Nitrogen release from five organic fertilizers commonly used in greenhouse organic horticulture with contrasting effects on bacterial communities

Organic fertilization in greenhouses relies on organic fertilizers with low carbon/nitrogen ratio. Nitrogen (N) availability thus depends on an efficient mineralization driven by microbial communities. However, data on the mineralization rate of such fertilizers are scarce, and their improper use can lead to either N deficiency, or N losses to the environment. Consequently, better knowledge of N availability following organic fertilization is crucial for the development of sustainable greenhouse organic horticulture. We investigated the effect of pelleted poultry manure (PM) and blood (BM), feather (FM), alfalfa (AM), and shrimp (SM) meals on N availability and bacterial communities in a peat-based organic growing medium and a mineral soil. Nitrogen and carbon (C) pools were measured periodically over a 52 wk incubation experiment. Bacterial communities were characterized by sequencing the regions V6–V8 of the 16S rRNA gene on the high-throughput Illumina MiSeq platform, 4 wk after the start of the incubation. Nitrogen mineralization plateaued for the mineral soil and the peat substrate at, respectively, 41% and 63% of applied N for PM, 56%–93% (BM), 54%–81% (FM), 34%–53% (AM), and 57%–73% (SM). Organic fertilizers supported markedly contrasted bacterial communities, closely linked to soil biochemical properties, especially mineral N, pH, and soluble C. Alfalfa meal promoted the highest Shannon diversity index in the mineral soil, whereas SM and PM increased it in the peat-based growing medium. Our results quantified the mineralization and highlighted the impact on bacterial communities of commonly used organic N fertilizers in conditions relevant to organic greenhouse horticulture.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Ligands act as pharmacological chaperones and increase the efficiency of δ opioid receptor maturation

The endoplasmic reticulum (ER) is recognized as an important site for regulating cell surface expression of membrane proteins. We recently reported that only a fraction of newly synthesized δ opioid receptors could leave the ER and reach the cell surface, the rest being degraded by proteasomes. Here, we demonstrate that membrane-permeable opioid ligands facilitate maturation and ER export of the receptor, thus acting as pharmacological chaperones. We propose that these ligands stabilize the newly synthesized receptor in the native or intermediate state of its folding pathway, possibly by inducing stabilizing conformational constrains within the hydrophobic core of the protein. The receptor precursors that are retained in the ER thus represent fully competent folding intermediates that can be targets for pharmacological intervention aimed at regulating receptor expression and cellular responsiveness. The pharmacological chaperone action is independent of the intrinsic signaling efficacy of the ligand, since both agonists and antagonists were found to promote receptor maturation. This novel property of G protein-coupled receptor ligands may have important implications when considering their effects on cellular responsiveness during therapeutic treatments

Increased production of active human \u3b2\u2082-adrenergic/G\u3b1s fusion receptor in Sf-9 cells using nutrient limiting conditions

Using the baculovirus/insect-cell expression vector system, we succeeded in obtaining a high yield of active human \u3b2\u2082-adrenergic/G\u3b1s fusion protein. This was achieved following high cell density production under nutrient-limiting conditions using a very low multiplicity of infection (MOI). This approach was found to significantly reduce inactive protein accumulation that occurred when production was done using conventional high MOI procedures. The maximum specific and volumetric yields of active receptor using this strategy increased by factors of two- and sixfold, respectively. Our results suggest that the increase in the ratio of active/total protein produced results from production under nutrient limitation. Since low multiplicity of infection offers many advantages for large-scale applications, we suggest that this simple production method should be considered when optimizing expression of G-protein-coupled receptors and other complex proteins.NRC publication: Ye

Export from the endoplasmic reticulum represents the limiting step in the maturation and cell surface expression of the human δ opioid receptor

Abstract Synthesis and maturation of G protein-coupled receptors are complex events that require an intricate combination of processes that include protein folding, post-translational modifications, and transport through distinct cellular compartments. Relatively little is known about the nature and kinetics of specific steps involved in these processes. Here, the human δ opioid receptor expressed in human embryonic kidney 293S cells is used as a model to delineate these steps and to establish the kinetics of receptor synthesis, glycosylation, and transport. We found that the receptor is synthesized as a core-glycosylatedM r 45,000 precursor that is converted to the fully mature M r 55,000 receptor with a half-time of about 120 min. In addition to trimming and processing of two N-linked oligosaccharides, maturation involves addition of O-glycans containing N-acetylgalactosamine, galactose, and sialic acid. In contrast to N-glycosylation, which is initiated co-translationally and is completed when the protein reaches the trans-Golgi network, O-glycosylation was found to occur only after the receptor exits from the endoplasmic reticulum (ER) and was terminated as early as thetrans-Golgi cisternae. Once the carbohydrates are fully processed and the receptor reaches the trans-Golgi network, it is transported to the cell surface in about 10 min. The exit from the ER was found to be the limiting step in overall processing of the receptor. This indicates that early events in the folding of the receptor are probably rate-limiting and that receptor folding intermediates are retained in the ER until they can adopt the correct conformation. The overall low efficiency of receptor maturation, less than 50% of the precursor being processed to the fully glycosylated protein, further suggests that only a fraction of the synthesized receptors attain properly folded conformation that allows exit from the ER. This indicates that folding and ER export are key events in control of receptor cell surface expression. Whether or not the low efficiency of the ER export is a general feature among G protein-coupled receptors remains to be investigated

