Toxin Mediated Diarrhea in the 21st Century: The Pathophysiology of Intestinal Ion Transport in the Course of ETEC, V. cholerae and Rotavirus Infection

An estimated 4 billion episodes of diarrhea occur each year. As a result, 2–3 million children and 0.5–1 million adults succumb to the consequences of this major healthcare concern. The majority of these deaths can be attributed to toxin mediated diarrhea by infectious agents, such as E. coli, V. cholerae or Rotavirus. Our understanding of the pathophysiological processes underlying these infectious diseases has notably improved over the last years. This review will focus on the cellular mechanism of action of the most common enterotoxins and the latest specific therapeutic approaches that have been developed to contain their lethal effects

Studying catabolism of protein ADP-Ribosylation

Protein ADP-ribosylation is a conserved posttranslational modification that regulates many major cellular functions, such as DNA repair, transcription, translation, signal transduction, stress response, cell division, aging, and cell death. Protein ADP-ribosyl transferases catalyze the transfer of an ADP-ribose (ADPr) group from the β-nicotinamide adenine dinucleotide (β-NAD+) cofactor onto a specific target protein with the subsequent release of nicotinamide. ADP-ribosylation leads to changes in protein structure, function, stability, and localization, thus defining the appropriate cellular response. Signaling processes that are mediated by modifications need to be finely tuned and eventually silenced and one of the ways to achieve this is through the action of enzymes that remove (reverse) protein ADP-ribosylation in a timely fashion such as PARG, TARG1, MACROD1, and MACROD2. Here, we describe several basic methods used to study the enzymatic activity of de-ADP-ribosylating enzymes

(ADP-ribosyl)hydrolases: Structural basis for differential substrate recognition and inhibition

Protein ADP-ribosylation is a highly dynamic post-translational modification. The rapid turnover is achieved, among others, by ADP-(ribosyl)hydrolases (ARHs), an ancient family of enzymes that reverses this modification. Recently ARHs came into focus due to their role as regulators of cellular stresses and tumor suppressors. Here we present a comprehensive structural analysis of the enzymatically active family members ARH1 and ARH3. These two enzymes have very distinct substrate requirements. Our data show that binding of the adenosine ribose moiety is highly diverged between the two enzymes, whereas the active sites harboring the distal ribose closely resemble each other. Despite this apparent similarity, we elucidate the structural basis for the selective inhibition of ARH3 by the ADP-ribose analogues ADP-HPD and arginine-ADP-ribose. Together, our biochemical and structural work provides important insights into the mode of enzyme-ligand interaction, helps to understand differences in their catalytic behavior, and provides useful tools for targeted drug design

The Cytopathic Activity Of Cholera Toxin Requires A Threshold Quantity Of Cytosolic Toxin.

