Stimulation of β-adrenergic receptors (β-AR) induces apoptosis in adult rat ventricular myocytes (ARVMs) via the JNK-dependent activation of mitochondrial death pathway. Recently, we have shown that inhibition of matrix metalloproteinase-2 (MMP-2) inhibits β-AR-stimulated apoptosis and that the apoptotic effects of MMP-2 are possibly mediated via its interaction with β1 integrins. Herein we tested the hypothesis that MMP-2 impairs β1 integrin-mediated survival signals, such as activation of focal adhesion kinase (FAK), and activates the JNK-dependent mitochondrial death pathway. Inhibition of MMP-2 using SB3CT, a selective gelatinase inhibitor, significantly increased FAK phosphorylation (Tyr-397 and Tyr-576). TIMP-2, tissue inhibitor of MMP-2, produced a similar increase in FAK phosphorylation, whereas treatment of ARVMs with purified active MMP-2 significantly inhibited FAK phosphorylation. Inhibition of MMP-2 using SB3CT inhibited β-AR-stimulated activation of JNKs and levels of cytosolic cytochrome c. Treatment of ARVMs with purified MMP-2 increased cytosolic cytochrome c release. Furthermore, inhibition of MMP-2 using SB3CT and TIMP-2 attenuated β-AR-stimulated decreases in mitochondrial membrane potential. Overexpression of β1 integrins using adenoviruses expressing the human β1A-integrin decreased β-AR-stimulated cytochrome c release and apoptosis. Overexpression of β1 integrins also inhibited apoptosis induced by purified active MMP-2. These data suggest that MMP-2 interferes with the β1 integrin survival signals and activates JNK-dependent mitochondrial death pathway leading to apoptosis.

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Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease characterized by progressive motor neuron degeneration in the brain and spinal cord leading to muscle atrophy, paralysis, and death. Mitochondrial dysfunction is a major contributor to motor neuron degeneration associated with ALS progression. Mitochondrial abnormalities have been determined in spinal cords of animal disease models and ALS patients. However, molecular mechanisms leading to mitochondrial dysfunction in sporadic ALS (sALS) patients remain unclear. Also, segmental or regional variation in mitochondrial activity in the spinal cord has not been extensively examined in ALS. In our study, the activity of mitochondrial electron transport chain complex IV was examined in post-mortem gray and white matter of the cervical and lumbar spinal cords from male and female sALS patients and controls. Mitochondrial distribution and density in spinal cord motor neurons, lateral funiculus, and capillaries in gray and white matter were analyzed by immunohistochemistry. Results showed that complex IV activity was significantly decreased only in gray matter in both cervical and lumbar spinal cords from ALS patients. In ALS cervical and lumbar spinal cords, significantly increased mitochondrial density and altered distribution were observed in motor neurons, lateral funiculus, and cervical white matter capillaries. Discrete decreased complex IV activity in addition to changes in mitochondria distribution and density determined in the spinal cord in sALS patients are novel findings. These explicit mitochondrial defects in the spinal cord may contribute to ALS pathogenesis and should be considered in development of therapeutic approaches for this disease.

Proteins are large biomolecules that have great therapeutic potential in treating many human diseases. However, chemical/enzymatic degradation, denaturation, and poor penetration into cells are some of the challenges for clinical use of intracellular proteins. Previously, our group has developed cationic lipid-coated magnesium phosphate nanoparticle (LP MgP NP-CAT) formulations to enhance the intracellular delivery of the negatively charged protein catalase. The goal of the current research is to develop a formulation to deliver cytochrome c (CytC), a positively charged protein into lung cancer cells A549. Specifically, this thesis research prepares and tests liposome-coated magnesium phosphate nanoparticle for delivery of cytochrome c (CytC LP/MgP). CytC LP/MgP was designed, prepared and characterized, showing that it had an average diameter around 150 nm and ζ-potential around +30 mV. The morphology of CytC LP/MgP was validated by transmission electron microscopy. CytC LP/MgP successfully led to the attachment of CytC to A549 cells, as supported by fluorescence imaging. Intracellular delivery of CytC alleviated the cytotoxicity of cationic lipids in A549 cells, as suggested by the MTS assay on cell viability, which could facilitate the clinical use of cationic lipids in drug delivery systems.

