| 1. |
- Ahooghalandari, Parvin, et al.
(författare)
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Mutations in Arg143 and Lys192 of the Human Mast Cell Chymase Markedly Affect the Activity of Five Potent Human Chymase Inhibitors
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:6, s. e65988-
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Tidskriftsartikel (refereegranskat)abstract
- Chymotrypsin-like serine proteases are found in high abundance in mast cell granules. By site-directed mutatgenesis, we have previously shown that basic amino acids in positions 143 and 192 (Arg and Lys respectively) of the human mast cell chymase are responsible for an acidic amino acid residue preference in the P2' position of substrates. In order to study the influence of these two residues in determining the specificity of chymase inhibitors, we have synthesized five different potent inhibitors of the human chymase. The inhibitory effects of these compounds were tested against the wild-type enzyme, against two single mutants Arg143Gln and Lys192Met and against a double mutant, Arg143Gln+Lys192Met. We observed a markedly reduced activity of all five inhibitors with the double mutant, indicating that these two basic residues are involved in conferring the specificity of these inhibitors. The single mutants showed an intermediate phenotype, with the strongest effect on the inhibitor by the mutation in Lys192. The Lys192 and the double mutations also affected the rate of cleavage of angiotensin I but did not seem to affect the specificity in the cleavage of the Tyr(4)-Ile(5) bond. A more detailed knowledge about which amino acids that confer the specificity of an enzyme can prove to be of major importance for development of highly specific inhibitors for the human chymase and other medically important enzymes.
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| 2. |
- Andersson, Mattias K., et al.
(författare)
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Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2′ of substrates
- 2010
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:10, s. 2255-2267
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Tidskriftsartikel (refereegranskat)abstract
- Chymases are chymotrypsin-like serine proteases that are found in large amounts in mast cell granules. So far, the extended cleavage specificities of eight such chymases have been determined, and four of these were shown to have a strong preference for acidic amino acids at position P2'. These enzymes have basic amino acids in positions 143 and 192 (Arg and Lys, respectively). We therefore hypothesized that Arg143 and Lys192 of human chymase mediate the preference for acidic amino acids at position P2' of substrates. In order to address this question, we performed site-directed mutagenesis of these two positions in human chymase. Analysis of the extended cleavage specificities of two single mutants (Arg143 -> Gln and Lys192 -> Met) and the combined double mutant revealed an altered specificity for P2' amino acids, whereas all other positions were essentially unaffected. A weakened preference for acidic amino acids at position P2' was observed for the two single mutants, whereas the double mutant lacked this preference. Therefore, we conclude that positions 143 and 192 in human chymase contribute to the strong preference for negatively charged amino acids at position P2'. This is the first time that a similar combined effect has been shown to influence the cleavage specificity, apart from position P1, among the chymases. Furthermore, the conservation of the preference for acidic P2' amino acids for several mast cell chymases clearly indicates that other substrates than angiotensin I may be major in vivo targets for these enzymes.
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| 3. |
- Gallwitz, Maike, et al.
(författare)
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The Extended Cleavage Specificity of Human Thrombin
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:2, s. e31756-
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Tidskriftsartikel (refereegranskat)abstract
- Thrombin is one of the most extensively studied of all proteases. Its central role in the coagulation cascade as well as several other areas has been thoroughly documented. Despite this, its consensus cleavage site has never been determined in detail. Here we have determined its extended substrate recognition profile using phage-display technology. The consensus recognition sequence was identified as, P2-Pro, P1-Arg, P1'-Ser/Ala/Gly/Thr, P2'-not acidic and P3'-Arg. Our analysis also identifies an important role for a P3'-arginine in thrombin substrates lacking a P2-proline. In order to study kinetics of this cooperative or additive effect we developed a system for insertion of various pre-selected cleavable sequences in a linker region between two thioredoxin molecules. Using this system we show that mutations of P2-Pro and P3'-Arg lead to an approximate 20-fold and 14-fold reduction, respectively in the rate of cleavage. Mutating both Pro and Arg results in a drop in cleavage of 200-400 times, which highlights the importance of these two positions for maximal substrate cleavage. Interestingly, no natural substrates display the obtained consensus sequence but represent sequences that show only 1-30% of the optimal cleavage rate for thrombin. This clearly indicates that maximal cleavage, excluding the help of exosite interactions, is not always desired, which may instead cause problems with dysregulated coagulation. It is likely exosite cooperativity has a central role in determining the specificity and rate of cleavage of many of these in vivo substrates. Major effects on cleavage efficiency were also observed for residues as far away as 4 amino acids from the cleavage site. Insertion of an aspartic acid in position P4 resulted in a drop in cleavage by a factor of almost 20 times.
