Abstract Lysyl hydroxylases (E.C. 1.14.11.4, LHs) have three isoenzymes that are found in humans and mice, and they hydroxylate lysine residues in collagens and other proteins containing collagenous sequences. The hydroxylysines formed are crucial for the intermolecular collagen crosslinks that stabilise collagen fibres, thereby providing the stiffness and stability required by various tissues. In addition, hydroxylysines serve as attachment sites for carbohydrates, whose functions on collagen molecules are not completely understood yet. In humans, lack of LH1 causes Ehlers-Danlos syndrome (EDS) VIA, which is characterised, for example, by severe progressive kyphoscoliosis and muscular hypotonia with joint laxity. Mutations in the LH2 gene are associated with Bruck syndrome, which is characterised by fragile bones with congenital joint contractures. In the present work recombinant human lysyl hydroxylases were produced in insect cells and purified to homogeneity. Limited proteolysis revealed that LHs consist of at least three structural domains. The N-terminal domain plays no role in the lysyl hydroxylase activity, but instead, is responsible for the recently reported glucosyltransferase activity of LH3, and the galactosyltransferase activity reported here for the first time. The LH polypeptide lacking the N-terminal domain is a fully active LH with Km values identical to those of full-length enzyme. In addition, direct evidence is shown that LH2, but not LH1 or LH3, hydroxylates the telopeptide lysine residues of fibrillar collagens. All three recombinant LHs were able to hydroxylate the synthetic peptides representing the helical hydroxylation sites in types I and IV collagens, with some differences in the Vmax and Km values. In addition, all three LHs hydroxylated the collagenous domain of coexpressed type I procollagen chain to similar extend. In this study mouse lines lacking LH3 or LH1 were created and analysed. Unexpectedly, the LH3 null mice died during the embryonal period due to fragmentation of basement membranes. Type IV collagen, one of the major components in basement membranes, aggregates on its way to extracellular space and is absent from the basement membranes making them fragile. This is most probably caused by abnormal processing of type IV collagen due to decreased glucosyltransferase activity of the LH3 null embryos. The first mouse model for human EDS VIA is presented here. The LH1 null mice did not have kyphoscoliosis characteristic of EDS VIA, but showed gait abnormalities due to muscular hypotonia and possible joint laxity, as also seen in EDS VIA patients. In addition, the null mice died occasionally from aortic ruptures. Ultra structural analysis revealed degradation of smooth muscle cells and abnormal collagen fibres even in non-ruptured aortas of LH1 null mice. The hydroxylation of lysine residues and crosslinking in LH1 null mice were also abnormal, as in human EDS VIA patients. The LH1 null mouse line provides an excellent tool for analysing several aspects of human EDS VIA, including muscular hypotonia, abnormalities in collagen fibres and their crosslinking.

Abstract Collagen prolyl 4-hydroxylase (C-P4H) plays a central role in the biosynthesis of collagens by hydroxylating proline residues. The enzyme has been a subject of intense interest as a target enzyme for drug development. The recombinant expression of human C-P4H in prokaryotes has not yet been described. This work reports on the development of an expression system for human C-P4H in E. coli. The vertebrate C-P4H enzymes are α2β2 tetramers, consisting of two β subunits which are identical to protein disulphide isomerase (PDI), aside from the two α subunits which have the catalytic activity. The function of PDI is to keep the α subunit in a soluble and active state. Therefore, the expression system should assure the expression of the β subunit in the cell before the α subunit by using two different promoters. An active C-P4H tetramer was obtained in the periplasm of E. coli. However, further optimization for production by stepwise regulated coexpression of its subunits in the cytoplasm of a thioredoxin reductase and glutathione reductase mutant E. coli strain resulted in large amounts of human C-P4H tetramer. The exchange of four rare E. coli codons of the pdi gene and the optimized distance between ribosome binding site and translation initiation, resulted in 50-fold P4H-activity and 25 mg/l purified enzyme. Comparison of the expression level of mRNA from the α and β subunits by Sandwich hybridization identified single induction with anhydrotetracycline in fed-batch fermentations as a limiting parameter. This caused an insufficient expression level of mRNA and thereby a low yield of C-P4H. A maximum yield was obtained by repeated addition of anhydrotetracycline that led to higher mRNA levels and increased productivity. A newly developed stochastic simulation model of translational ribosome traffic in bacteria assesses the effect of codon usage to ribosome traffic and to the overall translation rate and mRNA stability. Using human PDI, it was shown that substitution of four 5' codons of the human PDI sequence that are rare in E. coli sequences, by synonymous codons preferred in E. coli led to a 2-fold increase of total PDI amount and even to a 10-fold increase of soluble PDI amount.

