Diss. (sammanfattning) Stockholm : Stockholms universitet, 2009. / Härtill 4 uppsatser.

Diss. (sammanfattning) Luleå : Luleå tekniska universitet, 2009. / Härtill 5 uppsatser.

Ph.D. afhandling, Roskilde universitetscenter 2004.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 7 uppsatser.

Lic.-avh. (sammanfattning) Luleå : Luleå tekniska univ., 2007. / Härtill 2 uppsatser.

Licentiatavhandling (sammanfattning) Karlstad : Karlstads universitet, 2009. / Härtill 2 uppsatser.

Diss. (sammanfattning) Karlstad : Karlstads universitet, 2008. / Härtill 5 uppsatser.

<p>Glutathione transferases (GSTs) make up a superfamily that is involved in the cellular defense against various reactive compounds by catalyzing the conjugation of glutathione to electrophilic centra. Members of this family have also been implicated in different facets of biological signaling. </p><p>The gene encoding human GST P1-1 is polymorphic, resulting in variant amino acid residues in positions 105 and 114. The role of the polymorphism in the active-site residue 105 on enzyme stability and activity with various substrates was investigated. A valine instead of an isoleucine in position 105 decreased the thermal stability of the enzyme. The effect on enzyme activity was dependent on the substrate and reaction studied. With some substrates tested, such as carcinogenic diolepoxides derived from polyaromatic hydrocarbons, GST P1-1/Val105 displayed the highest catalytic efficiency. In contrast, with 1-chloro-2,4-dinitrobenzene, the GST P1-1/Ile105 showed higher activity. Residue 105 was mutated to alanine and tryptophan to investigate the role of size and hydrophobicity of residue 105 on enzyme properties. Generally, a smaller amino acid in position 105 gave increased activity with large substrates. Clearly, residue 105 of GST P1-1 helps to determine the substrate selectivity of the enzyme. In addition, more voluminous amino acids in position 105 increase the thermal stability of the enzyme. </p><p>GST P1-1 is believed to contribute to the development of drug resistance in cancer cells. The affinity of GST P1-1 for TER 117, designed to inhibit GST P1-1 in tumors, was not affected by the variability in position 105. TER 117 was found to be a potent inhibitor of glyoxalase I as well.</p><p>The cDNA encoding GST A3-3 was isolated from a placental cDNA library. GST A3-3 was heterologously expressed, purified and found to catalyze efficiently the double-bond isomerization of Δ⁵-androstene-3,17-dione and Δ⁵-pregnene-3,20-dione, reactions taking place in the biosynthesis of the steroid hormones testosterone and progesterone, respectively. GST A3-3 was found to be selectively expressed in steroidogenic tissues, suggesting that this enzyme is involved in the production of steroid hormones. The presence of both the hydroxyl group of the active-site tyrosine 9 and the thiolate form of glutathione, acting as a cofactor, is important for high double-bond isomerase activity. A leucine in position 111 appears to have a major role in productive binding of the steroid substrate but also residues F10 and A216 are determinants for the high isomerase activity. </p><p>GST A2-2 is a poor catalyst of the steroid double-bond isomerization of Δ⁵-androstene-3,17-dione as compared to GST A3-3, despite 88% sequence identity. GST A2-2 was redesigned to a highly efficient double-bond isomerase by mutating five active-site residues to the corresponding residues of GST A3-3. This demonstrates the functional plasticity of GSTs and the power of a rational approach to redesign of these enzymes. </p>

