has been uploaded to NCBI database (ID: SUB7714154 and SUB7765255). The raw data of proteome has been uploaded to ProteomeXchange database (PXD020425). The raw data of metabolome has been uploaded to Metabolight (ID: MTBLS1888).
Assuntos
Peixes-Gato , Proteoma , Animais , Proteoma/genética , Proteoma/metabolismo , Multiômica , Hipóxia/genética , Peixes-Gato/metabolismo , Metabolismo Energético , Encéfalo/metabolismo , TranscriptomaRESUMO
Blood redistribution occurs in mammals under hypoxia but has not been reported in fish. This study investigated the tissue damage, hypoxia-inducible factor (HIF) activation level, and blood flow changes in the brain, liver, and muscle of Pelteobagrus vachelli during the hypoxia process for normoxia-hypoxia-asphyxia. The results showed that P. vachelli has tissue specificity in response to hypoxic stress. Cerebral blood flow increased with less damage than in the liver and muscle, suggesting that P. vachelli may also have a blood redistribution mechanism in response to hypoxia. It is worth noting that severe hypoxia can lead to a sudden increase in the degree of brain tissue damage. In addition, higher dissolved oxygen levels activate HIF and may have contributed to the reduced damage observed in the brain. This study provides basic data for investigating hypoxic stress in fish.
Assuntos
Estruturas Animais , Peixes-Gato , Hipóxia , Fluxo Sanguíneo Regional , Peixes-Gato/fisiologia , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Músculos/química , Músculos/patologia , Fígado/irrigação sanguínea , Fígado/patologia , Estruturas Animais/irrigação sanguínea , Estruturas Animais/patologia , Hipóxia/fisiopatologia , Estresse Fisiológico/genética , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Genes Mitocondriais/genética , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismo , Fluxo Sanguíneo Regional/fisiologia , AnimaisRESUMO
Exosomes are membranous vesicles actively secreted by almost all cells and they deliver certain intracellular molecules, including nucleic acids, proteins, and lipids, to target cells. They are also considered to be good carriers for drug delivery due to their biocompatibility, high permeability, low immunogenicity, and low toxicity. Exosomes from immune cells were also reported to have immunomodulatory activities. Herein we evaluated the application of exosomes derived from expanded natural killer cells (eNK-EXO) for the treatment of ovarian cancer (OC). We demonstrate that eNK-EXO express typical protein markers of natural killer (NK) cells, can be preferentially uptaken by SKOV3 cells, and display cytotoxicity against OC cells. Furthermore, eNK-EXO loaded with cisplatin could sensitize drug-resistant OC cells to the anti-proliferation effect of cisplatin. In addition, we show that eNK-EXO could activate NK cells from immunosuppressive tumor microenvironment, the mechanism of which is explored by transcriptional analysis. In summary, eNK-EXO exhibit anti-tumor activity against OC on its own, could be used to deliver cisplatin and enhance its cytotoxic effect against drug-resistant OC cells and also reverse the immunosuppression of NK cells, which may lead to great prospect of using eNK-EXO in the treatment of OC in the clinic. Our work also builds a strong foundation for further evaluation of eNK-EXO in other solid tumor therapies.