Coq6 est une enzyme impliquée dans la biosynthèse du coenzyme Q (aussi nommé ubiquinone, ou Q), un lipide benzoquinone polyprenylé essentiel à la fonction de la chaîne respiratoire mitochondriale. Dans la levure Saccharomyces cerevisiae, cette monooxygénase flavine-dépendante putatif est proposé pour hydroxyler le noyau benzénique d' un précurseur du coenzyme Q à la position C5. Nous montrons ici à travers des études biochimiques que Coq6 est une flavoprotéine utilisant le FAD comme cofacteur. Des modèles d'homologie du complexe Coq6-FAD ont étés réalisés et étudiés par dynamique moléculaire et arrimage moléculaire du 3-hexaprenyl-4-hydroxyphényl (4-HP6), un substrat modèle hydrophobe et volumineux. Nous identifions un canal d'accès putatif pour Coq6 dans un modèle de la forme sauvage et proposons des mutations in silico positionnés à l'entrée capable de partiellement (les mutations simples G248R et L382E) ou complètement (une double-mutation G248R-L382E) bloquer l'accès du substrat au site actif via le canal d' accès. Des essais in vivo soutiennent les prédictions in silico, qui expliquent l'abrogation ou la diminution des enzymes mutées. Ce travail fournit la première information structurale détaillée d'une enzyme importante et hautement conservée de biosynthèse de l'ubiquinone. / Coq6 is an enzyme involved in the biosynthesis of coenzyme Q, a polyisoprenylated benzoquinone lipid essential to the function of the mitochondrial respiratory chain. In the yeast Saccharomyces cerevisiae, this putative flavin-dependent monooxygenase is proposed to hydroxylate the benzene ring of coenzyme Q (ubiquinone) precursor at position C5. We show here through biochemical studies that Coq6 is a flavoprotein using FAD as a cofactor. Homology models of the Coq6-FAD complex are constructed and studied through molecular dynamics and substrate docking calculations of 3-hexaprenyl-4-hydroxyphenol (4-HP6), a bulky hydrophobic model substrate. We identify a putative access channel for Coq6 in a wild type model and propose in silico mutations positioned at its entrance capable of partially (G248R and L382E single mutations) or completely (a G248R-L382E double-mutation) blocking access of the substrate to thechannel . Further in vivo assays support the computational predictions, thus explaining the decreased activities or inactivation of the mutated enzymes. This work provides the first detailed structural information of an important and highly conserved enzyme of ubiquinone biosynthesis.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra and motor deficits. Although the majority of PD cases are sporadic, several genetic defects in rare familial cases have been identified. Animal models of these genetic defects have been created and have provided unique insights into the molecular mechanisms of the pathogenesis of PD. However, the etiology of PD is still not well understood. Here, taking advantage of the unique features offered by zebrafish, I characterized the functions of PINK1 (PTEN-induced kinase 1) gene, which is associated with recessive familial PD, in the development and survival of DA neurons. In zebrafish, antisense morpholino knockdown of pink1 did not cause a large loss of DA neurons in the ventral diencephalon (vDC), but the patterning of these neurons and their projections were perturbed. The pink1 morphants also showed impaired response to touch stimuli and reduced swimming behaviour. Moreover, the pink1 knockdown caused a significant reduction in the number of mitochondria, as well as mitochondrial morphological defects such as smaller size or loss of cristae, thus affecting mitochondrial function. These results suggest that zebrafish pink1 plays conserved important roles in the development of DA neurons and in the mitochondrial morphology and function. To better follow DA neurons after injury or administration of toxins, I generated a transgenic zebrafish line, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of dopamine transporter (dat). In Tg(dat:EGFP) fish, all major groups of DA neurons are correctly labeled with GFP, especially the ones in the vDC, which are analogous to the ascending midbrain DA neurons in mammals. In addition, we observed that the DA neurons in the vDC could partially be replaced after severe laser cell ablation. This suggests that zebrafish may have the unique capacity of regenerating DA neurons after injury. Taken together, my studies suggested that zebrafish could be a useful alternative animal model for the study of the molecular mechanisms underlying PD and for the screening of potential therapeutic compounds for PD.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder which affects over a million people in the United States. This disease is marked by the selective loss of dopaminergic neurons in the substantia nigra, leading to a decrease in the important neurotransmitter dopamine (DA), which is essential for the initiation and execution of coordinated movement. Currently, the pathogenesis behind PD is unknown, but there is evidence that both exogenous causes, such as pesticides and metals, as well as endogenous causes, such as reactive oxygen species or reactive metabolism intermediates, may play a role in the onset and progression of the disease. DA is catabolized by monoamine oxidase to 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is further metabolized by aldehyde dehydrogenase and aldehyde reductase to the acid and alcohol products, respectively. Studies have demonstrated the reactivity of DOPAL with peptides and proteins, leading to covalent modification which may be detrimental to protein action. Furthermore, studies have shown that DOPAL is toxic, leading to a decrease in cell viability. Due to this, it was of interest to further study DOPAL and how it may play a role in the onset and progression of PD. It was of particular interest to determine protein targets of DOPAL modification. Until recently, no protein targets were identified and the cellular consequence of elevated DOPAL had not been fully studied. It has been previously shown that the important enzyme, tyrosine hydroxylase (TH) is inhibited by other catechols, including DA. This enzyme catalyzes the rate-limiting step in DA synthesis, oxidizing tyrosine to L-DOPA which is further metabolized to DA. Therefore, it was of interest to determine the effect of DOPAL on TH activity. It was hypothesized that DOPAL modifies and inhibits TH, leading to a decrease in the production of L-DOPA and DA. This work employed the use of a dopaminergic cell model (PC6-3 cells), to positively identify TH as a protein target of DOPAL modification. It also used both cell lysate as well as PC6-3 cell studies to investigate the effect of DOPAL modification on TH activity. Mass spectrometry was also utilized to determine sites of protein modification on TH. Results show that TH is potently inhibited by DOPAL modification, leading to a significant decrease in both L-DOPA and DA. Furthermore, DOPAL inhibition appears to be slowly-irreversible, with enzyme activity showing a time- and concentration dependent in recovery after preincubation with DOPAL. A novel cloning and purification procedure was used to clone human recombinant TH, which was used in mass spectrometry studies in which five sites of DOPAL modification were discovered. Furthermore, a real-time assay for TH activity was developed using a plate reader to spectrophotometrically observe the formation of L-DOPA over time. These data demonstrate the toxicity and potent enzyme inhibition by DOPAL and implicate DOPAL as a neurotoxin relevant in the pathogenesis of PD.

