title: Mitochondrial m-calpain plays a role in the release of truncated apoptosis-inducing factor from the mitochondria.
abstract: Calpains, calcium-dependent cysteine proteases, are involved in a variety of cellular processes. We have reported on the characteristics of mitochondrial mu-calpain and have shown that ERp57-associated mitochondrial mu-calpain cleaves the apoptosis-inducing factor (AIF) to a truncated form (tAIF). In addition, we found an unknown mitochondrial calpain. In this study, we identified and characterized this undescribed mitochondrial calpain in rat liver mitochondrial intermembrane space. The mitochondrial mu- and unknown calpains were separated by DEAE-Sepharose column chromatography. We immunoprecipitated the unknown calpain with anti-calpain small subunit and identified it as calpain 2 (m-calpain large subunit) by nanoflow-LC-MS/MS analysis and database searching. Because the identified mitochondrial calpain was stained with anti-m-calpain large subunit antibody, we named it mitochondrial m-calpain. The Ca(2+) dependency of mitochondrial m-calpain was similar to that of cytosolic m-calpain. Immunoprecipitation analyses showed that mitochondrial m-calpain is associated with a 75-kDa glucose-regulated protein, a member of the heat shock protein 70 family. We also investigated the involvement of mitochondrial m-calpain in the release of tAIF from mitochondria. Calpain inhibitor, PD150606, an anti-voltage-dependent anion channel (VDAC), and anti-Bax antibodies prevented the release of tAIF from mitochondria. In addition, we found that mitochondrial m-calpain truncated VDAC in Ca(2+)-dependent manner. This cleavage of VDAC promotes the mitochondrial accumulation of Bax and the release of tAIF from mitochondria. The accumulated Bax in mitochondrial outer membrane was co-immunoprecipitated with VDAC. Our results demonstrated that mitochondrial m-calpain plays a role in the release of tAIF from mitochondria by cleaving VDAC, and tAIF is released through VDAC-Bax pores.
authors.affiliation: Department of Biochemistry and Biotechnology, Hirosaki University, Hirosaki 036-8561, Japan.
authors.forename: Taku
authors.initials: T
authors.lastname: Ozaki
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Yamashita
authors.forename: Sei-Ichi
authors.initials: S
authors.lastname: Ishiguro
mesh_terms: D000179:Acrylates / Q000494:pharmacology; D000818:Animals; D051033:Apoptosis Inducing Factor / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D050994:Voltage-Dependent Anion Channels / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D000179:Acrylates; D051033:Apoptosis Inducing Factor; C494828:Bax protein, rat; D018840:HSP70 Heat-Shock Proteins; D024101:Mitochondrial Proteins; C100442:PD 150606; D050994:Voltage-Dependent Anion Channels; D051028:bcl-2-Associated X Protein; D002154:Calpain; C449000:m-calpain; C513196:PDIA3 protein, rat; D019704:Protein Disulfide-Isomerases; D002118:Calcium