Peptidyl-prolyl isomerization is an important post-translational modification of protein because proline is the only amino acid that can stably exist as cis and trans, while other amino acids are in the trans conformation in protein backbones. This makes prolyl isomerization a unique mechanism for cells to control many cellular processes. Isomerization is a rate-limiting process that requires a peptidyl-prolyl cis/trans isomerase (PPIase) to overcome the energy barrier between cis and trans isomeric forms. Pin1, a key PPIase in the cell, recognizes a phosphorylated Ser/Thr-Pro motif to catalyze peptidyl-prolyl isomerization in proteins. The significance of the phosphorylation-dependent Pin1 activity was recently highlighted for isomerization of ATR (ataxia telangiectasia- and Rad3-related). ATR, a PIKK protein kinase, plays a crucial role in DNA damage responses (DDR) by phosphorylating hundreds of proteins. ATR can form cis or trans isomers in the cytoplasm depending on Pin1 which isomerizes cis-ATR to trans-ATR. Trans-ATR functions primarily in the nucleus. The cis-ATR, containing an exposed BH3 domain, is anti-apoptotic at mitochondria by binding to tBid, preventing activation of pro-apoptotic Bax. Given the roles of apoptosis in many human diseases, particularly cancer, we propose that cytoplasmic cis-ATR enables cells to evade apoptosis, thus addicting cancer cells to cis-ATR formation for survival. But in normal DDR, a predominance of trans-ATR in the nucleus coordinates with a minimal level of cytoplasmic cis-ATR to promote DNA repair while preventing cell death; however, cells can die when DNA repair fails. Therefore, a delicate balance/equilibrium of the levels of cis- and trans-ATR is required to ensure the cellular homeostasis. In this review, we make a case that this anti-apoptotic role of cis-ATR supports oncogenesis, while Pin1 that drives the formation of trans-ATR suppresses tumor growth. We offer a potential, novel target that can be specifically targeted in cancer cells, without killing normal cells, to significantly reduce the adverse effects usually seen in cancer treatment. We also raise important issues regarding the roles of phosphorylation-dependent Pin1 isomerization of ATR in diseases and propose areas of future studies that would shed more understanding on this important cellular mechanism.

Long-term postnatal treatment of rats with SCH 23390 is associated with a reduction in the development of dopamine D1 receptors in the striatum. Because the tripeptide, l-prolyl-l-leucylglycinamide (PLG) attenuates the neuroleptic-induced increase in D2 receptors in the striatum in adult rats, this study was undertaken with the objective of determining whether PLG could modulate a developmental alteration in the D1 subtype of receptor. Rats were treated with the dopamine D1 receptor antagonist, SCH 23390 (R[+]-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1-H-3benzazepine) (0.30 mg/kg/d i.p.) for 32 successive days from birth, while D1 receptors in the striatum were assessed at 5 and 8 weeks from birth. Postnatal treatment with SCH 23390 reduced in vitro binding of [3H]SCH 23390 to homogenates in the striatum by 70% at 8 weeks. Scatchard analysis at 5 weeks determined that the Bmax for the binding of [3H]SCH 23390 was reduced by 78%, while the Kd was unaltered. When PLG (1.0 mg/kg/d i.p.) was administered together with SCH 23390 for the initial 32 days from birth, the binding of [3H]SCH 23390 to homogenates of the striatum was unchanged from that of the control group at 8 weeks. Also, at 5 weeks the Bmax and Kd were unaltered from control in the group that was treated with both SCH 23390 and PLG. The binding of [3H]SCH 23390 was not altered from control in the group treated with PLG alone. Also, PLG given in vitro did not alter the binding of [3H]SCH 23390 to control homogenates of the striatum. These findings indicate that PLG is able to attenuate neuroleptic-induced alterations in dopamine d1 receptors in the striatum.

Long-term postnatal treatment of rats with the dopamine D2 receptor antagonist, spiroperidol, results in the impaired development of striatal D2 receptors. Because the tripeptide prolyl-leucyl-glycinamide (MIF-1) attenuates haloperidol-induced up-regulation of striatal dopamine D2 receptors in adult rats, we studied the effect of MIF-1 on the spiroperidol-induced alteration of striatal D2 ontogeny. Postnatal treatment of rats with spiroperidol (1.0 mg/kg/day, IP, ×32 days from birth) resulted in a 74% decrease in the Bmax for [3H]spiroperidol binding with no change in the Kd at 5 weeks. When rats were studied at 8 weeks, in the absence of additional treatment, total specific [3H]spiroperidol binding was reduced by 59%. While MIF-1 alone (1.0 mg/kg/day, IP, ×32 days from birth) had no effect on [3H]spiroperidol binding, MIF-1 completely attenuated the ontogenic impairment of striatal D2 receptors that was produced by spiroperidol treatment. At 5 weeks the Bmax for [3H]spiroperidol binding was at the saline control level in the group of rats cotreated with spiroperidol and MIF-1. At 8 weeks, with no additional treatments, the specific binding of [3H]spiroperidol to striatum was also at control levels in the group cotreated with spiroperidol and MIF-1. These findings demonstrate that MIF-1 attenuates spiroperidol-induced impairment of development of striatal dopamine D2 receptors in rats.

