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1.
Nature ; 574(7779): 565-570, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645726

RESUMO

Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer1,2. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies3. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement.


Assuntos
Antígenos B7/química , Antígenos B7/metabolismo , Glicoproteínas de Membrana/metabolismo , Linfócitos T/metabolismo , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Antígenos B7/antagonistas & inibidores , Antígenos B7/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Cristalografia por Raios X , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Feminino , Histidina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Linfócitos T/citologia , Linfócitos T/imunologia , Microambiente Tumoral/imunologia
2.
Electrophoresis ; 43(9-10): 1059-1067, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35362108

RESUMO

Antibody-based therapeutic proteins have highly complex molecular structures. The final therapeutic protein product may contain a wide range of charge variants. Accurate analysis of this charge variant composition is critical to determine manufacturing process consistency and protein stability and ultimately helps to ensure that patients receive a safe and efficacious product. Here, a highly sialylated bispecific antibody (bsAb-1) challenged the ability to monitor stability by imaged capillary isoelectric focusing (iCIEF). This challenge was overcome by optimization of the iCIEF master mix buffer (adjustment of urea concentration, addition of l-arginine) and enzymatic removal of sialic acid. The method was qualified by assessing linearity, precision, LOD, LOQ, accuracy, and robustness in accordance with ICH guidance. Main species loss detectability increased up to approximately fivefold compared to the iCIEF method without desialylation when monitoring changes in stressed samples. Importantly, the results of the iCIEF method with desialylation correlated with results obtained through LC-MS tryptic peptide mapping and enabled analysis of formulation development stability samples. Finally, this analytical method shows the potential to assess low-concentration formulation development samples down to a sample concentration of 0.1 mg/ml.


Assuntos
Eletroforese Capilar , Ácido N-Acetilneuramínico , Cromatografia Líquida , Eletroforese Capilar/métodos , Humanos , Focalização Isoelétrica/métodos , Espectrometria de Massas
3.
Electrophoresis ; 42(4): 460-464, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32885501

RESUMO

Protein glycosylation can impact the efficacy, safety, and pharmacokinetics of therapeutic proteins. Achieving uniform and consistent protein glycosylation is an important requirement for product quality control at all stages of therapeutic protein drug discovery and development. The development of a new microfluidic CE device compatible with MS offers a fast and sensitive orthogonal mode of high-resolution separation with MS characterization. Here, we describe a fast and robust chip-based CE-MS method for intact glycosylation fingerprinting of a therapeutic fusion protein with complex sialylated N and O-linked glycoforms. The method effectively separates multiple sialylated glycoforms and offers a rapid detection of changes in glycosylation profile in 6 min.


Assuntos
Eletroforese Capilar/instrumentação , Dispositivos Lab-On-A-Chip , Espectrometria de Massas/instrumentação , Polissacarídeos/análise , Proteínas Recombinantes de Fusão , Glicosilação , Mapeamento de Peptídeos/instrumentação , Mapeamento de Peptídeos/métodos , Polissacarídeos/química , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação
4.
J Am Chem Soc ; 142(51): 21260-21266, 2020 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-33290649

RESUMO

Bioconjugation technologies have revolutionized the practice of biology and medicine by allowing access to novel biomolecular scaffolds. New methods for residue-selective bioconjugation are highly sought to expand the toolbox for a variety of bioconjugation applications. Herein we report a site-selective methionine bioconjugation protocol that uses photoexcited lumiflavin to generate open-shell intermediates. This reduction-potential-gated strategy enables access to residues unavailable with traditional nucleophilicity-based conjugation methods. To demonstrate the versatility and robustness of this new protocol, we have modified various proteins and further utilized this functional handle to append diverse biological payloads.


