Studies on the {\it in vivo} function of a phosphatidylinositol transfer protein from {\it Saccharomyces cerevisiae}

Phosphatidylinositol transfer proteins (PI-TP's) catalyze the transfer of phosphatidylinositol and phosphatidylcholine between membranes in vitro. However the in vivo function of these proteins is unknown. In this thesis we have used a combined biochemical and genetic approach to determine the importance of PI-TP in vivo. An oligonucleotide based on the amino terminal sequence of the PI-TP from Saccharomyces cerevisiae, was used to screen a yeast genomic library for the gene encoding PI-TP (PIT1 gene). Yeast strains transformed with the positive clones showed overproduction of transfer activities and transfer protein in the 100,000 x g supernatants. The 5$\sp\prime$ terminus of the PIT1 gene correlates with the predicted codons for residues 3-30 of the determined protein sequence. Tetrad analysis of a heterozygous diploid (PIT1/pit1::LEU2) revealed that the PIT1 gene is essential for cell growth. Non-viable spores could be rescued by transformation of the above diploid prior to sporulation, with a plasmid borne copy of the wild type gene. Sequencing of the entire PIT1 gene has revealed that the PIT1 gene is identical to the SEC14 gene. The sec14 ts mutant which exhibits conditional defects at the Golgi stage of protein secretion, is also temperature sensitive for PI-TP activity in vitro. These findings represent the first instance in which a physiological function has been assigned to any phospholipid transfer protein. ^

Regulation of vascular endothelial growth factor expression in human pancreatic cancer

Pancreatic adenocarcinoma is currently the fifth-leading cause of cancer-related death in the United States. Like with other solid tumors, the growth and metastasis of pancreatic adenocarcinoma are dependent on angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic molecule that plays an important role in angiogenesis, growth and metastasis of many types of human cancer, including pancreatic adenocarcinoma. However, the expression and regulation of VEGF in human pancreatic cancer cells are mostly unknown. ^ To examine the hypothesis that VEGF is constitutively expressed in human pancreatic cancer cells, and can be further induced by tumor environment factors such as nitric oxide, a panel of human pancreatic cancer cell lines were studied for constitutive and inducible VEGF expression. All the cell lines examined were shown to constitutively express various levels of VEGF. To identify the mechanisms responsible for the elevated expression of VEGF, its rates of turnover and transcription were then investigated. While the half-live of VEGF was unaffected, higher transcription rates and increased VEGF promoter activity were observed in tumor cells that constitutively expressed elevated levels of VEGF. Detailed VEGF promoter analyses revealed that the region from −267 to +50, which contains five putative Sp1 binding sites, was responsible for this VEGF promoter activity. Further deletion and point mutation analyses indicated that deletion of any of the four proximal Sp1 binding sites significantly diminished VEGF promoter activity and when all four binding sites were mutated, it was completely abrogated. Consistent with these observations, high levels of constitutive Sp1 expression and DNA binding activities were detected in pancreatic cancer cells expressing high levels of VEGF. Collectively, our data indicates that constitutively expressed Sp1 leads to the constitutive expression of VEGF, and implicates that both molecules involve in the aggressive pathogenesis of human pancreatic cancer. ^ Although constitutively expressed in pancreatic cancer cells, VEGF can be further induced. In human pancreatic cancer specimens, we found that in addition to VEGF, both inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were overexpressed, suggesting that nitric oxide might upregulate VEGF expression. Indeed, a nitric oxide donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) significantly induced VEGF mRNA expression and protein secretion in pancreatic adenocarcinoma cells in a time- and dose-dependant manner. Using a luciferase reporter containing both the VEGF promoter and the 3′ -UTR, we showed that SNAP significantly increased luciferase activity in human pancreatic cancer cells. Notwithstanding its ability to induce VEGF in vitro, pancreatic cancer cells genetically engineered to produce NO did not exhibit increased tumor growth. This inability of NO to promote tumor growth appears to be related to NO-mediated cytotoxicity. The balance between NO mediated effects on pro-angiogenesis and cytotoxicity would determine the biological outcome of NO action on tumor cells. ^ In summary, we have demonstrated that VEGF is constitutively expressed in human pancreatic cancer cells, and that overexpression of transcription factor Sp1 is primarily responsible. Although constitutively expressed in these cells, VEGF can be further induced by NO. However, using a mouse model, we have shown that NO inhibited tumor growth by promoting cytotoxicity. These studies suggest that both Sp1 and NO may be important targets for designing potentially effective therapies of human pancreatic cancer and warrant further investigation. ^

