Computational Insights on the Competing Effects of Nitric Oxide in Regulating Apoptosis

Despite the establishment of the important role of nitric oxide (NO) on apoptosis, a molecular- level understanding of the origin of its dichotomous pro- and anti-apoptotic effects has been elusive. We propose a new mathematical model for simulating the effects of nitric oxide (NO) on apoptosis. The new model integrates mitochondria-dependent apoptotic pathways with NO-related reactions, to gain insights into the regulatory effect of the reactive NO species N2O3, non-heme iron nitrosyl species (FeLnNO), and peroxynitrite (ONOO−). The biochemical pathways of apoptosis coupled with NO-related reactions are described by ordinary differential equations using mass-action kinetics. In the absence of NO, the model predicts either cell survival or apoptosis (a bistable behavior) with shifts in the onset time of apoptotic response depending on the strength of extracellular stimuli. Computations demonstrate that the relative concentrations of anti- and pro-apoptotic reactive NO species, and their interplay with glutathione, determine the net anti- or pro-apoptotic effects at long time points. Interestingly, transient effects on apoptosis are also observed in these simulations, the duration of which may reach up to hours, despite the eventual convergence to an anti-apoptotic state. Our computations point to the importance of precise timing of NO production and external stimulation in determining the eventual pro- or anti-apoptotic role of NO

Studies on Memo, an important ErbB2 receptor-mediated component of the cellular migratory machinery

The ErbB2 receptor tyrosine kinase has been shown to play an important role in cancer cell motility and metastases formation. This receptor is often overexpressed in human tumors of diverse origins, including breast and ovarian cancer. Individuals with ErbB2 over expressing tumors have shown poor clinical outcome. Our studies are focused on signaling molecules that interact with autophosphorylated tyrosine residues of the cytoplasmic tail of the receptor. Two of the sites, Tyr 1201 (YC) and Tyr 1227 (YD) are fully able to restore the migratory phenotype of breast carcinoma cells. Studies of the functional role of ErbB2 phosphorylation sites identified PLCγ1 as an interacting partner of the YC autophosphorylation site, and Memo (Mediator of ErbB2-driven cell Motility) as a binding partner of the YD site that is required for ErbB2 induced cell motility. Memo is encoded by a unique gene that is found in all branches of life, from bacteria to humans. Memo has no characterized domains, nor does it have obvious catalytic activity. Various approaches were used to position Memo in a signaling pathway and to uncover its biochemical function. Memo was initially detected based on its important role in ErbB2- induced cell motility. In fact, tumor cells with a specific knock-down (KD) of Memo failed to grow microtubules in response to Heregulin (HRG)-induced ErbB2 activation and were impaired in their migration. Cell migration proceeds in distinct steps. In response to a chemotactic stimulus, cells extend protrusions at the front that help in attachment. This is followed by contraction of the cell body and tail detachment at the rear allowing movement in the direction of the ligand. The initial event in the process is sensing of the ligand in response to activation of cellular receptors like EGFR or ErbB2. Their activation initiates signaling pathways that lead to polymerization of new actin at the leading edge, which is necessary for generating the protrusive force allowing migration. An important goal of my thesis work has been to investigate the step(s) of the migratory process that require Memo. In the first study, we explored migration using Dunn chambers and analyzed the chemotactic response of tumor cells in a shallow gradient of ligand. By tracing HRG-stimulated cell migration in time-lapse video microscopy, we found that Memo or PLCγ1 KD strongly impairs cell directionality, reflecting an important role for Memo and PLCγ1 in orchestrating directional cell migration. We also demonstrated that depletion of Memo or PLCγ1 resulted in very similar phenotypes, with a strong