Assuntos
Metionina/química , Processos Fotoquímicos , Sítios de Ligação , Catálise , Oxirredução , Proteínas/química , Especificidade por Substrato
5.
Anal Chem ; 92(13): 9086-9094, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32441507

RESUMO

Programmed cell death-1 (PD-1), an antigen co-receptor on cell surfaces, is one of the conspicuous immune checkpoints. Nivolumab, a monoclonal antibody therapeutic approved by the FDA, binds to PD-1 and efficiently blocks its pathways. In this study, an integrated approach was developed to map the epitope/paratope of PD-1/nivolumab. The approach includes hydrogen-deuterium exchange mass spectrometry (HDX-MS) followed by electron-transfer dissociation (ETD), chemical cross-linking, and molecular docking. HDX-ETD offers some binding-site characterization with amino acid resolution. Chemical cross-linking provides complementary information on one additional epitope (i.e., the BC-loop) and a potential paratope at the N-terminus of the heavy chain. Furthermore, cross-linking identifies another loop region (i.e., the C'D-loop) that undergoes a remote conformational change. The distance restraints derived from the cross-links enable building high-confidence models of PD-1/nivolumab, evaluated with respect to a resolved crystal structure. This integrated strategy is an opportunity to characterize comprehensively other antigen-antibody interactions, to enable the understanding of binding mechanisms, and to design future antibody therapeutics.


Assuntos
Medição da Troca de Deutério , Mapeamento de Epitopos/métodos , Epitopos/análise , Nivolumabe/imunologia , Receptor de Morte Celular Programada 1/imunologia , Sequência de Aminoácidos , Complexo Antígeno-Anticorpo/química , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Epitopos/química , Epitopos/imunologia , Humanos , Simulação de Acoplamento Molecular , Nivolumabe/metabolismo , Receptor de Morte Celular Programada 1/química , Receptor de Morte Celular Programada 1/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Espectrometria de Massas em Tandem
6.
Anal Chem ; 92(15): 10709-10716, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32639723

RESUMO

Bispecific antibodies (BsAbs), with a unique mechanism of recognizing two different epitopes or antigens, have shown potential in various therapeutic areas. Molecular characterization of BsAbs' epitopes not only allows for detailed understanding of their mechanism of actions but also guides the design and selection of drug candidate molecules. In this study, we illustrate the practical utility of an integrated approach, including size exclusion chromatography with multiangle light scattering and native mass spectrometry (MS) for the biophysical characterization of complex formation of a BsAb with two target antigens, cluster of differentiation 3 (CD3) and B-cell maturation antigen (BCMA). MS-based protein footprinting strategies, including hydrogen/deuterium exchange MS, fast photochemical oxidation of proteins, and carboxyl group footprinting with glycine ethyl ester, were further applied to determine BsAb's binding epitopes. This combination approach provides molecular details on the binding mechanisms of BsAb to the two distinct antigens with rapid output and high resolution.


Assuntos
Anticorpos Biespecíficos/imunologia , Antígenos/imunologia , Cromatografia em Gel , Mapeamento de Epitopos/métodos , Espectrometria de Massas , Pegadas de Proteínas , Anticorpos Biespecíficos/química , Modelos Moleculares , Conformação Proteica
7.
Bioconjug Chem ; 31(8): 1908-1916, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32687313

RESUMO

Chemoselective methionine bioconjugation with alkyne-bearing oxaziridine and alkyne-bearing iodonium salts was investigated as a new platform for site-selective radiolabeling of proteins and peptides with fluorine-18. Alkyne-bearing sulfimide conjugates, resulting from oxaziridine modification, underwent copper-assisted alkyne-azide cycloaddition (CuAAC) with an 18F-labeled PEGylated azide to afford 18F-labeled triazoles in excellent radiochemical yields. Diazoester sulfonium salt bioconjugates, formed from alkyne-bearing 2-diazoiodonium salts, gave low yields of 18F-labeled triazoles and were shown to be unstable to CuAAC conditions. Photolytic removal of the diazo group, however, afforded the trialkylsulfonium salt which smoothly underwent CuAAC with the 18F-labeled PEGylated azide to afford high radiochemical yields of the desired 18F-labeled click product. Overall, the results establish the viability of chemoselective methionine bioconjugation as a method for preparing site-selective 18F-labeled PET radioligands.


