Fluorescence assay for simultaneous quantification of CFTR ion-channel function and plasma membrane proximity [Methods and Resources]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane anion channel that plays a key role in controlling transepithelial fluid movement. Excessive activation results in intestinal fluid loss during secretory diarrheas, whereas CFTR mutations underlie cystic fibrosis (CF). Anion permeability depends both on how well CFTR channels work (permeation/gating) and on how many are present at the membrane. Recently, treatments with two drug classes targeting CFTR—one boosting ion-channel function (potentiators) and the other increasing plasma membrane density (correctors)—have provided significant ...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Stella Prins, Emily Langron, Cato Hastings, Emily J. Hill, Andra C. Stefan, Lewis D. Griffin, Paola Vergani Tags: Molecular Bases of Disease Source Type: research
Integrin and autocrine IGF2 pathways control fasting insulin secretion in {beta}-cells [Signal Transduction]
Elevated levels of fasting insulin release and insufficient glucose-stimulated insulin secretion (GSIS) are hallmarks of diabetes. Studies have established cross-talk between integrin signaling and insulin activity, but more details of how integrin-dependent signaling impacts the pathophysiology of diabetes are needed. Here, we dissected integrin-dependent signaling pathways involved in the regulation of insulin secretion in β-cells and studied their link to the still debated autocrine regulation of insulin secretion by insulin/insulin-like growth factor (IGF) 2–AKT signaling. We observed for the first time a cooperatio...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Caroline Arous, Maria Luisa Mizgier, Katharina Rickenbach, Michel Pinget, Karim Bouzakri, Bernhard Wehrle-Haller Tags: Cell Biology Source Type: research
Polydisperse molecular architecture of connexin 26/30 heteromeric hemichannels revealed by atomic force microscopy imaging [Protein Structure and Folding]
Connexin (Cx) protein forms hemichannels and gap junctional channels, which play diverse and profound roles in human physiology and diseases. Gap junctions are arrays of intercellular channels formed by the docking of two hemichannels from adjacent cells. Each hexameric hemichannel contains the same or different Cx isoform. Although homomeric Cxs forms have been largely described functionally and structurally, the stoichiometry and arrangement of heteromeric Cx channels remain unknown. The latter, however, are widely expressed in human tissues and variation might have important implications on channel function. Investigati...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Pamela A. Naulin, Benjamin Lozano, Christian Fuentes, Yu Liu, Carla Schmidt, Jorge E. Contreras, Nelson P. Barrera Tags: Molecular Biophysics Source Type: research
Cutting out the fat: Site-specific deacylation of an ion channel [Membrane Biology]
S-Acylation, a reversible post-translational lipid modification of proteins, controls the properties and function of various proteins, including ion channels. Large conductance Ca2+-activated potassium (BK) channels are S-acylated at two sites that impart distinct functional effects. Whereas the enzymes that attach lipid groups are known, the enzymes mediating lipid removal (i.e. deacylation) are largely unknown. Here, McClafferty et al. identify two enzymes, ABHD17a and ABHD17c, that excise BK channel lipid groups with remarkable precision. These findings lend insights into mechanisms that orchestrate the (de)acylation th...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Pedro J. del Rivero Morfin, Manu Ben-Johny Tags: Editors ' Picks Highlights Source Type: research
Site-specific deacylation by ABHD17a controls BK channel splice variant activity [Signal Transduction]
S-Acylation, the reversible post-translational lipid modification of proteins, is an important mechanism to control the properties and function of ion channels and other polytopic transmembrane proteins. However, although increasing evidence reveals the role of diverse acyl protein transferases (zDHHC) in controlling ion channel S-acylation, the acyl protein thioesterases that control ion channel deacylation are very poorly defined. Here we show that ABHD17a (α/β-hydrolase domain-containing protein 17a) deacylates the stress-regulated exon domain of large conductance voltage- and calcium-activated potassium (BK) channels...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Heather McClafferty, Hamish Runciman, Michael J. Shipston Tags: Editors ' Picks Source Type: research
