Hydrophobic interactions at subsite S1′ of human dipeptidyl peptidase IV contribute significantly to the inhibitory effect of tripeptides

Functional inhibitory peptides of human dipeptidyl peptidase 4 (hDPP4) have been highly anticipated as the active ingredient of functional food for type II diabetes; however, the molecular mechanism of hDPP4 inhibition remains unclear. In this study, we focused on dipeptides and tripeptides, which display structure-function correlations that are relatively easy to analyze, and examined their interactions with hDPP4 on an atomic level using a combination of docking studies and an hDPP4 inhibition assay. First, we performed comprehensive binding mode analysis of the dipeptide library and demonstrated that the formation of a tight interaction with the S1 subsite composing part of the substrate pocket is essential for dipeptides to compete with the substrate and strongly inhibit hDPP4. Next, we synthesized tripeptides by adding various amino acids to the C-terminus of Ile-Pro and Val-Pro, which have especially high inhibitory activity among compounds in the dipeptide library, and measured the hDPP4 inhibitory activity of the tripeptides. When hydrophobic amino acids (Ile, Met, Val, Trp) were added, the inhibitory activity increased several-fold. This phenomenon could be explained as follows: the C-terminal amino acid of the tripeptide formed hydrophobic interactions with Tyr547 and Trp629, which compose the S1′ subsite located relatively outside the substrate pocket, thereby stabilizing the hDPP4-peptide binding. The structural information on the interaction between hDPP4 and peptide inhibitors attained in this study is anticipated to be useful in the development of a more potent hDPP4 competitive inhibitor

Synthesis of Photoreactive 2-Phenethylamine Derivatives : Synthesis of Adenosine Derivatives Enabling Functional Analysis of Adenosine Receptors by Photoaffinity Labeling

2-Phenylethylamine is well known as a substructure of many biologically active compounds, and the synthesis of its photoreactive derivatives to allow the analysis of biological functions is reported. This allowed us to synthesise ligands for adenosine receptors, which have many functional roles in biology and have been extensively studied for their many roles in maintaining homeostasis. Adenosine is one of the most common biochemical compounds, but photoaffinity labeling has not yet been used to study adenosine receptors. Synthetic methods for producing photoreactive adenosine derivatives that can be used with adenosine receptors were established for the photophores phenyl azide and benzophenone. The effect of the introduction of the photoreactive components was determined using an adenosine receptor assay

Enhanced Cytotoxic Effects of Arenite in Combination with Active Bufadienolide Compounds against Human Glioblastoma Cell Line U-87

The cytotoxicity of a trivalent arsenic derivative (arsenite, AsIII) combined with arenobufagin or gamabufotalin was evaluated in human U-87 glioblastoma cells. Synergistic cytotoxicity with upregulated intracellular arsenic levels was observed, when treated with AsIII combined with arenobufagin instead of gamabufotalin. Apoptosis and the activation of caspase-9/-8/-3 were induced by AsIII and further strengthened by arenobufagin. The magnitude of increase in the activities of caspase-9/-3 was much greater than that of caspase-8, suggesting that the intrinsic pathway played a much more important role in the apoptosis. An increase in the number of necrotic cells, enhanced LDH leakage, and intensified G2/M phase arrest were observed. A remarkable increase in the expression level of γH2AX, a DNA damage marker, was induced by AsIII+arenobufagin. Concomitantly, the activation of autophagy was observed, suggesting that autophagic cell death associated with DNA damage was partially attributed to the cytotoxicity of AsIII+arenobufagin. Suppression of Notch signaling was confirmed in the combined regimen-treated cells, suggesting that inactivation of Jagged1/Notch signaling would probably contribute to the synergistic cytotoxic effect of AsIII+arenobufagin. Given that both AsIII and arenobufagin are capable of penetrating into the blood–brain barrier, our findings may provide fundamental insight into the clinical application of the combined regimen for glioblastoma

Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin.

BACKGROUND: Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown. METHODS: Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure-function relationships. The Saccharomyces cerevisiae expression system has advantages as a high-throughput analysis system, but compared to other hosts it is characterized by a relatively low level of recombinant protein expression. To alleviate this weakness, in this study we optimized the codon usage and signal-sequence as the first step. Recombinant MCL (rMCL) was expressed and purified, and the sensory taste was analyzed. RESULTS: As a result, a 2 mg/l yield of rMCL was successfully obtained. Although sensory taste evaluation showed that rMCL was flat in taste under all the pH conditions employed, taste-modifying activity similar to that of native MCL was recovered after deglycosylation. Mutagenetic analysis revealed that the N-glycan attached to Asn42 was bulky in rMCL. CONCLUSIONS: The high-mannose-type N-glycan attached in yeast blocks the taste-modifying activity of rMCL. GENERAL SIGNIFICANCE: The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3