| 1. |
- Carlsson, Karin, 1975-, et al.
(författare)
-
Inhibitors of factor VIIa affect the interface between the protease domain and tissue factor
- 2006
-
Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 349:3, s. 1111-1116
-
Tidskriftsartikel (refereegranskat)abstract
- Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). The γ-carboxyglutamic acid-rich domain of FVIIa docks with the C-terminal domain of TF, the EGF1 domain of FVIIa contacts both domains of TF, and the EGF2 domain and protease domain (PD) form a continuous surface that sits on the N-terminal domain of TF. Our aim was to investigate the conformational changes that occur in the sTF·PD binding region when different types of inhibitors, i.e., one active-site inhibitor (FFR-chloromethyl ketone (FFR)), two different peptide exosite inhibitors (E-76 and A-183), and the natural inhibitor tissue factor pathway inhibitor (TFPI), were allowed to bind to FVIIa. For this purpose, we constructed two sTF mutants (Q37C and E91C). By the aid of site-directed labeling technique, a fluorescent label was attached to the free cysteine. The sTF·PD interface was affected in position 37 by the binding of FFR, TFPI, and E-76, i.e., a more compact structure was sensed by the probe, while for position 91 located in the same region no change in the surrounding structure was observed. Thus, the active site inhibitors FFR and TFPI, and the exosite inhibitor E-76 have similar effects on the probe in position 37 of sTF, despite their differences in size and inhibition mechanism. The allosteric changes at the active site caused by binding of the exosite inhibitor E-76 in turn induce similar conformational changes in the sTF·PD interface as does the binding of the active site inhibitors. A-183, on the other hand, did not affect position 37 in sTF, indicating that the A-183 inhibition mechanism is different from that of E-76.
|
|
| 2. |
- Wirehn, J., et al.
(författare)
-
Activity, folding, misfolding, and aggregation in vitro of the naturally occurring human tissue factor mutant R200W
- 2005
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 44:18, s. 6755-6763
-
Tidskriftsartikel (refereegranskat)abstract
- Tissue factor (TF), a small transmembrane receptor, binds factor VIIa (FVIIa), and the formed complex initiates blood coagulation by proteolytic activation of substrate factors IX and X. A naturally occurring mutation in the human TF gene was recently reported, where a single-base substitution results in an R200W mutation in the TF extracellular domain [Zawadzki, C., Preudhomme, C., Gavériaux, V., Amouyel, P., and Jude, B. (2002) Thromb. Haemost. 87, 540-541]. This mutation appears to be associated with low monocyte TF expression and may protect against thrombosis but has not been associated with any pathological condition, and individuals who present the heterozygous trait appear healthy. Here, we report the activity, folding, and aggregation behavior of the R200W mutant of the 219-residue soluble extracellular domain of TF (sTFR200W) compared to that of the wild-type protein (sTF wt). No differences in stability or FVIIa cofactor activity but an impaired ability to promote FX activation at physiological conditions between the sTFR200W mutant and sTFwt were evident. Increased binding of 1-anilino-8-naphthalene-sulfonic acid (ANS) to sTFR200W indicated a population of partially folded intermediates during denaturation. sTFR200W showed a dramatically increased propensity for aggregate formation compared to sTFwt at mildly acidic pHs, with an increased rate of aggregation during conditions, promoting the intermediate state. The lowered pH resistance could explain the loss of sTFR200W in vivo because of aggregation of the mutant. The intrinsic structure of the sTF aggregates appears reminiscent of amyloid fibrils, as revealed by thioflavin T fluorescence, atomic force microscopy, and transmission electron microscopy. We conclude that the lowered activity for FX activation and the propensity of the mutant protein to misfold and aggregate will both contribute to decreased coagulation activity in TFR200W carriers, which could protect from thrombotic disease. © 2005 American Chemical Society.
|
|
| 3. |
- Almstedt, Karin, et al.
(författare)
-
Small-Molecule Suppression of Misfolding of Mutated Human Carbonic Anhydrase II Linked to Marble Brain Disease
- 2009
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 48:23, s. 5358-5364
-
Tidskriftsartikel (refereegranskat)abstract
- Carbonic anhydrase II deficiency syndrome or Marble brain disease (MBD) is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. Here we report a small-molecule stabilization study of the exceptionally destabilized HCA II mutant H107Y employing inhibitors based on p-aminobenzoyisulfonamide compounds and 1,3,4-thiadiazolylsulfonamides as well as amino acid activators. Protein stability assays showed a significant stabilization by the aromatic sulfonamide inhibitors when present at 10 mu M concentration, providing shifts of the midpoint of thermal denaturation between 10 degrees C and 16 degrees C and increasing the free energies of denaturation 0.5-3.0 kcal/mol as deduced from GuHCl denaturation. This study could be used as a starting point for the design of small-molecule folding modulators and possibly autoactivatable molecules for suppression of misfolding of destabilized HCA II mutants.
|
|
| 4. |
- Almstedt, Karin, 1980-, et al.
