RESUMO
mAbs have revolutionized the treatment of autoimmune disorders. Even though mAbs have shown impressive efficacy in blocking T cell or B cell activation and/or recruitment to sites of inflammation, this group of biologicals are not devoid of adverse effects. The most serious adverse effects include infusion reactions, including the activation of the complement pathway. In this study, we present a detailed structure-function study of an anti-CCL20 humanized IgG1 mAb that neutralizes CCL20 chemokine and prevents the recruitment of Th17 cells to sites of inflammation. We demonstrate that the anti-CCL20 Ab changes significantly following administration to humans and monkeys and exposure to human serum. Analysis of the drug product revealed that the anti-CCL20 Ab has unexpectedly high C1q binding. This high binding was linked to immune complex formation in vivo but not during in vitro serum incubation. The immune complex contained multiple complement components. Anti-CCL20 Ab-mediated, complement-dependent cytotoxicity occurred when the Ab bound to CCL20 tethered to the cell membrane of target cells. Taken together, these results provide a likely cause for the animal toxicity observed. In addition, anti-CCL20 revealed progressive acidification because of N100 (located in CDR) deamidation over time, which did not directly impact Ag binding. Our study demonstrates that the safety profiling of mAbs should include the evaluation of effector functions in addition to typical stressed conditions.
Assuntos
Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/imunologia , Quimiocina CCL20/imunologia , Animais , Doenças Autoimunes/imunologia , Membrana Celular/imunologia , Proteínas do Sistema Complemento/imunologia , Humanos , Imunoglobulina G/imunologia , Inflamação/imunologia , Macaca fascicularis , Células Th17/imunologiaRESUMO
Despite the potential for the chemokine class as therapeutic targets in immune mediated disease, success has been limited. Many chemokines can bind to multiple receptors and many receptors have multiple ligands, with few exceptions. One of those exceptions is CCL20, which exclusively pairs to CCR6 and is associated with several immunologic conditions, thus providing a promising therapeutic target. Following successful evaluation in a single dose, first time in human clinical study, GSK3050002-a humanized IgG1 monoclonal antibody against human CCL20-was evaluated in a 26-week cynomolgus monkey toxicology study. A high incidence of unexpected vascular and organ inflammation was observed microscopically, leading to the decision to halt clinical development. Here we report a dose-responsive increase in the incidence and severity of inflammation in multiple organs from monkeys receiving 30 and 300 mg/kg/week by either subcutaneous or intravenous injection. Histomorphological changes resembled an immune complex-mediated pathology, which is often due to formation of anti-drug antibodies in monkeys receiving a human protein therapeutic and thus not predictive of clinical outcome. However, the presentation was atypical in that there was a clear dose response with a very high incidence of inflammation with a low incidence of ADA that did not correlate well individually. Additionally, the immunohistologic presentation was atypical in that the severity and distribution of tissue inflammation was greater than the numbers of associated immune complexes (i.e., granular deposits). An extensive ex vivo analysis of large molecular weight protein complexes in monkey serum from this study and in human serum samples demonstrated a time-dependent aggregation of GSK3050002, that was not predicted by in vitro assays. The aggregates also contained complement components. These findings support the hypothesis that immune complexes of drug aggregates, not necessarily including anti-drug antibodies, can fix complement, accumulate over time, and trigger immune complex disease. A situation which may have increased clinical relevance than typical anti-drug antibody-associated immune complex disease in monkeys administered human antibody proteins.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Quimiocina CCL20/imunologia , Proteínas do Sistema Complemento/imunologia , Doenças do Complexo Imune/tratamento farmacológico , Doenças do Complexo Imune/imunologia , Imunoconjugados/uso terapêutico , Animais , Anticorpos Monoclonais/toxicidade , Doença Crônica , Cristalização , Determinação de Ponto Final , Feminino , Humanos , Inflamação/imunologia , Inflamação/patologia , Macaca fascicularisRESUMO
Biotransformation monitoring involves tracking drug modification occurring during in-life studies. Critical Quality Attribute monitoring from forced degraded drug material or in-life sample sets can provide an in-depth assessment of product quality for support in early- or late-stage drug development. For Critical Quality Attribute analysis, biotherapeutic monoclonal antibody (mAb) subunit analysis and peptide mapping liquid chromatography-mass spectrometry (LC-MS) approaches are used, although typically from an in vitro setting (e.g., formulation buffer) not involving biological samples or material. Here, samples from a high-dose rat study (in vivo) are subjected to analysis by ligand binding assay, mAb subunit LC-MS, and peptide mapping by LC-MS. Taken together, data from the 3 analytical approaches provide information regarding drug concentration in circulation, biotransformation, and biotherapeutic drug product quality. The concept of a multitier workflow for preclinical or clinical sample sets can be applied to other biotherapeutic mAb products such as bispecific mAbs, fusions proteins, or antibody-drug conjugates.
