1
BACKGROUND METHODS RESULTS CONCLUSIONS REFERENCES ACKNOWLEDGMENTS Response of Sah1 in Saccaromyces cerevisiae to H 2 O 2 Induced Oxidative Stress Greggory Perry, Department of Environmental and Health Sciences, Johnson State College, Johnson, VT 05656 Twelve sites were located on the two dimensional gels through visual inspection. Different intensities were sought out to determine if changes occurred due to oxidative stress. Of these twelve sites seven were analyzed through mass spectrometry. One spot was chosen (see Fig. 2), and the results of the mass spectrometry further investigated through bioinformatics which con- ducted image analysis through the use of an algorithm. Seven peptides were produced by trypsin, 21 peptides were from Uba1and 29 peptides from Sah1 when exposed to H2O2. Sah1 was chosen due to its connection with glutathione in the transmethylation metabolic pathway, which is known to relieve oxidative stress in eukaryotes (see Fig. 3). PROTEOMICS Proteomics is a technology that allows the researcher to observe the structure and function of the entire pro- teome of an organism, tissue, or cell. It is particularly useful as a comparative study examining the affects of some type of stress. OXIDATIVE STRESS Oxidative stress is caused by the accumulation of reactive oxygen species (ROS) in the cells, or by an imbalance of the cellular redox state. ROS include H 2 O 2 , O 2 ¬ and oxygen species with unpaired valence electrons; more com- monly known as free radicals. Responses can be enzymatic or nonenzymatic. Enzymatic responses include superox- ide dismutase, catalase, and peroxidases. Glutathione is generally considered nonenymatic (Mager, de Boer, Sid- erius, & Voss, 2000). These proteins and nonenymatic molecules, in particular glutathione, acts as an electron donor to stabilize the ROS (see Fig. 1). Stress due to ROS may cause transcriptional changes or severe damage to cell DNA, protein, membranes, and organelles. This can ultimately result in apoptosis (cell death) if exposure is sufficient. PROTEOMICS The proteome is defined as the set of proteins that an organism can produce. Proteomics therefore, is the study of all of the proteins in an organism or tissue. The technology is powerful because it is pos- sible to connect certain proteins to functions that may have been previously unknown. The basic func- tion of many proteins is known, but the interactions in a metabolic pathway may be far more wide- spread. For example, the enzyme Sah1 is capable of producing glutathione indirectly through the trans- methylation metabolic pathway. Sah1 Sah1 regulates transmethylation reactions by catalyzing the degradation of S-adenosyl-L homo- cysteine (AdoHcy) (Malanovic, et al., 2008). In this way Sah1 activity has a pleitrophic effect on lipid bio- synthesis, signal transduction, and gene expression. Transmethylation reactions are also reversible which compounds the intricacy of the reactions (see Fig. 3). This allows Sah1 to form AdoHcy from ad- enosine and homocysteine. Metabolism of adenosine to ATP and inosine, and homocysteine to methion- ine and cysteine produces glutathione (Hoffman, Marion, Cornatzer, & Duerre, 1982). REGULATION It is possible that a greater quantity of the Sah1 enzyme was produced in response to oxidative stress. An increase in the enzyme Sah1 will synthesize a greater amount of glutathione; a known com- pound that cells use to circumvent oxidative stress (Mager, de Boer, Siderius, & Voss, 2000)(see Fig.1). OXIDIZING GLUTATHIONE Glutathione has been identified as playing a major role in protecting cells against free radicals, ra- diation, carcinogens, and xenobiotics. Three amino acids are used in the production of glutathione, in- cluding glutamate, cysteine and glycine (Izawa, Inoue, & Kimura, 1995). The sulfhydryl group of cysteine serves as an electron donor and is responsible for the reducing capacity of glutathione (Edited by Fis- cher & Schillberg, 2004). The defense mechanisms result in the oxidized form (GSSG), which is converted and recycled back into glutathione (see Fig 3). IMPLICATIONS Research of oxidative stress has far reaching effects well beyond what happens in S. cerevisiae. Oxi- dative stress has been implicated in many human diseases including neurodegenerative disorders and pathogenesis. (Diwakar & Ravindranath, 2007). Free radicals have been linked to heart disease and aging. The enzyme Sah1 may provide a tremendous opportunity to develop drugs that would be effec- tive against any number of age and neurologically related ailments that lower the quality of life. FIGURE 3. Role of Sah1 glutathione production Hoffman, Marion, Cornatzer, & Duerre, 1982 Diwakar, L., & Ravindranath, V. (2007). Inhibition of cystathionine-y-lyase leads to loss of glutathione and aggravation of mitochondrial dysfunction mediated by excitatory amino acid in the CNS. Neurochemistry International , 50, 418–426. Edited by Fischer, R., & Schillberg, S. (2004). Molecular Farming: Plant-made Pharmaceuticals and Technical Proteins. Weinheim: Wiley-VCH Verlag GmbH & Co. Hoffman, D. R., Marion, D., Cornatzer, W. E., & Duerre, J. A. (1982). Reduced availability of endogenously synthesized methionine for S-adenosylmethio- nine formation in methionine-dependen cancer cells (simian virus 40 transformation/human fibroblasts/S-adenosylhomocysteine). Journal of Bio- chemistry , Vol. 79, pp. 4248-4251, July 1982. Izawa, S., Inoue, S., & Kimura, A. (1995). Oxidative Stress Response in Yeast: Effect of Glutathione on Adaptation to Hydrogen Peroxide Stress in Saccha- romyces cerevisiae. FEBS Letters 368 , 73-76. Mager, W. H., de Boer, A. H., Siderius, M. H., & Voss, H.-P. (2000). Cellular responses to oxidative and asmotic stress. Cell Stress & Chaperones (pp. 73-75). Amsterdam: Cell Stress Society International 2000. Malanovic, N., Streith, I., Heimo, W., Gerald, R., Kohlwein, S. D., & Tehlivets, O. (n.d.). S-Adenosyl-L-homocysteine Hydrolase, Key Enzyme of Methylation Metabolism, Regulates Phosphatidylcholine Synthesis and Triacylglycerol Homeostasis in Yeast IMPLICATIONS FOR HOMOCYSTEINE AS A RISK FACTOR OF ATHEROSCLEROSIS*. THE JOURNAL OF BIOLOGICAL CHEMISTRY , VOL. 283, NO. 35, pp. 23989–23999,. I thank VGN and the staff from UVM: Bryan Ballif, Tim Hunter, Scott Tighe, Pat Reed, and Janet Murray for their support and materials. I also thanks Elizabeth Dolci from JSC for her guidance and enthusiasm. FIGURE 1. glutathione oxidation bcn.boulder.co.us/health/rmeha/rmehztra.htm PURPOSE The purpose of this study was to examine changes in protein expression of Saccharomyces cerevisiae after H 2 O 2 induced oxidative stress. Control Gel Experiment Gel Red = Site explored by author Yellow = Possible sites not put into mass spectrometry Blue = Sites investigated by others involved in the study FIGURE 2. Comparative 2 dimensional electrophoresis gel of total soluble proteins after oxidative stress. 2D Gels Saccharomyces cerevisiae (ATTC 18824) Baking Yeast TREATMENT 5,0 mM H 2 O 2 CONTROL CELL LYSIS ISOELECTRIC FOCUSING SDS-PAGE SPOT IDENTIFICATION BIOINFORMATICS TWO DIMENSIONAL ELECTROPHORESIS TRYPSINIZATION MASS SPECTROMETRY

