Takahiro Kihara

Identification of Novel γ-Secretase-associated Proteins in Detergent-resistant Membranes from Brain

Background: In AD, APP can be processed in lipid rafts, and γ-secretase-associated proteins (GSAPs) can affect Aβ production. Results: We identify novel GSAPs in detergent-resistant membranes (DRMs). Conclusion: VDAC1 and CNTNAP1 associate with γ-secretase in DRMs and affect APP processing with less effect on Notch processing. Significance: Novel GSAPs that regulate Aβ production can be used as AD therapeutic targets. In Alzheimer disease, oligomeric amyloid β-peptide (Aβ) species lead to synapse loss and neuronal death. γ-Secretase, the transmembrane protease complex that mediates the final catalytic step that liberates Aβ from its precursor protein (APP), has a multitude of substrates, and therapeutics aimed at reducing Aβ production should ideally be specific for APP cleavage. It has been shown that APP can be processed in lipid rafts, and γ-secretase-associated proteins can affect Aβ production. Here, we use a biotinylated inhibitor for affinity purification of γ-secretase and associated proteins and mass spectrometry for identification of the purified proteins, and we identify novel γ-secretase-associated proteins in detergent-resistant membranes from brain. Furthermore, we show by small interfering RNA-mediated knockdown of gene expression that a subset of the γ-secretase-associated proteins, in particular voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1), reduced Aβ production (Aβ40 and Aβ42) by around 70%, whereas knockdown of presenilin 1, one of the essential γ-secretase complex components, reduced Aβ production by 50%. Importantly, these proteins had a less pronounced effect on Notch processing. We conclude that VDAC1 and CNTNAP1 associate with γ-secretase in detergent-resistant membranes and affect APP processing and suggest that molecules that interfere with this interaction could be of therapeutic use for Alzheimer disease.

Localization of Bradykinin B2 Receptor in the Follicles of Porcine Ovary and Increased Expression of Matrix Metalloproteinase-3 and -20 in Cultured Granulosa Cells by Bradykinin Treatment

Abstract We have recently shown that not only bradykinin, but also all components for the production of bradykinin, can be detected within the follicle of porcine ovaries. To elucidate the relevance of the intrafollicular bradykinin-producing system to its physiological role, we investigated the distribution of bradykinin receptor (B2R) mRNA and the protein in porcine ovaries. A cDNA encoding porcine B2R was first cloned from a porcine uterus cDNA library. The receptor mRNA was scarcely detected in the ovary by Northern blot analysis. Polymerase chain reaction analysis with total RNAs isolated from the ovary and from granulosa cells of small and large follicles demonstrated the ovarian expression of B2R mRNA. The B2R protein was detected by Western blot analysis in extracts of isolated granulosa cells. In situ hybridization of B2R mRNA and immunohistochemical analysis of the protein revealed that the receptor is expressed in the theca and granulosa cells of all growing follicles. The effect of bradykinin on the expression of some matrix metalloproteinase (MMP) genes was examined using isolated granulosa cells. Bradykinin treatment induced MMP-3 and MMP-20 gene expression to an extreme degree. The expression of MT1-MMP was also affected by bradykinin treatment. These results suggest that MMPs play a role in follicle rupture during ovulation. The present study provides new information regarding the mechanisms of bradykinin-induced ovulation in porcine ovaries.

Identification of Components of the Intrafollicular Bradykinin-Producing System in the Porcine Ovary

Abstract As a step in elucidating the biological role of plasma kallikrein (PK) present in the follicular fluid of mammalian ovaries, we examined pig ovary fluid to determine its constituent activators and substrates. Using the inactive precursor form of plasma kallikrein (prePK) as a substrate, we purified an enzyme capable of activating this protein. The prePK-activating enzyme was shown to be the active enzyme blood coagulation factor XIIa. We also isolated high molecular weight kininogen (HMW-K) from the same fluid. Incubation of HMW-K with the ovarian follicular fluid PK resulted in the production of the nanopeptide bradykinin (BK). Expression of prePK, blood coagulation factor XII, and HMW-K was examined by Northern blot analysis using ovary and liver poly(A)+ RNA. All these transcripts were found in the liver, but none were found in the ovary. In addition, it was found that BK levels in the fluid derived from the small follicles were approximately 6 times higher than those from medium and large follicles. These results demonstrate the presence of a BK-producing system in the ovarian follicles and suggest the physiological importance of this peptide hormone in the early stages of follicular development and at ovulation.

Identification of porcine follipsin as plasma kallikrein, and its possible involvement in the production of bradykinin within the follicles of porcine ovaries

To determine the identity of porcine follipsin, a plasma kallikrein cDNA clone was isolated from a porcine liver cDNA library. The clone encoded a protein of 643 amino acids, exhibiting identities 79.7, 72.9, and 74.4% homologous to human, rat, and mouse plasma prekallikrein, respectively. The amino acid sequences of four internal peptides isolated from the tryptic digest of follipsin were all found in the deduced sequence. Authentic plasma kallikrein was purified from porcine plasma and compared directly with follipsin. Actions on synthetic substrates and behaviors with proteinase inhibitors were indistinguishable between these two enzymes. The cDNA was expressed in COS‐7 cells and the recombinant protein was prepared from the culture medium of these cells. No active enzyme could be obtained, but the expressed protein was reacted with anti‐porcine plasma kallikrein antibody. The mRNA was detected only in the liver in northern blot analysis. RT‐PCR analysis of RNAs revealed that porcine testis, in addition to the liver, expressed the corresponding mRNA. In the ovary, plasma kallikrein was detected as a main band of the active form (Mr = 85,000) and the band of the minor inactive precursor form (Mr = 80,000), respectively. In contrast, the liver extract contained only the precursor form. Incubation of high molecular weight kininogen with follicular fluid plasma kallikrein resulted in an increased production of bradykinin. Further, the fresh fluid of large‐sized follicles of porcine ovaries was found to contain this peptide hormone at a detectable level. These results indicate that porcine follipsin is plasma kallikrein, and that the enzyme may be involved in the production of bradykinin within ovarian follicles. Mol. Reprod. Dev. 57:79–87, 2000.