Assuntos
Anticorpos Monoclonais/farmacocinética , Mapeamento de Peptídeos/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Anticorpos Monoclonais/química , Biotransformação , Química Farmacêutica/métodos , Cromatografia Líquida de Alta Pressão/métodos , Controle de Qualidade , RatosRESUMO
The molecular mechanism that determines under physiological conditions transmissibility of the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD) is unknown. We report the synthesis of new human prion from the recombinant human prion protein expressed in bacteria in reaction seeded with sCJD MM1 prions and cofactor, ganglioside GM1. These synthetic human prions were infectious to transgenic mice expressing non-glycosylated human prion protein, causing neurologic dysfunction after 459 and 224 days in the first and second passage, respectively. The neuropathology, replication potency, and biophysical profiling suggest that a novel, particularly neurotoxic human prion strain was created. Distinct biological and structural characteristics of our synthetic human prions suggest that subtle changes in the structural organization of critical domains, some linked to posttranslational modifications of the pathogenic prion protein (PrPSc), play a crucial role as a determinant of human prion infectivity, host range, and targetting of specific brain structures in mice models.
Assuntos
Síndrome de Creutzfeldt-Jakob/metabolismo , Proteínas PrPSc/metabolismo , Proteínas Priônicas/metabolismo , Príons/metabolismo , Animais , Encéfalo/metabolismo , Síndrome de Creutzfeldt-Jakob/genética , Modelos Animais de Doenças , Humanos , Camundongos Transgênicos , Proteínas PrPSc/genética , Proteínas Priônicas/genética , Príons/genética , Análise de SobrevidaRESUMO
The infectious pathogen responsible for prion diseases is the misfolded, aggregated form of the prion protein, PrPSc. In contrast to recent progress in studies of laboratory rodent-adapted prions, current understanding of the molecular basis of human prion diseases and, especially, their vast phenotypic diversity is very limited. Here, we have purified proteinase resistant PrPSc aggregates from two major phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD), determined their conformational stability and replication tempo in vitro, as well as characterized structural organization using recently emerged approaches based on hydrogen/deuterium (H/D) exchange coupled with mass spectrometry. Our data clearly demonstrate that these phenotypically distant prions differ in a major way with regard to their structural organization, both at the level of the polypeptide backbone (as indicated by backbone amide H/D exchange data) as well as the quaternary packing arrangements (as indicated by H/D exchange kinetics for histidine side chains). Furthermore, these data indicate that, in contrast to previous observations on yeast and some murine prion strains, the replication rate of sCJD prions is primarily determined not by conformational stability but by specific structural features that control the growth rate of prion protein aggregates.
