S-Nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner

BACKGROUND: Recent studies indicate that the G protein-coupled receptor (GPCR) signaling machinery can serve as a direct target of reactive oxygen species, including nitric oxide (NO) and S-nitrosothiols (RSNOs). To gain a broader view into the way that receptor-dependent G protein activation – an early step in signal transduction – might be affected by RSNOs, we have studied several receptors coupling to the G(i )family of G proteins in their native cellular environment using the powerful functional approach of [(35)S]GTPγS autoradiography with brain cryostat sections in combination with classical G protein activation assays. RESULTS: We demonstrate that RSNOs, like S-nitrosoglutathione (GSNO) and S-nitrosocysteine (CysNO), can modulate GPCR signaling via reversible, thiol-sensitive mechanisms probably involving S-nitrosylation. RSNOs are capable of very targeted regulation, as they potentiate the signaling of some receptors (exemplified by the M2/M4 muscarinic cholinergic receptors), inhibit others (P2Y(12 )purinergic, LPA(1)lysophosphatidic acid, and cannabinoid CB(1 )receptors), but may only marginally affect signaling of others, such as adenosine A(1), μ-opioid, and opiate related receptors. Amplification of M2/M4 muscarinic responses is explained by an accelerated rate of guanine nucleotide exchange, as well as an increased number of high-affinity [(35)S]GTPγS binding sites available for the agonist-activated receptor. GSNO amplified human M4 receptor signaling also under heterologous expression in CHO cells, but the effect diminished with increasing constitutive receptor activity. RSNOs markedly inhibited P2Y(12 )receptor signaling in native tissues (rat brain and human platelets), but failed to affect human P2Y(12 )receptor signaling under heterologous expression in CHO cells, indicating that the native cellular signaling partners, rather than the P2Y(12 )receptor protein, act as a molecular target for this action. CONCLUSION: These in vitro studies show for the first time in a broader general context that RSNOs are capable of modulating GPCR signaling in a reversible and highly receptor-specific manner. Given that the enzymatic machinery responsible for endogenous NO production is located in close proximity with the GPCR signaling complex, especially with that for several receptors whose signaling is shown here to be modulated by exogenous RSNOs, our data suggest that GPCR signaling in vivo is likely to be subject to substantial, and highly receptor-specific modulation by NO-derived RSNOs

Discovery of 12-thiazole abietanes as selective inhibitors of the human metabolic serine hydrolase hABHD16A

Screening of an in-house library of compounds identified 12-thiazole abietanes as a new class of reversible inhibitors of the human metabolic serine hydrolase. Further optimization of the first hit compound lead to the 2-methylthiazole derivative 18, with an IC50 value of 3.4 ± 0.2 µM and promising selectivity. ABHD16A has been highlighted as a new target for in-flammation-mediated pain, although selective inhibitors of hABHD16A (human ABHD16A) have not yet been reported. Our study presents abietane-type diterpenoids as an attractive starting point for the design of selective ABHD16A inhibitors, which will contribute towards understanding the significance of hABHD16A inhibition in vivo

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections - Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils

Background Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. Results Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Conclusions Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.Peer reviewe

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections - Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils

Background Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. Results Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Conclusions Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.Peer reviewe

Discovery of Triterpenoids as Reversible Inhibitors of α/β hydrolase Domain Containing 12 (ABHD12)

Peer reviewe

Nuoren päänsärkypotilaan elämäntapaohjaus

Tämän opinnäytetyön tarkoituksena oli selvittää millä keinoilla nuori saadaan ohjattua päänsäryn hoitoon omia elämäntapojaan muuttamalla. Työmme tavoitteena oli vahvistaa nuoren, hänen perheensä sekä hoitotyössä työskentelevien asiantuntijuutta ja yhteistyötä. Työmme kuuluu tulevaisuus lasten erikoisairaanhoidossa hankkeeseen vuosina 2011- 2014. Se tehtiin yhteistyössä Metropolia ammattikorkeakoulun ja HYKS Naisten- ja lasten-tautien tulosyksikön kanssa. Hankkeen tavoitteena on kehittää potilaslähtöisiä, lasten ja perheiden tarpeita vastaavia hoitotyön käytäntöjä. Tavoite on kehittää yhtenäisiä hoitome-netelmiä, saumatonta hoitoketjun toimintaa ja lapsen ja perheen voimavaroja vahvistavaa verkostomaista työskentelyä. Etsimme teoriatietoa päänsäryn syistä sekä elämäntapojen merkityksestä nuoren ohjauk-sessa ja hoidon suunnittelussa. Lisätietoa haimme tapaamalla työelämän yhteistyökump-paneita saadaksemme käytännönläheistä tietoa. Kysyimme palautetta työelämän asian-tuntijoilta työmme loppuvaiheessa. Näitä tietoja yhdistämällä pyrimme luomaan käytettävissä olevan ehdotuksen toiminnan parantamiseksi. Opinnäytetyömme käsittelee 12- 16 -vuotiaiden nuorten elämäntapaohjausta. Tarkoituksenamme oli selvittää, millä keinoilla nuori saadaan sitoutumaan päänsäryn hoitoon omilla elämäntavoilla. Elämäntavoilla on suuri merkitys migreenin hoidossa sekä kohtausten estämisessä. Työmme johtopäätös on, että ensitapaamista tulee korostaa nuoren hoidon aloituksessa. Tapaamisessa tulee antaa ajantasaista, asianmukaista sekä oikea-aikaista tietoa. Vanhempien mukanaolon tärkeys korostuu perheen elämäntapoja selvitettäessä sekä nuoren tukemisessa mahdollisessa elämäntapamuutoksessa. Nuoren hoitoon sitoutumista tuetaan hyvällä elämäntapaohjauksella kohtaamalla nuori yksilöllisesti sekä ottamalla huomioon hänen senhetkiset voimavaransa. Jälkiseurannan merkitys korostuu hoitoon sitoutumista selvitettäessä.The purpose of this thesis was to clarify the ways, how a young person could be guided for treatment of headache by changing his/her own way of life. The aim of our work was to strengthen of expertise and co-operation of young people, of their families and of the per-sons who work in care. Our thesis belongs to the project “The Future of Specialised Health Care for Children” in years 2011 – 2014”. It was done in cooperation with Metropolia university of applied sci-ences and HYKS Women's and children's diseases business unit. The objective of this project is to develop such methods of treatment that are patient-oriented and that corre-spond to the needs of the children and their families. The goal is to develop integrated methods of treatment, a seamless chain of care activities and the network-like work that strengthens the resources of a child and his/her family. We looked for theoretical information about the reasons of headache. We also investigated how the way of life should be noticed in the guidance of a young and in the planning of the treatment. More information we got by interviewing the cooperation partners in order to get practical information. We also asked feedback of some experts of work life. By combining this information we aimed to create a proposal that could be used in practice. Our thesis handles lifestyle guidance of young people in age 12 – 16 years. Our purpose was to find out the means by which a young can be committed to treatment of headaches, by changing his/her own way of life. Way of life has a big effect in the treatment of mi-graine and in the preventing of the attacks. The conclusion of our work is that it is important to emphasize the first appointment, when the treatment of a young is started. In the appointment should be given actual, appropriate and timely information. Also it is important that parents are available in the appointments, when the way of life of the family is being clarified. Parents are also needed in supporting of young if his/her lifestyle should change. Young’s commitment to the treatment is sup-ported by good control of life by meeting a young individually and taking into account his current resources in his life. Further follow-ups have a high meaning when the commitment to the treatment is being investigated