Theses and Dissertations from UMD
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Item THE POTENTIAL DICHOTOMOUS ROLE OF ACTIVATING TRANSCRIPTION FACTOR 3 (ATF3) IN COLON CANCER(2015) Jiang, Xiaojing; Lee, Seong-Ho; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Colorectal cancer (CRC) is the third leading cause of cancer-related death in the United States. During the tumorigenesis and metastasis of CRC, cells encounter numerous cellular and molecular events. ATF3, a member of the ATF/CREB transcription factor family, plays an important role on regulation of apoptosis and is regarded as a potential molecular target for chemoprevention and chemotherapy of colon cancer. The current study was performed to investigate cellular and molecular mechanisms by which ATF3 affects colon cancer-related phenotypes including apoptosis and metastasis. Here, we demonstrated that knockdown of ATF3 using small interfering RNA (siRNA) promotes the expression of anti-apoptotic protein, B-cell lymphoma 2 (Bcl-2), in colon cancer cells, while overexpression of ATF3 resulted in a dramatic decrease in Bcl-2 protein. Gain of function of ATF3 in colon cancer cell line HCT116 led to an increase of pro-apoptotic protein Bcl-2 homologous antagonist killer (Bak), followed by the induction of apoptosis. Furthermore, we observed that ATF3 overexpression downregulated expression of epithelial-mesenchymal transition (EMT)-related transcription factors. However, mammosphere forming assay indicated that ATF3 overexpressed colon cancer cells form larger and more budding sites compared to control, which is associated with an increase of cluster of differentiation 44 (CD44) expression and a decrease of retinoblastoma (Rb) and tight junction protein zonula occludens (ZO)-1. This study suggested that ATF3 may play a dichotomous role in regulation of apoptosis and metastasis in colon cancer.Item BIOPHYSICAL STUDIES OF THE MECHANISM OF CERAMIDE CHANNEL DESTABLIZATION BY BCL-XL IN APOPTOSIS AND THE USE OF RECTIFICATION TO PROBE THE STRUCTURE AND DYNAMICS OF A NOVEL ESCHERICHIA COLI CHANNEL(2015) Chang, Kai-Ti; Colombini, Marco; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ceramide forms a novel type of channel in the mitochondrial outer membrane and these channels are involved the release of intermembrane space proteins from mitochondria, a decision-making step in the apoptotic process. An antiapoptotic protein, Bcl-xL, regulates the apoptotic process and inhibits the formation of ceramide channels. However, there is no precedent to indicate how a protein regulates a lipid channel. We investigated the mechanism of this regulation and identified the hydrophobic groove of the Bcl-xL as the binding site by which Bcl-xL binds to the channel. This was demonstrated by using a combination of experimental and modeling methods, including site-directed mutagenesis, a fluorescence quenching assay, a mitochondrial outer membrane permeability assay, and molecular dynamic simulations. Interestingly, the hydrophobic groove serves to inhibit another channel former, Bax. We found that the binding sites for Bax and ceramide on Bcl-xL are distinct but overlapping. We used that fact to generate mutants that have differential abilities to inhibit one or the other of these channels. These are useful because although ceramide is important in apoptosis, it is still controversial that whether ceramide channels result in apoptosis in vivo. To probe the relative importance of these two channels in apoptosis, Bcl-xL mutant proteins were expressed in Bcl-xL deficient cells. Weakening the inhibitory potency of Bcl-xL on either Bax or ceramide channels resulted in cells being more sensitive to the induction of apoptosis. This is the first evidence for the role of ceramide channels in the apoptotic process in vivo. In a separate investigation, a novel voltage-gated channel unit was found in E. coli extracts. The unit is consistent with three channels forming the functional triplet. These channels are highly voltage gated and highly cooperative. Those results indicated that one of the channels is oriented in an antiparallel fashion compared to the rest. This arrangement is very rare in protein channels. Rectification of the current flowing through the channels was used to identify the orientation of the channels to provide evidence for or against the antiparallel hypothesis. The results favor the antiparallel hypothesis but also reveal an unexpected asymmetry in the transmembrane electrostatics.Item Ceramide Channels and the Induction of Apoptosis: Structural Insights on Channel Formation and Regulation by Bcl-2 Family Proteins(2012) Perera, Meenu Nadisha; Colombini, Marco; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A critical event in apoptosis is the release of intermembrane space proteins from mitochondria following mitochondrial outer membrane (MOM) permeabilization (MOMP). The Bcl-2 family of proteins regulates MOM integrity and includes pro- and anti-apoptotic members, like Bax and Bcl-xL respectively. Preceding MOMP, the MOM becomes enriched with the sphingolipid, ceramide, which can self-assemble to form ceramide channels, contributing to MOMP. Bax and ceramide channels were