Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide (O2 • −) have been shown to serve as messengers in biological signal transduction, and many prokaryotic and eukaryotic proteins are now known to have their function controlled via ROS-mediated oxidation reactions occurring on critical cysteine residues. The tumour-suppressor protein p53 is involved in the regulation of a diverse range of cellular processes including apoptosis, differentiation, senescence, DNArepair, cell-cycle arrest, autophagy, glycolysis and oxidative stress. However, little is understood about the specific molecular mechanisms that allow p53 to discriminate between these various different functions. p53 is a multiple cysteine-containing protein and there is mounting evidence to suggest that redox-modification of p53 Cys residues participate in control of its biological activity. Furthermore, p53 activity has been linked to intracellular ROS levels. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers superior mass resolving power and mass measurement accuracy, which is beneficial for the study of intact proteins and the characterisation of their posttranslational modifications (PTMs). The primary goal of the work described in this thesis was to employ FT-ICR mass spectrometry to investigate the molecular details of p53 redox-regulation. The relative reactivity of each of the ten cysteine residues in the DNA-binding core domain of recombinant human p53 was characterised by treatment with the Cys-alkylating reagent N-ethylmaleimide (NEM) under various conditions. A combination of top-down and middle-down FT-ICR MS was used to unambiguously identify Cys182 and Cys277 as sites of preferential alkylation. These results were confirmed by site-directed mutagenesis. Interestingly, Cys182 and Cys277 have previously been implicated in p53 redox-regulation. Alkylation beyond these two residues was found to trigger rapid alkylation of the remaining Cys residues, presumably accompanied by protein unfolding. These observations have implications for the re-activation of mutant p53 with Cys-targeting compounds which result in the death of cancer-cells. Furthermore, the molecular interaction between p53 and the ROS hydrogen peroxide was investigated. p53 was found to form two disulfide bonds upon treatment with H2O2. An enrichment strategy was developed to purify oxidised p53 and top-down FT-ICR mass spectrometry revealed unambiguously that Cys176, 182, 238 and 242 were the oxidised residues. Interestingly, Cys176, 238 and 242 are Zn2+- binding residues suggesting that p53 contains a zinc-redox switch. The mechanism of H2O2 oxidation was investigated, and revealed that oxidation via an alternative pathway results in indiscriminate over-oxidation of p53. Moreover, Cys176, 238 or 242 was shown to act as a nucleophile, and the intracellular antioxidant glutathione (GSH) did not prevent oxidation of the Zn2+-binding Cys residues, providing further evidence for a role in p53 redox-regulation. This study has revealed hitherto unknown details regarding the chemistry of cysteine residues within the important tumour-suppressor protein p53. Furthermore, the analytical power of FT-ICR MS for the study of multiple Cys-containing proteins has been very clearly demonstrated.

At birth, the mammalian ovary has a finite number of dormant primordial follicles. Repeated daily dosing of rats with the occupational chemical, 4- vinylcyclohexene diepoxide (VCD), depletes the ovary of small pre-antral follicles (primordial and primary follicles) through an increase in the natural process of atresia (apoptosis). In addition, in vitro exposure of postnatal day 4 (PND4) rat ovaries to VCD causes a similar depletion of ovarian follicles. Since many growth factors play crucial roles in the promotion of early folliculogenesis and follicle survival, it is possible that any number of factors and subsequent signaling pathways could be disrupted in response to VCD exposure. Therefore, the studies in this work address the hypothesis that VCD disrupts oocyte-granulosa cell survival pathways in the rat ovary, thereby compromising cell-cell communication and causing follicle cell death. The results from the first aim reveal that through the use of genomic analyses a subset of genes were determined to be affected via in vivo and in vitro exposure routes to VCD. The results of the second aim show that two transforming growth factor β (TGFβ) growth factors, growth and differentiation factor-9 (GDF-9), and bone morphogenetic factor-4 (BMP-4), are not likely involved in VCD-induced ovotoxicity as they were unable to prevent ovarian follicle loss in the presence of VCD. The results of the third aim reveal that expression of the c-Kit receptor, present on the oocytes, is decreased and its ligand, Kit Ligand (KL), produced from the granulosa cells, is increased in response to in vitro VCD exposure. In addition, attenuation of VCD-induced follicle loss occurs in the presence of exogenous KL. Finally, the results of the fourth aim examines the involvement of the AKT signaling molecule in response to VCD exposure, in which the active phosphorylated AKT is determined to be down-regulated by VCD. Taken together, these studies show that VCD is able to disrupt at least one of the cellular survival pathways that are crucial to maintain the ovarian follicle. As a result, a breakdown in cell-cell communication may occur at that level and contribute to an increase in follicular atresia and eventual cell death.

