The Helicobacter pylori vacuolating cytotoxin: from cellular vacuolation to immunosuppressive activities

Helicobacter pylori is a highly successful bacterial pathogen of humans, infecting the stomach of more than half of the worlds population. The H. pylori infection results in chronic gastritis, eventually followed by peptic ulceration and, more rarely, gastric cancer. H. pylori has developed a unique set of virulence factors, actively supporting its survival in the special ecological niche of the human stomach. Vacuolating cytotoxin (VacA) and cytotoxin-associated antigen A (CagA) are two major bacterial virulence factors involved in host cell modulation. VacA, so far mainly regarded as a cytotoxin of the gastric epithelial cell layer, now turns out to be a potent immunomodulatory toxin, targeting the adapted immune system. Thus, in addition to the well-known vacuolating activity, VacA has been reported to induce apoptosis in epithelial cells, to affect B lymphocyte antigen presentation, to inhibit the activation and proliferation of T lymphocytes, and to modulate the T cell-mediated cytokine response.     

Ubiquitin‐specific protease‐like 1 (USPL1) is a SUMO isopeptidase with essential,non‐catalytic functions

Isopeptidases are essential regulators of protein ubiquitination and sumoylation. However, only two families of SUMO isopeptidases are at present known. Here, we report an activity‐based search with the suicide inhibitor haemagglutinin (HA)‐SUMO‐vinylmethylester that led to the identification of a surprising new SUMO protease, ubiquitin‐specific protease‐like 1 (USPL1). Indeed, USPL1 neither binds nor cleaves ubiquitin, but is a potent SUMO isopeptidase both in vitro and in cells. C13orf22l—an essential but distant zebrafish homologue of USPL1—also acts on SUMO, indicating functional conservation. We have identified invariant USPL1 residues required for SUMO binding and cleavage. USPL1 is a low‐abundance protein that colocalizes with coilin in Cajal bodies. Its depletion does not affect global sumoylation, but causes striking coilin mislocalization and impairs cell proliferation, functions that are not dependent on USPL1 catalytic activity. Thus, USPL1 represents a third type of SUMO protease, with essential functions in Cajal body biology.     

The dynamics of ubiquitin pools within cultured human lung fibroblasts

The dynamics of ubiquitin pools within the cultured human lung fibroblast line IMR-90 were examined using solid phase immunochemical methods to quantitate free and conjugated polypeptide. Fetal calf serum was found to contain a nondialyzable factor that induced a transient accumulation of ubiquitin. During the induction, free and conjugated ubiquitin pools changed in concert so that the fraction conjugated remained constant. The induction of ubiquitin by the serum factor resulted from an enhanced rate of protein synthesis. Within experimental error no change in the first order rate constant for intracellular ubiquitin degradation was observed. Pulse-chase studies revealed ubiquitin to turn over with a half-life of 28-31 h in conditioned and freshly fed cultures. Withdrawal of serum from cultures led to a rapid decline in total ubiquitin during which the fractional level of conjugation remained constant. The accelerated ubiquitin turnover following removal of serum likely involves lysosomal autophagy since 10 mM NH4Cl led to an accumulation of the polypeptide. Since no similar effect of the lysosomotropic compound was observed in conditioned or freshly fed cultures, nonlysosomal processes are probably responsible for ubiquitin turnover under nutritional balance. The dynamics of these intracellular pools suggests that the ubiquitin ligation system is subject to regulatory constraints not previously suspected. The short half-life for ubiquitin is consistent with the apparent ability of cells to alter ubiquitin levels in response to external stimuli and stress.     

Direct analysis of α- and β-chains of hemoglobins from mammalian blood samples by nanoESI mass spectrometry during in-capillary proteolytic digestion

α- and β-chains of hemoglobins derived from several species were analyzed directly from diluted blood samples by simultaneous in-capillary proteolytic digestion and nanoESI MS and MS/MS analysis. Starting from fresh or frozen and thawed blood samples, sequence coverages of >80% were usually obtained. Only 2 h after resuspension of a dried blood spot, human origin could be demonstrated from data obtained by in-capillary tryptic digestion, nanoESI mass spectrometric analysis, and data base search. A fast and facile differentiation of closely related species by hemoglobin-derived proteolytic “marker peptides” was demonstrated for Asian (Elephas maximus) and African elephants (Loxodonta africana). Finally, amino acid sequences deduced from collision-induced dissociation experiments during in-capillary proteolytic digestion of the corresponding blood samples allowed de novo sequencing of previously unknown sequences of hemoglobin chains of the Patagonian cavy (Dolichotum patagona) and the Persian gazelle (Gazella subgutturosa subgutturosa). 100% of the α-chain sequences and more than 85% of the β-chain sequences were covered for both the species. Additionally, sequence data derived from tandem MS experiments obtained with the Q-Tof analyzer were confirmed by high resolution Fourier-transform ion cyclotron resonance mass spectrometric experiments. Accurate protein mass determination of the intact hemoglobin chains directly from the corresponding blood samples by use of a Fourier-transform ion cyclotron resonance mass spectrometer corroborated the deduced sequences of the respective α-chains. The present study demonstrates that in-capillary digestion allows fast characterization and/or sequencing of hemoglobin chains directly from blood samples.     

Biotin-Binding Proteins in the Defense of Mushrooms against Predators and Parasites

Tamavidins are fungal biotin-binding proteins (BBPs) displaying antifungal activity against phytopathogens. Here we show high toxicity of tamavidins toward nematodes, insects, and amoebae. As these organisms represent important phyla of fungal predators and parasites, we propose that BBPs are part of the chemical defense system of fungi.