Browsing by Subject "proteolysis"

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  • Luoto, Sanna (Helsingfors universitet, 2011)
    The literature review dealt with celiac-toxic Triticeae prolamins and their enzymatic degradation. Also the immunochemical methods for prolamin analysis were introduced. The gluten-derived immunogenic peptides are proline-rich and thereby remarkably resistant to proteolytic degradation. Most of the triggering prolamins can, however, be degraded by combining endogenous cereal enzyme activity with acidic incubation. Despite of this residual prolamins still exist and their concentration exceeds the threshold considered to be safe for gluten intolerants. The objective of the experimental work was to further hydrolyse the residual prolamins present in malt autolysates of wheat, barley and rye, with a food grade proline endopeptidase from Aspergillus niger (AN-PEP). Size-exclusion chromatography (SEC), free amino nitrogen (FAN) and SDS-PAGE analysis determined the extent of protein hydrolysis. Actual prolamin degradation was observed with immunological methods. Hydrolysis of residual prolamins was extensive in all malt systems – more than 96% of the prolamins were hydrolysed. The SEC and FAN data revealed that continuation of the hydrolysis overnight converted the polypeptides into smaller hydrolysis products. According to enzyme-linked immunosorbent assay analyses, 22 h incubation decreased the prolamin contents of wheat and rye malt hydrolysates below the level of 100 mg/kg. This level was achieved with AN-PEP concentration of 35 ?L/g in relation to freeze-dried autolysate. According to the Codex Alimentarius, food products containing gluten up to 100 mg/kg can be labelled 'very low gluten' and thus included in coeliac diet. AN-PEP treated rye malt ingredient could especially be a promising low-gluten ingredient to enhance the flavour of often poor-quality gluten-free bread. Before commercial applications can be devised the potential as a flavouring agent as well as the clinical safety of the product must be evaluated.
  • Kareinen, Ilona; Baumann, Marc; Su Duy Nguyen,; Maaninka, Katariina; Anisimov, Andrey; Tozuka, Minoru; Jauhiainen, Matti; Lee-Rueckert, Miriam; Kovanen, Petri T. (2018)
    ApoA-I, the main structural and functional protein of HDL particles, is cardioprotective, but also highly sensitive to proteolytic cleavage. Here, we investigated the effect of cardiac mast cell activation and ensuing chymase secretion on apoA-I degradation using isolated rat hearts in the Langendorff perfusion system. Cardiac mast cells were activated by injection of compound 48/80 into the coronary circulation or by low-flow myocardial ischemia, after which lipid-free apoA-I was injected and collected in the coronary effluent for cleavage analysis. Mast cell activation by 48/80 resulted in apoA-I cleavage at sites Tyr(192) and Phe(229), but hypoxic activation at Tyr(192) only. In vitro, the proteolytic end-product of apoA-I with either rat or human chymase was the Tyr(192)-truncated fragment. This fragment, when compared with intact apoA-I, showed reduced ability to promote migration of cultured human coronary artery endothelial cells in a wound-healing assay. We propose that C-terminal truncation of apoA-I by chymase released from cardiac mast cells during ischemia impairs the ability of apoA-I to heal damaged endothelium in the ischemic myocardium.
  • Feleciano, Diogo R.; Juenemann, Katrin; Iburg, Manuel; Bras, Ines C.; Holmberg, Carina; Kirstein, Janine (2019)
    A functional protein quality control machinery is crucial to maintain cellular and organismal physiology. Perturbation in the protein homeostasis network can lead to the formation of misfolded and aggregated proteins that are a hallmark of protein conformational disorders and aging. Protein aggregation is counteracted by the action of chaperones that can resolubilize aggregated proteins. An alternative protein aggregation clearance strategy is the elimination by proteolysis employing the ubiquitin proteasome system (UPS) or autophagy. Little is known how these three protein aggregate clearance strategies are regulated and coordinated in an organism with the progression of aging or upon expression of disease-associated proteins. To unravel the crosstalk between the protein aggregate clearance options, we investigated how autophagy and the UPS respond to perturbations in protein disaggregation capacity. We found that autophagy is induced as a potential compensatory mechanism, whereas the UPS exhibits reduced capacity upon depletion of disaggregating chaperones in C. elegans and HEK293 cells. The expression of amyloid proteins A beta(3-42) and Q(40) result in an impairment of autophagy as well as the UPS within the same and even across tissues. Our data indicate a tight coordination between the different nodes of the proteostasis network (PN) with the progression of aging and upon imbalances of the capacity of each clearance mechanism.
