Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography-tandem mass spectrometry

Saliva is a crucial biofluid for oral health and is also of increasing importance as a non-invasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high diversity of post-translational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid sample preparation and non-targeted LC-ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme diversity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of post-translational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva. (C) 2012 Elsevier B.V. All rights reserved.

Mycobacterium tuberculosis RsdA provides a conformational rationale for selective regulation of sigma-factor activity by proteolysis

The relative levels of different sigma factors dictate the expression profile of a bacterium. Extracytoplasmic function sigma factors synchronize the transcriptional profile with environmental conditions. The cellular concentration of free extracytoplasmic function sigma factors is regulated by the localization of this protein in a sigma/anti-sigma complex. Anti-sigma factors are multi-domain proteins with a receptor to sense environmental stimuli and a conserved anti-sigma domain (ASD) that binds a sigma factor. Here we describe the structure of Mycobacterium tuberculosis anti-sigma(D) (RsdA) in complex with the -35 promoter binding domain of sigma(D) (sigma(D)(4)). We note distinct conformational features that enable the release of sigma(D) by the selective proteolysis of the ASD in RsdA. The structural and biochemical features of the sigma(D)/RsdA complex provide a basis to reconcile diverse regulatory mechanisms that govern sigma/anti-sigma interactions despite high overall structural similarity. Multiple regulatory mechanisms embedded in an ASD scaffold thus provide an elegant route to rapidly re-engineer the expression profile of a bacterium in response to an environmental stimulus.

Evidence in support of a role for plant-mediated proteolysis in the rumens of grazing animals

Kingston-Smith, A. H., Merry, R. J., Leemans, D. K., Thomas, Howard, Theodorou, M. K. (2005). Evidence in support of a role for plant-mediated proteolysis in the rumens of grazing animals. British Journal of Nutrition, 93(1), 73-79. Sponsorship: DEFRA / BBSRC RAE2008

Primary proteolysis of caseins in cheddar cheese

Studies were undertaken to investigate proteolysis of the caseins during the initial stages of maturation of Cheddar cheese. Isolated caseins were hydrolyzed by enzymes thought to be of importance during cheese ripening and the resulting peptides isolated and identified. Large peptides were also isolated from Cheddar cheese and identified, thus enabling the extent to which casein degradation studies could be extrapolated to cheese to be established. The proteolytic specificity of chymosin on bovine αs1- and αs2-caseins and of plasmin on bovine αs1-casein were determined. The action of cathepsin D, the principal indigenous acid milk proteinase, on caseins was studied and its pH optimum and sensitivity to NaCI determined. The action of cathepsin D on αs1-, αs2-, β- and κ-caseins was compared with that of chymosin and was found to be generally similar for the hydrolysis of αs1- and κ-caseins but to differ for αs2-and β- caseins. β-Casein in solution was hydrolyzed by cell wall-associated proteinases from three strains of Lactococcus lactis; comparison of electrophoretograms of the hydrolyzates with those of Cheddar cheese indicated that no peptides produced by cell wall-associated proteinases were detectable in the cheeses. All the major peptides in the water-insoluble fraction of Cheddar cheese were isolated and identified. It was found that β-casein was degraded primarily by plasmin and αs1 -casein by chymosin. Initial chymosin and plasmin cleavage sites in αs1-, and β-casein, respectively, identified in these and other studies corresponded to the peptides isolated from cheese. The importance of non-starter lactic acid bacteria (NSLAB) to the maturation of Cheddar was studied in cheeses manufactured from raw, pasteurized or microfiltered milks. NSLAB were found to strongly influence the quality and patterns of proteolysis. Results presented in this thesis are consistent with the hypothesis that primary proteolysis in Cheddar is catalysed primarily by the action of chymosin and plasmin on intact αs1- and β-caseins, respectively. The resulting large peptides so produced are subsequently degraded by these enzymes and by proteinases and peptidases from the starter and NSLAB.

