Inverse Agonistic Action of 3-Iodothyronamine at the Human Trace Amine-Associated Receptor 5

ObjectiveApplication of 3-iodothyronamine (3-T₁AM) results in decreased body temperature and body weight in rodents. The trace amine-associated receptor (TAAR) 1, a family A G protein-coupled receptor, is a target of 3-T₁AM. However, 3-T₁AM effects still persist in mTaar1 knockout mice, which suggest so far unknown further receptor targets that are of physiological relevance. TAAR5 is a highly conserved TAAR subtype among mammals and we here tested TAAR5 as a potential 3-T₁AM target. First, we investigated mouse Taar5 (mTaar5) expression in several brain regions of the mouse in comparison to mTaar1. Secondly, to unravel the full spectrum of signaling capacities, we examined the distinct G_s-, G_i/o-, G_12/13-, G_q/11- and MAP kinase-mediated signaling pathways of mouse and human TAAR5 under ligand-independent conditions and after application of 3-T₁AM. We found overlapping localization of mTaar1 and mTaar5 in the amygdala and ventromedial hypothalamus of the mouse brain. Second, the murine and human TAAR5 (hTAAR5) display significant basal activity in the G_q/11 pathway but show differences in the basal activity in G_s and MAP kinase signaling. In contrast to mTaar5, 3-T₁AM application at hTAAR5 resulted in significant reduction in basal IP₃ formation and MAP kinase signaling. In conclusion, our data suggest that the human TAAR5 is a target for 3-T₁AM, exhibiting inhibitory effects on IP₃ formation and MAP kinase signaling pathways, but does not mediate G_s signaling effects as observed for TAAR1. This study also indicates differences between TAAR5 orthologs with respect to their signaling profile. In consequence, 3-T₁AM-mediated effects may differ between rodents and humans.     

Identification of Regulatory and Cargo Proteins of Endosomal and Secretory Pathways in Arabidopsis thaliana by Proteomic Dissection