Assuntos
Síndrome de Creutzfeldt-Jakob , Proteínas PrPSc/química , Western Blotting , Humanos , Imunoensaio , Espectrometria de Massas , Fenótipo , Estabilidade Proteica , Estrutura Quaternária de ProteínaRESUMO
Recent studies indicate that the pathogenesis of Alzheimer disease may be related to the interaction between prion protein (PrP) and certain oligomeric species of Aß peptide. However, the mechanism of this interaction remains unclear and controversial. Here we provide direct experimental evidence that, in addition to previously demonstrated binding to Aß oligomers, PrP also interacts with mature Aß fibrils. However, contrary to the recent claim that PrP causes fragmentation of Aß fibrils into oligomeric species, no evidence for such a disassembly could be detected in the present study. In contrast, our data indicate that the addition of PrP to preformed Aß fibrils results in a lateral association of individual fibrils into larger bundles. These findings have potentially important implications for understanding the mechanism by which PrP might impact Aß toxicity as well as for the emerging efforts to use PrP-derived compounds as inhibitors of Aß-induced neurodegeneration.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Príons/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/farmacologia , Animais , Humanos , Príons/química , Príons/farmacologia , Ligação Proteica/efeitos dos fármacosRESUMO
Mammalian prion strains are believed to arise from the propagation of distinct conformations of the misfolded prion protein PrP(Sc). One key operational parameter used to define differences between strains has been conformational stability of PrP(Sc) as defined by resistance to thermal and/or chemical denaturation. However, the structural basis of these stability differences is unknown. To bridge this gap, we have generated two strains of recombinant human prion protein amyloid fibrils that show dramatic differences in conformational stability and have characterized them by a number of biophysical methods. Backbone amide hydrogen/deuterium exchange experiments revealed that, in sharp contrast to previously studied strains of infectious amyloid formed from the yeast prion protein Sup35, differences in ß-sheet core size do not underlie differences in conformational stability between strains of mammalian prion protein amyloid. Instead, these stability differences appear to be dictated by distinct packing arrangements (i.e. steric zipper interfaces) within the amyloid core, as indicated by distinct x-ray fiber diffraction patterns and large strain-dependent differences in hydrogen/deuterium exchange kinetics for histidine side chains within the core region. Although this study was limited to synthetic prion protein amyloid fibrils, a similar structural basis for strain-dependent conformational stability may apply to brain-derived PrP(Sc), especially because large strain-specific differences in PrP(Sc) stability are often observed despite a similar size of the PrP(Sc) core region.
Assuntos
Amiloide/química , Proteínas PrPSc/química , Doenças Priônicas/genética , Deficiências na Proteostase/genética , Amiloide/genética , Amiloide/metabolismo , Animais , Humanos , Mamíferos , Polimorfismo Genético , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Recent studies revealed that elk-like S170N/N174T mutation in mouse prion protein (moPrP), which results in an increased rigidity of ß2-α2 loop, leads to a prion disease in transgenic mice. Here we characterized the effect of this mutation on biophysical properties of moPrP. Despite similar thermodynamic stabilities of wild type and mutant proteins, the latter was found to have markedly higher propensity to form amyloid fibrils. Importantly, this effect was observed even under fully denaturing conditions, indicating that the increased conversion propensity of the mutant protein is not due to loop rigidity but rather results from greater amyloidogenic potential of the amino acid sequence within the loop region of S170N/N174T moPrP.
Assuntos
Proteínas Amiloidogênicas/química , Modelos Moleculares , Proteínas PrPC/química , Motivos de Aminoácidos , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/ultraestrutura , Animais , Expressão Gênica , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica , Mutação , Proteínas PrPC/genética , Proteínas PrPC/ultraestrutura , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/ultraestrutura , Soluções , Eletricidade Estática , TermodinâmicaRESUMO
Many proteins do not exist in a single rigid conformation. Protein motions, or dynamics, exist and in many cases are important for protein function. The analysis of protein dynamics relies on biophysical techniques that can distinguish simultaneously existing populations of molecules and their rates of interconversion. Hydrogen exchange (HX) detected by mass spectrometry (MS) is contributing to our understanding of protein motions by revealing unfolding and dynamics on a wide timescale, ranging from seconds to hours to days. In this review we discuss HX MS-based analyses of protein dynamics, using our studies of multi-domain kinases as examples. Using HX MS, we have successfully probed protein dynamics and unfolding in the isolated SH3, SH2 and kinase domains of the c-Src and Abl kinase families, as well as the role of inter- and intra-molecular interactions in the global control of kinase function. Coupled with high-resolution structural information, HX MS has proved to be a powerful and versatile tool for the analysis of the conformational dynamics in these kinase systems, and has provided fresh insight regarding the regulatory control of these important signaling proteins. HX MS studies of dynamics are applicable not only to the proteins we illustrate here, but to a very wide range of proteins and protein systems, and should play a role in both classification of and greater understanding of the prevalence of protein motion.