Because of conflicting reports of the actions of the antiparkinsonian agent L-prolyl-L-leucyl-glycine amide (PLG, MIF-I) on the turnover of Striatal dopamine (DA), this process was reinvestigated. In the present series of studies, it was found that neither our MIF-I (200 ng ICV) nor the MIF-I used by Versteeg et al. [25]was effective in altering the rate of decline of endogenous DA in the caudate nucleus of rats pretreated with α-methyl-p-tyrosine (300 mg/kg IP). In addition, our MIF-I (1 mg/kg IP) did not change endogenous dihydroxyphenylacetic acid (DOPAC) or homovanillic acid (HVA) in rat striatum. These studies indicate that MIF-I does not alter the turnover rate of DA in nigrostriatal neurons. It is possible that MIF-I or some substance released by MIF-I acts at a posfsynaptic receptor site.

Since prolyl-leucyl-glycinamide (MIF-1) modifies the behavior of adult rats after treatment with neuroleptics, we examined whether MIF-1 would also modify adult behavior after treatment of neonatal rats with 6-hydroxydopamine (6-OHDA). Rats received 6-OHDA (100 μg i.c.v.) or diluent at 3 days after birth and either MIF-1 (2.0 mg/kg per day s.c. × 10 days) or diluent beginning at 28 or 29 days after birth. At 5 weeks, a low dose (0.1 mg/kg s.c.) of apomorphine increased the distance traveled, time in ambulation, number of stereotypic movements, and number of movements per time in stereotypy, but decreased the time in stereotypy in the 6-OHDA group. MIF-1 (× 7 or 8 days) showed a tendency to attenuate the increased number of movements and significantly (P < 0.05) reduced all of the other effects of neonatal 6-OHDA. Behavior induced by higher doses of apomorphine in the 6-OHDA group (reduced licking and head nodding; increased paw treading, taffy pulling and self-biting) were not attenuated by MIF-1. At 38 or 39 days, total in vitro binding of [3H]SCH-23390 and [3H]spiroperidol to striatal homogenates was not altered in any of the groups. The findings demonstrate that specific early developmental alterations in apomorphine-induced behaviors can be modified by treatment of adult rats with MIF-1, even in the absence of overt changes in the binding of striatal dopamine D-1 and D-2 receptors.

No description available.

京都大学 / 新制・論文博士 / 博士(農学) / 乙第13488号 / 論農博第2900号 / 新制||農||1093(附属図書館) / 学位論文||R4||N5367(農学部図書室) / (主査)教授 井上 和生, 教授 谷 史人, 教授 保川 清 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