Cholera toxin (CT), secreted from Vibrio cholerae, causes a massive fluid and electrolyte efflux in the small intestine that results in life-threatening diarrhea and dehydration which impacts 3-5 million people per year. CT is secreted into the intestinal lumen but acts within the cytosol of intestinal epithelial cells. CT is an AB5 toxin that has a catalytic A1 subunit and a cell binding B subunit. CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde transport. Much of the toxin is transported to the lysosomes for degradation, but a secondary pool of toxin is diverted to the Golgi apparatus and then to the ER. Here the A1 subunit detaches from the rest of the toxin and enters the cytosol. The disordered conformation of free CTA1 facilitates toxin export to the cytosol by activating a quality control mechanism known as ER-associated degradation. The return to a folded structure in the cytosol allows CTA1 to attain an active conformation for modification of its Gsα target through ADP-ribosylation. This modification locks the protein in an active state which stimulates adenylate cyclase and leads to elevated levels of cAMP. A chloride channel located in the apical enterocyte membrane opens in response to signaling events induced by these elevated cAMP levels. The osmotic movement of water into the intestinal lumen that results from the chloride efflux produces the characteristic profuse watery diarrhea that is seen in intoxicated individuals. The current model of intoxication proposes only one molecule of cytosolic toxin is required to affect host cells, making therapeutic treatment nearly impossible. However, based on emerging evidence, we hypothesize a threshold quantity of toxin must be present within the cytosol of the target cell in order to elicit a cytopathic effect. Using the method of surface plasmon resonance along with toxicity assays, I have, for the first time, directly measured the efficiency of toxin delivery to the cytosol and correlated the levels of cytosolic toxin to toxin iv activity. I have shown CTA1 delivery from the cell surface to the cytosol is an inefficient process with only 2.3 % of the surface bound CTA1 appearing in the cytosol after 2 hours of intoxication. I have also determined and a cytosolic quantity of more than approximately .05ng of cytosolic CTA1 must be reached in order to elicit a cytopathic effect. Furthermore, CTA1 must be continually delivered from the cell surface to the cytosol in order to overcome the constant proteasome-mediated clearance of cytosolic toxin. When toxin delivery to the cytosol was blocked, this allowed the host cell to de-activate Gs, lower cAMP levels, and recover from intoxication. Our work thus indicates it is possible to treat cholera even after the onset of disease. These findings challenge the idea of irreversible cellular toxicity and open the possibility of postintoxication treatment options

A Therapeutic Chemical Chaperone Inhibits Cholera Intoxication and Unfolding/Translocation of the Cholera Toxin A1 Subunit

Cholera toxin (CT) travels as an intact AB5 protein toxin from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. In the ER, the catalytic A1 subunit dissociates from the rest of the toxin. Translocation of CTA1 from the ER to the cytosol is then facilitated by the quality control mechanism of ER-associated degradation (ERAD). Thermal instability in the isolated CTA1 subunit generates an unfolded toxin conformation that acts as the trigger for ERAD-mediated translocation to the cytosol. In this work, we show by circular dichroism and fluorescence spectroscopy that exposure to 4-phenylbutyric acid (PBA) inhibited the thermal unfolding of CTA1. This, in turn, blocked the ER-to-cytosol export of CTA1 and productive intoxication of either cultured cells or rat ileal loops. In cell culture studies PBA did not affect CT trafficking to the ER, CTA1 dissociation from the holotoxin, or functioning of the ERAD system. PBA is currently used as a therapeutic agent to treat urea cycle disorders. Our data suggest PBA could also be used in a new application to prevent or possibly treat cholera

A Therapeutic Chemical Chaperone Inhibits Cholera Intoxication and Unfolding/Translocation of the Cholera Toxin A1 Subunit

Cholera toxin (CT) travels as an intact AB(5) protein toxin from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. In the ER, the catalytic A1 subunit dissociates from the rest of the toxin. Translocation of CTA1 from the ER to the cytosol is then facilitated by the quality control mechanism of ER-associated degradation (ERAD). Thermal instability in the isolated CTA1 subunit generates an unfolded toxin conformation that acts as the trigger for ERAD-mediated translocation to the cytosol. In this work, we show by circular dichroism and fluorescence spectroscopy that exposure to 4-phenylbutyric acid (PBA) inhibited the thermal unfolding of CTA1. This, in turn, blocked the ER-to-cytosol export of CTA1 and productive intoxication of either cultured cells or rat ileal loops. In cell culture studies PBA did not affect CT trafficking to the ER, CTA1 dissociation from the holotoxin, or functioning of the ERAD system. PBA is currently used as a therapeutic agent to treat urea cycle disorders. Our data suggest PBA could also be used in a new application to prevent or possibly treat cholera

Reconstitution of the DTX3L-PARP9 complex reveals determinants for high affinity heterodimerization and multimeric assembly