Cholesterol 7-hydroxylase (CYP7A1) is the rate limiting enzyme of the classical pathway of bile acid (BA) synthesis, which catabolizes approximately half of cholesterol in man. Determination of CYP7A enzymatic activity is a key subject of lipid metabolism research. Direct determination of CYP7A1 activity in hepatic biopsy is mostly not allowed for ethical reasons, so indirect methods are used with serum markers such as 7α-hydroxy-4-cholestene-3- one (C4). The first, methodical aim of the work was to convert the introduced HPLC method for the determination of C4 to LC-MS in order to increase the sensitivity. We focused on the solid phase extraction step, adjusting the composition and volumes of the washing and elution solution. By converting the method from HPLC to LC-MS, the sensitivity was increased approximately 7 times (LD = 1.39 ng/ml). In the second, clinical part of our work, we attempted to confirm the preliminary results of our laboratory on the distribution of C4 in lipoprotein fractions (LPP) in order to find parameter that would correlate with CYP7A1 activity better than C4 level itself. Preliminary results (performed in healthy individuals) showed that most of C4 is carried on HDL, and that the C4 distribution within LPP fractions is similar among examined subjects. We repeated the...

Trichloroethylene (TCE) is a toxic pollutant that has become a widespread problem by seeping into groundwater across the developed world. Clean-up of sites contaminated with TCE is extremely difficult due to the absence of an efficient and cost-effective method for clean-up. Bioremediation efforts include a variety of potential microbial candidates with various metabolic capabilities as clean up options of contaminated sites. Cupriavidus necator, a soil bacterium was found to possess the ability to degrade TCE via a phenol-dependent pathway. Previous research by Ayoubi and Harker (1998) created a strain (MM02) capable of constitutive TCE degradation but the underlying genetic alteration causing constitutive production of the phenol hydroxylase pathway (PHL) and TCE breakdown was poorly characterized. We attempted to gain further understanding of the alterations that occurred in the PHL pathway to cause TCE to break down and replicate constitutive TCE degradation in a new strain with reduced foreign elements that may be introduced into the environment. Strain MM02 possessing this constitutive degradation activity and strain MM01were sequenced and compared to discover the source of this variation. A 210 base-pair deletion in the beginning of the PHL operon was identified and is likely the cause of this altered activity. The new strain of C. necator (MM14) was created using traditional bacterial mating methods and included a cleanly introduced kanamycin resistance gene and its associated promoter which could drive constitutive expression of the PHL pathway. The TCE degradation abilities of strains MM01, MM02, and MM14 were evaluated through the TCE degradation assay and gas chromatography. We had difficulty accurately measuring the concentration of TCE due to its volatile nature and dramatically altered the method ultimately reducing variation and capturing TCE concentrations in assays. When accurate readings were obtained, none of the strains measured exhibited quantifiable TCE degradation activity when compared to controls. Our results showed .08% of the degradation by strain MM02 measured previously (P. J. Ayoubi, 1997). Based on our findings, we were unable to replicate the TCE degradation caused my MM02 and our genetically modified strain also failed to breakdown TCE.

