Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references (leaves 104-122).

No description available.

Selective ‘super-specific’ inhibitors of Protein Phosphatase 1 (PP1) are not available. Several natural product toxins possessing marginal selectivity between PP1 and the closely related Protein Serine/Threonine Phosphatase (PSTP), Protein Phosphatase 2A (PP2A) have been used to study the role of PP1 and PP2A in cellular signaling processes, such as the cyclic peptide inhibitors (microcystins and nodularins); terpenoid (cantharidin); polyketides (okadaic acid, calyculin, and tautomycin). The organic molecule tautomycetin is a natural product which has the highest selectivity for PP1 compared to the closely related PSTP PP2A, albeit slightly so (about 39 times more selective). Calyculin A is equally selective to PP1 and PP2A. On the other hand, okadaic acid is about 100 times more selective towards PP2A compared to PP1. Specific protein inhibitors are not suitable for cell-based assay due to low, intrinsic cellular permeability of proteins. A si-RNA mediated knockdown approach though feasible, is not ‘fast-acting’. The knockdown often lasts for an extended time period and cannot be modulated (turned on or off) as desired. Also, analysis of knockdown data is complex as the system can regulate itself in complex ways, making any effort to interpret the data liable to misinterpretation. The ultimate goal of this project is to develop a cell-permeable, potent, and selective inhibitor for PP1 (which does not target the related protein phosphatases PP2A, PP2B and PP5) whose activity inside cells can be modulated as desired so that spatiotemporal control over the activity of PP1 can be achieved. Development of such an inhibitor can be used as a chemical tool to study the cellular signaling of PP1 and not by the related PSTP PP2A. To address the problem of a lack of inhibitor targeting Protein Phosphatase 1 selectively over the closely related PSTP, PP2A; design of a peptide based inhibitor has been envisioned which targets the acidic groove and hydrophobic groove of Protein Phosphatase 1 in addition to targeting the active site (triple approach combination). The parent peptide (V6.2.10) of this study has been designed using a co-crystal structure of rat PP1cγ complexed with mouse inhibitor-2 (PDB ID: 2O8A). The parent peptide V6.2.10 has an IC50 value of 4.2 µM, which has been confirmed in the present study. A combination of single site mutations has been made using N-terminus arginine scanning, C-terminus arginine scanning, active site mutations, cyclohexylalanine scanning, and miscellaneous site-specific mutations. A hydrophobic pocket present in Protein Phosphatase 1 has been probed using ortho and meta fluorophenyalanine residue to increase potency and metabolic stability of the peptide. The rationale for such mutations was based upon a combination of approaches: mutagenesis in PyMOL, calculation of binding energies in FoldX, suitability of parent residues to be mutated, and how important are parent and substituent residues for cellular permeability and metabolic stability. Several peptides were identified from single-site mutations which had lower (improved) IC50 compared to the parent peptide of the study, V6.2.10. Several double mutations combining potent single-mutant peptides identified from this study has lower (improved) IC50 values than either of the single mutant peptides. #30 (combination of #15 and #4.2) has an IC50 value of about 334 nM and #36 (combination of #15 and 4-Fluoro Phenylalanine at the F5 position) has an IC50 value of 531 nM. #30 is the optimized peptide inhibitor from this study which is currently being utilized for crystallization trails in the laboratory. Far UV Circular dichroism study of #4.2 peptide shows mostly random coil conformation along with contributions from other secondary structures. Moreover, #4.2 is capable of adopting an alpha helical conformation in the presence of the well-known helix inducer chemical trifluoroethanol. Purification of PP1α protein using affinity chromatography has been optimized in order to increase the yield of pure protein phosphatase 1. Attempts to express and purify PP1α protein in BL21 (DE3) bacterial cells gave low yield. Thus, expression and purification of PP1α protein derived from human genomic sequence has been attempted in BL21 (RIL) codon-optimized cells which resulted in increased production of pure protein.

