Mitochondria are eukaryotic organelles derived in evolution from within the ? subdivision of Proteobacteria. Although mitochondria are structurally and metabolically complex, modern-day mitochondrial genomes (mtDNA) encode only a small number of RNAs and proteins predominantly involved in adenosine triphosphate (ATP) formation through electron transport coupled to oxidative phosphorylation, as well as translation of mtDNA-encoded proteins. In humans, only 13 of the >1000 polypeptides that constitute the complete mitochondrial protein complement (proteome) are encoded in mtDNA; the remainder is encoded by nuclear DNA (nuDNA). It is therefore imperative to comprehensively catalog nuDNA-encoded mitochondrial proteins in order to understand holistically the evolution of mitochondria. Mitochondrial proteome investigations of animals, fungi and land plants have dramatically altered our conception of mitochondrial evolution: in contrast to mtDNA-encoded proteins, few nuDNA-encoded mitochondrial proteins are demonstrably derived from the eubacterial progenitor of mitochondria, and many are found only in eukaryotes. Notably, however, little is known about the mitochondria of eukaryotic microbes (protists), which constitute the bulk of biochemical and genetic diversity within the domain Eucarya. The proteomic characterization of protist mitochondria is therefore crucial to fully elucidating mitochondrial function and evolution. Employing tandem mass spectrometry (MS/MS), I have analyzed highly purified mitochondria from Acanthamoeba castellanii (Amoebozoa). In combination, nearly 750 nuDNA- and mtDNA-encoded proteins were identified. These data were used to catalog metabolic pathways and protein complexes, and to infer functional and evolutionary profiles of A. castellanii mitochondria. My analyses suggest that while A. castellanii mitochondria have many features in common with other eukaryotes, they possess several novel attributes and pronounced metabolic versatility. An analysis of the A. castellanii electron transport chain (ETC) was also performed, utilizing a combination of blue native polyacrylamide gel electrophoresis (BN-PAGE), MS/MS and bioinformatic queries. A significant proportion of A. castellanii ETC proteins was identified, yielding several insights into ETC evolution in eukaryotes. Lastly, I present two unusual cases of ‘split’ mitochondrial proteins: the iron-sulfur subunit SdhB of succinate:ubiquinone oxidoreductase (Complex II), in the phylum Euglenozoa and Cox1 of cytochrome c:O2 oxidoreductase (Complex IV) in various eukaryotes, including A. castellanii. Functional and evolutionary implications of these findings are discussed.

Melanoma incidence is increasing faster than any other cancer in the world today. Disease detected early can be cured by surgery, but once melanoma progresses to the metastatic stage it is lethal, with an overall median survival of less than one year. The poor prognosis for late stage melanoma patients is attributed to the intrinsic resistance of melanoma to all Federal Drug Administration approved melanoma therapies. Therefore, there is a critical need for novel treatment approaches that circumvent melanoma therapy resistance. Emerging evidence suggests that differences in melanoma metabolism relative to non-malignant cells represents a potential target for therapeutic intervention. The research presented here demonstrates the potential for using triphenylphosphonium-based compounds as a new therapeutic platform for metastatic melanoma that is designed to take advantage of these metabolic differences. In vitro experiments demonstrate that triphenylphosphonium-based compounds modified with an aliphatic side chain target melanoma cell mitochondria and promote melanoma cell death via mitochondria metabolism inhibition and subsequent reactive oxygen species production. Increased reactive oxygen species production results in decreased glutathione levels and an oxidized cellular state. There is also a structure-activity relationship between side chain length, metabolic disruption, and melanoma cell cytotoxicity. Further, results demonstrate that traditional in vivo triphenylphosphonium drug administration routes such as oral gavage, intraperitoneal injection, and intravenous injection do not result in significant tumor accumulation of triphenylphosphonium drugs. However, the use of a thermosensitive hydrogel delivery system localizes triphenylphosphonium drugs directly at the melanoma tumor site and decreases melanoma tumor growth rate. These results suggest that a hydrogel-based triphenlyphosphonium delivery system could potentially be a therapeutic strategy that circumvents melanoma resistance mechanisms in order to provide durable therapy for an increasing number of metastatic melanoma patients worldwide.