Redox reactions are crucial to biological processes that protect organisms against oxidative stress. Metalloenzymes, such as cytochrome c peroxidases which reduce excess hydrogen peroxide into water in the periplasm of multiple bacterial organisms, play a key role in detoxification mechanisms. While accurate computational tools can be used to simulate ground state redox potentials in biomolecules, adapting such approaches to properly describe redox reactions in transition metal complexes, particularly in hemes in heterogeneous protein environments, remains a significant challenge. Here we present the results of polarizable hybrid QM/MM studies of the reduction potentials of two heme sites in the cytochrome c peroxidase of Nitrosomonas europaea. The simulated redox potential of the catalytic site Low Potential (LP) is in good agreement with the experiment, while for the High Potential (HP) heme the computational estimate significantly overestimate the experimental value. We have found that environment polarization shifts the computed value of the redox potential of the catalytic LP heme by 1.3 V, while it does not affect that of the non-catalytic High Potential (HP) heme. We demonstrate that it is necessary to account for mutual polarization of heme site and the protein environment when describing redox processes, particularly those that involve more charged heme sites. We have explored the role of various factors such as heme geometries, axial ligands, propionate side chains, and electrostatic field of the protein in tuning the redox potentials of hemes in NeCcP. The fluctuations in computed vertical ionization and electron attachment energies are predominantly affected by fluctuations in the electrostatic field of the environment but not by fluctuations in heme geometries. We attribute the difference in computed LP and HP heme reduction potentials of 0.05 V and 1.15 V, respectively, to different axial ligands and electrostatic interactions of the hemes with the protein environment. / 2023-06-30T00:00:00Z

The adsorption of cytochrome c onto mesoporous silica (MCM-41) was investigated in this study. MCM-41 was synthesized and characterized by different methods. The pore size of MCM-41 was calculated from each method and all were in agreement with each other. Result from SAXS method showed a well ordered 2D hexagonal structure of MCM- 41. To investigate the effect of pH on adsorption process, different buffers were used with various pH in the range from 3.8 to 10.7. It was observed that the maximum adsorption occurs at pH near the isoelectric point of cytochrome c. The surface charges of cytochrome c and MCM-41 play an essential role for the process of adsorption. Desorption of cytochrome c from MCM-41 was investigated as well. Pure water and buffers with pH 7.1 and 10.7 were used to study the desorption. The result shows that desorption of cytochrome c from MCM-41 takes place at a pH above its isoelectric point.

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<p>Cytochrome c (Cyt c) is a heme-containing protein that is a component of the electron transport chain as well as the mitochondrial apoptotic pathway. It is released from the mitochondrial intermembrane space to the cytosol during apoptosis and is also thought to be a biomarker for cancer and liver disease. Therefore, an efficient Cyt c biosensor would be a very useful tool for studying apoptosis. Here we show the process of development of Cyt c-dependent aptazymes, derived by <em>in vitro </em>selection. These aptazymes consist of 3 components: 1) a substrate with a cleavage site that consists of a single ribonucleotide flanked by a fluorophore and quencher; 2) a DNAzyme (catalytic DNA) motif capable of cleaving the substrate; 3) an aptamer, a short piece of single- stranded DNA that can specifically bind Cyt c. When Cyt c is absent, the aptamer occludes the catalytic core of the DNAzyme and the fluorophore of the intact substrate is quenched. However, when Cyt c is present, the aptamer binds Cyt c, allowing the DNAzyme to cleave the embedded ribonucleotide, separating the fluorophore and quencher, resulting in a fluorescent signal. Simulations of <em>in vitro </em>selection of Cyt c- dependent aptazymes were also performed. The simulations revealed several methods that can improve the success rate of future <em>in vitro </em>selections of aptazymes.</p> <p>Further analysis of the previously derived DNAzyme DEC22-18 was also performed. A detailed understanding of this DNAzyme will allow it to be developed into a biosensor.</p> / Master of Science (MSc)