No description available.

Methyl-accepting chemotaxis proteins MCPs can bind one or more receptor- specific ligands. In the case of the Tar MCP of Escherichia coli (TarEc), a primary attractant ligand is aspartate. Its binding to the periplasmic domain of Tar generates a conformational change that is transmitted via helix 4 transmembrane helix 2 (TM2). An inward movement of TM2 initiates a transmembrane signal to the cytoplasmic HAMP (histidine kinases, adenyl cyclases, methyl-accepting proteins, phosphatases) domain. Baseline CheA kinase-stimulating activity and ligand-induced responses are both strongly influenced by residues at the C-terminus of transmembrane helix 2 (TM2). The cytoplasmic aromatic anchor, composed of residues Trp-209 and Tyr-210 in TarEc, is of particular importance. These residues are not highly conserved among transmembrane receptors having a HAMP domain, although there are almost always some aromatic residues in this region. The question thus becomes what properties of this aromatic anchor are necessary for proper signal transduction. In this dissertation, I studied the effect on TarEc function by substituting all possible combinations of Ala, Phe, Tyr, and Trp at positions 209 and 210. This library of TarEc variants allowed the direct assessment of the effect of the residue composition of the aromatic anchor and led to a model of how the wild-type anchor maintains the base-line signaling state in TarEc. Additional receptor variants containing double aromatic tandems and Ala substitutions for the periplasmic Trp residue were created, and the aromatic residues were also shifted in position within the six residues 207-212. Trp, Tyr, and Phe, in that order, had the greatest effect on function when they were moved to novel positions. It was also discovered that Gly-211 plays a critical role in maintaining receptor function. A model was generated that proposes that Gly-211 plays a role in maintaining the flexibility of the TM2-HAMP domain connector. The results suggest that the signaling properties of the transmembrane sensor kinases of two-component systems can be predicted by the nature of their TM2-HAMP connections. It may also be possible to modulate their activity in a controlled way by manipulating the amino acid sequences that comprise those connections.

Noonan syndrome (NS) is one of several autosomal dominant “RASopathies” caused by mutations in components of the RAS-RAF-MEK-ERK MAPK pathway. Germ line mutations in RAF1 (encoding the serine-threonine kinase for MEK1/2) account for ~3-5% of NS, and unlike other NS alleles, RAF1 mutations that confer increased kinase activity are highly associated with hypertrophic cardiomyopathy (HCM). Notably, some NS-associated RAF1 mutations show normal or decreased kinase activity. To explore the pathogenesis of such mutations, I generated “knock-in” mice that express kinase-activating (L613V) or -impaired (D486N) Raf1 mutants, respectively. Knock-in mice expressing the kinase-activated allele Raf1L613V developed typical NS features (short stature, facial dysmorphia, haematological abnormalities), as well as HCM. As expected, agonist-evoked Mek/Erk activation was enhanced in multiple cell types expressing Raf1L613V. Moreover, postnatal Mek inhibition normalized the growth, facial, and cardiac defects in L613V/+ mice, showing that enhanced Mek/Erk activation by Raf1 mutant is critical for evoking NS phenotypes. D486N/+ female mice exhibited a mild growth defect. Male and female D486N/D486N mice developed concentric cardiac hypertrophy and incompletely penetrant, but severe, growth defects. Remarkably, Mek/Erk activation was enhanced in Raf1D486N-expressing cells compared with controls. In both mouse and human cells, RAF1D486N, as well as other kinase-impaired RAF1 mutants, show increased heterodimerization with BRAF, which is necessary and sufficient to promote increased MEK/ERK activation. Furthermore, kinase-activating RAF1 mutants also require heterodimerization to enhance MEK/ERK activation. Our results suggest that increased heterodimerization ability is the common pathogenic mechanism for NS-associated RAF1 mutations.