<p>Hyaluronan, a component of the extracellular matrix, is synthesized by either of three hyaluronan-synthesizing enzymes termed Has1, Has2 and Has3. The expression level of each <i>Has</i> gene varies between cell types of mesenchymal origin and is differentially regulated in response to external stimuli. For example, stimulation of mesothelial cells with PDGF-BB induced an up-regulation of the <i>Has2</i> gene, whereas the <i>Has1</i> and <i>Has3</i> genes remained unaffected. The induction of <i>Has2</i> gene expression correlated well with increased Has2 protein levels and accumulation of hyaluronan. Moreover, treatment of mesothelial cells with hydrocortisone suppressed hyaluronan synthesis in cell culture primarily through down-regulation of the <i>Has2 </i>gene. Thus, among the <i>Has</i> isoforms, <i>Has2</i> seems to be most markedly regulated in response to external stimuli.</p><p>In an attempt to investigate the importance of hyaluronan in tumor progression, the hyaluronan synthesizing enzyme Has2 and the hyaluronan degrading enzyme Hyal1 were over-expressed in a rat colon adenocarcinoma cell line, PROb. We found that <i>Has2</i> gene over-expression in colon carcinoma cells promoted cell growth <i>in vitro</i> and progression of transplantable tumors. In contrast, over-expression of <i>Hyal1 </i>lead to a considerable reduction of growth rates both <i>in vivo</i> and <i>in vitro</i>. A linear correlation between tumor growth rate and hyaluronan amount in tumor tissue was observed. In another tumor model, experimental anaplastic thyroid carcinoma, the effects of TGF-β inhibition on hyaluronan and collagen contents in tumor xenografts were investigated. We found that inhibition of TGF-β, a stimulator of hyaluronan and collagen synthesis, lead to reduced collagen deposition whereas the hyaluronan levels in stromal tissue only marginally differed. Our results indicate that a high ratio of collagen to hyaluronan may be characteristic of a pathogenic mechanism that leads to elevated interstitual tumor pressure.</p>

<p>The present work focuses on the glutathione transferase (GST) Alpha-class enzymes, their characteristics as steroid isomerases and structural plasticity as malleable scaffolds for protein design. The GSTs are a family of detoxication enzymes that appears to have a wider variety of additional functions.</p><p>Kinetic steady-state parameters for human GST A1-1 with the steroid isomerase substrate Δ5-androstene-3,17-dione (AD), an intermediate in steroid hormone biosynthesis, were determined. It was established that GST A1-1 is a highly efficient steroid isomerase with a 30-fold higher catalytic efficiency, in terms of <i>k</i>_cat/<i>K</i>_m, than 3β-hydroxysteroid dehydrogenase/Δ⁵→⁴-isomerase, the enzyme regarded as the mammalian Δ⁵→⁴-isomerase in steroid hormone biosynthesis. Kinetic parameters were also determined for GST A2-2, GST A4-4 and the GST A1-1 mutant Y9F. From the dependency on pH of the kinetic parameters it was established that efficient catalysis requires glutathione (GSH) in its deprotonated form and it is suggested that the GSH-thiolate acts as a base in the catalysis of the Δ⁵→⁴-3-ketosteroid isomerase reaction.</p><p>GST A2-2 is a poor catalyst of the steroid isomerase reaction while GST A3-3 is highly efficient. Their catalytic efficiencies (<i>k</i>_cat/<i>K</i>_m) differ 5000-fold. Stepwise point mutations were performed to GST A2-2 in order to insert the amino acid residues from the active-site of GST A3-3 that distinguishes the two isoenzymes. The result was that GST A2-2 was redesigned to a highly efficient double-bond isomerase with both the catalytic constant (<i>k</i>_cat) and catalytic efficiency (<i>k</i>_cat/<i>K</i>_m) in the same order as for GST A3-3. Furthermore, this was done by only exchanging amino-acid residues with first-sphere interactions, providing empirical proof-of principle for knowledge-based enzyme design.</p><p>Kinetic studies on GST A1-1 and a T68E mutant of GST A1-1 were also performed with a GSH analog lacking the g-glutamate a-carboxylate (dGSH), and using three different electrophilic substrates (AD; 1-chloro-2,4-dinitrobenzene, CDNB; 4-nitrocinnamaldehyde). Deletion of the a-carboxylate from the GSH glutamate had a severe impact on all reaction constants and it changed the rate-limiting step for the CDNB reaction as well as changed the pKa value for the enzyme-bound GSH thiol. The loss in activity caused by dGSH could in part be compensated by the T68E mutant contributing an enzyme-bound carboxylate instead.</p><p>The C-terminus of GST A1-1 is flexible and folds over the active site when the enzyme binds a substrate. Phenylalanine residues in the C-terminal end, known to interact with active-site residues tyrosine 9 and phenylalanine 10, were mutated to abolish those interactions. Studies of viscosity dependence for CDNB and AD with regard to <i>k</i>_cat and <i>k</i>_cat/<i>K</i>_m showed that the dynamic C-terminal segment influence rate-determining steps for both the larger isomerase substrate, AD, as well as for the smaller conjugation substrate, CDNB.</p>