Ile-Ile-OH

Eukaryotic precursor tRNAs (pre-tRNAs) often have an intron between positions 37 and 38 of the anticodon loop. However, atypical introns are found in some eukaryotes and archaea. In an early-diverged red alga Cyanidioschyzon merolae, the tRNAIle(UAU) gene contains three intron coding regions, located in the D-, anticodon, and T-arms. In this study, we focused on the relationship between the intron removal and formation of pseudouridine (Ψ), one of the most universally modified nucleosides. It had been reported that yeast Pus1 is a multiple-site-specific enzyme that synthesizes Ψ34 and Ψ36 in tRNAIle(UAU) in an intron-dependent manner. Unexpectedly, our biochemical experiments showed that the C. merolae ortholog of Pus1 pseudouridylated an intronless tRNAIle(UAU) and that the modification position was determined to be 55 which is the target of Pus4 but not Pus1 in yeast. Furthermore, unlike yeast Pus1, cmPus1 mediates Ψ modification at positions 34, 36, and/or 55 only in some specific intron-containing pre-tRNAIle(UAU) variants. cmPus4 was confirmed to be a single-site-specific enzyme that only converts U55 to Ψ, in a similar manner to yeast Pus4. cmPus4 did not catalyze the pseudouridine formation in pre-tRNAs containing an intron in the T-arm.

In this study, pyrralylisoleucine (Pyrr-Ile) and pyrralylalanine (Pyrr-Ala), two dipeptide-bound pyrralines with different C-termini were synthesized as the representatives of dietary advanced glycation end products (dAGEs). The structures of Pyrr-Ile and Pyrr-Ala were characterized by high-resolution mass spectrometry, nuclear magnetic resonance, and Fourier transform infrared spectroscopy. Then, the transport of Pyrr-Ile and Pyrr-Ala across intestinal epithelial cells was investigated using Caco-2 cell monolayers, and their interaction with aminopeptidase N (APN) was analyzed. The results showed that the apparent permeability coefficient (Papp) of Pyrr-Ala was (14.1 ± 2.26) × 10-7 cm·s-1 calculated by free pyrraline, while the Papp values of Pyrr-Ile were (32.4 ± 5.35) × 10-7 and (19.1 ± 1.46) × 10-7 cm·s-1 when they were, respectively, calculated according to their dipeptide-bound or free form. Both Pyrr-Ala and Pyrr-Ile were potential substrates of APN, and their hydrolysis by APN may make the intact transmembrane transport of Pyrr-Ala and Pyrr-Ile more difficult, especially for Pyrr-Ala. Besides, the occurrence of product inhibition in hydrolysis of Pyrr-Ile was possible. Pyrr-Ile and Pyrr-Ala were different in Papp values and transport forms, which suggested that the C-terminus may play an important role in their transport across the Caco-2 cell monolayers. In addition, the results highlight the intact transmembrane transport of dipeptide-bound pyrraline.

Expression takes place for most of the jasmonic acid (JA)-induced genes in a COI1-dependent manner via perception of its conjugate JA-Ile in the SCFCOI1-JAZ co-receptor complex. There are, however, numerous genes and processes, which are preferentially induced COI1-independently by the precursor of JA, 12-oxo-phytodienoic acid (OPDA). After recent identification of the Ile-conjugate of OPDA, OPDA-Ile, biological activity of this compound could be unequivocally proven in terms of gene expression. Any interference of OPDA, JA, or JA-Ile in OPDA-Ile-induced gene expression could be excluded by using different genetic background. The data suggest individual signaling properties of OPDA-Ile. Future studies for analysis of an SCFCOI1-JAZ co-receptor-independent route of signaling are proposed.