Akiko Terauchi

Amino acid sequence of a peptide with molt-inhibiting activity from the kuruma prawn Penaeus japonicus

Six major peptides (Pej-SGP-I-VI) that belong to the CHH family have been isolated from the sinus gland extracts of the kuruma prawn Penaeus japonicus. By in vitro assay using the Y-organ of the crayfish Procambarus clarkii, Pej-SGP-IV was found to be active in inhibiting ecdysteroid synthesis. We determined the complete amino acid sequence. Pej-SGP-IV consists of 77 amino acid residues, with both free aimno- and carboxyl-termini. The sequence of Pej-SGP-IV shows considerable similarity to that of MIH of the shore crab Carcinus maenas and less similarity to Pej-SGP-III, whose sequence has been previously determined.

Three-dimensional Rearrangements within Inositol 1,4,5-Trisphosphate Receptor by Calcium

Allosteric binding of calcium ion (Ca2+) to inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) controls channel gating within IP3R. Here, we present biochemical and electron microscopic evidence of Ca2+-sensitive structural changes in the three-dimensional structure of type 1 IP3R (IP3R1). Low concentrations of Ca2+ and high concentrations of Sr2+ and Ba2+ were shown to be effective for the limited proteolysis of IP3R1, but Mg2+ had no effect on the proteolysis. The electron microscopy and the limited proteolysis consistently demonstrated that the effective concentration of Ca2+ for conformational changes in IP3R1 was <10-7 m and that the IP3 scarcely affected the conformational states. The structure of IP3R1 without Ca2+, as reconstructed by three-dimensional electron microscopy, had a “mushroom-like” appearance consisting of a large square-shaped head and a small channel domain linked by four thin bridges. The projection image of the “head-to-head” assembly comprising two particles confirmed the mushroom-like side view. The “windmill-like” form of IP3R1 with Ca2+ also contains the four bridges connecting from the IP3-binding domain toward the channel domain. These data suggest that the Ca2+-specific conformational change structurally regulates the IP3-triggered channel opening within IP3R1.

Potent transglutaminase inhibitors, dithio β-aminoethyl ketones

Aryl beta-aminoethyl ketones were discovered as potent inhibitors of tissue transglutaminase. Heteroaryl-like thiophene groups and N-benzyl N-t-butyl aminoethyl group are critical to the strong inhibitory activity of aryl beta-aminoethyl ketones.

Aberrant calcium signaling by transglutaminase-mediated posttranslational modification of inositol 1,4,5-trisphosphate receptors

Significance Reversible and repetitive structural changes are essential for ligand-gated ion channels to mediate biological signaling. The inositol 1,4,5-trisphosphate receptor (IP3R) assembles ligand-gated ion channels that mediate calcium signaling. IP3 activates channels at a distance by reversible allosteric changes in the IP3R tetramer. Here we show a new mode of posttranslational modification that irreversibly blocks allosteric changes in the IP3R. We identified an IP3R-modifying enzyme as tissue transglutaminase that inhibits IP3R function by locking subunit configurations. This modification chronically impaired calcium signaling and autophagy regulation in living cells, and up-regulated modification was observed in Huntington disease models. To our knowledge, this is the first demonstration of transglutaminase-catalyzed posttranslational modification in ligand-gated channel allostery and provides a new framework for enzymatic regulation of allostery. The inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum mediates calcium signaling that impinges on intracellular processes. IP3Rs are allosteric proteins comprising four subunits that form an ion channel activated by binding of IP3 at a distance. Defective allostery in IP3R is considered crucial to cellular dysfunction, but the specific mechanism remains unknown. Here we demonstrate that a pleiotropic enzyme transglutaminase type 2 targets the allosteric coupling domain of IP3R type 1 (IP3R1) and negatively regulates IP3R1-mediated calcium signaling and autophagy by locking the subunit configurations. The control point of this regulation is the covalent posttranslational modification of the Gln2746 residue that transglutaminase type 2 tethers to the adjacent subunit. Modification of Gln2746 and IP3R1 function was observed in Huntington disease models, suggesting a pathological role of this modification in the neurodegenerative disease. Our study reveals that cellular signaling is regulated by a new mode of posttranslational modification that chronically and enzymatically blocks allosteric changes in the ligand-gated channels that relate to disease states.

