RESUMO
The directional movement of cells in a gradient of external stimulus is termed chemotaxis and is important in many aspects of development and differentiated cell function. Phophoinositide 3-kinases (PI(3)Ks) are thought to have critical roles within the gradient-sensing machinery of a variety of highly motile cells, such as mammalian phagocytes, allowing these cells to respond quickly and efficiently to shallow gradients of soluble stimuli. Our analysis of mammalian neutrophil migration towards ligands such as fMLP shows that, although PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) accumulate in a PI(3)Kgamma-dependent fashion at the up-gradient leading-edge, this signal is not required for efficient gradient-sensing and gradient-biased movement. PI(3)Kgamma activity is however, a critical determinant of the proportion of cells that can move, that is, respond chemokinetically, in reaction to fMLP. Furthermore, this dependence of chemokinesis on PI(3)Kgamma activity is context dependent, both with respect to the state of priming of the neutrophils and the type of surface on which they are migrating. We propose this effect of PI(3)Kgamma is through roles in the regulation of some aspects of neutrophil polarization that are relevant to movement, such as integrin-based adhesion and the accumulation of polymerized (F)-actin at the leading-edge.
Assuntos
Neutrófilos/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Actinas/química , Animais , Movimento Celular , Células Cultivadas , Classe Ib de Fosfatidilinositol 3-Quinase , Relação Dose-Resposta a Droga , Humanos , Integrinas/fisiologia , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/enzimologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
Light chain amyloidosis (AL), is classified as a plasma cell dyscrasia, whereby a mutant plasma cell multiplies uncontrollably and secretes enormous amounts of immunoglobulin-free light chain (FLC) fragments. These FLCs undergo a process of misfolding and aggregation into amyloid fibrils, that can cause irreversible system-wide damage. Current treatments that focus on depleting the underlying plasma cell clone are often poorly tolerated, particularly in patients with severe cardiac involvement, meaning patient prognosis is poor. An alternative treatment approach currently being explored is the inhibition of FLC aggregation by stabilisation of the native conformer. Here, we aimed to identify and characterise antibody fragments that target FLC domains and promote their stabilisation. Using phage-display screening methods, we identified a variable heavy (VH) domain, termed VH1, targeted towards the FLC. Using differential scanning fluorimetry and surface plasmon resonance, VH1 was characterised to bind and kinetically stabilise an amyloidogenic FLC, whereby a > 5.5 °C increase in thermal stability was noted. This improved stability corresponded to the inhibition of fibril formation, where 10 : 1 LC : VH1 concentration reduced aggregation to baseline levels. X-ray crystallographic structures of the LC : VH1 complex at atomic resolution revealed binding in a 1 : 1 ratio, mimicking the dimeric antigen binding sites of the native immunoglobulin molecule and the native LC homodimer.
RESUMO
Owing to its widespread activation in inflammation and cancer, a growing appreciation of the therapeutic potential of inhibitors of the phosphoinositide 3-kinase (PI3K) pathway has stimulated intense interest in compounds with suitable pharmacological profiles. These are primarily directed toward PI3K itself. However, as class I PI3Ks are also essential for a range of normal physiological processes, broad spectrum PI3K inhibition could be poorly tolerated. In recent years, patents describing a new generation of PI3K inhibitors have started to appear, with a particular focus on the development of compounds with enhanced isoform selectivity for use as anti-cancer and anti-inflammatory therapies. However, challenges remain for the efforts to pharmacologically target this enzyme family in a successful manner.
