Thesis--Wisconsin. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18703号 / 生博第322号 / 新制||生||43(附属図書館) / 31636 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 豊島 文子, 教授 西田 栄介, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

The first step in the synthesis of steroid hormones occurs in the mitochondria where cholesterol is converted to pregnenolone by cytochrome P450scc (CYP11A1). Cholesterol is insoluble in water and is supplied to the CYP11A1 directly from the inner mitochondrial membrane to which the enzyme is bound. The aim of this study was to characterise the interaction of bovine CYP11A1 with the phospholipid membrane. The effect of osmotic stress provided by glycerol on the spin-state, activity and degree of hydration of CYP11A1 was also investigated. Multiple sequence alignment of mitochondrial P450s revealed that there are 46 absolutely conserved residues, with the highest conservation in the heme-binding region at the C-terminal. The greatest variablility between subfamilies is in the regions believed to be involved in substrate binding (SRSs), as defined for the CYP2B family. The secondary structure prediction for CYP11A1 suggests that there is strong similarity in secondary structure to P450s of known structure. A model structure of CYP11A1 was built from primary sequence alignment to template P450 structures using the SwissModel automated server. From the model and other bioinformatic analyses, the distal face of the P450 which includes the A’ helix, F-G loop and beta sheet 1 regions, were predicted to interact with the membrane. Tryptic digests of CYP11A1 were performed with the aim of identifying membrane bound peptides that may be protected from protease activity. HPLC tryptic maps were similar in profile between soluble and vesicle-bound P450 which suggests that there is not a large region of CYP11A1 protected from protease digestion when the enzyme is attached to a membrane. Mass spectrometric analysis of peptides resulting from tryptic digestion revealed a number of peptides in the soluble digest that were not present in the digest of vesicle-bound P450. These peptides were located at the N-terminal and the J to J’ helix and interestingly, there was an absence of C-terminal peptides for both digests. This C-terminal peptide could be detected in digests of vesicle-bound P450 but not in digests of soluble P450 by tricine SDS polyacrylamide gel electrophoresis, Western transfer and N-terminal sequence analysis. Based upon the bioinfomatic and tryptic digestion data, a set of N- and C-terminal deletion mutants of CYP11A1 were expressed in E. coli and fractionated based on their association with the soluble or membrane fraction of the cells. The N-terminal deletion of the A’ helix resulted in an increase in the proportion of CYP11A1 in the soluble fraction while the C-terminal deletion did not alter membrane localisation. There are eight tryptophan residues in mature CYP11A1. The accessibility of these tryptophans to a water-soluble fluorescence quencher was determined for soluble and vesicle-bound enzyme. When CYP11A1 was associated with the vesicle membrane an average of 2 tryptophan residues were protected from quenching compared to soluble CYP11A1. This suggests that these tryptophan residues become buried within the membrane following association of CYP11A1 with the vesicles and are no longer accessible to quencher. The only free cysteine (C265S) of bovine CYP11A1 was removed by site directed mutagenesis and new cysteine residues introduced at selected sites based upon earlier results and the modelled CYP11A1 structure. The cysteine mutants were expressed, purified and labelled with the environmentally sensitive fluorescent probe, N-(7-nitrobenz-2-oxal-3-diazol-4-yl)ethylenediamine (NBD). There was an increase in the hydrophobicity of the NBD environment following the association of CYP11A1 with vesicles for the labeled mutants V212C and L219C. This indicates that these residues which are in the F-G loop, become localized to a more hydrophobic environment following membrane binding. Labeled cysteine residues introduced into the A’, B’ and G helices and β4-2 did not show major changes in hydrophobicity following membrane integration of CYP11A1. Osmotic stress of CYP11A1 induced by glycerol resulted in a low-spin spectral response and inhibition of activity. The change to low spin correlated with the dissociation of five or six water molecules from CYP11A1 and the inhibition of activity with cholesterol as substrate correlated with the dissociation of two molecules of water. In conclusion, this study shows that CYP11A1 is held to the membrane, at least in part, by the F-G loop region, and that the removal of water from the active site of CYP11A1 by osmotic stress causes a low spin spectral response and inhibition of activity.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Thesis (M. S.)--Biology, Georgia Institute of Technology, 2006. / Donald Doyle, Committee Member ; Harish Radhakrishna, Committee Member ; Alfred Merrill, Committee Member ; Marion Sewer, Committee Chair Includes bibliographical references.

