1. 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
BACKGROUND RESULTS CONCLUSIONS
PROTEOMICS Twelve sites were located on the two dimensional gels through visual inspection. Di erent
Proteomics is a technology that allows the researcher to observe the structure and function of the entire pro- intensities were sought out to determine if changes occurred due to oxidative stress. Of these PROTEOMICS
teome of an organism, tissue, or cell. It is particularly useful as a comparative study examining the a ects of some twelve sites seven were analyzed through mass spectrometry. One spot was chosen (see Fig. 2),
The proteome is de ned as the set of proteins that an organism can produce. Proteomics therefore,
type of stress. and the results of the mass spectrometry further investigated through bioinformatics which con-
is the study of all of the proteins in an organism or tissue. The technology is powerful because it is pos-
ducted image analysis through the use of an algorithm. Seven peptides were produced by
sible to connect certain proteins to functions that may have been previously unknown. The basic func-
trypsin, 21 peptides were from Uba1and 29 peptides from Sah1 when exposed to H2O2. Sah1
OXIDATIVE STRESS tion of many proteins is known, but the interactions in a metabolic pathway may be far more wide-
was chosen due to its connection with glutathione in the transmethylation metabolic pathway,
Oxidative stress is caused by the accumulation of reactive oxygen species (ROS) in the cells, or by an imbalance spread. For example, the enzyme Sah1 is capable of producing glutathione indirectly through the trans-
which is known to relieve oxidative stress in eukaryotes (see Fig. 3).
of the cellular redox state. ROS include H2O2, O2 ¬ and oxygen species with unpaired valence electrons; more com- methylation metabolic pathway.
monly known as free radicals. Responses can be enzymatic or nonenzymatic. Enzymatic responses include superox- 2D Gels
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,
Sah1
protein, membranes, and organelles. This can ultimately result in apoptosis (cell death) if exposure is su cient.
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-
PURPOSE synthesis, signal transduction, and gene expression. Transmethylation reactions are also reversible
The purpose of this study was to examine
which compounds the intricacy of the reactions (see Fig. 3). This allows Sah1 to form AdoHcy from ad-
changes in protein expression of Saccharomyces
enosine and homocysteine. Metabolism of adenosine to ATP and inosine, and homocysteine to methion-
cerevisiae after H2O2 induced oxidative stress.
Control Gel ine and cysteine produces glutathione (Ho man, Marion, Cornatzer, & Duerre, 1982).
FIGURE 3. Role of Sah1 glutathione production
Ho man, Marion, Cornatzer, & Duerre, 1982
REGULATION
It is possible that a greater quantity of the Sah1 enzyme was produced in response to oxidative
FIGURE 1. glutathione oxidation stress. An increase in the enzyme Sah1 will synthesize a greater amount of glutathione; a known com-
bcn.boulder.co.us/health/rmeha/rmehztra.htm
pound that cells use to circumvent oxidative stress (Mager, de Boer, Siderius, & Voss, 2000)(see Fig.1).
METHODS
Red = Site explored by author Experiment Gel
Yellow = Possible sites not put into mass spectrometry FIGURE 2. Comparative 2
Blue = Sites investigated by others involved in the study dimensional electrophoresis gel
of total soluble proteins after
oxidative stress. OXIDIZING GLUTATHIONE
Glutathione has been identi ed as playing a major role in protecting cells against free radicals, ra-
Saccharomyces REFERENCES diation, carcinogens, and xenobiotics. Three amino acids are used in the production of glutathione, in-
cerevisiae Diwakar, L., & Ravindranath, V. (2007). Inhibition of cystathionine-y-lyase leads to loss of glutathione and aggravation of mitochondrial dysfunction cluding glutamate, cysteine and glycine (Izawa, Inoue, & Kimura, 1995). The sulfhydryl group of cysteine
mediated by excitatory amino acid in the CNS. Neurochemistry International , 50, 418–426.
(ATTC 18824) serves as an electron donor and is responsible for the reducing capacity of glutathione (Edited by Fis-
Baking Yeast BIOINFORMATICS Edited by Fischer, R., & Schillberg, S. (2004). Molecular Farming: Plant-made Pharmaceuticals and Technical Proteins. Weinheim: Wiley-VCH Verlag
GmbH & Co.
cher & Schillberg, 2004). The defense mechanisms result in the oxidized form (GSSG), which is converted
Ho man, D. R., Marion, D., Cornatzer, W. E., & Duerre, J. A. (1982). Reduced availability of endogenously synthesized methionine for S-adenosylmethio-
TREATMENT nine formation in methionine-dependen cancer cells (simian virus 40 transformation/human broblasts/S-adenosylhomocysteine). Journal of Bio- and recycled back into glutathione (see Fig 3).
CONTROL chemistry , Vol. 79, pp. 4248-4251, July 1982.
5,0 mM H2O2
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.
CELL LYSIS MASS 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.
SPECTROMETRY Malanovic, N., Streith, I., Heimo, W., Gerald, R., Kohlwein, S. D., & Tehlivets, O. (n.d.). S-Adenosyl-L-homocysteine Hydrolase, Key Enzyme of Methylation
ISOELECTRIC
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,. IMPLICATIONS
FOCUSING Research of oxidative stress has far reaching e ects well beyond what happens in S. cerevisiae. Oxi-
TRYPSINIZATION dative stress has been implicated in many human diseases including neurodegenerative disorders and
SDS-PAGE ACKNOWLEDGMENTS 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-
I thank VGN and the sta from UVM: Bryan Ballif, Tim Hunter, Scott Tighe, Pat Reed,
TWO DIMENSIONAL SPOT tive against any number of age and neurologically related ailments that lower the quality of life.
and Janet Murray for their support and materials. I also thanks Elizabeth Dolci from JSC
ELECTROPHORESIS IDENTIFICATION
for her guidance and enthusiasm.