Tomoya Kinumi

Phosrestin I undergoes the earliest light-induced phosphorylation by a calcium/calmodulin-dependent protein kinase in drosophila photoreceptors

Activation of PI-PLC initiates two independent branches of protein phosphorylation cascades catalyzed by either PKC or Ca2+/calmodulin-dependent protein kinase (CaMK). We find that phosrestin I (PRI), a Drosophila homolog of vertebrate photoreceptor arrestin, undergoes light-induced phosphorylation on a subsecond time scale which is faster than that of any other protein in vivo. We determine that a CaMK activity is responsible for in vitro PRI phosphorylation at Ser366 in the C-terminal tryptic segment, MetLysSer(P)IleGluGlnHisArg, in which Ser(P) represents phosphoserine366. We also demonstrate that Ser366 is the phosphorylation site of PRI in vivo by identifying the molecular species resulting from in-gel tryptic digestion of purified phospho-PRI using HPLC-electrospray ionization tandem quadrupole mass spectroscopy. From these data, we conclude that the CaMK pathway, not the PKC pathway, is responsible for the earliest protein phosphorylation event following activation of PI-PLC in living Drosophila photoreceptors.

Multi-valent polymer of vancomycin : enhanced antibacterial activity against VRE

A multivalent polymer of vancomycin, synthesized via ring-opening metathesis polymerization (ROMP), exhibited significant enhancement of antibacterial activity against vancomycin-resistant enterococi (VRE).

Affinity of a vancomycin polymer with bacterial surface models

The affinity between a vancomycin polymer (3) and cell wall intermediate mimics of vancomycin resistant bacteria (VRE) was determined by use of surface plasmon resonance (SPR). The increased affinity of 3 over monomeric vancomycin derivatives 1 and 2 suggests the importance of tighter binding to VRE surfaces in the enhanced antibacterial activities of 3.

The phosphorylation site and desmethionyl N-terminus of Drosophila phosrestin I in vivo determined by mass spectrometric analysis of proteins separated by two-dimensional gel electrophoresis

Post-translational modifications of proteins play crucial roles in modulating many cellular processes. In order to understand the physiological roles of post-translational protein modifications it is imperative to determine the nature of the change in chemical structure involved in each protein modification. In our earlier work, we developed a method for the study of protein modification through a streamlined combination of two-dimensional gel electrophoresis, in-gel digestion, high performance liquid chromatography and electrospray ionization tandem quadrupole mass spectrometry. Using this method we determined the in vivo phosphorylation site of phosrestin I to be Ser366. Since our earlier work described the method only briefly, we will present a full description of the method in this paper. In addition, by using this method, we also show that the N-terminus of phosrestin I is desmethionylated in vivo. These techniques are easily adapted to the study of other proteins and will serve as powerful tools for the study of post-translational protein modifications in general.

Phosrestide-1, a peptide derived from the Drosophila photoreceptor protein phosrestin I, is a potent substrate for Ca2+/calmodulin-dependent protein kinase II from rat brain.

Multifunctional Ca2+/calmodulin-dependent protein kinase type II (CaMK II) plays a crucial role in mediation of cellular responses to rising cytosolic Ca2+ levels. We find that the novel peptide substrate PGTIEKKRSNAMKKMKSIEQHR serves as a highly potent substrate for CaMK II enzymes purified from both Drosophila and rat. The peptide is derived from a photoreceptor-specific protein, phosrestin I, of the Drosophila compound eye and is designated as phosrestide-1. Using saturating substrate concentrations, the enzymes from both species transfer the gamma-phosphoryl group of ATP to phosrestide-1 at a level three to ten times greater than to the commercially available mammalian-derived CaMK II substrates, autocamtide-3 and syntide-2. This indicates a conservation of substrate preferences for CaMK II derived from distantly related species, a dipteran fly and a mammal. Although phosrestide-1 contains two potential serine residues for CaMK II phosphorylation, we find that only the C-terminal serine is phosphorylated by rat CaMK II. However, removal of the upstream sequence containing the N-terminal serine substantially reduced the potency of phosrestide-1 as a CaMK II substrate to a level comparable to that of syntide-2 or autocamtide-3. We also find that a peptide representing the N-terminal segment of phosrestide-1 does not inhibit either CaMK II. Therefore, the enhanced potency of phosrestide-1 as a CaMK II substrate is likely to be due to a preferred conformation of the peptide induced by the N-terminal segment rather than to a specific binding of the enzymes to the N-terminus of the peptide. To the best of our knowledge, phosrestide-1 is the first CaMK II substrate which is designed based on an invertebrate sequence. The high phosphorylation level of phosrestide-1 by CaMK II of mammalian origin may reflect highly conserved CaMK II signaling cascades between vertebrates and invertebrates.

Synthesis of 818-ethanoretinal and its interaction with apo-retinochrome

Abstract All-(E)-8,18-ethanoretinal was synthesized from 2,2-dimethylcyclohexanone, and its binding experiment with apo-retinochrome afforded the new retinochrome analog, whose opsin shift exhibited fairly similar to that of the natural retinochrome.

THE CONFORMATIONAL ANALYSIS AND PHOTOISOMERIZATION OF RETINOCHROME ANALOGS WITH POLYENALS

Abstract— 3, 7‐Dimethyl‐2, 4, 6, 8, 10‐dodecapentaenal was synthesized for reconstitution of the retinochrome analog. Its opsin shift was 1000 cm 1 smaller than that of native retinochrome, whose chromophore contains the same number of double bonds. The conformational change from 6‐s‐trans to 6‐s‐cis, as figured in a retinal molecule, plays an important role in the formation of the retinochrome analog, based on the estimation of opsin shifts for retinal analogs locked in the 6‐s conformation. Thus the conformation of the 6–7 single bond in the native retinochrome was suggested to be 6 ‐cis. Analysis of the circular dichroic spectra of retinochrome analogs revealed that the 6‐s conformation is independent of the appearance of the β‐band. The stereoselectivity in the photoisomerization of the retinal analogs by a retinochrome template depends on the hydrophobic binding in the region of the β‐ionone ring.