Currently, the intensive chemotherapy remains the first line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although these regimens have significantly improved patient outcomes, their use is associated with debilitating morbidities and fatal relapses, highlighting the great need in new agents that target essential survival signals in leukemia. Thus, the overall goal of my project was to gain insights into the signaling abnormalities that regulate aberrant proliferation and survival of B-ALL cells in an effort to identify novel targets in this malignancy. This study demonstrated that pre-B cell receptor (pre-BCR)-independent spleen tyrosine kinase (SYK) activity was required for the survival and proliferation of a p53-/-PrkdcSCID/SCID mouse model of B-ALL. I extended this discovery to human disease, demonstrating that SYK was activated in primary B-ALL, independent of the pre-BCR expression. The small molecule SYK inhibitor fostamatinib (fosta) significantly attenuated proliferation of 79 primary diagnostic B-ALL samples at clinically achievable concentrations. Importantly, fosta treatment reduced dissemination of engrafting B-ALL cells into the spleen, liver, kidney and central nervous system (CNS) in a NOD.Prkdcscid/scidIl2rgtm1Wjl/SzJ xenotransplant model of B-ALL. Analysis of signaling abnormalities using a high-throughput phospho-flow cytometry platform demonstrated that pediatric and adult B-ALL samples exhibit variable basal activation of BCR, iii PI3K/AKT/mTOR, MAPK and JAK/STAT pathways. Importantly, we identified that fosta-mediated inhibition of SYK, PLC2, CRKL and EIF4E phosphorylation in B-ALL was predictive of its anti-leukemic activity, and was distinct from the cellular actions of other small molecule inhibitors of key nodal signaling pathways. Examination of molecular mechanism of fosta action by gene expression profiling revealed transcriptional effects of fosta treatment that included, most notably, potent inhibition of pathways involved in lymphocyte activation and inflammation. In conclusion, this study demonstrates that SYK signaling is crucial for B-ALL survival and provides detailed characterization of cellular and molecular mechanisms of fosta action in B-ALL. These data argue in favor of testing small molecule SYK inhibitors in pediatric and adult B-ALL.

B lymphocytes are an important type of immune cell that contributes to pathogen clearance. When dysregulated, these cells contribute significantly to diseases such as autoimmunity, allergy and B cell cancers. Here we examine an important regulatory circuit that involves the lipid phosphatase SHIP, a key regulator of the PI3K signaling pathway. SHIP was first described as the major effector of inhibitory IgG receptor FcγRIIB, which downregulates B cell antigen receptor (BCR) signaling pathways when co-engaged. However, it is also known to inhibit signaling downstream of several other receptors, both activating and inhibitory. Here we examine the regulation and function of SHIP in B cells, focusing on the inter-related influences of binding partners, tyrosine phosphorylation and subcellular localization dynamics. First, we assess interaction of SHIP to selected candidate binding partners using an in vitro screening approach. The two most robust interactions were further characterized with respect to dissociation constant. These were: a novel interaction between SHIP phospho-Tyr944 and the SH2 domain of Nck, and a known interaction between the SH2 domain of SHIP and FcγRIIB phospho-Y292. Next, we perform the first examination of SHIP Tyr944. We provide evidence that it contributes to interaction with Nck after BCR engagement and is required for inhibition of actin turnover by SHIP. Finally, we perform the first detailed examination of the mechanisms controlling SHIP localization in human B cells stimulated through the BCR with and without co-engagement of FcγRIIB. We discover that SHIP is recruited to the plasma membrane equally in both stimulation contexts, however FcγRIIB co-ligation results in reduced mobility of SHIP molecules at the cell periphery. We identify a novel and essential role for Syk kinase in promoting SHIP membrane localization, tyrosine phosphorylation, and interaction with known binding partner Shc1. Together, these results provide significant and exploitable insight into the molecular control of a clinically important regulator of B cell responses. / February 2017

No description available.

