Senior Scholar Award in Aging
Art Gafni, Ph.D.
University of Michigan

Single Molecule Studies of Age-Related Alterations in Heat Shock Factor 1

A decline in the capacity of living organisms to respond vigorously to external stresses (elevated temperature, damaging radiation, reactive chemical agents, etc.) and to maintain homeostasis is a hallmark of aging and is largely responsible for the age-related increase in mortality. Young cells respond to environmental stresses by vigorously expressing a group of special proteins, collectively called heat shock proteins (HSPs), that protect existing cellular proteins from becoming damaged and assist newly synthesized proteins to fold correctly. A prominent heat shock protein is HSP70 whose production in young cells in response to stress is elevated many fold. Cells of senescent organisms show a marked decline in their ability to elevate the expression of HSP70 and, therefore, possess a much-reduced ability to cope with external challenges. Previous research has revealed that the age-related reduction in HSP70 production correlates with a decreased fidelity of binding of the HSP70 transcription factor (called HSF1) to a specific region on the DNA, the heat shock element (HSE).

Exposure of young cells to stress triggers a series of events: the HSF1 monomer is converted to a DNA–binding trimer that migrates from the cytosol to the nucleus, binds to the HSE, undergoes a phosphorylation reaction and elicits HSP70 expression. Why this process becomes defective during aging is not known, and previous work in this area has been hindered by two experimental constraints: 1. the low abundance of HSF1 in mammalian cells (from which the old form of the protein has to be extracted); 2. the structural and functional heterogeneity of HSF1 molecules in old tissues. These challenges limit the effectiveness of traditional biochemical or biophysical tools. We plan to overcome these difficulties by using novel methodologies that enable studies at the single molecule level. Single molecule spectroscopy (SMS) allows us to work with minute amounts of material (tens to hundreds of molecules), and to discern mechanistic aspects which, when using conventional approaches, are masked by ensemble averaging. Our long-term goal is to elucidate the molecular origin of the age related decline in the functional fidelity of HSF1 and to test the hypothesis that its reduced DNA binding affinity, with the resultant reduction in HSP70 production, is brought about by conformational changes in the HSF1 monomer/trimer. Specifically our research is directed towards the following two aims:

  1. To characterize the structural changes responsible for HSF1 activation and trimerization during heat sensing. We work to characterize the structural changes that take place in monomeric HSF1 upon exposure to elevated temperature, to identify the domains in this heat-induced conformation responsible for trimer formation and the interactions that feature in stabilizing the trimer.
  2. To identify and characterize the age-related alterations in HSF1 and to determine how these affect its activation, trimerization and HSE binding. This study will be done using HSF1 purified from young and old rats and fluorescently labeled. Using SMS, we will determine what structural changes in old-HSF1 are responsible for its reduced binding affinity to the HSE. These studies will also reveal whether old-HSF1 is structurally heterogeneous leading to the formation of several classes of trimers.

This collaborative research proposal will apply the power of single molecule manipulation to determine the structural basis for the altered behavior of old HSF1, and will provide new insight into a critical aging phenomenon, the severe attenuation of the stress response. More generally, we expect that this research will provide a molecular explanation for how protein aging negatively impacts transcriptional activation of target genes.

Contact Dr. Gafni.