| 1. |
- Backman, Dan, 1972-
(författare)
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Interaction Studies of Secreted Aspartic Proteases (Saps) from Candida albicans : Application for Drug Discovery
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis is focused on enzymatic studies of the secreted aspartic proteases (Saps) from Candida albicans as a tool for discovery of anti-candida drugs. C. albicans causes infections in a number of different locations, which differ widely in the protein substrates available and pH. Since C. albicans needs Saps during virulent growth, these enzymes are good targets for drug development.In order to investigate the catalytic characteristics of Saps and their inhibitor affinities, substrate-based kinetic assays were developed. Due to the low sensitivity of these assays, especially at the sub-optimal pH required to mimic the different locations of infections, these assays were not satisfactory. Therefore, a biosensor assay was developed whereby, it was possible to study interaction between Saps and inhibitors without the need to optimise catalytic efficacy. Furthermore, the biosensor assay allowed determination of affinity, as well as the individual association and dissociation rates for inhibitor interactions.Knowledge about substrate specificity, Sap subsite adaptivity, and the pH dependencies of catalytic efficacy has been accumulated. Also, screening of transition-state analogue inhibitors designed for HIV-1 protease has revealed inhibitors with affinity for Saps. Furthermore, the kinetics and pH dependencies of their interaction with Saps have been investigated. One of these inhibitors, BEA-440, displayed a complex interaction with Saps, indicating a conformational change upon binding and a very slow dissociation rate. A time dependent interaction was further supported by inhibition measurements. The structural information obtained affords possibilities for design of new more potent inhibitors that might ultimately become drugs against candidiasis. The strategy to combine substrate specificity studies with inhibitor screening has led to complementary results that generate a framework for further development of potent inhibitors.
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| 2. |
- Eklund, Sandra, et al.
(författare)
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Characterization of the endopeptidase activity of tripeptidyl-peptidase II
- 2012
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 424:3, s. 503-507
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Tidskriftsartikel (refereegranskat)abstract
- Tripeptidyl-peptidase II (TPP II) is a giant cytosolic peptidase with a proposed role in cellular protein degradation and protection against apoptosis. Beside its well-characterised exopeptidase activity, TPP II also has an endopeptidase activity. Little is known about this activity, and since it could be important for the physiological role of TPP II, we have investigated it in more detail. Two peptides, Nef(69-87) and LL37, were incubated with wild-type murine TPP II and variants thereof as well as TPP II from human and Drosophila melanogaster. Two intrinsically disordered proteins were also included in the study. We conclude that the endopeptidase activity is more promiscuous than previously reported. It is also clear that TPP II can attack longer disordered peptides up to 75 amino acid residues. Using a novel FRET substrate, the catalytic efficiency of the endopeptidase activity could be determined to be 5 orders of magnitude lower than for the exopeptidase activity.
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| 3. |
- Eklund, Sandra, et al.
(författare)
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Exploring the active site of tripeptidyl-peptidase II through studies of pH dependence of reaction kinetics
- 2012
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639 .- 1878-1454. ; 1824:4, s. 561-570
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Tidskriftsartikel (refereegranskat)abstract
- Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine protease which forms a large enzyme complex (> 4 MDa). It is considered a potential drug target due to its involvement in specific physiological processes. However, information is scarce concerning the kinetic characteristics of TPP II and its active site features, which are important for design of efficient inhibitors. To amend this, we probed the active site by determining the pH dependence of TPP II catalysis. Access to pure enzyme is a prerequisite for kinetic investigations and herein we introduce the first efficient purification system for heterologously expressed mammalian TPP II. The pH dependence of kinetic parameters for hydrolysis of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was determined for murine, human and Drosophila melanogaster TPP II as well as mutant variants thereof. The investigation demonstrated that TPP II, in contrast to subtilisin, has a bell-shaped pH dependence of kcatapp/KM probably due to deprotonation of the N-terminal amino group of the substrate at higher pH. Since both the KM and kcatapp are lower for cleavage of AAA-pNA than for AAF-pNA we propose that the former can bind non-productively to the active site of the enzyme, a phenomenon previously observed with some substrates for subtilisin. Two mutant variants, H267A and D387G, showed bell-shaped pH-dependence of kcatapp, possibly due to an impaired protonation of the leaving group. This work reveals previously unknown differences between TPP II orthologues and subtilisin as well as features that might be conserved within the entire family of subtilisin-like serine peptidases.
