Structural analysis of the human cannabinoid receptor one carboxyl‐terminus identifies two amphipathic helices

Recent research has implicated the C‐terminus of G‐protein coupled receptors in key events such as receptor activation and subsequent intracellular sorting, yet obtaining structural information of the entire C‐tail has proven a formidable task. Here, a peptide corresponding to the full‐length C‐tail of the human CB1 receptor (residues 400–472) was expressed in E.coli and purified in a soluble form. Circular dichroism (CD) spectroscopy revealed that the peptide adopts an α‐helical conformation in negatively charged and zwitterionic detergents (48–51% and 36–38%, respectively), whereas it exhibited the CD signature of unordered structure at low concentration in aqueous solution. Interestingly, 27% helicity was displayed at high peptide concentration suggesting that self‐association induces helix formation in the absence of a membrane mimetic. NMR spectroscopy of the doubly labeled (¹⁵N‐ and ¹³C‐) C‐terminus in dodecylphosphocholine (DPC) identified two amphipathic α‐helical domains. The first domain, S401‐F412, corresponds to the helix 8 common to G protein‐coupled receptors while the second domain, A440‐M461, is a newly identified structural motif in the distal region of the carboxyl‐terminus of the receptor. Molecular modeling of the C‐tail in DPC indicates that both helices lie parallel to the plane of the membrane with their hydrophobic and hydrophilic faces poised for critical interactions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 565–573, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Corneal Dystrophy-associated R124H Mutation Disrupts TGFBI Interaction with Periostin and Causes Mislocalization to the Lysosome