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BACKGROUND

METHODS

RESULTS CONCLUSIONS

REFERENCES

ACKNOWLEDGMENTS

Response of Sah1 in Saccaromyces cerevisiae to H2O2 Induced Oxidative Stress

Greggory Perry, Department of Environmental and Health Sciences, Johnson State College,Johnson, VT 05656

Twelve sites were located on the two dimensional gels through visual inspection. Di�erent

intensities were sought out to determine if changes occurred due to oxidative stress. Of these

twelve sites seven were analyzed through mass spectrometry. One spot was chosen (see Fig. 2),

and the results of the mass spectrometry further investigated through bioinformatics which con-

ducted image analysis through the use of an algorithm. Seven peptides were produced by

trypsin, 21 peptides were from Uba1and 29 peptides from Sah1 when exposed to H2O2. Sah1

was chosen due to its connection with glutathione in the transmethylation metabolic pathway,

which is known to relieve oxidative stress in eukaryotes (see Fig. 3).

PROTEOMICS Proteomics is a technology that allows the researcher to observe the structure and function of the entire pro-

teome of an organism, tissue, or cell. It is particularly useful as a comparative study examining the a�ects of some

type of stress.

OXIDATIVE STRESS Oxidative stress is caused by the accumulation of reactive oxygen species (ROS) in the cells, or by an imbalance

of the cellular redox state. ROS include H2O2, O2 ¬ and oxygen species with unpaired valence electrons; more com-

monly known as free radicals. Responses can be enzymatic or nonenzymatic. Enzymatic responses include superox-

ide dismutase, catalase, and peroxidases. Glutathione is generally considered nonenymatic (Mager, de Boer, Sid-

erius, & Voss, 2000). These proteins and nonenymatic molecules, in particular glutathione, acts as an electron donor

to stabilize the ROS (see Fig. 1). Stress due to ROS may cause transcriptional changes or severe damage to cell DNA,

protein, membranes, and organelles. This can ultimately result in apoptosis (cell death) if exposure is su�cient.

PROTEOMICS The proteome is de�ned as the set of proteins that an organism can produce. Proteomics therefore,

is the study of all of the proteins in an organism or tissue. The technology is powerful because it is pos-

sible to connect certain proteins to functions that may have been previously unknown. The basic func-

tion of many proteins is known, but the interactions in a metabolic pathway may be far more wide-

spread. For example, the enzyme Sah1 is capable of producing glutathione indirectly through the trans-

methylation metabolic pathway.

