A Dibasic Amino Acid Pair Conserved in the Activation Loop Directs Plasma Membrane Localization and Is Necessary for Activity of Plant Type I/II Phosphatidylinositol Phosphate Kinase

Phosphatidylinositol phosphate kinase (PIPK) is an enzyme involved in the regulation of cellular levels of phosphoinositides involved in various physiological processes, such as cytoskeletal organization, ion channel activation, and vesicle trafficking. In animals, research has focused on the modes of activation and function of PIPKs, providing an understanding of the importance of plasma membrane localization. However, it still remains unclear how this issue is regulated in plant PIPKs. Here, we demonstrate that the carboxyl-terminal catalytic domain, which contains the activation loop, is sufficient for plasma membrane localization of PpPIPK1, a type I/II B PIPK from the moss Physcomitrella patens. The importance of the carboxyl-terminal catalytic domain for plasma membrane localization was confirmed with Arabidopsis (Arabidopsis thaliana) AtPIP5K1. Our findings, in which substitution of a conserved dibasic amino acid pair in the activation loop of PpPIPK1 completely prevented plasma membrane targeting and abolished enzymatic activity, demonstrate its critical role in these processes. Placing our results in the context of studies of eukaryotic PIPKs led us to conclude that the function of the dibasic amino acid pair in the activation loop in type I/II PIPKs is plant specific.Phosphoinositides (PIs) are minor lipids found in membrane fractions but implicated in a wide variety of physiological regulations in eukaryotes (Di Paolo and De Camilli, 2006; Zonia and Munnik, 2006). Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] is a major PI in animal plasma membranes, affecting the localization and activity of various kinds of proteins carrying phosphatidylinositol-binding domains, which in turn affect the regulation of cytoskeletal organization, vesicle trafficking, cell proliferation, and cell growth during development and stress responses (Doughman et al., 2003; Downes et al., 2005; Di Paolo and De Camilli, 2006; Zonia and Munnik, 2006; Heck et al., 2007). In addition, PtdIns(4,5)P₂ is also a well-known substrate of phospholipase C, producing second messengers such as diacylglycerol, phosphatidic acid (PA), and inositol-1,4,5-trisphosphate, which are involved in the activation of intracellular signal transduction pathways (Zonia and Munnik, 2006). Transient accumulation of PtdIns(4,5)P₂ has also been observed under various kinds of environmental stress (Pical et al., 1999; DeWald et al., 2001), suggesting an important role of this lipid in the regulation of stress signal transduction pathways also in plants. These findings indicate that PtdIns(4,5)P₂ is multifunctional and involved in a variety of cellular processes. Therefore, elucidation of the mechanisms controlling the cellular levels of PtdIns(4,5)P₂ is important in understanding the significance of PI signaling in eukaryotes.PtdIns(4,5)P₂ is synthesized by phosphatidylinositol phosphate kinases (PIPKs; Anderson et al., 1999; Doughman et al., 2003; Heck et al., 2007). Physiological roles of several plant PIPKs have been reported. In Arabidopsis (Arabidopsis thaliana), AtPIP5K3 is an essential regulator of tip growth of root hairs (Kusano et al., 2008; Stenzel et al., 2008), while AtPIPK4 and AtPIPK5 are essential for pollen germination and pollen tube elongation (Ischebeck et al., 2008; Sousa et al., 2008). In addition, AtPIP5K9 was shown to interact with the cytosolic invertase CINV1 to regulate sugar-mediated root cell elongation negatively (Lou et al., 2007). Rice (Oryza sativa) OsPIPK1 is proposed to be involved in shoot growth and floral initiation through the regulation of floral induction genes (Ma et al., 2004). In animals, membrane-associated type I PIPK mainly phosphorylates the D-5 hydroxyl group of PtdIns4P to produce PtdIns(4,5)P₂ but also produces PtdIns(3,4)P₂ and PtdIns(3,5)P₂ from PtdIns3P with 5- and 4-kinase activity (Anderson et al., 1999; Heck et al., 2007), whereas type II PIPK prefers the D-4 position of PtdIns5P, producing PtdIns(4,5)P₂ in the nucleus and at the endoplasmic reticulum (Clarke et al., 2007). Thus, in animals, type I and II PIPKs are involved in the generation of PtdIns(4,5)P₂ via different pathways. Molecular biological analysis of plant PIPKs was initiated with AtPIP5K1 from Arabidopsis (Mikami et al., 1998), which phosphorylates PtdIns3P, PtdIns4P, and PtdIns(4,5)P₂ to produce PtdIns(3,4)P₂, PtdIns(4,5)P₂, and PtdIns (3,4,5)P₃, respectively, with D-4- and D-5-kinase activity (Elge et al., 2001; Westergren et al., 2001; Im et al., 2007). Similar enzymatic activity was also reported for other PIPKs from Arabidopsis (Ischebeck et al., 2008; Kusano et al., 2008; Stenzel et al., 2008). In addition, a PIPK from the moss Physcomitrella patens (designated as PpPIPK1) preferred PtdIns4P, PtdIns3P, and PtdIns(3,4)P₂ as substrates, but not PtdIns5P, producing PtdIns(4,5)P₂, PtdIns(3,4)P₂, and PtdIns(3,4,5)P₃, respectively (Saavedra