Of the considerable challenges researchers face in the control and elimination of malaria, the development of antimalarial drug resistance in parasite populations remains a significant hurdle to progress worldwide. Atovaquone is used in combination with proguanil (Malarone) as an antimalarial treatment in uncomplicated malaria, but is rendered ineffective by the rapid development of atovaquone resistance during treatment. Previous studies have established that de novo mutant parasites confer resistance to atovaquone with a substitution in amino acid 268 in the cytochrome b gene encoded by the parasite mitochondrial genome, yet much is still unknown about how this resistance develops, and whether parasites are inherently predisposed to resistance development. Here we report phenotypic characterization of isolates from patients that failed treatment in the original atovaquone Phase II studies in Thailand by using a diverse series of chemotypes that target mitochondrial functions. We defined their structure-activity relationships and observed broad resistance (5-30,000 fold in atovaquone), suggesting that cytochrome b mutations alone are not sufficient to explain this spectrum of resistance. We also report the first known in vitro selection that recapitulates the clinical Y268S mutation using the TM90-C2A genetic background, the pre-treatment parent for TM90-C2B. Selection of the Y268S mutation in TM90-C2A and others indicates that the parasite genetic background is critical in the selection of clinical atovaquone resistance, since selection attempts in multiple other genetic backgrounds results in mutations at positions other than amino acid 268. We implicate mitochondrial heteroplasmy in the development of sporadic, rapid resistance to atovaquone, where pre-existing low-level mutations in the multi-copy mitochondrial DNA can be quickly selected for in parasite populations. High-coverage mitochondrial deep-sequencing data showed that low-level Y268S mutants were present in admission parasites from the atovaquone Phase II clinical trials in Thailand, and recrudescent parasites either maintained high level Y268S mutation frequencies or gradually returned to cryptic Y268S levels. The phenomenon of gradual heteroplasmic conversion back to wild-type was noted in some ex vivo patient isolated parasites as well as some in vitro selected lines, which suggests that other factors are at play that influence heteroplasmy stability. In addition to mitochondrial heteroplasmy, the total mtDNA copy number is likely influencing phenotypes in a gene dose-dependent fashion. Further, pressure on the DHODH enzyme that results in DHODH copy number amplifications/mutations has been shown to influence mitochondrial heteroplasmy directly. Last, mitochondrial diversity was shown to be vastly underestimated without heteroplasmic loci being taken into account, as seen in the re-analysis of the Pf3K MalariaGEN genome dataset we performed. The complex interactions between these drug resistance mechanisms reveal the phenotypic and genotypic plasticity that the Plasmodium falciparum parasite utilizes are a clear fitness advantage in the face of mitochondrial stress, and further studies are required to determine the impact of this wide-ranging phenotype on the development of new mitochondria-targeted drugs.

In neurons, mitochondrial quantity and basal cellular respiration are maintained with age, but alterations in other key functions and quantities make these cells susceptible to the pathology of age-related neurodegenerative disease. We observed age-related decreases in cytochrome C, cardiolipin, cytochrome C oxidase (CCO) function, and glutamate response that render cells less capable of responding to stress. Rescue experiments showed that estrogen is a promising treatment in restoring neuron function with age. After finding key differences in CCO, we examined the electron transport chain more closely and found age-related deficits in quantity or function for each individual complex. Our experiments support a lack of endogenous substrates or a failure of upstream complexes to transport electrons to complex IV with age, ultimately leading to age-related neurodegeneration. Reactive oxygen species production may add to the problem by degrading macromolecules such as nucleic acid, cardiolipin, and proteins. Increased ROS may also lead to a redox imbalance in the neuron, reducing the potential for energy production. Also, epigenetic controls such as DNA methylation, histone acetylation ubiquitination and phosphorylation that persist in culture independent of aging hormone levels, vasculature, and immune system may be partly responsible for the observed age-related deficiencies as has been previously observed in aging human muscle (Ronn et al., 2008). This compelling cumulative evidence suggests an age-related deficiency in electron transport via quinones from complexes I to III, and age-related deficiencies in substrates, cofactors, and quantity or function for complex IV. These studies add to the growing body of evidence that dysfunction in the enzyme complexes of the electron transport chain lead to neurodegeneration in senescence-related diseases. In an attempt to integrate our age-related findings with Alzheimer's Disease (AD) pathology, we sequentially isolated the electron transport chain complexes using selective mitochondrial inhibitors in cortical neurons removed from the 3xTg-AD mouse model, which harbors mutations in the PS1, APPSwe and tauP301L genes and follows the proposed temporal development of human AD pathology (Oddo et al., 2003a; 2003b). Overall, we did not detect 3xTg-AD cortical neuron deficits at the four electron transport complexes of mitochondria or in NAD(P)H oxidase (NOX), an extramitochondrial oxygen consumer and regulator of NAD(P)+/NAD(P)H homeostasis (Morre et al., 2000).

