The Release of Outer Membrane Vesicles from the Strains of Enterotoxigenic Escherichia coli

The clinically isolated heat labile enterotoxin (LT)-producing strains of Escherichia coli can be separated into two groups, namely spontaneous LT-releasing strain and non-spontaneous LT-releasing strain, based on their phenotypes of spontaneous release of LT into the culture medium. The phenotype of spontaneous LT release was observed to correlate closely with the phenotype of the release of numerous small vesicles into the culture medium. Both morphological and biological examinations of the vesicle showed that the vesicle was released from the outer membrane. It can, therefore, be assumed that LT may be released from the cell at the time the vesicles form.     

Novel proteases from the genome of the carnivorous plant Drosera capensis: Structural prediction and comparative analysis

In his 1875 monograph on insectivorous plants, Darwin described the feeding reactions of Drosera flypaper traps and predicted that their secretions contained a “ferment” similar to mammalian pepsin, an aspartic protease. Here we report a high‐quality draft genome sequence for the cape sundew, Drosera capensis, the first genome of a carnivorous plant from order Caryophyllales, which also includes the Venus flytrap (Dionaea) and the tropical pitcher plants (Nepenthes). This species was selected in part for its hardiness and ease of cultivation, making it an excellent model organism for further investigations of plant carnivory. Analysis of predicted protein sequences yields genes encoding proteases homologous to those found in other plants, some of which display sequence and structural features that suggest novel functionalities. Because the sequence similarity to proteins of known structure is in most cases too low for traditional homology modeling, 3D structures of representative proteases are predicted using comparative modeling with all‐atom refinement. Although the overall folds and active residues for these proteins are conserved, we find structural and sequence differences consistent with a diversity of substrate recognition patterns. Finally, we predict differences in substrate specificities using in silico experiments, providing targets for structure/function studies of novel enzymes with biological and technological significance. Proteins 2016; 84:1517–1533. © 2016 Wiley Periodicals, Inc.     

An improved fluorescent substrate for assaying soluble and membrane-associated ADAM family member activities

A fluorescent resonance energy transfer substrate with improved sensitivity for ADAM17, −10, and −9 (where ADAM represents a disintegrin and metalloproteinase) has been designed. The new substrate, Dabcyl-Pro-Arg-Ala-Ala-Ala-Homophe-Thr-Ser-Pro-Lys(FAM)-NH₂, has specificity constants of 6.3 (±0.3) × 10⁴ M⁻¹ s⁻¹ and 2.4 (±0.3) × 10³ M⁻¹ s⁻¹ for ADAM17 and ADAM10, respectively. The substrate is more sensitive than widely used peptides based on the precursor tumor necrosis factor-alpha (TNF-alpha) cleavage site, PEPDAB010 or Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(FAM)-NH₂ and Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Arg-NH₂. ADAM9 also processes the new peptide more than 18-fold better than the TNF-alpha-based substrates. The new substrate has a unique selectivity profile because it is processed less efficiently by ADAM8 and MMP1, −2, −3, −8, −9, −12, and −14. This substrate provides a unique tool in which to assess ADAM17, −10, and −9 activities.     

Proteasome dynamics between proliferation and quiescence stages of Saccharomyces cerevisiae

The ubiquitin-proteasome system (UPS) plays a critical role in cellular protein homeostasis and is required for the turnover of short-lived and unwanted proteins, which are targeted by poly-ubiquitination for degradation. Proteasome is the key protease of UPS and consists of multiple subunits, which are organized into a catalytic core particle (CP) and a regulatory particle (RP). In Saccharomyces cerevisiae, proteasome holo-enzymes are engaged in degrading poly-ubiquitinated substrates and are mostly localized in the nucleus during cell proliferation. While in quiescence, the RP and CP are sequestered into motile and reversible storage granules in the cytoplasm, called proteasome storage granules (PSGs). The reversible nature of PSGs allows the proteasomes to be transported back into the nucleus upon exit from quiescence. Nuclear import of RP and CP through nuclear pores occurs via the canonical pathway that includes the importin-αβ heterodimer and takes advantage of the Ran-GTP gradient across the nuclear membrane. Dependent on the growth stage, either inactive precursor complexes or mature holo-enzymes are imported into the nucleus. The present review discusses the dynamics of proteasomes including their assembly, nucleo-cytoplasmic transport during proliferation and the sequestration of proteasomes into PSGs during quiescence.

     

Vibrio cholerae hemolysin: The β-trefoil domain is required for folding to the native conformation

Vibrio cholerae cytolysin/hemolysin (VCC) is a 65 kDa β-pore-forming toxin causing lysis and death of eukaryotic cells. Apart from the core cytolysin domain, VCC has two lectin domains with β-trefoil and β-prism folds. The β-prism domain binds to cell surface carbohydrate receptors; the role of the β-trefoil domain is unknown. Here, we show that the pro-VCC mutant without the β-trefoil domain formed aggregates highly susceptible to proteolysis, suggesting lack of a properly folded compact structure. The VCC variants with Trp532Ala or Trp534Ala mutation in the β-trefoil domain formed hemolytically inactive, protease-resistant, ring-shaped SDS-labile oligomers with diameters of ~19 nm. The Trp mutation induced a dramatic change in the global conformation of VCC, as indicated by: (a) the change in surface polarity from hydrophobic to hydrophilic; (b) movement of core Trp residues to the protein-water interface; and (c) decrease in reactivity to the anti-VCC antibody by >100-fold. In fact, the mutant VCC had little similarity to the wild toxin. However, the association constant for the carbohydrate-dependent interaction mediated by the β-prism domain decreased marginally from ~3×10⁸ to ~5×10⁷ M^?1. We interpret the observations by proposing: (a) the β-trefoil domain is critical to the folding of the cytolysin domain to its active conformation; (b) the β-prism domain is an autonomous folding unit.     

Production of yeast killer toxin in experimentally infected animals

The ability of a killer yeast (Pichia anomala, UCSC 25F) to produce toxin in vivo was demonstrated, for the first time, in tissues of normal and immunosuppressed experimentally infected mice by means of a fluorescent antibody technique and a killer toxin specific monoclonal antibody. The possible significance of the findings is discussed.     

Killer toxin of Hanseniaspora uvarum

The yeast Hanseniaspora uvarum liberates a killer toxin lethal to sensitive strains of the species Saccharomyces cerevisiae. Secretion of this killer toxin was inhibited by tunicamycin, an inhibitor of N-glycosylation, although the mature killer protein did not show any detectable carbohydrate structures. Culture supernatants of the killer strain were concentrated by ultrafiltration and the extracellular killer toxin was precipitated with ethanol and purified by ion exchange chromatography. SDS-PAGE of the electrophoretically homogenous killer protein indicated an apparent molecular mass of 18,000.Additional investigations of the primary toxin binding sites within the cell wall of sensitive yeast strains showed that the killer toxin of Hanseniaspora uvarum is bound by -1, 6-d-glucans.