(författare)
-
Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease.
- 2008
-
Ingår i: Biochemistry. - Washington : ACS. - 0006-2960 .- 1520-4995. ; 47:5, s. 1288-1298
-
Tidskriftsartikel (refereegranskat)abstract
- Marble brain disease (MBD) also known as Guibaud−Vainsel syndrome is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. HCA II is a 259 amino acid single domain enzyme and is dominated by a 10-stranded β-sheet. One mutation associated with MBD entails the H107Y substitution where H107 is a highly conserved residue in the carbonic anhydrase protein family. We have previously demonstrated that the H107Y mutation is a remarkably destabilizing folding mutation [Almstedt et al. (2004) J. Mol. Biol. 342, 619−633]. Here, the exceptional destabilization by the H107Y mutation has been further investigated. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II toward populating ensembles of intermediates of molten globule type under physiological conditions. The native state stability of the mutants was in the following order: wt > H107N > E117A > H107A > H107F > H107Y > H107N/E117A > H107A/E117A. In conclusion: (i) H107N is least destabilizing likely due to compensatory H-bonding ability of the introduced Asn residue. (ii) Double mutant cycles surprisingly reveal additive destabilization of H107N and E117A showing that H107 and E117 are independently stabilizing the folded protein. (iii) H107Y and H107F are exceptionally destabilizing due to bulkiness of the side chains whereas H107A is more accommodating, indicating long-range destabilizing effects of the natural pathogenic H107Y mutation.
|
|
| 5. |
|
|
| 6. |
- Forsberg, Ole, 1979-, et al.
(författare)
-
High frequency of prostate antigen-directed T cells in cancer patients compared to healthy age-matched individuals
- 2009
-
Ingår i: The Prostate. - : Wiley. - 0270-4137 .- 1097-0045. ; 69:1, s. 70-81
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND. In order to obtain a sustained cytotoxic T lymphocyte (CTL) response against cancer cells it is preferable to have CTLs directed against multiple peptide epitopes from numerous tumor-associated antigens. METHODS. We used a flow cytometry-based interferon (IFN)-g secretion assay to analyze whether CD8+ T cells directed against any of 24 HLA-A*0201-binding peptides from 15 prostate-associated proteins can be found in the peripheral blood of patients with localized prostate cancer. We also investigated whether multiple prostate antigen-specific CD8+ T cells can be generated simultaneously, from a naïve T cell repertoire. In that case, dendritic cells (DCs) from peripheral blood of healthy donors were divided in six portions and separately pulsed with six peptides. The peptide-pulsed DCs were then pooled and used to stimulate autologous T cells. The T cells were re-stimulated with peptide-pulsed monocytes. RESULTS. We found prostate antigen-restricted CD8+ T cells in the peripheral blood in 48 out of 184 (26.1%) analyzed samples from 25 cancer patients. This is significantly higher than 17 out of 214 analyzed samples (7.9%) from 10 healthy age-matched male individuals (p = 0.0249). In the cases when antigen-specific T cells could not be detected, we were able to generate IFN-g-producing CD8+ T cells specific for up to three prostate antigens simultaneously from a naïve T cell repertoire. CONCLUSIONS. CD8+ T cells directed against prostate antigen peptides can be found in, or generated from, peripheral blood. This indicates that such T cells could be expanded ex vivo for adoptive transfer to prostate cancer patients.
|
|
| 7. |
|
|
| 8. |
- Gårdmark, Truls, et al.
