GENERATION OF MITOCHONDRIAL HSP70 TRANSGENIC MICE





Radhika Puttagunta and Tomas Prolla

Department of Genetics and Medical Genetics, University of Wisconsin-Madison, 445 Henry Mall, Room 118, Madison, WI. 55706



Emerging research indicates that mitochondria play a central role in the aging process. The epicenter of oxidative damage is the mitochondrion, where radical oxygen species (ROS) are created as byproducts of energy metabolism. ROS can damage proteins, lipids and DNA. Severe damage can lead to mitochondrial dysfunction, such as alterations in mitochondria structure, membrane potential, release of cytochrome c from the inner membrane matrix and induction of the apoptotic pathway. Cytosolic inducible HSP70 (heat shock protein of 70kDa) has shown a protective role against protein damage, oxidative stress, ischemia, apoptosis and mitochondrial dysfunction in cell lines, drosophila, and/or mice. The HSP70 family is large and highly conserved throughout evolution. First discovered as a response to heat shock, HSP70 is now known to have multiple functions. Of the human HSP70 family, mitochondrial HSP70 (MTHSP75) is most homologous to bacterial Hsp70 (DnaK). MTHSP75 is constitutively transcribed in the nucleus but localized to the inner membrane of the mitochondria, where it serves as an ATPase transporter of proteins into the mitochondria. Once proteins arrive inside the mitochondria, MTHSP75 is able to fold them into their proper conformation. It also serves as a chaperone of proteins synthesized on mitochondrial ribosomes and can help degrade misfolded proteins within the mitochondrion. Under stress, MTHSP75 refolds denatured proteins properly and prevents aggregation. MTHSP75 is also known as glucose regulated protein of 75kD (GRP75). Another grp family member, GRP78, is a resident endoplasmic reticulum HSP70 family member involved in Ca++ homeostasis. GRP75 (MTHSP75) may contain a calcium binding domain and also be involved in mitochondrial Ca++ homeostasis. By creating a transgenic mouse overexpressing human MTHSP75 we are studying the role of this protein in protection against protein oxidative damage/aging and maintaining the integrity of the mitochondrion. The human MTHSP75 cDNA was cloned into the pCAGGS vector. Expression is driven at high levels in the heart and muscle, and at lower levels in the brain, by the chicken Beta-actin promoter and CMV enhancer. Five transgenic mice were generated, two of which died after birth prior to weaning. A high copy number of MTHSP75 leads to smaller animals and in some cases an early death. A low copy number founder of MTHSP75 has thus far shown no phenotype varying from wildtype C57Bl/6 mice. Northern analysis shows expression in muscle, heart and brain. Western analysis and immunohistochemistry confirm these results. Electron microscopy has shown that the protein is localizing to the mitochondria. These mice will be further analyzed for resistance to protein oxidative damage, retarding of aging and/or resistance to mitochondrial dysfunction under stressful conditions.




Key words: mitochondrial HSP70, oxidative damage, aging







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