Many mutations in superoxide dismutase 1 (SOD1) cause destabilization and misfolding of the protein and are implicated in amyotrophic lateral sclerosis. Likewise, a few post-translational modifications (PTMs) on SOD1 have been shown to cause the same phenotype. However, relatively few PTMs on SOD1 have been studied in depth and, in particular, very few studies have demonstrated how these PTMs affect SOD1's various biological roles. SOD1 is traditionally known for its role in reactive oxygen species (ROS)-scavenging but has also been found to have a few other biological roles, including transcription factor activity to promote genomic stability, preservation of cytoskeletal activity, maintaining zinc and copper homeostasis, and suppressing respiration. We have used the computational analysis tool, SAPH-ire, to find PTM 'hotspots' on SOD1 that have a high likelihood of affecting its biological functions. Interestingly, the top seven ranked PTM 'hotspots' were found in a small region of SOD1, between S98-K128. We focused our studies on one of the PTM 'hotspots' found in this region, lysine-122 (K122). K122 is found in the electrostatic loop of SOD1, a loop that is important for shuttling in superoxide radicals to be neutralized. According to our data, and other studies, this lysine is both succinylated and acetylated. We found that acetyl and succinyl-mimetics (K122Q and K122E, respectively) of this site do not affect its ROS scavenging activity but do prevent SOD1 from suppressing respiration and decrease its localization to the mitochondria. Further, when cells are depleted of SIRT5 (the desuccinylase for K122), SOD1 can no longer suppress respiration. Additionally, we found that SOD1 appears to suppress respiration at complex I, whether directly or through an indirect pathway is unknown. When HCT116 colon cancer cells were depleted of endogenous SOD1, the overexpressed succinyl K122-mimetic (K122E) could not recover growth as well as overexpressed WT SOD1. The K122E SOD1 expressing cells also exhibited increased mitochondrial ROS and unhealthier mitochondria. We propose a mechanism whereby SOD1 suppression of respiration acts as an additional regulator of oxidative stress: SOD1 suppresses the electron transport chain to decrease reactive oxygen species leakage and to promote healthier mitochondria and growth.

Modulation of mitochondrial complex I during reperfusion reduces cardiac injury. Complex I exists in two structural states: active (A) and deactive (D) with transition from A→D during ischemia. Reperfusion reactivates D→A with an increase in ROS production. Metformin preserves the D-Form. Our aim was to study the contribution of maintenance of deactivation of complex I during early reperfusion by metformin to protect against ischemia reperfusion injury. Our results showed that metformin decreased H9c2 cardiomyoblast apoptosis and total cell death following simulated ischemia for six hours followed by reoxygenation for twenty four hours compared to untreated cells. Reactive oxygen species (ROS) generation was reduced at the onset of reoxygenation with metformin treatment. Metformin also prevented the acute reactivation of complex I during reoxygenation following 10 minutes of hypoxia accompanied by decreased ROS generation. In addition, the content of C/EBP homologous protein was decreased in metformin treated cells, suggesting that metformin treatment decreased endoplasmic reticulum stress. 5' adenosine monophosphate-activated protein kinase was activated in our model independent of metformin treatment. Intriguingly, metformin protects in 5' adenosine monophosphate-activated protein kinase knock down system. Surprisingly, we found that metformin successfully downregulated p53 compared to untreated simulated ischemia reoxygenation. We sought potential metformin related impact on anti-apoptotic protein B-cell lymphoma 2. Our results showed the expression of the anti-apoptotic protein B-cell lymphoma 2 was markedly decreased in SI6/RO24 and metformin increased expression of B-cell lymphoma 2. Metformin, likely by partial inhibition of complex I with decreased ROS generation, resulted in less sulfhydryl modification and decreased modification of thiol groups by nitrosylation. We propose that the slowing down of activation of complex I at early stage of reperfusion by acute use of high dose metformin would be protective in cells and hearts against ischemia reperfusion injury. This potential new mechanism of protection is relevant at the onset of reperfusion to directly modulate electron transport to achieve cardiac protection and to decrease cardiac cell injury. Modulation of mitochondrial complex I during reperfusion reduces cardiac injury. Complex I exists in two structural states: active (A) and deactive (D) with transition from A→D during ischemia. Reperfusion reactivates D→A with an increase in ROS production. Metformin preserves the D-Form. Our aim was to study the contribution of maintenance