RESUMO
For decades, formaldehyde has been routinely used to cross-link proteins in cells, tissue, and in some instances, even entire organisms. Due to its small size, formaldehyde can readily permeate cell walls and membranes, resulting in efficient cross-linking, i.e. the formation of covalent bonds between proteins, DNA, and other reactive molecules. Indeed, formaldehyde cross-linking is an instrumental component of many mainstream analytical/cell biology techniques including chromatin immunoprecipitation (ChIP) of protein-DNA complexes found in nuclei; immunohistological analysis of protein expression and localization within cells, tissues, and organs; and mass spectrometry (MS)-compatible silver-staining methodologies used to visualize low abundance proteins in polyacrylamide gels. However, despite its exquisite suitability for use in the analysis of protein environments within cells, formaldehyde has yet to be commonly employed in the directed analysis of protein-protein interactions and cellular networks. The general purpose of this article is to discuss recent advancements in the use of formaldehyde cross-linking in combination with MS-based methodologies. Key advantages and limitations to the use of formaldehyde over other cross-linkers and technologies currently used to study protein-protein interactions are highlighted, and formaldehyde-based experimental approaches that are proving very promising in their ability to accurately and efficiently identify novel protein-protein and multiprotein interaction complexes are presented.
Assuntos
Reagentes de Ligações Cruzadas/química , Formaldeído/química , Espectrometria de Massas/métodos , Proteômica/métodos , DNA/química , Ligação Proteica , Proteínas/químicaRESUMO
BACKGROUND: The efficacy of AQX-1125, a small-molecule SH2-containing inositol-5'-phosphatase 1 (SHIP1) activator and clinical development candidate, is investigated in rodent models of inflammation. EXPERIMENTAL APPROACH: AQX-1125 was administered orally in a mouse model of passive cutaneous anaphylaxis (PCA) and a number of rodent models of respiratory inflammation including: cigarette smoke, LPS and ovalbumin (OVA)-mediated airway inflammation. SHIP1 dependency of the AQX-1125 mechanism of action was investigated by comparing the efficacy in wild-type and SHIP1-deficient mice subjected to an intrapulmonary LPS challenge. RESULTS: AQX-1125 exerted anti-inflammatory effects in all of the models studied. AQX-1125 decreased the PCA response at all doses tested. Using bronchoalveolar lavage (BAL) cell counts as an end point, oral or aerosolized AQX-1125 dose dependently decreased the LPS-mediated pulmonary neutrophilic infiltration at 3-30 mg kg⻹ and 0.15-15 µg kg⻹ respectively. AQX-1125 suppressed the OVA-mediated airway inflammation at 0.1-10 mg kg⻹. In the smoke-induced airway inflammation model, AQX-1125 was tested at 30 mg kg⻹ and significantly reduced the neutrophil infiltration of the BAL fluid. AQX-1125 (10 mg kg⻹) decreased LPS-induced pulmonary neutrophilia in wild-type mice but not in SHIP1-deficient mice. CONCLUSIONS: The SHIP1 activator, AQX-1125, suppresses leukocyte accumulation and inflammatory mediator release in rodent models of pulmonary inflammation and allergy. As shown in the mouse model of LPS-induced lung inflammation, the efficacy of the compound is dependent on the presence of SHIP1. Pharmacological SHIP1 activation may have clinical potential for the treatment of pulmonary inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Asma/tratamento farmacológico , Cicloexanóis/uso terapêutico , Dermatite Alérgica de Contato/tratamento farmacológico , Ativadores de Enzimas/uso terapêutico , Indanos/uso terapêutico , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/sangue , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/farmacocinética , Asma/sangue , Asma/imunologia , Asma/metabolismo , Cicloexanóis/sangue , Cicloexanóis/metabolismo , Cicloexanóis/farmacocinética , Dermatite Alérgica de Contato/sangue , Dermatite Alérgica de Contato/imunologia , Dermatite Alérgica de Contato/metabolismo , Modelos Animais de Doenças , Ativadores de Enzimas/sangue , Ativadores de Enzimas/metabolismo , Ativadores de Enzimas/farmacocinética , Feminino , Indanos/sangue , Indanos/metabolismo , Indanos/farmacocinética , Inositol Polifosfato 5-Fosfatases , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Infiltração de Neutrófilos/efeitos dos fármacos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Ratos , Ratos Endogâmicos BN , Ratos Sprague-Dawley , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Doenças Respiratórias/sangue , Doenças Respiratórias/imunologia , Doenças Respiratórias/metabolismo , Doenças Respiratórias/prevenção & controleRESUMO