AGROBACTERIUM VIRB10 CONTRIBUTIONS TO TYPE IV SUBSTRATE SECRETION, T-PILUS BIOGENESIS, AND OUTER MEMBRANE PORE FORMATION

The VirB/D4 type IV secretion system (T4SS) of Agrobacterium tumefaciens functions to transfer substrates to infected plant cells through assembly of a translocation channel and a surface structure termed a T-pilus. This thesis is focused on identifying contributions of VirB10 to substrate transfer and T-pilus formation through a mutational analysis. VirB10 is a bitopic protein with several domains, including a: (i) cytoplasmic N-terminus, (ii) single transmembrane (TM) α-helix, (iii) proline-rich region (PRR), and (iv) large C-terminal modified β-barrel. I introduced cysteine insertion and substitution mutations throughout the length of VirB10 in order to: (i) test a predicted transmembrane topology, (ii) identify residues/domains contributing to VirB10 stability, oligomerization, and function, and (iii) monitor structural changes accompanying energy activation or substrate translocation. These studies were aided by recent structural resolution of a periplasmic domain of a VirB10 homolog and a ‘core’ complex composed of homologs of VirB10 and two outer membrane associated subunits, VirB7 and VirB9. By use of the substituted cysteine accessibility method (SCAM), I confirmed the bitopic topology of VirB10. Through phenotypic studies of Ala-Cys insertion mutations, I identified “uncoupling” mutations in the TM and β-barrel domains that blocked T-pilus assembly but permitted substrate transfer. I showed that cysteine replacements in the C-terminal periplasmic domain yielded a variety of phenotypes in relation to protein accumulation, oligomerization, substrate transfer, and T-pilus formation. By SCAM, I also gained further evidence that VirB10 adopts different structural states during machine biogenesis. Finally, I showed that VirB10 supports substrate transfer even when its TM domain is extensively mutagenized or substituted with heterologous TM domains. By contrast, specific residues most probably involved in oligomerization of the TM domain are required for biogenesis of the T-pilus.

NMR structure of a complex between the VirB9/VirB7 interaction domains of the pKM101 type IV secretion system.

Type IV secretion (T4S) systems translocate DNA and protein effectors through the double membrane of Gram-negative bacteria. The paradigmatic T4S system in Agrobacterium tumefaciens is assembled from 11 VirB subunits and VirD4. Two subunits, VirB9 and VirB7, form an important stabilizing complex in the outer membrane. We describe here the NMR structure of a complex between the C-terminal domain of the VirB9 homolog TraO (TraO(CT)), bound to VirB7-like TraN from plasmid pKM101. TraO(CT) forms a beta-sandwich around which TraN winds. Structure-based mutations in VirB7 and VirB9 of A. tumefaciens show that the heterodimer interface is conserved. Opposite this interface, the TraO structure shows a protruding three-stranded beta-appendage, and here, we supply evidence that the corresponding region of VirB9 of A. tumefaciens inserts in the membrane and protrudes extracellularly. This complex structure elucidates the molecular basis for the interaction between two essential components of a T4S system.

Biological diversity of prokaryotic type IV secretion systems.

Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.

Biological diversity of prokaryotic type IV secretion systems.

Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.