L'unité structurale de la chromatine, nommée nucléosome, est composée d'un double brin d'ADN enroulé autour d'un octamère d'histone, et subit une pléthore de modifications post-traductionnelles. Les conséquences biologiques de l’acétylation des lysines et de l’isomérisation des liaisons peptidyl-prolyl ont été étudiées à travers une analyse à l’échelle atomique par RMN de systèmes d'intérêt reconstitués in vitro. Les liaisons peptidyl-prolyl du domaine N-terminal de l'histone H3 sont substrats in vitro d’une isomérase chez S. cerevisiae nommée Fpr4p, laquelle exerce un contrôle catalyse-dépendant de la transcription. La résolution de la structure du domaine catalytique de Fpr4p, à partir de contraintes géométriques mesurées par RMN, révéla un domaine canonique de la famille FKBP (FK506-binding protein). Grâce à l'analyse de la séquence primaire et aux expériences RMN, nous proposons un modèle structural préliminaire de Fpr4p entière. L'analyse fonctionnelle est réalisée grâce à trois décapeptides construits à partir de la séquence primaire de H3 chez S. cerevisiae. Ils sont tous substrats de Fpr4p et la catalyse est équivalente pour Pro16 et Pro30. La proportion à l'équilibre du conformère cis fut déterminée pour les trois peptides et celle-ci n'est pas affectée par l'activité catalytique de Fpr4p. Les structures en solution des substrats en conformation trans ont été résolues par spectroscopie RMN, et seront utilisées pour des appariements moléculaires in silico sur le domaine catalytique de Fpr4p. Pour étudier le rôle biologique de l'acétylation des histones, une méthodologie de production de protéines acétylées a été développée. Le protocole repose sur la mutation d'une lysine en cystéine d'une protéine recombinante, suivie d'une alkylation contrôlée exploitant la nucléophilie du groupe thiol préalablement introduit. La production de l'agent alkylant adéquat est simple, rapide, réalisable dans un laboratoire de biologie et permet différents marquages isotopiques du groupe acétyle. L'alkylation d'une protéine repliée fut réalisée avec succès en conditions natives. Le dimère d'histone H2A-H2B, un intermédiaire de l'assemblage du nucléosome et siège d'acétylation in vivo, fut reconstruit in vitro. Les déplacements chimiques des domaines N et C-terminaux de H2A sont cohérents avec un état intrinsèquement déstructuré bien que leurs dynamiques moléculaires ne soient pas équivalentes. / The structural unit of chromatin, the nucleosome, is composed of double-stranded DNA wrapped around a histone octamer and is subject to a plethora of post-translational modifications. The biological consequences of peptidyl-prolyl isomerization and lysine acetylation were investigated at atomic scale through analysis of in vitro reconstituted systems by NMR. Peptidyl-prolyl bonds of histone H3 N-terminal domain are substrates in vitro of an isomerase from S. cerevisiae named Fpr4p, which underlies transcriptional control dependent on its catalytic activity. The solution structure of the catalytic domain of Fpr4p was calculated based on restraints from NMR spectroscopy, and reveals a canonical catalytic domain belonging to the FK506-binding protein (FKBP) family. Based on primary sequence analysis and NMR experiments, a preliminary structural model of full length Fpr4p is also presented. Functional analyses were performed with three decapeptides designed from the primary sequence from the N-terminal tail of S. cerevisiae histone H3. All three constitute substrates of Fpr4p, with equivalent catalysis observed for Pro16 and Pro30. The equilibrium proportion of the cis-proline conformer has been determined for all three decapeptides, and these populations are unaffected by Fpr4p catalytic activity. Structural ensembles of the substrates with proline in the trans conformation were determined by using NMR spectroscopy, and will be subsequently used for in silico molecular docking onto Fpr4p. To study a second form of histone regulation, a semi-synthetic method to produce acetylated protein was developed. The protocol relies on the site-specific mutation of lysine to cysteine in recombinant proteins followed by controlled alkylation thanks to nucleophilicity of the introduced thiol. The production of the required alkylation reagent is easy, quick, and suitable for biology laboratory and allows diverse isotopic labeling within the acetyl group. Alkylation of folded proteins has also been achieved in native conditions. As one target of acetylation in vivo, the histone H2A-H2B dimer is an intermediate of nucleosome assembly and was reconstituted in vitro. Chemical shift values of the N- and C-terminal domains of H2A are in agreement with an intrinsically disordered state although they display differences in dynamic mobility.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Toxaphene is reported to cause defects in the collagen of fish. Chronic exposure to toxhaphene weakens the backbone of fish by decreasing the amount of collagen and usually increasing the amount of calcium in the bone which results in a more brittle and fragile bone. We investigated the possible direct action of toxaphene on collagen synthesis by exposing vertebral and swim bladder organ cultures obtained from unexposed rainbow trout (Salmo gairdneri) fingerlings to the same lot of toxaphene found to cause this defect in vivio. Collagen produced by these organ cultures was measured by: (1) total 3H-proline incorporated into the matrix; (2) 3H-proline released during collagenase digestion of acid-precipitated protein; (3) 3H-hydroxyproline extracted from the acid hydrolysate, and (4) tritiated water produced during the hydroxylation of 4-3H-proline. At a relatively high concentration of toxaphene (2.4 mM) these indices of collagen production were reduced, but this was probably caused by a decrease in tissue viability rather than by a direct affect on collagen synthesis. At 240 μM cellular protein synthesis was reduced. Generally no effects were found at toxaphene concentrations below 240 μM. From these studies it was concluded that toxaphene does not have a direct inhibitory effect upon collagen production at the tissue level. For comparison to the in vivo were measured. All three of these parameters were significantly reduced in comparison to controls (α = 0.05) in those fish exposed to the highest concentration of toxaphene (200 ng/1). fish exposed to 150 ng/l toxaphene also had reduced prolyl hydroxylase activity. These results indicated that vertebral prolyl hydroxylase activity may be a sensitive indicator of toxaphene exposure in fish, and inhibition of that enzyme may be involved in the mechanism of toxaphene-induced collagen effects.