Characterization of Photosynthetic Reaction Centers from Bradyrhizobium Strain BTAi 1

Photosynthetic rhizobia have been studied for about 15 years now. They are now considered to be metabolically aligned with a relatively recently discovered group of bacteria, the anoxygenic aerobic phototrophs (AAP’s).Rhizobia form symbiotic relationships with plants from the Fabaceae family. Photosynthetic rhizobia not only nodulate the roots, as most other rhizobia do, but they also form nodules on the stems of certain leguminous plants. The plant provides carbon to the bacteria and the bacteria provides the plant with soluble nitrogen fixed from the biologically inert but abundant atmospheric N2. A key question regarding photosynthetic rhizobia and other AAP’s derives from the observation that photosynthesis in these organisms shuts down under anaerobic conditions. It has been proposed, and is the hypothesis of this thesis that the primary electron acceptor (QA) in the photosynthetic reaction center has a higher midpoint potential than in reaction centers found in the AAP’s counterparts, the anaerobic purple bacteria. If QA had a higher midpoint potential, it would be more labile to overreduction under anoxic conditions, and if QA is reduced, then photosynthetic electron transport is blocked. A redox titration was done to measure the midpoint potential of Q in the reaction centers of BTAi 1. This was done by observing the level of P (primary electron donor) bleaching upon excitation with bright light at different ambient redox potentials. The level of P bleaching is proportional to the fraction of QA that is not reduced, since P cannot bleach and donate an electron if QA is already reduced. Reaction centers from BTAi 1 were purified using two techniques, both involving ion exchange chromatography and one involving ammonium sulfate precipitation. Reaction centers were characterized by spectrophotometric studies, mass spectroscopy studies (MALDI TOF) and the cofactor composition was determined.Themidpoint potential of QA in BTAi 1 is –44 mV vs. SHE. The molecular weights of the subunits are very comparable to other photosynthetic reaction centers, from both aerobic and anaerobic bacteria. The pigment stoichiometry of reaction centers from BTAi1 is 2:1 bacteriochlorophyll:bacteriopheophytin. Both absorbance and light minus dark absorbance spectra are nearly identical to that found in anaerobic photosynthetic bacteria.Photosynthetic reaction centers in BTAi 1 are very similar to reaction centers of anaerobic photosynthetic bacteria. The midpoint potential of QA cannot account for its overreduction under anaerobic conditions. It is likely that AAP’s lack a key enzyme that would participate in redox homeostasis of the photosynthetic electron transport chain

Superoxide production in respiratory electron transport minimization and utilization

The oxidation of ubiquinol by the cytochrome bc1 complex is usually a tightly controlled process, however there are instances when the reaction is compromised by mutation or other external factors such as inhibitors that leads to superoxide production. The first part of this dissertation compares the electron transfer reactions between when the enzyme is performing efficiently and when superoxide production is taking place. The results of this section suggest that the same rate limiting step governs the two reactions, which allows for the elimination of several models explaining these critical reactions

Targeting Mitochondria in Cancer Therapy Could Provide a Basis for the Selective Anticancer Activity

To determine the target of the recently identified lead compound NSC130362 that is responsible for its selective anti-cancer efficacy and safety in normal cells, structure-activity relationship (SAR) studies were conducted. First, NSC13062 was validated as a starting compound for the described SAR studies in a variety of cell-based viability assays. Then, a small library of 1,4-naphthoquinines (1,4-NQs) and quinoline-5,8-diones was tested in cell viability assays using pancreatic cancer MIA PaCa-2 cells and normal human hepatocytes. The obtained data allowed us to select a set of both non-toxic compounds that preferentially induced apoptosis in cancer cells and toxic compounds that induced apoptosis in both cancer and normal cells. Anti-cancer activity of the selected non-toxic compounds was confirmed in viability assays using breast cancer HCC1187 cells. Consequently, the two sets of compounds were tested in multiple cell-based and in vitro activity assays to identify key factors responsible for the observed activity. Inhibition of the mitochondrial electron transfer chain (ETC) is a key distinguishing activity between the non-toxic and toxic compounds. Finally, we developed a mathematical model that was able to distinguish these two sets of compounds. The development of this model supports our conclusion that appropriate quantitative SAR (QSAR) models have the potential to be employed to develop anti-cancer compounds with improved potency while maintaining non-toxicity to normal cells

Targeting mitochondria in cancer therapy could provide a basis for the selective anti-cancer activity.

