SKGQA, a Peptide Derived from the ANA/BTG3 Protein, Cleaves Amyloid-ß with Proteolytic Activity.

Despite the extensive research conducted on Alzheimer's disease (AD) over the years, no effective drug for AD treatment has been found. Therefore, the development of new drugs for the treatment of AD is of the utmost importance. We recently reported the proteolytic activities of JAL-TA9 (YKGSGFRMI) and ANA-TA9 (SKGQAYRMA), synthetic peptides of nine amino acids each, derived from the Box A region of Tob1 and ANA/BTG3 proteins, respectively. Furthermore, two components of ANA-TA9, ANA-YA4 (YRMI) at the C-terminus end and ANA-SA5 (SKGQA) at the N-terminus end of ANA-TA9, exhibited proteolytic activity against amyloid-ß (Aß) fragment peptides. In this study, we identified the active center of ANA-SA5 using AEBSF, a serine protease inhibitor, and a peptide in which the Ser residue of ANA-SA5 was replaced with Leu. In addition, we demonstrate the proteolytic activity of ANA-SA5 against the soluble form Aß42 (a-Aß42) and solid insoluble form s-Aß42. Furthermore, ANA-SA5 was not cytotoxic to A549 cells. These results indicate that ANA-SA5 is a promising Catalytide and a potential candidate for the development of new peptide drugs targeting Aß42 for AD treatment.

Five-mer peptides prevent short-term spatial memory deficits in Aß25-35-induced Alzheimer's model mouse by suppressing Aß25-35 aggregation and resolving its aggregate form.

BACKGROUND: The development of drugs for Alzheimer's disease (AD), which is related to the misfolding and aggregation of amyloid-ß (Aß), is high in demand due to the growing number of AD patients. In this study, we screened 22 kinds of 5-mer synthetic peptides derived from the Box A region of Tob1 protein to find a peptide effective against Aß aggregation. METHODS: A Thioflavin T (ThT) assay was performed to evaluate aggregation and screen aggregation inhibitors. Male ICR mice (6 weeks old) were administered saline, 9 nmol Aß25-35, or a mixture of 9 nmol Aß25-35 and 9 nmol GSGFK in the right lateral ventricle. Short-term spatial memory was assessed through Y-maze. Microglia cells (BV-)2 cells were plated on 24-well plates (4 × 104 cells/well) and incubated for 48 h, and then, the cells were treated with 0.01, 0.05, 0.1, 0.2, or 0.5 mM GSGFK. After incubation for 24 h, bead uptake was evaluated using a laser confocal microscope and Cytation 5. RESULTS: We found two kinds of peptides, GSGNR and GSGFK, that were not only suppressed by aggregation of Aß25-35 but also resolved the aggregated Aß25-35. Results obtained from the Y-maze test on an Aß25-35-induced AD model mouse indicated that GSGFK prevents the deficits in short-term memory induced by Aß25-35. The effect of GSGFK on phagocytosis in BV-2 cells proved that GSGFK activates the phagocytic ability of microglia. CONCLUSIONS: In conclusion, 5-mer peptides prevent short-term memory deficit in Aß25-35 induced AD model mouse by reducing the aggregated Aß25-35. They may also upregulate the phagocytic ability of microglia, which makes 5-mer peptides suitable candidates as therapeutic drugs against AD.

Structure-Activity Relationship of 5-mer Catalytides, GSGYR and RYGSG.

We recently discovered JAL-TA9 (YKGSGFRMI), a short hydrolytic peptide that we termed a Catalytide. The catalytic center of JAL-TA9 was modeled using MM2 and MMFF94 parameters and identified as GSGFR. Additionally, a structure-activity relationship study showed that GSGYR cleaved Aß11-29. Here, we developed a novel Catalytide in silico. Molecular dynamics simulations of GSGYR and RYGSG using MM2 and MMFF94 parameters suggested that both peptides may form catalytic triads and oxyanion holes. The hydrolytic potency of RYGSG was five times higher than that of GSGYR. Moreover, both peptides showed three common cleavage positions for Aß11-29; namely, L17-V18, V18-F19, and E22-D23. The aggregation ratio analyzed by the thioflavin-T assay correlated well with proteolytic activity, suggesting that the aggregation of Aß11-29 was suppressed by the cleavage reaction. Docking simulations with the carbonyl carbon of L17 or the carbonyl carbon of E22 in Aß11-29 were conducted using the secondary structures of GSGYR and RYGSG. The distance between the hydroxyl group of serine and the carbonyl carbon of the two cleavage sites proved that RYGSG was closer to Aß11-29 than to GSGYR. This study demonstrated that Catalytides are useful for understanding structure-activity relationships.

