72 research outputs found
Identification and Characterization of a Bacterial Catalytic Scaffold with Specificity for Host Endomembrane Traffic
No full textThe fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signaling circuits. These circuits can be inhibited by bacterial effector proteins that post-translationally modify individual pathway components. However, there is emerging evidence that pathogens directly organize higher order signaling networks through enzyme scaffolding, and the identity of the effectors or their mechanisms of action are poorly understood. Here, we used a functional screen to identify the EHEC O157:H7 type III effector EspG as a regulator of endomembrane trafficking and we report ADP-ribosylation factor (ARF) GTPases and p21-activated kinases (PAK) as its relevant host substrates. The 2.5 Å crystal structure of EspG in complex with ARF6 shows how EspG blocks GAP-assisted GTP hydrolysis, revealing a potent mechanism of GTPase signaling inhibition at membrane organelles. In addition, the 2.8 Å crystal structure of EspG in complex with the autoinhibitory Iα3-helix of PAK2 defines a previously unknown catalytic site in EspG and provides an allosteric mechanism of kinase activation by a bacterial effector. Unexpectedly, ARF and PAK are organized on adjacent surfaces of EspG, suggesting its dual role as a “catalytic scaffold” that effectively reprograms cellular events through the functional assembly of GTPase-kinase signaling complex. Bidirectional vesicular transport between ER and Golgi is mediated largely by ARF and Rab GTPases, which orchestrate vesicle fission and fusion, respectively. How their activities are coordinated to define the successive steps of the secretory pathway and preserve traffic directionality is not well understood, in part due to the scarcity of molecular tools that simultaneously target ARF and Rab signaling. Here, we take advantage of the unique scaffolding properties of E.coli Secreted Protein G (EspG) to describe the critical role of ARF1/Rab1 spatiotemporal coordination in vesicular transport at the ER-Golgi Intermediate Compartment. Structural modeling and cellular studies show that EspG induces bidirectional traffic arrest by tethering vesicles through select ARF1-GTP/effector complexes and local inactivation of Rab1. Mechanistic insights presented in this study establish the effectiveness of a small bacterial catalytic scaffold in studying complex processes and reveal an alternative mechanism of immune regulation by an important human pathogen
Activation of PAK by a bacterial type III effector EspG reveals alternative mechanisms of GTPase pathway regulation.
No full textTargeting the Early Endosome-to-Golgi Transport of Shiga Toxins as a Therapeutic Strategy
No full textShiga toxin (STx) produced by Shigella and closely related Shiga toxin 1 and 2 (STx1 and STx2) synthesized by Shiga toxin-producing Escherichia coli (STEC) are bacterial AB5 toxins. All three toxins target kidney cells and may cause life-threatening renal disease. While Shigella infections can be treated with antibiotics, resistance is increasing. Moreover, antibiotic therapy is contraindicated for STEC, and there are no definitive treatments for STEC-induced disease. To exert cellular toxicity, STx, STx1, and STx2 must undergo retrograde trafficking to reach their cytosolic target, ribosomes. Direct transport from early endosomes to the Golgi apparatus is an essential step that allows the toxins to bypass degradative late endosomes and lysosomes. The essentiality of this transport step also makes it an ideal target for the development of small-molecule inhibitors of toxin trafficking as potential therapeutics. Here, we review the recent advances in understanding the molecular mechanisms of the early endosome-to-Golgi transport of STx, STx1, and STx2, as well as the development of small-molecule inhibitors of toxin trafficking that act at the endosome/Golgi interface
Genome-wide siRNA screen identifies UNC50 as a regulator of Shiga toxin 2 trafficking
No full textShiga toxins 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol. Early endosome-to-Golgi transport allows the toxins to evade degradation in lysosomes. Targeting this trafficking step has therapeutic promise, but the mechanism of trafficking for the more potent toxin STx2 is unclear. To identify host factors required for early endosome-to-Golgi trafficking of STx2, we performed a viability-based genome-wide siRNA screen in HeLa cells. 564, 535, and 196 genes were found to be required for toxicity induced by STx1 only, STx2 only, and both toxins, respectively. We focused on validating endosome/Golgi-localized hits specific for STx2 and found that depletion of UNC50 blocked early endosome-to-Golgi trafficking and induced lysosomal degradation of STx2. UNC50 acted by recruiting GBF1, an ADP ribosylation factor–guanine nucleotide exchange factor (ARF-GEF), to the Golgi. These results provide new information about STx2 trafficking mechanisms and may advance efforts to generate therapeutically viable toxin-trafficking inhibitors.</jats:p
Tamoxifen Derivatives Alter Retromer-Dependent Endosomal Tubulation and Sorting to Block Retrograde Trafficking of Shiga Toxins