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| 4. |
- Eklund, Sandra, et al.
(författare)
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Inter-species variation in the pH dependence of tripeptidyl-peptidase II
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Tripeptidyl-peptidase II (TPP II) is a large enzyme complex (>4 MDa) participating in the general protein turn-over in the cell downstream of the proteasome. In addition, there have been reports of involvement of TPP II in different physiological situations. To facilitate further investigations of the physiological role of TPP II and its enzymatic properties, a characterization at protein level is necessary. Therefore, an expression system for murine TPP II using Escherichia coli has been developed. The pH-optimum for cleavage of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was investigated for mTPP II, and compared with human TPP II and TPP II from Drosophila melanogaster. It was shown that the mouse enzyme had similar pH dependence as the human enzyme, while dTPP II had a slightly lower optimum. Surprisingly, the investigation also demonstrated that TPP II from all sources showed a different pH-profile for hydrolysis of AAA-pNA compared to AAF-pNA. To investigate this observation further, steady-state kinetic parameters were determined at various pH. Since both the KM and Vmax are lower for cleavage of AAA-pNA, a potential explanation could be that the substrate AAA-pNA is non-productively bound to the active site of the enzyme.
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| 5. |
- Eklund, Sandra, 1982-
(författare)
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Interpreting a Giant : Studies of Structure and Function of Tripeptidyl-peptidase II
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine peptidase that forms a gigantic homooligomeric complex, and is involved in the degradation of peptides in the cytosol. In addition, TPP II has been implicated in specific cellular processes, such as apoptosis control and adipogenesis, but if this is dependent on its endo- or exopeptidase activity remains to be determined. This work is devoted to the structure and function of TPP II, and to finding connections between the two. Evolutionarily conserved regions of TPP II have been identified, and sequence signatures have been constructed as an aid in identification of TPP II homologues. The conserved regions highlight amino acid residues of potential importance to structure, function or both. In addition, the first TPP II homologue in a prokaryote has been documented, which was likely the result of a horizontal gene transfer. Substrate binding for the exopeptidase activity of TPP II has been studied through mutagenesis of Glu-331, which revealed a molecular ruler mechanism that positions substrates for cleavage at the third peptide bond from the N-terminus. Thus, the well-known tripeptidyl-releasing property of TPP II could be explained. The exopeptidase activity was also probed by pH dependence studies, which revealed that a substrate with a smaller residue in the P1 position could bind non-productively to the active site. Furthermore, a difference in the pH dependence of KM between TPP II from Drosophila and homologues from mammals indicated a difference in the configuration of the binding pockets between these species. The endopeptidase activity of TPP II has also been investigated, and was found to differ from the exopeptidase activity. The endopeptidase activity appeared to be promiscuous and the preference for basic amino acid residues in the P1 position reported earlier could not be substantiated. In conclusion, many structural and mechanistic features have been observed in this work. This might be of value to future drug discovery efforts towards TPP II, and in elucidating the physiological role of this gigantic enzyme.
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| 6. |
- Eklund, Sandra, et al.
(författare)
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Structure, Function and Evolution of a Giant Enzyme, Tripeptidyl-Peptidase II
- 2012
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Ingår i: Serine proteases. - : Nova Science Publishers, Inc.. - 9781619426696 ; , s. 55-70
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Bokkapitel (refereegranskat)abstract
- Tripeptidyl-peptidase II (TPP II) is a giant exopeptidase with an active site of the subtilisin-type. Its main function is to remove tripeptides from a free N-terminal end of longer peptides. TPP II is active at neutral pH and is dependent on the same catalytic triad as other subtilases, i.e. Asp-44, His-264 and Ser-449 (numbering for murine TPP II). Furthermore, Glu-331 has been shown to be important for binding the N-terminal amino group of the substrate. Besides its exopeptidase activity, TPP II also appears to have a low endopeptidase activity. The large subunit (138 kDa in humans) forms a >4 MDa. Oligomerisation is essential for full enzymatic activity. The recently determined hybrid structure of the TPP II spindle from Drosophila melanogaster demonstrated that the active site is localized inside the spindle and that it is a self-compartmentalizing enzyme. TPP II is present in most eukaryotes, but has not been detected in archea and the homologous genes that appear in prokaryotes are suggested to be the result of a horizontal gene transfer. A role for TPP II in degradation of the neuropeptide cholecystokinin has been suggested, and the enzyme appears to be involved in trimming of some substrates for antigen presentation. However, considering its widespread distribution, this is probably not its main physiological function. A more reasonable assumption is that the enzyme has evolved to participate in a general protein turnover in the cytosol of most cells, presumably together with the proteasome and other peptidases.
