Effects of Δtor1 on Yeast Longevity via Chronological Lifespan Assay

TOR1 is known to extend chronological lifespan in S. cerevisiae. Δtor1 deletion was studied via chronological lifespan assay and environmental oxidative stress test. This deletion was confirmed via growth in selective media and through deletion check primers. The Δtor1 deletion created demonstrated similar growth and cellular response to environmental oxidative stress as previously put forth by Bonawitz et al. and Pan et al. In this work, partial inhibition of TOR by deletion of TOR1 is studied through S. cerevisiae longevity via a chronological lifespan assay, with intention of studying the double gene deletion strains of Δlys7/Δtor1, Δsod1/Δtor1, Δsod2/Δtor1, and Δctr1/Δtor1. Double gene deletion strains of Δlys7/Δtor1, Δsod1/Δtor1, Δsod2/Δtor1, and Δctr1/Δtor1 are not well-characterized for their effects on S. cerevisiae longevity

Fluorescent Detection of Reactive Oxygen Species in Saccharomyces Cerevisiae Applied to Chronological Lifespan

During the course of normal aerobic metabolism, cells are exposed to a wide range of reactive oxygen species such as the superoxide anion, hydrogen peroxide, and the hydroxyl radical. These reactive oxygen species (ROS) are highly reactive metabolites of oxygen and can damage a wide range of macromolecules in the cell, including nucleic acids, proteins, and lipids, and can even, in some severe cases, lead to cell death. Normally, molecular oxygen is relatively unreactive and harmless in its ground state; however, it can undergo partial reduction via electrons that are leaked from the electron transport chain to form both the superoxide anion and hydrogen peroxide, both of which can react further to form the dangerously reactive hydroxyl radical. In order to combat the toxic and potentially deadly effects of ROS, cells are equipped with various antioxidant defense mechanisms, which include enzymes like superoxide dismutase 1 (Sod1p). Our objective is to observe these various reactive oxygen species using yeast (Saccharomyces cerevisiae) as a model organism and explore different biochemical staining assays such as Amplex Red (AR) and Dihydroethidium (DHE). These stains can both be used to track live cells and quantify ROS levels. This will allow us to study how ROS changes during chronological yeast lifespan. Although there are many types of reactive oxygen species that exist in various parts of the cell, our work thus far has aimed to track extracellular hydrogen peroxide via AR and superoxide generation in the mitochondria via DHE. Our initial results indicate that we are able to track superoxide production using DHE in wild type cell and sod1∆ yeast strains spectroscopically. Ultimately, we will use both fluorescence spectroscopy and live cell imaging via fluorescence microscopy to assess superoxide levels in multiple yeast strains. Our results will provide insight into the role of ROS in aging as we quantify levels during yeast lifespan

Separation of Nucleic Acids by Ion Pair Reversed Phase High Performance Liquid Chromatography (IP RP HPLC)

Ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC) is shown to be an effective method to reliably analyze DNA. IP RP HPLC is a much faster and safer alternative to conventional methods of DNA separation and quantification. The method described here utilized a two-buffer effluent system consisting of triethylammonium acetate (TEAA) and acetonitrile (ACN). The method reliably separated and quantified DNA samples of 54 and 58 nt. This method will be used and optimized to separate similarly sized RNA samples. The ultimate goal is to separate mixtures of nucleotides generated from in-vitro transcription reactions

Fluorescent Detection of Reactive Oxygen Species in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e Applied to Chronological Lifespan

During the course of normal aerobic metabolism, cells are exposed to a wide range of reactive oxygen species such as the superoxide anion, hydrogen peroxide, and the hydroxyl radical. These reactive oxygen species (ROS) are highly reactive metabolites of oxygen and can damage a wide range of macromolecules in the cell, including nucleic acids, proteins, and lipids, and can even, in some severe cases, lead to cell death. Normally, molecular oxygen is relatively unreactive and harmless in its ground state; however, it can undergo partial reduction via electrons that are leaked from the electron transport chain to form both the superoxide anion and hydrogen peroxide, both of which can react further to form the dangerously reactive hydroxyl radical. In order to combat the toxic and potentially deadly effects of ROS, cells are equipped with various antioxidant defense mechanisms, which include enzymes like superoxide dismutase 1 (∆sod1). Our objective is to observe these various reactive oxygen species using yeast (Saccharomyces cerevisiae) as a model organism and explore different biochemical staining assays such as Amplex Red (AR) and Dihydroethidium (DHE). These stains can both be used to track live cells and quantify ROS levels. This will allow us to study how ROS changes during chronological yeast lifespan. Although there are many types of reactive oxygen species that exist in various parts of the cell, our work thus far has aimed to track extracellular hydrogen peroxide via AR and superoxide generation in the mitochondria via DHE. Our initial results indicate that we are able to track superoxide production using DHE in wild type cell and ∆sod1 yeast strains spectroscopically. Ultimately, we will use both fluorescence spectroscopy and live cell imaging via fluorescence microscopy to assess superoxide levels in multiple yeast strains. Our results will provide insight into the role of ROS in aging as we quantify levels during yeast lifespan

