Browsing by Subject "PROTEASES"

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  • Pitkänen, H. H.; Kärki, M.; Niinikoski, H.; Tanner, L.; Näntö-Salonen, K.; Pikta, M.; Kopatz, W. F.; Zuurveld, M.; Meijers, J. C. M.; Brinkman, H. J. M.; Lassila, R. (2018)
    Introduction: Lysinuric protein intolerance (LPI), a rare autosomal recessive transport disorder of cationic amino acids lysine, arginine and ornithine, affects intestines, lungs, liver and kidneys. LPI patients may display potentially life-threatening bleeding events, which are poorly understood. Aims: To characterize alterations in haemostatic and fibrinolytic variables associated with LPI. Methods: We enrolled 15 adult patients (8 female) and assessed the clinical ISTH/ SSC-BAT bleeding score (BS). A variety of metabolic and coagulation assays, including fibrin generation test derivatives, clotting time (CT) and clot lysis time (CLT), thromboelastometry (ROTEM), and PFA-100 and Calibrated Automated Thrombogram (CAT), were used. Results: All patients had mild-to-moderate renal insufficiency, and moderate bleeding tendency (BS 4) without spontaneous bleeds. Mild anaemia and thrombocytopenia occurred. Traditional clotting times were normal, but in contrast, CT in fibrin generation test, and especially ROTEM FIBTEM was abnormal. The patients showed impaired primary haemostasis in PFA, irrespective of normal von Willebrand factor activity, but together with lowered fibrinogen and FXIII. Thrombin generation (TG) was reduced in vitro, according to CAT-derived endogenous thrombin potential, but in vivo TG was enhanced in the form of circulating prothrombin fragment 1 and 2 values. Very high D-dimer and plasmin-alpha 2-antiplasmin (PAP) complex levels coincided with shortened CLT in vitro. Conclusions: Defective primary haemostasis, coagulopathy, fibrin abnormality (FIBTEM, CT and CLT), low TG in vitro and clearly augmented fibrinolysis (PAP and D-dimer) in vivo were all detected in LPI. Altered fibrin generation and hyperfibrinolysis were associated with the metabolic and renal defect, suggesting a pathogenetic link in LPI.
  • Bitu, Carolina C.; Kauppila, Joonas H.; Bufalino, Andreia; Nurmenniemi, Sini; Teppo, Susanna; Keinanen, Meeri; Vilen, Suvi-Tuuli; Lehenkari, Petri; Nyberg, Pia; Coletta, Ricardo D.; Salo, Tuula (2013)
  • Hilander, Taru; Zhou, Xiao-Long; Konovalova, Svetlana; Zhang, Fu-Ping; Euro, Liliya; Shilov, Dmitri; Poutanen, Matti; Chihade, Joseph; Wang, En-Duo; Tyynismaa, Henna (2018)
    Accuracy of protein synthesis is enabled by the selection of amino acids for tRNA charging by aminoacyl-tRNA synthetases (ARSs), and further enhanced by the proofreading functions of some of these enzymes for eliminating tRNAs mischarged with noncognate amino acids. Mouse models of editing-defective cytoplasmic alanyl-tRNA synthetase (AlaRS) have previously demonstrated the importance of proofreading for cytoplasmic protein synthesis, with embryonic lethal and progressive neurodegeneration phenotypes. Mammalian mitochondria import their own set of nuclear-encoded ARSs for translating critical polypeptides of the oxidative phosphorylation system, but the importance of editing by the mitochondrial ARSs for mitochondrial proteostasis has not been known. We demonstrate here that the human mitochondrial AlaRS is capable of editing mischarged tRNAs in vitro, and that loss of the proofreading activity causes embryonic lethality in mice. These results indicate that tRNA proofreading is essential in mammalian mitochondria, and cannot be overcome by other quality control mechanisms.
  • Lorey, Martina B.; Rossi, Katriina; Eklund, Kari; Nyman, Tuula A.; Matikainen, Sampsa (2017)
    Gram-negative bacteria are associated with a wide spectrum of infectious diseases in humans. Inflammasomes are cytosolic protein complexes that are assembled when the cell encounters pathogens or other harmful agents. The non-canonical caspase-4/5 inflammasome is activated by Gram-negative bacteria-derived lipopolysaccharide (LPS) and by endogenous oxidized phospholipids. Protein secretion is a critical component of the innate immune response. Here, we have used label-free quantitative proteomics to characterize global protein secretion in response to non-canonical inflammasome activation upon intracellular LPS recognition in human primary macrophages. Before proteomics, the total secretome was separated into two fractions, enriched extracellular vesicle (EV) fraction and rest-secretome (RS) fraction using size-exclusion centrifugation. We identified 1048 proteins from the EV fraction and 1223 proteins from the RS fraction. From these, 640 were identified from both fractions suggesting that the non-canonical inflammasome activates multiple, partly overlapping protein secretion pathways. We identified several secreted proteins that have a critical role in host response against severe Gram-negative bacterial infection. The soluble secretome (RS fraction) was highly enriched with inflammation-associated proteins upon intracellular LPS recognition. Several ribosomal proteins were highly abundant in the EV fraction upon infection, and our data strongly suggest that secretion of translational machinery and concomitant inhibition of translation are important parts of host response against Gram-negative bacteria sensing caspase-4/5 inflammasome. Intracellular recognition of LPS resulted in the secretion of two metalloproteinases, a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and MMP14, in the enriched EV fraction. ADAM10 release was associated with the secretion of TNF, a key inflammatory cytokine, and M-CSF, an important growth factor for myeloid cells probably through ADAM10-dependent membrane shedding of these cytokines. Caspase-4/5 inflammasome activation also resulted in secretion of danger-associated molecules S100A8 and prothymosin- in the enriched EV fraction. Both S100A8 and prothymosin- are ligands for toll-like receptor 4 recognizing extracellular LPS, and they may contribute to endotoxic shock during non-canonical inflammasome activation.
