Sec12 Binds to Sec16 at Transitional ER Sites

COPII vesicles bud from an ER domain known as the transitional ER (tER). Assembly of the COPII coat is initiated by the transmembrane guanine nucleotide exchange factor Sec12. In the budding yeast Pichia pastoris, Sec12 is concentrated at tER sites. Previously, we found that the tER localization of P. pastoris Sec12 requires a saturable binding partner. We now show that this binding partner is Sec16, a peripheral membrane protein that functions in ER export and tER organization. One line of evidence is that overexpression of Sec12 delocalizes Sec12 to the general ER, but simultaneous overexpression of Sec16 retains overexpressed Sec12 at tER sites. Additionally, when P. pastoris Sec12 is expressed in S. cerevisiae, the exogenous Sec12 localizes to the general ER, but when P. pastoris Sec16 is expressed in the same cells, the exogenous Sec12 is recruited to tER sites. In both of these experimental systems, the ability of Sec16 to recruit Sec12 to tER sites is abolished by deleting a C-terminal fragment of Sec16. Biochemical experiments confirm that this C-terminal fragment of Sec16 binds to the cytosolic domain of Sec12. Similarly, we demonstrate that human Sec12 is concentrated at tER sites, likely due to association with a C-terminal fragment of Sec16A. These results suggest that a Sec12–Sec16 interaction has a conserved role in ER export

Bacteria-mediated anti-cancer therapy

Traditional cancer treatments like chemotherapy and radiation therapy continue to have limited efficacy due to phenomena like tumor hypoxia and multi-drug resistance. Bacterial cancer therapy has the potential to overcome these problems, using anaerobic spores of bacteria such as the proteolytic Clostridium sporogenes. However, the use of spores or live bacteria comes with the risks of toxicity, infection and tumor recurrence. This thesis is motivated by the research question, “How can bacterial therapy be made safer while retaining the natural anti-cancer properties of bacteria?”. To address this question, heat-inactivated bacteria (IB) and conditioned media (CM) with the secreted products of C. sporogenes were generated to minimize the safety and efficacy risks of live bacteria. These derivatives of C. sporogenes, which are cannot replicate or metabolise, were tested for their anti-cancer properties for the first time. It was hypothesized that these non-viable derivatives of C. sporogenes can exert a direct anti-cancer effect and elicit an immune response against tumors. The direct anti-cancer effect of IB and CM was investigated using a range of human cancer cells of various tissue origin: colorectal (HCT116), breast (MCF7), renal (A498), hepatic (HepG2), bladder (RT4) and melanoma (G361). These in vitro studies were conducted on 2-Dimensional (2D) monolayer and 3-Dimensional (3D) spheroid culture models. The results showed that IB and CM have a selective anti-cancer effect on the different cell lines. Colorectal cancer cells were found to be the susceptible to IB and CM treatment in both in vitro models, resulting in significant decreases in cell proliferation and spheroid size. Subsequent studies were conducted on colorectal cancer cell lines HCT116 (human) and CT26 (murine) to understand the nature of IB- and CM- mediated inhibition. The inhibition of HCT116 and CT26 cells when kept in cell culture-conditions showed that the anti-cancer efficacy of IB and CM was hypoxia-independent. This would minimize the risk of tumor recurrence associated with hypoxia-dependent spores and live anaerobic bacteria. Scanning electron microscopy imaging showed that the IB adherent to the surface of cancer cells and restricted-culture studies proved that physical contact between IB and cancer cells was crucial for inhibition. IB was found to mediate necrosis in colorectal cancer cells. On the other hand, CM was found to mediate apoptosis in the cancer cells. To understanding the mechanism of CM-mediated cancer inhibition, the active component of CM was examined. Size fractionation of CM showed that the fraction of size smaller than 3 kDa in molecular weight retained the inhibitive of CM. The findings suggested that IB and CM have intrinsically different mechanisms of cancer inhibition. After successfully inhibiting the growth of CT26 and HCT116 colorectal cancer cells in vitro, the efficacy of IB and CM was tested in mouse models of colorectal tumors. Sub-cutaneous tumors of CT26 and HCT116 cells were established in BALB/cARC and athymic BALB/c-Foxn1nu/ARC (nude) mice, respectively. IB and CM were administered through intra-tumoral injections over a period of 12 days, during which the tumor volume was measured. CM had limited efficacy in decreasing the tumor-burden of the mice and did not elicit an immune response. However, IB significantly inhibited the growth of the tumors and improved the survival rate of immunocompetent CT26 tumor-bearing mice. Histological analysis showed that IB increased the necrotic area in HCT116 tumors. Immunohistochemical analysis of the tumor samples showed that IB elicited an immune response against the tumor cells. Notably, when the IB-treated mice were subsequently re-challenged with CT26 cells, the immune mediated-response prevented the establishment of a second tumor in the mice. Taken together, the results show in vivo efficacy of the non-viable derivatives of C. sporogenes. In summary, the work of this thesis demonstrates the potential of IB and CM of C. sporogenes as anti-cancer agents which retain the natural anti-cancer properties of the bacteria and advantages over conventional therapy, while minimizing the safety risks.Doctor of Philosoph