of deactivation of complex I during early reperfusion by metformin to protect against ischemia reperfusion injury. Our results showed that metformin decreased H9c2 cardiomyoblast apoptosis and total cell death following simulated ischemia for six hours followed by reoxygenation for twenty four hours compared to untreated cells. Reactive oxygen species (ROS) generation was reduced at the onset of reoxygenation with metformin treatment. Metformin also prevented the acute reactivation of complex I during reoxygenation following 10 minutes of hypoxia accompanied by decreased ROS generation. In addition, the content of C/EBP homologous protein was decreased in metformin treated cells, suggesting that metformin treatment decreased endoplasmic reticulum stress. 5' adenosine monophosphate-activated protein kinase was activated in our model independent of metformin treatment. Intriguingly, metformin protects in 5' adenosine monophosphate-activated protein kinase knock down system. Surprisingly, we found that metformin successfully downregulated p53 compared to untreated simulated ischemia reoxygenation. We sought potential metformin related impact on anti-apoptotic protein B-cell lymphoma 2. Our results showed the expression of the anti-apoptotic protein B-cell lymphoma 2 was markedly decreased in SI6/RO24 and metformin increased expression of B-cell lymphoma 2. Metformin, likely by partial inhibition of complex I with decreased ROS generation, resulted in less sulfhydryl modification and decreased modification of thiol groups by nitrosylation. We propose that the slowing down of activation of complex I at early stage of reperfusion by acute use of high dose metformin would be protective in cells and hearts against ischemia reperfusion injury. This potential new mechanism of protection is relevant at the onset of reperfusion to directly modulate electron transport to achieve cardiac protection and to decrease cardiac cell injury.

Phenylarsine oxide (PAO) is a membrane-permeable trivalent arsenic compounds, which interfere the biochemical activity of intracellular enzymes or proteins through reacting specifically with sulfhydryl and vicinal dithiol groups in the protein structure. Although the deleterious effects of arsenic compounds in bioorganisms have been extensively studied, however its role in the synaptogenesis is still obscure. Here we test the role of PAO on the synaptic activity at developing Xenopus neuromuscular synapse by using whole-cell patch clamp recording. Bath application of PAO dose-dependently increases the frequency of spontaneous synaptic currents (SSC frequency) and reaches its maximal effect at 10 £gM. The SSC frequency is robustly facilitated in 10~15 minutes after PAO application and then the release of neurotransmitter were abruptly ceased due to the degenerative collapse of the presynaptic motoneuron. Pretreatment of the culture with Ca2+ chelator BAPTA-AM significantly blunted the SSC frequency facilitation induced by PAO, suggesting a rise in Ca2+ in presynaptic motoneuron is a prerequisite. The PAO-induced SSC frequency facilitation is unaffected even that Ca2+ is eliminated from culture medium or addition of pharmacological Ca2+ channel inhibitor cadmium, indicating the influx of extracellular Ca2+ is not needed for the rise of [Ca2+]i. Depletion of endoplasmic reticulum Ca2+ pool with thapsigargin effectively hampered the PAO-induced SSC frequency facilitation. Pretreatment of ryanodine receptor inhibitor TMB-8 but not IP3 receptor inhibitor XeC significantly occluded the increase of SSC frequency elicited by PAO. Furthermore, bath application of the culture with either mitochondria oxidative phosphorylation uncoupler FCCP or mitochondrial permeability transition pore inhibitor cyclosporin A significantly abolished the SSC facilitating effect of PAO. Pretreatment the culture with TMB-8 and cyclosporin A have no addictive effects on the occlusion of PAO-induced SSC frequency facilitation, suggesting a consecutively released Ca2+ from internal store through ryanodine receptor and mitochondria is responsible for PAO-induced SSC frequency facilitation. The synaptic facilitating effect of PAO is eliminated while incubated with free radical scavenger n-acetylcysteine. Furthermore, treating cultures with complex III of electron transport chain (ETC) inhibitor antimycin A, but not complex I inhibitor rotenone, abolished PAO-induced facilitation of synaptic transmission. PAO elicited no facilitation effects on SSC frequency when pretreatment the culture with either thiol-modifying agent NEM or thiol-reducing agent DTT. Overall, results from our current study provide evidences that reactive oxygen species derived from PAO inhibition on complex III of ETC induce the open of MPT pore in mitochondria, the accompanied Ca2+ leak from mitochondria and Ca2+-induced Ca2+ release from endoplasmic reticulum resulted in a robustly release of neurotransmitter and a destructive damage on the neuron.

