Functionally diverse ligands modulate different activation states of the formyl peptide receptor 2, a G protein-coupled receptor / 中国药理学与毒理学杂志

OBJECTIVE To identify the mechanisms by which the formyl peptide receptor 2 (FPR2) mediates both inflammatory and anti-inflammatory signaling in an agonist-dependent manner. METHODS Cells expressing FPR2 were incubated with weak agonists, Aβ42 and Ac2-26, before stimulation with a strong agonist, WKYMVm. Calcium mobilization, cAMP inhibition and MAP kinase activation were measured. Intramolecular FRET were determined using FPR2 constructs with an ECFP attached to the C- terminus and a FlAsH binding motif embedded in the first or third intracellular loop (IL1 or IL3, respectively). RESULTS Aβ42 did not induce significant Ca2 + mobilization, but positively modulated WKYMVm-induced Ca2 + mobilization and cAMP reduction in a dose-variable manner within a narrow range of ligand concentrations. Treating FPR2-expressing cells with Ac2-26, a peptide with anti-inflam?matory activity, negatively modulated WKYMVm-induced Ca2 + mobilization and cAMP reduction. Intra?molecular FRET assay showed that stimulation of the receptor constructs with Aβ42 brought the C-terminal domain closer to IL1 but away from IL3. An opposite conformational change was induced by Ac2-26. The FPR2 conformation induced by Aβ42 corresponded to enhanced ERK phosphorylation and attenuated p38 MAPK phosphorylation, whereas Ac2-26 induced FPR2 conformational change corresponding to elevated p38 MAPK phosphorylation and reduced ERK phosphorylation. CONCLUSION Aβ42 and Ac2-26 induce different conformational changes in FPR2. These findings provide a structural basis for FPR2 mediation of inflammatory vs anti-inflammatory functions and identify a type of receptor modulation that differs from the classic positive and negative allosteric modulation.

Biphasic modulation of chemerin peptide-induced calcium flux and ERK phosphorylation by amyloid beta peptide / 中国药理学与毒理学杂志

OBJECTIVE The chemokine-like receptor 1 (CMKLR1, ChemR23) is a functional receptor for chemerin, the chemerin-derived nonapeptide (C9), and the amyloid β peptide 1-42 (Aβ42). Because these peptides share little sequence homology, studies were conducted to investigate their pharmaco?logical properties and regulation at CMKLR1. METHODS Cells expressing CMKLR1 were incubated with Aβ42 before stimulation with a strong agonist, the C9 peptide. Calcium mobilization, cAMP inhibition and MAP kinase activation were measured. Intramolecular FRET were determined using CMKLR1 constructs with an ECFP attached to the C- terminus and a FlAsH binding motif embedded in the first intracellular loop (IL1). RESULTS Binding of both Aβ42 and the C9 peptide induced CMKLR1 internal?ization, but only the Aβ42-induced receptor internalization involved clathrin-coated pits. Likewise, Aβ42 but not C9 stimulated β-arrestin 2 translocation to plasma membranes. A robust Ca2+ flux was observed following C9 stimulation, whereas Aβ42 was ineffective even at micromolar concentrations. Despite its low potency in calcium mobilization assay, Aβ42 was able to alter C9 -induced Ca2+ flux in dose-dependent manner: a potentiation effect at 100 pmol·L-1 of Aβ42 was followed by a suppression at 10 nmol·L-1 and further potentiation at 1 μmol·L-1. This unusual and biphasic modulatory effect was also seen in the C9-induced ERK phosphorylation but the dose curve was opposite to that of Ca2+ flux and cAMP inhibition, suggesting a reciprocal regulatory mechanism. Intramolecular FRET assay confirmed that Aβ42 modulates CMKLR1 rather than its downstream signaling pathways. CONCLUSION These findings suggest Aβ42 as an allosteric modulator that can both positively and negatively regulate the activation state of CMKLR1 in a manner that differs from existing allosteric modulatory mechanisms.

