Mitochondrial import of PKCepsilon is mediated by HSP90: a role in cardioprotection from ischaemia and reperfusion injury.
OBJECTIVE
Protein kinase C epsilon (PKCepsilon) is critical for cardiac protection from ischaemia and reperfusion (IR) injury. PKCepsilon substrates that mediate cytoprotection reside in the mitochondria. However, the mechanism enabling mitochondrial translocation and import of PKCepsilon to enable phosphorylation of these substrates is not known. Heat shock protein 90 (HSP90) is a cytoprotective protein chaperone that participates in mitochondrial import of a number of proteins. Here, we investigated the role of HSP90 in mitochondrial import of PKCepsilon.


RESULTS
Using an ex vivo perfused rat heart model of IR, we found that PKCepsilon translocates from the cytosol to the mitochondrial fraction following IR. Immunogold electron microscopy and mitochondrial fractionation demonstrated that following IR, mitochondrial PKCepsilon is localized within the mitochondria, on the inner mitochondrial membrane. Pharmacological inhibition of HSP90 prevented IR-induced interaction between PKCepsilon and the translocase of the outer membrane (Tom20), reduced mitochondrial import of PKCepsilon, and increased necrotic cell death by approximately 70%. Using a rational approach, we designed a 7-amino acid peptide activator of PKCepsilon, derived from an HSP90 homologous sequence located in the C2 domain of PKCepsilon (termed psiepsilonHSP90). Treatment with this peptide (conjugated to the cell permeating TAT protein-derived peptide, TAT(47-57)) increased PKCepsilon-HSP90 protein-protein interaction, enhanced mitochondrial translocation of PKCepsilon, increased phosphorylation and activity of an intra-mitochondrial PKCepsilon substrate, aldehyde dehydrogenase 2, and reduced cardiac injury in ex vivo and in vivo models of myocardial infarction.


CONCLUSIONS
Our results suggest that HSP90-mediated mitochondrial import of PKCepsilon plays an important role in the protection of the myocardium from IR injury.
Budas
Grant R
GR
Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305-5174, USA.
Churchill
Eric N
EN
Disatnik
Marie-Hélène
MH
Sun
Lihan
L
Mochly-Rosen
Daria
D
D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D000595:Amino Acid Sequence; D000818:Animals; D019610:Cytoprotection; D004195:Disease Models, Animal; D015195:Drug Design; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000737:chemistry / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008297:Male; D026901:Membrane Transport Proteins; D008929:Mitochondria, Heart / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D051336:Mitochondrial Membranes / Q000201:enzymology; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009200:Myocardial Contraction; D015428:Myocardial Reperfusion Injury / Q000201:enzymology / Q000473:pathology / Q000503:physiopathology / Q000517:prevention & control*; D009206:Myocardium / Q000201:enzymology* / Q000473:pathology; D009336:Necrosis; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D021381:Protein Transport; D051381:Rats; D017208:Rats, Wistar; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D020127:Recovery of Function; D016415:Sequence Alignment
D020536:Enzyme Activators; D018841:HSP90 Heat-Shock Proteins; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D009842:Oligopeptides; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; C489998:Tomm20 protein, rat; D000444:Aldehyde Dehydrogenase; D000071396:Aldehyde Dehydrogenase, Mitochondrial; C505112:Aldh2 protein, rat; C497257:Prkce protein, rat; D051744:Protein Kinase C-epsilon