Stress, Aging and Molecular Chaperones
Studies from a number of laboratories, including ours, have provided clear evidence of an age-dependent attenuation in induction of the heat shock transcriptional response in a variety of model aging systems. In other words, the ability of a cell (or organism) to mount a genomic response to stress in inversely related to its biological age. We are currently working on two complementary research projects in the laboratory: one is aimed at understanding the mechanism of the age-dependent changes in the regulation and function of the heat shock transcription factor (human HSF1) in human diploid fibroblasts, and the other is to evaluate the role of the MAP kinase signaling cascades, notably Jun-N-terminal kinase (JNK), in modulating the function and activity of hHSF1. Our hypothesis is that redox is an important mechanism in regulating the structure and function of HSF1, and that oxidation and disulfide crosslinking of cysteines is an off-switch for the activation and trimerization of hHSF1. Pharmacological, genetic, and biophysical means are used to determine if and how changes in the redox status of cysteines of HSF1 modulate its structure and function. To this end, we have recently developed the necessary reagents and protocols that allow for the resolution, detection, and quantitation of the reduced versus the disulfide crosslinked conformers of hHSF1 and have learned that disulfide crosslinking locks the hHSF1 in a monomeric conformation that is resistant to activation and trimerization. We intend to further map the cysteine disulfide crosslink using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Changes in the relative abundance and distribution of the redox conformers of hHSF1 of cells maintained under various experimental conditions will be determined and correlated to changes in the function of hHSF1.
Activation of hHSF1 is a multistep process and includes hyperphosphorylation of the protein. In a separate research project, we have been investigating the role of the Jun-N-terminal kinase (JNK) in phosphorylating and regulating the function of hHSF1. It may be noteworthy that JNK is activated by heat stress (in fact JNK is aka stress kinase), and that the activity of JNK is down regulated in aging cells. In our studies, we showed that activators of JNK enhance, and inhibitor blocked hsp70 promoter-driven reporter gene expression. Furthermore, the C-terminal transcription activation domain of HSF1 contains a serine motif that conforms to the conserved phospho-acceptor sites of JNK substrates, which was phosphorylated by activated JNK in vitro and in vivo. We are interested in understanding the functional consequence and biological relevance of this phosphorylation event.