RESUMO
The c-abl proto-oncogene encodes a unique protein-tyrosine kinase (Abl) distinct from c-Src, c-Fes, and other cytoplasmic tyrosine kinases. In normal cells, Abl plays prominent roles in cellular responses to genotoxic stress as well as in the regulation of the actin cytoskeleton. Abl is also well known in the context of Bcr-Abl, the oncogenic fusion protein characteristic of chronic myelogenous leukemia. Selective inhibitors of Bcr-Abl, of which imatinib is the prototype, have had a tremendous impact on clinical outcomes in chronic myelogenous leukemia and revolutionized the field of targeted cancer therapy. In this minireview, we focus on the structural organization and dynamics of Abl kinases and how these features influence inhibitor sensitivity.
Assuntos
Proteínas de Fusão bcr-abl/biossíntese , Proteínas de Fusão bcr-abl/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes abl/genética , Benzamidas , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Modelos Biológicos , Modelos Químicos , Conformação Molecular , Mutação , Ácido Mirístico/química , Piperazinas/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Proto-Oncogene Mas , Pirimidinas/farmacologia , Domínios de Homologia de srcRESUMO
Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control.
Assuntos
Ácido Mirístico/química , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/química , Sítios de Ligação , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/química , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Domínios de Homologia de srcRESUMO
The pathogenesis of Alzheimer disease appears to be strongly linked to the aggregation of amyloid-ß (Aß) peptide and, especially, formation of soluble Aß1-42 oligomers. It was recently demonstrated that the cellular prion protein, PrP(C), binds with high affinity to these oligomers, acting as a putative receptor that mediates at least some of their neurotoxic effects. Here we show that the soluble (i.e. glycophosphatidylinositol anchor-free) prion protein and its N-terminal fragment have a strong effect on the aggregation pathway of Aß1-42, inhibiting its assembly into amyloid fibrils. Furthermore, the prion protein prevents formation of spherical oligomers that normally occur during Aß fibrillogenesis, acting as a potent inhibitor of Aß1-42 toxicity as assessed in experiments with neuronal cell culture. These findings may provide a molecular level foundation to explain the reported protective action of the physiologically released N-terminal N1 fragment of PrP(C) against Aß neurotoxicity. They also suggest a novel approach to pharmacological intervention in Alzheimer disease.
Assuntos
Peptídeos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/química , Doença de Alzheimer/metabolismo , Linhagem Celular Tumoral , Humanos , Microscopia de Força Atômica/métodos , Neuroblastoma/metabolismo , Fragmentos de Peptídeos/química , Peptídeos/química , Príons/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The C-terminally-truncated human prion protein variant Y145Stop (or PrP23-144), associated with a familial prion disease, provides a valuable model for studying the fundamental properties of protein amyloids. In previous solid-state NMR experiments, we established that the ß-sheet core of the PrP23-144 amyloid is composed of two ß-strand regions encompassing residues â¼113-125 and â¼130-140. The former segment contains a highly conserved hydrophobic palindrome sequence, (113)AGAAAAGA(120), which has been considered essential to PrP conformational conversion. Here, we examine the role of this segment in fibrillization of PrP23-144 using a deletion variant, Δ113-120 PrP23-144, in which the palindrome sequence is missing. Surprisingly, we find that deletion of the palindrome sequence affects neither the amyloidogenicity nor the polymerization kinetics of PrP23-144, although it does alter amyloid conformation and morphology. Using two-dimensional and three-dimensional solid-state NMR methods, we find that Δ113-120 PrP23-144 fibrils contain an altered ß-core extended N-terminally to residue â¼106, encompassing residues not present in the core of wild-type PrP23-144 fibrils. The C-terminal ß-strand of the core, however, is similar in both fibril types. Collectively, these data indicate that amyloid cores of PrP23-144 variants contain "essential" (i.e. nucleation-determining) and "nonessential" regions, with the latter being "movable" in amino acid sequence space. These findings reveal an intriguing new mechanism for structural polymorphism in amyloids and suggest a potential means for modulating the physicochemical properties of amyloid fibrils without compromising their polymerization characteristics.