This study has investigated the involvement of photosynthetic electron transport chain on the regulation of gene expression of methionine sulfoxide reductase (UfMSR) in the marine macroalga Ulva fasciata Delile.UfMSRA is from copper stress and UfMSRB ir from hypersalinity stress. UfMSRA is similar to Arabidopsis AtMSRA4 and UfMSRB is similar to AtMSRB1. UfMSRA is specific to the MetSO S-enantiomer and UfMSRB catalytically reduces the MetSO R-enantiomer. Both enzymes are required, since in the cell oxidation of Met residues at the sulfur atom results in a racemic mixture of the two stereoisomers. UfMSRA and UfMSRB transcripts were increased by white light, blue light and red light with the maximum at 1 h following a decline, but kept constant in the dark. The magnitude of UfMSRA and UfMSRB transcript increase showed a positive linear correlation to increasing light intensity from 0-1200 u mole¡Pm-2¡Ps-1. The treatment with linear electron transport chain inhibitors, hydroxylamine, 3-(3,4-dichlorophenyl) -1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and stigmatellin, effectively inhibited PS II activity under 300 u mole¡Pm-2¡Ps-1 irradiance. DBMIB and stigmatellin can increase UfMSRA transcript that was reversed by 2,6-dichlorophenolindophenol (DCPIP), a PS I electron donor. It indicates that the block of electron transport of the downstream of cytochrome b6f indeuces UfMSRA gene expression. Hydroxylamine, DCMU and DBMIB decreased UfMSRB transcript that was not reversed by DCPIP while stigmatellin increased UfMSRB mRNA level, reflecting a role of reduced state with Qo site located at cytochrome b6f on the induction of UfMSRB gene expression. The cyclic electron transport chain inhibitors, antimycin A that inhibited photosynthetic electron transport, can inhibit the increase of UfMSRA and UfMSRB transcripts by irradiance. UfMSRA and UfMSRB gene expression were both modulated by cyclic electron transport chain and linear electron transport chain. These results reveal that photosynthetic electron transport chain modulates UfMSRA and UfMSRB gene expression by change its redox state.

