Multiple Cellular Transport and Binding Processes of Unesterified Docosahexaenoic Acid in Outer Blood-Retinal Barrier Retinal Pigment Epithelial Cells.

Docosahexaenoic acid (DHA, 22 : 6) is an essential omega-3 long-chain polyunsaturated fatty acid that plays a pivotal role in vision. The purpose of this study was to clarify the cellular uptake and binding processes of free and protein-bound unesterified DHA in retinal pigment epithelial cell (RPE) line ARPE-19 as a model of the human outer blood-retinal barrier and isolated porcine RPE cell fractions. Uptake of free [C]DHA by ARPE-19 cells was saturable with a Michaelis-Menten constant of 283 µM, and was significantly inhibited by eicosapentaenoic acid, arachidonic acid, and linoleic acid, but not by oleic acid.

Behavioral impairment in SHATI/NAT8L knockout mice via dysfunction of myelination development.

We have identified SHATI/NAT8L in the brain of mice treated with methamphetamine. Recently, it has been reported that SHATI is N-acetyltransferase 8-like protein (NAT8L) that produces N-acetylaspatate (NAA) from aspartate and acetyl-CoA. We have generated SHATI/NAT8L knockout (Shati ) mouse which demonstrates behavioral deficits that are not rescued by single NAA supplementation, although the reason for which is still not clarified.

Photoaffinity electrophoretic mobility shift assay using photoreactive DNA bearing 3-trifluoromethyl-3-phenyldiazirine in its phosphate backbone.

Photoaffinity cross-linking enables the analysis of interactions between DNA and proteins even under denaturing conditions. We present a photoaffinity electrophoretic mobility shift assay (EMSA) in which two heterogeneous techniques-photoaffinity cross-linking using DNA bearing 3-trifluoromethyl-3-phenyldiazirine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis-are combined. To prepare the photoreactive DNA, which is an essential tool for photoaffinity EMSA, we first determined the optimal conditions for the integration of 4-(3-trifluoromethyl-3H-diazirin-3-yl)benzyl bromide to the specific site of oligonucleotide where phosphodiester linkage was replaced with phosphorothioate linkage.

Syndecan-4 Is a Receptor for Clathrin-Mediated Endocytosis of Arginine-Rich Cell-Penetrating Peptides.

Arginine-rich cell-penetrating peptides (CPPs) such as Tat and oligoarginine peptides have been widely used as carriers for intracellular delivery of bioactive molecules. Despite accumulating evidence for involvement of endocytosis in the cellular uptake of arginine-rich CPPs, the primary cell-surface receptors for these peptide carriers that would initiate endocytic processes leading to intracellular delivery of bioactive cargoes have remained poorly understood. Our previous attempt to identify membrane receptors for octa-arginine (R8) peptide, one of the representative arginine-rich CPPs, using the photo-cross-linking probe bearing a photoreactive diazirine was not successful due to considerable amounts of cellular proteins nonspecifically bound to the affinity beads.

Development and leading-edge application of innovative photoaffinity labeling.

Photoaffinity labeling has become increasingly important with the development of powerful specific probes that are synthesized by installing a photo-activatable functional group (photophore) on the framework of biological ligands. The present review summarizes the development of diazirine-based photoaffinity labeling by focusing on its application to the structural elucidation of ligand-accepting sites within proteins.

Alternative one-pot synthesis of (trifluoromethyl)phenyldiazirines from tosyloxime derivatives: application for new synthesis of optically pure diazirinylphenylalanines for photoaffinity labeling.

Alternative one-pot synthesis of 3-(trifluoromethyl)-3-phenyldiazirine derivatives from corresponding tosyloximes is developed. The deprotonation of intermediate diaziridine by NH2(-) is a new approach for construction of diazirine. Moreover, a novel synthesis of optically pure (trifluoromethyl)diazirinylphenylalanine derivatives was attempted involving these methods.

An isotope-coded fluorogenic cross-linker for high-performance target identification based on photoaffinity labeling.

