FLOURESCENT TOOLS FOR THE STUDY OF METALLOTHIONEIN

DESCRIPTION (provided by applicant): We will deliver to life scientists fluorescent tools for the study of metallothionein and metallothionein- zinc signaling. Metallothionein-zinc signals have recently emerged as central step in many life-or-death signal pathways, including proliferation, apoptosis and metastasis. Arguably, MT-zinc signaling rivals calcium-calmodulin, IP3, and nitric oxide in ubiquity and essentiality. In action, the metallated protein, metallothionein (MT) can release 7 zinc ions into solution under oxidative or nitrosative stimulation (Fig 1). Conversely, The non-metalated protein, thionein (T) can sequester 7 zinc ions, terminating a zinc signal. Even more intriguing, both T and MT show evidence of moving through both plasma and organelle membranes, thus allowing zinc signals to be transposed among and between those compartments. We will deliver T and MT proteins that are labeled with fluorescent reporters at specific positions. These reporters constitute fluorescence-resonance-energy-transfer (FRET) systems that change color (emission spectra) when zinc is bound or released (Fig. 2). Because these proteins can be coaxed into tissues and thence into cytosol from the circulation, they can be used for both in vitro and in vivo research. With appropriate endoscopic and transdermal confocal or two-photon microscopy MT/T signaling can potentially be imaged in vivo and in vitro. MT/T and zinc signaling are integral to cell injury and death pathways as well as both metastatic and .healthy cell growth. Our tools will facilitate research in all of those areas and contribute to the advancement and development of diagnostic and therapeutic medical science.