55 research outputs found

    Crystal Structures of the Type III Effector Protein AvrPphF and Its Chaperone Reveal Residues Required for Plant Pathogenesis

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    The avrPphF locus from Pseudomonas syringae pv. phaseolicola, the causative agent of bean halo-blight disease, encodes proteins which either enhance virulence on susceptible hosts or elicit defense responses on hosts carrying the R1 resistance gene. Here we present the crystal structures of the two proteins from the avrPphF operon. The structure of AvrPphF ORF1 is strikingly reminiscent of type III chaperones from bacterial pathogens of animals, indicating structural conservation of these specialized chaperones, despite high sequence divergence. The AvrPphF ORF2 effector adopts a novel "mushroom"-like structure containing "head" and "stalk" subdomains. The head subdomain possesses limited structural homology to the catalytic domain of bacterial ADP-ribosyltransferases (ADP-RTs), though no ADP-RT activity was detected for AvrPphF ORF2 in standard assays. Nonetheless, this structural similarity identified two clusters of conserved surface-exposed residues important for both virulence mediated by AvrPphF ORF2 and recognition of this effector by bean plants expressing the R1 resistance gene

    The DH and PH Domains of Trio Coordinately Engage Rho GTPases for their Efficient Activation

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    Rho-family GTPases are activated by the exchange of bound GDP for GTP, a process that is catalyzed by Dbl-family guanine nucleotide exchange factors (GEFs). The catalytic unit of Dbl-family GEFs consists of a Dbl-homology (DH) domain followed almost invariantly by a pleckstrin-homology (PH) domain. The majority of the catalytic interface forms between the switch regions of the GTPase and the DH domain, but full catalytic activity often requires the associated PH domain. Although PH domains are usually characterized as lipid binding regions, they also participate in protein-protein interactions. For example, the DH-associated PH domain of Dbs must contact its cognate GTPases for efficient exchange. Similarly, the N-terminal DH/PH fragment of Trio, which catalyzes exchange on both Rac1 and RhoG, is four-fold more active in vitro than the isolated DH domain. Given continued uncertainty regarding functional roles of DH-associated PH domains, we have undertaken structural and functional analyses of the N-terminal DH/PH cassette of Trio. The crystal structure of this fragment of Trio bound to nucleotide-depleted Rac1 highlights the engagement of the PH domain with Rac1 and substitution of residues involved in this interface substantially diminishes activation of Rac1 and RhoG. Also, these mutations significantly reduce the ability of full-length Trio to induce neurite outgrowth dependent on RhoG activation in PC-12 cells. Overall, these studies substantiate a general role for DH-associated PH domains in directly engaging Rho GTPases for efficient guanine nucleotide exchange and support a parsimonious explanation for the essentially invariant linkage between DH and PH domains

    Rapid, directed transport of DC-SIGN clusters in the plasma membrane

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    C-type lectins, including dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN), are all-purpose pathogen receptors that exist in nanoclusters in plasma membranes of dendritic cells. A small fraction of these clusters, obvious from the videos, can undergo rapid, directed transport in the plane of the plasma membrane at average speeds of more than 1 μm/s in both dendritic cells and MX DC-SIGN murine fibroblasts ectopically expressing DC-SIGN. Surprisingly, instantaneous speeds can be considerably greater. In MX DC-SIGN cells, many cluster trajectories are colinear with microtubules that reside close to the ventral membrane, and the microtubule-depolymerizing drug, nocodazole, markedly reduced the areal density of directed movement trajectories, suggesting a microtubule motor–driven transport mechanism; by contrast, latrunculin A, which affects the actin network, did not depress this movement. Rapid, retrograde movement of DC-SIGN may be an efficient mechanism for bringing bound pathogen on the leading edge and projections of dendritic cells to the perinuclear region for internalization and processing. Dengue virus bound to DC-SIGN on dendritic projections was rapidly transported toward the cell center. The existence of this movement within the plasma membrane points to an unexpected lateral transport mechanism in mammalian cells and challenges our current concepts of cortex-membrane interactions