(författare)
-
Analysis of HER2 expression in primary urinary bladder carcinoma and corresponding metastases
- 2005
-
Ingår i: BJU International. - 1464-4096 .- 1464-410X. ; 95:7, s. 982-986
-
Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: To evaluate the expression of HER2 receptors (previously reported to be over-expressed in malignant urothelium) in both primary tumours and metastases of transitional cell cancer, using two different staining methods and two different scoring techniques, considering the potential use of these receptors as targets for planned systemic anti-HER2 nuclide-based treatment. MATERIALS AND METHODS: HER2 expression was evaluated with two different immunohistochemical methods in 90 patients with primary urinary bladder cancer tumours and corresponding metastases. Sections were first stained with the commercially available breast cancer test kit (HercepTest, Dako, Glostrup, Denmark). Parallel sections were then stained with a modified HercepTest procedure. Two different evaluation criteria were compared; the HercepTest score that requires > or = 10% stained tumour cells (as for breast cancer) and a proposed 'Target score' that requires > 67% stained tumour cells. The latter score is assumed to be preferable for HER2-targeted radionuclide therapy. RESULTS: Using the HercepTest kit, the Target score gave lower fractions of positive primary tumours and metastases than the HercepTest score. The modified HercepTest staining procedure and Target score gave high HER2 values in 80% of primary tumours and 62% of metastases, which is considerably more than that obtained with the HercepTest staining and score. There was a significant decrease in HER2 positivity with increasing distance from the primary tumour. In nine sentinel-node metastases assessed, all but one were HER2-positive. Considering all regional metastases, 74% were positive, and of distant metastases, 47%; 72% of the patients with positive primary tumours also expressed HER2 in their metastases. CONCLUSIONS: When combining the modified HercepTest with customised evaluation criteria, more HER2-positive tumours were diagnosed. The degree of HER2 down-regulation was significantly higher in distant than in regional metastases. HER2-targeted therapy may be an alternative or complementary to other methods in the future treatment of metastatic urinary bladder carcinoma.
|
|
| 9. |
- Gårdmark, Truls, 1965-
(författare)
-
Urinary Bladder Carcinoma – Studies of Outcome of Current Management and Experimental Therapy
- 2006
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The thesis concerns the epidemiology, current and possible future treatment of urothelial cancer of the urinary bladder. The Swedish National Quality Registry for Bladder Cancer 1997-2001 was used to explore epidemiology, current therapies and outcome. More common in men, the incidence for Ta and T1 tumours peaks in the age range 70-79 years. There were differences in treatment activity between the reporting regions. An increasing activity was seen. Older patients received less intravesical treatment, which was also a tendency for women. The five year relative survival for all stages (Ta-T4) was 70%; 93% for Ta and 75% for T1. For Ta or T1 survival did not differ significantly between regions. Because the registry has only been running since 1997 a long term follow-up (ten years) of 250 patients comparing Bacillus Calmette-Guerin and Mitomycin-C, was performed. No differences regarding complementary treatment, progression or survival (overall or disease specific) were shown. Looking for new drugs, gemcitabine was tried for intravesical instillations. Patients were randomised to one of three dose schedules. The effect on a marker tumour lesion was evaluated after nine weeks. The overall complete response rate was 31% (9/29). Side effects were more common in women but generally mild; the most common was nausea. One patient stopped instillations (nausea and fever). No patients were excluded due to pathological changes in laboratory parameters. For metastasised disease, over-expression of the growth factor receptor HER2 on urothelial cancer cells was explored in primary tumours and metastases, aiming at radionuclide target therapy. With a new antigen retrieval procedure and evaluation protocol 80% of primary tumours overexpressed the receptor and 72% remained so in the metastases. In conclusion current therapies were increasingly used by clinicians. Superiority for BCG could not be proven. Prerequisites for new therapies have been explored and the way has been paved for future studies.
|
|
| 10. |
- Karlsson, Martin, 1965-, et al.
(författare)
-
Adsorption at the liquid-solid Interface - Influence of protein stability on conformational changes
- 2007. - 2
-
Ingår i: Encyclopedia of surfaces and colloid science. - : Taylor & Francis. - 9780849396090
-
Bokkapitel (refereegranskat)abstract
- Protein adsorption has large implications in a variety of fields and can be both a problem and an asset. Most often protein adsorption is accompanied by structural changes in the adsorbed protein. The degree and rate of these changes are dependent on the surface, conditions during adsorption and experimental set up as well as of intrinsic properties of the protein. The effect of conformational changes influences both practical applications and experimental results in studies of protein adsorption at the liquid/solid interface. The intrinsic property of the protein that is most instrumental for conformational changes upon adsorption is the stability of the protein. Hence, large efforts have been directed towards analysis of how both the nature of surfaces and conditions influence the stability of proteins upon adsorption. Less work has been focused on the reversed view, i.e. how the stability of proteins influences adsorption, the rate and degree of the subsequent conformational changes as well as the effects of these changes. However, the increasing use of proteins in a variety of medical and biotechnological applications requires a deeper knowledge of the importance and effects of stabilizing interactions in the protein structure. Engineered stabilized proteins that are less affected by surface interactions should be of potential use for various practical purposes.
|
|
