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.

Mechanism of silica -induced apoptosis in human alveolar macrophages

The mechanisms involved in the development of pulmonary silicosis have not been well defined, however most current evidence implicates a central role for alveolar macrophages in this process. We propose that the fibrotic potential of a particulate depends upon its ability to cause apoptosis in alveolar macrophage (AM). The overall goal of this study was to determine the mechanism of silica-induced apoptosis of AM. Human AM were treated with fibrogenic, poorly fibrogenic and nonfibrogenic model particulates, such as, silica, amorphous silica and titanium dioxide, respectively (equal surface area). Treatment with silica resulted in apoptosis in human AM as observed by morphology, DNA fragmentation and Cell Death ELISA assays. In contrast, amorphous silica and titanium dioxide demonstrated no significant apoptotic potential. To elucidate the possible mechanism by which silica causes apoptosis, we investigated the role of the scavenger receptor (SR) in silica-induced apoptosis. Cells were pretreated with and without SR ligand binding inhibitors, polyinosinic acid (Poly I), fucoidan and high density lipoprotein (HDL), prior to silica treatment. Pretreatment with Poly I and fucoidan resulted in significant inhibition of silica-induced apoptosis suggesting that silica-induced AM apoptosis is mediated via the SR. Further, we examined the involvement of interleukin converting enzyme (ICE) family of proteases in silica-mediated apoptosis. Silica activated ICE, Ich-1L, cpp32 beta and cleavage of PARP. Taken together, these results suggested that (1) fibrogenic particulates, such as, silica caused apoptosis of alveolar macrophages, (2) this apoptotic potential of fibrogenic particulates may be a critical factor in initiating an inflammatory response resulting in fibrosis, (3) silica-induced apoptosis of alveolar macrophages may be due to the interaction of silica particulates with the SR, and (4) silica-induced apoptosis involves the activation of the ICE family of proteases. An understanding of the molecular events involved in fibrogenic particulate-induced apoptosis may provide a useful insight into the mechanism involved in particulate-induced fibrosis. ^

Endogenous nitric oxide regulates p53 signaling and DNA damage-induced apoptosis in melanoma cells

Nitric oxide is involved in a multitude of processes including regulation of vascular tone, neurotransmission, immunity, and cancer. Evidence suggests that nitric oxide exhibits anti-apoptotic activity in melanoma cells. Our laboratory showed that tumor expression of inducible nitric oxide synthase correlated strongly with poor survival in stage III and IV melanoma patients, suggesting an antagonistic role for nitric oxide in melanoma response to therapy. Therefore, the hypothesis that endogenously produced nitric oxide antagonizes chemotherapy-induced apoptosis was formed. Using cisplatin as a model for DNA damage in melanoma cell lines, the capacity of nitric oxide to regulate cell growth and apoptotic responses to cisplatin treatment was examined. The depletion of endogenously generated nitric oxide resulted in changes in cell cycle regulation and enhanced cisplatin-induced apoptosis in melanoma cells. Since nitric oxide was shown to be involved in the regulation of p53 stability, conformation and DNA binding activity, whether signaling through wild-type p53 in melanoma cells is regulated by nitric oxide was tested. Cisplatin-induced p53 accumulation and p21Waf1/Cip1/Sdi1 expression in nitric oxide-depleted melanoma cells were found to be strongly suppressed. When p53 binding to the p21Waf1/Cip1/Sdi1 promoter was examined, it was found that nitric oxide depletion significantly reduced the cisplatin-induced formation of p53-DNA complexes. These results suggest that nitric oxide is required for activation of wild-type p53 after DNA damage in melanoma cells. Finally, whether signaling through p53 controls melanoma response to DNA damage was examined. Transfection of a plasmid containing a dominant negative form of mutated p53 inhibited p21 Waf1/Cip1/Sdi1 expression and concomitantly enhanced apoptosis after cisplatin treatment. These data suggest that the induction of wild-type p53 protects melanoma cells against DNA damage via the up-regulation of p21 Waf1/Cip1/Sdi1. Together, these data strongly support the model that endogenous nitric oxide is required for p53 activation and p21Waf1/Cip1/Sdi1 expression after DNA damage, which can enhance melanoma resistance to therapy. Thus, in context of melanoma cells with wild-type p53 , low levels of endogenous constitutively-produced nitric oxide appear to facilitate the activation of p53 in response to DNA damage, thereby allowing for cell cycle arrest via p21Waf1/Cip1/Sdi1 induction, adequate DNA repair, and ultimately enhanced resistance to apoptosis. ^