Sec12 Binds to Sec16 at Transitional ER Sites

COPII vesicles bud from an ER domain known as the transitional ER (tER). Assembly of the COPII coat is initiated by the transmembrane guanine nucleotide exchange factor Sec12. In the budding yeast Pichia pastoris, Sec12 is concentrated at tER sites. Previously, we found that the tER localization of P. pastoris Sec12 requires a saturable binding partner. We now show that this binding partner is Sec16, a peripheral membrane protein that functions in ER export and tER organization. One line of evidence is that overexpression of Sec12 delocalizes Sec12 to the general ER, but simultaneous overexpression of Sec16 retains overexpressed Sec12 at tER sites. Additionally, when P. pastoris Sec12 is expressed in S. cerevisiae, the exogenous Sec12 localizes to the general ER, but when P. pastoris Sec16 is expressed in the same cells, the exogenous Sec12 is recruited to tER sites. In both of these experimental systems, the ability of Sec16 to recruit Sec12 to tER sites is abolished by deleting a C-terminal fragment of Sec16. Biochemical experiments confirm that this C-terminal fragment of Sec16 binds to the cytosolic domain of Sec12. Similarly, we demonstrate that human Sec12 is concentrated at tER sites, likely due to association with a C-terminal fragment of Sec16A. These results suggest that a Sec12–Sec16 interaction has a conserved role in ER export.</p

Effect of Heat-Inactivated Clostridium sporogenes and Its Conditioned Media on 3-Dimensional Colorectal Cancer Cell Models

Traditional cancer treatments, such as chemotherapy and radiation therapy continue to have limited efficacy due to tumor hypoxia. While bacterial cancer therapy has the potential to overcome this problem, it comes with the risk of toxicity and infection. To circumvent these issues, this paper investigates the anti-tumor effects of non-viable bacterial derivatives of Clostridium sporogenes. These non-viable derivatives are heat-inactivated C. sporogenes bacteria (IB) and the secreted bacterial proteins in culture media, known as conditioned media (CM). In this project, the effects of IB and CM on CT26 and HCT116 colorectal cancer cells were examined on a 2-Dimensional (2D) and 3-Dimensional (3D) platform. IB significantly inhibited cell proliferation of CT26 to 6.3% of the control in 72 hours for the 2D monolayer culture. In the 3D spheroid culture, cell proliferation of HCT116 spheroids notably dropped to 26.2%. Similarly the CM also remarkably reduced the cell-proliferation of the CT26 cells to 2.4% and 20% in the 2D and 3D models, respectively. Interestingly the effect of boiled conditioned media (BCM) on the cells in the 3D model was less inhibitory than that of CM. Thus, the inhibitive effect of inactivated C. sporogenes and its conditioned media on colorectal cancer cells is established.Published versio

Evaluation and comparison of color change in two commercially shade guides after disinfection

Background: Due to the close proximity to the oral cavity, shade guides are prone to contamination with saliva. Hence, disinfection of shade guides is important to prevent cross infection in clinics. This study has compared and evaluated the color change in VITA shade guide and the WALDENT shade guide after disinfecting with 70% isopropyl alcohol. Objectives: The objective of this study was to compare and evaluate the amount of color change in the VITA shade guide and WALDENT shade guide after disinfecting them with 70% isopropyl alcohol for the simulated time of 6 months and 1 year. Materials and Methods: Four commonly used shade tabs, namely A2, A3.5, B2, and C1 of two shade guides – VITA Classical Shade Guide and Waldent shade guides – were chosen for this study. VITA Easyshade spectrophotometer measured the shade, and the values were recorded in the International Commission on Illumination L*a*b* Scale. Baseline values of 8 shade tabs were obtained followed by surface disinfection using 70% isopropyl alcohol using gauze. The testing process was done to simulate a 6-month period and 1-year period. Results: Unpaired t-test was used for statistical analysis. It was found that shade tabs underwent significant changes in short period of time. Conclusion: Within the limitations of this study, it was found that shade guides undergo significant changes in color when disinfected with 70% isopropyl alcohol during a simulated period of 6 months. Clinical Significance: If shade guides are not periodically evaluated using a control (preferably unused) shade guide, they may have a profound impact on resulting restoration