Assuntos
Antineoplásicos , Exossomos , Neoplasias Ovarianas , Humanos , Feminino , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Exossomos/metabolismo , Células Matadoras Naturais , Neoplasias Ovarianas/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Microambiente TumoralRESUMO
Hemophilia A is a bleeding disorder resulting from deficient factor VIII (FVIII), which normally functions as a cofactor to activated factor IX (FIXa) that facilitates activation of factor X (FX). To mimic this property in a bispecific antibody format, a screening was conducted to identify functional pairs of anti-FIXa and anti-FX antibodies, followed by optimization of functional and biophysical properties. The resulting bispecific antibody (Mim8) assembled efficiently with FIXa and FX on membranes, and supported activation with an apparent equilibrium dissociation constant of 16 nM. Binding affinity with FIXa and FX in solution was much lower, with equilibrium dissociation constant values for FIXa and FX of 2.3 and 1.5 µM, respectively. In addition, the activity of Mim8 was dependent on stimulatory activity contributed by the anti-FIXa arm, which enhanced the proteolytic activity of FIXa by 4 orders of magnitude. In hemophilia A plasma and whole blood, Mim8 normalized thrombin generation and clot formation, with potencies 13 and 18 times higher than a sequence-identical analogue of emicizumab. A similar potency difference was observed in a tail vein transection model in hemophilia A mice, whereas reduction of bleeding in a severe tail-clip model was observed only for Mim8. Furthermore, the pharmacokinetic parameters of Mim8 were investigated and a half-life of 14 days shown in cynomolgus monkeys. In conclusion, Mim8 is an activated FVIII mimetic with a potent and efficacious hemostatic effect based on preclinical data.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Hemofilia A/tratamento farmacológico , Hemorragia/tratamento farmacológico , Animais , Fator IXa/antagonistas & inibidores , Fator VIIIa/uso terapêutico , Fator X/antagonistas & inibidores , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BLRESUMO
The insulin epitopes for two monoclonal antibodies (mAbs), OXI-005 and HUI-018, commonly used in combination for insulin concentration determination in sandwich assays, were determined using X-ray crystallography. The crystal structure of the HUI-018 Fab in complex with human insulin (HI) was determined and OXI-005 Fab crystal structures were determined in complex with HI and porcine insulin (PI) as well as on its own. The OXI-005 epitope comprises insulin residues 1,3,4,19-21 (A-chain) and 25-30 (B-chain) and for HUI-018 residues 7,8,10-14,17 (A-chain) and 5-7, 10, 14 (B-chain). The areas of insulin involved in interactions with the mAb are 20% (OXI-005) and 24% (HUI-018) of the total insulin surface. Based on the Fab complex crystal structures with the insulins a molecular model for simultaneous binding of the Fabs to PI was built and this model was validated by small angle X-ray scattering measurements for the ternary complex. The epitopes for the mAbs on insulin were found well separated from each other as expected from luminiscent oxygen channeling immunoassay results for different insulins (HI, PI, bovine insulin, DesB30 HI, insulin glargine, insulin lispro). The affinities of the OXI-005 and HUI-018 Fabs for HI, PI, and DesB30 HI were determined using surface plasmon resonance. The KD s were found to be in the range of 1-4 nM for the HUI-018 Fab, while more different for the OXI-005 Fab (50 nM for HI, 20 nM for PI and 400 nM for DesB30 HI) supporting the importance of residue B30 for binding to OXI-005.
Assuntos
Anticorpos Monoclonais/química , Epitopos/química , Fragmentos Fab das Imunoglobulinas/química , Insulina/química , Modelos Moleculares , Cristalografia por Raios X , Mapeamento de Epitopos , HumanosRESUMO
Microstructures are applied to various hydrophobic/hydrophilic surfaces due to the role of adjusting the surface wettability. In this paper, a 1064 nm pulsed picosecond laser was applied to prepare a micro/nano hierarchical structure on the surface of the titanium alloy (Ti-6Al-4V). The microstructures consist of dimple arrays with various diameters, depths, and areal densities. They are obtained by controlling the pulse energy and the number of pulses. The nanostructures are periodic ripples, which are defined as laser-induced periodic surface structure (LIPSS), and the dimensional parameter of LIPSS can be adjusted by changing the laser energy density and scanning speed. The contact angles of various laser textured surfaces were measured. It is found that the contact angle increases with the density of micro-textured surface increases, and the wetting state of textured surfaces conforms to the Cassie model. Some laser processed samples were subjected to low-temperature annealing treatment. It is observed that the low-temperature annealing process can accelerate the surface wettability transition significantly, which is attributed to the change of the hydroxyl groups on the surface. Finally, a superhydrophobic surface with the maximum contact angle of 144.58° is obtained.