Co-crystal structures of HIV TAR RNA bound to lab-evolved proteins show key roles for arginine relevant to the design of cyclic peptide TAR inhibitors [Molecular Biophysics]
RNA-protein interfaces control key replication events during the HIV-1 life cycle. The viral trans-activator of transcription (Tat) protein uses an archetypal arginine-rich motif (ARM) to recruit the host positive transcription elongation factor b (pTEFb) complex onto the viral trans-activation response (TAR) RNA, leading to activation of HIV transcription. Efforts to block this interaction have stimulated production of biologics designed to disrupt this essential RNA-protein interface. Here, we present four co-crystal structures of lab-evolved TAR-binding proteins (TBPs) in complex with HIV-1 TAR. Our results reveal that ...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Sai Shashank Chavali, Sachitanand M. Mali, Jermaine L. Jenkins, Rudi Fasan, Joseph E. Wedekind Tags: Editors ' Picks Source Type: research
Correction: Targeting the unique methylation pattern of androgen receptor (AR) promoter in prostate stem/progenitor cells with 5-aza-2'-deoxycytidine (5-AZA) leads to suppressed prostate tumorigenesis. [Additions and Corrections]
VOLUME 287 (2012) PAGES 39954–39966The control GAPDH immunoblot in Fig. 4B (b) was accidentally flipped and used in the control immunoblot in Fig. 1B. For Fig. 6G, the Oct4 5-AZA panel was mistakenly inserted into the CK8 control panel during figure preparation. The correct images are presented in the correction figures. We sincerely apologize for the error, and we assure the reader that the correction of the images will not alter the original results or conclusions.jbc;295/48/16469/F1F1F1Figure 1B.jbc;295/48/16469/F6F2F6Figure 6G. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - November 27, 2020 Category: Chemistry Authors: Jing Tian, Soo Ok Lee, Liang Liang, Jie Luo, Chiung-Kuei Huang, Lei Li, Yuanjie Niu, Chawnshang Chang Tags: Additions and Corrections Source Type: research
Correction: MicroRNA-27 (miR-27) targets prohibitin and impairs adipocyte differentiation and mitochondrial function in human adipose-derived stem cells. [Additions and Corrections]
VOLUME 288 (2013) PAGES 34394–34402The immunoblots in Figs. 2C and 4A had undeclared splices. We have repeated the experiments, and the corrected figures contain no splices. This correction does not affect the results or conclusions of this work. In the eighth line from the last in the right column of page 34396, “Prohibitin Targets miR-27a and miR-27b in ASC” should read “miR-27a and miR-27b Target Prohibitin in ASC.” The authors sincerely apologize for any inconvenience these errors may have caused readers.jbc;295/48/16468/F2F1F2Figure 2C.jbc;295/48/16468/F4F2F4Figure 4A. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - November 27, 2020 Category: Chemistry Authors: Ting Kang, Wan Lu, Wei Xu, Leonard Anderson, Methode Bacanamwo, Winston Thompson, Y. Eugene Chen, Dong Liu Tags: Additions and Corrections Source Type: research
Correction: Mechanistic insights into cancer cell killing through interaction of phosphodiesterase 3A and schlafen family member 12. [Additions and Corrections]
VOLUME 295 (2020) PAGES 3431–3446In response to a reviewer's request to change the x axis units of our dose-response curves from µm to m, we mistakenly changed the numbers to mm while labeling as m. The numbers in the original submission were accurate, Table 1 has remained accurate, and we have now corrected the x axis numbers in the graphs of the print version to reflect the label, “m”. The authors regret the error, which does not affect the conclusions of the paper.jbc;295/48/16464/F1F1F1Figure 1A.jbc;295/48/16464/F2F2F2Figure 2B.jbc;295/48/16464/F5AF3F5AFigure 5A.jbc;295/48/16464/F5CF4F5CFigure 5C.jbc;295/48/1646...
Source: Journal of Biological Chemistry - November 27, 2020 Category: Chemistry Authors: Xiaoyun Wu, Gavin R. Schnitzler, Galen F. Gao, Brett Diamond, Andrew R. Baker, Bethany Kaplan, Kaylyn Williamson, Lindsay Westlake, Selena Lorrey, Timothy A. Lewis, Colin W. Garvie, Martin Lange, Sikander Hayat, Henrik Seidel, John Doench, Andrew D. Chern Tags: Additions and Corrections Source Type: research
Biosynthesis of GlcNAc-rich N- and O-glycans in the Golgi apparatus does not require the nucleotide sugar transporter SLC35A3 [Cell Biology]
Nucleotide sugar transporters, encoded by the SLC35 gene family, deliver nucleotide sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. GlcNAc, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T, and HepG2 cell lines including WT cells, SLC35A2 knockouts, SLC35A3 knockouts, and double-knockout cel...
