Proteins are intimately involved in almost every cellular phenomenon, from life to death. Understanding the interactions of proteins with each other and other macromolecules and the ability to rationally redesign them to improve their activities or control their function are of considerable current interest. Split-protein methodologies provide an avenue for achieving many of these goals. Since the original discovery of conditionally activated split-ubiquitin, the field has grown exponentially to include the activities of over a dozen different proteins. The flexibility of the systems has resulted in their use across a wide spectrum, both literally and figuratively, to primarily screen, visualize and quantitate macromolecular interactions in a variety of biological systems. In another arena, there is significant interest the apoptosis-regulating proteins: the Bcl-2 family. These proteins are found in many cell types and control, through expression levels as well as other mechanisms, the apoptotic state of a protein as governed by intrinsic death signals generated from such sources as DNA damage and viral infection. The apoptotic function of these proteins are mainly governed by a single type of interaction: the helix:receptor binding of the BH3-Only helices to the anti-apoptotic receptor proteins. While this often promiscuous helix:receptor interaction has received much scrutiny, the nature of the anti-apoptotic binding pocket, especially with regard to the specific residues that govern the interaction, has been lacking. With the high sensitivity and rapid analysis platform afforded by the cell-free split-luciferase analysis methodology, we devised and carried out the first systematic and large scale alanine mutagenesis of all five major anti-apoptotic members of the Bcl-2 family, validated these results both with biophysical methods as well as correlation with previous studies. Our results help explain how different receptors can bind a wide range of helices and also uncovered details regarding binding that are not possible with structural or computational analysis alone. In a second area of research, we have utilized the interaction of BH3 helices and their receptors for designing small molecule controlled protein kinases and phosphatases. In this protein design area, BH3-Only helices were inserted using a knowledge based approach into particular loops within both a protein kinase and a protein phosphatase. The BH3-Only helix interaction with added receptors, such as Bcl-xL provided an allosteric switch for turning-off the activity of the helix-inserted enzymes. The activity of the enzymes could then be turned-on by the addition of a cell-permeable small molecule that is known to bind the receptor. This plug-and-play design was demonstrated to be successful for two very different enzyme classes and likely provides a general and tunable biological element for controlling the activity of one or more proteins and enzymes in a biochemical networks.

The nature of DNA has captivated scientists for more than fifty years. The discovery of the double-helix model of DNA by Watson and Crick in 1953 not only established the primary structure of DNA, but also provided the mechanism behind DNA function. Since then, the demonstration of DNA secondary structure formation has allowed for the proposal that the dynamics of DNA itself can function to modulate transcription. We demonstrate for the first time the i-motif DNA secondary structure formed from an element within the Bcl-2 promoter region has potential to serve as a cellular molecular target for modulation of gene expression. Unlike typical oncogenes, Bcl-2 acts by promoting cellular survival rather than increasing cellular proliferation. The over-expression of Bcl-2, most notably in lymphomas, has been associated with the development of chemoresistance.Transcriptional regulation of Bcl-2 is highly complex and a guanine- and cytosine-rich (GC-rich) region directly upstream of the P1 site has been shown to be integral to Bcl-2 promoter activity. We have demonstrated that the C-rich strand is capable of forming an intramolecular i-motif DNA secondary structure with a transition pH of 6.6 and a predominant 8:5:7 loop using mutational studies coupled with circular dichroic spectra and thermal stability analyses. In addition, a novel assay involving the sequential incorporation of a fluorescent thymine analog at each thymine position provided evidence of a capping structure within the top loop region of the i-motif. Two different classes of steroids either stabilize or destabilize the i-motif structure and this differential interaction results in the activation or repression of Bcl-2 expression. The i-motif stabilizing steroid significantly up-regulated Bcl-2 gene and protein expression in BJAB Burkitt's lymphoma cells while the destabilizing steroid down-regulated Bcl-2 expression in B95.8 Burkitt's and Granta-519 mantle cell lymphoma cells, as well as in a SCID mouse lymphoma model. More importantly, the down-regulation of Bcl-2 led to chemosensitization of etoposide-resistant lymphoma cells demonstrating that Bcl-2 i-motif interactive small molecules can act as chemosensitizing agents. Conversely, compounds that up-regulate Bcl-2 by stabilization of the i-motif have potential for use as neuroprotective agents.

The inflammatory response caused by the deposition of crystals of monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) in the synovial fluid of joints, results from the interaction of the crystals with neutrophils. Neutrophils (whose function in the body is to remove hazardous microorganisms and inflammatory debris) are activated by the binding of the crystals to the neutrophil cellular membrane, which leads to respiratory burst activity, engulfment of the crystals and release of proteolytic enzymes. Furthermore, we have found that crystals delay the normal “cell death program” or apoptosis, thus allowing for the accumulation of these cells, and extended inflammatory responses. Very little is known about the mechanisms of activation and delay of apoptosis, however, bcl-2 family proteins have been implicated in the control of neutrophil apoptosis. This study helps to define the role of several bcl-2 family proteins (both pro- and anti-apoptotic) by examining the differential expression of these proteins upon stimulation with crystals. Subsequent identification of signaling targets that function to regulate this process in response to crystals could lead to potential therapeutics for crystal-induced inflammatory diseases.

No description available.