RESUMO
A modified Ni-rich Li[Ni0.8Co0.1Mn0.1]O2 cathode material with exposed {010} planes is successfully synthesized for lithium-ion batteries. The scanning electron microscopy images have demonstrated that by tuning the ammonia concentration during the synthesis of precursors, the primary nanosheets could be successfully stacked along the [001] crystal axis predominantly, self-assembling like multilayers. According to the high-resolution transmission electron microscopy results, such a morphology benefits the growth of the {010} active planes of final layered cathodes during calcination treatment, resulting in the increased area of the exposed {010} active planes, a well-ordered layer structure, and a lower cation mixing disorder. The Li-ion diffusion coefficient has also been improved after the modification based on the results of potentiostatic intermittent titration technique. As a consequence, the modified Li[Ni0.8Co0.1Mn0.1]O2 material exhibits superior initial discharges of 201.6 mA h g-1 at 0.2 C and 185.7 mA h g-1 at 1 C within 2.8-4.3 V (vs Li+/Li), and their capacity retentions after 100 cycles reach 90 and 90.6%, respectively. The capacity at 10 C also increases from 98.3 to 146.5 mA h g-1 after the modification. Our work proposes a novel approach for exposing high-energy {010} active planes of the layered cathode material and again confirms its validity in improving electrochemical properties.
RESUMO
Interactions between protein ligands and receptors play crucial roles in cell-cell signalling. Most of the human cell surface receptors have been identified in the post-Human Genome Project era but many of their corresponding ligands remain unknown. To facilitate the pairing of orphan receptors, 2762 sequences encoding all human single-pass transmembrane proteins were selected for inclusion into a mammalian-cell expression library. This expression library, consisting of all the individual extracellular domains (ECDs), was constructed as a Fab fusion for each protein. In this format, individual ECD can be produced as a soluble protein or displayed on cell surface, depending on the applied heavy-chain Fab configuration. The unique design of the Fab fusion concept used in the library led to not only superior success rate of protein production, but also versatile applications in various high-throughput screening paradigms including protein-protein binding assays as well as cell binding assays, which were not possible for any other existing expression libraries. The protein library was screened against human coagulation factor VIIa (FVIIa), an approved therapeutic for the treatment of hemophilia, for binding partners by AlphaScreen and ForteBio assays. Two previously known physiological ligands of FVIIa, tissue factor (TF) and endothelial protein C receptor (EPCR) were identified by both assays. The cell surface displayed library was screened against V-domain Ig suppressor of T-cell activation (VISTA), an important immune-checkpoint regulator. Immunoglobulin superfamily member 11 (IgSF11), a potential target for cancer immunotherapy, was identified as a new and previously undescribed binding partner for VISTA. The specificity of the binding was confirmed and validated by both fluorescence-activated cell sorting (FACS) and surface plasmon resonance (SPR) assays in different experimental setups.
Assuntos
Proteínas de Membrana , Biblioteca de Peptídeos , Receptores de Superfície Celular , Proteínas Recombinantes de Fusão , Clonagem Molecular , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Growth differentiation factor 15 (GDF15; also known as MIC-1) is a divergent member of the TGF-ß superfamily and is associated with body-weight regulation in humans and rodents. However, the cognate receptor of GDF15 is unknown. Here we show that GDF15 binds specifically to GDNF family receptor α-like (GFRAL) with high affinity, and that GFRAL requires association with the coreceptor RET to elicit intracellular signaling in response to GDF15 stimulation. We also found that GDF15-mediated reductions in food intake and body weight of mice with obesity were abolished in GFRAL-knockout mice. We further found that GFRAL expression was limited to hindbrain neurons and not present in peripheral tissues, which suggests that GDF15-GFRAL-mediated regulation of food intake is by a central mechanism. Lastly, given that GDF15 did not increase energy expenditure in treated mice with obesity, the anti-obesity actions of the cytokine are likely driven primarily by a reduction in food intake.