Degeneration of the noradrenergic locus coeruleus (LC) in aging and neurodegenerative diseases is well documented. Slowing or reversing this effect may have therapeutic implications. Phox2a and Phox2b are homeodomain transcriptional factors that function as determinants of the noradrenergic phenotype during embryogenesis. In the present study, recombinant lentiviral eGFP-Phox2a and -Phox2b (vPhox2a and vPhox2b) were constructed to study the effects of Phox2a/2b over-expression on dopamine β-hydroxylase (DBH) and norepinephrine transporter (NET) levels in central noradrenergic neurons. Microinjection of vPhox2 into the LC of adult rats significantly increased Phox2 mRNA levels in the LC region. Over-expression of either Phox2a or Phox2b in the LC was paralleled by significant increases in mRNA and protein levels of DBH and NET in the LC. Similar increases in DBH and NET protein levels were observed in the hippocampus following vPhox2 microinjection. In the frontal cortex, only NET protein levels were significantly increased by vPhox2 microinjection. Over-expression of Phox2 genes resulted in a significant increase in BrdU-positive cells in the hippocampal dentate gyrus. The present study demonstrates an upregulatory effect of Phox2a and Phox2b on the expression of DBH and NET in noradrenergic neurons of rat brains, an effect not previously shown in adult animals. Phox2 genes may play an important role in maintaining the function of the noradrenergic neurons after birth, and regulation of Phox2 gene expression may have therapeutic utility in aging or disorders involving degeneration of noradrenergic neurons.

Petunia hybrida and Citrus paradisi have significantly different flavonoid accumulation patterns. Petunia sp. tend to accumulate flavonol glycosides and anthocyanins while Citrus paradisi is known for its accumulation of flavanone diglycosides. One possible point of regulation of flavanone metabolism is flavanone 3-hydroxylase (F3H) expression. To test whether this is a key factor in the different flavanone usage by Petunia hybrida and Citrus paradisi, F3H mRNA expression in seedlings of different developmental stages was measured using semi-quantitative RT-PCR. Primers were designed to conserved regions of F3H and used to amplify an approximately 350 bp segment for quantitation by PhosphorImaging. Primary leaves of 32 day old grapefruit seedlings and a grapefruit flower bud had the highest levels of F3H mRNA expression. Petunia seedlings had much lower levels of F3H mRNA expression relative to grapefruit. The highest expression in petunia was in primary leaves and roots of 65 day old seedlings. These results indicate that preferential use of naringenin for production of high levels of flavanone glycosides in young grapefruit leaves cannot be attributed to decreased F3H mRNA expression.

Dopamine β-hydroxylase (DBH) and norepinephrine (NE) transporter (NET) are the noradrenergic phenotypes for their functional importance to noradrenergic neurons. It is known that in vivo N-(2-chloroethyl)-N-ethyl-2- bromobenzylamine (DSP4) treatment induces degeneration of noradrenergic terminals by interacting with NET and depleting intracellular NE. However, DSP4's precise mechanism of action remains unclear. In this study various biochemical approaches were employed to test the hypothesis that DSP4 down-regulates the expression of DBH and NET, and to determine molecular mechanisms that may be involved. The results showed that treatment of SH-SY5Y neuroblastoma cells with DSP4 significantly decreased mRNA and protein levels of DBH and NET. DSP4-induced reduction of DBH mRNA and proteins, as well as NET proteins showed a time- and concentration-dependent manner. Flow cytometric analysis demonstrated that DSP4-treated cells were arrested predominantly in the S-phase, which was reversible. The arrest was confirmed by several DNA damage response markers (phosphorylation of H2AX and p53), suggesting that DSP4 causes replication stress which triggers cell cycle arrest via the S-phase checkpoints. Moreover, the comet assay verified that DSP4 induced single-strand DNA breaks. In summary, the present study demonstrated that DSP4 down-regulates the noradrenergic phenotypes, which may be mediated by its actions on DNA replication, leading to replication stress and cell cycle arrest. These action mechanisms of DSP4 may account for its degenerative consequence after systematic administration for animal models.