Direct Delivery of ANA-TA9, a Peptide Capable of Aß Hydrolysis, to the Brain by Intranasal Administration.

We have recently reported Catalytides (Catalytic peptides) JAL-TA9 (YKGSGFRMI) and ANA-TA9 (SKGQAYRMI), which are the first Catalytides found to cleave Aß42. Although the Catalytides must be delivered to the brain parenchyma to treat Alzheimer's disease, the blood-brain barrier (BBB) limits their entry into the brain from the systemic circulation. To avoid the BBB, the direct route from the nasal cavity to the brain was used in this study. The animal studies using rats and mice clarified that the plasma clearance of ANA-TA9 was more rapid than in vitro degradation in the plasma, whole blood, and the cerebrospinal fluid (CSF). The brain concentrations of ANA-TA9 were higher after nasal administration than those after intraperitoneal administration, despite a much lower plasma concentration after nasal administration, suggesting the direct delivery of ANA-TA9 to the brain from the nasal cavity. Similar findings were observed for its transport to CSF after nasal and intravenous administration. The concentration of ANA-TA9 in the olfactory bulb reached the peak at 5 min, whereas those in the frontal and occipital brains was 30 min, suggesting the sequential backward translocation of ANA-TA9 in the brain. In conclusion, ANA-TA9 was efficiently delivered to the brain by nasal application, as compared to other routes.

Amyloid beta cleavage by ANA-TA9, a synthetic peptide from the ANA/BTG3 Box A region.

INTRODUCTION: We recently discovered a short synthetic peptide derived from the ANA/BTG3 protein Box A region called ANA-TA9 (SKGQAYRMI), which possesses catalytic activity. Herein we demonstrated the proteolytic activity of ANA-TA9 against amyloid beta 42 (Aß42). METHODS: The proteolytic activity of ANA-TA9 against both the authentic soluble form Aß42 (a-Aß42) and the solid insoluble form Aß42 (s-Aß42) was analyzed by high-performance liquid chromatography and mass spectrometry. Plasma clearance, brain uptake, and cell viability were examined. RESULTS: ANA-TA9 cleaved not only a-Aß42 but also s-Aß42. Proteolytic activity was partially inhibited by 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, a serine protease inhibitor. Plasma clearance was very rapid, and the brain concentration indicated efficient brain delivery of ANA-TA9 via nasal application. Cell viability analysis indicated that ANA-TA9 did not display toxicity. DISCUSSION: ANA-TA9 is an attractive potential candidate for the development of novel peptide drugs in Alzheimer's disease treatment.

Catalytides derived from the Box A region in the ANA/BTG3 protein cleave amyloid-ß fragment peptide.

We have recently reported about shorter proteolytic peptides termed Catalytide as general name. JAL-TA9 (YKGSGFRMI), a fragment peptide derived from Box A region of Tob1 protein, is the first Catalytide and cleaves Aß42 and its fragment peptides. Herein, we demonstrate the enzymatic properties of ANA-TA9 corresponding region to JAL-TA9 in ANA/BTG3 protein. ANA-TA9 showed the auto-proteolytic activity and cleaved 3 kinds of synthetic fragment peptides derived from Aß42, especially on the central region of Aß42 with a serine protease like activity. Interestingly, 2 kinds of components, ANA-SA5 (SKGQA) and ANA-YA4 (YRMI), also showed similar proteolytic activity. These results indicate that ANA-TA9 is composed of two different Catalytides.