Assuntos
Radioisótopos de Flúor , Metionina/química , Peptídeos/química , Proteínas/química , Química Click/métodos , Compostos Radiofarmacêuticos , Soroalbumina Bovina/química
8.
Anal Bioanal Chem ; 412(7): 1693-1700, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31993727

RESUMO

Major histocompatibility complex class I chain-related A and B (MICA/B) are cell-surface proteins that act as ligands to natural killer cell receptors, NKG2D, expressed on immune cells. Prevention of proteolytic shedding of MICA/B to retain their integrity on the cell surface has become a therapeutic strategy in immuno-oncology. Given the unique mechanism of MICA/B shedding, structural characterization of MICA/B and therapeutic agent interaction is important in the drug discovery process. In this study, we describe the practical utility of hydrogen/deuterium exchange mass spectrometry (HDX-MS) in epitope mapping studies of a cohort of four monoclonal antibodies targeting MICA in a rapid manner. HDX-MS followed by electron-transfer dissociation allows high-resolution refinement of binding epitopes. This integrated strategy offers, for the first time, molecular-level understanding of MICA's conformational dynamics in solution as well as the unique mechanism of actions of these antibodies in targeting MICA. Graphical abstract.


Assuntos
Anticorpos Monoclonais/imunologia , Medição da Troca de Deutério/métodos , Mapeamento de Epitopos/métodos , Antígenos de Histocompatibilidade Classe I/imunologia , Espectrometria de Massas/métodos , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Transporte de Elétrons , Humanos
9.
Anal Chem ; 91(24): 15709-15717, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31710208

RESUMO

We describe an integrated approach of using hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking mass spectrometry (XL-MS), and molecular docking to characterize the binding interface and to predict the three-dimensional quaternary structure of a protein-protein complex in solution. Interleukin 7 (IL-7) and its α-receptor, IL-7Rα, serving as essential mediators in the immune system, are the model system. HDX kinetics reports widespread protection on IL-7Rα but shows no differential evidence of binding-induced protection or remote conformational change. Cross-linking with reagents that differ in spacer lengths and targeting residues increases the spatial resolution. Using five cross-links as distance restraints for protein-protein docking, we generated a high-confidence model of the IL-7/IL-7Rα complex. Both the predicted binding interface and regions with direct contacts agree well with those in the solid-state structure, as confirmed by previous X-ray crystallography. An additional binding region was revealed to be the C-terminus of helix B of IL-7, highlighting the value of solution-based characterization. To generalize the integrated approach, protein-protein docking was executed with a different number of cross-links. Combining cluster analysis and HDX kinetics adjudication, we found that two intermolecular cross-link-derived restraints are sufficient to generate a high-confidence model with root-mean-square distance (rmsd) value of all alpha carbons below 2.0 Å relative to the crystal structure. The remarkable results of binding-interface determination and quaternary structure prediction highlight the effectiveness and capability of the integrated approach, which will allow more efficient and comprehensive analysis of interprotein interactions with broad applications in the multiple stages of design, implementation, and evaluation for protein therapeutics.


Assuntos
Medição da Troca de Deutério/métodos , Hidrogênio/química , Interleucina-7/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Domínios e Motivos de Interação entre Proteínas , Receptores de Interleucina-7/metabolismo , Humanos , Cinética , Ligação Proteica , Conformação Proteica
10.
Anal Chem ; 91(11): 7336-7345, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31045344

RESUMO

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A ) from 15 laboratories. Laboratories reported ∼89 800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (∼78 900 centroids), giving ∼100% coverage, and ∼10 900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87%) exhibited centroid mass laboratory repeatability precisions of ⟨ sLab⟩ ≤ (0.15 ± 0.01) Da (1σx̅). All laboratories achieved ⟨sLab⟩ ≤ 0.4 Da. For immersions of protein at THDX = (3.6 to 25) °C and for D2O exchange times of tHDX = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σreproducibility15 Laboratories( tHDX) = (9.0 ± 0.9) % (1σ). A nine laboratory cohort that immersed samples at THDX = 25 °C exhibited reproducibility of σreproducibility25C cohort( tHDX) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements.