Cell penetrating peptides (CPPs) have drawn the attention of researchers due to their ability to internalize large cargos into cells including cancer cells. The mechanism(s) with which the peptides enter the cell, however, is/are not clear and full of controversy. The peptide conformations and their microenvironment in live cells had been unknown until the development of a technique developed in our lab. As a first demonstration of principle, penetratin, a 16-residue CPP derived from the Antennapedia homeodomain protein of Drosophila, was measured in single, living melanoma cells. Carbon-13 labeling of the Phe residue of penetratin was used to shift the intense aromatic ring-breathing vibrational mode from 1003 to 967 cm-1, thereby enabling the peptide to be traced in cells. Difference spectroscopy and principal components analysis (PCA) were used independently to resolve the Raman spectrum of the peptide from the background cellular Raman signals. / On the basis of the position of the amide I vibrational band in the Raman spectra, the secondary structure of the peptide was found to be mainly random coil and b-strand in the cytoplasm, and possibly assembling as b-sheets in the nucleus. Next, label-free transportan was studied with the same methodology. The peptide, besides predominantly a-helix, adopted a significant portion of b-sheet conformation in the cytoplasm and nucleolus, which is different from the peptide in aqueous solution. The peptide microenvironment was also probed through H-bonding reported by the tyrosine Fermi doublet. Transportan displayed a tendency to accumulate in the cytoplasm over time which was unlike penetratin, which concentrated in the nucleus. The relative concentration of CPPs in various locations of live melanoma cells was directly estimated from the Raman spectra using average Phe concentration in the cell as an internal standard. / The rapid entry and almost uniform cellular distribution of both peptides, as well as the lack of correlation between peptide and lipid Raman signatures, indicated that the mechanism of CPP internalization under the conditions of study was probably non-endocytotic. Last, transportan and penetratin were studied using polarized Raman spectroscopy for more detailed vibrational spectroscopic information of the two peptides in water and TFE solutions. The majority of the bands in the Raman spectra of the peptides were highly polarized, consistent with the high symmetry of aromatic ring side chain vibrational bands dispersed throughout the spectra. This work has provided new insights into the structure of CPPs in live cells and in solutions. / by Jing Ye. / Thesis (Ph.D.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

The turtle is a unique model of anoxic survival. The turtle's brain can tolerate total oxygen deprivation for hours to days as well as prevent high levels of mitochondrial-derived free radicals upon re-oxygenation. Because of its ability to prevent elevated free radical generation, the turtle has also become recognized as a model of exceptional longevity. We are employing the turtle model for an investigation into the regulation of a key antioxidant enzyme system - methionine sulfoxide reductases (Msrs), primarily MsrA and MsrB. The Msr system is capable of reversing oxidation of methionines in proteins and Msr subtypes have been implicated in protecting tissues against oxidative stress, as well as, enhancing the longevity of organisms from yeast to mammals. Preliminary data, unpublished results, indicate that MsrA protein and transcripts are elevated by anoxia. A recent study on Caenorhabditis elegans demonstrated that FOXO is involved in activation of the MsrA promoter. Using the turtle MsrA promoter sequence we worked to determine which regions in the promoter are necessary for activation by anoxia. The results of the present study were 1) to prepare a TAT-FOXO3a fusion protein which could penetrate animal cells and 2) to construct a FOXO3a expression vector for transcription studies on MsrA expression. / by Diana Navarro. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.