BACKGROUND: The SH2-containing inositol-5'-phosphatase 1 (SHIP1) metabolizes PI(3,4,5)P3 to PI(3,4)P2. SHIP1-deficient mice exhibit progressive inflammation. Pharmacological activation of SHIP1 is emerging as a potential therapy for pulmonary inflammatory diseases. Here we characterize the efficacy of AQX-1125, a small-molecule SHIP1 activator currently in clinical development. EXPERIMENTAL APPROACH: The effects of AQX-1125 were tested in several in vitro assays: on enzyme catalytic activity utilizing recombinant human SHIP1, on Akt phosphorylation in SHIP1-proficient and SHIP1-deficient cell lines, on cytokine release in murine splenocytes, on human leukocyte chemotaxis using modified Boyden chambers and on ß-hexosaminidase release from murine mast cells. In addition, pharmacokinetic and drug distribution studies were performed in rats and dogs. RESULTS: AQX-1125 increased the catalytic activity of human recombinant SHIP1, an effect, which was absent after deletion of the C2 region. AQX-1125 inhibited Akt phosphorylation in SHIP1-proficient but not in SHIP1-deficient cells, reduced cytokine production in splenocytes, inhibited the activation of mast cells and inhibited human leukocyte chemotaxis. In vivo, AQX-1125 exhibited >80% oral bioavailability and >5 h terminal half-life. CONCLUSIONS: Consistent with the role of SHIP1 in cell activation and chemotaxis, the SHIP1 activator AQX-1125 inhibits Akt phosphorylation, inflammatory mediator production and leukocyte chemotaxis in vitro. The in vitro effects and the pharmacokinetic properties of the compound make it a suitable candidate for in vivo testing in various models of inflammation.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Cicloexanóis/farmacologia , Ativadores de Enzimas/farmacologia , Indanos/farmacologia , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Anti-Inflamatórios não Esteroides/sangue , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/farmacocinética , Linhagem Celular , Células Cultivadas , Cicloexanóis/sangue , Cicloexanóis/metabolismo , Cicloexanóis/farmacocinética , Cães , Ativadores de Enzimas/sangue , Ativadores de Enzimas/metabolismo , Ativadores de Enzimas/farmacocinética , Feminino , Humanos , Indanos/sangue , Indanos/metabolismo , Indanos/farmacocinética , Inositol Polifosfato 5-Fosfatases , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Baço/citologia , Baço/efeitos dos fármacos , Baço/metabolismoRESUMO
Cross-linking of proteins in a complex requires the chemical modification of the proteins in order to form a covalent link. This can be achieved in vivo using formaldehyde as it is small and rapidly permeates the cell membrane. Previous model studies of the speed and specificity of the first step of this reaction on peptides have suggested that residue accessibility and sequence micro-environment play a significant role in the production of the reactive intermediate necessary for cross-linking. This dependency was therefore further investigated on model proteins, which contain a more complex tertiary structure. Under mild reaction conditions, similar to those used for in vivo protein cross-linking, it was found that the vast majority of modification occurred on lysines, tertiary structure and solvent accessible surface area played a major role in regulating the extent of formaldehyde-induced modifications, and that the modifications on a folded protein did not significantly affect its tertiary structural stability.
Assuntos
Formaldeído/química , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Reagentes de Ligações Cruzadas/química , Mioglobina/química , Estrutura Terciária de Proteína , Serina Endopeptidases/metabolismoRESUMO
Formaldehyde cross-linking of proteins is emerging as a novel approach to study protein-protein interactions in living cells. It has been shown to be compatible with standard techniques used in functional proteomics such as affinity-based protein enrichment, enzymatic digestion, and mass spectrometric protein identification. So far, the lack of knowledge on formaldehyde-induced protein modifications and suitable mass spectrometric methods for their targeted detection has impeded the identification of the different types of cross-linked peptides in these samples. In particular, it has remained unclear whether in vitro studies that identified a multitude of amino acid residues reacting with formaldehyde over the course of several days are suitable substitutes for the much shorter reaction times of 10-20 min used in cross-linking experiments in living cells. The current study on model peptides identifies amino-termini as well as lysine, tryptophan, and cysteine side chains, i.e. a small subset of those modified after several days, as the major reactive sites under such conditions, and suggests relative position in the peptide sequence as well as sequence microenvironment to be important factors that govern reactivity. Using MALDI-MS, mass increases of 12 Da on amino groups and 30 Da on cysteines were detected as the major reaction products, while peptide fragment ion analysis by tandem mass spectrometry was used to localize the actual modification sites on a peptide. Non-specific cross-linking was absent, and could only be detected with low yield at elevated peptide concentrations. The detailed knowledge on the constraints and products of the formaldehyde reaction with peptides after short incubation times presented in this study is expected to facilitate the targeted mass spectrometric analysis of proteins after in vivo formaldehyde cross-linking.