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<p>Semicarbazide-sensitive amine oxidase (SSAO, EC 1.4.3.6) belongs to a family of copper-containing amine oxidases. SSAO exists as a membrane bound protein in endothelial-, smooth muscle-, and adipose cells as well as soluble in plasma. SSAO catalyses oxidative deamination of primary monoamines, which results in the production of corresponding aldehydes, hydrogen peroxide and ammonia. These compounds are very reactive and potentially cytotoxic, and are able to induce vascular damage if produced in high levels. Patients with diabetes mellitus, and with diabetic complications in particular, have a higher SSAO activity in plasma compared to healthy controls. It has therefore been speculated that high SSAO activity is involved in the development of vascular complications associated with diabetes. The aim of this thesis is to investigate the importance of SSAO in the development of disorders of a vascular origin. We have studied the transcriptional regulation of the SSAO gene, by inducing diabetes in NMRI and in transgenic mice, overexpressing the human form of SSAO in smooth muscle cells. We found that the increase in SSAO activity in diabetes is accompanied by reduced mRNA levels of the endogenous mouse gene, suggesting a negative feedback on the transcription of the SSAO gene. In addition, the transgenic mice exhibited an abnormal phenotype in the elastic tissue of aorta and renal artery. These mice have a lower mean artery pressure and an elevated pulse pressure. These results indicate that high SSAO activity in smooth muscle cells is associated with a change in the morphology of large arteries. This is likely contributing to the development of vascular complications in diabetes.</p>

Semicarbazide-sensitive amine oxidase (SSAO, EC 1.4.3.6) belongs to a family of copper-containing amine oxidases. SSAO exists as a membrane bound protein in endothelial-, smooth muscle-, and adipose cells as well as soluble in plasma. SSAO catalyses oxidative deamination of primary monoamines, which results in the production of corresponding aldehydes, hydrogen peroxide and ammonia. These compounds are very reactive and potentially cytotoxic, and are able to induce vascular damage if produced in high levels. Patients with diabetes mellitus, and with diabetic complications in particular, have a higher SSAO activity in plasma compared to healthy controls. It has therefore been speculated that high SSAO activity is involved in the development of vascular complications associated with diabetes. The aim of this thesis is to investigate the importance of SSAO in the development of disorders of a vascular origin. We have studied the transcriptional regulation of the SSAO gene, by inducing diabetes in NMRI and in transgenic mice, overexpressing the human form of SSAO in smooth muscle cells. We found that the increase in SSAO activity in diabetes is accompanied by reduced mRNA levels of the endogenous mouse gene, suggesting a negative feedback on the transcription of the SSAO gene. In addition, the transgenic mice exhibited an abnormal phenotype in the elastic tissue of aorta and renal artery. These mice have a lower mean artery pressure and an elevated pulse pressure. These results indicate that high SSAO activity in smooth muscle cells is associated with a change in the morphology of large arteries. This is likely contributing to the development of vascular complications in diabetes.

The amine oxidase family of enzymes has been the center of numerous mechanistic studies because of the medical relevance of the reactions they catalyze. This study describes transient and steady-state kinetic analyses of two flavin amine oxidases, mouse polyamine oxidase (PAO) and human lysine specific demethylase (LSD1), to determine the mechanisms of amine oxidation. PAO is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyzes the oxidation of N1-acetylated polyamines. The pH-dependence of the kcat/Kamine indicates that the monoprotonated form of the substrate is required for catalysis, with the N4 nitrogen next to the site of CH bond cleavage being unprotonated. Stopped-flow spectroscopy shows that the pH-dependence of the rate constant for flavin reduction, kred, displays a pKa of 7.3 with a decrease in activity at acidic pH. This is consistent with an uncharged nitrogen being required for catalysis. Mutating Lys315 to methionine has no effect on the kcat/Kamine-pH profile with the substrate spermine, and the kred value only shows a 1.5-fold decrease with respect to wild-type PAO. The mutation results in a 30- fold decrease in kcat/KO2. Solvent isotope effects and proton inventories are consistent with Lys315 accepting a proton from a water molecule hydrogen-bonded to the flavin N5 during flavin oxidation. Steady-state and transient kinetic studies of para-substituted N,N'-dibenzyl-1,4- diaminobutanes as substrates for PAO show that the kred values for each correlate with the van der Waals volume (VW) and the value. The coefficient for VW is the same at pH 8.6 and 6.6, whereas the p value increases from -0.59 at pH 8.6 to -0.09 at pH 6.6. These results are most consistent with a hydride transfer mechanism. The kinetics of oxidation of a peptide substrate by human lysine specific demethylase (LSD1) were also studied. The kcat/KM pH-profile is bell-shaped, indicating the need for one unprotonated nitrogen next to the site of CH bond cleavage and another protonated nitrogen. The kcat and kred values are equal, and identical isotope effects are observed on kred, kcat, and kcat/KM, indicating that CH bond cleavage is rate-limiting with this substrate.

Several members of the lysyl oxidase family of copper-dependent amine oxidases have been implicated in tumor development. The Iysyl oxidase (LOX) and LOX-like 2 (LOXL2) genes have been mapped to chromosomal regions affected by loss of heterozygosity (LOH) in several cancers, including those of the colon and esophagus. Indeed, there have been numerous reports of reduced LOX and a few reports of reduced LOXL2 expression in various cancers. Identification of microsatellite markers within the LOX locus and the LOXL2 gene allowed for evaluation ofthe status of these gene alleles in colon and esophageal tumors. There was significant LOH of the LOX locus in colon tumors that was accompanied by reduced mRNA expression and a spectrum of alterations and mutations affecting the LOX gene. This study demonstrated, for the first time, that genetic events, namely LOH, deletions and mutations ofthe LOX gene, were responsible, at least partly, for the reduction of LOX gene expression. There was also significant LOH of the LOXL2 gene in both colon and esophageal tumors. However, instead of a reduction of LOXL2 expression, there was increased expression that correlated with less differentiated tumors and absent elastosis, both indicators of poor prognosis. Further studies indicated that both LOX and LOXL2 are absent in non-invasive tumor cell lines but re-expressed in invasive cell lines, likely as part of the thelial-mesenchymal transition that occurs in the last steps of tumorigenesis to facilitate metastasis. The results presented and research strategy outlined in this dissertation will define the importance of LOXL2 amine oxidase activity and protein interactions in the critical but poorly understood process oftumor cell migration and invasion.