Newly synthesized human δ opioid receptors retained in the endoplasmic reticulum are retrotranslocated to the cytosol, deglycosylated, ubiquitinated, and degraded by the proteasome

Abstract We have previously shown that only a fraction of the newly synthesized human δ opioid receptors is able to leave the endoplasmic reticulum (ER) and reach the cell surface (Petäjä-Repo, U. E, Hogue, M., Laperrière, A., Walker, P., and Bouvier, M. (2000) J. Biol. Chem. 275, 13727–13736). In the present study, we investigated the fate of those receptors that are retained intracellularly. Pulse-chase experiments revealed that the disappearance of the receptor precursor form (M r 45,000) and of two smaller species (Mr 42,000 and 39,000) is inhibited by the proteasome blocker, lactacystin. The treatment also promoted accumulation of the mature receptor form (Mr 55,000), indicating that the ER quality control actively routes a significant proportion of rescuable receptors for proteasome degradation. In addition, degradation intermediates that included full-length deglycosylated (Mr 39,000) and ubiquitinated forms of the receptor were found to accumulate in the cytosol upon inhibition of proteasome function. Finally, coimmunoprecipitation experiments with the β-subunit of the Sec61 translocon complex revealed that the receptor precursor and its deglycosylated degradation intermediates interact with the translocon. Taken together, these results support a model in which misfolded or incompletely folded receptors are transported to the cytoplasmic side of the ER membrane via the Sec61 translocon, deglycosylated and conjugated with ubiquitin prior to degradation by the cytoplasmic 26 S proteasomes

N-glycan-dependent and -independent quality control of human δ opioid receptor N-terminal variants

Abstract Quality control (QC) in the endoplasmic reticulum (ER) scrutinizes newly synthesized proteins and directs them either to ER export or ER-associated degradation (ERAD). Here, we demonstrate that the human δ-opioid receptor (hδOR) is subjected to ERQC in both N-glycan-dependent and -independent manners. This was shown by investigating the biosynthesis and trafficking of wild-type and non-N-glycosylated F27C variants in metabolic pulse-chase assays coupled with flow cytometry and cell surface biotinylation. Both QC mechanisms distinguished the minute one-amino acid difference between the variants, targeting a large fraction of hδOR-Cys²⁷ to ERAD. However, the N-glycan-independent QC was unable to compensate the N-glycan-dependent pathway, and some incompletely folded non-N-glycosylated hδOR-Cys²⁷ reached the cell surface in conformation incompatible with ligand binding. The turnover of receptors associating with the molecular chaperone calnexin (CNX) was significantly slower for the hδOR-Cys²⁷, pointing to an important role of CNX in the hδOR N-glycan-dependent QC. This was further supported by the fact that inhibiting the co-translational interaction of hδOR-Cys²⁷ precursors with CNX led to their ERAD. Opioid receptor pharmacological chaperones released the CNX-bound receptors to ER export and, furthermore, were able to rescue the Cys²⁷ variant from polyubiquitination and retrotranslocation to the cytosol whether carrying N-glycans or not. Taken together, the hδOR appears to rely primarily on the CNX-mediated N-glycan-dependent QC that has the capacity to assist in folding, whereas the N-glycan-independent mechanism constitutes an alternative, although less accurate, system for directing misfolded/incompletely folded receptors to ERAD, possibly in altered cellular conditions

Ackr3-Venus knock-in mouse lights up brain vasculature.

