scholarly journals Salmonella SipA mimics a cognate SNARE for host Syntaxin8 to promote fusion with early endosomes

2018 ◽  
Vol 217 (12) ◽  
pp. 4199-4214 ◽  
Author(s):  
Pawan Kishor Singh ◽  
Anjali Kapoor ◽  
Richa Madan Lomash ◽  
Kamal Kumar ◽  
Sukrut C. Kamerkar ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Enteric Fever ◽  
Caspase 3 ◽  
Arginine Residue ◽  
Terminal Domain ◽  
Trafficking Pathway ◽  
Early Endosomes ◽  
N Terminus ◽  
Snare Motif ◽  
Salmonella Survival

SipA is a major effector of Salmonella, which causes gastroenteritis and enteric fever. Caspase-3 cleaves SipA into two domains: the C-terminal domain regulates actin polymerization, whereas the function of the N terminus is unknown. We show that the cleaved SipA N terminus binds and recruits host Syntaxin8 (Syn8) to Salmonella-containing vacuoles (SCVs). The SipA N terminus contains a SNARE motif with a conserved arginine residue like mammalian R-SNAREs. SipAR204Q and SipA1–435R204Q do not bind Syn8, demonstrating that SipA mimics a cognate R-SNARE for Syn8. Consequently, Salmonella lacking SipA or that express the SipA1–435R204Q SNARE mutant are unable to recruit Syn8 to SCVs. Finally, we show that SipA mimicking an R-SNARE recruits Syn8, Syn13, and Syn7 to the SCV and promotes its fusion with early endosomes to potentially arrest its maturation. Our results reveal that SipA functionally substitutes endogenous SNAREs in order to hijack the host trafficking pathway and promote Salmonella survival.

On mechanisms of colicin import: the outer membrane quandary

2018 ◽  
Vol 475 (23) ◽  
pp. 3903-3915 ◽  
Author(s):  
William A. Cramer ◽  
Onkar Sharma ◽  
S.D. Zakharov
Keyword(s):  
Outer Membrane ◽  
Catalytic Domain ◽  
Efflux Pump ◽  
Crystal Structure Analysis ◽  
Multidrug Efflux Pump ◽  
Terminal Domain ◽  
Colicin E1 ◽  
N Terminus ◽  
T Domain

Current problems in the understanding of colicin import across the Escherichia coli outer membrane (OM), involving a range of cytotoxic mechanisms, are discussed: (I) Crystal structure analysis of colicin E3 (RNAase) with bound OM vitamin B12 receptor, BtuB, and of the N-terminal translocation (T) domain of E3 and E9 (DNAase) inserted into the OM OmpF porin, provide details of the initial interaction of the colicin central receptor (R)- and N-terminal T-domain with OM receptors/translocators. (II) Features of the translocon include: (a) high-affinity (Kd ≈ 10−9 M) binding of the E3 receptor-binding R-domain E3 to BtuB; (b) insertion of disordered colicin N-terminal domain into the OmpF trimer; (c) binding of the N-terminus, documented for colicin E9, to the TolB protein on the periplasmic side of OmpF. Reinsertion of the colicin N-terminus into the second of the three pores in OmpF implies a colicin anchor site on the periplasmic side of OmpF. (III) Studies on the insertion of nuclease colicins into the cytoplasmic compartment imply that translocation proceeds via the C-terminal catalytic domain, proposed here to insert through the unoccupied third pore of the OmpF trimer, consistent with in vitro occlusion of OmpF channels by the isolated E3 C-terminal domain. (IV) Discussion of channel-forming colicins focuses mainly on colicin E1 for which BtuB is receptor and the OM TolC protein the proposed translocator. The ability of TolC, part of a multidrug efflux pump, for which there is no precedent for an import function, to provide a trans-periplasmic import pathway for colicin E1, is questioned on the basis of an unfavorable hairpin conformation of colicin N-terminal peptides inserted into TolC.

scholarly journals Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition

2008 ◽  
Vol 411 (3) ◽  
pp. 523-530 ◽  
Author(s):  
Gary S. Laco ◽  
Yves Pommier
Keyword(s):  
Amino Acids ◽  
Topoisomerase I ◽  
Electrostatic Interactions ◽  
Functional Domain ◽  
Site Mutation ◽  
Supercoiled Dna ◽  
Terminal Domain ◽  
Tryptophan Residues ◽  
N Terminus