The 5q31-linked corneal dystrophies are heterogeneous autosomal-dominant eye disorders pathologically characterized by the progressive accumulation of aggregated proteinaceous deposits in the cornea, which manifests clinically as severe vision impairment. The 5q31-linked corneal dystrophies are commonly caused by mutations in the TGFBI (transforming growth factor-β-induced) gene. However, despite the identification of the culprit gene, the cellular roles of TGFBI and the molecular mechanisms underlying the pathogenesis of corneal dystrophy remain poorly understood. Here we report the identification of periostin, a molecule that is highly related to TGFBI, as a specific TGFBI-binding partner. The association of TGFBI and periostin is mediated by the amino-terminal cysteine-rich EMI domains of TGFBI and periostin. Our results indicate that the endogenous TGFBI and periostin colocalize within the trans-Golgi network and associate prior to secretion. The corneal dystrophy-associated R124H mutation in TGFBI severely impairs interaction with periostin in vivo. In addition, the R124H mutation causes aberrant redistribution of the mutant TGFBI into lysosomes. We also find that the periostin-TGFBI interaction is disrupted in corneal fibroblasts cultured from granular corneal dystrophy type II patients and that periostin accumulates in TGFBI-positive corneal deposits in granular corneal dystrophy type II (also known as Avellino corneal dystrophy). Together, our findings suggest that TGFBI and periostin may play cooperative cellular roles and that periostin may be involved in the pathogenesis of 5q31-linked corneal dystrophies.Corneal dystrophies are characterized by the progressive loss of corneal transparency as a result of extracellular amyloid and non-amyloid deposits, which accumulate in different layers of corneal tissues. 5q31-linked corneal dystrophies are pathologically heterogeneous, autosomal-dominant disorders caused by mutations in the TGFBI (transforming growth factor-β-induced) gene, which encodes the TGFBI protein (also known as keratoepithelin or Big-H3) (1, 2). To date, more than 30 different mutations leading to corneal dystrophies have been attributed to mutations in TGFBI, the most frequent of which are mutations within exons 4 and 12, which result in amino acid substitutions in Arg¹²⁴ and Arg⁵⁵⁵, respectively (3, 4). The different mutations in TGFBI cause clinically distinct types of corneal dystrophies, which are classified according to the accumulation patterns of the deposits, including lattice corneal dystrophies type I and IIIA, deep stromal lattice corneal dystrophy, granular corneal dystrophies (GCDs)² type I and II (also known as Avellino corneal dystrophy), Reis-Bucklers corneal dystrophy (also known as corneal dystrophy of Bowman''s layer type I), or Thiel-Behnke corneal dystrophy (also known as corneal dystrophy of Bowman''s layer type II) (reviewed in Refs. 5 and 6). Histological examinations of corneal tissues demonstrate the presence of amyloid deposits in lattice corneal dystrophies and GCD II, hyaline accumulations in GCDs, and subepithelial fibrous material in Reis-Bucklers corneal dystrophy and Thiel-Behnke corneal dystrophy (7–14).TGFBI was originally identified as a gene induced by transforming growth factor-β stimulation in adenocarcinoma cells and is expressed in many tissues (15). The human TGFBI consists of 683 amino acids, with the mature protein predicted to have a molecular mass of ∼68 kDa. As shown in Fig. 1A, TGFBI contains an NH₂-terminal signal peptide that targets it for insertion into the lumen of the endoplasmic reticulum (ER) for eventual secretion, a cysteine-rich EMI domain, four tandem repeats of fasciclin-1 like (FAS1) domains, and a COOH-terminal RGD sequence (15–19). The FAS1 domains of TGFBI display homology to the cell adhesion protein fasciclin-I in Drosophila, an axon guidance protein that is involved in neuronal development (20). Based on the presence of multiple FAS1 domains, TGFBI has been assigned to a larger family of proteins, which includes periostin, stabilin-1, and stabilin-2 (16, 21). To date, many TGFBI homologues have been reported in various vertebrates, including mouse, chicken, pig, and zebrafish, but no homologues have been identified in invertebrates (16, 19, 21). TGFBI has been shown to interact with a number of extracellular matrix (ECM) proteins, including fibronectin, biglycan, decorin, and several types of collagen (19, 22–25). Furthermore, TGFBI also functions as a ligand for several integrins, including α3β1, αvβ5, αvβ3, and αmβ2 (26–29). The COOH-terminal RGD domain of TGFBI is the putative integrin-binding motif. However, several studies have suggested that the interactions between TGFBI and integrins are mediated via the YH (tyrosine-histidine) motifs and DI (aspartate-isoleucine) motifs present in the TGFBI FAS1 domains (30). Although the precise roles of TGFBI are not fully understood yet, emerging evidence suggests a role for TGFBI as a secreted factor involved in cell adhesion, proliferation, and migration.Open in a separate windowFIGURE 1.Periostin is expressed in human cornea. A, schematic representation of TGFBI and periostin. TGFBI and periostin contain NH₂-terminal signal peptides, followed by a cysteine-rich EMI domain and four tandem FAS1 domains. TGFBI also contains a COOH-terminal RGD motif that is not present in periostin, which instead contains a COOH-terminal hydrophilic region. The following domains of TGFBI and periostin are indicated: EMI, FAS1, fasciclin 1, and Arg-Gly-Asp (RGD). Antigenic regions of antibodies used in this study (ab14041 and C-20) are represented by black lines. B, cell and tissue lysates were separated by SDS-PAGE and Western blotted using the indicated antibodies. Anti-periostin antibody (C-20) recognizes an ∼85-kDa protein in several cell lines and corneal fibroblast cell lines. In contrast, anti-periostin antibody recognizes a ∼60-kDa form in corneal epithelial cell lines and corneal epithelium tissues (top, black arrow). The specificity of periostin antibody (C-20) was confirmed by preabsorption with 10 μg of C-20 antigen peptide (second panel). WB, Western blot.TGFBI and periostin show a high degree of similarity in amino acid sequence and in overall domain structure, diverging primarily at the COOH terminus (Fig. 1A) (16, 21). Similar to TGFBI, periostin contains a NH₂-terminal secretory signal peptide followed by a cysteine-rich EMI domain, four tandem repeats of FAS1 domains, and a hydrophilic region in its COOH terminus (Fig. 1A) (16, 17, 31, 32). Periostin has been found to be ubiquitously expressed in multiple tissues in mammals (31, 33, 34). In addition, the expression of periostin has been implicated in the development of variety of cancers, including neuroblastoma, head and neck cancer, and non-small cell lung cancer, possibly by regulating the metastatic growth (32, 35). Periostin is also associated with epithelial-mesenchymal transition during cardiac development (36) and is induced during the proliferation of cardiomyocytes, thereby promoting cardiac repair after heart failure (37, 38). In addition, interlukin-4 and -13 have been found to induce the secretion of periostin from lung fibroblasts, implicating periostin in subepithelial fibrosis in bronchial asthma (39).Despite the similarities between TGFBI and periostin, it is not known whether periostin is involved in the pathogenesis of 5q31-linked corneal dystrophies. In this study, we find that periostin specifically interacts with TGFBI via the NH₂-terminal cysteine-rich EMI domain and colocalizes with TGFBI in the trans-Golgi network of COS-7 and corneal fibroblast cells. In addition, corneal dystrophy-linked mutations in TGFBI disrupt its subcellular localization and impair its interaction with periostin. Furthermore, we find that periostin accumulates in extracellular corneal deposits in GCD II patients bearing homozygous R124H mutations in TGFBI. These findings provide new insights into the pathogenic mechanisms of TGFBI mutations in 5q31-linked corneal dystrophies and have important implications for understanding and treating corneal dystrophies.     

