Paramecium, a unicellular ciliate, can be attracted by various chemical stimuli. Chemoattractants such as glutamate, folate, cAMP, and acetate activate different receptor mediated signal transduction pathways. The final event in these signal transductions is a hyperpolarization of membrane potential, which makes Paramecium swim smoothly and fast. There is evidence that the effecter of this hyperpolarization is the plasma membrane calcium ATPase (PMCA), that when activated, expels Ca2+ from the cell. In Paramecium three PMCA isoforms, named PMCA2, 3, and 4, have been cloned. PMCA2 is associated with lipid rafts, which is demonstrated by its resistance to cold detergent solubilization and distribution in sucrose density gradients in ultracentrifugation. PMCA3 and 4 are not associated with lipid rafts. On the cell surface, PMCAs are localized to the bases of cilia. Sterol-depletion by methyl-ß-cyclodextrin (MßCD) treatment disrupts the distribution of PMCA2 in sucrose density gradients and ciliary base-localization on the cell surface. MßCD treatment also decreases the chemoattraction to glutamate and cAMP. This indicates that PMCA2 and its association with lipid rafts are essential in the chemoattraction signal transduction pathways. Based on these results, a model of membrane domains incorporating three signal transduction pathways is proposed.

Heart failure is responsible for one in twenty deaths in the UK, and as the average age of the general population increases that number is predicted to rise over the coming years. Hypertrophic growth is believed to be an adaptive response to a chronic increase in workload under circumstances such as hypertension, yet it is also known to contribute to the pathological progression into heart failure. Abnormal calcium handling is known to play a critical role in determining disease progression, not only through its function as the driving force behind myocardial contraction and relaxation but also through directing the signals which regulate hypertrophic growth. Both isoforms 1 and 4 of the diastolic calcium extrusion pump plasma membrane calcium ATPase (PMCA) are present in the heart, yet unlike in other cell types their contribution to overall calcium clearance is only small; however their role in the disease process is yet to be defined. A novel mouse line was generated in which both PMCA1 and 4 were deleted from the myocardium (PMCA1:4dcko mice). Through comparison with PMCA1 knockout mice (PMCA1cko) this thesis set out to identify the specific function of each pump under normal conditions and during the development of pathological hypertrophy induced by pressure overload through transverse aortic constriction (TAC).Under basal conditions each isoform functioned independently, PMCA1 to extrude calcium during diastole and PMCA4 to regulate calcium levels during systole; however the loss of neither isoform impacted significantly on cardiac function. In response to TAC, PMCA1cko mice progressed rapidly into decompensation and displayed signs of systolic failure after just 2 weeks, whilst cardiac function was preserved in TAC controls. Calcium handling analysis revealed that prior to the onset of failure PMCA1cko mice displayed a distinct lack of adaptive changes to calcium cycling which were present in controls. In stark contrast, the additional loss of PMCA4 led to an attenuated hypertrophic response to TAC in PMCA1:4dcko mice which remarkably preserved cardiac function despite the absence of PMCA1. This adds to accumulating evidence which suggests that the inhibition of PMCA4 may be protective during the development of pathological hypertrophy, whilst highlighting the possibility for a novel role for PMCA1 in coordinating essential adaptations required to enhance calcium cycling in response to the increased demands imposed on the left ventricle during pressure overload.

The extensive relationship between modulation of intracellular Ca2+ content and the control of cell proliferation (Boynton et al. 1974; Whitfield et al. 1979; Berridge and Irvine 1984), differentiation (Bridges et al. 1981; Holliday et al. 1991) and death (Orrenius et al. 2003) has led to much examination into the relationship between Ca2+ signaling pathways and the onset of various pathological conditions, including cancer, cardiac hypertrophy, immunodeficiency, neurodegeneration. Control of Ca2+ signals is achieved via an extensive combination of pumps, channels and exchangers which regulate the concentration of Ca2+ within not only the cytosol but also all intracellular compartments. Accordingly, a great deal of research has focused on the mechanisms which regulate these channels and pumps, and recently the primary mechanism for Ca2+ influx in non-excitable cells has been identified. This process, termed Store-operated calcium entry (SOCe), is a key evolutionarily conserved mechanism whereby decreases in endoplasmic reticulum Ca2+ content (sensed by the ER Ca2+ sensor, STIM1) leads to the influx of Ca2+ across the plasma membrane through the Orai family of Ca2+ channels. However, many questions remain about how this Ca2+ signaling pathway is regulated. In this thesis, I provide evidence regarding the transcriptional and molecular mechanisms regulating SOCe. Initial studies in my thesis work aimed to identify some of the key events leading to dysregulation of Ca2+ homeostasis in the kidney specific pediatric malignancy, Wilms' Tumor. I found that STIM1 expression levels and SOCe signals are significantly reduced in Primary Wilms' Tumor samples. Subsequent analysis of these phenomena led me to the finding that STIM1 expression is under the control of the transcription factors Wilms' Tumor Suppressor 1 (WT1) and Early Growth Response 1 (EGR1). Subsequent investigations were carried out with the purpose to assess how activation of the EGR1 transcription factor alters long term Ca2+ signals. Indeed, I found that receptor-mediated activation of EGR1 leads to induction of STIM1 expression and increases in SOCe. However, unexpectedly through these analyses, I propose a novel role for STIM1 that STIM1 interacts with the Plasma Membrane Ca2+ ATPase (PMCA) through its C-terminal proline-rich domain and reduces PMCA-mediated Ca2+ clearance, effectively creating local, augmented Ca2+ gradients. This coordinated control of Ca2+ entry and exit from the cell has wide-ranging implications for Ca2+ signaling in multiple cell types. / Biochemistry