impairment of HRG-induced cytoskeletal organization. To gain more insight into Memo’s function, we carried out a Yeast-2-Hybrid (YTH) analysis and found a number of interesting new partners of interaction for Memo. Of particular interest is the small protein cofilin, one of the major cellular actin severing and depolymerizing factors that is known to have an essential role in directional sensing during chemotaxis. This interaction was confirmed in vitro using recombinant proteins and in vivo in coimmunoprecipitation experiments where Memo was detected in complexes with cofilin, ErbB2 and PLCγ1. Interestingly, we also found that HRG-induced PLCγ1 phosphorylation was decreased in Memo KD cells, suggesting that Memo regulates PLCγ1 activation. Furthermore, by introducing GFP-tagged cofilin into control, Memo or PLCγ1 siRNA transfected breast tumor cells, we showed that HRG-induced recruitment of GFP-cofilin to lamellipodia is impaired in Memo- and in PLCγ1 KD cells, suggesting that both proteins lie upstream of cofilin in models of ErbB2-driven tumor cell migration. Finally, we examined the effect of Memo on cofilin binding and severing/depolymerizing properties. In vitro F-actin binding assays showed that Memo does not impair cofilin binding to F-actin, and revealed that Memo is a novel F-actin binding protein. In vitro F-actin depolymerization assays indicated that Memo promotes cofilin depolymerizing/severing activity. Altogether, these data suggest a novel role for Memo during the migratory process and its implication in the regulation of actin dynamics through cofilin binding. In the second study, we used two different Memo-defective cellular models to examine Memo’s function in more detail. We demonstrated that inhibition of Memo impairs activation of a number of signaling molecules including Src, Shc, ERK and PLCγ1. We also provide evidence that Memo interacts with the three Shc isoforms, p46shc, p52shc, and p66shc, and showed that Shc is required for Memo binding to the ErbB2 receptor. Control and Memodeficient cells were also scored for their migration and adhesion properties. These assays indicated that Memo is important in both cell migration and adhesion processes. Also, morphological and biochemical analyses of control and Memo-deficient cells suggested that Memo is involved in focal adhesion organization and rear cell deadhesion during the migratory process. Altogether, these two studies revealed important roles for Memo at different steps of cell migration and metastasis, making it a potential interesting target for cancer therapy. Genetic approaches in model organisms have been important for gaining insight into the function of evolutionarily conserved proteins. To position Memo within a genetic network, experiments in the model organism S. cerevisae that lends itself to rapid genetic screening were performed. We investigated cellular localization of Memo in yeast and found that Memo is located in the nucleus and cytoplasm of the cell. A S. cerevisae memo Δ strain has been generated and is viable. Considering the role of Memo in the microtubule and actin networks that we described in mammalian cells, we examined the memo Δ strain for defects in different cytoskeletal dynamics. No significant effect was observed. We also performed a Synthetic Lethal Screen of genetic interactions between a memo Δ strain and an ordered array of 4700 Yeast strains containing non-essential gene deletions. This analysis revealed a limited number of synthetic interactions. Lethality was observed in combination with the plc1Δ strain. PLC1 encodes for the unique isoform of phosphatidylinositol-specific phospholipase C of S. cerevisiae. The results are intriguing and exciting considering the data obtained in the mammalian models; in fact, we demonstrated that Memo and PLCγ1 interact with ErbB2 autophosphorylation sites and are essential for directional migration. We also showed that Memo is found in a complex with PLCγ1 and ErbB2 and that Memo is likely contributing to PLCγ1 activation. We hypothesize that in Yeast, Memo and PLC1 act in the same or in distinct but related pathways, and suggest that the connection between PLC and Memo induced-pathways is also conserved through evolution