AbstractUbiquitination and ADP-ribosylation are post-translational modifications that play major roles in pathways including the DNA damage response and viral infection. The enzymes responsible for these modifications are therefore potential targets for therapeutic intervention. DTX3L is an E3 Ubiquitin ligase that forms a heterodimer with PARP9. In addition to its ubiquitin ligase activity, DTX3L–PARP9 also acts as an ADP-ribosyl transferase for Gly76 on the C-terminus of ubiquitin. NAD⁺-dependent ADP-ribosylation of ubiquitin by DTX3L–PARP9 prevents ubiquitin from conjugating to protein substrates. To gain insight into how DTX3L–PARP9 generates these post-translational modifications, we produced recombinant forms of DTX3L and PARP9 and studied their physical interactions. We show the DTX3L D3 domain (230–510) mediates the interaction with PARP9 with nanomolar affinity and an apparent 1 : 1 stoichiometry. We also show that DTX3L and PARP9 assemble into a higher molecular weight oligomer, and that this is mediated by the DTX3L N-terminal region (1–200). Lastly, we show that ADP-ribosylation of ubiquitin at Gly76 is reversible in vitro by several Macrodomain-type hydrolases. Our study provides a framework to understand how DTX3L–PARP9 mediates ADP-ribosylation and ubiquitination through both intra- and inter-subunit interactions.Abstract Ubiquitination and ADP-ribosylation are post-translational modifications that play major roles in pathways including the DNA damage response and viral infection. The enzymes responsible for these modifications are therefore potential targets for therapeutic intervention. DTX3L is an E3 Ubiquitin ligase that forms a heterodimer with PARP9. In addition to its ubiquitin ligase activity, DTX3L–PARP9 also acts as an ADP-ribosyl transferase for Gly76 on the C-terminus of ubiquitin. NAD⁺-dependent ADP-ribosylation of ubiquitin by DTX3L–PARP9 prevents ubiquitin from conjugating to protein substrates. To gain insight into how DTX3L–PARP9 generates these post-translational modifications, we produced recombinant forms of DTX3L and PARP9 and studied their physical interactions. We show the DTX3L D3 domain (230–510) mediates the interaction with PARP9 with nanomolar affinity and an apparent 1 : 1 stoichiometry. We also show that DTX3L and PARP9 assemble into a higher molecular weight oligomer, and that this is mediated by the DTX3L N-terminal region (1–200). Lastly, we show that ADP-ribosylation of ubiquitin at Gly76 is reversible in vitro by several Macrodomain-type hydrolases. Our study provides a framework to understand how DTX3L–PARP9 mediates ADP-ribosylation and ubiquitination through both intra- and inter-subunit interactions

Cellular mechanisms that regulate the endogenous mono-ADP-ribosylation of the G protein βγ subunit.

Mono-ADP-ribosylation is a reversible, post-translational modification of cellular proteins that has been implicated in regulation of signal transduction, muscle cell differentiation, and protein trafficking and secretion. The reaction is catalysed by mono-ADP-ribosyltransferases that transfer a single ADP-ribose moiety from p-NAD+ to a specific amino-acid of acceptor proteins. An ADP-ribosylation reaction occurs in intact cells on the p subunit of heterotrimeric G proteins that is carried out by an arginine- specific, plasma-membrane-associated, mono-ADP-ribosyltransferase. This modification is reversed by a cytosolic ADP-ribosylhydrolase that regenerates native Py dimer by releasing the bound ADP-ribose. Once ADP-ribosylated, the py dimer is inactive towards its effector enzymes, such as adenylyl cyclase, phosphoinositide 3-kinase and phospholipase C. It thus appears that endogenous P subunit mono-ADP-ribosylation might represent a novel cellular mechanism for the modulation of the G-protein-mediated signal transduction machinery through a direct regulation of the py dimer. In this study, the mechanisms that regulate endogenous mono-ADP-ribosylation of the p subunit have been investigated. The reaction appears to be under hormonal control both in vitro and in vivo, since the levels of ADP-ribosylated p are increased upon activation of certain G-protein-coupled receptors (GPCRs), such as thrombin, serotonin and cholecystokinin receptors. Conversely, hormonal stimulation by additional GPCRs, such as the GnRH receptor, can lead to a decrease in p subunit mono-ADP-ribosylation. Thus, ADP-ribosylation of the py dimer can be differentially regulated by different GPCRs in a receptor-type-dependent manner. In addition, the involvement of the ADP-ribosylating factor ARF6 in GnRH-mediated regulation of p subunit mono-ADP-ribosylation is demonstrated. Indeed, removal of ARF6 from plasma membranes results in loss of GnRH-mediated inhibition of p subunit mono-ADP-ribosylation, which can be fully restored by re-addition of purified ARF6. In conclusion, the results reported in this thesis allow the definition of the mechanisms that regulated endogenous ADP-ribosylation of the p subunit, and demonstrate a novel role for ARF6 in hormonal regulation of p subunit mono-ADP-ribosylation