Plasma membrane calcium/calmodulin-dependent calcium ATPases (PMCAs) are high affinity calcium pumps regulating many calcium-dependent processes and advances in its characterisation have discovered that it may play a novel role in signal transduction pathways. It was the aim of this work to further characterise and confirm the role PMCA plays in regulating calcium/calmodulin-dependent signal transduction pathways. PMCA4 has already been shown to inhibit the NFAT family of transcription factors by its interaction with calcineurin A in mammalian cells when ectopically expressed. This prompted the investigation into other isoforms of PMCA that may interact with the calcium/calmodulin-dependent calcineurin, to determine if this interaction was isoform-specific in a variety of cell lines. Endogenous proteins were isolated by immunoprecipitation with calcineurin A antibody and the presence of PMCA isoforms was determined by western blot using isoform-specific antibodies. This work has demonstrated that the PMCA and calcineurin interaction occurs in vitro at endogenous levels in MCF-7 human breast adenocarcinoma cells and endothelial cells and is isoform specific, predominantly for PMCA2. The characterisation of the PMCA2b-calcineurin A interactive domain was performed and it was demonstrated that PMCA2b significantly inhibits the NFAT/calcineurin pathway. These results indicate that PMCA2 is important in regulating the calcineurin/NFAT pathway in tissues where it is highly expressed. This work also demonstrates that the Flag-tagged, characterised interaction domain of PMCA2 with calcineurin, F-PMCA(462-684) when overexpressed, can disrupt the inhibitory PMCA2/calcineurin interaction in endothelial cells and significantly increase calcineurin activity. The expression of PMCA in endothelial cells prompted the investigation of calcium/calmodulin-dependent proteins in endothelial cells as evidence for the important role of PMCA in regulating signal transduction pathways. Nitric oxide synthases have been shown to be regulated by PMCA4 in cardiac cells. To further characterise the regulation of NOS by PMCA, this work shows that there is a novel molecular interaction between endogenous eNOS and the plasma membrane calcium ATPase (PMCA) in HUVEC primary endothelial cells. PMCA2 has been identified as the major isoform interacting with eNOS in endothelial cells. The interaction between the two proteins has been mapped to the region 735-934 of eNOS and 462-684 of human PMCA2b. NO production was found to be inhibited by ectopic expression of PMCA2b in HUVEC cells. Moreover, disruption of the interaction between endogenous PMCA and eNOS by overexpression of theFlag-tagged, PMCA2b interaction domain, F-PMCA2(462-684), significantly increased NO levels in activated HUVEC endothelial cells. In summary, these results offer strong evidence for a novel functional interaction between endogenous PMCA and eNOS in endothelial cells, suggesting a role for endothelial PMCA2 as a negative modulator of eNOS activity, and, therefore, NO-dependent signal transduction pathways. Overall this is a novel discovery which clearly demonstrates that PMCA is an important regulator of calcium/calmodulin-dependent signal transduction pathways in various cell types. Parts of this work have been published; ‘Holton, M., Yang, D., Wang, W., Mohamed, T.M., Neyses, L. and Armesilla, A. (2007) The interaction between endogenous calcineurin and the plasma membrane calcium-dependent ATPase is isoform specific in breast cancer cells. FEBS letter. 581(21), 4115-4119.’ and presented at ‘The 14th congress of calcium binding proteins, La Palma, Canary Islands, Spain. 2007’ and ‘The 25th Conference of the European Society on Microcirculation (August 26-29, 2008, Budapest, Hungary).’