Variations in total and differential milk somatic cell counts and plasmin levels and their role in proteolysis and quality of milk and cheese

Increased plasmin and plasminogen levels and elevated somatic cell counts (SCC) and polymorphonuclear leucocyte levels (PMN) were evident in late lactation milk. Compositional changes in these milks were associated with increased SCC. The quality of late lactation milks was related to nutritional status of herds, with milks from herds on a high plane of nutrition having composition and clotting properties similar to, or superior to, early-mid lactation milks. Nutritionally-deficient cows had elevated numbers of polymorphonuclear leucocytes (PMNs) in their milk, elevated plasmin levels and increased overall proteolytic activity. The dominant effect of plasmin on proteolysis in milks of low SCC was established. When present in elevated numbers, somatic cells and PMNs in particular had a more significant influence on the proteolysis of both raw and pasteurised milks than plasmin. PMN protease action on the caseins showed proteolysis products of two specific enzymes, cathepsin B and elastase, which were also shown in high SCC milk. Crude extracts of somatic cells had a high specificity on αs1-casein. Cheeses made from late lactation milks had increased breakdown of αs1-casein, suggestive of the action of somatic cell proteinases, which may be linked to textural defects in cheese. Late lactation cheeses also showed decreased production of small peptides and amino acids, the reason for which is unknown. Plasmin, which is elevated in activity in late lactation milk, accelerated the ripening of Gouda-type cheese, but was not associated with defects of texture or flavour. The retention of somatic cell enzymes in cheese curd was confirmed, and a potential role in production of bitter peptides identified. Cheeses made from milks containing high levels of PMNs had accelerated αs1-casein breakdown relative to cheeses made from low PMN milk of the same total SCC, consistent with the demonstrated action of PMN proteinases. The two types of cheese were determined significantly different by blind triangle testing.

Ubiquitin-dependent proteolysis of trihydrophobin 1 (TH1) by the human papilloma virus E6-associated protein (E6-AP).

Human Papilloma virus E6-associated protein (E6-AP), which is known as an E3 ubiquitin ligase, mediates ubiquitination and subsequent degradation of a series of cellular proteins. In this paper, we identify here trihydrophobin 1 (TH1), an integral subunit of the human negative transcription elongation factor (NELF) complex, as a novel E6-AP interaction protein and a target of E6-AP-mediated degradation. Overexpression of E6-AP results in degradation of TH1 in a dose-dependent manner, whereas knock-down of endogenous E6-AP elevates the TH1 protein level. TH1 protein turnover is substantially faster, compared to controls, in cells that overexpressed E6-AP. Wild-type E6-AP promotes the ubiquitination of TH1, while a catalytically inactive point mutant of E6-AP abolishes its ubiquitination. Furthermore, in vitro ubiquitination assay also demonstrates that TH1 can be ubiquitinated by E6-AP. The degradation is blocked by treatment with proteasome inhibitor MG132. Herein, we provide strong evidence that TH1 is a specific substrate that is targeted for degradation through E6-AP-catalyzed polyubiquitination.

Evaluation of Serpa cheese proteolysis by nitrogen content and capillary zone electrophoresis

Proteolysis of Serpa cheese produced traditionally (B) and semi-industrially (C) was evaluated for the first time by determination of nitrogen content and capillary zone electrophoresis (CZE). A citrate dispersion of cheese was fractionated to determine the nitrogen in pH 4.4, trichloroacetic and phosphotungstic acid soluble fractions (pH 4.4-SN, TCA-SN and PTA-SN, respectively). The pH 4.4-SN was significantly higher for B ( P <0.001), while TCA-SN was significantly higher for C ( P <0.001). PTA-SN was also higher for C but at 60 days ripening no significant difference was found between B and C. Degradation of alpha(s1) - and beta-caseins evaluated by CZE was in good agreement with the maturation index (pH 4.4-SN/TN).