Interferon (IFN) regulatory factor 7 (IRF7) plays a key role in the production of IFN‐α in response to viral infection, and phosphorylation at IRF7 C‐terminal serine sites is prelude to its function. However, phosphatases that negatively regulate IRF7 phosphorylation and activity have not been reported. In this study, we have identified a conserved protein phosphatase 1 (PP1)‐binding motif in human and mouse IRF7 proteins, and shown that PP1 physically interacts with IRF7. Exogenous expression of PP1 subunits (PP1α, β, or γ) ablates IKKε‐stimulated IRF7 phosphorylation and dramatically attenuates IRF7 transcriptional activity. Inhibition of PP1 activity significantly increases IRF7 phosphorylation and IRF7‐mediated IFN‐α production in response to Newcastle disease virus (NDV) infection or Toll‐like receptor 7 (TLR7) challenge, leading to impaired viral replication. In addition, IFN treatment, TLR challenges and viral infection induce PP1 expression. Our findings disclose for the first time a pivotal role for PP1 in impeding IRF7‐mediated IFN‐α production in host immune responses.

No description available.

No description available.

Gene expression profiles obtained by means of semi-quantitative RT-PCR showed that both PPP1R1B and PPP1R3C were frequently silenced in multiple carcinomas. Bisulfite treated tumor DNA was subjected to Methylation-specific PCR (MSP) using primers flanking across the ∼130bp CpG island of the promoter of the particular gene of interest. It was revealed that PPP1R1B and PPP1R3C gene silencing in the carcinoma cell lines were due to promoter CpG island hypermethylation. Such claim was further confirmed by bisulfite genomic sequencing (BGS). Treatment with 5' azacytidine and TSA restored PPP1R1B and PPP1R3C expression in carcinoma cells through demethylating the hypermethylated promoter. In terms of cancer growth inhibition, ectopic expression of PPP1R1B and PPP1R3C could significantly inhibit the proliferation of carcinoma cell lines by 40--50% and 50--60%, respectively, according to the result of anchorage-dependent colony formation assay. / Overall, we believed that PPP1R1B and PPP1R3C are the putative tumor suppressor genes in which their expression silencing through promoter CpG island hypermethylation may be strongly linked to the development of cancer. / Protein Phosphatase 1 regulatory subunits are a family of small molecules which define the substrate specificity and subcellular localization of protein phosphatase-1 upon their interactions. Downregulation of Protein Phosphatase 1 regulatory subunits were often associated with tumor initiation and progression, for example, ASPP family (PPP1R13A and PPP1R13B). In the present study, PPP1R1B and PPP1R3C were identified in which their tumor suppressor functions had been investigated. / Reduction in the level of p-ser473 Akt and p-ser552 beta-catenin could be observed when PPP1R1B expression was restored in respective carcinoma cells. In addition, the transcription activity of AP-1 decreased in the presence of full-length PPP1R1B expression as determined by Dual-Luciferase reporter assay system. Ectopic expression of PPP1R3C increased the amount of inactive pSer9-GSK-3beta as shown in the western blot analysis and a concomitant increased in p53 level was observed in colorectal carcinoma HCT116 cells. Transcription activity of NF-kappaB in HCT116 cells was increased but decreased in KYSE150 cells (ESCC) in the presence of PPP1R3C expression. Subcellular localization study using the GFP-fusion protein revealed that PPP1R1B protein was distributed throughout the cytoplasm while PPP1R3C protein was mainly localized around the nuclear membrane. / Leung, Ching Hei. / Adviser: Tak Cheung Chan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 160-183). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Due to heavy nutrient load and adverse climate change the occurrence of toxic cyanobacterial blooms have significantly increased during the last decades. Nodularia spumigena is one of the dominant toxic cyanobacteria which produces massive and inherent blooms in brackish water body, the Baltic Sea, particularly in late summer. Nodularia spp. are known to produce nodularins (NOD) and a range of other bioactive peptides such as spumigins and nodulopeptins, all of which have unclear function. In a recent study, three new nodulopeptins with molecular weight of 899, 901 and 917 were characterised from N. spumigena KAC 66. In the present study, N. spumigena KAC 66 was fractionated by reversed phase flash chromatography and their toxicity was determined by their lethality to Daphnia pulex and D. magna along with inhibition of protein phosphatase 1 assay (PP1). All fractions showed lethality to Daphnids and inhibitory activity against PP1, the toxicity was due to additional compounds as NOD and nodulopeptin 901 were only detected in 7 fractions. Pure NOD was lethal to D. pulex and D. magna LC50= 8.4 μg/mL and 5.0 μg/mL, respectively. The newly characterised nodulopeptin 901 was also tested against D. magna (LC50=>100 μg/mL). NOD and nodulopeptin 901 inhibited PP1 with IC50 0.038 μg/mL and 25 μg/mL, respectively. In common with many studies, the maximum amount of NOD was retained within the cells during the seven week growth experiment. In contrast, as much as ~50% of nodulopeptin 901 was detected in the growth media throughout the duration of experiments. To gain further insight on the effects of environmental stress on growth and production of bioactive metabolites in N. spumigena KAC 66, a range of parameters were investigated which included; temperature, salinity, nitrate and phosphorus. In the present study it was investigated that extreme growth conditions have a considerable effect on biomass and toxin levels by N. spumigena KAC 66. The light intensity ranged from 17.35-17.47 μmol/s/m2, 22°C and 11-20 ‰ of salinity were the optimal growth conditions to obtain maximum biomasses, intra and extracellular peptide contents. At 6.5 mg/L nitrate the maximum growth, as indicated by Chl-a and maximum concentrations of intracellular NOD and nodulopeptin 901 were detected found in week 5 and 4, respectively. Temperature had the greatest effect on peptide production. Whilst growth was similar at 22°C, 25°C and 30°C, increase in temperature had a profound effect on NOD production in that an increase from 22°C to 25°C resulted in a 50% decrease in intracellular NOD levels. At 30°C little or no NOD was detected. In contrast, whilst concentrations of nodulopeptin 901 decreased with increasing temperature, they were still detected at consistent levels suggesting they play an important role. The results from phosphate experiment showed Chl-a, cell biomass and peptide production did not show clear dependency on availability of PO-3 4. This is the first study to evaluate the effects of selected environmental parameters on NOD/nodulopeptin 901 production which ultimately may be helpful to explain the distribution, control of natural blooms and toxin levels of N. spumigena in the Baltic Sea and as well as laboratory based experiments. In an attempt further exploit cyanobacterial diversity, 20 strains were isolated from the Dian Lake and 6 from the Dead Sea. The UPLC-PDA-MS analysis of isolates, Microcystis spp. from Dian Lake, China indicated the presence of several peptides namely MC-LR, cyanopeptolin A and aerucyclamides A-D. These new isolates will be examined for biological activity and chemical characterisation in future studies.