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| 4. |
- Gallwitz, Maike, et al.
(författare)
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The extended substrate recognition profile of the dog mast cell chymase reveals similarities and differences to the human chymase
- 2010
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Ingår i: International Immunology. - : Oxford University Press (OUP). - 0953-8178 .- 1460-2377. ; 22:6, s. 421-431
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Tidskriftsartikel (refereegranskat)abstract
- Human chymase (HC) constitutes a major granule protease in one of the two human mast cell (MC) types. The main biological role of this haematopoietic serine protease is probably not yet known, although it has been implicated in a large number of functions. Dogs, like humans, have only one chymase. This enzyme is closely related to its human homologue, and the MC subtypes of human and dog appear to be similar as well. Therefore, the functions of the dog chymase (DC) may closely reflect the functions of the HC. Moreover, dogs may serve as good models for studies of human MC functions and MC-related diseases. To reveal functional similarities and differences between the DC and HC, we have determined the extended cleavage specificity of the DC by substrate phage display. This method allows the simultaneous permutation of primed and unprimed substrate positions. The DC was found to have very similar preferences to its human counterpart for substrate positions P1, P3, P4 and P3', whereas their preferences differ at positions P2, P1' and P2'. Therefore, the HC and DC may have co-evolved with a substrate where positions P1, P3, P4 and P3' are conserved between dogs and humans, whereas positions P2 and P1' are not and P2' differs to a minor extent. The differences observed between these two enzymes suggest that results obtained from dog models cannot be directly extrapolated to human clinical settings but need to be evaluated carefully concerning potential differences in substrate preferences.
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| 5. |
- Hellman, Lars, et al.
(författare)
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Granule proteases of hematopoietic cells, a family of versatile inflammatory mediators - an update on their cleavage specificity, in vivo substrates, and evolution
- 2014
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Ingår i: Biological chemistry (Print). - : Walter de Gruyter GmbH. - 1431-6730 .- 1437-4315. ; 395:1, s. 15-49
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Forskningsöversikt (refereegranskat)abstract
- Cells from several of the hematopoietic cell lineages including mast cells, basophils, neutrophils, cytotoxic T cells, and natural killer (NK) cells store proteases at very high levels within their cytoplasmic granules. In mast cells, these proteases can account for up to 35% of the total cellular protein, and the absolute majority of these belong to the chymotrypsin-related serine protease family. A number of very diverse functions have been identified for these proteases, including apoptosis induction, blood pressure regulation, inactivation of insect and snake toxins, intestinal parasite expulsion, killing of bacteria and fungi, induction, mobilization, or degradation of cytokines, and the degradation of connective tissue components. A very broad spectrum of primary cleavage specificities has also been observed, including chymase, tryptase, asp-ase, elastase, and met-ase specificities, which highlights the large flexibility in the active site of these proteases. Mast cells primarily express chymases and tryptases with chymotryptic or tryptic primary cleavage specificities, respectively. Neutrophils have several enzymes with chymase, elastase, and tryptase specificities. T cells and NK cells express between 5 and 14 different granzymes, depending on the species, and these enzymes have tryptase, asp-ase, chymase, and met-ase specificities. This review focuses on the appearance of these proteases during vertebrate evolution, their primary and extended cleavage specificities, and their potential in vivo substrates. The in vivo substrates and functions are a particular challenging issue because several of these enzymes have a relatively broad specificity and may therefore cleave a wide range of different substrates.
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| 6. |
- Korkmaz, Brice, et al.