The atypical chemokine receptor 3, ACKR3, is a G protein-coupled receptor, which does not couple to G proteins but recruits βarrestins. At present, ACKR3 is considered a target for cancer and cardiovascular disorders, but less is known about the potential of ACKR3 as a target for brain disease. Further, mouse lines have been created to identify cells expressing the receptor, but there is no tool to visualize and study the receptor itself under physiological conditions. Here, we engineered a knock-in (KI) mouse expressing a functional ACKR3-Venus fusion protein to directly detect the receptor, particularly in the adult brain. In HEK-293 cells, native and fused receptors showed similar membrane expression, ligand induced trafficking and signaling profiles, indicating that the Venus fusion does not alter receptor signaling. We also found that ACKR3-Venus enables direct real-time monitoring of receptor trafficking using resonance energy transfer. In ACKR3-Venus knock-in mice, we found normal ACKR3 mRNA levels in the brain, suggesting intact gene transcription. We fully mapped receptor expression across 14 peripheral organs and 112 brain areas and found that ACKR3 is primarily localized to the vasculature in these tissues. In the periphery, receptor distribution aligns with previous reports. In the brain there is notable ACKR3 expression in endothelial vascular cells, hippocampal GABAergic interneurons and neuroblast neighboring cells. In conclusion, we have generated Ackr3-Venus knock-in mice with a traceable ACKR3 receptor, which will be a useful tool to the research community for interrogations about ACKR3 biology and related diseases

Distinct subcellular localization for constitutive and agonist-modulated palmitoylation of the human delta opioid receptor

Abstract Protein palmitoylation is a reversible lipid modification that plays important roles for many proteins involved in signal transduction, but relatively little is known about the regulation of this modification and the cellular location where it occurs. We demonstrate that the humanδ opioid receptor is palmitoylated at two distinct cellular locations in human embryonic kidney 293 cells and undergoes dynamic regulation at one of these sites. Although palmitoylation could be readily observed for the mature receptor (Mr 55,000), [3H]palmitate incorporation into the receptor precursor (Mr 45,000) could be detected only following transport blockade with brefeldin A, nocodazole, and monensin, indicating that the modification occurs initially during or shortly after export from the endoplasmic reticulum. Blocking of palmitoylation with 2-bromopalmitate inhibited receptor cell surface expression, indicating that it is needed for efficient intracellular transport. However, cell surface biotinylation experiments showed that receptors can also be palmitoylated once they have reached the plasma membrane. At this location, palmitoylation is regulated in a receptor activation-dependent manner, as was indicated by the opioid agonist-promoted increase in the turnover of receptor-bound palmitate. This agonist-mediated effect did not require receptor-G protein coupling and occurred at the cell surface without the need for internalization or recycling. The activation-dependent modulation of receptor palmitoylation may thus contribute to the regulation of receptor function at the plasma membrane

Biased Signaling of the Mu Opioid Receptor Revealed in Native Neurons

Summary: G protein-coupled receptors are key signaling molecules and major targets for pharmaceuticals. The concept of ligand-dependent biased signaling raises the possibility of developing drugs with improved efficacy and safety profiles, yet translating this concept to native tissues remains a major challenge. Whether drug activity profiling in recombinant cell-based assays, traditionally used for drug discovery, has any relevance to physiology is unknown. Here we focused on the mu opioid receptor, the unrivalled target for pain treatment and also the key driver for the current opioid crisis. We selected a set of clinical and novel mu agonists, and profiled their activities in transfected cell assays using advanced biosensors and in native neurons from knock-in mice expressing traceable receptors endogenously. Our data identify Gi-biased agonists, including buprenorphine, and further show highly correlated drug activities in the two otherwise very distinct experimental systems, supporting in vivo translatability of biased signaling for mu opioid drugs. : Biological Sciences; Physiology; Molecular Biology; Neuroscience; Bioengineering; Cell Biology Subject Areas: Biological Sciences, Physiology, Molecular Biology, Neuroscience, Bioengineering, Cell Biolog

Computationally designed GPCR quaternary structures bias signaling pathway activation

Communication across membranes controls critical cellular processes and is achieved by receptors translating extracellular signals into selective cytoplasmic responses. While receptor tertiary structures can be readily characterized, receptor associations into quaternary structures are challenging to study and their implications in signal transduction remain poorly understood. Here, we report a computational approach for predicting receptor self-associations, and designing receptor oligomers with various quaternary structures and signaling properties. Using this approach, we designed chemokine receptor CXCR4 dimers with reprogrammed binding interactions, conformations, and abilities to activate distinct intracellular signaling proteins. In agreement with our predictions, the designed CXCR4s dimerized through distinct conformations and displayed different quaternary structural changes upon activation. Consistent with the active state models, all engineered CXCR4 oligomers activated the G protein Gi, but only specific dimer structures also recruited β-arrestins. Overall, we demonstrate that quaternary structures represent an important unforeseen mechanism of receptor biased signaling and reveal the existence of a bias switch at the dimer interface of several G protein-coupled receptors including CXCR4, mu-Opioid and type-2 Vasopressin receptors that selectively control the activation of G proteins vs β-arrestin-mediated pathways. The approach should prove useful for predicting and designing receptor associations to uncover and reprogram selective cellular signaling functions