Preclinical results support the use of N-methyl D-aspartate receptor (NMDAR) modulators for cognition enhancement therapeutics. Pregnenolone sulfate (PregS) is a neuroactive steroid derived from cholesterol that augments long term potentiation (LTP) in hippocampal slices and improves memory performance in rats and mice. At micromolar concentrations, PregS is a subtype selective positive allosteric modulator of NMDARs at NR2A and NR2B containing receptors, and at concentrations ranging from pM - nM induces NMDAR-dependent dopamine release in the striatum and from striatal synaptosomes. In this report, we observe that micromolar [PregS] induces an increase in levels of neuronal intracellular calcium ([Ca^2+]i) and surface NMDARs in cortical neurons. Moreover, our results show that PregS stimulated upregulation of surface NR1 subunits in cortical neurons is dependent on NMDARs but independent of channel activity. As PregS has been detected in brain at bulk concentrations of 0.1 nM to 5 nM, we asked whether low, picomolar concentrations of PregS might alter [Ca^2+] levels. We report here that PregS increases [Ca^2+]i signal in cortical neurons in a voltage-gated Na^+ channel and NMDAR-NR2B dependent manner with an EC50 of ~2 pM, at least 6 orders of magnitude higher affinity than its rapid potentiating effect upon the NMDAR-mediated ionotropic response, and within the range of PregS detected in bulk brain tissue. Additionally, calcium (Ca^2+) activation of cyclic AMP response element binding protein (CREB) is critical to the protein synthesis-dependent component of LTP and important in associated behavioral measures of learning and memory. Increased [Ca^2+]i levels are known to induce CREB activation and we now show that 50 pM PregS induces a 44 ± 13% increase in the ratio of pCREB to total CREB that is dependent upon ERK signaling and canonical excitatory synaptic transmission: this includes voltage gated Na+ channels, NMDARs, and voltage-gated Ca^2+ channel activation. The results taken together indicate that PregS may be a useful platform for the development of high-affinity positive modulators of NMDAR-signaling that can be used as cognitive enhancers to treat a variety of neurological disorders: such as Alzheimer's disease, Parkinson's disease, and schizophrenia.

Steroidogenic factor (SF1) is an orphan nuclear receptor that is essential for steroid hormone-biosynthesis and endocrine development. Recent studies have demonstrated that phospholipids are ligands for SF1. In the present study our aim was to identify endogenous ligands for SF1 and characterize their functional significance in mediating cAMP-dependent transcription of human CYP17. Using mass spectrometry we show that in H295R adrenocortical cells SF1 is bound to sphingosine (SPH) under basal conditions and that cAMP stimulation decreases the amount of SPH bound to the receptor. We also show that silencing both acid and neutral ceramidases using siRNA induces CYP17 mRNA expression, suggesting that SPH acts as an inhibitory ligand. In vitro analysis of ligand binding using scintillation proximity assays show that several sphingolipids and phospholipids, including phosphatidic acid (PA), can compete with [3H]SPH for binding to SF1, suggesting that SF1 may have more than one ligand and binding specificity may change with the changes in intracellular fluxes of phospholipids. Further, phosphatidic acid (PA) induces SF1-dependent transcription of CYP17 reporter constructs. Inhibition of diacyglycerol kinase (DAGK) activity using R59949 and silencing DAGK- expression attenuates SF1-dependent CYP17 transcriptional. We propose that PA is an activating ligand for SF1 and that cAMP-stimulated activation of SF1 takes place by displacement of SPH.

Pregnenolone (PREG), the precursor of all neurosteroids, is synthesized in the nervous system from cholesterol and recent clinical studies indicate that reduced cognitive symptoms of schizophrenia correlate with elevated serum levels of pregnenolone sulfate (PregS), its immediate sulfated metabolite. PregS fulfills most of the classical criteria for an endogenous modulator of excitatory synaptic transmission, including: presence in nervous tissue at physiologically relevant concentrations, potentiation of N-methyl-D-aspartate receptor (NMDAR) mediated synaptic activity, and a mechanism for its inactivation. As NMDAR hypoactivity has been implicated in the pathophysiology of schizophrenia, defects in neurosteroid metabolism might play a role in its associated cognitive dysfunction. PregS improves memory performance in rodents and augments long-term potentiation (LTP), an electrophysiological correlate of synaptic plasticity that is stabilized by phosphorylation of the cAMP response element binding protein (CREB). We have previously demonstrated that PregS at low picomolar (pM) concentrations increases intracellular Ca2+ and CREB via synaptic NMDARs. Therefore, we hypothesized that low pM concentrations of PregS might potentiate spontaneous excitatory postsynaptic currents (sEPSCs) and promote molecular events underlying synaptic plasticity. Here, using whole-cell patch clamp recordings, we report that PregS enhances the frequency of sEPSCs of cultured hippocampal neurons by about 2-fold while not altering their amplitude or passive membrane properties. This suggests that PregS acts presynaptically by increasing the frequency of neurotransmitter release or postsynaptically by activating silent synapses. We then investigated the hypothesis that PregS increases α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and NMDAR subtypes at synapses as a molecular switch for this enhancement. We measured receptor redistribution and phosphorylation using fluorescence imaging and Western blot technology. The results demonstrate that PregS (50pM, 10min): (1) Increases AMPAR (GluA1)/PSD95 colocalization (dependent on L-type voltage-gated Ca+2 channel and synaptic NMDAR activity), and increases phosphorylation of GluA1 at serine-831/845; (2) Increases casein kinase 2 (CK2) dependent surface NMDAR2A (GluN2A) but not GluN1 or GluN2B; and (3) Increases GluN2B serine-1480 phosphorylation. The results show that PregS increases the frequency of excitatory synaptic transmission and increases surface/synaptic AMPARs and surface GluN2A (but not GluN1 or GluN2B) NMDARs, shifting the molecular composition of young glutamatergic synapses toward the adult GluN2A enriched synaptic phenotype.