(författare)
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Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates
- 2011
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 278:15, s. 2635-2646
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Tidskriftsartikel (refereegranskat)abstract
- Cathepsin G (CG) (EC 3.4.21.20) and chymase (EC 3.4.21.39) are two closely-related chymotrypsin-like proteases that are released from cytoplasmic granules of activated mast cells and/or neutrophils. We investigated the potential for their substrate-binding subsites to discriminate between their substrate specificities, aiming to better understand their respective role during the progression of inflammatory diseases. In addition to their preference for large aromatic residues at P1, both preferentially accommodate small hydrophilic residues at the S1' subsite. Despite significant structural differences in the S2' subsite, both prefer an acidic residue at that position. The Ala226/Glu substitution at the bottom of the CG S1 pocket, which allows CG but not chymase to accommodate a Lys residue at P1, is the main structural difference, allowing discrimination between the activities of these two proteases. However, a Lys at P1 is accommodated much less efficiently than a Phe, and the corresponding substrate is cleaved by beta 2-tryptase (EC 3.4.21.59). We optimized a P1 Lys-containing substrate to enhance sensitivity towards CG and prevent cleavage by chymase and beta 2-tryptase. The resulting substrate (ABZ-GIEPKSDPMPEQ-EDDnp) [ where ABZ is O-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] was cleaved by CG but not by chymase and tryptase, with a specificity constant of 190 mM(-1).s(-1). This allows the quantification of active CG in cells or tissue extracts where it may be present together with chymase and tryptase, as we have shown using a HMC-1 cell homogenate and a sputum sample from a patient with severe asthma.
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| 7. |
- Thorpe, Michael, et al.
(författare)
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Extended cleavage specificity of the mast cell chymase from the crab-eating macaque (Macaca fascicularis) : an interesting animal model for the analysis of the function of the human mast cell chymase
- 2012
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Ingår i: International Immunology. - : Oxford University Press (OUP). - 0953-8178 .- 1460-2377. ; 24:12, s. 771-782
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Tidskriftsartikel (refereegranskat)abstract
- Serine proteases are the major protein constituents within mast cell secretory granules. These proteases are subdivided into chymases and tryptases depending on their primary cleavage specificity. Here, we present the extended cleavage specificity of the macaque mast cell chymase and compare the specificity with human chymase (HC) and dog chymase (DC) that were produced in the same insect cell expression host. The macaque chymase (MC) shows almost identical characteristics as the HC, including both primary and extended cleavage specificities as well as sensitivity to protease inhibitors, whereas the DC differs in several of these characteristics. Although previous studies have shown that mouse mast cell protease-4 (mMCP-4) is similar in its hydrolytic specificity to the HC, mouse mast cells contain several related enzymes. Thus mice may not be the most appropriate model organism for studying HC activity and inhibition. Importantly, macaques express only one chymase and, as primates, are closely related to human general physiology. In addition, the human and macaque enzymes both cleave angiotensin I (Ang I) in the same way, generating primarily angiotensin II (Ang II) and they do not further degrade the peptide like most rodent enzymes do. Both enzymes also cleave two additional potential in vivo substrates, fibronectin and secretory leukocyte protease inhibitor (SLPI) in a similar way. Given the fact that both HC and MC are encoded by a single gene with high sequence homology and that many physiological processes are similar between these species, the macaque may be a very interesting model to study the physiological role of the chymase and to determine the potency and potential side-effects of various chymase inhibitors designed for therapeutic human use.