XIAP and Bcl-xL are critical anti-apoptotic molecules that directly inhibit caspases and block mitochondrial membrane permeabilization, respectively. In addition to preventing apoptosis, both XIAP and Bcl-xL can be generated by cap-independent translation via the utilization of an IRES in the 5'-UTR of their mRNAs. In recent years it has been shown that activation of S6K2 induces the translational upregulation of these two apoptotic regulators. Here I have determined that activation of S6K2 enhances IRES-mediated translation of XIAP and Bcl-xL by inducing the degradation of PDCD4, which I have identified as a novel regulator of XIAP and Bcl-xL IRES elements. Furthermore, I have shown that PDCD4 is a positive modulator of the Apaf-1 IRES element. The concurrent regulation of XIAP, Bcl-xL and Apaf-1 by PDCD4 suggests a model in which the level of PDCD4 expression alters the apoptotic threshold by specifically impacting IRES-mediated translation of the XIAP, Bcl-xL and Apaf-1 mRNAs.

The RNA-binding protein HuR controls key cellular processes by binding target mRNAs and regulating them at various post-transcriptional levels. HuR can function as an Internal Ribosome Entry Site (IRES) trans-acting factor that regulates the IRES-mediated translation of XIAP. Since XIAP and Bcl-xL expression was reported to be co-regulated, we investigated whether HuR is also a regulat or of Bcl-xL expression. We found that HuR binds the 3’end of the Bcl-xL 5’UTR in-vitro. In U2OS cells, we showed that loss of HuR by siRNA significantly increased Bcl-xL protein expression while Bcl-2 and Mcl-1 levels remained unchanged. We found that the HuR-dependent Bcl-xL increase was through translation, shown by polysome profiling. Possible transcriptional, stability and splicing changes were eliminated. At the physiological level HuR levels did not impact cell survival but altered mitochondrial morphology, partially through Bcl-xL. Thus, HuR may be involved in maintaining proper mitochondrial function by controlling Bcl-xL expression.

The inflammatory response caused by the deposition of crystals of monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) in the synovial fluid of joints, results from the interaction of the crystals with neutrophils. Neutrophils (whose function in the body is to remove hazardous microorganisms and inflammatory debris) are activated by the binding of the crystals to the neutrophil cellular membrane, which leads to respiratory burst activity, engulfment of the crystals and release of proteolytic enzymes. Furthermore, we have found that crystals delay the normal “cell death program” or apoptosis, thus allowing for the accumulation of these cells, and extended inflammatory responses. Very little is known about the mechanisms of activation and delay of apoptosis, however, bcl-2 family proteins have been implicated in the control of neutrophil apoptosis. This study helps to define the role of several bcl-2 family proteins (both pro- and anti-apoptotic) by examining the differential expression of these proteins upon stimulation with crystals. Subsequent identification of signaling targets that function to regulate this process in response to crystals could lead to potential therapeutics for crystal-induced inflammatory diseases.

Exposure of cells to hydrogen peroxide can cause oxidative damage to cellular constituents including lipids, protein, and DNA. At elevated concentrations, hydrogen peroxide can trigger cell death by apoptosis or necrosis. Apoptotic cell death can be prevented by overexpression of the oncoprotein Bcl-2. The exact mechanism by which Bcl-2 blocks cell death is controversial. Some researchers believe that Bcl-2 possesses antioxidant properties that protect cells from apoptosis. The purpose of this thesis was to assess oxidative stress and apoptosis following hydrogen peroxide exposure in Jurkat T cells overexpressing Bcl-2. One of the major objectives was to ascertain whether or not Bcl-2 overexpression elevated the antioxidant capacity of Jurkat T cells to provide protection from oxidant-induced cell death. Hydrogen peroxide treated Jurkat cells became apoptotic at moderate levels of oxidant (25-100 uM H2O2), and necrotic at higher doses (greater than 200 uM H2O2). Bcl-2 overexpression prevented caspase activation and cell death at the apoptotic doses of H2O2, but not the necrotic doses. Caspase inhibition studies demonstrated that Bcl-2 overexpression provided a greater level of resistance from H2O2-induced cell death than the broad-spectrum caspase inhibitor z-VAD.fmk. A systematic study was carried out examining the antioxidant status of Jurkat cells overexpressing Bcl-2. Several Bcl-2 transfectants were utilised for the study, so that any differences seen could be correlated to the level of Bcl-2 expression. Surprisingly, there were no statistically significant differences among the Bcl-2 transfectants for any of the antioxidant enzymes. Jurkat cells overexpressing Bcl-2 exhibited the same level of oxidative damage to lipids and protein in response to H2O2 exposure as the parental Jurkat cells. Interestingly, Jurkat cells overexpressing Bcl-2 continued to grow in culture after H2O2 exposure, despite harboring damage to cellular constituents. Consistent with these results, H2O2 treated Jurkat cells overexpressing Bcl-2, which failed to undergo apoptosis, were more prone to genomic instability. Together, these findings suggest that Bcl-2 overexpression protects Jurkat cells from H2O2-induced cell death by blocking apoptosis. Jurkat cells overexpressing Bcl-2 were no better at detoxifying oxidants and showed the same level of oxidative damage following H2O2 exposure. As a result, the overexpression of Bcl-2 considerably enhanced the mutagenicity of H2O2.

Thesis (Ph.D.). / Written for the Dept. of Medicine. Title from title page of PDF (viewed 2008/05/09). Includes bibliographical references.

Thesis (M.Sc.). / Written for the Dept. of Neurology & Neurosurgery. Title from title page of PDF (viewed 2008/05/14). Includes bibliographical references.