Assuntos
Amiloide/química , Amiloide/genética , Polimorfismo Genético , Príons/química , Escherichia coli/metabolismo , Deleção de Genes , Humanos , Cinética , Espectroscopia de Ressonância Magnética/métodos , Microscopia de Força Atômica/métodos , Doenças Priônicas/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
Soluble oligomers of Abeta42 peptide are believed to play a major role in the pathogenesis of Alzheimer disease (AD). It was recently found that at least some of the neurotoxic effects of these oligomers may be mediated by specific binding to the prion protein, PrP(C), on the cell surface (Laurén, J., Gimbel, D. A., Nygaard, H. B., Gilbert, J. W., and Strittmatter, S. M. (2009) Nature 457, 1128-1132). Here we characterized the interaction between synthetic Abeta42 oligomers and the recombinant human prion protein (PrP) using two biophysical techniques: site-directed spin labeling and surface plasmon resonance. Our data indicate that this binding is highly specific for a particular conformation adopted by the peptide in soluble oligomeric species. The binding appears to be essentially identical for the Met(129) and Val(129) polymorphic forms of human PrP, suggesting that the role of PrP codon 129 polymorphism as a risk factor in AD is due to factors unrelated to the interaction with Abeta oligomers. It was also found that in addition to the previously identified approximately 95-110 segment, the second region of critical importance for the interaction with Abeta42 oligomers is a cluster of basic residues at the extreme N terminus of PrP (residues 23-27). The deletion of any of these segments results in a major loss of the binding function, indicating that these two regions likely act in concert to provide a high affinity binding site for Abeta42 oligomers. This insight may help explain the interplay between the postulated protective and pathogenic roles of PrP in AD and may contribute to the development of novel therapeutic strategies as well.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Proteínas PrPC/metabolismo , Doença de Alzheimer/etiologia , Peptídeos beta-Amiloides/química , Sítios de Ligação , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Polimorfismo Genético , Proteínas PrPC/química , Proteínas PrPC/genética , Príons/química , Príons/metabolismo , Ligação Proteica , Marcadores de Spin , Ressonância de Plasmônio de SuperfícieRESUMO
The epitope of the 3F4 antibody most commonly used in human prion disease diagnosis is believed to consist of residues Met-Lys-His-Met (MKHM) corresponding to human PrP-(109-112). This assumption is based mainly on the observation that 3F4 reacts with human and hamster PrP but not with PrP from mouse, sheep, and cervids, in which Met at residue 112 is replaced by Val. Here we report that, by brain histoblotting, 3F4 did not react with PrP of uninfected transgenic mice expressing elk PrP; however, it did show distinct immunoreactivity in transgenic mice infected with chronic wasting disease. Compared with human PrP, the 3F4 reactivity with the recombinant elk PrP was 2 orders of magnitude weaker, as indicated by both Western blotting and surface plasmon resonance. To investigate the molecular basis of these species- and conformer-dependent preferences of 3F4, the epitope was probed by peptide membrane array and antigen competition experiments. Remarkably, the 3F4 antibody did not react with MKHM but reacted strongly with KTNMK (corresponding to human PrP-(106-110)), a sequence that is also present in cervids, sheep, and cattle. 3F4 also reacted with elk PrP peptides containing KTNMKHV. We concluded that the minimal sequence for the 3F4 epitope consists of residues KTNMK, and the species- and conformer-dependent preferences of 3F4 arise largely from the interactions between Met(112) (human PrP) or Val(115) (cervid PrP) and adjacent residues.
