Enzymatic activity and immunoreactivity of Aca s 4, an alpha-amylase allergen from the storage mite Acarus siro

<p>Abstract</p> <p>Background</p> <p>Enzymatic allergens of storage mites that contaminate stored food products are poorly characterized. We describe biochemical and immunological properties of the native alpha-amylase allergen Aca s 4 from <it>Acarus siro</it>, a medically important storage mite.</p> <p>Results</p> <p><it>A. siro </it>produced a high level of alpha-amylase activity attributed to Aca s 4. This enzyme was purified and identified by protein sequencing and LC-MS/MS analysis. Aca s 4 showed a distinct inhibition pattern and an unusual alpha-amylolytic activity with low sensitivity to activation by chloride ions. Homology modeling of Aca s 4 revealed a structural change in the chloride-binding site that may account for this activation pattern. Aca s 4 was recognized by IgE from house dust mite-sensitive patients, and potential epitopes for cross-reactivity with house dust mite group 4 allergens were found.</p> <p>Conclusions</p> <p>We present the first protein-level characterization of a group 4 allergen from storage mites. Due to its high production and IgE reactivity, Aca s 4 is potentially relevant to allergic hypersensitivity.</p

Cathepsin L from the hard tick Ixodes ricinus

Ticks are globally important parasites involved in transmission of a wide variety of infectious agents. The most common tick species found in Europe is the hard tick Ixodes ricinus, which transmits bacterium Borrelia burgdorferi (a causative agent of Lyme disease) or tick-borne encephalitis virus. Cathepsin proteases are important in the process of digestion of blood proteins in the tick gut. This work is focused on cathepsin L, an important digestive cysteine protease of ticks. Recombinant I. ricinus cathepsin L was expressed in Pichia pastoris and separated from the culture medium by chromatographic purification. N-terminal protein sequencing and labeling by activity-based probe Green-DCG-04 were used for characterization of purified cathepsin L. Substrate and inhibitor specificity were analyzed using peptide substrates and inhibitors. This analysis showed that Z-FR-AMC is a suitable substrate with pH optimum 3.5, and that Z-FF-DMK is an efficient inhibitor. It was demonstrated that cathepsin L cleaves protein substrates in strongly acidic environment (pH 3.5-4.5). Cathepsin L-like proteolytic activity was demonstrated in salivary gland extract and in saliva of the I. ricinus tick. The presence of a cathepsin protease in tick saliva is reported here for the first time. This finding suggests that..

Cathepsin L from the hard tick Ixodes ricinus: analysis of proteolytic activity and its regulation

The hard tick Ixodes ricinus is an important blood-feeding parasite that transmits tick- borne diseases, such as tick-borne encephalitis and Lyme disease. Ticks employ a battery of proteolytic enzymes, including cathepsins, to digest their bloodmeal. These proteins are potential targets for the development of anti-tick vaccines. This work is focused on cathepsin L from I. ricinus (IrCL), namely its isoenzymes IrCL1 and IrCL3. IrCL1 was expressed in Pichia pastoris and chromatographically purified. Its substrate specifity was determined by the cleavage of (a) peptide fluorogenic substrates and (b) protein substrates analyzed by mass spectrometry. The proteolytic activity of IrCL1 was modulated by its interaction with glycosaminoglycans, which affected the pH optimum value. Futhermore, a proteolytically active mutant of IrCL1 with reduced number of N-glycosylation sites was prepared; this form will be used for crystallization experiments. IrCL3 was expressed in Escherichia coli, refolded and activated to its active form. The proteolytic activity of IrCL3 is in many ascpects similar to that of IrCL1, including substrate specifity, acidic pH optimum and activity modulation by glycosaminoglycans. Key words: cysteine proteases, cathepsin L, hard tick I. ricinus, substrate specifity, proteolytic activity..