Anticorpos conformacionais para PKCs clássicas e suas aplicações / Conformational antibodies against classical PKCs and their applications

A família proteína quinases C (PKC) é composta por dez isoenzimas, as quais são capazes de fosforilar resíduos de serina e treonina. A ativação dessas quinases envolve mudanças conformacionais, como a remoção do pseudo-substrato do sítio ativo e associação dessas enzimas com lipídeos em membranas biológicas. Além disso, três fosforilações são importantes para a maturação/ enovelamento da enzima e não estão associadas com o estado de ativação das cPKCs. Apesar dessas quinases estarem envolvidas em vários processos patológicos, como carcinogênese e doenças cardiovasculares, ainda não se estabeleceu a relação entre estado de ativação das PKCs com essas doenças. Isso se deve, em parte, à ausência de ferramentas que possibilitam a distinção das formas ativas e inativas das PKCs. Na presente tese, baseando-se em mudanças conformacionais sofridas pelas PKCs durante o processo de ativação, dois anticorpos contra cPKCs ativas foram racionalmente desenvolvidos, sendo um anticorpo policlonal (anti-C2Cat) e outro monoclonal (4.8E). O anticorpo anti-C2Cat foi desenvolvido a partir de imunização de coelhos com um peptídeo localizado na região de interação entre os domínios C2 e catalítico na PKC inativa. Já o anticorpo monoclonal 4.8E foi produzido após a imunização de camundongos Balb/ C com extrato de proteínas proveniente de células HEK293T superexpressando formas constitutivamente ativas da PKCßI. A seletividade de anti-C2Cat e 4.8E por cPKCs ativas foi demonstrada por ensaios de ELISA e de imunoprecipitação, sendo que os anticorpos sempre apresentaram maior afinidade por cPKCs ativas purificadas, superexpressas ou mesmo as endógenas. O anticorpo anti-C2Cat foi capaz de monitorar a dinâmica espaço-temporal da ativação das cPKCs em linhagens de neuroblastoma (Neuro-2A e SK-N-SH) estimuladas com PMA, morfina, ATP ou glutamato por diferentes tempos. Ainda, um maior conteúdo de cPKCs ativas foi detectado por anti-C2Cat na linhagem de câncer de mama MDA-MB-231 (triplo- negativa) do que em células MCF-7 (ER+). Em acordo com esses dados, anti-C2Cat identificou uma maior ativação de cPKCs em tumores mais agressivos de câncer de mama (subtipo triplo-negativo) do que em tumores menos agressivos (ER+, subtipo luminal). Os anticorpos conformacionais anti-C2Cat e 4.8E foram aplicados para elucidar vias de sinalização que levam à carcinogênese em células MDA-MB-231, por meio da realização de ensaios de co-imunoprecipitação, seguida pela identificação das proteínas por espectrometria de massas. Usando essa abordagem, os resultados sugerem que as cPKCs ativas possam estar envolvidas com a tradução de proteínas envolvidas na migração celular, como actina. Em conjunto, os resultado obtidos na presente tese demonstram duas formas racionais de desenvolver anticorpos contra cPKCs ativas, sendo que algumas aplicações para estas ferramentas foram demonstradas. Estratégias baseadas em mudanças conformacionais, similares às apresentadas aqui, poderão ser utilizadas para a produção racional de anticorpos contra outras quinases ou proteínas