The contribution of A3 adenosine receptor signaling to pulmonary inflammation and mucus secretion in the mouse

Adenosine has been implicated in chronic lung diseases such as asthma and COPD. Most physiological actions of adenosine are mediated through G-protein coupled adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B, and A 3. However, the specific roles of the various adenosine receptors in processes central to asthma and COPD are not well understood in part due to the lack of adequate animal models that examine the effect of adenosine on the development of lung disease. In this study we have investigated the expression and function of the A3 adenosine receptor in pulmonary eosinophilia and mucus production/secretion in adenosine deaminase (ADA)-deficient mice in which adenosine levels are elevated. ADA-deficient mice develop features of asthma and COPD, including lung eosinophilia and mucus hyperplasia in association with elevated lung adenosine levels. The A3 receptor was found to be expressed in eosinophils and mucus producing cells in the airways of ADA-deficient. Disruption of A3 receptor signaling in ADA-deficient mice by genetic removal of the receptor or treatment with MRS 1523, a selective A3 adenosine receptor antagonist, prevented airway eosinophilia and mucus production. Although eosinophils were decreased in the airways of ADA-deficient mice with disrupted A3 receptor signaling, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3 receptor is needed for the migration of eosinophils into the airways. Further examination of the role of the A3 receptor in mucus biology demonstrated that the A3 receptor is neither required nor is overexpression of the receptor in clara cells sufficient for mucus production in naive mice. Transgenic overexpression of the A3 receptor did elucidate a role for the A3 receptor in the secretion of mucus into the airways of ovalbumin challenged mice. These findings identify an important role for the A3 adenosine receptor in regulating lung eosinophilia and mucus secretion in inflammatory lung diseases. Therefore, the A3 adenosine receptor may represent a novel therapeutic target for the treatment and prevention of asthma. ^

Analysis of Agrobacterium tumefaciens VirB6 and VirB9 of the VirB /D4 type IV secretion system

Agrobacterium tumefaciens uses the VirB/D4 type IV secretion system (T4SS) to translocate oncogenic DNA (T-DNA) and protein substrates to plant cells. Independent of VirD4, the eleven VirB proteins are also essential for elaboration of a conjugative pilus termed the T pilus. The focus of this thesis is the characterization and analysis of two VirB proteins, VirB6 and VirB9, with respect to substrate translocation and T pilus biogenesis. Observed stabilizing effects of VirB6 on other VirB subunits and results of protein-protein interaction studies suggest that VirB6 mediates assembly of the secretion machine and T pilus through interactions with VirB7 and VirB9. Topology studies support a model for VirB6 as a polytopic membrane protein with a periplasmic N terminus, a large internal periplasmic loop, five transmembrane segments, and a cytoplasmic C terminus. Topology studies and Transfer DNA immunoprecipitation (TrIP) assays identified several important VirB6 functional domains: (i) the large internal periplasmic loop mediates interaction of VirB6 with the T-DNA, (ii) the membrane spanning region carboxyl-terminal to the large periplasmic loop mediates substrate transfer from VirB6 to VirB8, and (iii) the terminal regions of VirB6 are required for substrate transfer to VirB2 and VirB9. To analyze structure-function relationships of VirB9, the phenotypic consequences of dipeptide insertion mutations were characterized. Substrate discriminating mutations were shown to selectively export the oncogenic T-DNA and VirE2 to plant cells or a mobilizable IncQ plasmid to bacterial cells. Mutations affecting VirB9 interactions with VirB7 and VirB10 were localized to the C- and N- terminal regions respectively. Additionally, “uncoupling” mutations identified in VirB11 and VirB6 that block T pilus assembly, but not substrate transfer to recipient cells, were also identified in VirB9. These results in conjunction with computer analysis establish that VirB9, like VirB6, is also composed of distinct regions or domains that contribute in various ways to secretion channel activity and T pilus assembly. Lastly, in vivo immunofluorescent studies suggest that VirB9 localizes to the outer membrane and may play a role similar to that of secretion/ushers of types II and III secretion systems to facilitate substrate translocation across this final bacterial barrier. ^