To determine the target of the recently identified lead compound NSC130362 that is responsible for its selective anti-cancer efficacy and safety in normal cells, structure-activity relationship (SAR) studies were conducted. First, NSC13062 was validated as a starting compound for the described SAR studies in a variety of cell-based viability assays. Then, a small library of 1,4-naphthoquinines (1,4-NQs) and quinoline-5,8-diones was tested in cell viability assays using pancreatic cancer MIA PaCa-2 cells and normal human hepatocytes. The obtained data allowed us to select a set of both non-toxic compounds that preferentially induced apoptosis in cancer cells and toxic compounds that induced apoptosis in both cancer and normal cells. Anti-cancer activity of the selected non-toxic compounds was confirmed in viability assays using breast cancer HCC1187 cells. Consequently, the two sets of compounds were tested in multiple cell-based and in vitro activity assays to identify key factors responsible for the observed activity. Inhibition of the mitochondrial electron transfer chain (ETC) is a key distinguishing activity between the non-toxic and toxic compounds. Finally, we developed a mathematical model that was able to distinguish these two sets of compounds. The development of this model supports our conclusion that appropriate quantitative SAR (QSAR) models have the potential to be employed to develop anti-cancer compounds with improved potency while maintaining non-toxicity to normal cells

Biochemical, Molecular, and Clinical Characterization of Succinate Dehydrogenase Subunit A Variants of Unknown Significance

Abstract Purpose: Patients who inherit a pathogenic loss-of-function genetic variant involving one of the four succinate dehydrogenase (SDH) subunit genes have up to an 86% chance of developing one or more cancers by the age of 50. If tumors are identified and removed early in these high-risk patients, they have a higher potential for cure. Unfortunately, many alterations identified in these genes are variants of unknown significance (VUS), confounding the identification of high-risk patients. If we could identify misclassified SDH VUS as benign or pathogenic SDH mutations, we could better select patients for cancer screening procedures and remove tumors at earlier stages. Experimental Design: In this study, we combine data from clinical observations, a functional yeast model, and a computational model to determine the pathogenicity of 22 SDHA VUS. We gathered SDHA VUS from two primary sources: The OHSU Knight Diagnostics Laboratory and the literature. We used a yeast model to identify the functional effect of a VUS on mitochondrial function with a variety of biochemical assays. The computational model was used to visualize variants' effect on protein structure. Results: We were able to draw conclusions on functional effects of variants using our three-prong approach to understanding VUS. We determined that 16 (73%) of the alterations are actually pathogenic, causing loss of SDH function, and six (27%) have no effect upon SDH function. Conclusions: We thus report the reclassification of the majority of the VUS tested as pathogenic, and highlight the need for more thorough functional assessment of inherited SDH variants. Clin Cancer Res; 23(21); 6733–43. ©2017 AACR.</jats:p

Structure of 1,4-NQ and quinoline-5,8-dione.

Structure of 1,4-NQ and quinoline-5,8-dione.</p

Targeting mitochondria in cancer therapy could provide a basis for the selective anti-cancer activity - Fig 1

A, Structure of NSC130362. B, Dose response curve of NSC130362 in MIA PaCa-2 cells. Cells were pre-incubated with NSC130362 for 2 h followed by addition of ATO (5 μM) and incubation for an additional 24 h. At the end of the treatment, the ratio of dead cells was determined by a CellTiterGlo reagent. P<0.05.</p