Colon cancer is the third most common type of cancer, with high mortality throughout the world. During tumorigenesis, normal cells transform into tumour cells following changes in the expression of oncogenes and/or tumour suppressor genes, which are involved in many processes including the cell cycle, differentiation and apoptosis. An imbalance in the regulation of proliferation and differentiation in colon epithelial cells is usually associated with the development of colon cancers. Uncontrolled proliferation with a lack of differentiation is one of the major characteristic features of cancer cells and a remodelling of the Ca2+ signalling is linked to these pathways. Among the Ca2+ transporting proteins, P-type Ca2+-ATPases, the plasma membrane Ca2+ ATPase (PMCA) pump, has a high-affinity for Ca2+ and is involved in the efflux of Ca2+ against the electrochemical gradient from the cytosol across the extracellular space. Four PMCA isoforms have been identified. PMCA1 and 4 are expressed in most tissues. Changes in the expression of PMCA have been documented in breast cancer cells, whereas the expression profile of PMCA isoforms in colon cancer cells remains unknown. Up-regulation of another P-type Ca2+-ATPase, expressed in the endoplasmic reticulum, SERCA3, occurs during the differentiation of colon cancer cell lines and is down-regulated in colon cancers. Changes in PMCA expression have not been assessed during colon cancer cell differentiation. The first part of this thesis describes the analysis of the expression profile of PMCA during colon cancer cell differentiation. Both PMCA mRNA and protein levels were assessed in differentiated HT-29 cells by real time RT-PCR and western blotting analysis, respectively. The results showed changes in PMCA4 expression, whereas changes in the expression of PMCA1 were not associated with differentiation of HT-29 cells. PMCA mRNA levels were also reduced in some colon cancers suggesting a remodelling of PMCA-mediated Ca2+ efflux during colon carcinogenesis. The second part of this thesis involved exploring the functional role of PMCA4 in Ca2+-mediated signalling pathways such as differentiation, proliferation and apoptosis. PMCA4 expression was altered in HT-29 colon cancer cells via transient and stable over-expression of a PMCA4 expressing plasmid or siRNA-mediated silencing of PMCA4. An increase in the PMCA4 level did not alter or induce differentiation of HT-29 cells. Hence, up-regulation of PMCA4 expression may be a consequence rather than a cause of HT-29 colon cancer cell differentiation. PMCA4-mediated reduction in proliferation was observed in HT-29 colon cancer cells where PMCA4 was stably over-expressed. Stable PMCA4 over-expression was also associated with the down-regulation of the transcription of the early response gene, FOS. Despite the apparent augmentation of cytosolic Ca2+ responses to G-protein coupled receptor Ca2+ mobilizing agents, the sensitivity of cells to the apoptotic inducing agents such as TRAIL and/or CCCP was not affected following siRNA-mediated PMCA4 inhibition in HT-29 cells. Collectively this thesis describes PMCA isoform-specific changes during differentiation of HT-29 colon cancer cells and alterations in PMCA levels in some colon cancers.Evidence is also presented to suggest that alterations in PMCA expression in colon cancer cells may provide a growth advantage by promoting proliferation without increasing sensitivity to apoptotic stimuli.