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| 8. |
- Thorpe, Michael
(författare)
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Haematopoietic Serine Proteases : A Cleavage Specificity Analysis
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mast cells are innate immune cells, historically involved in allergy responses involving IgE. Through this, they have earned a reputation as a fairly detrimental cell type. Their beneficial roles remain somewhat enigmatic although they clearly have the ability to modulate the immune system. This is due to their ability to synthesise many cytokines and chemokines as well as immediately release potent granule-stored mediators. One such mediator is a serine protease, chymase, which has been targeted by pharmaceutical companies developing inhibitors for use in inflammatory conditions.In order to address roles of the proteases, information regarding their cleavage specificity using substrate phage display can help find potential in vivo substrates. The human chymase cleaves substrates with aromatic amino acids in the P1 position and has a preference for negatively charged amino acids in the P2’ position. The molecular interactions mediating this P2’ preference was investigated by site-directed mutagenesis, where Arg143 and Lys192 had a clear effect in this selectivity.As humans express one chymase and rodents express multiple chymases, extrapolating data between species is difficult. Here, the crab-eating macaque was characterised, which showed many similarities to the human chymase including a near identical extended cleavage specificity and effects of human chymase inhibitors. Appropriate models are needed when developing human inhibitors for therapeutic use in inflammatory conditions.The effects of five specific chymase inhibitors in development were also tested. The selectivity of inhibitors was dependent on both Arg143 and Lys192, with a greater effect of Lys192. Identification of residues involved in specific inhibitor interactions is important for selective inhibitor development.Another innate cell type, the NK cell, is important in virus and tumour defence. In the channel catfish, a serine protease from an NK-like cell, granzyme-like I, was characterised. A strict preference for Met in the P1 position was seen, and caspase 6 was identified as a potential in vivo target. This may highlight a novel apoptosis-inducing mechanism from a similar cell type has been conserved for approximately 400 myr.Here, important residues mediating chymases’ specificity and interactions with inhibitors has been addressed, as well as finding a new animal model for providing ways to combat their roles in pathological settings.
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| 9. |
- Thorpe, Michael J., et al.
(författare)
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Frequency stabilization to 6 x 10(-16) via spectral-hole burning
- 2011
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Ingår i: Nature Photonics. - 1749-4885. ; 5:11, s. 689-694
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate two-stage laser stabilization based on a combination of Fabry-Perot and spectral-hole burning techniques. The laser is first pre-stabilized by the Fabry-Perot cavity to a fractional-frequency stability of sigma(y)(tau) < 1 x 10(-13). A pattern of multiple spectral holes written in the absorption spectrum of Eu3+:Y2SiO5 serves to further stabilize the laser to sigma(y)(tau) <= 6 x 10(-16) for 2 s <= tau <= 8 s. We also measure the frequency sensitivity of Eu3+:Y2SiO5 spectral holes to environmental perturbations including temperature (16 kHz K-2), pressure (211.4 Hz Pa-1) and acceleration (7 x 10(-12) g(-1)). Each spectral hole sensitivity parameter is lower than the corresponding parameter for Fabry-Perot cavities, suggesting that spectral holes can be more frequency-stable.
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| 10. |
- Waern, Ida, et al.
(författare)
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Mast cells limit extracellular levels of IL-13 via a serglycin proteoglycan-serine protease axis
- 2012
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Ingår i: Biological chemistry (Print). - : Walter de Gruyter GmbH. - 1431-6730 .- 1437-4315. ; 393:12, s. 1555-1567
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Tidskriftsartikel (refereegranskat)abstract
- Mast cell (MC) granules contain large amounts of proteases of the chymase, tryptase and carboxypeptidase A (MC-CPA) type that are stored in complex with serglycin, a proteoglycan with heparin side chains. Hence, serglycin-protease complexes are released upon MC degranulation and may influence local inflammation. Here we explored the possibility that a serglycin-protease axis may regulate levels of IL-13, a cytokine involved in allergic asthma. Indeed, we found that wild-type MCs efficiently degraded exogenous or endogenously produced IL-13 upon degranulation, whereas serglycin(-/-) MCs completely lacked this ability. Moreover, MC-mediated IL-13 degradation was blocked both by a serine protease inhibitor and by a heparin antagonist, which suggests that IL-13 degradation is catalyzed by serglycin-dependent serine proteases and that optimal IL-13 degradation is dependent on both the serglycin and the protease component of the serglycin-protease complex. Moreover, IL-13 degradation was abrogated in MC-CPA(-/-) MC cultures, but was normal in cultures of MCs with an inactivating mutation of MC-CPA, which suggests that the IL-13-degrading serine proteases rely on MC-CPA protein. Together, our data implicate a serglycin-serine protease axis in the regulation of extracellular levels of IL-13. Reduction of IL-13 levels through this mechanism possibly can provide a protective function in the context of allergic inflammation.
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