    Structural analyses to identify selective inhibitors of glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme

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    Detailed structural comparisons of sperm-specific glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS) and the somatic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) isozyme should facilitate the identification of selective GAPDHS inhibitors for contraceptive development

    Cell Therapy in Veterinary Medicine as a Proof-of-Concept for Human Therapies: Perspectives From the North American Veterinary Regenerative Medicine Association

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    In the past decade, the potential to translate scientific discoveries in the area of regenerative therapeutics in veterinary species to novel, effective human therapies has gained interest from the scientific and public domains. Translational research using a One Health approach provides a fundamental link between basic biomedical research and medical clinical practice, with the goal of developing strategies for curing or preventing disease and ameliorating pain and suffering in companion animals and humans alike. Veterinary clinical trials in client-owned companion animals affected with naturally occurring, spontaneous disease can inform human clinical trials and significantly improve their outcomes. Innovative cell therapies are an area of rapid development that can benefit from non-traditional and clinically relevant animal models of disease. This manuscript outlines cell types and therapeutic applications that are currently being investigated in companion animals that are affected by naturally occurring diseases. We further discuss how such investigations impact translational efforts into the human medical field, including a critical evaluation of their benefits and shortcomings. Here, leaders in the field of veterinary regenerative medicine argue that experience gained through the use of cell therapies in companion animals with naturally occurring diseases represent a unique and under-utilized resource that could serve as a critical bridge between laboratory/preclinical models and successful human clinical trials through a One-Health approach

    Release of autoinhibition of ASEF by APC leads to CDC42 activation and tumor suppression

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    Autoinhibition of the Rho guanine nucleotide exchange factor ASEF is relieved by interaction with the APC tumor suppressor. Here we show that binding of the armadillo repeats of APC to a ‘core APC-binding’ (CAB) motif within ASEF, or truncation of the SH3 domain of ASEF, relieves autoinhibition, allowing the specific activation of CDC42. Structural determination of autoinhibited ASEF reveals that the SH3 domain forms an extensive interface with the catalytic DH and PH domains to obstruct binding and activation of CDC42, and the CAB motif is positioned adjacent to the SH3 domain to facilitate activation by APC. In colorectal cancer cell lines, full-length, but not truncated, APC activates CDC42 in an ASEF-dependent manner to suppress anchorage-independent growth. We therefore propose a model in which ASEF acts as a tumor suppressor when activated by APC and inactivation of ASEF by mutation or APC truncation promotes tumorigenesis

    Structural and Functional Analysis of the Human Nuclear Xenobiotic Receptor PXR in Complex with RXRα

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    The human nuclear xenobiotic receptor PXR recognizes a range of potentially harmful drugs and endobiotic chemicals, but must complex with the nuclear receptor RXRα to control the expression of numerous drug metabolism genes. To date, the structural basis and functional consequences of this interaction have remained unclear. Here we present 2.8 Å resolution crystal structures of the heterodimeric complex formed between the ligand binding domains (LBDs) of human PXR and RXRα. These structures establish that PXR and RXRα form a heterotetramer unprecedented in the nuclear receptor family of ligand-regulated transcription factors. We further show that both PXR and RXRα bind to the transcriptional coregulator SRC-1 with higher affinity when they are part of the PXR-RXRα heterotetramer complex than they do when each LBD is examined alone. Furthermore, we purify the full-length forms of each receptor from recombinant bacterial expression systems, and characterize their interactions with a range of direct and everted repeat DNA elements. Taken together, these data advance our understanding of PXR, the master regulator of drug metabolism gene expression in humans, in its functional partnership with RXRα

