Orexin Signalling in Brown Adipose Tissue Precursor Cells

Orexins are neuropeptides for which signalling effects have been noted in multiple functions of the central nervous system, and also potentially in the periphery of the body. Orexin receptors couple to a number of different proteins eliciting cellular responses such as activation/inhibition of ion channels, kinase activation, and second messenger generation, and downstream effects such as neuronal excitation, synaptic plasticity, and cell death. The majority of knowledge on orexin signalling has been obtained from recombinant expression systems, thus studies of signalling responses in specific cell or tissue types are desired. The goal of this master’s thesis project was to investigate orexin signalling in two mouse brown adipocyte precursor cell lines (C3H10T1/2 and HIB1b) with native OX1 receptor expression. p38 mitogen-activated protein kinase (MAPK) activation was assessed by western blot analysis, while phospholipase D (PLD) activity, arachidonic acid (AA) and 2-arachidonoylglycerol (2-AG) release, and adenylyl cyclase activity were assessed by radioactive prelabelling, extraction and separation of the molecular species, and quantification of radioactivity. p38 was activated by orexin in C3H10T1/2 cells, but not HIB1b cells; PLD, AA, and 2-AG showed no response to orexin; and adenylyl cyclase appears to be both stimulated and inhibited by orexin, at different concentrations. The results indicate that orexin signalling in these cell lines significantly differs from recombinantly expressed orexin receptors. This raises interesting questions regarding the variability of responses to orexin in different tissue types

The cysteine-rich receptor-like kinase CRK2 during stress responses in Arabidopsis thaliana

In order to maintain health, growth, and productivity, plants must be able to adapt to increasingly variable environmental conditions. Plants are continuously flooded with information from their surrounding environment, which must be sensed, incorporated, and responded to accordingly. Much of the communication between plant cells and the extracellular environment is carried out by the receptor-like protein kinases (RLKs), including the cysteine-rich receptor-like kinase (CRK) subfamily. Despite the large size of the CRK gene family, their physiological roles and functions on a biochemical and cellular level remain largely uncharacterized. We performed large scale phenotyping of a crk T-DNA mutant collection in Arabidopsis thaliana (Arabidopsis), which suggested roles for the CRKs in several developmental processes, as well as during abiotic and biotic stress responses. CRK2 emerged as an important CRK, with several strong loss-of-function phenotypes and a notable phylogenetic position. We established that CRK2 enhances salt tolerance through the regulation of callose synthase 1 (CALS1) dependent callose deposition at plasmodesmata. This revealed a previously uncharacterized role for callose deposition in response to high salinity. We showed that this callose deposition has an effect on plasmodesmal permeability, and therefore a potential impact on intercellular signalling. Additionally, CRK2 was found to regulate the formation of an unknown vesicle type during salt stress, which could possibly be involved in cell-to-cell signalling as well. We have described how CRK2 regulates ROS production during immunity by regulation of RBOHD via C-terminal phosphorylation. We observed highly specific changes in the subcellular localization of CRK2 in response to various stress treatments, and demonstrated that these localization patterns are critical for protein function and interactions. The subcellular localization and many of the cellular functions of CRK2 were dependent on phospholipase D alpha 1 (PLDɑ1) activity, and PLDɑ1 was consistently identified as one of the top proteins to interact with CRK2. Thus, we propose that CRK2 is a fundamental CRK, which acts in connection with PLDɑ1 to regulate several cellular processes during the response to environmental stimuli.Voidakseen pitää yllä terveyttä, kasvua ja tuottavuutta, kasvien täytyy kyetä sopeutumaan lisääntyvästi muuttuviin ympäristöolosuhteisiin. Kasvit altistuvat jatkuvasti niiden ympäristöstä tulevalle informaatiolle, joka täytyy aistia ja välittää soluihin sopivan vasteen aikaansaamiseksi. Suuri määrä kommunikaatiosta kasvisolujen ja solun ulkoisen ympäristön välillä on reseptorinkaltaisten proteiinikinaasien (RLK:en) suorittamaa. Kysteiinirikkaat reseptorinkaltaiset kinaasit (CRK:t) muodostavat yhden RLK:en alaryhmän. Huolimatta CRK-geeniperheen suuresta koosta, niiden fysiologiset roolit ja toiminnot sekä biokemiallisella- että solutasolla ovat suurelta osin karakterisoimatta. Suoritimme laajamittaisen fenotyypityksen crk T-DNA -lituruohomutanttikokoelmalla ja tämä antoi viitteitä siitä, että CRK:illa on rooleja lukuisissa kehittymisprosesseissa sekä abioottisissa että bioottisissa stressivasteissa. Kysteiinirikas reseptorinkaltainen kinaasi 2 (CRK2) osoittautui tärkeäksi CRK:ksi; sillä on useita vahvoja puuttuvan toiminnan fenotyyppejä ja huomattava fylogeneettinen sijainti. Osoitimme, että CRK2 parantaa suolatoleranssia sääntelemällä kalloosisyntaasi 1:stä (CALS1:stä) riippuvaista kalloosikertymää plasmodesmilla. Tämä paljasti aiemmin karakterisoimattoman roolin kalloosikertymälle vasteessa korkeaan suolapitoisuuteen. Osoitimme, että tämä kalloosikertymä vaikuttaa plasmodesmin läpäisevyyteen ja siten mahdollisesti myös solujen väliseen viestintään. Lisäksi, suolastressin aikana CRK2:n huomattiin sääntelevän tuntemattoman vesikkelityypin muodostumista, tällä voi mahdollisesti olla osuus myös solusta soluun tapahtuvaan viestintään. Olemme kuvailleet, kuinka CRK2 sääntelee reaktiivisten happilajien tuottoa immuniteetin aikana sääntelemällä RBOHD:tä C-terminaalisen fosforylaation kautta. Vasteena erilaisiin stressikäsittelyihin havaitsimme erittäin spesifisiä muutoksia CRK2:n lokalisaatiossa soluissa ja osoitimme näiden lokalisaatiokuvioiden olevan kriittisiä proteiinin toiminnalle ja vuorovaikutuksille. CRK2:n lokalisaatio soluissa sekä sen monet solutoiminnot olivat riippuvaisia fosfolipaasi D alpha 1:n (PLDɑ1:n) aktiivisuudesta ja PLDɑ1 tunnistettiin johdonmukaisesti yhdeksi yleisimmäksi CRK2:n kanssa vuorovaikutuksessa olevaksi proteiiniksi. Täten esitämme CRK2:n olevan keskeinen CRK, joka toimii yhteydessä PLDɑ1:n kanssa säännelläkseen useita solun prosesseja vasteessa ympäristön ärsykkeisiin