Assuntos
Inflamação/tratamento farmacológico , Neoplasias/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/uso terapêutico , Ativação Enzimática , Inflamação/enzimologia , Neoplasias/enzimologiaRESUMO
14-3-3 proteins regulate many intracellular processes and their ability to bind in subtly different fashions to their numerous partner proteins provides attractive drug-targeting points for a range of diseases. Schnurri-3 is a suppressor of mouse bone formation and a candidate target for novel osteoporosis therapeutics, and thus it is of interest to determine whether it interacts with 14-3-3. In this work, potential 14-3-3 interaction sites on mammalian Schnurri-3 were identified by an in silico analysis of its protein sequence. Using fluorescence polarization, isothermal titration calorimetry and X-ray crystallography, it is shown that synthetic peptides containing either phosphorylated Thr869 or Ser542 can indeed interact with 14-3-3, with the latter capable of forming an interprotein disulfide bond with 14-3-3σ: a hitherto unreported phenomenon.
Assuntos
Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Osteogênese/fisiologia , Proteínas 14-3-3/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/fisiologia , Cristalografia por Raios X/métodos , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Dedos de Zinco/fisiologiaRESUMO
Expansion of the polyglutamine tract in the N terminus of Ataxin-1 is the main cause of the neurodegenerative disease, spinocerebellar ataxia type 1 (SCA1). However, the C-terminal part of the protein - including its AXH domain and a phosphorylation on residue serine 776 - also plays a crucial role in disease development. This phosphorylation event is known to be crucial for the interaction of Ataxin-1 with the 14-3-3 adaptor proteins and has been shown to indirectly contribute to Ataxin-1 stability. Here we show that 14-3-3 also has a direct anti-aggregation or "chaperone" effect on Ataxin-1. Furthermore, we provide structural and biophysical information revealing how phosphorylated S776 in the intrinsically disordered C terminus of Ataxin-1 mediates the cytoplasmic interaction with 14-3-3 proteins. Based on these findings, we propose that 14-3-3 exerts the observed chaperone effect by interfering with Ataxin-1 dimerization through its AXH domain, reducing further self-association. The chaperone effect is particularly important in the context of SCA1, as it was previously shown that a soluble form of mutant Ataxin-1 is the major driver of pathology.
Assuntos
Proteínas 14-3-3/metabolismo , Ataxina-1/química , Ataxina-1/metabolismo , Citoplasma/metabolismo , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Células HEK293 , Humanos , Fosforilação , Domínios Proteicos , Multimerização Proteica , Estabilidade ProteicaRESUMO
Synthetic biology and metabolic engineering offer potentially green and attractive routes to the production of high value compounds. The provision of high-quality parts and pathways is crucial in enabling the biosynthesis of chemicals using synthetic biology. While a number of regulatory parts that provide control at the transcriptional and translational level have been developed, relatively few exist at the protein level. Single domain antibodies (sdAb) such as camelid heavy chain variable fragments (VHH) possess binding characteristics which could be exploited for their development and use as novel parts for regulating metabolic pathways at the protein level in microbial cell factories. Here, a platform for the use of VHH as tools in Escherichia coli is developed and subsequently used to modulate linalool production in E. coli. The coproduction of a Design of Experiments (DoE) optimized pBbE8k His6-VHHCyDisCo system alongside a heterologous linalool production pathway facilitated the identification of anti-bLinS VHH that functioned as modulators of bLinS. This resulted in altered product profiles and significant variation in the titers of linalool, geraniol, nerolidol, and indole obtained. The ability to alter the production levels of high value terpenoids, such as linalool, in a tunable manner at the protein level could represent a significant step forward for the development of improved microbial cell factories. This study serves as a proof of principle indicating that VHH can be used to modulate enzyme activity in engineered pathways within E. coli. Given their almost limitless binding potential, we posit that single domain antibodies could emerge as powerful regulatory parts in synthetic biology applications.