A photoaffinity labeling (PAL)-based method for the rapid identification of target proteins is presented in which a high-performance chemical tag, an isotope-coded fluorescent tag (IsoFT), can be attached to the interacting site by irradiation. Labeled peptides can be easily distinguished among numerous proteolytic digests by sequential detection with highly sensitive fluorescence spectroscopy and mass spectrometry. Subsequent MS/MS analysis provides amino acid sequence information with a higher depth of coverage.

Hydrogen/deuterium exchange of cross-linkable α-amino acid derivatives in deuterated triflic acid.

In this paper we report here a hydrogen/deuterium exchange (H/D exchange) of cross-linkable α-amino acid derivatives with deuterated trifluoromethanesulfonic acid (TfOD). H/D exchange with TfOD was easily applied to o-catechol containing phenylalanine (DOPA) within an hour. A partial H/D exchange was observed for trifluoromethyldiazirinyl (TFMD) phenylalanine derivatives.

Coupling reaction of thioamides with sulfonyl azides: an efficient catalyst-free click-type ligation under mild conditions.

We report a coupling reaction of thioamides and sulfonyl azides to generate sulfonyl amidines in the absence of any activation additives. The reaction progresses in various solvents under mild conditions. Water exhibits the highest performance with respect to efficiency.

Affinity-based fluorogenic labeling of ATP-binding proteins with sequential photoactivatable cross-linkers.

A specific illumination approach has been developed for identification of adenosine triphosphate (ATP)-binding proteins. This strategy utilizes a tandem photoactivatable unit that consists of a diazirine group as a carbene precursor and an o-hydroxycinnamate moiety as a coumarin precursor. The photolysis of diazirine induces a specific cross-link on target proteins and is followed by photoactivation of coumarin generation with a concomitant release of the pre-installed affinity ligand.

Distinct metabolites for photoreactive L-phenylalanine derivatives in Klebsiella sp. CK6 isolated from rhizosphere of a wild dipterocarp sapling.

Photoaffinity labeling is a reliable analytical method for biological functional analysis. Three major photophores--aryl azide, benzophenone and trifluoromethyldiazirine--are utilized in analysis. Photophore-bearing L-phenylalanine derivatives, which are used for biological functional analysis, were inoculated into a Klebsiella sp.

Identification of cellular proteins interacting with octaarginine (R8) cell-penetrating peptide by photo-crosslinking.

Octaarginine (R8) is a representative cell-penetrating peptide. Lanthionine synthetase component C-like protein 1 (LanCL1) was identified as a potential intracellular target of R8 by using a photo-crosslinking assay that utilized a phenyl-trifluoromethyl diazirine moiety and peptide mass fingerprinting. Increased cellular uptake of R8 by LanCL1-overexpressing cells was observed.

Photoaffinity casting of a coumarin flag for rapid identification of ligand-binding sites within protein.

A photo-switchable fluorescent flagging approach has been developed to identify photoaffinity-labeled peptides in target protein. Upon photochemical release of the ligand, the protein was newly modified with a coumarin in place of the previously attached biotin. It allowed us to simplify complex identification processes for labeled sites.

CXCR4 stimulates macropinocytosis: implications for cellular uptake of arginine-rich cell-penetrating peptides and HIV.

CXCR4 is a coreceptor of HIV-1 infection in host cells. Through a photocrosslinking study to identify receptors involved in internalization of oligoarginine cell-penetrating peptides (CPPs), we found that CXCR4 serves as a receptor that stimulates macropinocytic uptake of the arginine 12-mer peptide (R12) but not of the 8-mer. We also found that stimulating CXCR4 with its intrinsic ligands, stromal cell-derived factor 1α and HIV-1 envelope glycoprotein 120, induced macropinocytosis.

Comprehensive synthesis of photoreactive (3-trifluoromethyl)diazirinyl indole derivatives from 5- and 6- trifluoroacetylindoles for photoaffinity labeling.

5- and 6-trifluoromethyldiazirinyl indoles were synthesized from corresponding bromoindole derivatives for the first time. They acted as mother skeletons for the comprehensive synthesis of various bioactive indole metabolites. These can be used in biological functional analysis as diazirine-based photoaffinity labels.