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| 7. |
- Engvall, Caroline, 1976-
(författare)
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Drug Partitioning into Natural and Artificial Membranes : Data Applicable in Predictions of Drug Absorption
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- When drug molecules are passively absorbed through the cell membrane in the small intestine, the first key step is partitioning of the drug into the membrane. Partition data can therefore be used to predict drug absorption. The partitioning of a solute can be analyzed by drug partition chromatography on immobilized model membranes, where the chromatographic retention of the solute reflects the partitioning. The aims of this thesis were to develop the model membranes used in drug partition chromatography and to study the effects of different membrane components and membrane structures on drug partitioning, in order to characterize drug–membrane interactions. Electrostatic effects were observed on the partitioning of charged drugs into liposomes containing charged detergent, lipid or phospholipid; bilayer disks; proteoliposomes and porcine intestinal brush border membrane vesicles (BBMVs), and on the retention of an oligonucleotide on positive liposomes. Biological membranes are naturally charged, which will affect drug partitioning in the human body. Proteoliposomes containing transmembrane proteins and cholesterol, BBMVs and bilayer disks were used as novel model membranes in drug partition chromatography. Partition data obtained on proteoliposomes and BBMVs demonstrated how cholesterol and transmembrane proteins interact with drug molecules. Such interactions will occur between drugs and natural cell membranes. In the use of immobilized BBMVs for drug partition chromatography, yet unsolved problems with the stability of the membrane were encountered. A comparison of partition data obtained on bilayer disks with data on multi- and unilamellar liposomes indicated that the structure of the membrane affect the partitioning. The most accurate partition values might be obtained on bilayer disks. Drug partition data obtained on immobilized model membranes include both hydrophobic and electrostatic interactions. Such partition data should preferably be used when deriving algorithms or computer programs for prediction of drug absorption.
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| 8. |
- Eriksson, Sandra, et al.
(författare)
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Development, evaluation and application of tripeptidyl-peptidase II sequence signatures
- 2009
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 484:1, s. 39-45
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Tidskriftsartikel (refereegranskat)abstract
- Tripeptidyl-peptidase II (TPP II) is a cytosolic peptidase that has been implicated in fat formation and cancer, apparently independent of the enzymatic activity. In search for alternative functional regions, conserved motifs were identified and eleven signatures were constructed. Seven of the signatures covered previously investigated residues, whereas the functional importance of the other motifs is unknown. This provides directions for future investigations of alternative activities of TPP II. The obtained signatures provide an efficient bioinformatic tool for the identification of TPP II homologues. Hence, a TPP II sequence homologue from fission yeast, Schizosaccharomyces pombe, was identified and demonstrated to encode the TPP II-like protein previously reported as multicorn. Furthermore, an homologous protein was found in the prokaryote Blastopirellula marina, albeit the TPP II function was apparently not conserved. This gene is probably the result of a rare gene transfer from eukaryote to prokaryote.
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| 9. |
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| 10. |
- Kessler, Jan H., et al.
(författare)
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Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes
- 2010
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Ingår i: Nature Immunology. - : Springer Science and Business Media LLC. - 1529-2908 .- 1529-2916. ; 12:1, s. 45-53
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Tidskriftsartikel (refereegranskat)abstract
- Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus protein EBNA3C, and a clinically important epitope from the melanoma protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing, and nardilysin contributed to both the C-terminal and N-terminal generation of CTL epitopes. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.
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