Interaction of Organic Cations with Organic Anion Transporters

Studies of the organic anion transporters (Oats) have focused mainly on their interactions with organic anionic substrates. However, as suggested when Oat1 was originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471–6478), since the Oats share close homology with organic cation transporters (Octs), it is possible that Oats interact with cations as well. We now show that mouse Oat1 (mOat1) and mOat3 and, to a lesser degree, mOat6 bind a number of “prototypical” Oct substrates, including 1-methyl-4-phenylpyridinium. In addition to oocyte expression assays, we have tested binding of organic cations to Oat1 and Oat3 in ex vivo assays by analyzing interactions in kidney organ cultures deficient in Oat1 and Oat3. We also demonstrate that mOat3 transports organic cations such as 1-methyl-4-phenylpyridinium and cimetidine. A pharmacophore based on the binding affinities of the tested organic cations for Oat3 was generated. Using this pharmacophore, we screened a chemical library and were able to identify novel cationic compounds that bound to Oat1 and Oat3. These compounds bound Oat3 with an affinity higher than the highest affinity compounds in the original set of prototypical Oct substrates. Thus, whereas Oat1, Oat3, and Oat6 appear to function largely in organic anion transport, they also bind and transport some organic cations. These findings could be of clinical significance, since drugs and metabolites that under normal physiological conditions do not bind to the Oats may undergo changes in charge and become Oat substrates during pathologic conditions wherein significant variations in body fluid pH occur.     

Enzymatic synthesis of alkyl glucosides using <Emphasis Type="Italic">Leuconostoc</Emphasis> <Emphasis Type="Italic">mesenteroides</Emphasis> dextransucrase

Alkyl glucosides were synthesized by the reaction of Leuconostoc mesenteroides dextransucrase with sucrose and various alcohols. Alkyl α-d-glucosides were obtained with a yield of 30% (mol/mol) with primary alcohols, but secondary alcohols or tertiary alcohols gave yields below 5%. The optimal yield was 50% using 1-butyl α-d-glucoside with 0.9 M 1-butanol. The acceptor products of methanol or ethanol were confirmed as methyl α-d-glucopyranoside and ethyl α-d-glucopyranoside via MALDI-TOF MS and NMR analysis. Thus, methyl or ethyl α-d-glucoside constituted half the emulsification activities of Triton X-100 as commercially available surfactants. Young-Min Kim and Byung-Hoon Kim contributed equally to this work.     

混交林测树因子概率分布模型的构建及应用

基于最大熵原理,针对目前对混交林测树因子概率分布模型研究的不足,提出了联合最大熵概率密度函数,该函数具有如下特点：1）函数的每一组成部分都是相互联系的最大熵函数,故可以综合混交林各主要组成树种测树因子的概率分布信息;2）函数是具有双权重的概率表达式,能体现混交林结构复杂的特点,在最大限度地利用混交林每一主要树种测树因子概率分布信息的同时,还能精确地全面反映混交林测树因子概率分布规律;3）函数的结构简洁、性能优良.用天目山自然保护区的混交林样地对混交林测树因子概率分布模型进行了应用与检验,结果表明：模型的拟合精度(R²=0.9655)与检验精度(R²=
0.9772)都较高.说明联合最大熵概率密度函数可以作为混交林测树因子概率分布模型,为全面了解混交林林分结构提供了一种可行的方法.     

成团泛菌YS19 symplasmata结构对菌体抵抗逆境的作用

成团泛菌（Pantoea agglomerans）YS19是从水稻“越富”品种中分离出的优势内生菌,其所形成的共质体（symplasmata）是一种与生物薄膜（biofilm）类似的多细胞聚集体结构,但细胞间联系比biofilm更加紧密。研究symplasmata结构对成团泛菌YS19抵抗逆境的贡献,有助于阐释内生菌与植物的相互作用的适应性。比较研究了symplasmata结构与散生菌体对于蔗糖渗透压冲击、重金属离子和干燥处理的抵抗能力差异,结果表明,与以散生状态存在的菌体相比,在面临逆境时形成symplasmata结构的菌体抗逆存活能力显著增强。