Assuntos
Anticorpos Monoclonais/química , Espectrometria de Massa com Troca Hidrogênio-Deutério , Fragmentos Fab das Imunoglobulinas/análise
11.
Anal Chem ; 90(8): 5162-5170, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29605994

RESUMO

Accurate quantification of soluble glypican-3 in clinical samples using immunoassays is challenging, because of the lack of appropriate antibody reagents to provide a full spectrum measurement of all potential soluble glypican-3 fragments in vivo. Glypican-3 SOMAmer (slow off-rate modified aptamer) is a novel reagent that binds, with high affinity, to a far distinct epitope of glypican-3, when compared to all available antibody reagents generated in-house. This paper describes an integrated analytical approach to rational selection of key reagents based on molecular characterization by epitope mapping, with the focus on our work using a SOMAmer as a new reagent to address development challenges with traditional antibody reagents for the soluble glypican-3 immunoassay. A qualified SOMAmer-based assay was developed and used for soluble glypican-3 quantification in hepatocellular carcinoma (HCC) patient samples. The assay demonstrated good sensitivity, accuracy, and precision. Data correlated with those obtained using the traditional antibody-based assay were used to confirm the clinically relevant soluble glypican-3 forms in vivo. This result was reinforced by a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay quantifying signature peptides generated from trypsin digestion. The work presented here offers an integrated strategy for qualifying aptamers as an alternative affinity platform for immunoassay reagents that can enable speedy assay development, especially when traditional antibody reagents cannot meet assay requirements.


Assuntos
Aptâmeros de Nucleotídeos/química , Carcinoma Hepatocelular/diagnóstico , Glipicanas/análise , Imunoensaio , Neoplasias Hepáticas/diagnóstico , Cromatografia Líquida , Humanos , Proteínas Recombinantes/análise , Solubilidade , Espectrometria de Massas em Tandem
12.
Anal Chem ; 89(14): 7742-7749, 2017 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-28621526

RESUMO

Higher-order structure (HOS) is a crucial determinant for the biological functions and quality attributes of protein therapeutics. Mass spectrometry (MS)-based protein footprinting approaches play an important role in elucidating the relationship between protein biophysical properties and structure. Here, we describe the use of a combined method including hydrogen-deuterium exchange (HDX), fast photochemical oxidation of proteins (FPOP), and site-specific carboxyl group footprinting to investigate the HOS of protein and protein complexes. The work focuses on implementing complementary solution-phase footprinting approaches that differ in time scale, specificity for protein residue side chains vs backbone as well as selectivity for different residue types to map integratively the epitope of human interleukin-6 receptor (IL-6R) for two adnectins with distinct affinities (Kd, Adnectin1 ∼ 6.2 pM vs Kd, Adnectin2 ∼ 46 nM). Furthermore, the study evaluates the resultant conformation/dynamic change of IL-6R. The suggested epitope, which is conserved for adnectin1 and adnectin2 binding, is a flexible loop that connects two ß-strands in the cytokine-binding domain (DII) of IL-6R. We also found that adnectin1, the more strongly binding ligand, induces structural perturbations on two unstructured loops that are distally located beyond the epitope. Those changes are either attenuated or not detected for the case of adnectin2 binding. In addition to providing credibility in epitope determination, utilization of those combined approaches reveals the structural effects that can differentiate protein therapeutics with apparently similar biophysical properties.