Rapidly photoactivatable ATP probes for specific labeling of tropomyosin within the actomyosin protein complex.

Tropomyosin-specific photoaffinity adenosine triphosphate (ATP) probes have been first developed, in which a diazirine moiety is incorporated into the γ-phosphate group as a rapidly carbene-generating photophore. These probes clearly labeled tropomyosin in the presence of other actomyosin components, that is, myosin, actin, and troponins. The specific labeling of tropomyosin was easily identified by selective trapping of the photo-incorporated ATP probe on Fe(3+)-immobilized metal ion affinity chromatography (IMAC) beads.

Photochemical construction of coumarin fluorophore on affinity-anchored protein.

A simple and unique construction of a coumarin fluorophore on a prey protein has been achieved by sequential photoreactions using a nonfluorescent bait protein. The bait protein was first modified with a novel diazirine-based photo-cross-linker containing an o-hydroxycinnamoyl unit. The strategy involves two wavelength-dependent photoreactions: photolysis of the diazirine group and E to Z photoisomerization of the cinnamoyl group.

Identification of a substrate-binding site in a peroxisomal beta-oxidation enzyme by photoaffinity labeling with a novel palmitoyl derivative.

Peroxisomes play an essential role in a number of important metabolic pathways including beta-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various beta-oxidation enzymes and specialized fatty acid transport systems. However, the molecular mechanisms of these proteins, especially in terms of substrate binding, are still unknown.

Synthesis and properties of diazirinyl organo-platinum compounds for manipulations of photoaffinity labeled components.

Synthesis of diazirinyl organo-platinum complexes, which specifically interact with purine base, characterization of photoreactivity and interaction between guanosine 5'-monophosphate (GMP) were examined. The results indicated that the diazirinyl organo-platinum complex was useful for manipulations of photoaffinity labeled components.

Efficient approach for profiling photoaffinity labeled peptides with a cleavable biotinyl photoprobe.

Based on the application of our recent biotinyl photoprobe with a cleavable N-acylsulfonamide, an efficient process has been developed for profiling photoaffinity labeled peptides among a large excess of unlabeled concomitants. N-acylsulfonamide group was found to be stable under the usual S-pyridylethylation condition of cysteine residues whereas the group was easily cleaved by N-alkylation with iodoacetic acid in acidic condition. The selective nature between two common protein alkylation reactions was evaluated with L-glutamate dehydrogenase (GDH) using an acidic amino acid photoprobe with biotinylated acylsulfonamide function.

Photoactive ligands probing the sweet taste receptor. Design and synthesis of highly potent diazirinyl D-phenylalanine derivatives.

Some D-amino acids such as d-tryptophan and D-phenylalanine are well known as naturally-occurring sweeteners. Photoreactive D-phenylalanine derivatives containing trifluoromethyldiazirinyl moiety at 3- or 4-position of phenylalanine, were designed as sweeteners for functional analysis with photoaffinity labeling. The trifluoromethyldiazirinyl D-phenylalanine derivatives were prepared effectively with chemo-enzymatic methods using L-amino acid oxidase and were found to have potent activity toward the human sweet taste receptor.

Effective synthesis of optically active trifluoromethyldiazirinyl homophenylalanine and aroylalanine derivatives with the Friedel-Crafts reaction in triflic acid.

The Friedel-Crafts reaction with 3-(3-methoxyphenyl)-3-(trifluoromethyl)-3H-diazirine and optically active N-TFA-Asp(Cl)-OMe in triflic acid afforded homophenylalanine derivatives without any loss of the optical purity.

Synthesis and evaluation of novel photoreactive alpha-amino acid analog carrying acidic and cleavable functions.

A novel photoreactive alpha-amino acid bearing an acidic residue and a cleavable diazirine was developed. To mimic common acidic alpha-amino acids, the residue was designed to be N-acylsulfonamide that possesses an acidic proton and is able to dissociate under the physiological conditions. The inhibition assay of its biotin-tagged derivative with glutamyl endopeptidase from Staphylococcus aureus V8 strain revealed a Ki(app) value of 162 microM, which is slightly higher than the K(m) value of a common substrate.