Assuntos
Anticorpos Monoclonais/química , Especificidade de Anticorpos , Epitopos/química , Príons/química , Animais , Bovinos , Cricetinae , Epitopos/genética , Epitopos/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Príons/genética , Príons/metabolismo , Conformação Proteica , Ovinos , Especificidade da EspécieRESUMO
Recent studies have shown that trans-phosphorylation of the Abl SH3 domain at Tyr89 by Src-family kinases is required for the full transforming activity of Bcr-Abl. Tyr89 localizes to a binding surface of the SH3 domain that engages the SH2-kinase linker in the crystal structure of the c-Abl core. Displacement of SH3 from the linker is likely to influence efficient downregulation of c-Abl. Hydrogen-deuterium exchange (HX) and mass spectrometry (MS) were used to investigate whether Tyr89 phosphorylation affects the ability of the SH3 domain to interact intramolecularly with the SH2-kinase linker in cis as well as other peptide ligands in trans. HX MS analysis of SH3 binding showed that when various Abl constructs were phosphorylated at Tyr89 by the Src-family kinase Hck, SH3 was unable to engage a high-affinity ligand in trans and that interaction with the linker in cis was reduced dramatically in a construct containing the SH3 and SH2 domains plus the linker. Phosphorylation of the Abl SH3 domain on Tyr89 also interfered with binding to the negative regulatory protein Abi-1 in trans. Site-directed mutagenesis of Tyr89 and Tyr245, another tyrosine phosphorylation site located in the linker that may also influence SH3 binding, implicated Tyr89 as the key residue necessary for disrupting regulation after phosphorylation. These results imply that phosphorylation at Tyr89 by Src-family kinases prevents engagement of the Abl SH3 domain with its intramolecular binding partner leading to enhanced Abl kinase activity and cellular signaling.
Assuntos
Proteínas Proto-Oncogênicas c-abl/química , Proteínas Proto-Oncogênicas c-abl/metabolismo , Tirosina/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Fusão bcr-abl/química , Proteínas de Fusão bcr-abl/metabolismo , Genes abl , Humanos , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Conformação Proteica , Transdução de Sinais , Tirosina/genética , Domínios de Homologia de src/genéticaRESUMO
Crystal structures and other biochemical data indicate that the N-terminal cap (NCap) region of the Abelson tyrosine kinase (c-Abl) is important for maintaining the downregulated conformation of the kinase domain. The exact contributions that the NCap makes in stabilizing the various intramolecular interactions within c-Abl are less clear. While the NCap appears to be important for locking the SH3 and SH2 domains to the back of the kinase domain, there may be other more subtle elements of regulation. Hydrogen exchange (HX) and mass spectrometry (MS) were used to determine if the NCap contributes to intramolecular interactions involving the Abl SH3 domain. Under physiological conditions, the Abl SH3 domain underwent partial unfolding and its unfolding half-life was slowed during binding to the SH2 kinase linker, providing a unique assay for testing NCap-induced stabilization of the SH3 domain in various constructs. The results showed that the NCap stabilizes the dynamics of the SH3 domain in certain constructs but does not increase the relative affinity of the SH3 domain for the native SH2 kinase linker. The stabilization effect was absent in constructs of just the NCap and SH3 but was obvious when the SH2 domain and the SH2 kinase linker were present. These results suggest that interactions between the NCap and the SH3 domain can contribute to c-Abl stabilization in constructs that contain at least the SH2 domain, an effect that may partially compensate for the absence of the negative regulatory C-terminal tail found in the related Src family of kinases.
Assuntos
Bioquímica/métodos , Proteínas Proto-Oncogênicas c-abl/química , Cristalografia por Raios X/métodos , Escherichia coli/metabolismo , Humanos , Hidrogênio/química , Ligantes , Espectrometria de Massas/métodos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Domínios de Homologia de srcRESUMO
The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.