TLR4/MD-2 is a receptor for extracellular nucleophosmin 1.

Nucleophosmin 1 (NPM1) primarily localizes to the nucleus and is passively released into the extracellular milieu by necrotic or damaged cells, or is secreted by monocytes and macrophages. Extracellular NPM1 acts as a potent inflammatory stimulator by promoting cytokine production [e.g., tumor necrosis factor-α (TNF-α)], which suggests that NPM1 acts as a damage-associated molecular pattern. However, the receptor of NPM1 is unknown. Evidence indicates that DAMPs, which include high mobility group box 1 and histones, may bind Toll-like receptors (TLRs). In the present study, it was shown that NPM1 signaling was mediated via the TLR4 pathway, which suggests that TLR4 is an NPM1 receptor. TLR4 binds myeloid differentiation protein-2 (MD-2), which is essential for intracellular signaling. Furthermore, the TLR4 antagonist, LPS-Rhodobacter sphaeroides (an MD-2 antagonist) and TAK-242 (a TLR4 signaling inhibitor) significantly inhibited NPM1-induced TNF-α production by differentiated THP-1 cells as well as reducing ERK1/2 activation. Far-western blot analysis revealed that NPM1 directly bound MD-2. Thus, the results of the present study provide compelling evidence that TLR4 binds NPM1, and it is hypothesized that inhibiting NPM1 activity may serve as a novel strategy for treating TLR4-related diseases.

Characterization of a novel class of glyoxylate reductase belonging to the ß-hydroxyacid dehydrogenase family in Acetobacter aceti.

Enzymes related to ß-hydroxyacid dehydrogenases/3-hydroxyisobutyrate dehydrogenases are ubiquitous, but most of them have not been characterized. An uncharacterized protein with moderate sequence similarities to Gluconobacter oxydans succinic semialdehyde reductase and plant glyoxylate reductases/succinic semialdehyde reductases was found in the genome of Acetobacter aceti JCM20276. The corresponding gene was cloned and expressed in Escherichia coli. The gene product was purified and identified as a glyoxylate reductase that exclusively catalyzed the NAD(P)H-dependent reduction of glyoxylate to glycolate. The strict substrate specificity of this enzyme to glyoxylate, the diverged sequence motifs for its binding sites with cofactors and substrates, and its phylogenetic relationship to homologous enzymes suggested that this enzyme represents a novel class of enzymes in the ß-hydroxyacid dehydrogenase family. This study may provide an important clue to clarify the metabolism of glyoxylate in bacteria. Abbreviations: GR: glyoxylate reductase; GRHPR: glyoxylate reductase/hydroxypyruvate reductase; HIBADH: 3-hydroxyisobutyrate dehydrogenase; SSA: succinic semialdehyde; SSAR: succinic semialdehyde reductase.

Identification of novel mammalian phospholipids containing threonine, aspartate, and glutamate as the base moiety.

In this study, we showed the occurrence of phosphatidyl-L-threonine (PThr), phosphatidyl-L-aspartate (PAsp), and phosphatidyl-L-glutamate (PGlu) in rat brain. Analyses using an HPLC-ESI-MS and an amino acid analyzer showed the presence of L-threonine, L-aspartate, and L-glutamate in the acid-hydrolysates of phospholipids from porcine cerebrum, rat cerebrum, and rat liver. Results of ESI-MS/MS analyses with neutral loss scanning and product ion scanning suggest the presence of PThr-(18:0, 18:1), PThr-(18:0, 22:6), PAsp-(18:0, 18:1), PAsp-(18:0, 22:6), PGlu-(18:0, 18:1), and PGlu-(18:0, 22:6) in rat brain. This is the first study to identify 2 novel phospholipids, PAsp and PGlu, with a carboxylate-phosphate anhydride bond, in living organisms.

The distribution of phosphatidyl-D-serine in the rat.

Phosphatidylserine plays an important role in cell membranes. We have reported the occurrence of phosphatidyl-D-serine (D-PS) in the rat cerebrum. Here, we describe the tissue distribution of D-PS in the rat. The D/D+L ratio of D-PS in the cerebrum was 0.9%, while no detectable amount of D-PS was detected in the cerebellum. D-PS was also found in the heart, spleen, lung, testis, liver, and kidney in a range of 0.05-0.7% (the D/D+L ratio). Thus, D-PS, even in small amounts, is localized to the cerebrum in the brain and is distributed to various tissues other than the brain in the rat.

Occurrence of phosphatidyl-D-serine in the rat cerebrum.

Phosphatidylserine (PS), a relatively abundant component of mammalian cell membranes, plays important roles in biological processes including apoptosis and cell signaling. It is believed that phosphatidyl-L-serine is the only naturally occurring PS. Here, we describe for the first time the occurrence of phosphatidyl-D-serine (D-PS) in rat cerebrum. Quantitative HPLC analysis of the derivatives of serine liberated from PS by hydrolysis revealed that the amount of D-PS was approximately 1% of the total PS in the cerebrum. Enzymatic cleavage of cerebrum PS with phospholipase D and phospholipase C resulted in the release of both isomers of serine and phosphoserine, respectively, providing additional evidence for the existence of D-PS. Free D-serine was incorporated into PS in an in vitro system using a cerebrum extract, and this activity was inhibited by EDTA, suggesting the occurrence of a divalent cation-dependent enzyme that synthesizes D-PS by a base-exchange reaction.

A novel zinc-containing alpha-keto ester reductase from actinomycete: an approach based on protein chemistry and bioinformatics.

We have achieved the purification of an alpha-keto ester reductase (SCKER) from S. coelicolor A3(2) whole cells. SCKER proved to be a homotetramer of 132 kDa containing one equivalent of zinc ion per subunit. The enzyme differed from other alpha-keto ester reductases from microorganisms with regard to subunit structure and metal ion dependency. From a computer search using the protein data banks, the N-terminal amino acid sequence of SCKER was consistent with that of a possible zinc containing alcohol dehydrogenase in S. coelicolor A3(2). None of three hypothetical proteins of S. coelocor A3(2) having a high homology sequence with those of already purified alpha-keto ester reductases from S. thermocyaneoviolaceus [Yamaguchi, H., et al., Biosci. Biotechnol. Biochem., 66, 588-597 (2002)] was identical with that of SCKER.