  • Daly, Paul; Peng, Mao; Di Falco, Marcos; Lipzen, Anna; Wang, Mei; Ng, Vivian; Grigoriev, Igor; Tsang, Adrian; Makela, Miia R.; de Vries, Ronald P. (2019)
    The extent of carbon catabolite repression (CCR) at a global level is unknown in wood-rotting fungi, which are critical to the carbon cycle and are a source of biotechnological enzymes. CCR occurs in the presence of sufficient concentrations of easily metabolizable carbon sources (e.g., glucose) and involves downregulation of the expression of genes encoding enzymes involved in the breakdown of complex carbon sources. We investigated this phenomenon in the white-rot fungus Dichomitus squalens using transcriptomics and exoproteomics. In D. squalens cultures, approximately 7% of genes were repressed in the presence of glucose compared to Avicel or xylan alone. The glucose-repressed genes included the essential components for utilization of plant biomass-carbohydrate-active enzyme (CAZyme) and carbon catabolic genes. The majority of polysaccharide-degrading CAZyme genes were repressed and included activities toward all major carbohydrate polymers present in plant cell walls, while repression of ligninolytic genes also occurred. The transcriptome-level repression of the CAZyme genes observed on the Avicel cultures was strongly supported by exoproteomics. Protease-encoding genes were generally not glucose repressed, indicating their likely dominant role in scavenging for nitrogen rather than carbon. The extent of CCR is surprising, given that D. squalens rarely experiences high free sugar concentrations in its woody environment, and it indicates that biotechnological use of D. squalens for modification of plant biomass would benefit from derepressed or constitutively CAZyme-expressing strains. IMPORTANCE White-rot fungi are critical to the carbon cycle because they can mineralize all wood components using enzymes that also have biotechnological potential. The occurrence of carbon catabolite repression (CCR) in white-rot fungi is poorly understood. Previously, CCR in wood-rotting fungi has only been demonstrated for a small number of genes. We demonstrated widespread glucose-mediated CCR of plant biomass utilization in the white-rot fungus Dichomitus squalens. This indicates that the CCR mechanism has been largely retained even though wood-rotting fungi rarely experience commonly considered CCR conditions in their woody environment. The general lack of repression of genes encoding proteases along with the reduction in secreted CAZymes during CCR suggested that the retention of CCR may be connected with the need to conserve nitrogen use during growth on nitrogen-scarce wood. The widespread repression indicates that derepressed strains could be beneficial for enzyme production.
  • Aaltonen, Niina; Singha, Prosanta K.; Jakupovic, Hermina; Wirth, Thomas; Samaranayake, Haritha; Pasonen-Seppanen, Sanna; Rilla, Kirsi; Varjosalo, Markku; Edgington-Mitchell, Laura E.; Kasperkiewicz, Paulina; Drag, Marcin; Kälvälä, Sara; Moisio, Eemeli; Savinainen, Juha R.; Laitinen, Jarmo T. (2020)
    Background Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. Results Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Conclusions Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.
  • Gürsoy, Ulvi K.; Könönen, Eija; Tervahartiala, Taina; Gürsoy, Mervi; Pitkänen, Jari; Torvi, Paula; Suominen, Anna-Liisa; Pussinen, Pirkko; Sorsa, Timo (2018)
    Aim To investigate the molecular forms of salivary matrix metalloproteinase (MMP)-8 in relation to periodontitis. Materials and Methods Molecular forms, degree of activation and fragmentation of neutrophilic and mesenchymal-type MMP-8 isoforms were analysed from salivary samples of 81 subjects with generalized periodontitis, 63 subjects with localized periodontitis and 79 subjects without pocket teeth, by using western-immunoblots with computer quantitation. In addition, human recombinant proMMP-8 was in vitro activated by Treponema denticola chymotrypsin-like protease (Td-CTLP), sodium hypochlorite (NaOCl, 1 mM, oxidant) or amino phenyl mercuric acetate (APMA, 1 mM). Results In saliva of periodontitis-affected individuals, MMP-8 is found in multiple forms, that is, complexes, active and pro-forms of neutrophilic and mesenchymal-type MMP-8, and especially 20-27 kDa fragments. The quantity of these fragments was elevated in both localized and generalized forms of periodontitis. Moreover, the tested activators (Td-CTLP, NaOCl and APMA) activated inactive proMMP-8, resulting in fragments of 20-27 kDa, in vitro, and salivary concentrations of T. denticola correlated significantly with salivary levels of fragmented MMP-8. Conclusion The present results indicate that during the development and progression of periodontitis, MMP-8 appears as activated and fragmented, and treponemal proteases most likely play role in this cascade.