Isolation and identification of novel neutrophil-activating cryptides hidden in mitochondrial cytochrome C.

Although it is known that neutrophils infiltrate damaged sites immediately after tissue injury, the endogenous factors that induce their acute transmigration and activation have not been thoroughly investigated. For the candidates of those factors, we recently discovered two novel neutrophil-activating cryptides, mitocryptide-1 (MCT-1) and mitocryptide-2 (MCT-2), hidden in mitochondrial proteins. In addition, many unknown neutrophil-activating peptides other than MCT-1 and MCT-2 were also observed during their purification.

Mitocryptide-2: purification, identification, and characterization of a novel cryptide that activates neutrophils.

Neutrophils are a class of leukocytes involved in innate immunity by monitoring and scavenging invading microorganisms and toxic substances. The actions of neutrophils in damaged tissues are still not well understood, particularly in the early stage of inflammation, and as-yet-unknown neutrophil-activating substances are proposed to induce their acute transmigration and activation. Here, we isolated and identified from porcine hearts a neutrophil-activating peptide.

Discovery of mitocryptide-1, a neutrophil-activating cryptide from healthy porcine heart.

Although neutrophils are known to migrate in response to various chemokines and complement factors, the substances involved in the early stages of their transmigration and activation have been poorly characterized to date. Here we report the discovery of a peptide isolated from healthy porcine hearts that activated neutrophils. Its primary structure is H-Leu-Ser-Phe-Leu-Ile-Pro-Ala-Gly-Trp-Val-Leu-Ser-His-Leu-Asp-His-Tyr-Lys-Arg-Ser-Ser-Ala-Ala-OH, and it was indicated to originate from mitochondrial cytochrome c oxidase subunit VIII.

Cryptide signaling: Amphiphilic peptide-induced exocytotic mechanisms in mast cells.

Amphiphilic peptides with positive charges such as substance P (SP) and mastoparan (MP) are known to induce exocytosis in rat peritoneal mast cells. To elucidate whether and how intracellular Ca(2+) signaling is involved in the peptide-induced exocytosis, here we investigated the relationships between an increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and exocytosis caused by SP and MP. SP and MP induced exocytosis coinciding with an initial rapid and transient [Ca(2+)](i) increase, but not with a sustained increase.

A mastoparan analog without lytic effects and its stimulatory mechanisms in mast cells.

Mastoparan, a tetradecapeptide isolated from wasp venom, is known to not only induce the secretion of histamine but also cause cell lysis in rat peritoneal mast cells. This lytic effect makes investigations concerning MP-induced signaling mechanisms difficult. Here, we report that a mastoparan derivative peptide, [Lys(10), Leu(13)]mastoparan, also designated "mas 11'', induces exocytosis with greater activity than mastoparan without the undesired lytic effect.

A selective requirement for copper-dependent activation of cytochrome c oxidase by Cox17p.

Cox17p is cloned from yeast as a chaperone to deliver copper to the mitochondria of assembly for cytochrome c oxidase (CCO). In mammals, CCO is a key enzyme for cellular respiration and a defect in its function is associated with severe neonatal or infantile lactic acidosis and early death. Recently, we found that Cox17p is not only required for mitochondrial oxidative phosphorylation but also is essential for embryonic growth and development in COX17 gene-deficient mice.

The chemical synthesis and binding affinity to the EGF receptor of the EGF-like domain of heparin-binding EGF-like growth factor (HB-EGF).

Heparin-binding EGF-like growth factor (HB-EGF), which belongs to the EGF-family of growth factors, was isolated from the conditioned medium of macrophage-like cells. To investigate the effect of N- and C-terminal residues of the EGF-like domain of HB-EGF in the binding affinity to the EGF receptor on A431 cell. We synthesized HB-EGF(44-86) corresponding to the EGF-like domain of HB-EGF and its N- or C-terminal truncated peptides.

Mammalian copper chaperone Cox17p has an essential role in activation of cytochrome C oxidase and embryonic development.

Cox17p is essential for the assembly of functional cytochrome c oxidase (CCO) and for delivery of copper ions to the mitochondrion for insertion into the enzyme in yeast. Although this small protein has already been cloned or purified from humans, mice, and pigs, the function of Cox17p in the mammalian system has not yet been elucidated. In vitro biochemical data for mammalian Cox17p indicate that the copper binds to the sequence -KPCCAC-.

Characterization and identification of promoter elements in the mouse COX17 gene.

Cox17p, essential for the assembly of functional cytochrome c oxidase (CCO) in Saccharomyces cerevisiae, has been believed to deliver copper ions to the mitochondrion for insertion into the enzyme. We have recently isolated an approximately 20 kb genomic fragment of the mouse COX17. Reporter assay experiments have shown that most of the promoter activity was restricted to a 0.