Effects of Cu2+ on conformational change and aggregation of hPrP180-192 with a V180I mutation of the prion protein.

Prion diseases are neurodegenerative disorders caused by misfolding of the prion protein (PrP) from a normal cellular protein (PrPC) to a protease-resistant isoform (PrPSc). However, the aggregation mechanism is not entirely understood because of the physical properties of PrP, such as its solubility or aggregation in vitro and conformational or mutation diversity. Recently, we reported the physical and physiological properties of a synthetic fragment peptide. In the present study, we assessed the importance of a point mutation at the C-terminal region of PrP in structural conversion and aggregation and evaluated the physical and physiological properties of the point-mutated human-PrP180-192 V180I (hPrP180-192 V180I) using circular dichroism spectra, high-performance liquid chromatography, Affinix QNµ, and thioflavin-T staining, including the effects of Cu2+. The secondary structure of hPrP180-192 V180I changed from a random coil to a ß-sheet in Cu2+ free buffer. In addition, we observed molecular interactions in hPrP180-192 V180I and aggregation with itself, which were inhibited by Cu2+. We conclude that the point mutation in the C-terminal region of PrP, including hPrP180-192 V180I, and Cu2+ may play an important role in the conversion of PrPC to PrPSc.

The discovery of shorter synthetic proteolytic peptides derived from Tob1 protein.

We screened nearly 1000 synthetic peptides and found that JAL-AK22 (KYEGHWYPEKPYKGSGFRCIHI), which is derived from the BoxA domain in the Tob1 protein, activates both unfolded and folded proMMP-7. Interestingly, the smaller derivative of JAL-AK22, termed JAL-TA9 (YKGSGFRMI) possessed auto-proteolytic activity and cleaved three synthetic peptides fragment (MMP18-33, MMP18-40, and Aß11-29) under physiological conditions. The kcat of JAL-TA9 was 4.58 × 10-4 min-1 against MMP18-33 and 6.5 × 10-4 min-1 against MMP18-40. These kinetic parameters are lower than those of general proteinases like trypsin, for which the kcat is 247.2 × 105 min-1 against benzoyl-l-arginine ethyl ester. In addition, a 5-mer peptide derived from JAL-TA9, GSGFR also cleaved Aß11-29. These proteolytic activities were inhibited by AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride), a serine protease inhibitor. Our results demonstrate that some small synthetic peptides have protease activity. Thus, we propose calling small peptides possessing with protease activity Catalytides (catalytic peptides). We expect that our findings will stimulate the development of novel Catalytides and related applications such as the development of strategic peptide drugs.

MMP-7 cleaves amyloid ß fragment peptides and copper ion inhibits the degradation.

The extracellular deposition of amyloid ß (Aß) is known to be the fundamental cause of Alzheimer's disease (AD). Aß1-42, generated by ß-secretases from the amyloid precursor protein (APP), is the main component of neuritic plaque, and the aggregation of this protein is shown to be dependent to an extent on metal ions such as copper and zinc. However, the mechanism by which Cu2+ affects the physicochemical properties of Aß1-42 or the central nervous system is still under debate. A recent series of studies have demonstrated that both the soluble-type matrix metalloproteinases (MMP-2 and MMP-9) and the membrane-type matrix metalloproteinase (MT1-MMP) are capable of degrading Aß peptides. MMP-7, one of the soluble-type matrix metalloproteinases, is expressed in hippocampal tissue; however, less information is available concerning the pathophysiological roles of this enzyme in the process and/or progress of Alzheimer's disease. In this study, we examined the degradation activity of MMP-7 against various Aß1-42's fragment peptides and the effect of Cu2+. Although Aß22-40 was degraded by MMP-7 regardless of Cu2+, Cu2+ inhibited the degradation of Aß1-19, Aß11-20, and Aß11-29 by MMP-7. These results indicate that MMP-7 is capable of degrading Aß1-42, and that Aß1-42 acquired resistance against MMP-7 cleavage through Cu2+-binding and structure changes. Our results demonstrate that MMP-7 may play an important role in the defensive mechanism against the aggregation of Aß1-42, which gives rise to the pathology of AD.