Perturbation of nucleo-cytoplasmic transport affects size of nucleus and nucleolus in human cells

Size regulation of human cell nucleus and nucleolus are poorly understood subjects. 3D reconstruction of live image shows that the karyoplasmic ratio (KR) increases by 30-80% in transformed cell lines compared to their immortalized counterpart. The attenuation of nucleo-cytoplasmic transport causes the KR value to increase by 30-50% in immortalized cell lines. Nucleolus volumes are significantly increased in transformed cell lines and the attenuation of nucleo-cytoplasmic transport causes a significant increase in the nucleolus volume of immortalized cell lines. A cytosol and nuclear fraction swapping experiment emphasizes the potential role of unknown cytosolic factors in nuclear and nucleolar size regulation

Viability of <i>S. cerevisiae</i> cells carrying <i>PpSEC12</i> as the only gene from the <i>SEC12</i> family.

<p>A plasmid shuffle was performed in <i>sed4</i>Δ <i>sec12</i>Δ cells, with <i>SED4</i> in a <i>URA3</i> plasmid plus either <i>ScSEC12</i> (top row) or <i>PpSEC12</i> (middle row) in a <i>LEU2</i> plasmid. Both strains grew on rich media (YPD) and also on media containing 5-FOA, indicating that <i>PpSEC12</i> could replace <i>ScSEC12</i> even in the absence of <i>SED4</i>. As a control, <i>sec12</i>Δ cells carrying <i>ScSEC12</i> on a <i>URA3</i> plasmid were plated on the same media, and no growth was seen in the presence of 5-FOA.</p

Biochemical interaction of the C-terminal portion of PpSec16 with the cytosolic domain of PpSec12.

<p>Glutathione-agarose beads were incubated with a bacterial lysate from cells expressing either GST alone, or GST fused to the C-terminal residues 1960–2550 of PpSec16. Sufficient lysate was used to saturate the binding sites on the glutathione-agarose. A second incubation was then performed with sub-saturating amounts of a bacterial lysate from cells expressing a hexahistidine-tagged version of the cytosolic domain of PpSec12 (PpSec12(cyto)-His6). The beads were centrifuged, and the unbound material in the supernatant was collected. Bound protein was eluted from the beads with 100 mM glutathione. I, input (100% relative to other lanes); U, unbound; B, bound. PpSec12(cyto)-His6 bound to the beads carrying GST-PpSec16(1960–2550) but not to the beads carrying GST alone.</p

Requirement of the C-terminal portion of PpSec16 for recruiting overexpressed PpSec12 to tER sites.

<p>As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031156#pone-0031156-g001" target="_blank">Fig. 1</a>, PpSec16-GFP was overexpressed in <i>P. pastoris</i> cells overexpressing PpSec12, except that deletions were introduced as indicated near the C-terminus of PpSec16. Two hundred randomly chosen cells from each of the indicated <i>P. pastoris</i> strains were examined by immunofluorescence and scored for colocalization of PpSec12-GG with PpSec16-GFP. Cells in which nearly all of the PpSec12-GG overlapped with PpSec16-GFP were scored as having strong colocalization (+). Cells in which PpSec12-GG showed clear concentration in the PpSec16-GFP puncta but also showed prominent staining outside of these puncta were scored as having partial colocalization (+/−). Cells showing no visible concentration of PpSec12-GG in the PpSec16-GFP puncta were scored as having no colocalization (−). Colocalization was virtually abolished by deleting the entire C-terminal portion of PpSec16, and was strongly reduced by deleting only the C-terminal conserved region (CTR).</p

Recruitment of overexpressed PpSec12 to tER sites in <i>P. pastoris</i> by simultaneous overexpression of PpSec16.

<p>PpSec12 was tagged with the Glu-Glu epitope (PpSec12-GG) by gene replacement, and a second untagged copy of PpSec12 was expressed in the same cells using the methanol-inducible <i>AOX1</i> promoter, resulting in a high total level of PpSec12 expression. Top row: in a strain overexpressing PpSec12, PpSec16 was expressed at normal levels after being tagged by gene replacement with GFP. A small fraction of the PpSec12-GG colocalized with PpSec16-GFP, but most of the PpSec12-GG was in the general ER as indicated by the prominent nuclear envelope signal. Bottom row: in a strain overexpressing PpSec12, PpSec16-GFP was overexpressed as a second copy using the <i>AOX1</i> promoter. Most of the PpSec12-GG colocalized with PpSec16-GFP in exaggerated tER sites. Scale bar, 2 µm.</p