Human Top1 (topoisomerase I) relaxes supercoiled DNA during cell division and transcription. Top1 is composed of 765 amino acids and contains an unstructured N-terminal domain of 200 amino acids, and a structured functional domain of 565 amino acids that binds and relaxes supercoiled DNA. In the present study we examined the region spanning the junction of the N-terminal domain and functional domain (junction region). Analysis of several published Top1 structures revealed that three tryptophan residues formed a network of aromatic stacking interactions and electrostatic interactions that anchored the N-terminus of the functional domain to sub-domains containing the nose cone and active site. Mutation of the three tryptophan residues (Trp203/Trp205/Trp206) to an alanine residue, either individually or together, in silico revealed that the individual tryptophan residue's contribution to the tryptophan ‘anchor’ was additive. When the three tryptophan residues were mutated to alanine in vitro, the resulting mutant Top1 differed from wild-type Top1 in that it lacked processivity, exhibited resistance to camptothecin and was inactivated by urea. The results indicated that the tryptophan anchor stabilized the N-terminus of the functional domain and prevented the loss of Top1 structure and function.

scholarly journals The C-terminal domain of Clostridioides difficile TcdC is exposed on the bacterial cell surface

2019 ◽  
Author(s):  
Ana M. Oliveira Paiva ◽  
Leen de Jong ◽  
Annemieke H. Friggen ◽  
Wiep Klaas Smits ◽  
Jeroen Corver
Keyword(s):  
Sigma Factor ◽  
Negative Regulator ◽  
Toxin Gene ◽  
Cytoplasmic Localization ◽  
Terminal Domain ◽  
Clostridioides Difficile ◽  
C Terminus ◽  
N Terminus ◽  
Ob Fold ◽  
Toxin Gene Expression

AbstractClostridioides difficile is an anaerobic gram-positive bacterium that can can produce the large clostridial toxins, Toxin A and Toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also encodes the sigma factor TcdR, that positively regulates toxin gene expression, and TcdC, a putative negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor, however, several studies failed to show an association between tcdC genotype and toxin production. Consequently, TcdC function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is mediated through the OB-fold domain present at the C-terminus of the protein. This domain was previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating a cytoplasmic localization of the C-terminal OB-fold.In this study we aimed to obtain topological information on the C-terminus of TcdC. Using Scanning Cysteine Accessibility Mutagenesis and a HiBiT-based system, we demonstrate that the C-terminus of TcdC is located extracellularly. The extracellular location of TcdC is not compatible with direct binding of the OB-fold domain to intracellular nucleic acid or protein targets, and suggests a mechanism of action that is different from characterized anti-sigma factors.ImportanceTranscription of the C. difficile large clostrididial toxins (TcdA and TcdB) is directed by the sigma factor TcdR. TcdC has been implicated as a negative regulator, possible acting as an anti-sigma factor.Activity of TcdC has been mapped to its C-terminal OB fold domain. TcdC is anchored in the bacterial membrane, through its hydrophobic N-terminus and acting as an anti-sigma factor would require cytoplasmic localization of the C-terminal domain.Remarkably, topology predictions for TcdC suggest the N-terminus to be membrane localized and the C-terminal domain to be located extracellularly. Using independent assays, we show that the C-terminus of TcdC indeed is located in the extracellular environment, which is incompatible with its proposed role as anti-sigma factor in toxin regulation.

scholarly journals A proline repeat domain in the Notch co-activator MAML1 is important for the p300-mediated acetylation of MAML1

2007 ◽  
Vol 404 (2) ◽  
pp. 289-298 ◽  
Author(s):  
Mariana Saint Just Ribeiro ◽  
Magnus L. Hansson ◽  
Annika E. Wallberg
Keyword(s):  
Target Genes ◽  
Repeat Motif ◽  
Terminal Domain ◽  
Evolutionarily Conserved ◽  
Repeat Domain ◽  
Intracellular Receptor ◽  
N Terminus ◽  
Lysine Residues ◽  
Receptor Domain