The Sli15-Ipl1-Bir1 chromosomal passenger complex is essential for proper kinetochore-microtubule attachment and spindle stability in the budding yeast Saccharomyces cerevisiae. Subcellular localization of this complex during anaphase is regulated by the Cdc14 protein phosphatase, which is kept inactive in the nucleolus until anaphase onset. I show here that the predominantly nucleolar ribosome biogenesis protein Utp7 is also present at kinetochores and is required for normal organization of kinetochore proteins and proper chromosome segregation. Utp7 associates with and regulates the localization of Sli15 and Cdc14. It prevents the abnormal localization of Sli15 on cytoplasmic microtubules, the premature concentration of Sli15 on the pre-anaphase spindle, and the premature nucleolar release of Cdc14 before anaphase onset. Utp7 regulates Sli15 localization not entirely through its effect on Cdc14. Furthermore, the mitotic exit block caused by Cdc14 inactivation is relieved partially by the simultaneous inactivation of Utp7. Thus, Utp7 is a multifunctional protein that plays essential roles in the vital cellular processes of ribosome biogenesis, chromosome segregation and cell cycle control. Protein phosphatase 1, Glc7 opposes in vivo functions of the Ipl1-Sli15-Bir1 kinase complex in budding yeast. I show here Scd5- a targeting subunit of Glc7 that regulates endocytosis/cortical actin organization and undergoes nuclear-cytoplasmic shuttling- is present at kinetochores. Ipl1 associates with both Glc7 and Scd5. The scd5-PP1[Delta]2 mutation, which disrupts the association between Glc7 and Scd5, also disrupts the association between Ipl1 and Scd5-Glc7 without affecting the kinetochore localization of these proteins. Genetic studies suggest that Scd5 may positively regulate both Glc7 phosphatase and the Ipl1 kinase complex. In accordance, Scd5 stimulates in vitro kinase activity of Ipl1. scd5-PP1[Delta]2 cells missegregate chromosomes severely due to several defects: i) at least one of sister kinetochores appears not attached to microtubule. ii) sister chromatids are persistently cohesed through anaphase. iii) Sli15 is hyperphosphorylated and less abundant on the anaphase spindle resulting in unstable mitotic spindle. These results together suggest that Scd5 functions in diverse processes that are essential for faithful chromosome segregation. How Scd5 coordinately regulates two apparently antagonistic enzymatic activities of Ipl1 and Glc7 remains to be determined. / text

No description available.