To detect and respond to the detrimental situation of hypoxia, metazoan cells employ O₂- sensing prolyl and asparaginyl hydroxylases which directly utilise O₂ to hydroxylate and regulate the Hypoxia Inducible transcription Factor-α (HIF-α). This thesis focuses upon the asparaginyl hydroxylase, ‘Factor Inhibiting HIF-1 (FIH-1), which represses HIF-α in normoxia by asparaginyl hydroxylation of its C-terminal trans-Activation Domain (CAD). During hypoxia FIH-1 is inhibited, allowing non-hydroxylated HIF-α to drive expression of over 70 target genes, leading to tissue and cellular changes that increase O₂ supply and reduce its consumption. This response is central to normal physiology and to the pathophysiology of diseases, including stroke and cancer. The pivotal role of FIH-1 in regulating these processes invites its characterisation, as a key cellular O₂-sensor and therapeutic target. This thesis contributes important information by elucidating a novel FIH-1 substrate and by defining numerous FIH-1 substrate recognition determinants. The first aim was to investigate the cell-fate regulator Notch1 as a potential FIH-1 substrate, due to myriad reports of Notch/hypoxic crosstalk and the discovery by collaborators that FIH- 1 represses Notch1 activity. Mutagenesis, hydroxylation assays, affinity-purification and mass spectrometry techniques enabled definition of two asparaginyl hydroxylations of mouse Notch 1 ankyrin repeat domain (N1945 and N2012), performed by FIH-1 in vitro. These residues were likewise detected to be hydroxylated in mNotch1 expressed in mammalian cells. FIH-1 kinetic assays comparing mNotch1 ankyrin domain with the unstructured hHIF- 1α CAD uncovered major distinctions between substrates; mNotch1 facilitated a 7-fold lower rate of cosubstrate turnover by FIH-1, but affinity was robust (>10-fold higher). Interrogation of the structure/affinity correlate implies FIH-1 binds unstable ankyrins preferentially. Functionally, a non-catalytic mechanism of Notch1 repression by FIH-1 is supported. The second aim derived from literature analyses implicating threonine and RLL motifs in HIF-α as critical hydroxylation determinants. T796 (hHIF-1α) contacts FIH-1 and is a likely phospho-acceptor, thus a mimetic T796D mutant was generated and its hydroxylation kinetics compared with wildtype hHIF-1α CAD. In vitro, the mutant exhibited a 6-fold greater apparent Km, explaining its constitutive activity in cell-based reporter assays, whereas wildtype hHIF-1α CAD is hydroxylated and thus repressed in normoxia by FIH-1. This indicates that phosphorylation reduces hydroxylation by FIH-1 in vitro and in vivo. The RLL motif does not contact FIH-1 in vitro however RLL-AAA mutant HIF-α proteins are constitutively active in normoxia, suggesting resilience to hydroxylation within cells. To reconcile these data I predicted that a cellular Factor X functionalises the RLL motif as an FIH-1 binding site. Reporter assays, in vitro kinetic assays and interaction assays +/- lysate confirmed this hypothesis and additionally showed the motif to increase HIF-α protein turnover 8-fold. Numerous mechanisms for Factor X including nuclear export, posttranslational modifications of FIH-1 or HIF-α, and involvement of small molecules, were experimentally examined, but deemed unlikely. Rather, the data imply Factor X to be a proteinaceous facilitator of a HIF-α/FIH-1 complex, thus proteomic capture screens are underway. This research provides novel insight into FIH-1; its role in Notch/hypoxic crosstalk, its substrate recognition requirements, and its potential functions in cellular O₂-sensing. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1326855 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

The catalytic and regulatory mechanisms of phenylalanine hydroxylase were investigated by structural studies of in this research. Phenylalanine hydroxylase (PheH) hydroxylates phenylalanine to produce tyrosine using tetrahydrobiopterin (BH4) and oxygen. The three ligands to the iron, His285, His290, and Glu330, were mutated to glutamine, glutamate, and histidine. All the mutants had low but measurable activity. Mutation of Glu330 had the greatest effect on activity and mutation of His290 the least. All the mutations resulted in an excess of tetrahydropterin oxidized relative to tyrosine formation, with mutation of His285 having the greatest effect on the coupling of the two partial reactions. All the mutants greatly decreased the affinity for iron, with mutation of Glu330 the most deleterious. The results complement previous results with tyrosine hydroxylase in establishing the plasticity of the individual iron ligands in this enzyme family. Hydrogen/deuterium exchange and mass spectrometry showed that peptides lying in the interface between the regulatory and catalytic domains display large increases of deuterium incorporation in the presence of phenylalanine. However, the effects of phenylalanine on a mutant enzyme lacking the regulatory domain are limited to peptides surrounding the binding site of phenylalanine. These results support the autoinhibitory function of the N-terminus of PheH. No peptides show a changed deuterium incorporation pattern in the presence of BH4, suggesting that BH4 binding does not change the structure significantly from the resting form. In phosphorylated PheH, three peptides show a deuterium incorporation pattern similar to that of unphosphorylated PheH plus phenylalanine, while the other peptides sensitive to phenylalanine binding in unphosphorylated PheH show the same pattern as that of unphosphorylated PheH without phenylalanine. Therefore, the conformational changes induced by phosphorylation are similar to but not identical to those associated with phenylalanine activation. The isolated regulatory domain (PheH1-117) was expressed and purified using a QSepharose column followed by a gel filtration column. Analytical gel filtration shows that PheH1-117 exists as a dimer in solution. In the presence of phenylalanine, the retention time of PheH1-117 is significantly changed. The 1H-15N NMR spectra of PheH1- 117 show that the cross-peaks of several residues are altered in the presence of phenylalanine. These results support the existence of a regulatory binding site for phenylalanine in the regulatory domain of PheH.