Plasma membrane calcium ATPase 4 (PMCA4) is a calcium extrusion pump which may also modulate Ca2+-triggered signal transduction pathways. Previous studies postulate that PMCA4 modulates signalling via an interaction with neuronal nitric oxide synthase (nNOS) in localised plasmalemmal microdomains. The effect of PMCA4 on vascular contractility is unclear. This project has utilised PMCA4 ablated mice (PMCA4 KO (-/-)) and a novel specific PMCA4 inhibitor (termed AP2) to study the role of PMCA4 in mouse resistance artery contractility.Immunohistochemistry, Western blotting and polymerase chain reaction (PCR) confirmed the absence of PMCA4 in the brain, vasculature and ear snips obtained from PMCA4 KO (-/-) mice whereas it was present in those from wild type (WT (+/+)) mice. Pressure myography was employed to assesss contractile function of isolated, pressurised (to 60 mmHg) mesenteric resistance arteries from 3 months old male PMCA4 KO (-/-) and WT (+/+) mice, in response to high K+ physiological salt solution (KPSS) (40mM & 100mM) and noradrenaline (NA) (Log[NA] -9.0 to -5.0M). Passive lumen diameter and left and right wall thicknesses of arteries from PMAC4 KO (-/-) and WT (+/+) mice were taken at transmural pressures of 5-140 mmHg. Effects of acute PMCA4 inhibition with AP2 (10µM and 1µM), nitric oxide synthase (NOS) inhibition with LNNA (100µM) and specific nNOS inhibition with Vinyl-L-Nio (10µM) were also investigated. Effects of PMCA4 ablation and AP2 (10µM) on global intracellular Ca2+ changes ([Ca2+]i) in pressurised mesenteric arteries were assessed after loading arteries with the Ca2+-sensitive indicator indo-1. PMCA4 ablation had no effect on the magnitude of arterial constrictions or on the changes of [Ca2+]i in response to KPSS (40mM & 100mM) or to noradrenaline. The passive intra-lumen diameter, wall thickness, wall to lumen diameter and cross sectional area of mesenteric arteries across the intravascular pressure range studied were also not modulated by PMCA4 ablation. A leftwards shift in the stress to strain relationship and significant increase in beta elastic modulus (β) were revealed in arteries from PMCA4 KO (-/-) mice compared to those from WT (+/+) mice, suggesting that PMCA4 ablation reduces mesenteric arterial distensibility. Acute PMCA4 inhibition with AP2, significantly reduced arterial constrictions and the increase in [Ca2+]i in response to noradrenaline in arteries from WT (+/+) mice, but had no effect on arterial constrictions elicited by arteries from PMCA4 KO (-/-) mice. Inhibitory effects of AP2 were not present in arteries after NOS inhibition by LNNA and also after nNOS inhibition with Vinly-L-Nio. Hence, PMCA4 inhibition with AP2 reduces vascular constriction by a nNOS-dependent mechanism.In conclusion, the main findings of the study were that ablation and acute inhibition of PMCA4 with AP2 have different effects on mouse mesenteric resistance arterial contractility. This study provides more insight into PMCA4 as a significant modulator of signalling within the vasculature via effects on nNOS.

Transmissible spongiform encephalopathies (TSEs) are a group of fatal, neurodegenerative diseases that can affect both humans and animals. TSEs can be sporadic, familial, or acquired diseases. The prion hypothesis states that a misfolded form of the host glycoprotein, PrPC, acts as the infectious agent in TSE disease. The misfolded form, PrPSc, is increased in β-sheet content, detergent insoluble and partially resistant to proteinase K (PK) digestion. Based on the prion hypothesis, most current post-mortem diagnostic tests rely on the presence of PrPSc as indicative of TSE disease. However, recently experimental cases of TSE disease have been identified where no PrPSc deposition is evident. One example of this is a murine transgenic model of Gerstmann Sträussler Scheinker (GSS) disease. GSS is a familial TSE disease, caused by a number of different mutations in human PrP including a point mutation from proline to leucine at residue 102. A murine model of GSS disease, produced through gene-targeting, contains the same point mutation at the equivalent residue, 101, in murine PrP. These mice do not develop spontaneous disease during their lifespan, but when inoculated intra-cerebrally with either human P102L GSS (101LL/GSS) or hamster 263K scrapie (101LL/263K); develop a clinical disease and vacuolar TSE-related pathology. Upon biochemical and immunohistochemical analysis, the brain tissues of these clinically ill mice contain little or no detectable PrPSc. However titration experiments have previously shown infectivity titres of 107-109IU/g of brain tissue. Standard PK digestion (at 37°C), NaPTA precipitation and isolation of PrPSc through detergent insolubility and differential centrifugation all confirmed the observation of little or no detectable PK-resistant PrP (PrP-res) in the 101LL/GSS and 101LL/263K brain tissues, despite the high levels of TSE infectivity. The presence of PrPSc and/or TSE infectivity in the spleen during disease pathogenesis is dependent upon TSE agent strain and host species. Previous studies utilising wild-type mice infected with ME7, have shown that the levels of infectivity observed in spleen tissue are 2- 3log10 lower than those observed in the brain tissue of the same mice. However, experiments conducted as part of this thesis showed that sub-passage of both the brain and spleen tissue from clinically ill 101LL/GSS and 101LL/263K mice into 101LL mice by intra-cerebral inoculation result in short incubation periods, indicating that infectivity levels were similarly high in both tissues. Biochemical analysis of the primary spleen tissue identified the presence of PrP-res, albeit at lower levels than those observed in wild-type spleens infected with a standard laboratory TSE strain, ME7 or 79A. However, the presence of PrP-res indicates that the spleen has a role in disease pathogenesis, which will require further investigation. Additionally, the spleen tissue maintains the discrepancy between PrP-res and TSE infectivity that is observed in the brain tissue of these models and further questions the prion hypothesis. As little or no PrP-res was detectable in the brain tissues of 101LL/GSS and 101LL/263K mice by standard biochemical and immunohistochemical techniques, it was hypothesised that an in vitro amplification technique, protein misfolding cyclic amplification (PMCA) could amplify PrPSc to detectable levels. A series of optimisation experiments were performed to produce a reliable positive control for amplification of mouse PrPSc from a standard laboratory mouse TSE strain, 79A or ME7, with a normal wild-type mouse brain homogenate substrate. While a wide range of technical and experimental conditions were investigated, consistent and reproducible amplification of mouse PrPSc was not achieved and therefore amplification of PrPSc from 101LL/GSS and 101LL/263K tissues could not be performed as interpretation of results would be complicated without the presence of a positive control. Previous research has shown that while other commercial assays, e.g. TeSeE (BioRad), identified tissues from these models as borderline positive or negative for TSE disease, one TSE diagnostic assay, the IDEXX HerdChek kit, that utilises the Seprion ligand, identified both the brain and spleen tissue from 101LL/GSS and 101LL/263K clinical mice as positive for TSE disease. In order to identify if TSE infectivity is associated with the target of the Seprion ligand, brain tissue homogenates from 101LL/GSS, 101LL/263K and a positive control wild-type/79A homogenate were depleted of the Seprion ligand target utilising a PAD-beads kit (Microsens Biotechnologies), which incorporates the Seprion ligand as the capture agent, in combination with magnetic beads. Upon inoculation, a single depletion of the homogenates produced no significant reduction in incubation period to clinical disease in either the depleted homogenates or the wash buffers produced, in comparison to a non-depleted brain homogenate. This result indicates that a single depletion with the Seprion ligand, did not remove enough of the aggregated protein to significantly alter the level of infectivity in the depleted homogenate and that any infectious agent, which was initially bound to the Seprion ligand due to non-specific interactions, was then released during the wash steps of the procedure. Proteomic differences between all components produced during a single depletion of an infected brain homogenate, wild-type/79A, or a normal uninfected brain homogenate were assessed to potentially identify the target of the Seprion ligand. In conclusion, these murine models of TSE disease, 101LL/GSS and 101LL/263K, which contain both high infectivity levels with little or no PrP-res in the brain tissue and similar high levels of infectivity with low levels of PrP-res in the spleen, questions the accepted correlation between levels of infectivity and PrP-res or PrPSc as proposed by the prion hypothesis. It is hypothesised that either an alternative form of PrP, which has not yet been identified is the infectious agent in these disease models, or that the TSE infectious agent is a component which associates with PrPSc rather than being PrPSc itself. The eventual identification of the infectious agent present in these unusual disease models will increase our understanding of these diseases, potentially offer improved diagnostics for infectivity, and perhaps identify novel therapeutic targets.