The protein kinase C family (PKC) is composed of ten isoenzymes, which are capable of phosphorylating serine and threonine amino acid residues. PKC activation involves conformational changes, such as removing the pseudo-substrate from the active site and binding of the enzyme to lipids in biological membranes. In addition, PKC undergoes three phosphorylations that are important for the maturation/ folding of the enzyme and are not linked with activation status. Despite the fact that these kinases are involved in various pathological processes, such as carcinogenesis and cardiovascular disease, a relationship between PKC activation status with these diseases has not yet been established. This is partly due to the lack of tools to detect active PKC in tissue samples. In this thesis, based on conformational changes suffered by PKC during its activation, two antibodies against active cPKCs were rationally developed; a polyclonal antibody (anti-C2Cat) and a monoclonal (4.8E). Anti-C2Cat was produced after immunization of rabbits with a peptide located at the interface between the C2 and catalytic domains of cPKCs in an inactive PKC. The monoclonal antibody 4.8E was produced after immunization of Balb/C mice with total lysates from HEK293T cells overexpressing constitutively active forms of PKCßI. The anti-C2Cat and 4.8E specificity by active cPKCs was demonstrated by ELISA and immunoprecipitation assays, where the antibodies always showed higher affinity to active cPKCs. Anti-C2Cat was able to detect the temporal and spatial dynamics of cPKC activation upon receptor (morphine, ATP or glutamate) or phorbol ester stimulation in neuroblastoma lines (Neuro-2A and SK-N-SH). Futhermore, anti-C2Cat is able to detect active PKC in human tissues. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Also, both antibodies were applied to study signaling pathways that lead to carcinogenesis in MDA-MB-231 cells by performing co-immunoprecipitation and mass spectrometry. Using this approach, the results suggest that active cPKCs may be involved in translation of proteins involved in cell migration, such as actin. Taken together, the results obtained in this thesis showed two rational ways to develop antibodies against active cPKCs and some applications for these tools were demonstrated. Strategies based on conformational changes, similar to those presented herein may be used for rational production of antibodies against other kinases and proteins

Allosteric serine hydroxymethyltransferase from monkey liver: Correlation of conformational changes caused by denaturants with the alterations in catalytic activity.

The far-ultraviolet region circular dichroic spectrumof serine hydroxymethyltransferase from monkey liver showed that the protein is in an α-helical conformation. The near ultraviolet circular dichoric spectrum revealed two negative bands originating from the tertiary conformational environment of the aromatic amino acid residues. Addition of urea or guanidinium chloride perturbed the characteristic fluorescence and far ultraviolet circular dichroic spectrum of the enzyme. The decrease in (θ)222 and enzyme activity followed identical patterns with increasing concentrations of urea, whereas with guanidinium chloride, the loss of enzyme activity preceded the loss of secondary structure. 2-Chloroethanol, trifluoroethanol and sodium dodecyl sulphate enhanced the mean residue ellipticity values. In addition, sodium dodecyl sulphate also caused a perturbation of the fluorescence emission spectrum of the enzyme. Extremes of pH decreased the – (θ)222 value. Plots of –(θ)222and enzyme activity as a function of pH showed maximal values at pH 7.4-7.5. These results suggested the prevalence of "conformational flexibility" in the structure of serine hydroxymethyltransferase.

Comparison of the conformation and stability of the native dimeric, monomelic, tetrameric and the desensitized forms of the nucleotide pyrophosphatase from mung bean (Phaseolus aureus) seedlings.

A homogenous and crystalline form of nucleotide pyrophosphatase (EC 3.6.1.9) from Phaseolus aureus (mung bean) seedlings was used for the study of the regulation of enzyme activity by adenine nucleotides. The native dimeric form of the enzyme had a helical content of about 65% which was reduced to almost zero values by the addition of AMP. In addition to this change in the helical content, AMP converted the native dimer to a tetramer. Desensitization of AMP regulation, without an alteration of the molecular weight, was achieved either by reversible denaturation with 6 Μ urea or by passage through a column of Blue Sepharose but additionof phydroxymercuribenzoate desensitized the enzyme by dissociating the native dimer to a monomer. The changes in the quaternary structure and conformation of the enzyme consequent to AMP interaction or desensitization were monitored by measuring the helical content, EDTA inactivation and Zn2+ reactivation, stability towards heat denaturation, profiles of urea denaturation and susceptibility towards proteolytic digestion. Based on these results and our earlier work on this enzyme, we propose a model for the regulation of the mung bean nucleotide pyrophosphatase by association-dissociation and conformational changes. The model emphasizes that multiple mechanisms are operative in the desensitization of regulatory proteins.