Expression of Bradykinin B2 Receptor in the Mouse Ovary

Abstract The amounts of [1-5]-bradykinin in ovary extracts were determined using gonadotropin-treated immature female mice. The bradykinin levels in the ovary were high at 2, 6, and 48 hr after injection of human chorionic gonadotropin (hCG) into pregnant mares serum gonadotropin (PMSG)-treated mice. Northern blot analysis of total RNAs isolated from the PMSG/hCG-treated mouse ovaries indicated that the B2 receptor mRNA was constitutively expressed. Bradykinin B2 receptor protein was detected by Western blot analysis of the ovary extracts. In situ hybridization analysis revealed that the B2 receptor mRNA is expressed in the granulosa cells of all growing follicles of ovaries from both gonadotropin-treated immature and mature female mice. The effect of bradykinin on the expression of the B2 receptor gene was examined by RT-PCR analysis with the ovary previously cultured in the presence of bradykinin. Bradykinin treatment of immature female, gonadotropin-treated immature female, and mature female mouse ovaries brought about no apparent changes in the B2 receptor mRNA level. The present data indicate that the level of B2 receptor expression in the ovary is fairly constant, and that the biological effect elicited by bradykinin in this organ may be dependent upon concentrations of the ligand produced by operation of the kinin-kallikrein system.

Identification and partial characterization of a metallopeptidase from porcine ovaries

The follicular fluid of porcine ovaries contains an EDTA-sensitive enzyme activity for the synthetic substrate benzyloxycarbonyl-Val-Lys-Met-4-methylcoumaryl-7-amide. To investigate its characteristics and its identification, the enzyme was partially purified by ammonium sulfate fractionation followed by column chromatographies on DEAE-Cellulose and chelating Cellulofine columns. The enzyme activity was strongly inhibited by typical chelators, such as EDTA and o-phenanthroline, but after inhibition by EDTA the activity was completely restored with an appropriate amount of Zn2+ and Co2+ ions. It showed enzyme activity solely for benzyloxycarbonyl-Val-Lys-Met-4-methylcoumaryl-7-amide among the substrates tested. The molecular weight of the enzyme was estimated to be 400,000 by gel filtration. The enzyme activity in the fluid obtained from large follicles of porcine ovaries was significantly higher than that from smaller follicles. It appeared that the granulosa cell extract did not contain the metalloenzyme activity. Similar enzyme activities were detected in follicular fluids from bovine and human ovaries. These results suggest that the present enzyme is distinct from any other metalloendopeptidases thus far reported.

Expression of Bradykinin B

Abstract The amounts of [1-5]-bradykinin in ovary extracts were determined using gonadotropin-treated immature female mice. The bradykinin levels in the ovary were high at 2, 6, and 48 hr after injection of human chorionic gonadotropin (hCG) into pregnant mares serum gonadotropin (PMSG)-treated mice. Northern blot analysis of total RNAs isolated from the PMSG/hCG-treated mouse ovaries indicated that the B2 receptor mRNA was constitutively expressed. Bradykinin B2 receptor protein was detected by Western blot analysis of the ovary extracts. In situ hybridization analysis revealed that the B2 receptor mRNA is expressed in the granulosa cells of all growing follicles of ovaries from both gonadotropin-treated immature and mature female mice. The effect of bradykinin on the expression of the B2 receptor gene was examined by RT-PCR analysis with the ovary previously cultured in the presence of bradykinin. Bradykinin treatment of immature female, gonadotropin-treated immature female, and mature female mouse ovaries brought about no apparent changes in the B2 receptor mRNA level. The present data indicate that the level of B2 receptor expression in the ovary is fairly constant, and that the biological effect elicited by bradykinin in this organ may be dependent upon concentrations of the ligand produced by operation of the kinin-kallikrein system.

Expression of Bradykinin B2Receptor in the Mouse Ovary

Abstract The amounts of [1-5]-bradykinin in ovary extracts were determined using gonadotropin-treated immature female mice. The bradykinin levels in the ovary were high at 2, 6, and 48 hr after injection of human chorionic gonadotropin (hCG) into pregnant mares serum gonadotropin (PMSG)-treated mice. Northern blot analysis of total RNAs isolated from the PMSG/hCG-treated mouse ovaries indicated that the B2 receptor mRNA was constitutively expressed. Bradykinin B2 receptor protein was detected by Western blot analysis of the ovary extracts. In situ hybridization analysis revealed that the B2 receptor mRNA is expressed in the granulosa cells of all growing follicles of ovaries from both gonadotropin-treated immature and mature female mice. The effect of bradykinin on the expression of the B2 receptor gene was examined by RT-PCR analysis with the ovary previously cultured in the presence of bradykinin. Bradykinin treatment of immature female, gonadotropin-treated immature female, and mature female mouse ovaries brought about no apparent changes in the B2 receptor mRNA level. The present data indicate that the level of B2 receptor expression in the ovary is fairly constant, and that the biological effect elicited by bradykinin in this organ may be dependent upon concentrations of the ligand produced by operation of the kinin-kallikrein system.