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 4 uppsatser.

Thesis (M. S.)--Wake Forest University Graduate School of Arts and Sciences, Molecular Genetics Program, May 2003. / Kent E. Vrana, advisor. Includes curriculum vita. Includes bibliographical references.

Thesis (doctoral)--Rijksuniversiteit te Groningen. / Description based on print version record.

Thesis supervisor: Stefan Strack. Includes bibliographic references (p. 77-88).

Interest in brain catecholamines has grown considerably in the last few years in view of their possible role as neurotransmitters. This investigation deals primarily with the enzyme tyrosine hydroxylase which is thought to be the rate limiting step in catecholamine synthesis. Using tyrosine hydroxylase measurements and catecholamine depletion techniques,attempts were made to determine the site of increased synthesis of catecholamines in animals exposed to cold. Brain, heart and spleen do not appear to be the organs involved in this change. Adrenals may be of significance but the results were suggestive rather than conclusive. Tyrosine hydroxylase distribution in brain was determined in various regions of rat, rabbit and cat brain, and activity was shown to be highest in the caudate, septal area, nucleus accombens, and anterior perforating substance, with much lower activities in other regions such as hippocampus, amygdala, hypothalamus, thalamus, cortex, cerebellum and brain stem. Using these distribution studies as indications of normal tyrosine hydroxylase activity in areas of rat brain, and electrolytic lesion techniques, studies were carried out to determine noradrenergic and dopanergic pathways in brain. Catecholamine fibers from their origin in the midbrain were traced in the midbrain and diencephalon to the caudate and septal area, and the relative positions of each group of fibers determined along their course. / Medicine, Faculty of / Graduate

No description available.

Serotonin is a very important neuromodulatory agent that affects many physiological and behavioral responses of both vertebrates and invertebrates. In helminths, especially parasitic ones, not much is known about the biosynthesis and mode of action of serotonin or any of the related biogenic amine neurotransmitters, such as catecholamines (dopamine and noradrenaline). In this study, we cloned two full length cDNAs from Schistosoma mansoni encoding tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH). TPH and TH catalyze the rate limiting steps in the biosynthesis of serotonin and catecholamines, respectively. Both enzymes were expressed in Escherichia coli and the purified proteins were shown to have TPH and TH activities. This indicates that S. mansoni, and possibly other parasitic helminths, may be capable of synthesizing serotonin and catecholamines endogenously. In the second part of our studies, we looked at the mode of action of serotonin in helminths, in particular the molecular properties of serotonergic G protein-coupled receptors (GPCR). We cloned two helminth GPCRs, one from the free living nematode Caenorhabditis elegans and the second from S. mansoni. The C. elegans receptor (5-HT2Ce) was shown to encode a functional serotonin receptor with structural and signaling properties similar to those of mammalian 5-HT2 receptors. However, its agonist I antagonist binding profile differed from previously characterized serotonin receptors. The cloned S. mansoni receptor (SmGPCRx) was found to represent a new structural class of receptor, which shared about the same level of amino acid sequence homology with various biogenic amines receptors, such as serotonin, catecholamines, and octopamine receptors. Additional sequence analysis and immunolocalization studies confirmed that SmGPCRx possesses structural characteristics of a GPCR. SmGPCRx is the first GPCR ever cloned from a parasitic flatworm. Taken together, these studies mark an important first step to

In helminths, including the free-living nematode, Caenorhabditis elegans, serotonin (5-HT) acts as an important neuroactive agent and is associated with carbohydrate metabolism, glucouse utilization, motility, feeding and reproductive behaviour. In mammals and other organisms, 5-HT is synthesized through the action of tryptophan hydroxylase (TPH). TPH is the rate limiting enzyme in the biosynthesis of 5-HT, and as such sets the pace for the formation of 5-HT. TPH is a member of a family of enzymes that hydroxylate aromatic amino acids and have an absolute requirement for the pterin cofactor, tetrahydrobiopterin (BH4). It is unknown if this same enzyme catalyzes the synthesis of 5-HT in C. elegans and other helminths. / Based on sequence information from the C. elegans Genome Data Base and RT-PCR, we have cloned a full-length C. elegans TPH cDNA (CeTPH) that shows high homology to mammalian TPH. The predicted coding sequence of CeTPH was subcloned into the prokaryotic expression vector, pET-15b, and the resulting construct was introduced into E. coli (BL21 DE3 pLys strain) for IPTG-inducible expression of CeTPH protein. Results show that CeTPH expressed in E. coli has TPH activity and also shows an absolute requirement for the cofactor, BH4, just as shown previously for the mammalian enzyme. It has been well established that 5-HT is present and is biologically active in the tissues of C. elegans. By way of characterizing furthur CeTPH, we examined the localization of TPH in whole mounts of C. elegans by immunofluoresence using a polyclonal antibody against TPH. / Taken together, the results of this thesis characterize at the structural, functional and in situ levels one of the most primitive forms of TPH enzyme ever cloned.