Bound by Fate : The Role of Reactive Oxygen Species in Receptor-Like Kinase Signaling

In plants, receptor-like kinases (RLKs) and extracellular reactive oxygen species (ROS) contribute to the communication between the environment and the interior of the cell. Apoplastic ROS production is a frequent result of RLK signaling in a multitude of cellular processes; thus, by their nature, these two signaling components are inherently linked. However, it is as yet unclear how ROS signaling downstream of receptor activation is executed. In this review, we provide a broad view of the intricate connections between RLKs and ROS signaling and describe the regulatory events that control and coordinate extracellular ROS production. We propose that concurrent initiation of ROS-dependent and -independent signaling linked to RLKs might be a critical element in establishing cellular responses. Furthermore, we discuss the possible ROS sensing mechanisms in the context of the biochemical environment in the apoplast. We suggest that RLK-dependent modulation of apoplastic and intracellular conditions facilitates ROS perception and signaling. Based on data from plant and animal models, we argue that specific RLKs could be components of the ROS sensing machinery or ROS sensors. The importance of the crosstalk between RLK and ROS signaling is discussed in the context of stomatal immunity. Finally, we highlight challenges in the understanding of these signaling processes and provide perspectives for future research.Peer reviewe

CRK2 enhances salt tolerance in Arabidopsis thaliana by regulating endocytosis and callose deposition in connection with PLDα1

Peer reviewe

The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity

Perception of biotic and abiotic stresses often leads to stomatal closure in plants 1,2. Rapid influx of calcium ions (Ca 2+) across the plasma membrane has an important role in this response, but the identity of the Ca 2+ channels involved has remained elusive 3,4. Here we report that the Arabidopsis thaliana Ca 2+-permeable channel OSCA1.3 controls stomatal closure during immune signalling. OSCA1.3 is rapidly phosphorylated upon perception of pathogen-associated molecular patterns (PAMPs). Biochemical and quantitative phosphoproteomics analyses reveal that the immune receptor-associated cytosolic kinase BIK1 interacts with and phosphorylates the N-terminal cytosolic loop of OSCA1.3 within minutes of treatment with the peptidic PAMP flg22, which is derived from bacterial flagellin. Genetic and electrophysiological data reveal that OSCA1.3 is permeable to Ca 2+, and that BIK1-mediated phosphorylation on its N terminus increases this channel activity. Notably, OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immune signalling, and OSCA1.3 does not regulate stomatal closure upon perception of abscisic acid—a plant hormone associated with abiotic stresses. This study thus identifies a plant Ca 2+ channel and its activation mechanisms underlying stomatal closure during immune signalling, and suggests specificity in Ca 2+ influx mechanisms in response to different stresses

Identification and Characterization of a Leucine-Rich Repeat Kinase 2 (LRRK2) Consensus Phosphorylation Motif