    Structure of the Claudin-binding Domain of Clostridium perfringens Enterotoxin

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    Clostridium perfringens enterotoxin is a common cause of food-borne and antibiotic-associated diarrhea. The toxin's receptors on intestinal epithelial cells include claudin-3 and -4, members of a large family of tight junction proteins. Toxin-induced cytolytic pore formation requires residues in the NH(2)-terminal half, whereas residues near the COOH terminus are required for binding to claudins. The claudin-binding COOH-terminal domain is not toxic and is currently under investigation as a potential drug absorption enhancer. Because claudin-4 is overexpressed on some human cancers, the toxin is also being investigated for targeting chemotherapy. Our aim was to solve the structure of the claudin-binding domain to advance its therapeutic applications. The structure of a 14-kDa fragment containing residues 194 to the native COOH terminus at position 319 was solved by x-ray diffraction to a resolution of 1.75A. The structure is a nine-strand beta sandwich with previously unappreciated similarity to the receptor-binding domains of several other toxins of spore-forming bacteria, including the collagen-binding domain of ColG from Clostridium histolyticum and the large Cry family of toxins (including Cry4Ba) of Bacillus thuringiensis. Correlations with previous studies suggest that the claudin-4 binding site is on a large surface loop between strands beta8 and beta9 or includes these strands. The sequence that was crystallized (residues 194-319) binds to purified human claudin-4 with a 1:1 stoichiometry and affinity in the submicromolar range similar to that observed for binding of native toxin to cells. Our results provide a structural framework to advance therapeutic applications of the toxin and suggest a common ancestor for several receptor-binding domains of bacterial toxins

    Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway ,

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    G protein-coupled receptors mediate cellular responses to a wide variety of stimuli, including taste, light and neurotransmitters. In the yeast Saccharomyces cerevisiae, activation of the pheromone pathway triggers events leading to mating. The view had long been held that the G protein-mediated signal occurs principally at the plasma membrane. Recently, it has been shown that the G protein α subunit Gpa1 can promote signaling at endosomes and requires two components of the sole phosphatidylinositol-3-kinase in yeast, Vps15 and Vps34. Vps15 contains multiple WD repeats and also binds to Gpa1 preferentially in the GDP-bound state; these observations led us to hypothesize that Vps15 may function as a G protein β subunit at the endosome. Here we show an X-ray crystal structure of the Vps15 WD domain that reveals a seven-bladed propeller resembling that of typical Gβ subunits. We show further that the WD domain is sufficient to bind Gpa1 as well as to Atg14, a potential Gγ protein that exists in a complex with Vps15. The Vps15 kinase domain together with the intermediate domain (linking the kinase and WD domains) also contributes to Gpa1 binding, and is necessary for Vps15 to sustain G protein signaling. These findings reveal that the Vps15 Gβ-like domain serves as a scaffold to assemble Gpa1 and Atg14, whereas the kinase and intermediate domains are required for proper signaling at the endosome

    Structural and Biochemical Characterization of CIB1 Delineates a New Family of EF-hand-containing Proteins

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    CIB1 (CIB) is an EF-hand-containing protein that binds multiple effector proteins, including the platelet alphaIIbbeta3 integrin and several serine/threonine kinases and potentially modulates their function. The crystal structure for Ca(2+)-bound CIB1 has been determined at 2.0 A resolution and reveals a compact alpha-helical protein containing four EF-hands, the last two of which bind calcium ions in the standard fashion seen in many other EF-hand proteins. CIB1 shares high structural similarity with calcineurin B and the neuronal calcium sensor (NCS) family of EF-hand-containing proteins. Most importantly, like calcineurin B and NCS proteins, which possess a large hydrophobic pocket necessary for ligand binding, CIB1 contains a hydrophobic pocket that has been implicated in ligand binding by previous mutational analysis. However, unlike several NCS proteins, Ca(2+)-bound CIB1 is largely monomeric whether bound to a relevant peptide ligand or ligand-free. Differences in structure, oligomeric state, and phylogeny define a new family of CIB1-related proteins that extends from arthropods to humans
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