Assuntos
Mapeamento de Epitopos , Pegadas de Proteínas , Receptores de Interleucina-6/química , Medição da Troca de Deutério , Humanos , Espectrometria de Massas , Ligação Proteica , Conformação Proteica
13.
Anal Chem ; 89(4): 2250-2258, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28193005

RESUMO

Epitope mapping the specific residues of an antibody/antigen interaction can be used to support mechanistic interpretation, antibody optimization, and epitope novelty assessment. Thus, there is a strong need for mapping methods, particularly integrative ones. Here, we report the identification of an energetic epitope by determining the interfacial hot-spot that dominates the binding affinity for an anti-interleukin-23 (anti-IL-23) antibody by using the complementary approaches of hydrogen/deuterium exchange mass spectrometry (HDX-MS), fast photochemical oxidation of proteins (FPOP), alanine shave mutagenesis, and binding analytics. Five peptide regions on IL-23 with reduced backbone amide solvent accessibility upon antibody binding were identified by HDX-MS, and five different peptides over the same three regions were identified by FPOP. In addition, FPOP analysis at the residue level reveals potentially key interacting residues. Mutants with 3-5 residues changed to alanine have no measurable differences from wild-type IL-23 except for binding of and signaling blockade by the 7B7 anti-IL-23 antibody. The M5 IL-23 mutant differs from wild-type by five alanine substitutions and represents the dominant energetic epitope of 7B7. M5 shows a dramatic decrease in binding to BMS-986010 (which contains the 7B7 Fab, where Fab is fragment antigen-binding region of an antibody), yet it maintains functional activity, binding to p40 and p19 specific reagents, and maintains biophysical properties similar to wild-type IL-23 (monomeric state, thermal stability, and secondary structural features).


Assuntos
Alanina/metabolismo , Anticorpos Monoclonais/metabolismo , Mapeamento de Epitopos/métodos , Epitopos/metabolismo , Interleucina-23/metabolismo , Reações Antígeno-Anticorpo , Clonagem Molecular , Medição da Troca de Deutério , Fragmentos Fab das Imunoglobulinas/metabolismo , Espectrometria de Massas , Modelos Moleculares , Mutagênese , Oxirredução , Ligação Proteica
14.
Bioorg Med Chem ; 25(20): 5407-5414, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28803798

RESUMO

Cancer immunotherapy, unlike traditional cytotoxic chemotherapeutic treatments, engages the immune system to identify cancer cells and stimulate immune responses. The Programmed Death-1 (PD-1) protein is an immunoinhibitory receptor expressed by activated cytotoxic T-lymphocytes (CTL) that seek out and destroy cancer cells. Multiple cancer types express and upregulate the Programmed Death-Ligand 1 (PD-L1) and 2 (PD-L2) which bind to PD-1 as an immune escape mechanism. Nivolumab is a fully human IgG4 anti-PD-1 monoclonal antibody (mAb) approved for treatment of multiple cancer types. This study reports the preparation and in vivo evaluation of 89Zr labeled nivolumab in healthy non-human primates (NHP) as a preliminary study of biodistribution and clearance. The radiochemical and in vivo stabilities of the 89Zr complex were shown to be acceptable for imaging. Three naïve NHPs were intravenously injected with tracer only or tracer co-injected with nivolumab followed by co-registered by positron emission tomography (PET) and magnetic resonance imaging (MRI), acquired for eight days following injection. Image-derived standardized uptake values (SUV) were quantified by region of interest (ROI) analysis. Radioactivity in the spleen was significantly reduced by addition of excess nivolumab compared to the tracer only study at all imaging time points. Liver uptake of the radiotracer was consistent as a clearance organ with minimal signal from other tissues: lung, muscle, brain, heart, and kidney. The results indicate specific biodistribution to the spleen, which can be blocked by co-administration of excess nivolumab. Distribution to other organs is consistent with elimination pathways of antibodies, with primary clearance through the liver.