Structure-activity relationship study at C9 position of kaitocephalin.

Kaitocephalin (KCP) isolated from Eupenicillium shearii PF1191 is an unusual amino acid natural product in which serine, proline, and alanine moieties are liked with carbon-carbon bonds. KCP exhibits potent and selective binding affinity for one of the ionotropic glutamate receptor subtypes, NMDA receptors (Ki=7.8nM). In this study, new structure-activity relationship studies at C9 of KCP were implemented. Eleven new KCP analogs with different substituents at C9 were prepared and employed for binding affinity tests using native ionotropic glutamate receptors. Replacement of the 3,5-dichloro-4-hydroxybenzoyl group of KCP with a 3-phenylpropionyl group resulted in significant loss of binding affinity for NMDARs (Ki=1300nM), indicating an indispensable role of the aromatic ring of KCP in the potent and selective binding to NMDARs. Other analogs showed potent binding affinity in a range of 11-270nM. These findings would directly link to develop useful chemical tools toward imaging and labeling of NMDARs.

(7S)-Kaitocephalin as a potent NMDA receptor selective ligand.

A structure-activity relationship (SAR) study of kaitocephalin (KCP), known to be a potent naturally occurring NMDA receptor ligand, was performed. This study led us to the discovery of (7S)-kaitocephalin as a highly selective NMDA receptor ligand. It displayed a 22-fold higher degree of selectivity for the NMDA receptor over KCP, though the binding affinity of (7S)-KCP [Ki = 29 nM] was 3.7-fold less potent than that of KCP [Ki = 7.8 nM].

[Studies Using Text Mining on the Differences in Learning Effects between the KJ and World Café Method as Learning Strategies].

The KJ method (named for developer Jiro Kawakita; also known as affinity diagramming) is widely used in participatory learning as a means to collect and organize information. In addition, the World Café (WC) has recently become popular. However, differences in the information obtained using each method have not been studied comprehensively. To determine the appropriate information selection criteria, we analyzed differences in the information generated by the WC and KJ methods. Two groups engaged in sessions to collect and organize information using either the WC or KJ method and small group discussions were held to create "proposals to improve first-year education". Both groups answered two pre- and post- session questionnaires that asked for free descriptions. Key words were extracted from the results of the two questionnaires and categorized using text mining. In the responses to questionnaire 1, which was directly related to the session theme, a significant increase in the number of key words was observed in the WC group (p=0.0050, Fisher's exact test). However, there was no significant increase in the number of key words in the responses to questionnaire 2, which was not directly related to the session theme (p=0.8347, Fisher's exact test). In the KJ method, participants extracted the most notable issues and progressed to a detailed discussion, whereas in the WC method, various information and problems were spread among the participants. The choice between the WC and KJ method should be made to reflect the educational objective and desired direction of discussion.

[A self-improvement and participatory career development education program involving internships and volunteer training experience for pharmacy students: results verified in a follow-up survey three years after participation].

The Faculty of Pharmaceutical Sciences, Setsunan University, offers the Self-improvement and Participatory Career Development Education Program: Internship and Volunteer Training Experience for Pharmacy Students to third-year students. We previously reported that the training experience was effective in cultivating important attributes among students, such as a willingness to learn the aims of pharmacists, an awareness of their own role as healthcare workers, and a desire to reflect on their future careers and lives. A follow-up survey of the participants was carried out three years after the training experience. The questionnaire verified that the training experience affected attendance at subsequent lectures and course determination after graduation. We confirmed the relationship between the participants' degree of satisfaction with the training experience and increased motivation for attending subsequent lectures. Through the training experience, participants discovered future targets and subjects of study. In addition, they became more interested in subsequent classroom lessons and their future. The greater the participants' degree of satisfaction with their training experience, the more interest they took in practical training and future courses. The present study clarified that the training experience was effective in cultivating important attributes such as a willingness to learn and an interest in future courses. Moreover, the training positively affected the course determination after graduation.

Prion fragment peptides are digested with membrane type matrix metalloproteinases and acquire enzyme resistance through Cu²âº-binding.