Mutations in LRRK2 (leucine-rich repeat kinase 2) have been identified as major genetic determinants of Parkinson's disease (PD). The most prevalent mutation, G2019S, increases LRRK2's kinase activity, therefore understanding the sites and substrates that LRRK2 phosphorylates is critical to understanding its role in disease aetiology. Since the physiological substrates of this kinase are unknown, we set out to reveal potential targets of LRRK2 G2019S by identifying its favored phosphorylation motif. A non-biased screen of an oriented peptide library elucidated F/Y-x-T-x-R/K as the core dependent substrate sequence. Bioinformatic analysis of the consensus phosphorylation motif identified several novel candidate substrates that potentially function in neuronal pathophysiology. Peptides corresponding to the most PD relevant proteins were efficiently phosphorylated by LRRK2 in vitro. Interestingly, the phosphomotif was also identified within LRRK2 itself. Autophosphorylation was detected by mass spectrometry and biochemical means at the only F-x-T-x-R site (Thr 1410) within LRRK2. The relevance of this site was assessed by measuring effects of mutations on autophosphorylation, kinase activity, GTP binding, GTP hydrolysis, and LRRK2 multimerization. These studies indicate that modification of Thr1410 subtly regulates GTP hydrolysis by LRRK2, but with minimal effects on other parameters measured. Together the identification of LRRK2's phosphorylation consensus motif, and the functional consequences of its phosphorylation, provide insights into downstream LRRK2-signaling pathways

Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors

Reactive oxygen species (ROS)-dependent signaling pathways from chloroplasts and mitochondria merge at the nuclear protein RADICAL INDUCED CELL DEATH 1 (RCD1). RCD1 interacts in vivo and suppresses the activity of the transcription factors ANAC013 and ANAC017, which mediate a ROS-related retrograde signal originating from mitochondrial complex III. Inactivation of RCD1 leads to increased expression of mitochondrial dysfunction stimulon (MDS) genes regulated by ANAC013 and ANAC017. Accumulating MDS gene products, including alternative oxidases (AOXs), affect redox status of the chloroplasts, leading to changes in chloroplast ROS processing and increased protection of photosynthetic apparatus. ROS alter the abundance, thiol redox state and oligomerization of the RCD1 protein in vivo, providing feedback control on its function. RCD1-dependent regulation is linked to chloroplast signaling by 3'-phosphoadenosine 5'-phosphate (PAP). Thus, RCD1 integrates organellar signaling from chloroplasts and mitochondria to establish transcriptional control over the metabolic processes in both organelles

Large-scale phenomics identifies primary and fine-tuning roles for CRKs in responses related to oxidative stress

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.Peer reviewe

Genetic diversity fuels gene discovery for tobacco and alcohol use

Tobacco and alcohol use are heritable behaviours associated with 15% and 5.3% of worldwide deaths, respectively, due largely to broad increased risk for disease and injury(1-4). These substances are used across the globe, yet genome-wide association studies have focused largely on individuals of European ancestries(5). Here we leveraged global genetic diversity across 3.4 million individuals from four major clines of global ancestry (approximately 21% non-European) to power the discovery and fine-mapping of genomic loci associated with tobacco and alcohol use, to inform function of these loci via ancestry-aware transcriptome-wide association studies, and to evaluate the genetic architecture and predictive power of polygenic risk within and across populations. We found that increases in sample size and genetic diversity improved locus identification and fine-mapping resolution, and that a large majority of the 3,823 associated variants (from 2,143 loci) showed consistent effect sizes across ancestry dimensions. However, polygenic risk scores developed in one ancestry performed poorly in others, highlighting the continued need to increase sample sizes of diverse ancestries to realize any potential benefit of polygenic prediction.Peer reviewe

Association of genetic variation with systolic and diastolic blood pressure among African Americans: the Candidate Gene Association Resource study

The prevalence of hypertension in African Americans (AAs) is higher than in other US groups; yet, few have performed genome-wide association studies (GWASs) in AA. Among people of European descent, GWASs have identified genetic variants at 13 loci that are associated with blood pressure. It is unknown if these variants confer susceptibility in people of African ancestry. Here, we examined genome-wide and candidate gene associations with systolic blood pressure (SBP) and diastolic blood pressure (DBP) using the Candidate Gene Association Resource (CARe) consortium consisting of 8591 AAs. Genotypes included genome-wide single-nucleotide polymorphism (SNP) data utilizing the Affymetrix 6.0 array with imputation to 2.5 million HapMap SNPs and candidate gene SNP data utilizing a 50K cardiovascular gene-centric array (ITMAT-Broad-CARe [IBC] array). For Affymetrix data, the strongest signal for DBP was rs10474346 (P= 3.6 × 10−8) located near GPR98 and ARRDC3. For SBP, the strongest signal was rs2258119 in C21orf91 (P= 4.7 × 10−8). The top IBC association for SBP was rs2012318 (P= 6.4 × 10−6) near SLC25A42 and for DBP was rs2523586 (P= 1.3 × 10−6) near HLA-B. None of the top variants replicated in additional AA (n = 11 882) or European-American (n = 69 899) cohorts. We replicated previously reported European-American blood pressure SNPs in our AA samples (SH2B3, P= 0.009; TBX3-TBX5, P= 0.03; and CSK-ULK3, P= 0.0004). These genetic loci represent the best evidence of genetic influences on SBP and DBP in AAs to date. More broadly, this work supports that notion that blood pressure among AAs is a trait with genetic underpinnings but also with significant complexit