Source: Journal of Biological Chemistry - November 27, 2020 Category: Chemistry Authors: Bozena Szulc, Paulina Sosicka, Dorota Maszczak-Seneczko, Edyta Skurska, Auhen Shauchuk, Teresa Olczak, Hudson H. Freeze, Mariusz Olczak Tags: Glycobiology and Extracellular Matrices Source Type: research
Rate-limiting pyrophosphate release by hepatitis C virus polymerase NS5B improves fidelity [Enzymology]
In this study, we used transient-state kinetics to examine the mechanistic basis for polymerase fidelity. We observe a wide range of efficiency for incorporation of various mismatched base pairs and have uncovered a mechanism in which the rate constant for pyrophosphate release is slowed for certain misincorporation events. This results in an increase in fidelity against these specific misincorporations. Furthermore, we discover that some mismatches are highly unfavorable and cannot be observed under the conditions used here. The calculated fidelity of NS5B ranges between 10−4–10−9 for different mismatches. (Source: ...
Source: Journal of Biological Chemistry - November 27, 2020 Category: Chemistry Authors: Brian Villalba, Kenneth A. Johnson Tags: Enzymology Source Type: research
Rubisco activase requires residues in the large subunit N terminus to remodel inhibited plant Rubisco [Enzymology]
The photosynthetic CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) forms dead-end inhibited complexes while binding multiple sugar phosphates, including its substrate ribulose 1,5-bisphosphate. Rubisco can be rescued from this inhibited form by molecular chaperones belonging to the ATPases associated with diverse cellular activities (AAA+ proteins) termed Rubisco activases (Rcas). The mechanism of green-type Rca found in higher plants has proved elusive, in part because until recently higher-plant Rubiscos could not be expressed recombinantly. Identifying the interaction sites between Rubisco an...
Source: Journal of Biological Chemistry - November 27, 2020 Category: Chemistry Authors: Jediael Ng, Zhijun Guo, Oliver Mueller-Cajar Tags: Plant Biology Source Type: research
Global reprogramming of virulence and antibiotic resistance in Pseudomonas aeruginosa by a single nucleotide polymorphism in elongation factor, fusA1 [Genomics and Proteomics]
Clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa from patients with cystic fibrosis (CF) frequently contain mutations in the gene encoding an elongation factor, FusA1. Recent work has shown that fusA1 mutants often display elevated aminoglycoside resistance due to increased expression of the efflux pump, MexXY. However, we wondered whether these mutants might also be affected in other virulence-associated phenotypes. Here, we isolated a spontaneous gentamicin-resistant fusA1 mutant (FusA1P443L) in which mexXY expression was increased. Proteomic and transcriptomic analyses revealed that the fusA1 mutan...
Source: Journal of Biological Chemistry - November 27, 2020 Category: Chemistry Authors: Eve A. Maunders, Rory C. Triniman, Joshua Western, Taufiq Rahman, Martin Welch Tags: Microbiology Source Type: research
Calnexin mediates the maturation of GPI-anchors through ER retention [Protein Synthesis and Degradation]
The protein folding and lipid moiety status of glycosylphosphatidylinositol-anchored proteins (GPI-APs) are monitored in the endoplasmic reticulum (ER), with calnexin playing dual roles in the maturation of GPI-APs. In the present study, we investigated the functions of calnexin in the quality control and lipid remodeling of GPI-APs in the ER. By directly binding the N-glycan on proteins, calnexin was observed to efficiently retain GPI-APs in the ER until they were correctly folded. In addition, sufficient ER retention time was crucial for GPI-inositol deacylation, which is mediated by post-GPI attachment protein 1 (PGAP1)...
Source: Journal of Biological Chemistry - November 27, 2020 Category: Chemistry Authors: Xin-Yu Guo, Yi-Shi Liu, Xiao-Dong Gao, Taroh Kinoshita, Morihisa Fujita Tags: Glycobiology and Extracellular Matrices Source Type: research
Structural and molecular basis for the substrate positioning mechanism of a new PL7 subfamily alginate lyase from the arctic [Metabolism]
This study provides a better understanding of the mechanisms of alginate degradation by alginate lyases. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - November 27, 2020 Category: Chemistry Authors: Fei Xu, Xiu-Lan Chen, Xiao-Hui Sun, Fang Dong, Chun-Yang Li, Ping-Yi Li, Haitao Ding, Yin Chen, Yu-Zhong Zhang, Peng Wang Tags: Enzymology Source Type: research