No description available.

Pregnenolona (PREG) e 17-alfa-hidroxipregnenolona (17OHPREG) são dois esteroides produzidos pela glândula adrenal e precursores de vários hormônios esteroidais. A dosagem desses compostos tem aplicações clínicas, como o diagnóstico de doenças relacionadas aos corticoesteroides e mineralocorticóides e especialmente na avaliação da atividade da enzima 3-β-hidroxidesidrogenase que é decisiva no diagnóstico de um dos tipos de hiperplasia da glândula adrenal que causa defeitos severos na síntese de esteroides. Métodos cromatográficos associados à espectrometria de massas superaram a especificidade reduzida dos imunoensaios e tem sido crescentemente utilizados na quantificação de esteroides. Os últimos anos tem sido marcados pela hegemonia da cromatografia líquida acoplada à espectrometria de massas em tandem (LC-MS/MS) em grande parte devido à velocidade e possibilidade da análise direta de vários analitos. Porém, no caso específico dos esteroides de tipo 3-hidroxi-5-eno, que apresentam baixa afinidade protônica e, portanto, baixa eficiência de ionização, são necessárias muitas etapas para a conversão em derivados mais detectáveis. Embora desfavorecida em relação ao LC-MS/MS nos últimos anos, a cromatografia gasosa acoplada à espectrometria de massas (CG-MS) apresenta várias características favoráveis para a análise de esteroides como a eficiência cromatográfica ainda insuperável. Adicionalmente, a incorporação da espectrometria de massas em tandem ao CG (CG-MS/MS) torna a técnica tão seletiva quanto LC-MS/MS. No presente trabalho, foi desenvolvido um novo método que permite a extração e derivatização simultâneas da PREG e 17OHPREG de amostras de soro tornando o método de preparo da amostra tão simples quanto os descritos para LC-MS/MS. O método de detecção desenvolvido baseado em ionização química no modo negativo obteve a sensibilidade necessária para o diagnóstico da deficiência da enzima 3-beta-hidroxidesidrogenase utilizando apenas 250 &#181:L de amostra. / Pregnenolone (PREG) and 17α-hydroxypregnenolone (17OHPREG) are two steroid precursors produced by the adrenal gland. The quantification of these compounds is essential for the evaluation of 3-β-hidroxidesidrogenase enzyme activity, which promotes the conversion of PREG in 17OHPREG. The 3-&#946:-hidroxidesidrogenase deficiency is a rare but severe type of adrenal hyperplasia that causes serious defects in steroid synthesis. Chromatographic methods coupled to mass spectrometry overcame immunoassays limitations such as reduced specificity, and have been widely used for steroids quantification. Recent years have been marked by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) hegemony due to the speed and possibility to analyze directly several analytes. However, in the case of type 3-hydroxy-5-ene steroids, which have low affinity for protons and, therefore, low ionization efficiency, many steps are required for conversion to detectable products. Notwithstanding, gas chromatography coupled to mass spectrometry (GC-MS) has some favorable features for steroid analysis such as unbeatable chromatographic efficiency. In addition, the incorporation of tandem mass spectrometry (GC-MS/MS) makes it as selective as LC-MS/MS. In this study, a new method for simultaneous extraction and derivatization of PREG and 17OHPREG from serum was developed. This procedure makes sample preparation for GC-MS/MS as simples as those described for LC-MS/MS. The detection method based on negative mode chemical ionization achieved the sensitivity required for the diagnosis of 3-β-hidroxidesidrogenase defficiency using only 250 µL of sample.