Ligand activation of Notch leads to the release of Notch IC (the intracellular receptor domain), which translocates to the nucleus and interacts with the DNA-binding protein CSL to control expression of specific target genes. In addition to ligand-mediated activation, Notch signalling can be further modulated by interactions of Notch IC with a number of other proteins. MAML1 has previously been shown to act co-operatively with the histone acetyltransferase p300 in Notch IC-mediated transcription. In the present study we show that the N-terminal domain of MAML1 directly interacts with both p300 and histones, and the p300–MAML1 complex specifically acetylates histone H3 and H4 tails in chromatin. Furthermore, p300 acetylates MAML1 and evolutionarily conserved lysine residues in the MAML1 N-terminus are direct substrates for p300-mediated acetylation. The N-terminal domain of MAML1 contains a proline repeat motif (PXPAAPAP) that was previously shown to be present in p53 and important for the p300–p53 interaction. We show that the MAML1 proline repeat motif interacts with p300 and enhances the activity of the MAML1 N-terminus in vivo. These findings suggest that the N-terminal domain of MAML1 plays an important role in Notch-regulated transcription, by direct interactions with Notch, p300 and histones.

Withaferin A binds covalently to the N-terminal domain of annexin A2

2012 ◽  
Vol 393 (10) ◽  
pp. 1151-1163 ◽  
Author(s):  
Gabriel Ozorowski ◽  
Christopher M. Ryan ◽  
Julian P. Whitelegge ◽  
Hartmut Luecke
Keyword(s):  
Actin Polymerization ◽  
Inhibitory Effect ◽  
Annexin A2 ◽  
Actin Dynamics ◽  
Withaferin A ◽  
Core Domain ◽  
Cancer Pathways ◽  
Terminal Domain ◽  
C Terminus

Abstract Annexin A2 (AnxA2), a 38-kDa member of the Ca2+-binding annexin family, has been implicated in numerous cancer pathways. Withaferin A (WithfA), a natural plant compound, has been reported previously to bind covalently to Cys133 of the AnxA2 core domain leading to a reduction of the invasive capabilities of cancer cells by altering their cytoskeleton. We show here that AnxA2 has an inhibitory effect on actin polymerization, and a modification with WithfA significantly increases this inhibitory role of AnxA2. Using mass spectrometry and single-site mutants, we localized the WithfA-AnxA2 interaction to the N-terminal domain of AnxA2 where WithfA binds covalently to Cys9. Whereas binding to F-actin filaments has been mapped to the C terminus of AnxA2, our results suggest that the N-terminal domain modified by WithfA may also play a role in the AnxA2-actin interaction. The binding of WithfA may regulate the AnxA2-mediated actin dynamics in two distinct ways: (i) the increase of F-actin bundling activity by the Anx2/p11 heterotetramer and (ii) the decrease of actin polymerization as a result of the increased affinity of AnxA2 to the barbed end of actin microfilaments. We demonstrate the susceptibility of Cys9 of AnxA2 to chemical modifications and exclude Cys133 as a binding site for WithfA.

scholarly journals A presynaptic endosomal trafficking pathway controls synaptic growth signaling

2011 ◽  
Vol 193 (1) ◽  
pp. 201-217 ◽  
Author(s):  
Avital A. Rodal ◽  
Aline D. Blunk ◽  
Yulia Akbergenova ◽  
Ramon A. Jorquera ◽  
Lauren K. Buhl ◽  
...  
Keyword(s):  
Endocytic Pathway ◽  
Physical Interaction ◽  
Endosomal Trafficking ◽  
Synaptic Growth ◽  
Recycling Endosomes ◽  
Sorting Nexin ◽  
Trafficking Pathway ◽  
Early Endosomes ◽  
Protein Receptors ◽  
Escrt Complex