Assuntos
Anticorpos Monoclonais/farmacocinética , Tomografia por Emissão de Pósitrons , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Relação Dose-Resposta a Droga , Macaca fascicularis , Imageamento por Ressonância Magnética , Masculino , Estrutura Molecular , Nivolumabe , Relação Estrutura-Atividade , Distribuição Tecidual
15.
Nucleic Acids Res ; 42(9): 5776-89, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24728986

RESUMO

Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that associates with and influences the activity of many proteins participating in DNA metabolic processes and cell cycle progression. Previously, an uncharacterized small protein, encoded by TK0808 in the archaeon Thermococcus kodakarensis, was shown to stably interact with PCNA in vivo. Here, we show that this protein, designated Thermococcales inhibitor of PCNA (TIP), binds to PCNA in vitro and inhibits PCNA-dependent activities likely by preventing PCNA trimerization. Using hydrogen/deuterium exchange mass spectrometry and site-directed mutagenesis, the interacting regions of PCNA and TIP were identified. Most proteins bind to PCNA via a PCNA-interacting peptide (PIP) motif that interacts with the inter domain connecting loop (IDCL) on PCNA. TIP, however, lacks any known PCNA-interacting motif, suggesting a new mechanism for PCNA binding and regulation of PCNA-dependent activities, which may support the development of a new subclass of therapeutic biomolecules for inhibiting PCNA.


Assuntos
Proteínas Arqueais/química , Antígeno Nuclear de Célula em Proliferação/química , Substituição de Aminoácidos , Proteínas Arqueais/genética , DNA Polimerase II/química , Medição da Troca de Deutério , Endonucleases Flap/química , Cinética , Viabilidade Microbiana , Modelos Moleculares , Mutagênese Sítio-Dirigida , Antígeno Nuclear de Célula em Proliferação/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Thermococcus/fisiologia
16.
Expert Rev Proteomics ; 12(2): 159-69, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25711416

RESUMO

IL-23 is an important therapeutic target for the treatment of inflammatory diseases. Adnectins are targeted protein therapeutics that are derived from domain III of human fibronectin and have a similar protein scaffold to antibodies. Adnectin 2 was found to bind to IL-23 and compete with the IL-23/IL-23R interaction, posing a potential protein therapeutic. Hydrogen/deuterium exchange mass spectrometry and computational methods were applied to probe the binding interactions between IL-23 and Adnectin 2 and to determine the correlation between the two orthogonal methods. This review summarizes the current structural knowledge about IL-23 and focuses on the applicability of hydrogen/deuterium exchange mass spectrometry to investigate the higher order structure of proteins, which plays an important role in the discovery of new and improved biotherapeutics.


Assuntos
Terapia Biológica , Deutério/química , Hidrogênio/química , Interleucina-23/química , Biologia Computacional , Humanos , Interleucina-23/metabolismo , Espectrometria de Massas/métodos , Ligação Proteica , Conformação Proteica , Receptores de Interleucina/química
17.
J Biol Chem ; 288(20): 14212-14220, 2013 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-23546881

RESUMO

The PSII repair cycle is required for sustainable photosynthesis in oxygenic photosynthetic organisms. In cyanobacteria and higher plants, proteolysis of the precursor D1 protein (pD1) to expose a C-terminal carboxylate group is an essential step leading to coordination of the Mn4CaO5 cluster, the site of water oxidation. Psb27 appears to associate with both pD1- and D1-containing PSII assembly intermediates by closely interacting with CP43. Here, we report that reduced binding affinity between CP43 and Psb27 is triggered by the removal of the C-terminal extension of the pD1 protein. A mass spectrometry-based footprinting strategy was adopted to probe solvent-exposed aspartic and glutamic acid residues on the CP43 protein. By comparing the extent of footprinting between HT3ΔctpAΔ27PSII and HT3ΔctpAPSII, two genetically modified PSII assembly complexes, we found that Psb27 binds to CP43 on the side of Loop E distal to the pseudo-symmetrical D1-D2 axis. By comparing a second pair of PSII assembly complexes, we discovered that Loop E of CP43 undergoes a significant conformational rearrangement due to the removal of the pD1 C-terminal extension, altering the Psb27-CP43 binding interface. The significance of this conformational rearrangement is discussed in the context of recruitment of the PSII lumenal extrinsic proteins and Mn4CaO5 cluster assembly. In addition to CP43's previously known function as one of the core PSII antenna proteins, this work demonstrates that Loop E of CP43 plays an important role in the functional assembly of the Water Oxidizing Center (WOC) during PSII biogenesis.