Quantification of DNA Products Using Ion-Pair Reverse Phase Liquid Chromatography

The transcription of DNA via RNA polymerases is a fundamental process in cellular systems. In eukaryotic cells, we observe transcription in the nucleus (via genomic DNA) as well as in the mitochondria (via mitochondrial DNA). There are many tools available to investigate nuclear transcription; however, few tools exist to study mitochondrial transcription even though the mitochondrial DNA encodes several essential proteins. Recently an in vitro transcription system using purified mitochondrial transcription proteins, including the mitochondrial RNA polymerase, and linear mitochondrial DNA templates has been developed. Quantitative analysis of the DNA templates can be done via ion-pair reverse-phase high performance liquid chromatography (IP-RP HPLC), a high-resolution technique in separating DNA based on size. Using IP-RP HPLC our aim is to assess the lower limits of separation, and our quantification method is based on measuring peak area and the peak height

Role of Copper and Tor Signaling in Reactive Oxygen Species Induced Cell Aging

Chronological lifespan assays in yeast rely on promoting a culture’s quiescent stationary phase by calorie restriction, a phase characterized by curtailed overall metabolic rate and a shift from fermentation to mitochondrial respiration. This shift in glucose utilization can be accomplished by reduced TOR (Target of Rapamycin) pathway signaling, a complex regulator of cell growth and cell cycle in which various environmental growth and nutrient signals are integrated to activate or inhibit the Ser/Thr kinase activity of Tor1 protein. Optimal growth conditions promote TOR signaling causing macromolecule biosynthesis, sugar fermentation, increased metabolism, and progression through cell cycle. Many downstream effects of TOR signaling can be silenced by cell treatment with Rapamycin, a drug which binds to Tor1 and inhibits kinase domain function, drastically increasing a cell culture’s chronological lifespan. Mitochondrial electron transport chain (ETC) machinery, whose activity and expression is modulated by TOR signaling, is the primary site of superoxide formation. Superoxide is a reactive oxygen species (ROS) produced by premature electron leakage directly to oxygen, producing dangerous hydroxyl radicals, via the Haber-Weiss reaction, that diffuse throughout the cell causing damage to DNA, lipid peroxidation, and mitochondrial dysfunction associated with premature cell aging and death most prevalent in neuronal tissue afflicted with neurodegenerative disease. A primary defense of free radical damage as a respiratory by-product is the neutralization of superoxide by superoxide dismutase 1 (SOD1), which requires copper and zinc as cofactors in the conversion of superoxide to less harmful hydrogen peroxide. Copper is additionally utilized in cytochrome c oxidase as an electron transferring group and is required for the continuous movement of high energy electrons through the ETC, thereby limiting the potential for electron leakage and ROS. Copper, given its two roles in defending against ROS production and induced cell aging, is here modulated by extracellular supplementation to further elucidate its functionality in the context of possible lifespan extension by Rapamycin treatment of SOD1 deletion strains