Cathepsin L from the hard tick Ixodes ricinus

Ticks are globally important parasites involved in transmission of a wide variety of infectious agents. The most common tick species found in Europe is the hard tick Ixodes ricinus, which transmits bacterium Borrelia burgdorferi (a causative agent of Lyme disease) or tick-borne encephalitis virus. Cathepsin proteases are important in the process of digestion of blood proteins in the tick gut. This work is focused on cathepsin L, an important digestive cysteine protease of ticks. Recombinant I. ricinus cathepsin L was expressed in Pichia pastoris and separated from the culture medium by chromatographic purification. N-terminal protein sequencing and labeling by activity-based probe Green-DCG-04 were used for characterization of purified cathepsin L. Substrate and inhibitor specificity were analyzed using peptide substrates and inhibitors. This analysis showed that Z-FR-AMC is a suitable substrate with pH optimum 3.5, and that Z-FF-DMK is an efficient inhibitor. It was demonstrated that cathepsin L cleaves protein substrates in strongly acidic environment (pH 3.5-4.5). Cathepsin L-like proteolytic activity was demonstrated in salivary gland extract and in saliva of the I. ricinus tick. The presence of a cathepsin protease in tick saliva is reported here for the first time. This finding suggests that..

The MEK-ERK-MST1 Axis Potentiates the Activation of the Extrinsic Apoptotic Pathway during GDC-0941 Treatment in Jurkat T Cells

The discrete activation of individual caspases is essential during T-cell development, activation, and apoptosis. Humans carrying nonfunctional caspase-8 and caspase-8 conditional knockout mice exhibit several defects in the progression of naive CD4+ T cells to the effector stage. MST1, a key kinase of the Hippo signaling pathway, is often presented as a substrate of caspases, and its cleavage by caspases potentiates its activity. Several studies have focused on the involvement of MST1 in caspase activation and also reported several defects in the immune system function caused by MST1 deficiency. Here, we show the rapid activation of the MEK-ERK-MST1 axis together with the cleavage and activation of caspase-3, -6, -7, -8, and -9 after PI3K signaling blockade by the selective inhibitor GDC-0941 in Jurkat T cells. We determined the phosphorylation pattern of MST1 using a phosphoproteomic approach and identified two amino acid residues phosphorylated in an ERK-dependent manner after GDC-0941 treatment together with a novel phosphorylation site at S21 residue, which was extensively phosphorylated in an ERK-independent manner during PI3K signaling blockade. Using caspase inhibitors and the inhibition of MST1 expression using siRNA, we identified an exclusive role of the MEK-ERK-MST1 axis in the activation of initiator caspase-8, which in turn activates executive caspase-3/-7 that finally potentiate MST1 proteolytic cleavage. This mechanism forms a positive feed-back loop that amplifies the activation of MST1 together with apoptotic response in Jurkat T cells during PI3K inhibition. Altogether, we propose a novel MEK-ERK-MST1-CASP8-CASP3/7 apoptotic pathway in Jurkat T cells and believe that the regulation of this pathway can open novel possibilities in systemic and cancer therapies

Variation in mouse chemical signals is genetically controlled and environmentally modulated

Abstract In most mammals and particularly in mice, chemical communication relies on the detection of ethologically relevant fitness-related cues from other individuals. In mice, urine is the primary source of these signals, so we employed proteomics and metabolomics to identify key components of chemical signalling. We show that there is a correspondence between urinary volatiles and proteins in the representation of genetic background, sex and environment in two house mouse subspecies Mus musculus musculus and M. m. domesticus. We found that environment has a strong influence upon proteomic and metabolomic variation and that volatile mixtures better represent males while females have surprisingly more sex-biased proteins. Using machine learning and combined-omics techniques, we identified mixtures of metabolites and proteins that are associated with biological features

Additional file 2 of Identification of potential molecular targets for the treatment of cluster 1 human pheochromocytoma and paraganglioma via comprehensive proteomic characterization

Additional File 2. Volacano plots for the Pitchfork method