The cell''s endomembranes comprise an intricate, highly dynamic and well-organized system. In plants, the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi network (TGN), early endosomes (EE), secretory vesicles, late endosomes (LE), multivesicular bodies (MVB), and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNAREs, and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA, and the recently defined TPLATE complex. The subcellular localization of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic dataset to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localization of proteins and identify the components of regulatory complexes and provides a useful tool for the identification of new protein markers of the endomembrane system.Membrane compartmentalization is an essential mechanism for eukaryotic life, by which cells separate and control biological processes. Plant growth, development, and adaptation to biotic and abiotic stress all rely on the highly dynamic endomembrane system, yet we know comparatively little about the proteins regulating these dynamic trafficking events. The plasma membrane (PM) provides the main interface between the cell and its environment, mediating the transfer of material to and from the cell and is a primary site for perception of external signals. Transmembrane proteins are synthesized in the endoplasmic reticulum (ER) and trafficked to the PM via the Golgi, although there are other secretory routes for soluble cargo (discussed in (1–4)). Post-Golgi trafficking is the main route by which newly synthesized transmembrane proteins and cell wall glycans are delivered to the PM. In plants, secretory and endocytic traffic converge at the trans-Golgi network (TGN), which also functions as an early endosome (EE). Multivesicular bodies (MVBs) are the other main endosomal compartment in plants and serve as prevacuolar compartments (PVCs) or late endosomes (LE) destined for vacuolar degradation (reviewed (1, 5, 6)).Recycling and sorting of plasma membrane proteins is essential for generating the polar localization of auxin efflux transporters (discussed in (7)), formation of the cell plate during cell division (8–11), and in defense such as localized deposition of papilla reviewed in (12, 13). Furthermore, the subcellular localization of transporters and receptors is dynamically regulated. For example, the boron transporter (BOR1) exhibits polar localization and is internalized and degraded under conditions of high boron to reduce toxicity (14, 15). Similarly the receptor-like kinases (RLKs) flagellin-sensing 2 (FLS2) and brassinosteroid insensitive 1 (BRI1), important transmembrane receptors in antibacterial immunity and plant development, respectively, are constitutively endocytosed and recycled to the PM (16–18). Both receptors and transporters are also cargoes of the LE/MVB trafficking route (16) and are probably sorted to the vacuole for degradation (19, 20). Importantly, FLS2 trafficking via the recycling endocytic or the late endocytic route depends on its activation status; inactive receptors are recycled while ligand-activated receptors are sorted to the late endosomal pathway (16). Similarly, the polar sorting of auxin efflux transporters depends on their phosphorylation status (21). These observations illustrate that membrane compartmentalization underpins important aspects of plant cell biology and has initiated a quest toward a better understanding of the endomembrane compartments and the routes and mechanisms by which cargo is trafficked and sorted within the cell.Membrane trafficking within the cell requires complex machinery consisting of a plethora of coat and adaptor proteins, small GTPases, targeting, tethering, and scission factors (reviewed in (22, 23)). Homologues of some animal and yeast and endomembrane regulators have been identified in plants, but the localization and function of many of these remain to be characterized. For example, members of the RAB GTPase family have been shown to have markedly different roles and localizations in plants compared with their animal and yeast homologs (24). Therefore, acquiring localization data for tethering complexes and other regulators in plant systems is essential. In Arabidopsis thaliana, some of these proteins have been developed as useful probes to visualize the different endomembrane compartments by fusion with fluorescent reporters (9, 25–27). These include regulators of trafficking events such as RAB GTPases that are molecular switches responsible for the assembly of tethering and docking complexes and compartment identity. RAB proteins are widely used markers of endomembrane compartments, for example RABD2a/ARA5 labels the Golgi and TGN/EE as well as post-Golgi vesicles (4, 24, 26, 28), RABF2b/ARA7 localizes to TGN/EE and LE (25), RABF1/ARA6 is a marker of the LE/MVB vesicles (25, 29), and RABG3f localizes to MVBs and the tonoplast (26, 30).Fluorescent-tagged marker lines for the live-cell imaging of plant cells have been invaluable in defining the location of proteins within and between organelles and endomembrane compartments (26). However, microscopic investigation of membrane trafficking is limited by throughput, as only few proteins can be studied simultaneously. A powerful approach to large-scale identification of proteins in endomembrane compartments is through subcellular fractionation based on physical properties to directly isolate or enrich for the subcellular compartment of interest. Subcellular fractionation-based proteomics have been successfully used to decipher the steady state and cargo proteomes of, including but not limited to, the ER, the vacuole, PM, mitochondria and chloroplasts, and smaller vesicle-like compartments such as peroxisomes and Golgi (31–41). However, the smaller, transitory vesicles of the secretory and endocytic pathways have proved challenging to purify for reliable proteomic analysis. To overcome this, affinity purification of vesicles was established in animal cells (42, 43) and recently successfully applied in plants in combination with subcellular fractionation. Affinity purification and mass spectrometry (MS) of syntaxin of plants 61 (SYP61)-positive TGN/EE compartments identified 145 proteins specifically enriched in (44), while affinity isolation of VHA-a1-GFP (vacuolar H⁺ ATPase A1) identified 105 proteins associated with the TGN/EE (45). The VHA-A1 affinity purification data were then further refined using density gradient centrifugation to differentiate cargo and steady-state proteins (45).We have further explored affinity purification of fluorescent-tagged markers localizing to defined compartments to identify proteins associated with trafficking. Our motivation was to dissect the trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with small GTPases in the RAB family. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for Golgi/TGN/EE/secretory vesicles, LE/MVB compartments, LE/MVB compartments and LE/MVB/tonoplast, respectively. Additionally, we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles (CCVs) and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast (26, 27, 29, 46, 47) as comparisons. Our objective was to identify transient cargo proteins, tethers, and docking factors associated with dynamic subdomains of the endomembrane system, to supplement better-characterized “steady-state” components, and to identify components of recycling and vacuolar trafficking pathways.     

Activation of Type I and III Interferon Response by Mitochondrial and Peroxisomal MAVS and Inhibition by Hepatitis C Virus