A Novel Neuropeptide with Molt-inhibiting Activity from the Sinus Gland of the Crayfish, Procamharus clarkii

Abstract Molt-inhibiting hormone (MIH) activity was tested in terms of the inhibitory activity of ecdysteroid secretion by cultured Y-organs of the crayfish, Procamharus clarkii. Several neuropeptides were separated from a crude extract of sinus glands of P. clarkii by means of reverse-phase high-performance liquid chromatography (HPLC) and tested for MIH activity. An extremely high level of activity was found in one fraction in reverse-phase HPLC, suggesting that the novel neuropeptide in this fraction may be a P. clarkii MIH.

IP3-mediated gating mechanism of the IP3 receptor revealed by mutagenesis and X-ray crystallography

Significance Cells use chemical signals for intracellular communication in our bodies. Inositol 1,4,5-trisphosphate (IP3) is a chemical signal that binds to the IP3 receptor (IP3R) to release calcium ions from the endoplasmic reticulum. The distance from ligand-binding sites to the channel within IP3R is the longest among known ligand-gated ion channels, and the fundamental question of how IP3-binding physically opens the channel remains unanswered. Here, we solved IP3-bound and unbound structures of large cytosolic domains of the IP3R by X-ray crystallography and clarified the IP3-dependent gating mechanism through a unique leaflet structure. These findings reveal a principle of long-range allosteric coupling in ligand-gated ion channels and provide drug targets for IP3R-regulated events, including autophagy, apoptosis, cancers, and brain disorders. The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is an IP3-gated ion channel that releases calcium ions (Ca2+) from the endoplasmic reticulum. The IP3-binding sites in the large cytosolic domain are distant from the Ca2+ conducting pore, and the allosteric mechanism of how IP3 opens the Ca2+ channel remains elusive. Here, we identify a long-range gating mechanism uncovered by channel mutagenesis and X-ray crystallography of the large cytosolic domain of mouse type 1 IP3R in the absence and presence of IP3. Analyses of two distinct space group crystals uncovered an IP3-dependent global translocation of the curvature α-helical domain interfacing with the cytosolic and channel domains. Mutagenesis of the IP3R channel revealed an essential role of a leaflet structure in the α-helical domain. These results suggest that the curvature α-helical domain relays IP3-controlled global conformational dynamics to the channel through the leaflet, conferring long-range allosteric coupling from IP3 binding to the Ca2+ channel.

Distinct roles of M1 and M3 muscarinic acetylcholine receptors controlling oscillatory and non-oscillatory [Ca2+]i increase.

We examined ACh-induced [Ca2+]i dynamics in pancreatic acinar cells prepared from mAChR subtype-specific knockout (KO) mice. ACh did not induce any [Ca2+]i increase in the cells isolated from M1/M3 double KO mice. In the cells from M3KO mice, ACh (0.3-3 μM) caused a monotonic [Ca2+]i increase. However, we found characteristic oscillatory [Ca2+]i increases in cells from M1KO mice in lower concentrations of ACh (0.03-0.3 μM). We investigated the receptor specific pattern of [Ca2+]i increase in COS-7 cells transfected with M1 or M3 receptors. ACh induced the oscillatory [Ca2+]i increase in M3 expressing cells, but not in cells expressing M1, which exhibited monotonic [Ca2+]i increases. IP3 production detected in fluorescent indicator co-transfected cells was higher in M1 than in M3 expressing cells. From the examination of four types of M1/M3 chimera receptors we found that the carboxyl-terminal region of M3 was responsible for the generation of Ca2+ oscillations. The present results suggest that the oscillatory Ca2+ increase in response to M3 stimulation is dependent upon a moderate IP3 increase, which is suitable for causing Ca(2+)-dependent IP3-induced Ca2+ release. The C-terminal domain of M3 may contribute as a regulator of the efficiency of Gq and PLC cooperation.