TARG1 protects against toxic DNA ADP-ribosylation

ADP-ribosylation is a modification that targets a variety of macromolecules and regulates a diverse array of important cellular processes. ADP-ribosylation is catalysed by ADP-ribosyltransferases and reversed by ADP-ribosylhydrolases. Recently, an ADP-ribosyltransferase toxin termed 'DarT' from bacteria, which is distantly related to human PARPs, was shown to modify thymidine in single-stranded DNA in a sequence specific manner. The antitoxin of DarT is the macrodomain containing ADP-ribosylhydrolase DarG, which shares striking structural homology with the human ADP-ribosylhydrolase TARG1. Here, we show that TARG1, like DarG, can reverse thymidine-linked DNA ADP-ribosylation. We find that TARG1-deficient human cells are extremely sensitive to DNA ADP-ribosylation. Furthermore, we also demonstrate the first detection of reversible ADP-ribosylation on genomic DNA in vivo from human cells. Collectively, our results elucidate the impact of DNA ADP-ribosylation in human cells and provides a molecular toolkit for future studies into this largely unknown facet of ADP-ribosylation

Study of the Cellular Role of GRP78/BiP mono-ADP-ribosylation in UPR and Cancer

Mono-ADP-ribosylation is a reversible post-translational protein modification that modulates the function of proteins involved in different cellular processes, including signal transduction, protein transport, transcription, cell cycle regulation, DNA (deoxyribonucleic acid) repair and apoptosis. In mammals, mono-ADPribosylation is catalyzed by three different classes of enzymes: ARTCs, ARTDs, and members of the sirtuin family. In the present study, hARTC1-mediated mono-ADP-ribosylation was investigated in terms of the cellular compartments involved, target(s) and roles. The collected results demonstrated that hARTC1 protein and enzymatic activity is mainly localized to the endoplasmic reticulum (ER), in contrast to other ARTCs, which are either typically GPI-anchored enzymes in the plasma membrane, or secreted enzymes. Previous studies in my laboratory demonstrated that a protein macro domain was useful for the study of APD-ribosylation. The data reported here indicate, for the first time, that the macro domain can be used for immunofluorescence, allowing visualization of ADP-ribosylated proteins in intact cells, and in far-Western Blotting, allowing the detection of specific ADPribosylated targets. These methodologies were employed to demonstrate that the ER-localized chaperone, GRP78/BiP, was a prime target of hARTC1. A doubly mutated hARTC1 mutant was designed, and used as a specific control for hARTC1 expression. The mutant enzyme localized to the ER, but did not catalyze GRP78/BiP ADP-ribosylation. The demonstration that GRP78/BiP was mono-ADP-ribosylated by hARTC1 suggested that hARTC1 could be a key regulator of GRP78/BiP-mediated functions. Consistent with the key role of GRP78/BiP in the ER stress response, it was found that hARTC1 was activated during short-term cell treatment with ER stressors, resulting in acute GRP78/BiP ADP-ribosylation. However, the monoADP-ribosylation of the chaperone did not trigger an unfolded protein response. Recently, hARTC1 has been associated with cancer, suggesting a possible role in cell proliferation. In line with these findings, the results presented here demonstrated that hARTC1 over-expression inhibited cell proliferation