et al., 2009). These findings indicate that the substrate specificity of plant PIPKs is essentially the same as that of type I PIPKs. However, AtPIP5K1 has yet to be classified as either type I or type II based on sequence comparisons of the catalytic domain (CD; Mikami et al., 1998). This was confirmed by a genome-wide analysis of PIPK genes in Arabidopsis in which all 11 PIPKs were classified as type I/II based on sequence comparisons of the CDs, which were further subdivided into subtypes A and B (Mueller-Roeber and Pical, 2002). Therefore, it is suggested that typical type I and II PIPKs are absent in plants, although further confirmation is needed.The conserved PIPK CD contains a short highly conserved region near its C-terminal end, designated the activation loop, which acts as the substrate-binding site and is responsible for the differences in substrate specificity and subcellular localization between animal type I and type II PIPKs (Kunz et al., 2000, 2002). Substrate specificities of animal type I and type II PIPKs, for example, are determined by a respective Glu and Ala at the corresponding positions in the activation loop. Moreover, it has been established that substitution of Glu to Ala results in a swap of substrate specificity and subcellular localization between the two types (Kunz et al., 2000, 2002). In contrast to animal PIPKs, a substitution in the activation loop of PpPIPK1 from Glu to Ala resulted in a nearly complete loss of type I/II activity; however, such a mutation did not fully convert the substrate specificity, although an enhancement of type II versus type I activity was observed (Saavedra et al., 2009). Since the corresponding amino acid residue is Glu in all plant PIPKs so far reported, it is suggested that there also is a plant-specific mode of substrate specificity regulation in plant type I/II PIPKs. However, enzymatic activity appears to be modified in similar ways between plant type I/II and animal type I PIPKs; that is, phosphorylation- and PA-dependent activation of PIPKs has been observed in both animals and plants (Moritz et al., 1992; Jenkins et al., 1994; Pical et al., 1999; Westergren et al., 2001; Perera et al., 2005; Saavedra et al., 2009).The regulation of plasma membrane localization of mammalian type I PIPKs remains confusing. In addition to the involvement of a Glu residue as mentioned above, the substitution of two Lys residues in the activation loop to Asn residues changes the subcellular localization from the plasma membrane to the cytosol (Kunz et al., 2000, 2002). However, Arioka et al. (2004) also showed that the plasma membrane localization of type I PIPKs is regulated by another basic amino acid pair localized downstream of the activation loop in the CD, which is not found in type II PIPKs. Interestingly, the mechanism behind plasma membrane localization of plant PIPKs seems to differ significantly from the animal one. The obvious structural feature of plant PIPKs is the presence of a repetition of membrane occupation and recognition nexus (MORN) motifs at the N-terminal half, which is conserved across the B subfamily of plant type I/II PIPKs (Mueller-Roeber and Pical, 2002). The MORN motif was first identified in mammalian junctophilin, an endoplasmic reticulum-membrane-bound component of the junctional complex between the plasma membrane and the endoplasmic reticulum (Takeshima et al., 2000). Since MORN motifs are not found in PIPKs from nonplant organisms, a plant-specific mode of PIPK activation is speculated. Indeed, a regulatory role of the MORN domain was reported in the enzymatic activation of AtPIP5K1 (Im et al., 2007) and in root hair formation, but not in enzymatic activation, of AtPIP5K3 (Stenzel et al., 2008). Moreover, the MORN domain may play a role in the plasma membrane localization of OsPIPK1 from rice and AtPIP5K1 and AtPIP5K3 from Arabidopsis (Ma et al., 2006; Im et al., 2007; Kusano et al., 2008). However, stable transformation of tobacco (Nicotiana tabacum) cells to express an AtPIP5K1 MORN domain-GFP fusion did not allow visualization of the plasma membrane localization of this protein (Im et al., 2007). Thus, it is not clear if the MORN domain functions as a plasma membrane-targeting module.Given the sequence conservation of the CD among eukaryotic PIPKs (Saavedra et al., 2009), we hypothesize that the CD is responsible for the plasma membrane localization of plant PIPKs. Thus, to gain further insight into the mechanisms regulating this issue, we dissected PpPIPK1 to determine the molecular determinants of plasma membrane localization. Here, we show that the MORN domain is not involved in the plasma membrane localization of PpPIPK1 and AtPIP5K1 in P. patens protoplasts and onion (Allium cepa) epidermal cells. We further demonstrate that two basic amino acids, but not Glu, conserved in the activation loop of the CD are required for plasma membrane localization. These findings demonstrate that the activation mode of type I/II PIPKs is plant specific and differs from that of the membrane-localized animal type I PIPKs.     

