International Journal of Molecular Sciences Review Toxins Utilize the Endoplasmic Reticulum-Associated Protein Degradation Pathway in Their Intoxication Process Jowita Nowakowska-Gołacka, Hanna Sominka, Natalia Sowa-Rogozi ´nskaand Monika Słomi ´nska-Wojewódzka * Department of Medical Biology and Genetics, Faculty of Biology, University of Gda´nsk,Wita Stwosza 59, 80-308 Gda´nsk,Poland; [email protected] (J.N.-G.); [email protected] (H.S.); [email protected] (N.S.-R.) * Correspondence: [email protected]; Tel.: +48-585-236-035 Received: 16 February 2019; Accepted: 10 March 2019; Published: 15 March 2019 Abstract: Several bacterial and plant AB-toxins are delivered by retrograde vesicular transport to the endoplasmic reticulum (ER), where the enzymatically active A subunit is disassembled from the holotoxin and transported to the cytosol. In this process, toxins subvert the ER-associated degradation (ERAD) pathway. ERAD is an important part of cellular regulatory mechanism that targets misfolded proteins to the ER channels, prior to their retrotranslocation to the cytosol, ubiquitination and subsequent degradation by a protein-degrading complex, the proteasome. In this article, we present an overview of current understanding of the ERAD-dependent transport of AB-toxins to the cytosol. We describe important components of ERAD and discuss their significance for toxin transport. Toxin recognition and disassembly in the ER, transport through ER translocons and finally cytosolic events that instead of overall proteasomal degradation provide proper folding and cytotoxic activity of AB-toxins are discussed as well. We also comment on recent reports presenting medical applications for toxin transport through the ER channels. Keywords: AB-toxins; endoplasmic reticulum (ER); ER-associated degradation (ERAD) 1. Introduction Endoplasmic reticulum (ER) is a major cellular protein folding compartment that regulates biosynthesis, assembly and trafficking of most secretory and membrane proteins [1]. In order to maintain the fidelity of essential cellular functions, the ER is dedicated to a stringent quality control system (ERQC) which enables folding and modification of proteins and eliminates terminally misfolded polypeptides through ER-associated degradation (ERAD) or autophagy [2]. ERAD can be divided in four primary phases: (1) recognition (substrate recognition within the ER and direction to the retrotranslocon), (2) retrotranslocation (substrate transport across the ER membrane), (3) membrane unload (release of the substrate from the ER membrane into the cytosol) and (4) degradation (ubiquitin–proteasome dependent degradation) [2–4]. During all of these steps ER molecular chaperones and associated factors, both luminal and membrane-bound, ER translocons, as well as diverse cytosolic factors, are crucial for substrate driving through ERAD. However, disposal of different types of substrates, for example, soluble, membrane-bound, glycosylated or non-glycosylated, can be regulated by distinct ERAD pathways that differ in the set of factors involved in ERAD [5–7]. Moreover, work on Saccharomyces cerevisiae had shown that misfolded ER proteins are degraded by three different ERAD pathways (ERAD-L, -M and -C), depending on whether their misfolded domain is localized in the ER lumen, within the membrane or on the cytosolic side of the membrane [8–10]. Int. J. Mol. Sci. 2019, 20, 1307; doi:10.3390/ijms20061307 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 1307 2 of 36 There is also evidence that ERAD controls degradation of certain folded proteins, including MHC I andInt. CD4. J. Mol. InSci.these 2019, 20 cases, x adaptor-mediated substrate recognition is employed, as MHC I molecules2 of 36 are bound by US11 protein encoded by the human cytomegalovirus, whereas CD4 are targeted for degradationincluding MHC in cells I and expressing CD4. In thethese HIV-encoded cases adaptor- ERmediated membrane substrate protein recogn Vpuition [9,11 is– 15employed,]. The ERAD as strategyMHC I of molecules using substrate-specific are bound by US11 adaptors protein isencoded not controlled by the human exclusively cytomegalovirus, by viral encoded whereas proteins. CD4 Rhomboidsare targeted are for classified degradation as serine in cells proteases, expressing conserved the HIV-encoded across allER kingdomsmembrane protein of life. Vpu A subgroup [9,11– of rhomboid-like15]. The ERAD proteinsstrategy thatof using lack substrate-specific essential catalytic adaptors residues, is “iRhoms”not controlled [16], exclusively can target epidermalby viral growthencoded factor proteins. receptor Rhomboids (EGFR) for are proteasomal classified as removal serine proteases, by ERAD conserved in Drosophila across[17 all]. Akingdoms