Yeast Copper Proteins and Reactive Oxygen Species in Effecting Lifespan

Mitochondria are essential organelles in most eukaryotic cells because of their role in metabolism and the production of ATP by the oxidative phosphorylation (OXPHOS) pathway, as well as other key cellular processes. Metal cofactors, such as copper (Cu) and iron (Fe), are incorporated into OXPHOS protein complexes of yeast located within the inner membrane of the mitochondria. Misincorporation or modulation of these available metals in mitochondrial enzymes leads to the production of reactive oxygen species (ROS). ROS are reactive molecules containing oxygen such as peroxides, superoxide, and hydroxyl radicals. Yeast are a good model for studying aging and the effect of ROS on lifespan because they are easy to grow, and many of the genes and proteins involved in determining yeast lifespan are conserved in humans and other mammals. Mitochondrial OXPHOS protein complexes are the primary site of superoxide formation. A primary defense of free radical damage is the neutralization of superoxide by the enzyme superoxide dismutase 1 (Sod1), which requires copper and zinc (Zn) as cofactors. Copper is additionally utilized in the OXPHOS complex cytochrome c oxidase as an electron transferring group. Our first aim was to investigate the role of copper in the production of mitochondrial ROS as a part of normal aerobic respiration utilizing a yeast model. Specifically, we worked to quantify the effect of exogenous copper treatment on the relative protein expression of copper-dependent cytochrome c oxidase (COX) subunits and overall COX complex assembly. Our previous work has indicated a protective behavior of copper treatment on yeast lifespan, and we propose this is due to copper inducing more robust electron movement through a more functional electron transport chain (ETC) complexes. Improved efficacy of the ETC is thought to minimize premature electron leakage to oxygen and lessen mitochondrial ROS levels. We showed that addition of 0.25 mM copper to yeast media increases lifespan of wild type (WT) and lys7Δ cells, but the addition of 0.25 mM copper to the media only effects the growth of sod1Δ yeast cells in the short term. During this granting period we performed protein analysis of mitochondrial proteins showing an increase in subunits 1, 2, and 4 of Complex IV (cytochrome c oxidase, CcO) of the ETC. Our second aim was to determine how copper levels, ROS levels, and enzyme activity are related during yeast chronological aging. We are able to use biochemical staining assays utilizing fluorescent molecules to detect and quantify ROS levels. Specifically, we can track superoxide generation via dihydroethidium (DHE) with some of our results presented in Figure 1. We are also able to use live cell imaging via fluorescent microscopy to assess superoxide levels again using DHE, and an additional mitochondrial specific stain MitoSox (Molecular Probes). Future directions are to increase the yeast lifespan experimental length and to determine effects of copper on protein activity via in-gel analysis, and at the level of transcription level for respiratory proteins. This work hopes to contribute to our understanding of the copper-utilizing components of this mitochondrial pathway, and this metal’s impact on local ROS production. Ultimately, we will also use both spectroscopy and live cell imaging via fluorescent microscopy to assess superoxide levels in multiple yeast strains as well as in the presence and absence of copper. Our results will provide insight into the role of ROS in aging as we quantify levels during yeast lifespan

Student-Faculty Collaborative Research Grant Report

Mitochondria are essential organelles in most eukaryotic cells because of their role in metabolism and the production of ATP by the oxidative phosphorylation (OXPHOS) pathway, as well as other key cellular processes. Metal cofactors, such as copper (Cu) and iron (Fe), are incorporated into OXPHOS protein complexes of yeast located within the inner membrane of the mitochondria. Misincorporation or modulation of these available metals in mitochondrial enzymes leads to the production of reactive oxygen species (ROS). ROS are reactive molecules containing oxygen such as peroxides, superoxide, and hydroxyl radicals. Yeast are a good model for studying aging and the effect of ROS on lifespan because they are easy to grow, and many of the genes and proteins involved in determining yeast lifespan are conserved in humans and other mammals. Mitochondrial OXPHOS protein complexes are the primary site of superoxide formation. A primary defense of free radical damage is the neutralization of superoxide by the enzyme superoxide dismutase 1 (Sod1), which requires copper and zinc (Zn) as cofactors. Copper is additionally utilized in the OXPHOS complex cytochrome c oxidase as an electron transferring group. Our first aim was to investigate the role of copper in the production of mitochondrial reactive oxygen species (ROS) as a part of normal aerobic respiration utilizing a yeast model. Specifically, we worked to quantify the effect of exogenous copper treatment on the relative protein expression of copper-dependent cytochrome c oxidase (COX) subunits and overall COX complex assembly. Our previous work has indicated a protective behavior of copper treatment on yeast lifespan, and we propose this is due to copper inducing more robust electron movement through a more functional electron transport chain (ETC) complexes. Improved efficacy of the ETC is thought to minimize premature electron leakage to oxygen and lessen mitochondrial ROS levels. During this granting period we showed that addition of 0.25 mM copper to yeast media increases lifespan of wild type (WT) and lys7∆ cells, but the addition of 0.25 mM copper to the media only affects the growth of sod1∆ yeast cells in the short term. As expected, the higher concentration of 2.0 mM copper addition to media was too high and became toxic, killing most of the yeast cells. We began performing protein analysis of mitochondrial proteins as shown in figure 1 (see Comments). Future directions are to increase the yeast lifespan experimental length and to determine effects of copper on protein expression (i.e., improve the western blot loading), protein activity, and at the level of transcription level for respiratory proteins. This work hopes to contribute to our understanding of the copper-utilizing components of this mitochondrial pathway, and this metal’s impact on local ROS production. Our second aim was to determine how copper levels, ROS levels, and enzyme activity are related during yeast chronological aging. We are able to use biochemical staining assays utilizing fluorescent molecules to quantify ROS levels and track live cells. Specifically, we can track extracellular hydrogen peroxide via Amplex Red, and superoxide generation in the mitochondria via dihydroethidium (DHE). Our initial results indicate that we are able to track superoxide production using DHE in wild type cells and sod1∆ yeast strains spectroscopically. Ultimately, we will use both spectroscopy and live cell imaging via microscopy to assess superoxide levels in multiple yeast strains as well as in the presence and absence of copper. We are currently working on utilizing the fluorescent dye MitoSox (Molecular Probes) to specifically identify and quantify superoxide species generated within the mitochondria. Our results will provide insight into the role of ROS in aging as we quantify levels during yeast lifespan

