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Cholera Toxin Inhibits SNX27-Retromer-Mediated Delivery

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Cholera Toxin Inhibits SNX27-Retromer-Mediated Delivery © 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs218610. doi:10.1242/jcs.218610 RESEARCH ARTICLE Cholera toxin inhibits SNX27-retromer-mediated delivery of cargo proteins to the plasma membrane Varsha Singh1,*, Jianbo Yang1, Jianyi Yin1, Robert Cole2, Ming Tse1, Diego E. Berman3, Scott A. Small3, Gregory Petsko4 and Mark Donowitz1,* ABSTRACT intestinal epithelial barrier function via cyclic AMP (cAMP)-induced Cholera toxin (CT) causes severe diarrhea by increasing intracellular disruption of Rab11- and exocyst-dependent delivery of endocytic – cAMP leading to a PKA-dependent increase in Cl− secretion through recycling cargo to cell cell junctions (Guichard et al., 2013). CFTR and decreased Na+ absorption through inhibition of Na+/H+ CT is a classic AB toxin. CT translocates from the plasma exchanger 3 (NHE3; also known as SLC9A3). The mechanism(s) by membrane (PM) through the trans-Golgi network (TGN) into the which CT inhibits NHE3 is partially understood, although no drug endoplasmic reticulum (ER) in a retrograde fashion by binding the therapy has been successful at reversing this inhibition. We now ganglioside GM1 via the B subunit of the native holotoxin (Orlandi describe that CT phosphorylates an amino acid in the PDZ domain of and Fishman, 1998; Wernick et al., 2010). Once it is released from the SNX27, which inhibits SNX27-mediated trafficking of NHE3 from the ER lumen, the enzymatic moiety CT-A subunit causes a pathological early endosomes to the plasma membrane (PM), and contributes to increase in cellular cAMP levels and PKA activity via the induction reduced basal NHE3 activity through a mechanism that involves of host PM adenylyl cyclase (De Haan and Hirst, 2004; Sack et al., reduced PM expression and reduced endocytic recycling. Importantly, 2004; Wernick et al., 2010). PKA then activates CFTR and inhibits + + mutagenesis studies (Ser to Asp) showed that the effect of this the brush border (BB) Na /H antiporter NHE3 (also known as + phosphorylation of SNX27 phenocopies the effects seen upon loss of SLC9A3), which is a major contributor to small intestinal Na SNX27 function, affecting PM trafficking of cargo proteins that bind absorption. Inhibition of NHE3 by CT and in other cAMP-related SNX27–retromer. Additionally, CT destabilizes retromer function by diarrheal diseases involves increased endocytosis, which reduces BB decreasing the amount of core retromer proteins. These effects of CT NHE3 expression (Musch et al., 2007, 2010). However, it is not can be partially rescued by enhancing retromer stability by using known whether changes in NHE3 exocytosis are also caused by CT, ‘pharmacological chaperones’. Moreover, pharmacological chaperones particularly from the early endosome (EE) to the PM. canbeusedtoincreasebasalandcholeratoxin-inhibitedNHE3activity An early endosomal PDZ domain-containing protein, sorting and fluid absorption by intestinal epithelial cells. nexin 27 (SNX27) binds and regulates exocytosis of NHE3 from the EE to the PM (Singh et al., 2015). SNX27 has important regulatory This article has an associated First Person interview with the first roles in EE-to-PM trafficking of multiple classes of proteins; this author of the paper. occurs via interacting with the retromer complex. The retromer complex is composed of the vacuolar protein sorting (VPS) trimer KEY WORDS: SNX27, Retromer, NHE3, PDZ, Secretory diarrhea, core sub-complex (VPS26–VPS29–VPS35) and a membrane- Apical trafficking, Exocytosis, Cholera toxin, Early endosomes associated sorting nexin (SNX) dimer (SNX1–SNX2 or SNX5– SNX6) (Seaman, 2005). The retromer complex is important in INTRODUCTION regulating transmembrane receptor recycling from the EE either to the To infect human cells, pathogenic microorganisms have evolved TGN or to the PM (Belenkaya et al., 2008; Seaman, 2007; Yang et al., numerous strategies to suppress host defenses and exploit host cellular 2008), with SNX27 involved in the EE-to-PM pathway (Joubert signaling machinery. Specific pathogen virulence factors disable, et al., 2004; Lauffer et al., 2010). In this function, SNX27 directly subvert or even stimulate vesicular trafficking pathways to and from binds VPS26 via the SNX27 PDZ domain. In regulating this aspect of the host cell surface, which promotes pathogen entry, replication or trafficking, the mammalian retromer complex binds other protein escape. One prominent trafficking pathway that pathogens modulate or complexes including Wiskott–Aldrich protein and SCAR homolog exploit by multiple mechanisms is the final step of endocytic recycling, (WASH) complex, actin, ankrin-repeat 50 domain, VPS-ankrin repeat at which cargo-containing vesicles dock at the cell surface. As one domain protein and FAM21 proteins (Burd and Cullen, 2014). example, cholera toxin (CT) secreted by Vibrio cholerae compromises Retromer-mediated trafficking defects has been implicated in a