substrate of specific life. adaptorA subgroup also functions of rhomboid-like in the ERAD proteins regulation that lack ofesse HMG-CoAntial catalytic reductase residues, (HMGCR), “iRhoms” a[16], key can enzyme target of epidermal growth factor receptor (EGFR) for proteasomal removal by ERAD in Drosophila [17]. A the sterol biosynthetic pathway [18]. It has been also reported that regulated degradation of IRE1α substrate specific adaptor also functions in the ERAD regulation of HMG-CoA reductase (HMGCR), and ATF6, important sensors of the unfolded protein response (UPR), is controlled by ERAD [19–21]. a key enzyme of the sterol biosynthetic pathway [18]. It has been also reported that regulated All of these observations highlight the role of ER-associated degradation in cellular homeostasis and degradation of IRE1α and ATF6, important sensors of the unfolded protein response (UPR), is indicate that this process may control the complexity of ER-related functions. Despite complicated and controlled by ERAD [19–21]. All of these observations highlight the role of ER-associated degradation diversein cellular ERAD homeostasis mechanisms and and indicate pathways, that cellular this process significance may control of this processthe complexity should beof consideredER-related in a muchfunctions. broader Despite spectrum. complicated A group and of diverse AB-toxins ERAD have mechanisms evolved mechanisms and pathways, to exploit cellular ERAD significance for their ownof this benefit process (Figure should1). Thesebe considered toxins havein a much an overall broader similar spectrum. structure, A group which of AB-toxins typically have consists evolved of a singlemechanisms enzymatically to exploit active ERAD A subunit for their (chain) own benefit and a single(Figure or 1). multiple These toxins membrane have an binding overall Bsimilar subunit recognizingstructure, which particular typically cell surface consists glycolipids, of a single enzymatically glycoproteins active or receptor A subunit proteins. (chain) After and a cell single binding or andmultiple endocytosis, membrane toxins arebinding trafficked B insubunit a retrograde recognizing manner throughparticular the Golgicell surface apparatus glycolipids, and into the ERglycoproteins before reaching the cytosol or the host cell nucleus. FigureFigure 1. 1.AB-toxins AB-toxins subvert subvert thethe endoplasmicendoplasmic reticulum-associatedreticulum-associated protein protein degradation degradation pathway pathway (ERAD) in their transport from the ER to the cytosol. (ERAD) in their transport from the ER to the cytosol. TheThe first first report report suggesting suggesting a a link link between between thethe cell cytosol entry entry of of toxins toxins and and the the ERAD ERAD pathway pathway camecame in in 1997 1997 [22 [22].]. Then, Then, Rapak Rapak and and co-workersco-workers establishedestablished an an excellent excellent assay assay for for analysing analysing transport transport ofof ricin, ricin, a planta plant toxin, toxin, from from the the ER ER to tothe the cytosolcytosol [[22].22]. However, However, at at that that time time they they were were not not able able to to addressaddress the the question question as as to to how how the the toxin toxin entersenters thethe pathway or or how how it it escapes escapes degradation. degradation. The The fact fact that toxins avoid effective ubiquitination and thus they are transported to the cytosol without being that toxins avoid effective ubiquitination and thus they are transported to the cytosol without being directed for proteasomal degradation makes them untypical ERAD substrates. Over the past two directed for proteasomal degradation makes them untypical ERAD substrates. Over the past two decades, our knowledge on ERAD-dependent toxin transport to the cytosol has significantly decades, our knowledge on ERAD-dependent toxin transport to the cytosol has significantly expanded. expanded. The first three ERAD steps: recognition, retrotranslocation and membrane unload are generally common for misfolded proteins, endogenous ER substrates and toxins. However, in the case of toxins, they fold properly after transport to the cytosol in order to express their cytotoxic Int. J. Mol. Sci. 2019, 20, 1307 3 of 36 The first three ERAD steps: recognition, retrotranslocation and membrane unload are generally common for misfolded proteins, endogenous ER substrates and toxins. However, in the case of toxins, they fold properly after transport to the cytosol in order to express their cytotoxic activity (Figure1). Toxins that hijack the host cell ERAD pathway for their transport from the ER to the cytosol include: the cholera toxin (CT) (Figure2A), E. coli heat-labile enterotoxin (LT), Shiga