The recognition of lysine-type peptidoglycans (PG) by the PG recognition complex has been suggested to cause activation of the serine protease cascade leading to the processing of Spätzle and subsequent activation of the Toll signaling pathway. So far, two serine proteases involved in the lysine-type PG Toll signaling pathway have been identified. One is a modular serine protease functioning as an initial enzyme to be recruited into the lysine-type PG recognition complex. The other is the Drosophila Spätzle processing enzyme (SPE), a terminal enzyme that converts Spätzle proprotein to its processed form capable of binding to the Toll receptor. However, it remains unclear how the initial PG recognition signal is transferred to Spätzle resulting in Toll pathway activation. Also, the biochemical characteristics and mechanism of action of a serine protease linking the modular serine protease and SPE have not been investigated. Here, we purified and cloned a novel upstream serine protease of SPE that we named SAE, SPE-activating enzyme, from the hemolymph of a large beetle, Tenebrio molitor larvae. This enzyme was activated by Tenebrio modular serine protease and in turn activated the Tenebrio SPE. The biochemical ordered functions of these three serine proteases were determined in vitro, suggesting that the activation of a three-step proteolytic cascade is necessary and sufficient for lysine-type PG recognition signaling. The processed Spätzle by this cascade induced antibacterial activity in vivo. These results demonstrate that the three-step proteolytic cascade linking the PG recognition complex and Spätzle processing is essential for the PG-dependent Toll signaling pathway.

Recognition of lysine-type peptidoglycan by peptidoglycan recognition protein (PGRP)-SA provokes the activation of the Toll and prophenoloxidase pathways. Here we reveal that a soluble fragment of lysine-type peptidoglycan, a long glycan chain with short stem peptides, is a potent activator of the Drosophila Toll pathway and the prophenoloxidase activation cascade in the beetle Tenebrio molitor. Using this peptidoglycan fragment, we present biochemical evidence that clustering of PGRP-SA molecules on the peptidoglycan is required for the activation of the prophenoloxidase cascade. We subsequently highlight that the lysozymemediated partial digestion of highly cross-linked lysine-type peptidoglycan dramatically increases the binding of PGRP-SA, presumably by inducing clustering of PGRP-SA, which then recruits the Gram-negative bacteria-binding protein 1 homologue and a modular serine protease containing low-density lipoprotein and complement control protein domains. The crucial role of lysozyme in the prophenoloxidase activation cascade is further confirmed in vivo by using a lysozyme inhibitor. Taken together, we propose a model whereby lysozyme presents a processed form of lysine-type peptidoglycan for clustering of PGRP-SA that recruits Gram-negative bacteria-binding protein 1 and the modular serine protease, which leads to the activation of both the Toll and prophenoloxidase pathways.innate immunity ͉ pattern ͉ prophenoloxidase ͉ Toll

Serpins are known to be necessary for the regulation of several serine protease cascades. However, the mechanisms of how serpins regulate the innate immune responses of invertebrates are not well understood due to the uncertainty of the identity of the serine proteases targeted by the serpins. We recently reported the molecular activation mechanisms of three serine protease-mediated Toll and melanin synthesis cascades in a large beetle, Tenebrio molitor. Here, we purified three novel serpins (SPN40, SPN55, and SPN48) from the hemolymph of T. molitor. These serpins made specific serpin-serine protease pairs with three Toll cascade-activating serine proteases, such as modular serine protease, Spätzle-processing enzyme-activating enzyme, and Spätzle-processing enzyme and cooperatively blocked the Toll signaling cascade and ␤-1,3-glucanmediated melanin biosynthesis. Also, the levels of SPN40 and SPN55 were dramatically increased in vivo by the injection of a Toll ligand, processed Spätzle, into Tenebrio larvae. This increase in SPN40 and SPN55 levels indicates that these serpins function as inducible negative feedback inhibitors. Unexpectedly, SPN55 and SPN48 were cleaved at Tyr and Glu residues in reactive center loops, respectively, despite being targeted by trypsin-like Spätzle-processing enzyme-activating enzyme and Spätzle-processing enzyme. These cleavage patterns are also highly similar to those of unusual mammalian serpins involved in blood coagulation and blood pressure regulation, and they may contribute to highly specific and timely inactivation of detrimental serine proteases during innate immune responses. Taken together, these results demonstrate the specific regulatory evidences of innate immune responses by three novel serpins.

2H-TaSe2 has been one of unique transition metal dichalcogenides exhibiting several phase transitions due to a delicate balance among competing electronic ground states. An unusual metallic state at high-T is sequentially followed by an incommensurate charge density wave (ICDW) state at ≈122 K and a commensurate charge density wave (CCDW) state at ≈90 K, and superconductivity at TC ~ 0.14 K. Upon systematic intercalation of Pd ions into TaSe2, we find that CCDW order is destabilized more rapidly than ICDW to indicate a hidden quantum phase transition point at x ~ 0.09–0.10. Moreover, TC shows a dramatic enhancement up to 3.3 K at x = 0.08, ~24 times of TC in 2H-TaSe2, in proportional to the density of states N(EF). Investigations of upper critical fields Hc2 in single crystals reveal evidences of multiband superconductivity as temperature-dependent anisotropy factor γH = , quasi-linear increase of , and an upward, positive-curvature in near TC. Furthermore, analysis of temperature-dependent electronic specific heat corroborates the presence of multiple superconducting gaps. Based on above findings and electronic phase diagram vs x, we propose that the increase of N(EF) and effective electron-phonon coupling in the vicinity of CDW quantum phase transition should be a key to the large enhancement of TC in PdxTaSe2.

No abstract