Phylogeny, biogeography, and host-plant association in the subfamily Apaturinae (Insecta: Lepidoptera: Nymphalidae) inferred from eight nuclear and seven mitochondrial genes

The subfamily Apaturinae consists of 20 genera and shows disjunct distributions and unique host-plant associations. Most genera of this subfamily are distributed in Eurasia South-East Asia and Africa, whereas the genera Doxocopa and Asterocampa are distributed mainly in South America and North America, respectively. Although the Apaturinae larvae mainly feed on the Cannabaceae, those of the genus Apatura are associated with Salix and Populus (Salicaceae), which are distantly related to the Cannabaceae. Here, we infer the phylogeny of Apaturinae and reconstruct the history of host shifting and of colonization in the New World. We analyzed 9761 bp of nuclear and mitochondrial DNA sequence data, including the genes encoding EF1a, Wg, ArgK, CAD, GAPDH, IDH, MDH, RpS5, COI, COII, ATPase8, ATPase6, COIII, ND3, and ND5 for 12 apaturine genera. We also inferred the phylogeny with six additional genera using mitochondrial sequence data alone. Within the Apaturinae, two major clades are recovered in all the datasets. These clades separate the New World genera, Doxocopa and Asterocampa, indicating that dispersal to the New World occurred at least twice. According to our divergence time estimates, these genera originated during the Early Oligocene to the Early Miocene, implying that they migrated across the Bering Land Bridge rather than the Atlantic Land Bridge. The temporal estimates also show that host shifting to Salix or Populus in Apatura occurred more than 15 million years after the divergence of their host plants. Our phylogenetic results are inconsistent with the previously accepted apaturine genus groups and indicate that their higher classification should be reconsidered.     

A microbial TLR2 agonist imparts macrophage-activating ability to apolipoprotein A-1

There is increasing epidemiologic evidence implying a role for chronic infection in atherosclerosis and that microbial TLR agonists may contribute to this disease. Mycoplasma arthritidis is an agent of acute and chronic inflammatory disease in rodents, and has been used extensively as a model for defining the mechanisms involved in arthritis and other inflammatory diseases. We have purified a 28-kDa, apolipoprotein A-1 (apoA-1)-like TLR2-dependent macrophage-activating moiety from a culture of a virulent strain of M. arthritidis. ApoA-1 similarly isolated from uninoculated mycoplasma medium was without bioactivity. The activity of the mycoplasma-derived molecule was resistant to heat and to digestion with proteinase K, but was susceptible to alkaline hydrolysis and H(2)O(2) oxidation. Infrared profiles of normal apoA-1 and that derived from mycoplasma were distinct. Unlike the activity of other mycoplasmal TLR2 agonists such as macrophage-activating lipopeptide-2, activity of the M. arthritidis-derived 28-kDa component was dependent upon CD14, a coreceptor for LPS. Finally, we showed that bioactive lipopeptides prepared from M. arthritidis grown in serum-free medium and also from a 41-kDa known bioactive lipoprotein of M. arthritidis, avidly bound to purified apoA-1 that separated out by SDS-PAGE, induced TNF-alpha and IL-12p40 both in vitro and in vivo. ApoA-1 is a key functional component of the high-density lipoprotein cholesterol complex by scavenging and removing unwanted lipids. Our finding that this molecule can acquire macrophage-activating properties from microbial TLR2-dependent agonists suggests a novel mechanism whereby some microbial agents might reverse the protective role of apoA-1, thus contributing to the genesis of atherosclerosis.     