Sensing viruses by pattern recognition receptors (PRR) triggers the innate immune system of the host cell and activates immune signaling cascades such as the RIG-I/IRF3 pathway. Mitochondrial antiviral-signaling protein (MAVS, also known as IPS-1, Cardif, and VISA) is the crucial adaptor protein of this pathway localized on mitochondria, peroxisomes and mitochondria-associated membranes of the endoplasmic reticulum. Activation of MAVS leads to the production of type I and type III interferons (IFN) as well as IFN stimulated genes (ISGs). To refine the role of MAVS subcellular localization for the induction of type I and III IFN responses in hepatocytes and its counteraction by the hepatitis C virus (HCV), we generated various functional and genetic knock-out cell systems that were reconstituted to express mitochondrial (mito) or peroxisomal (pex) MAVS, exclusively. Upon infection with diverse RNA viruses we found that cells exclusively expressing pexMAVS mounted sustained expression of type I and III IFNs to levels comparable to cells exclusively expressing mitoMAVS. To determine whether viral counteraction of MAVS is affected by its subcellular localization we employed infection of cells with HCV, a major causative agent of chronic liver disease with a high propensity to establish persistence. This virus efficiently cleaves MAVS via a viral protease residing in its nonstructural protein 3 (NS3) and this strategy is thought to contribute to the high persistence of this virus. We found that both mito- and pexMAVS were efficiently cleaved by NS3 and this cleavage was required to suppress activation of the IFN response. Taken together, our findings indicate comparable activation of the IFN response by pex- and mitoMAVS in hepatocytes and efficient counteraction of both MAVS species by the HCV NS3 protease.     

Epidemiology of Pelvic Fractures in Germany: Considerably High Incidence Rates among Older People

Epidemiological data about pelvic fractures are limited. Until today, most studies only analyzed inpatient data. The purpose of this study was to estimate incidence rates of pelvic fractures in the German population aged 60 years or older, based on outpatient and inpatient data. We conducted a retrospective population-based observational study based on routine data from a large health insurance company in Germany. Age and sex-specific incidence rates of first fractures between 2008 and 2011 were calculated. We also standardized incidence rates with respect to age and sex in the German population. Multiple Poisson regression models were used to evaluate the association between the risk of first pelvic fracture as outcome and sex, age, calendar year and region as independent variables. The total number of patients with a first pelvic fracture corresponded to 8,041 and during the study period 5,978 insured persons needed inpatient treatment. Overall, the standardized incidence rate of all first pelvic fractures was 22.4 [95% CI 22.0–22.9] per 10,000 person-years, and the standardized incidence rate of inpatient treated fractures 16.5 [16.1–16.9]. Our adjusted regression analysis confirmed a significant sex (RR 2.38 [2.23–2.55], p < 0.001, men as reference) and age effect (higher risk with increasing age, p < 0.001) on first fracture risk. We found a slight association between calendar year (higher risk in later years compared to 2008, p = 0.0162) and first fracture risk and a further significant association with region (RR 0.92 [0.87–0.98], p = 0.006, Westfalen-Lippe as reference). The observed incidences are considerably higher than incidences described in the international literature, even if only inpatient treated pelvic fractures are regarded. Besides which, non-inclusion of outpatient data means that a relevant proportion of pelvic fractures are not taken into account. Prevention of low energy trauma among older people remains an important issue.     

High-Throughput Sequencing—The Key to Rapid Biodiversity Assessment of Marine Metazoa?

The applications of traditional morphological and molecular methods for species identification are greatly restricted by processing speed and on a regional or greater scale are generally considered unfeasible. In this context, high-throughput sequencing, or metagenetics, has been proposed as an efficient tool to document biodiversity. Here we evaluated the effectiveness of 454 pyrosequencing in marine metazoan community analysis using the 18S rDNA: V1-V2 region. Multiplex pyrosequencing of the V1-V2 region was used to analyze two pooled samples of DNA, one comprising 118 and the other 37 morphologically identified species, and one natural sample taken directly from a North Sea zooplankton community. A DNA reference library comprising all species represented in the pooled samples was created by Sanger sequencing, and this was then used to determine the optimal similarity threshold for species delineation. The optimal threshold was found at 99% species similarity, with 85% identification success. Pyrosequencing was able to identify between fewer species: 67% and 78% of the species in the two pooled samples. Also, a large number of sequences for three species that were not included in the pooled samples were amplified by pyrosequencing, suggesting preferential amplification of some genotypes and the sensitivity of this approach to even low levels of contamination. Conversely, metagenetic analysis of the natural zooplankton sample identified many more species (particularly gelatinous zooplankton and meroplankton) than morphological analysis of a formalin-fixed sample from the same sampling site, suggesting an increased level of taxonomic resolution with pyrosequencing. The study demonstrated that, based on the V1-V2 region, 454 sequencing does not provide accurate species differentiation and reliable taxonomic classification, as it is required in most biodiversity monitoring. The analysis of artificially prepared samples indicated that species detection in pyrosequencing datasets is complicated by potential PCR-based biases and that the V1-V2 marker is poorly resolved for some taxa.     