No description available.

The PMCA assay was optimized for adaptation to low level detection of PrPSc in hamster plasma. Evaluation of numerous key variables of the PMCA assay led to an optimized protocol capable of ~3 log10 amplification after 32 cycles (two 16 hour rounds). When commercially purchased normal hamster plasma was added to the PMCA reaction an accentuation in PrPSc amplification was observed (>6.75 log10 after 32 cycles). Only con-specific plasma appeared to enhance the conversion of PrPC to PrPSc, suggesting that a species-specific co-factor may be involved in assembly of protein aggregates. Serial PMCA in the presence of low level (10%) contiguous conspecific plasma resulted in the generation of de novo PrPSc after several rounds of PMCA. Although plasma significantly accentuated PrPSc amplification by PMCA, the formation of de novo PrPSc interfered with the ability of using the PMCA assay to detect prion infections in hamsters experimentally infected with 263K scrapie. / Physiology, Cell and Developmental Biology

神経変性疾患で蓄積する異常タンパク質の1つであるα-synは、PD、DLBおよびMSAの脳内に主に蓄積する。DLBやMSAの患者脳から解析可能な量のα-syn凝集体の増幅に成功した。増幅前後の凝集体のプロテイナーゼKコアのMS分析結果から、増幅による変化はないもののマウスとヒトのα-syn凝集体で切断パターンが異なることがわかった。これらの結果から、この方法が神経変性疾患の異常タンパク質研究の発展に貢献できることを示唆した。 / Pathological proteins related to neurodegenerative diseases are misfolded, aggregating to form amyloid fibrils. One of the pathological proteins, α-syn, accumulates in the brains of PD, DLB and MSA. We first performed amplification of α-syn aggregates. We successfully amplified enough α-syn aggregates derived from α-syncleinopathies. We found that the MS analysis results of proteinase K-resistant cores of the aggregates before and after the amplification differ between mouse and human α-syn aggregates. The results suggest that structural properties of amplified α-syn fibrils are preserved and these methods can be applicable in the study of pathological proteins of the neurodegenerative disorders. / 博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University