Biological implications of the occurrence of 32 members of the XTH (xyloglucan endotransglucosylase/hydrolase) family of proteins in the bryophyte Physcomitrella patens

This comprehensive overview of the xyloglucan endotransglucosylase/hydrolase (XTH) family of genes and proteins in bryophytes, based on research using genomic resources that are newly available for the moss Physcomitrella patens, provides new insights into plant evolution. In angiosperms, the XTH genes are found in large multi‐gene families, probably reflecting the diverse roles of individual XTHs in various cell types. As there are fewer cell types in P. patens than in angiosperms such as Arabidopsis and rice, it is tempting to deduce that there are fewer XTH family genes in bryophytes. However, the present study unexpectedly identified as many as 32 genes that potentially encode XTH family proteins in the genome of P. patens, constituting a fairly large multi‐gene family that is comparable in size with those of Arabidopsis and rice. In situ localization of xyloglucan endotransglucosylase activity in this moss indicates that some P. patens XTH proteins exhibit biochemical functions similar to those found in angiosperms, and that their expression profiles are tissue‐dependent. However, comparison of structural features of families of XTH genes between P. patens and angiosperms demonstrated the existence of several bryophyte‐specific XTH genes with distinct structural and functional features that are not found in angiosperms. These bryophyte‐specific XTH genes might have evolved to meet morphological and functional needs specific to the bryophyte. These findings raise interesting questions about the biological implications of the XTH family of proteins in non‐seed plants.     

Drug design and synthesis of a novel κ opioid receptor agonist with an oxabicyclo[2.2.2]octane skeleton and its pharmacology

A conformational analysis of κ opioid receptor agonists, TRK-820 and U-50,488H indicated an active conformation of TRK-820 in which the C-ring was in the boat form with the 14-OH interacting with the amide nitrogen. Based on the obtained active conformation of TRK-820, we designed and synthesized a novel κ agonist KNT-63 with oxabicyclo[2.2.2]octane skeleton. KNT-63 showed profound antinociceptive effects via the κ receptor which were as potent as that of TRK-820.     

Identification of an EMS-induced causal mutation in a gene required for boron-mediated root development by low-coverage genome re-sequencing in Arabidopsis

Next-generation sequencing (NGS) technologies enable the rapid production of an enormous quantity of sequence data. These powerful new technologies allow the identification of mutations by whole-genome sequencing. However, most reported NGS-based mapping methods, which are based on bulked segregant analysis, are costly and laborious. To address these limitations, we designed a versatile NGS-based mapping method that consists of a combination of low- to medium-coverage multiplex SOLiD (Sequencing by Oligonucleotide Ligation and Detection) and classical genetic rough mapping. Using only low to medium coverage reduces the SOLiD sequencing costs and, since just 10 to 20 mutant F₂ plants are required for rough mapping, the operation is simple enough to handle in a laboratory with limited space and funding. As a proof of principle, we successfully applied this method to identify the CTR1, which is involved in boron-mediated root development, from among a population of high boron requiring Arabidopsis thaliana mutants. Our work demonstrates that this NGS-based mapping method is a moderately priced and versatile method that can readily be applied to other model organisms.     

Label-free THz sensing of living body-related molecular binding using a metallic mesh

We have demonstrated label-free THz sensing of living body-related molecular binding using a thin metallic mesh and a polyvinylidene difluoride (PVDF) membrane. Metallic meshes in the THz region are designed for anomalous transmission phenomena derived from a resonant excitation of surface waves. Additionally, they are designed to have a sharp dip in transmittance. The metallic mesh is very sensitive to a change of the refractive index of materials attached to the metallic mesh. In this paper, we report sensing of interactions between lectin and sugar using this technique. We found that the dip frequency shift, transmittance attenuation of the dip frequency, and peak shift of the derivative spectrum of the phase shift depend on the bonding amount of lectin–sugar interactions. We also applied this technique to detect avidin–biotin interactions, leading to the detection of a small amount of biotin (0.17 pg/mm²).     

Hydrolyzed eggshell membrane immobilized on phosphorylcholine polymer supplies extracellular matrix environment for human dermal fibroblasts

We have found that a water-soluble alkaline-digested form of eggshell membrane (ASESM) can provide an extracellular matrix (ECM) environment for human dermal fibroblast cells (HDF) in vitro. Avian eggshell membrane (ESM) has a fibrous-meshwork structure and has long been utilized as a Chinese medicine for recovery from burn injuries and wounds in Asian countries. Therefore, ESM is expected to provide an excellent natural material for biomedical use. However, such applications have been hampered by the insolubility of ESM proteins. We have used a recently developed artificial cell membrane biointerface, 2-methacryloyloxyethyl phosphorylcholine polymer (PMBN) to immobilize ASESM proteins. The surface shows a fibrous structure under the atomic force microscope, and adhesion of HDF to ASESM is ASESM-dose-dependent. Quantitative mRNA analysis has revealed that the expression of type III collagen, matrix metalloproteinase-2, and decorin mRNAs is more than two-fold higher when HDF come into contact with a lower dose ASESM proteins immobilized on PMBN surface. A particle-exclusion assay with fixed erythrocytes has visualized secreted water-binding molecules around the cells. Thus, HDF seems to possess an ECM environment on the newly designed PMBN-ASESM surface, and future applications of the ASESM-PMBN system for biomedical use should be of great interest.