Prions are the cause of neurodegenerative disease in humans and other mammals. The structural conversion of the prion protein (PrP) from a normal cellular protein (PrPC) to a protease-resistant isoform (PrPSc) is thought to relate to Cu2+ binding to histidine residues. In this study, we focused on the membrane-type matrix metalloproteinases (MT-MMPs) such as MT1-MMP and MT3-MMP, which are expressed in the brain as PrPC-degrading proteases. We synthesized 21 prion fragment peptides. Each purified peptide was individually incubated with recombinant MT1-MMP or MT3-MMP in the presence or absence of Cu2+ and the cleavage sites determined by LC-ESI-MS analysis. Recombinant MMP-7 and human serum (HS) were also tested as control. hPrP61-90, from the octapeptide-repeat region, was cleaved by HS but not by the MMPs tested here. On the other hand, hPrP92-168 from the central region was cleaved by MT1-MMP and MT3-MMP at various sites. These cleavages were inhibited by treatment with Cu2+. The C-terminal peptides had higher resistance than the central region. The data obtained from this study suggest that MT-MMPs expressed in the brain might possess PrPC-degrading activity.

[Practical chemistry education provided by team-based learning (TBL) and peer evaluation].

Learning chemistry is cumulative: basic knowledge and chemical calculation skills are required to gain understanding of higher content. However, we often suffer from students' lack of learning skills to acquire these concepts. One of the reasons is the lack of adequate training in the knowledge and skills of chemistry, and one of the reasons for this lack is the lack of adequate evaluation of training procedures and content. Team-based learning (TBL) is a strong method for providing training in the knowledge and skills of chemistry and reaffirms the knowledge and skills of students of various levels. In our faculty, TBL exercises are provided for first-year students concurrently with lectures in physical chemistry and analytical chemistry. In this study, we researched the adoption of a peer evaluation process for this participatory learning model. Questionnaires taken after TBL exercises in the previous year showed a positive response to TBL. Further, a questionnaire taken after TBL exercises in the spring semester of the current year also yielded a positive response not only to TBL but also to peer evaluation. In addition, a significant correlation was observed between the improvement of students' grades in chemistry classes and the feeling the percentage (20%) of peer evaluation in overall evaluation low (logistic regression analysis, p=0.022). On the basis of the findings, we argue that TBL provides a generic, practical learning environment including an effective focus on learning strategy and evaluation of knowledge, skills, and attitudes, and studies on the educational effects of TBL and peer evaluation.

Metal-binding ability of human prion protein fragment peptides analyzed by column switch HPLC.

The structural conversion of the prion protein (PrP) from the normal cellular isoform (PrP(C)) to the posttranslationally modified form (PrP(Sc)) is thought to relate to Cu²âº binding to histidine (H) residues. Traditionally, the binding of metals to PrP has been investigated by monitoring the conformational conversion using circular dichroism (CD). In this study, the metal-binding ability of 21 synthetic peptides representing regions of human PrP(C) was investigated by column switch high-performance liquid chromatography (CS-HPLC). The CS-HPLC system is composed of a metal chelate affinity column and an octadecylsilica (ODS) reversed-phase column that together enable the identification of metal-binding regardless of conformational conversion. Synthetic peptides were designed with respect to the position of H residues as well as the secondary structure of human PrP (hPrP). The ability of the octapeptide (PHGGGWGQ)-repeating region (OP-repeat) to bind metals was analyzed by CS-HPLC and supported by CD analysis, and indicated that CS-HPLC is a reliable and useful method for measuring peptide metal-binding. Peptides from the middle region of hPrP showed a high affinity for Cu²âº, but binding to Zn²âº, Ni²âº, and Co²âº was dependent on peptide length. C-Terminal peptides had a lower affinity for Cu²âº, Zn²âº, Ni²âº, and Co²âº than OP-repeat region peptides. Interestingly, hPrP193-230, which contained no H residues, also bound to Cu²âº, Zn²âº, Ni²âº, and Co²âº, indicating that this region is a novel metal-binding site in the C-terminal region of PrP(C). The CS-HPLC method described in this study is useful and convenient for assessing metal-binding affinity and characterizing metal-binding peptides or proteins.