  • Turunen, S. Pauliina; Tatti-Bugaeva, Olga; Lehti, Kaisa (2017)
    Membrane-type matrix metalloproteases (MT-MMP) are pivotal regulators of cell invasion, growth and survival. Tethered to the cell membranes by a transmembrane domain or GPI-anchor, the six MT-MMPs can exert these functions via cell surface-associated extracellular matrix degradation or proteolytic protein processing, including shedding or release of signaling receptors, adhesion molecules, growth factors and other pericellular proteins. By interactions with signaling scaffold or cytoskeleton, the C-terminal cytoplasmic tail of the transmembrane MT-MMPs further extends their functionality to signaling or structural relay. MT-MMPs are differentially expressed in cancer. The most extensively studied MMP14/MT1-MMP is induced in various cancers along malignant transformation via pathways activated by mutations in tumor suppressors or proto-oncogenes and changes in tumor microenvironment including cellular heterogeneity, extracellular matrix composition, tissue oxygenation, and inflammation. Classically such induction involves transcriptional programs related to epithelial-to-mesenchymal transition. Besides inhibition by endogenous tissue inhibitors, MT-MMP activities are spatially and timely regulated at multiple levels by microtubular vesicular trafficking, dimerization/oligomerization, other interactions and localization in the actin-based invadosomes, in both tumor and the stroma. The functions of MT-MMPs are multifaceted within reciprocal cellular responses in the evolving tumor microenvironment, which poses the importance of these proteases beyond the central function as matrix scissors, and necessitates us to rethink MT-MMPs as dynamic signaling proteases of cancer. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.
  • Leskelä, Mariia (Helsingin yliopisto, 2018)
    Plasmin is an enzyme which is an important factor affecting both flavor and shelf life of UHT milk. Plasmin hydrolyzes mainly β, αs1- and αs2-caseins into γ-caseins and proteose peptones. During storage, proteolysis by plasmin causes bitterness and gelation. The objective of present study was to investigate time-temperature combinations for preheat treatment of UHT milk to decrease plasmin activity thus improving the shelf life and quality of existing product. In experimental research, 14 different UHT milk samples were manufactured with 7 different preheat processes and two different types of milk: prehydrolyzed (A) and posthydrolyzed (B). Samples were analyzed for their extent of proteolysis by two different methods: SDS-PAGE and RP-HPLC. SDS-PAGE gave qualitative information on the casein degradation and RP-HPLC gave quantitative information. Also, sensory analyses were made by eight trained employees at Arla Ltd (Sipoo, Finland). There were significant differences in casein degradation of samples at 2 and 4 months. All samples had some proteolysis after 4 months of storage. The largest amount of casein degradation was found in samples 5, 7 and 11, whereas the least proteolysis found in samples 1, 2, 13 and 14. Same results came from both SDS-PAGE and RP-HPLC. The sensory evaluation showed differences between the samples regarding bitterness at 2 and 3 months. The samples which were evaluated most bitter in sensory analysis, showed also most proteolysis in other analyses (samples 5, 7 and 11). The rest of the samples were quite equal in all evaluated features. The impact of hydrolysis point of lactose was unclear in this study. The aim of this study was accomplished since optimal processing conditions were found: 1, 2, 13 and 14 and the optimization of the process was possible based on the results from this study.
  • Judström, Ilona (Helsingfors universitet, 2009)
    In atherosclerosis, cholesterol accumulates in cholesterol-loaded macrophages (foam cells) forming cholesterol plaques in the arterial intima. Reverse cholesterol transport (RCT) is a mechanism in which HDL and its major structural protein apolipoprotein-A-1 (apoA-1) remove cholesterol from the foam cells and take it to the liver for its final excretion from the body in the faeces. An impaired removal of cholesterol from the foam cells is a potential contributor to a reduced RCT, which is related to a higher incidence of coronary heart disease. Chymase, a neutral protease of mast cells (MCs), is widely distributed in the connective tissue of most vertebrates and able to degrade apoA-1. After the degradation, HDL-particles are unable to interact with the ABCA-1 transporter protein on the surface of macrophages, which mediates efflux of cholesterol from the macrophage foam cells to HDL particles. It has been shown that chymase derived from rat peritoneal MCs is able to degrade apoA-1 even in the presence of blood plasma which contains natural inhibitors for chymase (α-2-macroglobulin and α-1-antichymotrypsin). In the present study we wanted to find out if mouse mast cell protease 4 (mMCP-4) isolated from peritoneal mast cells is able to maintain its enzymatic activity even in the presence of mouse serum and intraperitoneal fluid. A small molecular weight compound (S-2586) was used as a substrate. In the in vitro experiments a sonicated MC preparation that contains active chymase was used and the activity of chymase was measured in the presence of varying concentrations of plasma and intraperitoneal fluid. In the in vivo experiments we evaluate whether mast cell-dependent proteolysis of HDL particles does occur, and whether such modification inhibits their efficiency in inducing cellular cholesterol efflux in vitro. We found that both serum and intraperitoneal fluid inhibited chymase activity, serum to a higher extent. Systemic activation of MCs in mast cell-competent mice, but not in mast cell-deficient mice, in vivo led to a decreased ability of plasma and intraperitoneal fluid to act as cholesterol acceptors from cultured cholesterol-loaded macrophages. Local activation of peritoneal mast cells also blocked the cholesterol efflux-inducing effect of intraperitoneally injected human apoA-1. This work was performed at the Wihuri Research Institute. Licenses for animal work were approved by the Finnish Laboratory Animal Experiment Committee (Suomen eläinkoelautakunta, ELLA). Laboratory animals (female NMRI mice) were from the Viikki Laboratory Animal Centre of the University of Helsinki and the mast cell deficient strain of mice W-sash c-kit mutant KitW-sh/W-sh were from the Jackson Laboratory (BarHarbor, Maine). The work was supervised by the director of the Research Institute Petri Kovanen MD PhD and Miriam Lee-Rueckert PhD. Laboratory assistance was perceived from the technicians of the Wihuri Research Institute.