found to act synergistically in the generation of MOMP and a direct interaction between these was observed in phospholipid membranes. The apparent affinity of activated Bax for ceramide channels increases with ceramide channel size, consistent with an induced fit mechanism; Bax drives the enlargement of ceramide channels to an optimum fit for the Bax binding site. A ceramide channel specific inhibitor prevented the enhanced MOMP in the presence of Bax and ceramide indicating ceramide channels were the primary permeabilizing entity. Analogs with changes to all the major structural features of ceramide were used to assess the molecular basis of stability of ceramide channels. Methylation of the C1- hydroxy group abrogated channel formation in mitochondria. Methylation of the amide nitrogen or a change in chirality at C2, which influences the C1-hydroxy group orientation, greatly reduced channel-forming ability whereas other changes were well tolerated. Competition experiments between ceramide and analogs resulted in synergism or antagonism, depending on compatibility of the analog structure with the ceramide channel model. The results provide evidence for ceramide channels being highly organized structures, stabilized by specific inter-molecular interactions. Analogs that retained channel-forming ability were used to assess the structural features of ceramide channels required for regulation by Bcl-2 family proteins. The stereochemistry of the ceramide head group and access to the amide nitrogen is indispensible for regulation by Bax, implicating the polar portion of the channel as the Bax binding site. Bcl-xL's ability to disassemble ceramide channels depends on the length of the hydrophobic chains of ceramide. Specific Bcl-xL inhibitors reveal that BclxL binds ceramide channels through its hydrophobic groove and this is supported by simulated docking. The opposite effects of pro-and anti-apoptotic proteins are achieved at different sites on the ceramide channel.Item Characterization of the immune response induced by non-pathogenic mycobacteria in macrophages and dendritic cells(2012) Bohsali, Amro Youssef; Briken, Volker; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The capacity of the host to mount an effective immune response (IR) is crucial for the protection against invading pathogens. The apoptotic and proinflammatory responses of infected cells are important innate immune mechanisms. Consequently, the ability of persistent intracellular pathogens, such as the human pathogen Mycobacterium tuberculosis (Mtb), to inhibit infection-induced apoptosis of macrophages is important for its virulence in the host. Facultative-pathogenic mycobacterial species, like M. kansasii (Mkan), can cause disseminating disease in individuals with immune deficiencies. In contrast, non-pathogenic mycobacteria, like M. smegmatis (Msme), are not known to cause disseminating disease even in immunocompromised individuals. We hypothesized that this difference in phenotype could be explained by the strong induction of an innate IR by non-pathogenic mycobacteria. Here we analyze the mechanisms by which non-pathogenic mycobacteria induce a strong IR in their macrophage and dendritic cell (DC) host, specifically the induction of host cell apoptosis and the host inflammasome response via the secretion of IL-1β. The comparison of two non-pathogenic mycobacterial species (Msme and Mkan) with two facultative-pathogenic mycobacterial species (M.kan and M. bovis BCG) demonstrated that only the non-pathogenic mycobacteria induce strong apoptosis in murine bone marrow derived macrophages (BMDM) and dendritic cells (BMDC), which was dependent upon caspase-3 activation and TNF secretion. Consistently, BMDMs responded by secreting relatively large amounts of TNF and by upregulating the expression of Il-12. We also demonstrated that Msme infection of BMDCs strongly induces the secretion of IL-1β. This induction was dependent upon the presence of functional ASC and was partially independent of NLRP3. Interestingly this induction was also partially dependent on AIM2 and IFN-β. This AIM2-dpendent induction was observed in infection with non-pathogenic and opportunistic mycobacteria, and attenuated but not virulent Mtb. Surprisingly, caspase-1/11 deficient BMDCs still secreted substantial but reduced amounts of IL-1β upon Msme infection. In conclusion, we demonstrate a strong induction of the innate IR by non-pathogenic mycobacteria as measured by host cell apoptosis, and IL-12 / TNF / IL-1β cytokine induction. We also demonstrate the partially caspase-1/11 and NLRP3 -independent, partially AIM2-dependent, but ASC-dependent IL-1β secretion in Msme infected BMDCs. Our findings support the hypothesis that the strong induction of the innate IR is a major reason for the lack of pathogenicity in non-pathogenic mycobacteria.Item VISUALIZATION OF CERAMIDE CHANNELS BY TRANSMISSION ELECTRON MICROSCOPY(2011) SAMANTA, SOUMYA; COLOMBINI, MARCO; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Functional studies have shown that the sphingolipid ceramide, self-assembles in phospholipid membranes to form large channels capable of allowing proteins to cross the membrane. Here