A Zebrafish Drug-Repurposing Screen Reveals sGC-Dependent and sGC-Independent Pro-Inflammatory Activities of Nitric Oxide

Tissue injury and infection trigger innate immune responses. However, dysregulation may result in chronic inflammation and is commonly treated with corticosteroids and non-steroidal anti-inflammatory drugs. Unfortunately, long-term administration of both therapeutic classes can cause unwanted side effects. To identify alternative immune-modulatory compounds we have previously established a novel screening method using zebrafish larvae. Using this method we here present results of an in vivo high-content drug-repurposing screen, identifying 63 potent anti-inflammatory drugs that are in clinical use for other indications. Our approach reveals a novel pro-inflammatory role of nitric oxide. Nitric oxide affects leukocyte recruitment upon peripheral sensory nervous system or epithelial injury in zebrafish larvae both via soluble guanylate cyclase and in a soluble guanylate cyclase -independent manner through protein S-nitrosylation. Together, we show that our screening method can help to identify novel immune-modulatory activities and provide new mechanistic insights into the regulation of inflammatory processes.     

Resonance Raman and FTIR spectroscopic characterization of the closed and open states of channelrhodopsin-1

Channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) is a light-activated cation channel, which is a promising optogenetic tool. We show by resonance Raman spectroscopy and retinal extraction followed by high pressure liquid chromatography (HPLC) that the isomeric ratio of all-trans to 13-cis of solubilized channelrhodopsin-1 is with 70:30 identical to channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Critical frequency shifts in the retinal vibrations are identified in the Raman spectrum upon transition to the open (conductive P₂³⁸⁰) state. Fourier transform infrared spectroscopy (FTIR) spectra indicate different structures of the open states in the two channelrhodopsins as reflected by the amide I bands and the protonation pattern of acidic amino acids.     

Peroxisomal membrane channel Pxmp2 in the mammary fat pad is essential for stromal lipid homeostasis and for development of mammary gland epithelium in mice

To understand the functional role of the peroxisomal membrane channel Pxmp2, mice with a targeted disruption of the Pxmp2 gene were generated. These mice were viable, grew and bred normally. However, Pxmp2^−/− female mice were unable to nurse their pups. Lactating mammary gland epithelium displayed secretory lipid droplets and milk proteins, but the size of the ductal system was greatly reduced. Examination of mammary gland development revealed that retarded mammary ductal outgrowth was due to reduced proliferation of epithelial cells during puberty. Transplantation experiments established the Pxmp2^−/− mammary stroma as a tissue responsible for suppression of epithelial growth. Morphological and biochemical examination confirmed the presence of peroxisomes in the mammary fat pad adipocytes, and functional Pxmp2 was detected in the stroma of wild-type mammary glands. Deletion of Pxmp2 led to an elevation in the expression of peroxisomal proteins in the mammary fat pad but not in liver or kidney of transgenic mice. Lipidomics of Pxmp2^−/−mammary fat pad showed a decrease in the content of myristic acid (C₁₄), a principal substrate for protein myristoylation and a potential peroxisomal β-oxidation product. Analysis of complex lipids revealed a reduced concentration of a variety of diacylglycerols and phospholipids containing mostly polyunsaturated fatty acids that may be caused by activation of lipid peroxidation. However, an antioxidant-containing diet did not stimulate mammary epithelial proliferation in Pxmp2^−/− mice.     

Systematic in vivo RNAi analysis identifies IAPs as NEDD8-E3 ligases

The intimate relationship between mediators of the ubiquitin (Ub)-signaling system and human diseases has sparked profound interest in how Ub influences cell death and survival. While the consequence of Ub attachment is intensely studied, little is known with regards to the effects of other Ub-like proteins (UBLs), and deconjugating enzymes that remove the Ub or UBL adduct. Systematic in vivo RNAi analysis identified three NEDD8-specific isopeptidases that, when knocked down, suppress apoptosis. Consistent with the notion that attachment of NEDD8 prevents cell death, genetic ablation of deneddylase 1 (DEN1) suppresses apoptosis. Unexpectedly, we find that Drosophila and human inhibitor of apoptosis (IAP) proteins can function as E3 ligases of the NEDD8 conjugation pathway, targeting effector caspases for neddylation and inactivation. Finally, we demonstrate that DEN1 reverses this effect by removing the NEDD8 modification. Altogether, our findings indicate that IAPs not only modulate cellular processes via ubiquitylation but also through attachment of NEDD8, thereby extending the complexity of IAP-mediated signaling.