Regioselective synthesis of methylated epigallocatechin gallate via nitrobenzenesulfonyl (Ns) protecting group.

Regioselective synthesis of methylated epigallocatechin gallate from epigallocatechin was accomplished using a 2-nitrobenzenesulfonyl (Ns) group as a protecting group for phenols. This methodology provided several methylated catechins, which are naturally scarce catechin derivatives.

Novel digitalis-like factor, marinobufotoxin, isolated from cultured Y-1 cells, and its hypertensive effect in rats.

Marinobufagenin and telecinobufagin have been identified as digitalis-like factors in mammals. In toads, marinobufagenin-related compounds, such as marinobufotoxin (MBT), have been isolated in some tissues but not in mammals, and its biological action has not been elucidated. Herein, we aimed to explore the possible production and/or secretion of MBT and the biological action in rats. First, the MBT in culture supernatant of the adrenocortical-originated cell line Y-1 was analyzed by high-performance liquid chromatography and sensitive ELISA for marinobufagenin-like immunoreactivity. Moreover, the structural information was obtained by mass spectrometry. To determine the biological action, MBT (9.6 and 0.96 microg/kg per day) was intraperitoneally infused via an osmotic minipump for 1 week. Blood pressure and renal excretion of marinobufagenin-like immunoreactivity were measured. Marinobufagenin-like immunoreactivity was found in Y-1 cell culture media, and the concentration increased until 24 hours. The structural analysis suggested that marinobufagenin-like immunoreactivities were marinobufagenin and MBT, and tandem mass spectrum analysis revealed them with the specific daughter ions. The highest sensitive ELISA-positive peak of marinobufagenin-like immunoreactivity in the media was MBT. Continuous administration of MBT in rats for 1 week significantly increased systolic blood pressure and renal excretion of marinobufagenin-like immunoreactivity compared with control rats (135+/-3.0 versus 126+/-2.0 mm Hg and 1.41+/-0.286 versus 0.34+/-0.064 ng/day, respectively). These data suggest that MBT, arginine-suberoyl ester of marinobufagenin, can be a novel digitalis-like factor with hypertensive action and is secreted from the adrenocortical cells.

Characterization of a novel metalloproteinase in Duvernoy's gland of Rhabdophis tigrinus tigrinus.

During the characterization of hemorrhagic factor in venom of Rhabdophis tigrinus tigrinus, so-called Yamakagashi in Japan, one of the Colubridae family, a novel metalloproteinase with molecular weight of 38 kDa in the Duvernoy's gland of Yamakagashi was identified by gelatin zymography and by monitoring its proteolytic activity using a fluorescence peptide substrate, MOCAc-PLGLA2pr(Dnp)AR-NH2, which was developed for measuring the well-known matrix metalloproteinase (MMP) activity. After purification by gel filtration HPLC and/or column switch HPLC system consisting of an affinity column, which was immobilized with a synthetic BS-10 peptide (MQKPRCGVPD) originating from propeptide domain of MMP-7 and a reversed-phase column, the N-terminal amino acid sequence of the 38 kDa metalloproteinase was identified as FNTFPGDLK which shared a high homology to Xenopus MMP-9. The 38 kDa metalloproteinase required Zn2+ and Ca2+ ions for its proteolytic activity. In addition, the proteolytic activity was almost completely inhibited by BS-10, a MMP inhibitor, but not by the serine proteinase inhibitors, cysteine proteinase inhibitors and aspartic proteinase inhibitors. Together these results demonstrated that the 38 kDa proteinase is a novel snake verom metalloproteinase (SVMP) containing HExGHxxGxxH motif which possesses high affinity to the BS-10 peptide, into its molecule, and the enzymatic properties are closed to that of MMPs. Based on the results obtained in the present study, we concluded that the 38 kDa metalloproteinase is a novel metalloproteinase whose activity may be regulated by the cysteine switch mechanism, and could be classified as one of the matrix metalloproteinases rather than snake venom metalloproteinases.