Assuntos
Monoterpenos Acíclicos/metabolismo , Proteínas de Bactérias/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroliases/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Engenharia Metabólica/métodos , Anticorpos de Domínio Único/imunologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Reatores Biológicos , Camelídeos Americanos/imunologia , Códon , Ativação Enzimática/imunologia , Hidroliases/genética , Hidroliases/metabolismo , Streptomyces/enzimologia , Streptomyces/genética , Biologia Sintética/métodosRESUMO
IL-17-producing Th cells (Th17) are a distinct subset of effector cells that bridge the innate and adaptive immune system and are implicated in autoimmune disease processes. CD4(+) splenocytes from DO11.10 mice were activated with OVA peptide(323-339) and maintained under Th17 polarization conditions, resulting in significantly higher proportions of IL-17(+) T cells compared with nonpolarized (Th0) cells. Th17-polarizing conditions significantly increased the proportion of cells expressing the chemokine receptors CCR2, CCR6, and CCR9 when compared with Th0 cells. In contrast, there was a significant decrease in the proportion of cells expressing CXCR3 under Th17-polarizing conditions compared with nonpolarizing conditions. The respective chemokine agonists for CCR2 (CCL2 and CCL12), CCR6 (CCL20), and CCR9 (CCL25) elicited migration and PI-3K-dependent signaling events in Th17-polarized cells, thus indicating that all three receptors were functionally and biochemically responsive. Furthermore, postmigration phenotypic analysis demonstrated that the agonists for CCR2 and CCR6, but not CCR9, stimulated a modest enrichment of IL-17(+) cells compared with the premigration population. Pan-isoform inhibitors of PI-3K/Akt signaling prevented CCR2- and CCR6-mediated, polarized Th17 cell migration in a concentration-dependent manner. The unique chemokine receptor expression pattern of Th17 cells and their corresponding PI-3K-dependent migratory responses are important for understanding the pathogenesis of autoimmune diseases and may provide opportunities for the application of CCR2 and CCR6 antagonists and PI-3K isoform-selective inhibitors in defined inflammatory settings.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CCR2/fisiologia , Receptores CCR6/fisiologia , Linfócitos T Auxiliares-Indutores/fisiologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/fisiologia , Movimento Celular , Separação Celular , Citocinas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Fosfatidilinositol 3-Quinases/imunologia , Receptores CCR2/agonistas , Receptores CCR6/agonistas , Receptores de Quimiocinas/genética , Baço/citologia , Baço/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Optimization of the cellular and pharmacological activity of a novel series of PI3 kinase inhibitors targeting multiple isoforms is described.
Assuntos
Benzoxazinas/síntese química , Benzoxazinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Pirazóis/síntese química , Pirazóis/farmacologia , Administração Oral , Animais , Benzoxazinas/química , Técnicas de Química Combinatória , Concentração Inibidora 50 , Isoenzimas/antagonistas & inibidores , Masculino , Estrutura Molecular , Pirazóis/química , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
4-(1,3-Thiazol-2-yl)morpholine derivatives have been identified as potent and selective inhibitors of phosphoinositide 3-kinase. The SAR data of selected examples are presented and the in vivo profiling of compound 18 is shown to demonstrate the utility of this class of compounds in xenograft models of tumor growth.
Assuntos
1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Morfolinas/farmacologia , Tiazóis/farmacologia , Animais , Técnicas de Química Combinatória , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Isoenzimas , Camundongos , Estrutura Molecular , Morfolinas/síntese química , Morfolinas/química , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The SAR and pharmacokinetic profiles of a series of multi-isoform PI3K inhibitors based on a 3,4-dihydro-2H-benzo[1,4]oxazine scaffold are disclosed.