“Do We Know Jack” About JAK? A Closer Look at JAK/STAT Signaling Pathway

Janus tyrosine kinase (JAK) family of proteins have been identified as crucial proteins in signal transduction initiated by a wide range of membrane receptors. Among the proteins in this family JAK2 has been associated with important downstream proteins, including signal transducers and activators of transcription (STATs), which in turn regulate the expression of a variety of proteins involved in induction or prevention of apoptosis. Therefore, the JAK/STAT signaling axis plays a major role in the proliferation and survival of different cancer cells, and may even be involved in resistance mechanisms against molecularly targeted drugs. Despite extensive research focused on the protein structure and mechanisms of activation of JAKs, and signal transduction through these proteins, their importance in cancer initiation and progression seem to be underestimated. This manuscript is an attempt to highlight the role of JAK proteins in cancer biology, the most recent developments in targeting JAKs, and the central role they play in intracellular cross-talks with other signaling cascades

ROLE OF NOTCH AND JAK/STAT SIGNALING PATHWAYS IN ACUTE MEGAKARYOBLASTIC LEUKEMIA

Ph.DPHD IN CANCER BIOLOG

Delineating the Steps of BAX Pore Activation

The BCL2 protein family is the primary gatekeeper of mitochondrial apoptosis and governs integrity of the organelles\u27 outer membranes. Permeabilization of mitochondrial outer membranes permits egress of cytochrome c and other apoptogenic factors, resulting in apoptosome formation, caspase activation, and subsequent proteolytic demolition of cells. Proapoptotic BAX & BAK effect the release of cytochrome c while their antiapoptotic counterparts like BCL-2, BCL-XL, & MCL-1 oppose this permeabilization. A third class of the BCL2 family, the prodeath BH3-only proteins, act as sentinels of cell stress and exert their influences by occupying antiapoptotic BCL2 members and/or activating BAX/BAK. Cell-free reconstitution assays have revealed that BAX/BAK undergo significant conformational changes to oligomerize and form pores in membranes. Previously unresolved was the basis for the outer cell membranes\u27 escape from BAX poration during apoptosis. Unlike outer cell membranes, which are roughly 40% cholesterol, mitochondrial outer membranes are only 5-10% cholesterol. Vesicle leakage assays demonstrated that BAX pore activation is severely inhibited by the sterol. Inclusion of the total enantiomer of cholesterol in our assays uncovered that this BAX functional suppression was due to bilayer structure alteration rather than a stereospecific protein-cholesterol interaction. Real-time observation of BAX-vesicle binding showed that cholesterol curbs membrane integration by the protein, thus suppressing oligomerization and pore formation. Oxysterols and bile acids are physiological derivatives of cholesterol. Further employment of our vesicle leakage regime revealed that 25-hydroxycholesterol at low micromolar concentrations accelerates BAX pore formation, suggesting a compensatory mechanism for BAX inhibition by cholesterol. Bile acids lithocholic and chenodeoxycholic acids are toxic and induce apoptosis at high concentrations, thus we reasoned that the physiological detergents may directly activate BAX. While truncated BAX: ΔC) was effectively activated by monomeric detergent, the full-length protein required micellar bile acids, implying that bile acids could play at most only an amplification role in BAX-mediated apoptosis. In nonstressed cells, BAX exists as a soluble, cytosolic monomer or loosely affiliated with mitochondria while cellular recognition of death signals induces BAX transition to a membrane-integral state. The physical basis of this translocation and locale of BAX activation are poorly characterized. Assemblage of a cell-free scheme comprising full-length BAX, antiapoptotic BCL-XL, and BH3-only activators cBID & BIMS, and synthetic vesicles revealed that BIMS more effectively activates BAX and is less suppressible by BCL-XL. Each of these four proteins can independently adsorb to membranes and that BCL-XL outraces BAX to their in-membrane functional sites. Membrane-bound cBID and BIMS robustly accelerate the bilayer integrations of BAX & BCL-XL; the two activators recruit equivalently at the membrane surface, however, suggesting that BIMS, unlike cBID, can activate BAX prior to interaction on a bilayer scaffold

Spatial Stochastic Modeling of the ErbB Receptor Family

ErbB transmembrane receptors are a family of 4 receptor tyrosine kinases that interact with one another through homo and heterodimer interactions. When these dimers form, the kinase domains on the receptor tails interact with one another, transphosphorylating one another, initiating a signal cascade. The signaling pathways these receptors participate in are responsible for many different cell functions including apoptosis, growth, and proliferation. The overexpression of these receptors has been linked to various forms of cancer, emphasizing the importance of understanding how these receptors interact with one another to trigger these cascades. Single Particle Tracking experiments have provided more precise and detailed measures of dimer lifetimes and diffusion. A major observation from the experiments is the anomalous diffusion of the receptors. One suggested contributor to this anomalous diffusion is confinement zones on the membrane. In this work, we develop, validate, and implement a spatial stochastic model to study these receptors and uncover how their kinetics and dynamics as well as the membrane landscape come together to impact erbB activation. We start by focusing on erbB1. Single particle tracking experiments show that receptor pairs interact repeatedly over a period of time. One possible explanation for these repeated interactions is to facilitate phosphorylation. An asymmetric phosphorylation model is proposed, where one receptor in the dimer pair is responsible for activating the other receptor, the receiver, which then in turn phosphorylates the original activator. The model shows that the confinement zones on the membrane play a critical role in causing repeated receptor interactions and reveals that receptors dynamically switch between different activation states over time. Our work continues by delving deeper into the membrane landscape. Single particle tracking data is analyzed to investigate the characteristics of the observed anomalous diffusion. The analysis gives an estimate for the size range of the confinement zones and shows that they are a series of domains, not corrals. Taking the single particle tracking analysis one step further, we develop a Domain Reconstruction Algorithm that reconstructs confinement zone shapes and sizes from single particle tracking trajectories. In the final study, we move on to erbB2 and erbB3 interactions. ErbB3, which is traditionally believed to be kinase dead, has recently been shown to have weak kinase activity. Through kinase assay experiments, we show in the presence of erbB2 and heregulin, erbB3 has measurable kinase activity. Using the reconstructed domains from erbB2 and erbB3 data to create a simulation space, and experimental data from the kinase assay and single particle tracking, we extend the erbB1 spatial stochastic model for this study. We show that erbB2 and erbB3 have significantly different interactions with the cellular membrane confinement zones, erbB3 is dependent on erbB2 activation, and erbB3 homodimer stability inhibits erbB3 activation. Extension of the model to investigate mutation behaviors in erbB3 receptors reveals insights into how a gain of function mutation in the erbB3 kinase domain impacts erbB2 and erbB3 interactions. Finally, discovery of a gain of function mutation in the kinase domain of erbB3 is connected to an uptick in erbB3 kinase activity. As a path forward from this work, we suggest using the spatial stochastic model to investigate more possible mutations in erbB3 receptors to give better insight into which mutations would be promising to explore

THE SMALL GTPASE RAC1 IS A NOVEL BINDING PARTNER OF BCL-2 AND STABILIZES ITS ANTI-APOPTOTIC ACTIVITY

Ph.DDOCTOR OF PHILOSOPH