The effect of low concentrations of copper on mitochondria and activity in yeast cells

Mitochondria are essential organelles in both yeast and human cells due to their role in metabolism, ATP production via the oxidative phosphorylation (OXPHOS) pathway, and other regulatory cellular processes. Using yeast as a model organism to study mitochondrial function in a chronological lifespan assay allows experiments to be conducted over a shorter timeframe and allows for connections to be made to human cells. Our aim is to investigate how exogenous copper in the mitochondria of yeast affects the production of reactive oxygen species (ROS), protein expression, and enzyme activity during yeast lifespan. Small amounts excess copper added to growth media (0.25 mM copper sulfate in restricted nutrient media) extend yeast chronological lifespan, but yeast lifespan is reduced when added copper levels are increased to 2.0 mM copper sulfate or higher. These results indicate that low levels of exogenous copper in the media is beneficial for yeast in these restricted media conditions. To extend these findings, we assessed how added copper changes mitochondria within these cells over the course of the yeast lifespan (14 days growth). Using MitoTracker Green and fluorescence detection we showed an increase in mitochondria in copper treated cells. This is consistent with previous studies showing mitochondria in mammal and yeast cells contain a labile copper pool located in the matrix, which is used in the metalation of the copper containing enzyme of the OXPHOS pathway Complex IV, Cytochrome C Oxidase (CcO), and superoxide dismutase (Sod1p). Our most recent work focuses on assessing CcO protein complex expression during yeast lifespan, specifically looking at cytochrome c oxidase subunits using western blotting; and assessing Sod1p activity using in-gel activity assays. The results of this research allow us to better understand the role of copper in mitochondrial activity across the lifespan of yeast

In Vitro Analysis of the Thyroid Hormone Receptor in Mitochondrial Transcription

The central dogma theory relates how DNA is transcribed into messenger RNA (mRNAs) and then translated into proteins. Since the nucleus contains the majority of the DNA in cells, research related to transcription and translation focuses on these processes within the nucleus and cytosol; however, these processes are also taking place within the mitochondrial organelle. Mitochondria are most widely known for their essential role in producing energy for the cell, but the organelle also contains its own small, circular genome. Transcription of mitochondrial DNA (mtDNA) follows similar mechanisms as does transcription of nuclear DNA. During this essential process, specific mitochondrial transcription factors, such as TFAM and TFB2M, regulate the attachment of the mitochondrial RNA polymerase (POLRMT) to the promoter and initiation of transcription. With a fully functioning mitochondrial RNA polymerase, transcription is properly conducted, and transcripts can be translated to protein by the mitochondrial ribosome. Mitochondrial transcription is a major regulatory process within the organelle, and determining transcription factors involved in this control point is important for understanding mitochondrial function and many diseases relating to mitochondrial dysfunction. Numerous transcription factors are found both in the nucleus as well as in the mitochondria where their function is not well understood. One such transcription factor is the thyroid hormone receptor. Previous research suggests that when the hormone triiodothyronine (T3) is present and taken up in cells, mitochondrial transcription increases. The mechanism behind the T3 stimulation of transcription is thought to be a coordinated effect by interacting with both the mitochondrial and nuclear thyroid hormone receptor. Our aim is to analyze the level of interaction that the mitochondrial thyroid hormone receptor (mt-TRalpha1) has with the mitochondrial DNA and other core mitochondrial transcription factors in the presence and absence of the T3 hormone. With this information, we further understand another component of mitochondrial transcription that could have implications in mitochondrial dysfunction and disease