Assuntos
Benzoxazinas/química , Química Farmacêutica/métodos , Oxazinas/síntese química , Oxazinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Área Sob a Curva , Benzoxazinas/farmacologia , Cristalografia por Raios X/métodos , Desenho de Fármacos , Humanos , Inflamação , Concentração Inibidora 50 , Modelos Químicos , Estrutura Molecular , Isoformas de Proteínas , Relação Estrutura-AtividadeRESUMO
The effective management of AIDS with HIV protease inhibitors, or the use of angiotensin-converting enzyme inhibitors to treat hypertension, indicates that proteases do make good drug targets. On the other hand, matrix metalloproteinase (MMP) inhibitors from several companies have failed in both cancer and rheumatoid arthritis clinical trials. Mindful of the MMP inhibitor experience, this chapter explores how tractable proteases are as drug targets from a chemistry perspective. It examines the recent success of other classes of drug for the treatment of rheumatoid arthritis, and highlights the need to consider where putative targets lie on pathophysiological pathways--regardless of what kind of therapeutic entity would be required to target them. With genome research yielding many possible new drug targets, it explores the likelihood of discovering proteolytic enzymes that are causally responsible for disease processes and that might therefore make better targets, especially if they lead to the development of drugs that can be administered orally. It also considers the impact that biologics are having on drug discovery, and in particular whether biologically derived therapeutics such as antibodies are likely to significantly alter the way we view proteases as targets and the methods used to discover therapeutic inhibitors.
Assuntos
Endopeptidases/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/enzimologia , Genoma Humano , Humanos , Hidrólise , Inibidores de Proteases/uso terapêuticoRESUMO
It is well established that preexposure of human neutrophils to proinflammatory cytokines markedly augments the production of reactive oxygen species (ROS) to subsequent stimuli. This priming event is thought to be critical for localizing ROS to the vicinity of the inflammation, maximizing their role in the resolution of the inflammation, and minimizing the damage to surrounding tissue. We have used a new generation of isoform-selective phosphoinositide 3-kinase (PI3K) inhibitors to show that ROS production under these circumstances is regulated by temporal control of class I PI3K activity. Stimulation of tumor necrosis factor-alpha (TNF-alpha)-primed human neutrophils with N-formyl-methionyl-leucyl-phenylalanine (fMLP) results in biphasic activation of PI3K; the first phase is largely dependent on PI3Kgamma, and the second phase is largely dependent on PI3Kdelta. The second phase of PI3K activation requires the first phase; it is this second phase that is augmented by TNF-alpha priming and that regulates parallel activation of ROS production. Surprisingly, although TNF-alpha-primed mouse bone marrow-derived neutrophils exhibit superficially similar patterns of PI3K activation and ROS production in response to fMLP, these responses are substantially lower and largely dependent on PI3Kgamma alone. These results start to define which PI3K isoforms are responsible for modulating neutrophil responsiveness to infection and inflammation.
Assuntos
Neutrófilos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Explosão Respiratória , Animais , Células Cultivadas , Classe I de Fosfatidilinositol 3-Quinases , Classe Ib de Fosfatidilinositol 3-Quinase , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Isoenzimas , Camundongos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Especificidade da Espécie , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
We report experiments to investigate the role of the physiologically relevant protein tyrosine kinase Lck in the ordered phosphorylation of the T-cell receptor zeta chain. Six synthetic peptides were designed based on the sequences of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the zeta chain. Preliminary 1H-NMR studies of recombinant zeta chain suggested that it is essentially unstructured and therefore that peptide mimics would serve as useful models for investigating individual ITAM tyrosines. Phosphorylation kinetics were determined for each tyrosine by assaying the transfer of 32P by recombinant Lck on to each of the peptides. The rates of phosphorylation were found to depend on the location of the tyrosine, leading to the proposal that Lck phosphorylates the six zeta chain ITAM tyrosines in the order 1N (first) > 3N > 3C > 2N > 1C > 2C (last) as a result of differences in the amino-acid sequence surrounding each tyrosine. This proposal was then tested on cytosolic, recombinant T-cell receptor zeta chain. After in vitro phosphorylation by Lck, the partially phosphorylated zeta chain was digested with trypsin. Separation and identification of the zeta chain fragments using LC-MS showed, as predicted by the peptide phosphorylation studies, that tyrosine 1N is indeed the first to be phosphorylated by Lck. We conclude that differences in the amino-acid context of the six zeta chain ITAM tyrosines affect the efficiency of their phosphorylation by the kinase Lck, which probably contributes to the distinct patterns of phosphorylation observed in vivo.