these channels are visualized by negative stain transmission electron microscopy. The images contain features consistent with stain-filled pores having a roughly circular profile. There is no indication of tilt, and the results are consistent with the formation of right cylinders. The sizes of the pores range from 5 to 40 nm in diameter with an asymmetric distribution indicating no apparent upper size limit. The size distribution matches well with the distribution of sizes calculated from electrophysiological measurements.Item FUNCTIONAL CHARACTERIZATION OF THE VIRAL FLICE INHIBITORY PROTEIN OF RHESUS MONKEY RHADINOVIRUS(2011) Ritthipichai, Krit; Zhang, Yanjin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus closely related to Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV is associated with several malignant diseases, including Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. Here we found that RRV viral FLICE inhibitory protein (vFLIP) inhibits apoptosis. In HeLa cells with vFLIP expression, cleavage of poly [ADP-ribose] polymerase 1 (PARP-1) and activities of caspase 3, 7, and 9 were much lower than controls. RRV vFLIP was able to enhance cell survival under starved condition or apoptosis induction. After apoptosis induction, autophagosome formation was enhanced in cells with vFLIP expression and when autophagy was inhibited, these cells underwent apoptosis. Moreover, RRV latent infection of BJAB B-lymphoblastoid cells protects the cells against apoptosis. Knockdown of vFLIP expression in the RRV-infected BJAB cells with siRNA abolished the protection against apoptosis. These findings indicate that RRV vFLIP protects cells against apoptosis by enhancing autophagosome formation.Item Molecular Mechanisms of the Inhibition of Apoptosis by Mycobacterium tuberculosis(2009) Miller, Jessica Lynn; Briken, Volker; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The capacity of infected cells to undergo apoptosis upon insult with a pathogen is an ancient innate immune defense mechanism. Consequently, the ability of persistent intracellular pathogens, such as the human pathogen Mycobacterium tuberculosis (Mtb), to inhibit infection-induced apoptosis of macrophages is important for virulence and to achieve persistence in the host. The nuoG gene of Mtb, which encodes the NuoG subunit of the type I NADH dehydrogenase NDH-1, is important in Mtb-mediated inhibition of host macrophage apoptosis. Here I determine the molecular mechanisms of this host-pathogen interaction. Apoptosis induced by the nuoG deletion mutant (nuoG ) is caspase-8 and TNF-α dependent. This cell death was also reduced in the presence of neutralizers and inhibitors of reactive oxygen species (ROS) and in macrophages derived from NOX2 deficient mice, suggesting that DnuoG induced death is dependent upon NOX2 derived ROS. Correlatively, nuoG infected macrophages also produced more phagosomal ROS than those infected with Mtb, or cells derived from NOX2 deficient mice. NuoG also inhibited apoptosis in human alveolar macrophages in a NOX2 dependant manner. These data suggest that reduction of phagosomal ROS is important for inhibition of apoptosis. Consistent with this hypothesis, Mtb deficient in the ROS neutralizing catalase, KatG, also accumulated ROS in the phagosome and was pro-apoptotic in macrophages. The specific mechanism by which NuoG reduces phagosomal ROS is still unknown. We could not detect secretion of NuoG, so direct neutralization of ROS is unlikely. Interestingly, preliminary data suggests that nuoG may be defective in secretion of SodA and KatG, enzymes known to be important for neutralizing ROS. In conclusion, these studies revealed that Mtb inhibits macrophage apoptosis by neutralizing phagosomal ROS via the NuoG dependent secretion of SodA and KatG. Furthermore, this research suggests a novel function for NOX2 activity in innate immunity, which is the sensing of persisting intracellular pathogens and subsequent induction of host cell apoptosis as a second line of defense for pathogens resistant to the respiratory burst.Item Developmental Programmed Cell Death In The Midline Glia Cells Of Drosophila Embryo(2004-05-14) Jaligam, Vanaja; Baehrecke, Dr. Eric H; Cell Biology & Molecular GeneticsApoptosis is conserved in worms, flies and mammals. My goal was to identify new Drosophila midline glia cell death genes. Caspase substrate Nuclear Lamin and the midline glia specific reporter slit-lacZ were in Drosophila embryonic development cell death studies. Midline glia cell death is prevented in H99 mutants that delete hid, reaper and grim, and in p35 flies that over-express the pan-caspase inhibtor. Homozygous deficiency Df(3R)E79 flies possess a defect in midline glia cell death. Df(3R)E79 midline glia appear to be phagocytosed but do not undergo Lamin degradation suggesting that a caspase regulator resides in this deleted region of the genome. Df(3R)E79 removes 112 genes, including several interesting candidates including mus309, a protein with similarity to a RING finger protease reported to regulate apoptosis in humans. Future studies will determine the nature of the cell death regulator in Df(3R)E79 and how this gene functions in apoptosis.