No full textShiga toxin 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol of cells where they target ribosomes. As retrograde trafficking is essential for disease, inhibiting STx1/STx2 trafficking is therapeutically promising. Recently, we discovered that the chemotherapeutic drug tamoxifen potently inhibits the trafficking of STx1/STx2 at the critical early endosome-to-Golgi step. We further reported that the activity of tamoxifen against STx1/STx2 is independent of its selective estrogen receptor modulator (SERM) property and instead depends on its weakly basic chemical nature, which allows tamoxifen to increase endolysosomal pH and alter the recruitment of retromer to endosomes. The goal of the current work was to obtain a better understanding of the mechanism of action of tamoxifen against the more disease-relevant toxin STx2, and to differentiate between the roles of changes in endolysosomal pH and retromer function. Structure activity relationship (SAR) analyses revealed that a weakly basic amine group was essential for anti-STx2 activity. However, ability to deacidify endolysosomes was not obligatorily necessary because a tamoxifen derivative that did not increase endolysosomal pH exerted reduced, but measurable, activity. Additional assays demonstrated that protective derivatives inhibited the formation of retromer-dependent, Golgi-directed, endosomal tubules, which mediate endosome-to-Golgi transport, and the sorting of STx2 into these tubules. These results identify retromer-mediated endosomal tubulation and sorting to be fundamental processes impacted by tamoxifen; provide an explanation for the inhibitory effect of tamoxifen on STx2; and have important implications for the therapeutic use of tamoxifen, including its development for treating Shiga toxicosis
Selective Protection of an ARF1-GTP Signaling Axis by a Bacterial Scaffold Induces Bidirectional Trafficking Arrest
No full textSummaryBidirectional vesicular transport between the endoplasmic reticulum (ER) and Golgi is mediated largely by ARF and Rab GTPases, which orchestrate vesicle fission and fusion, respectively. How their activities are coordinated in order to define the successive steps of the secretory pathway and preserve traffic directionality is not well understood in part due to the scarcity of molecular tools that simultaneously target ARF and Rab signaling. Here, we take advantage of the unique scaffolding properties of E. coli secreted protein G (EspG) to describe the critical role of ARF1/Rab1 spatiotemporal coordination in vesicular transport at the ER-Golgi intermediate compartment. Structural modeling and cellular studies show that EspG induces bidirectional traffic arrest by tethering vesicles through select ARF1-GTP/effector complexes and local inactivation of Rab1. The mechanistic insights presented here establish the effectiveness of a small bacterial catalytic scaffold for studying complex processes and reveal an alternative mechanism of immune regulation by an important human pathogen
Proteolytic elimination of N-myristoyl modifications by the Shigella virulence factor IpaJ
No full textProtein N-myristoylation is a 14-carbon fatty-acid modification that is conserved across eukaryotic species and occurs on nearly 1% of the cellular proteome. The ability of the myristoyl group to facilitate dynamic protein-protein and protein-membrane interactions (known as the myristoyl switch) makes it an essential feature of many signal transduction systems. Thus pathogenic strategies that facilitate protein demyristoylation would markedly alter the signalling landscape of infected host cells. Here we describe an irreversible mechanism of protein demyristoylation catalysed by invasion plasmid antigen J (IpaJ), a previously uncharacterized Shigella flexneri type III effector protein with cysteine protease activity. A yeast genetic screen for IpaJ substrates identified ADP-ribosylation factor (ARF)1p and ARF2p, small molecular mass GTPases that regulate cargo transport through the Golgi apparatus. Mass spectrometry showed that IpaJ cleaved the peptide bond between N-myristoylated glycine-2 and asparagine-3 of human ARF1, thereby providing a new mechanism for host secretory inhibition by a bacterial pathogen. We further demonstrate that IpaJ cleaves an array of N-myristoylated proteins involved in cellular growth, signal transduction, autophagasome maturation and organelle function. Taken together, these findings show a previously unrecognized pathogenic mechanism for the site-specific elimination of N-myristoyl protein modification
JUNO Sensitivity on Proton Decay Searches
No full textThe Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid
scintillator detector designed to explore many topics in fundamental physics.
In this paper, the potential on searching for proton decay in mode with JUNO is investigated.The kaon and its decay particles feature a
clear three-fold coincidence signature that results in a high efficiency for
identification. Moreover, the excellent energy resolution of JUNO permits to
suppress the sizable background caused by other delayed signals. Based on these
advantages, the detection efficiency for the proton decay via is 36.9% with a background level of 0.2 events after 10 years of data
taking. The estimated sensitivity based on 200 kton-years exposure is years, competitive with the current best limits on the proton
lifetime in this channel.Comment: 14 pages, 12 figures, an author adde
TAO Conceptual Design Report: A Precision Measurement of the Reactor Antineutrino Spectrum with Sub-percent Energy Resolution
No full textInternational audienceThe Taishan Antineutrino Observatory (TAO, also known as JUNO-TAO) is a satellite experiment of the Jiangmen Underground Neutrino Observatory (JUNO). A ton-level liquid scintillator detector will be placed at about 30 m from a core of the Taishan Nuclear Power Plant. The reactor antineutrino spectrum will be measured with sub-percent energy resolution, to provide a reference spectrum for future reactor neutrino experiments, and to provide a benchmark measurement to test nuclear databases. A spherical acrylic vessel containing 2.8 ton gadolinium-doped liquid scintillator will be viewed by 10 m^2 Silicon Photomultipliers (SiPMs) of >50% photon detection efficiency with almost full coverage. The photoelectron yield is about 4500 per MeV, an order higher than any existing large-scale liquid scintillator detectors. The detector operates at -50 degree C to lower the dark noise of SiPMs to an acceptable level. The detector will measure about 2000 reactor antineutrinos per day, and is designed to be well shielded from cosmogenic backgrounds and ambient radioactivities to have about 10% background-to-signal ratio. The experiment is expected to start operation in 2022
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