title: Intravitreal injection or topical eye-drop application of a μ-calpain C2L domain peptide protects against photoreceptor cell death in Royal College of Surgeons' rats, a model of retinitis pigmentosa.
abstract: Mitochondrial μ-calpain initiates apoptosis-inducing factor (AIF)-dependent apoptosis in retinal photoreceptor degeneration. Mitochondrial μ-calpain inhibitors may represent therapeutic targets for the disease. Therefore, we sought to identify inhibitors of mitochondrial calpains and determine their effects in Royal College of Surgeons' (RCS) rats, an animal model of retinitis pigmentosa (RP). We synthesized 20-mer peptides of the C2-like (C2L) domain of μ-calpain. Two μ-calpain peptides N2 and N9 inhibited mitochondrial μ-calpain activity (IC(50); 892 and 498nM, respectively), but not other proteases. Western blotting showed that 50μM of both μ-calpain peptides caused specific degradation of mitochondrial μ-calpain. Three-dimensional structure of calpains suggested that the peptides N2 and N9 corresponded to the regions forming salt bridges between the protease core domain 2 and the C2L domain. We determined the inhibitory regions of μ-calpain peptides N2 and N9 using 10-mers, and one peptide, N2-10-2, inhibited the activity of mitochondrial μ-calpain (IC(50); 112nM). We next conjugated the peptide N2-10-2 to the C-terminal of HIV-1 tat (HIV), a cell-penetrating peptide. Using isolated rat liver mitochondria, 50μM HIV-conjugated μ-calpain N2-10-2 peptide (HIV-Nμ, IC(50); 285nM) significantly inhibited AIF truncation. The intravitreal injection of 20mM HIV-Nμ also prevented retinal photoreceptor apoptosis determined by TUNEL staining, and preserved retinal function assessed by electroretinography in RCS rats. Topical application of 40mM HIV-Nμ also prevented apoptosis of retinal photoreceptors in RCS rats. Our results demonstrate that HIV-Nμ, a peptide inhibitor of mitochondrial μ-calpain, offers a new modality for treating RP.
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Japan.
authors.forename: Taku
authors.initials: T
authors.lastname: Ozaki
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Nakazawa
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Yamashita
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sorimachi
authors.forename: Shoji
authors.initials: S
authors.lastname: Hata
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tomita
authors.forename: Hitomi
authors.initials: H
authors.lastname: Isago
authors.forename: Ayaka
authors.initials: A
authors.lastname: Baba
authors.forename: Sei-Ichi
authors.initials: S
authors.lastname: Ishiguro
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002154:Calpain / Q000008:administration & dosage / Q000138:chemical synthesis / Q000737:chemistry; D004195:Disease Models, Animal; D006801:Humans; D058449:Intravitreal Injections; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D009883:Ophthalmic Solutions; D010455:Peptides / Q000008:administration & dosage / Q000138:chemical synthesis / Q000737:chemistry; D010786:Photoreceptor Cells / Q000166:cytology / Q000187:drug effects / Q000473:pathology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D012174:Retinitis Pigmentosa / Q000188:drug therapy / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000008:administration & dosage / Q000737:chemistry
chemical_list: D009883:Ophthalmic Solutions; D010455:Peptides; D054322:tat Gene Products, Human Immunodeficiency Virus; D002154:Calpain; C449002:mu-calpain