Sah1 Sah1 regulates transmethylation reactions by catalyzing the degradation of S-adenosyl-L homo-

cysteine (AdoHcy) (Malanovic, et al., 2008). In this way Sah1 activity has a pleitrophic e�ect on lipid bio-

synthesis, signal transduction, and gene expression. Transmethylation reactions are also reversible

which compounds the intricacy of the reactions (see Fig. 3). This allows Sah1 to form AdoHcy from ad-

enosine and homocysteine. Metabolism of adenosine to ATP and inosine, and homocysteine to methion-

ine and cysteine produces glutathione (Ho�man, Marion, Cornatzer, & Duerre, 1982).

REGULATION It is possible that a greater quantity of the Sah1 enzyme was produced in response to oxidative

stress. An increase in the enzyme Sah1 will synthesize a greater amount of glutathione; a known com-

pound that cells use to circumvent oxidative stress (Mager, de Boer, Siderius, & Voss, 2000)(see Fig.1).

OXIDIZING GLUTATHIONE Glutathione has been identi�ed as playing a major role in protecting cells against free radicals, ra-

diation, carcinogens, and xenobiotics. Three amino acids are used in the production of glutathione, in-

cluding glutamate, cysteine and glycine (Izawa, Inoue, & Kimura, 1995). The sulfhydryl group of cysteine

serves as an electron donor and is responsible for the reducing capacity of glutathione (Edited by Fis-

cher & Schillberg, 2004). The defense mechanisms result in the oxidized form (GSSG), which is converted

and recycled back into glutathione (see Fig 3).

IMPLICATIONS Research of oxidative stress has far reaching e�ects well beyond what happens in S. cerevisiae. Oxi-

dative stress has been implicated in many human diseases including neurodegenerative disorders and

pathogenesis. (Diwakar & Ravindranath, 2007). Free radicals have been linked to heart disease and

aging. The enzyme Sah1 may provide a tremendous opportunity to develop drugs that would be e�ec-

tive against any number of age and neurologically related ailments that lower the quality of life.

FIGURE 3. Role of Sah1 glutathione productionHo�man, Marion, Cornatzer, & Duerre, 1982

Diwakar, L., & Ravindranath, V. (2007). Inhibition of cystathionine-y-lyase leads to loss of glutathione and aggravation of mitochondrial dysfunction mediated by excitatory amino acid in the CNS. Neurochemistry International , 50, 418–426.

Edited by Fischer, R., & Schillberg, S. (2004). Molecular Farming: Plant-made Pharmaceuticals and Technical Proteins. Weinheim: Wiley-VCH Verlag GmbH & Co.

Ho�man, D. R., Marion, D., Cornatzer, W. E., & Duerre, J. A. (1982). Reduced availability of endogenously synthesized methionine for S-adenosylmethio-nine formation in methionine-dependen cancer cells (simian virus 40 transformation/human �broblasts/S-adenosylhomocysteine). Journal of Bio-chemistry , Vol. 79, pp. 4248-4251, July 1982.

Izawa, S., Inoue, S., & Kimura, A. (1995). Oxidative Stress Response in Yeast: E�ect of Glutathione on Adaptation to Hydrogen Peroxide Stress in Saccha-romyces cerevisiae. FEBS Letters 368 , 73-76.

Mager, W. H., de Boer, A. H., Siderius, M. H., & Voss, H.-P. (2000). Cellular responses to oxidative and asmotic stress. Cell Stress & Chaperones (pp. 73-75). Amsterdam: Cell Stress Society International 2000.

Malanovic, N., Streith, I., Heimo, W., Gerald, R., Kohlwein, S. D., & Tehlivets, O. (n.d.). S-Adenosyl-L-homocysteine Hydrolase, Key Enzyme of Methylation Metabolism, Regulates Phosphatidylcholine Synthesis and Triacylglycerol Homeostasis in Yeast IMPLICATIONS FOR HOMOCYSTEINE AS A RISK FACTOR OF ATHEROSCLEROSIS*. THE JOURNAL OF BIOLOGICAL CHEMISTRY , VOL. 283, NO. 35, pp. 23989–23999,.

I thank VGN and the sta� from UVM: Bryan Ballif, Tim Hunter, Scott Tighe, Pat Reed, and Janet Murray for their support and materials. I also thanks Elizabeth Dolci from JSC for her guidance and enthusiasm.

FIGURE 1. glutathione oxidation

bcn.boulder.co.us/health/rmeha/rmehztra.htm

PURPOSE The purpose of this study was to examine

changes in protein expression of Saccharomyces

cerevisiae after H2O2 induced oxidative stress.Control Gel

Experiment GelRed = Site explored by authorYellow = Possible sites not put into mass spectrometryBlue = Sites investigated by others involved in the study

FIGURE 2. Comparative 2 dimensional electrophoresis gel of total soluble proteins after oxidative stress.

2D Gels

Saccharomyces cerevisiae

(ATTC 18824)Baking Yeast

TREATMENT5,0 mM H2O2

CONTROL

CELL LYSIS

ISOELECTRIC FOCUSING

SDS-PAGE

SPOT IDENTIFICATION

BIOINFORMATICS

TWO DIMENSIONAL ELECTROPHORESIS

TRYPSINIZATION

MASS SPECTROMETRY