Assuntos
Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/efeitos dos fármacos , Fator 15 de Diferenciação de Crescimento/farmacologia , Obesidade/metabolismo , Redução de Peso/efeitos dos fármacos , Animais , Ingestão de Alimentos/genética , Metabolismo Energético/genética , Citometria de Fluxo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Camundongos Knockout , Ratos , Ratos Sprague-Dawley , Ressonância de Plasmônio de Superfície , Redução de Peso/genéticaRESUMO
Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS characterized by demyelination and axonal damage. Experimental autoimmune encephalomyelitis (EAE) is a well-established animal model for human MS. Although Th17 cells are important for disease induction, Th2 cells are inhibitory in this process. In this article, we report the effect of a Th2 cell product, extracellular matrix protein 1 (ECM1), on the differentiation of Th17 cells and the development of EAE. Our results demonstrated that ECM1 administration from day 1 to day 7 following the EAE induction could ameliorate the Th17 cell responses and EAE development in vivo. Further study of the mechanism revealed that ECM1 could interact with αv integrin on dendritic cells and block the αv integrin-mediated activation of latent TGF-ß, resulting in an inhibition of Th17 cell differentiation at an early stage of EAE induction. Furthermore, overexpression of ECM1 in vivo significantly inhibited the Th17 cell response and EAE induction in ECM1 transgenic mice. Overall, our work has identified a novel function of ECM1 in inhibiting Th17 cell differentiation in the EAE model, suggesting that ECM1 may have the potential to be used in clinical applications for understanding the pathogenesis of MS and its diagnosis.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Proteínas da Matriz Extracelular/imunologia , Células Th17/imunologia , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Ensaio de Imunoadsorção Enzimática , Proteínas da Matriz Extracelular/farmacologia , Imuno-Histoquímica , Imunoprecipitação , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
IgG subclass-specific differences in biological function and in vitro stability are often referred to variations in the conformational flexibility, while this flexibility has rarely been characterized. Here, small-angle X-ray scattering data from IgG1, IgG2 and IgG4 antibodies, which were designed with identical variable regions, were thoroughly analysed by the ensemble optimization method. The extended analysis of the optimized ensembles through shape clustering reveals distinct subclass-specific conformational preferences, which provide new insights for understanding the variations in physical/chemical stability and biological function of therapeutic antibodies. Importantly, the way that specific differences in the linker region correlate with the solution structure of intact antibodies is revealed, thereby visualizing future potential for the rational design of antibodies with designated physicochemical properties and tailored effector functions. In addition, this advanced computational approach is applicable to other flexible multi-domain systems and extends the potential for investigating flexibility in solutions of macromolecules by small-angle X-ray scattering.
RESUMO
This study aimed to investigate the feasibility of nanostructured 3D poly(lactide-co-glycolide) (PLGA) constructs, which are loaded with dexamethasone (DEX) and growth factor embedded hepaiin/poly(L-lysine) nanoparticles by a layer-by-layer system, to serve as an effective scaffold for nucleus pulposus (NP) tissue engineering. Our results demonstrated that the microsphere constructs were capable of simultaneously releasing basic fibroblast growth factor and DEX with approximately zero-order kinetics. The dual bead microspheres showed no cytotoxicity, and promoted the proliferation of the rat mesenchymal stem cells (rMSCs) by lactate dehydrogenase assay and CCK-8 assay. After 4 weeks of culture in vitro, the rMSCs- scaffold hybrids contained significantly higher levels of sulfated GAG/DNA and type-II collagen than the control samples. Moreover, quantity real-time PCR analysis revealed that the expression of disc-matrix proteins, including type-II collagen, aggrecan and versican, in the rMSCs-scaffold hybrids was significantly higher than the control group, whereas the expression of osteogenic differentiation marker type-I collagen was decreased. Taken together, these data indicate that the heparin bound bFGF-coated and DEX-loaded PLGA microsphere constructs is an effective bioactive scaffold for the regeneration of NP tissue.