growing number of neurological diseases (Follett et al., 2014; Small 1Departments of Medicine and Physiology, School of Medicine, Johns Hopkins et al., 2005; Zimprich et al., 2011). Until now, the effect of CT on University, Baltimore, MD 21205, USA. 2Department of Biological Chemistry, retromer-mediated movement of cargo proteins in the intestine has School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA. 3The Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, not been described. In the present study, we demonstrate that CT Department of Pathology, Columbia University College of Physicians and increases phosphorylation of a serine residue in the PDZ domain of Surgeons, New York, NY 10032, USA. 4Helen and Robert Appel Alzheimer’s SNX27, which inhibits SNX27 binding to NHE3 and reduces NHE3 Disease Research Institute and Department of Neurology, Weill Cornell Medical College, New York, NY 10021, USA. exocytosis to the PM. Moreover, CT destabilizes retromer function by reducing the expression of two core components of the retromer *Authors for correspondence ([email protected]; [email protected]) complex – VPS35 and VPS26. This previously undescribed activity V.S., 0000-0003-0796-2619; M.D., 0000-0003-0477-8824 of CT identifies a site at which CT affects PM transporters, including NHE3, and identifies a step in trafficking for the potential targeting of Received 2 April 2018; Accepted 22 June 2018 drug development to treat diarrheal diseases. Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218610. doi:10.1242/jcs.218610 RESULTS the level of BB NHE3 was reduced by CT exposure; however, this CT inhibits exocytosis of NHE3 in intestinal epithelial occurred in a time-dependent manner. Cells treated with CT Caco-2/bbe cells for 8 h had more NHE3 on the membrane compared to cells Basal NHE3 activity is known to be inhibited after CT exposure, treated for 24 h or 48 h, and the percentage of total NHE3 on the with the effect involving reduced PM NHE3 expression and PM at all three time points of CT exposure was significantly stimulation of endocytosis (Subramanya et al., 2007). However, reduced compared to the untreated control (10, 7 and 5% characterization of the effects of CT on both rates of endocytosis respectively for 8, 24 and 48 h of CT exposure, respectively, and exocytosis of NHE3 in the same model has not been reported. versus 15% in untreated control cells) (Fig. 1B,C). To define the To extend this characterization, we initially tested the timecourse effect of CT on NHE3 trafficking, we measured the rate of of CT-mediated inhibition of NHE3 activity. Polarized endocytosis and exocytosis of NHE3 in CT-treated Caco-2/bbe- monolayers of Caco-2/bbe cells expressing HA–NHE3 (Caco-2/ HA-NHE3 cells. Endocytosis was determined using a biotinylation- bbe-HA-NHE3) were treated with CT for 8, 24 and 48 h (CT added based assay (Singh et al., 2015). This demonstrated similar increased every 12 h), and NHE3 activity was determined in the presence of rates of NHE3 endocytosis with 8 and 48 h of CT exposure (Fig. 1D) 50 µM HOE694 to inhibit endogenous NHE1 and NHE2 activity A cell surface biotinylation-based exocytosis assay showed that the (Ikuma et al., 1999). Consistent with previous studies, an 8 h CT rate of exocytosis of NHE3 in 8 and 48 h CT-treated cells was treatment significantly decreased basal NHE3 activity (∼40%), significantly less than that of control cells (Fig. 1E; Fig. S1). These and the effect became more pronounced at 24 h and 48 h, at which results indicate that CT, in addition to its known effect on stimulating times there was a similar extent of inhibition (Fig. 1A). To further NHE3 endocytosis, also inhibits NHE3 exocytosis, which delineate the mechanism of inhibited NHE3 activity in response to contributes to a lower abundance of NHE3 on the PM and to lower CT, we determined the surface abundance of NHE3. Biotinylation- NHE3 activity. Since there were slightly greater CT-mediated based surface abundance analysis showed that at all times studied, effects on NHE3 activity and PM expression at 48 h compared to Fig. 1. CT inhibits NHE3 activity, stimulates endocytosis and inhibits exocytosis in intestinal epithelial cells. (A) Initial rates of Na+/H+ exchange were + measured in either untreated or CT-treated (100 ng/0.5 ml for 8 h, 24 h or 48 h) Caco-2/bbe-HA-NHE3 cells as the Na -dependent pHi recovery by using the pH-sensitive dye BCECF. Results are means±s.e.m. with results from individual experiments shown; n=4 separate experiments. *P<0.05, comparison between control and CT-treated cells; #P<0.05, comparisons between 8 h and 24 h or 48 h CT-treated cells. NS, not significant. (B) Caco-2/bbe-HA-NHE3 cells were treated with CT (8 h, 24 h or 48 h) and surface NHE3 levels were analyzed. A representative western blot (IB) analysis is shown illustrating changes in PM expression of NHE3 in response to CT. (C) The quantification from at least three independent experiments, as in B, is shown for individual experiments expressed asthe percentage of total along with the mean±s.e.m.
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