Assuntos
Espectrometria de Massas/métodos , Mutação , Complexo de Proteína do Fotossistema II/química , Synechocystis/química , Reagentes de Ligações Cruzadas/farmacologia , Modelos Moleculares , Oxigênio/química , Fotossíntese , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Espectrofotometria/métodos
18.
Anal Bioanal Chem ; 406(26): 6541-58, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24948090

RESUMO

Characterization of therapeutic drugs is a crucial step in drug development in the biopharmaceutical industry. Analysis of protein therapeutics is a challenging task because of the complexities associated with large molecular size and 3D structures. Recent advances in hydrogen/deuterium-exchange mass spectrometry (HDX-MS) have provided a means to assess higher-order structure of protein therapeutics in solution. In this review, the principles and procedures of HDX-MS for protein therapeutics characterization are presented, focusing on specific applications of epitope mapping for protein-protein interactions and higher-order structure comparison studies for conformational dynamics of protein therapeutics.


Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Proteínas/química , Animais , Mapeamento de Epitopos/métodos , Humanos , Modelos Moleculares , Conformação Proteica
19.
Proc Natl Acad Sci U S A ; 108(45): 18536-41, 2011 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-22031695

RESUMO

Photosystem II (PSII), a large multisubunit pigment-protein complex localized in the thylakoid membrane of cyanobacteria and chloroplasts, mediates light-driven evolution of oxygen from water. Recently, a high-resolution X-ray structure of the mature PSII complex has become available. Two PSII polypeptides, D1 and CP43, provide many of the ligands to an inorganic Mn(4)Ca center that is essential for water oxidation. Because of its unusual redox chemistry, PSII often undergoes degradation followed by stepwise assembly. Psb27, a small luminal polypeptide, functions as an important accessory factor in this elaborate assembly pathway. However, the structural location of Psb27 within PSII assembly intermediates has remained elusive. Here we report that Psb27 binds to CP43 in such assembly intermediates. We treated purified genetically tagged PSII assembly intermediate complexes from the cyanobacterium Synechocystis 6803 with chemical cross-linkers to examine intermolecular interactions between Psb27 and various PSII proteins. First, the water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used to cross-link proteins with complementary charged groups in close association to one another. In the His27△ctpAPSII preparation, a 58-kDa cross-linked species containing Psb27 and CP43 was identified. This species was not formed in the HT3△ctpA△psb27PSII complex in which Psb27 was absent. Second, the homobifunctional thiol-cleavable cross-linker 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) was used to reversibly cross-link Psb27 to CP43 in His27△ctpAPSII preparations, which allowed the use of liquid chromatography/tandem MS to map the cross-linking sites as Psb27K(63)↔CP43D(321) (trypsin) and CP43K(215)↔Psb27D(58)AGGLK(63)↔CP43D(321) (chymotrypsin), respectively. Our data suggest that Psb27 acts as an important regulatory protein during PSII assembly through specific interactions with the luminal domain of CP43.


Assuntos
Proteínas de Bactérias/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Synechocystis/metabolismo , Proteínas de Bactérias/química , Modelos Moleculares , Proteólise , Espectrometria de Massas em Tandem
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