Assuntos
Portadores de Fármacos/química , Disco Intervertebral/fisiologia , Ácido Láctico/química , Microesferas , Nanoestruturas/química , Nanotecnologia , Ácido Poliglicólico/química , Regeneração , Animais , Proliferação de Células/efeitos dos fármacos , Colágeno/metabolismo , DNA/metabolismo , Dexametasona/química , Dexametasona/farmacologia , Estudos de Viabilidade , Feminino , Fatores de Crescimento de Fibroblastos/química , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosaminoglicanos/metabolismo , Heparina/química , Disco Intervertebral/citologia , Disco Intervertebral/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Nanopartículas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polilisina/química , Ratos , Ratos Sprague-Dawley , Regeneração/efeitos dos fármacos , Engenharia Tecidual , Alicerces Teciduais/químicaRESUMO
The poxvirus 2L protein binds tumor necrosis factor-alpha (TNFalpha) to inhibit host antiviral and immune responses. The 2.8-A 2L-TNFalpha structure reveals three symmetrically arranged 2L molecules per TNFalpha trimer. 2L resembles class I major histocompatibility complex (MHC) molecules but lacks a peptide-binding groove and beta2-microglobulin light chain. Overlap between the 2L and host TNF receptor-binding sites on TNFalpha rationalizes 2L inhibition of TNFalpha-TNF receptor interactions and prevention of TNFalpha-induced immune responses.
Assuntos
Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Poxviridae/metabolismo , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Sítios de Ligação , Humanos , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
UL18 is a human cytomegalovirus class I MHC (MHCI) homolog that binds the host inhibitory receptor LIR-1 and the only known viral MHC homolog that presents peptides. The 2.2-A structure of a LIR-1/UL18/peptide complex reveals increased contacts and optimal surface complementarity in the LIR-1/UL18 interface compared with LIR/MHCI interfaces, resulting in a >1,000-fold higher affinity. Despite sharing only approximately 25% sequence identity, UL18's structure and peptide binding are surprisingly similar to host MHCI. The crystal structure suggests that most of the UL18 surface, except where LIR-1 and the host-derived light chain bind, is covered by carbohydrates attached to 13 potential N-glycosylation sites, thereby preventing access to bound peptide and association with most MHCI-binding proteins. The LIR-1/UL18 structure demonstrates how a viral protein evolves from its host ancestor to impede unwanted interactions while preserving and improving its receptor-binding site.
Assuntos
Antígenos CD/química , Antígenos CD/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Citomegalovirus/química , Antígeno HLA-A2/química , Antígeno HLA-A2/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Receptores Virais/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Proteínas do Capsídeo/genética , Cristalografia por Raios X , Citomegalovirus/genética , Citomegalovirus/metabolismo , Glicosilação , Antígeno HLA-A2/genética , Humanos , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Modelos Moleculares , Mimetismo Molecular , Dados de Sequência Molecular , Complexos Multiproteicos , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
Malic enzymes catalyze the oxidative decarboxylation of L-malate to pyruvate and CO(2) with the reduction of the NAD(P)(+) cofactor in the presence of divalent cations. We report the crystal structures at up to 2.1 A resolution of human mitochondrial NAD(P)(+)-dependent malic enzyme in different pentary complexes with the natural substrate malate or pyruvate, the dinucleotide cofactor NAD(+) or NADH, the divalent cation Mn(2+), and the allosteric activator fumarate. Malate is bound deep in the active site, providing two ligands for the cation, and its C4 carboxylate group is out of plane with the C1-C2-C3 atoms, facilitating decarboxylation. The divalent cation is positioned optimally to catalyze the entire reaction. Lys183 is the general base for the oxidation step, extracting the proton from the C2 hydroxyl of malate. Tyr112-Lys183 functions as the general acid-base pair to catalyze the tautomerization of the enolpyruvate product from decarboxylation to pyruvate.
Assuntos
Malato Desidrogenase/química , Malato Desidrogenase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Ativação Enzimática , Humanos , Dados de Sequência Molecular , Oxirredução , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Piruvatos/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Acetyl-coenzyme A carboxylases (ACCs) are required for the biosynthesis and oxidation of long-chain fatty acids. They are targets for therapeutics against obesity and diabetes, and several herbicides function by inhibiting their carboxyltransferase (CT) domain. We determined the crystal structure of the free enzyme and the coenzyme A complex of yeast CT at 2.7 angstrom resolution and found that it comprises two domains, both belonging to the crotonase/ClpP superfamily. The active site is at the interface of a dimer. Mutagenesis and kinetic studies reveal the functional roles of conserved residues here. The herbicides target the active site of CT, providing a lead for inhibitor development against human ACCs.