A Solvent-Exposed Patch in Chaperone-Bound YopE Is Required for Translocation by the Type III Secretion System

Most effector proteins of bacterial type III secretion (T3S) systems require chaperone proteins for translocation into host cells. Such effectors are bound by chaperones in a conserved and characteristic manner, with the chaperone-binding (Cb) region of the effector wound around the chaperone in a highly extended conformation. This conformation has been suggested to serve as a translocation signal in promoting the association between the chaperone-effector complex and a bacterial component required for translocation. We sought to test a prediction of this model by identifying a potential association site for the Yersinia pseudotuberculosis chaperone-effector pair SycE-YopE. We identified a set of residues in the YopE Cb region that are required for translocation but are dispensable for expression, SycE binding, secretion into the extrabacterial milieu, and stability in mammalian cells. These residues form a solvent-exposed patch on the surface of the chaperone-bound Cb region, and thus their effect on translocation is consistent with the structure of the chaperone-bound Cb region serving as a signal for translocation.The type III secretion (T3S) system is crucial to the virulence of many Gram-negative bacterial pathogens (14, 18). These pathogens use the T3S system to translocate effector proteins from the bacterial cytosol directly into the interior of host cells. Typically, effectors contribute to the virulence of the pathogen by modifying or interacting with specific host cell targets. Effector proteins are arranged in a modular fashion, with sequence elements required for translocation located within their N-terminal ∼100 to 150 residues and with domains that interact with host components following (20, 49). For most effectors, including the extensively characterized Yersinia effector YopE (23 kDa), two different N-terminal sequence elements are required for translocation into host cells. The first, termed signal 1 (S1), occurs at the very N terminus and spans ∼10 to 15 residues (Fig. ​(Fig.1A).1A). The S1 element is highly degenerate in sequence (29, 36), and in YopE the S1 region has been shown to be structurally disordered (37). While the S1 element is sufficient for the nonphysiological process of secretion of effectors into the extrabacterial milieu, it is not sufficient for translocation into host cells (41, 44).Open in a separate windowFIG. 1.The SycE-YopE chaperone-effector complex. (A) Schematic of YopE domains. S1, signal 1; Cb, chaperone-binding region; RhoGAP, Rho GTPase activating protein domain. Residue numbers for domain boundaries are indicated. (B) Structure of the SycE-YopE(Cb) complex. The Cb region of YopE (red) and the SycE dimer (gray) are shown in C-α stick representation. Side chains that were mutated in the YopE Cb region are depicted for the following residues: V23 (green), E25 (blue), S27 (yellow), R29 (cyan), and S32 (magenta). Molecular figures were made with PyMol (http://pymol.sourceforge.net). (C) Enlarged view of boxed region in panel B. (D) Molecular surface representation of the SycE-YopE(Cb) complex. The YopE(Cb) surface is shown in red, except for surfaces formed by V23 (green), E25 (blue), S27 (yellow), R29 (cyan), and S32 (magenta). The SycE homodimer is shown in gray.For translocation, a second sequence element, termed the chaperone-binding (Cb) region, is required (Fig. ​(Fig.1A)1A) (44). The Cb region, which consists of ∼50 to 100 residues downstream of S1, promotes translocation only when bound by a member of the effector-dedicated chaperone protein family (49). The chaperone protein is dissociated from the effector by a T3S ATPase (2), as shown in Salmonella, and remains in the bacterial cytosol upon transport of the effector (17). There are a large number of such effector-dedicated chaperones, as each individual chaperone protein binds just a single effector, or in some cases a few effectors. These chaperones are divergent in sequence (≤20% identity) but have similar protein folds, dimeric oligomerization states, and effector-binding modes (4, 5, 10, 28, 30, 31, 35, 42, 43, 46, 47). In the binding mode, the Cb region of the effector winds around the surface of the dimeric chaperone in a highly extended fashion (Fig. 1B and C). The chaperone-bound Cb region lacks independent tertiary structure but has short α-helical, β-strand, and random coil stretches that contact the chaperone. This mode has been observed for the Cb region of YopE (residues 23 to 78) bound to its homodimeric chaperone, SycE (29 kDa per dimer) (5), as well as for a number of other chaperone-effector complexes (28, 35, 42, 46). The structural conservation among these chaperone-effector pairs is especially striking because the various effectors have no obvious sequence relationship to one another in their Cb regions. However, recent work showed that the chaperone-Cb-region binding mode can be predicted from de novo models (22).The functional significance of the conserved chaperone-Cb-region structure is not yet clear, but this structure has been suggested to constitute a translocation signal (5). It has been proposed that the conformation of the chaperone-bound Cb region promotes association between the chaperone-effector complex and a bacterial component required for translocation, i.e., a receptor that recognizes this three-dimensional translocation signal. Consistent with this hypothesis, recent evidence indicated that the chaperone SycE brings about the structuring of an otherwise unstructured Cb region in YopE (37). Nuclear magnetic resonance studies demonstrated that the Cb region of YopE in its free state is unstructured and flexible and that it undergoes a pronounced disorder-to-order transition upon SycE binding. The effect of SycE was strictly localized to the Cb region, while other portions of YopE, including the S1 region and the C-terminal RhoGAP domain, were impervious to SycE binding. Additional lines of evidence also support the translocation signal model of chaperone action (8, 13, 50).We sought here to test a prediction of the translocation signal model. This is the prediction that the surface of the chaperone-Cb-region complex ought to provide a receptor-binding site. We surmised that mutation of such a solvent-exposed site in the Cb region of YopE would affect translocation but not chaperone binding. Mutations of residues in the Cb region that contact the chaperone have already been shown to reduce translocation (5, 28, 41). These are simple to explain, as they diminish the affinity of the effector for the chaperone. In contrast, residues in the Cb region that are exposed in the chaperone-Cb-region complex, and hence available to form a receptor-binding site, have not yet been shown to be important for translocation. We now report the identification of such residues in the YopE Cb region. These residues are crucial for translocation but not for other aspects of YopE function, including steady-state expression, binding to SycE, secretion, and stability in mammalian cells. These results are consistent with a translocation signal model of action for the chaperone-bound Cb region and identify a potential receptor-binding site.     

