Nicotine up-regulates α4β2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning

The up-regulation of α4β2* nicotinic acetylcholine receptors (nAChRs) by chronic nicotine is a cell-delimited process and may be necessary and sufficient for the initial events of nicotine dependence. Clinical literature documents an inverse relationship between a person’s history of tobacco use and his or her susceptibility to Parkinson’s disease; this may also result from up-regulation. This study visualizes and quantifies the subcellular mechanisms involved in nicotine-induced nAChR up-regulation by using transfected fluorescent protein (FP)-tagged α4 nAChR subunits and an FP-tagged Sec24D endoplasmic reticulum (ER) exit site marker. Total internal reflection fluorescence microscopy shows that nicotine (0.1 µM for 48 h) up-regulates α4β2 nAChRs at the plasma membrane (PM), despite increasing the fraction of α4β2 nAChRs that remain in near-PM ER. Pixel-resolved normalized Förster resonance energy transfer microscopy between α4-FP subunits shows that nicotine stabilizes the (α4)2(β2)3 stoichiometry before the nAChRs reach the trans-Golgi apparatus. Nicotine also induces the formation of additional ER exit sites (ERES). To aid in the mechanistic analysis of these phenomena, we generated a β2enhanced-ER-export mutant subunit that mimics two regions of the β4 subunit sequence: the presence of an ER export motif and the absence of an ER retention/retrieval motif. The α4β2enhanced-ER-export nAChR resembles nicotine-exposed nAChRs with regard to stoichiometry, intracellular mobility, ERES enhancement, and PM localization. Nicotine produces only small additional PM up-regulation of α4β2enhanced-ER-export receptors. The experimental data are simulated with a model incorporating two mechanisms: (1) nicotine acts as a stabilizing pharmacological chaperone for nascent α4β2 nAChRs in the ER, eventually increasing PM receptors despite a bottleneck(s) in ER export; and (2) removal of the bottleneck (e.g., by expression of the β2enhanced-ER-export subunit) is sufficient to increase PM nAChR numbers, even without nicotine. The data also suggest that pharmacological chaperoning of nAChRs by nicotine can alter the physiology of ER processes

An overview of activity-based probes for glycosidases

As the scope of modern genomics technologies increases, so does the need for informative chemical tools to study functional biology. Activity-based probes (ABPs) provide a powerful suite of reagents to probe the biochemistry of living organisms. These probes, featuring a specificity motif, a reactive chemical group and a reporter tag, are opening-up large swathes of protein chemistry to investigation in vitro, as well as in cellular extracts, cells and living organisms in vivo. Glycoside hydrolases, by virtue of their prominent biological and applied roles, provide a broad canvas on which ABPs may illustrate their functions. Here we provide an overview of glycosidase ABP mechanisms, and review recent ABP work in the glycoside hydrolase field, encompassing their use in medical diagnosis, their application for generating chemical genetic disease models, their fine-tuning through conformational and reactivity insight, their use for high-throughput inhibitor discovery, and their deployment for enzyme discovery and dynamic characterization.Medical BiochemistryBio-organic Synthesi

Site-selective incorporation and ligation of protein aldehydes

The incorporation of aldehyde handles into proteins, and subsequent chemical reactions thereof, is rapidly proving to be an effective way of generating homogeneous, covalently linked protein constructs that can display a vast array of functionality. In this review, we discuss methods for introducing aldehydes into target proteins, and summarise the ligation strategies for site-selective modification of proteins containing this class of functional handles

The Hepatitis B Virus Ribonuclease H Is Sensitive to Inhibitors of the Human Immunodeficiency Virus Ribonuclease H and Integrase Enzymes

Nucleos(t)ide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV) reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(t)ide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(t)ide analogs. The HBV ribonuclease H (RNAseH) is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 μM, the best compounds had low micromolar IC50 values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 μM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug development. © 2013 Tavis et al

New factors for protein transport identified by a genome-wide CRISPRi screen in mammalian cells

Protein and membrane trafficking pathways are critical for cell and tissue homeostasis. Traditional genetic and biochemical approaches have shed light on basic principles underlying these processes. However, the list of factors required for secretory pathway function remains incomplete, and mechanisms involved in their adaptation poorly understood. Here, we present a powerful strategy based on a pooled genome-wide CRISPRi screen that allowed the identification of new factors involved in protein transport. Two newly identified factors, TTC17 and CCDC157, localized along the secretory pathway and were found to interact with resident proteins of ER-Golgi membranes. In addition, we uncovered that upon TTC17 knockdown, the polarized organization of Golgi cisternae was altered, creating glycosylation defects, and that CCDC157 is an important factor for the fusion of transport carriers to Golgi membranes. In conclusion, our work identified and characterized new actors in the mechanisms of protein transport and secretion, and opens stimulating perspectives for the use of our platform in physiological and pathological contexts.Includes Wellcome Trust, MRC and H202

CK2 Phosphorylates Sec31 and Regulates ER-To-Golgi Trafficking

Protein export from the endoplasmic reticulum (ER) is an initial and rate-limiting step of molecular trafficking and secretion. This is mediated by coat protein II (COPII)-coated vesicles, whose formation requires small GTPase Sar1 and 6 Sec proteins including Sec23 and Sec31. Sec31 is a component of the outer layer of COPII coat and has been identified as a phosphoprotein. The initiation and promotion of COPII vesicle formation is regulated by Sar1; however, the mechanism regulating the completion of COPII vesicle formation followed by vesicle release is largely unknown. Hypothesizing that the Sec31 phosphorylation may be such a mechanism, we identified phosphorylation sites in the middle linker region of Sec31. Sec31 phosphorylation appeared to decrease its association with ER membranes and Sec23. Non-phosphorylatable mutant of Sec31 stayed longer at ER exit sites and bound more strongly to Sec23. We also found that CK2 is one of the kinases responsible for Sec31 phosphorylation because CK2 knockdown decreased Sec31 phosphorylation, whereas CK2 overexpression increased Sec31 phosphorylation. Furthermore, CK2 knockdown increased affinity of Sec31 for Sec23 and inhibited ER-to-Golgi trafficking. These results suggest that Sec31 phosphorylation by CK2 controls the duration of COPII vesicle formation, which regulates ER-to-Golgi trafficking

Coriolis-induced instabilities in centrifuge modeling of granular flow

Granular flows are typically studied in laboratory flumes based on common similarity scaling, which create stress fields that only roughly approximate field conditions. The geotechnical centrifuge produces stress conditions that are closer to those observed in the field, but steady conditions can be hardly achieved. Moreover, secondary effects induced by the apparent Coriolis acceleration, which can either dilate or compress the flow, often obscure scaling. This work aims at studying a set of numerical experiments where the effects of the Coriolis acceleration are measured and analyzed. Three flow states are observed: dense, dilute, and unstable. It is found that flows generated under the influence of dilative Coriolis accelerations are likely to become unstable. Nevertheless, a steady dense flow can still be obtained if a large centrifuge is used. A parametric group is proposed to predict the insurgence of instabilities; this parameter can guide experimental designs and could help to avoid damage to the experimental apparatus and model instrumentation