Structural remodeling of synapses in response to growth signals leads to long-lasting alterations in neuronal function in many systems. Synaptic growth factor receptors alter their signaling properties during transit through the endocytic pathway, but the mechanisms controlling cargo traffic between endocytic compartments remain unclear. Nwk (Nervous Wreck) is a presynaptic F-BAR/SH3 protein that regulates synaptic growth signaling in Drosophila melanogaster. In this paper, we show that Nwk acts through a physical interaction with sorting nexin 16 (SNX16). SNX16 promotes synaptic growth signaling by activated bone morphogenic protein receptors, and live imaging in neurons reveals that SNX16-positive early endosomes undergo transient interactions with Nwk-containing recycling endosomes. We identify an alternative signal termination pathway in the absence of Snx16 that is controlled by endosomal sorting complex required for transport (ESCRT)–mediated internalization of receptors into the endosomal lumen. Our results define a presynaptic trafficking pathway mediated by SNX16, NWK, and the ESCRT complex that functions to control synaptic growth signaling at the interface between endosomal compartments.

scholarly journals SipA Activation of Caspase-3 Is a Decisive Mediator of Host Cell Survival at Early Stages of Salmonella enterica Serovar Typhimurium Infection

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Anne McIntosh ◽  
Lynsey M. Meikle ◽  
Michael J. Ormsby ◽  
Beth A. McCormick ◽  
John M. Christie ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Actin Polymerization ◽  
Caspase 3 ◽  
Ex Vivo ◽  
Cell Types ◽  
Laser Scanning Microscopy ◽  
Limiting Factor ◽  
Dual Function ◽  
Content Type

ABSTRACT Salmonella invasion protein A (SipA) is a dual-function effector protein that plays roles in both actin polymerization and caspase-3 activation in intestinal epithelial cells. To date its function in other cell types has remained largely unknown despite its expression in multiple cell types and its extracellular secretion during infection. Here we show that in macrophages SipA induces increased caspase-3 activation early in infection. This activation required a threshold level of SipA linked to multiplicity of infection and may be a limiting factor controlling bacterial numbers in infected macrophages. In polymorphonuclear leukocytes, SipA or other Salmonella pathogenicity island 1 effectors had no effect on induction of caspase-3 activation either alone or in the presence of whole bacteria. Tagging of SipA with the small fluorescent phiLOV tag, which can pass through the type three secretion system, allowed visualization and quantification of caspase-3 activation by SipA-phiLOV in macrophages. Additionally, SipA-phiLOV activation of caspase-3 could be tracked in the intestine through multiphoton laser scanning microscopy in an ex vivo intestinal model. This allowed visualization of areas where the intestinal epithelium had been compromised and demonstrated the potential use of this fluorescent tag for in vivo tracking of individual effectors.

scholarly journals Atg15 in Saccharomyces cerevisiae consists of two functionally distinct domains

2021 ◽  
pp. mbc.E20-07-0500
Author(s):  
Eri Hirata ◽  
Kyo Shirai ◽  
Tatsuya Kawaoka ◽  
Kosuke Sato ◽  
Fumito Kodama ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Multivesicular Body ◽  
Catalytic Triad ◽  
Double Membrane ◽  
Terminal Domain ◽  
C Terminus ◽  
N Terminus ◽  
Membrane Bound ◽  
Important Residue

Autophagy is a cellular degradation system widely conserved among eukaryotes. During autophagy, cytoplasmic materials fated for degradation are compartmentalized in double membrane–bound organelles called autophagosomes. After fusing with the vacuole, their inner membrane–bound structures are released into the vacuolar lumen to become autophagic bodies and eventually degraded by vacuolar hydrolases. Atg15 is a lipase essential for disintegration of autophagic body membranes and has a transmembrane domain at the N-terminus and a lipase domain at the C-terminus. However, the roles of both domains in vivo are not well understood. In this study, we found that the N-terminal domain alone can travel to the vacuole via the multivesicular body pathway, and that targeting of the C-terminal lipase domain to the vacuole is required for degradation of autophagic bodies. Moreover, we found that the C-terminal domain could disintegrate autophagic bodies when it was transported to the vacuole via the Pho8 pathway instead of the multivesicular body pathway. Finally, we identified H435 as one of the residues composing the putative catalytic triad, and W466 as an important residue for degradation of autophagic bodies. This study may provide a clue to understanding how the C-terminal lipase domain recognizes autophagic bodies to degrade them. [Media: see text] [Media: see text]

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