The long-term objective is to identify initial signals that are capable of inducing stress responses. The hypothesis to be tested is that modification of protein thiols (eg., oxidation, alkylation, carboxylation, etc.) can initiate changes in protein structure that can act as signals for the induction of heat shock proteins (Hsp), thermotolerance, and the genes which code for gamma-glutamylcysteine synthetase, the rate limiting enzyme for glutathione (GSH) synthesis. Cellular resistance to hyperthermia, radiation, and chemotherapeutic drugs can be increased by expression of Hsp, thermotolerance, and increased GSH. An understanding of the molecular regulation of this response may lead to strategies which increase therapeutic effectiveness.
Aim 1 will determine if the initial events that occur when thermotolerance is induced by modification of protein thiols are changes in either protein stability or solubility that subsequently lead to alterations in the state of Hsp 27 phosphorylation, and/or cause proteins to bind to Hsc/Hsp 70. Human cells will be exposed to various stresses to produce various types of modified thiols. Protein denaturation will be assessed by differential scanning calorimetry (DSC). The phosphorylation state of Hsp 27 will be determined by immunoprecipitation, and isoelectric focusing. Differential centrifugation will be used to determine the subcellular location of modified proteins. Immunoprecipitation and indirect immunofluoroscopy will be used to determine if modified proteins are recognized by Hsc/Hsp 70.
Aim 2 will determine if specific types of protein thiol modifications, that are capable of inducing transcription of Hsp 70, can initiate an alteration in protein conformation, as measured by protein stability, solubility, and hydrophobicity such that the protein now becomes a substrate for binding to Hsc/Hsp 70. Gel mobility shift assays, and nuclear run-ons will be used to demonstrate that the chemical stresses used in aim 1 induce transcription of Hsp 70. These same stresses will then be used to produce specific types of thiol modifications in BSA, beta-lactoglobulin, and fatty acid synthase. The modified proteins will be isolated based on the type of modification. The stability and hydrophobicity of these modified proteins will be compared to heat denatured and native protein using DSC,ethanol solubility, and reverse phase HPLC. A cell free gel mobility shift assay system will be used to determine if isolated, modified proteins activate the heat shock transcription factor.
Aims 3 & 4 will determine if modification of protein thiols can induce the transcription of the genes that code for the catalytic (aim 3) and regulatory (aim 4) subunits of gamma-glutamylcysteine synthetase. As part of these aims, the 5 prime DNA flanking sequences responsible for transcriptional induction will be identified. RT-PCR will be used to obtain cDNA, which in turn will be used to screen a genomic library. After identification of the 5 prime mRNA terminus, the 5 prime DNA flanking sequences will be cloned and sequenced. This will be used to construct deletion mutants that will be ligated into a pCAT expression vector. CAT expression, gel mobility shift and DNase foot print analysis will be used to positively demonstrate that a chemical stress which produces protein thiol modification, induces transcription of gamma-glutamylcysteine synthetase subunits.
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