abstract: Mitochondria produce the majority portion of ATP required in eukaryotic cells. ATP is generated through a process known as oxidative phosphorylation, through an pathway consisting five multi subunit proteins (complex I-IV and ATP synthase), embedded inside the mitochondrial membrane. Mitochondrial electron transport chain dysfunction increases reactive oxygen species in the cell and causes several serious disorders. Described herein are the synthesis of antioxidant molecules to reduce the effects in an already dysfunctional system. Also described is the study of the mitochondrial electron transport chain to understand the mechanism of action of a library of antioxidants. Illustrated in chapter 1 is the general history of research on mitochondrial dysfunction and reported ways to ameliorate them. Chapter 2 describes the design and synthesis of a series of compounds closely resembling the redox-active quinone core of the natural product geldanamycin. Geldanamycin has been reported to confer cytoprotection to FRDA lymphocytes in a dose dependent manner under conditions of induced oxidative stress. A library of rationally designed derivatives has been synthesized as a part of our pursuit of a better neuroprotective drug. Chapter 3 describes the design and synthesis of a library of pyrimidinol analogues. Compounds of this type have demonstrated the ability to quench reactive oxygen species and sustain mitochondrial membrane potential. Described herein are our efforts to increase their metabolic stability and total ATP production. It is crucial to understand the nature of interaction between a potential drug molecule and the mitochondrial electron transport chain to enable the design and synthesis a better therapeutic candidates. Chapter 4 describes a part of the enzymatic binding studies between a molecular library synthesized in our laboratory and the mitochondrial electron transport chain using sub mitochondrial particles (SMP). / Dissertation/Thesis / Doctoral Dissertation Chemistry 2015

Mitochondria are essential for the viability of mammalian cells and provide a compartment for specific chemical reactions. Cellular respiration -- the main mitochondrial function -- is tightly connected with ROS production: the mitochondrial electron transport chain complexes I and III are the main ROS sources in mammalian cells. It has been reported that complex I and complex III activities are essential for cell cycle, apoptosis and stem cell differentiation (Spitkovsky et al., 2004; Varum et al., 2009; Lee et al., 2011; Ma et al., 2011; Tormos et al., 2012). In our work, we aimed to investigate the role of mitochondrial electron transport chain activity in the regulation of the differentiation potential and to unravel signaling pathways that could participate in this regulation. As a model, we used the P19 pluripotent stem cell line that can be easily differentiated into trophoblasts, expressing intermediate filaments cytokeratin 8/18, and neurons, which express cytoskeleton protein beta-III-tubulin. We first showed that both trophoblast and neural differentiation of P19 cells were accompanied by activation of cellular respiration. The analysis of respiratory chain complexes and supercomplexes, however, showed that undifferentiated P19 cells, as well as their differentiated derivatives did not differ in their respiratory machinery, including functional respirasomes. While undifferentiated cells did not use respiration as the main energy source, cellular respiration was activated during differentiation, indicating that oxidative metabolism was important for efficient differentiation. To investigate the potential role of mitochondrial electron transport chain activity we monitored the influence of a disrupted electron flow on the differentiation of P19 cells. We found that the activity of complex I and complex III influenced the differentiation potential of the pluripotent P19 cell line: the presence of complex I and complex III inhibitors rotenone, antimycin A, or myxothiazol increased the amount of cytokeratin 8/18+ cells during trophoblast differentiation, but almost completely prevented the formation of neuron-like beta-III-tubulin+ cells during neuron differentiation. Moreover, a low oxygen level (1 % O2 vs 21 % O2 in atmosphere) - the final electron acceptor - had the same effect on differentiation. These data suggest that mitochondrial electron transport chain activity contributes to the regulation of differentiation. The presence of complex I and complex III inhibitors, as well as oxygen scarcity, increase ROS production. We suggested that increased ROS level could explain the observed effects. By visualizing mitochondrial superoxide production with a specific dye – MitoSox - we confirmed that rotenone, antimycin A, myxothiazol, as well as low oxygen conditions, increased the superoxide level. These results suggest that the observed changes of the differentiation potential of P19 cells are associated with ROS production. To prove this idea, we differentiated P19 cells in presence of paraquat – a known ROS inducer. In line with our hypothesis paraquat promoted trophoblast differentiation. The received results suggest that the mitochondrial electron transport chain activity regulates differentiation through the ROS level. ROS are secondary messengers that participate in numerous processes including cell proliferation and differentiation. We aimed to predict the signal pathway that connects ROS level in stem cells and their differentiation potential. For this purpose, we performed a microarray analysis and compared the gene expression profiles of cells grown under hypoxia or in the presence of the complex III inhibitor myxothiazol with untreated control cells. The expression analysis revealed p53 as a transcriptional factor that impacts the differentiation potential in treated cells. p53 is a known redox-sensing molecule (Bigarella et al., 2014) that influences the differentiation potential through cell cycle control (Maimets et al., 2008). This observation is in line with our results and suggests that p53 may regulate the differentiation potential of P19 cells. We are planning to investigate the role of p53 signaling in the regulation of cell cycle and differentiation potential of P19 cell line.