Bioconversion of para-substituted monophenolic compounds into corresponding catechols by alginate entrapped cells ofMucuna pruriens

Alginate-entrapped cells ofM. pruriens were able to convert a number of parasubstituted monophenolic compounds into the corresponding catechols. All catechols produced were released into the medium, which offered the opportunity to isolate these products via a relatively simple procedure. Prepurification was performed on a Sephadex G10 gel and catechols were concentrated on Affigel 601. The identity of all products was confirmed with combined liquid chromatography/mass spectrometry (LC/MS) or MS using the desorption chemical ionization technique, depending on the catechol. For the entrapped cells and for a cell homogenate prepared of the same cell line ofM. pruriens the substrate specificities were qualitatively identical when judged on initial rates of synthesis calculated on protein basis.     

Bioconversion of naturally occurring precursors and related synthetic compounds using plant cell cultures.

The nearly unlimited enzymatic potential of cultured plant cells can basically be employed for bioconversion purposes. Plant enzymes are able to catalyze regio- and stereospecific reactions and can therefore be applied to the production of compounds of pharmaceutical interest. Naturally occurring as well as related synthetic compounds may be used as precursors. A review of the current status of such bioconversions is given. It includes the performance of bioconversions by freely suspended and immobilized plant cells or enzyme preparations. In addition, the kinetic aspects of immobilized plant cells are discussed. Special attention is paid to the bioconversion of poorly or water insoluble precursors. Finally, a model scheme for the development of a commercially available drug, produced by bioconversion, and perspectives are discussed.     

Refined mapping of the CSNU3 gene to a 1.8-Mb region on chromosome 19q13.1 using historical recombinants in Libyan Jewish cystinuria patients.

Cystinuria is a genetic disease manifested by the development of kidney stones. In some patients, the disease is caused by mutations in the SLC3A1 gene located on chromosome 2p. In others, the disease is caused by a gene that maps to chromosome 19q, but has not yet been cloned. Cystinuria is very common among Jews of Libyan ancestry living in Israel. Previously we have shown that the disease-causing gene in Libyan Jews maps to an 8-cM interval on chromosome 19q between the markers D19S409 and D19S208. Several markers from chromosome 19q showed strong linkage disequilibrium, and a specific haplotype was found in more than half of the carrier chromosomes. In this study we have analyzed Libyan Jewish cystinuria families with eight markers from within the interval containing the gene. Seven of these markers showed significant linkage disequilibrium. A common haplotype was found in 16 of the 17 carrier chromosomes. Analysis of historical recombinants placed the gene in a 1.8-Mb interval between the markers D19S430 and D19S874. Two segments of the historical carrier chromosome used to calculate the mutation's age revealed that the disease-causing mutation was introduced into this population 7-16 generations ago.     

MYC Promotes Bone Marrow Stem Cell Dysfunction in Fanconi Anemia

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Cyclodextrin-facilitated bioconversion of 17 beta-estradiol by a phenoloxidase from Mucuna pruriens cell cultures.

After complexation with beta-cyclodextrin, the phenolic steroid 17 beta-estradiol could be ortho-hydroxylated into a catechol, mainly 4-hydroxyestradiol, by a phenoloxidase from in vitro grown cells of Mucuna pruriens. By complexation with beta-cyclodextrin the solubility of the steroid increased from almost insoluble to 660 microM. The bioconversion efficiency after 72 hr increased in the following order: freely suspended cells (0%), immobilized cells (1%), cell homogenate (6%), phenoloxidase preparation (40%). Mushroom tyrosinase converted 17 beta-estradiol, as a complex with beta-cyclodextrin, solely into 2-hydroxyestradiol, with a maximal yield of 30% after 6-8 hr. Uncomplexed 17 beta-estradiol was not converted at all in any of these systems.