Special Issue "Feature Papers"

Guest Editor
Prof. Dr. Jürg Bähler
Department of Genetics, Evolution & Environment and UCL Cancer Institute, University College London, Darwin Building, Gower Street, London, WC1E 6BT, UK
Website: http://www.bahlerlab.info
E-Mail: j.bahler@ucl.ac.uk
Phone: +44 2031 081602
Fax: +44 2076 797096
Interests: gene regulation; genomics; transcriptomics; next-generation sequencing; non-coding RNAs; genome evolution; fission yeast; oxidative stress response; cellular quiescence and ageing

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Roger S. Holmes and Ujjwal K. Rout Review: Comparative Studies of Vertebrate Beta Integrin Genes and Proteins: Ancient Genes in Vertebrate Evolution Biomolecules 2011, 1(1), 3-31; doi:10.3390/biom1010003 Received: 18 July 2011; in revised form: 14 August 2011 / Accepted: 15 August 2011 / Published: 23 August 2011 Show/Hide Abstract | Download PDF Full-text (1277 KB) Abstract: Intregins are heterodimeric α- and β-subunit containing membrane receptor proteins which serve various cell adhesion roles in tissue repair, hemostasis, immune response, embryogenesis and metastasis. At least 18 α- (ITA or ITGA) and 8 β-integrin subunits (ITB or ITGB) are encoded on mammalian genomes. Comparative ITB amino acid sequences and protein structures and ITB gene locations were examined using data from several vertebrate genome projects. Vertebrate ITB genes usually contained 13–16 coding exons and encoded protein subunits with ~800 amino acids, whereas vertebrate ITB4 genes contained 36-39 coding exons and encoded larger proteins with ~1800 amino acids. The ITB sequences exhibited several conserved domains including signal peptide, extracellular β-integrin, β-tail domain and integrin β-cytoplasmic domains. Sequence alignments of the integrin β-cytoplasmic domains revealed highly conserved regions possibly for performing essential functions and its maintenance during vertebrate evolution. With the exception of the human ITB8 sequence, the other ITB sequences shared a predicted 19 residue α-helix for this region. Potential sites for regulating human ITB gene expression were identified which included CpG islands, transcription factor binding sites and microRNA binding sites within the 3’-UTR of human ITB genes. Phylogenetic analyses examined the relationships of vertebrate beta-integrin genes which were consistent with four major groups: 1: ITB1, ITB2, ITB7; 2: ITB3, ITB5, ITB6; 3: ITB4; and 4: ITB8 and a common evolutionary origin from an ancestral gene, prior to the appearance of fish during vertebrate evolution. The phylogenetic analyses revealed that ITB4 is the most likely primordial form of the vertebrate β integrin subunit encoding genes, that is the only β subunit expressed as a constituent of the sole integrin receptor ‘α6β4’ in the hemidesmosomes of unicellular organisms.

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Tomonobu Yokomichi, Kyoko Morimoto, Nana Oshima, Yuriko Yamada, Liwei Fu, Shigeru Taketani, Masayoshi Ando and Takao Kataoka Article: Ursolic Acid Inhibits Na+/K+-ATPase Activity and Prevents TNF-α-Induced Gene Expression by Blocking Amino Acid Transport and Cellular Protein Synthesis Biomolecules 2011, 1(1), 32-47; doi:10.3390/biom1010032 Received: 20 August 2011; in revised form: 26 October 2011 / Accepted: 27 October 2011 / Published: 7 November 2011 Show/Hide Abstract | Download PDF Full-text (476 KB) Abstract: Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, induce the expression of a wide variety of genes, including intercellular adhesion molecule-1 (ICAM-1). Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) was identified to inhibit the cell-surface ICAM-1 expression induced by pro-inflammatory cytokines in human lung carcinoma A549 cells. Ursolic acid was found to inhibit the TNF-α-induced ICAM-1 protein expression almost completely, whereas the TNF-α-induced ICAM-1 mRNA expression and NF-κB signaling pathway were decreased only partially by ursolic acid. In line with these findings, ursolic acid prevented cellular protein synthesis as well as amino acid uptake, but did not obviously affect nucleoside uptake and the subsequent DNA/RNA syntheses. This inhibitory profile of ursolic acid was similar to that of the Na+/K+-ATPase inhibitor, ouabain, but not the translation inhibitor, cycloheximide. Consistent with this notion, ursolic acid was found to inhibit the catalytic activity of Na+/K+-ATPase. Thus, our present study reveals a novel molecular mechanism in which ursolic acid inhibits Na+/K+-ATPase activity and prevents the TNF-α-induced gene expression by blocking amino acid transport and cellular protein synthesis.

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Hirokazu Yagi, Erina Ohno, Sachiko Kondo, Atsuhiro Yoshida and Koichi Kato Article: Development and Application of Multidimensional HPLC Mapping Method for O-linked Oligosaccharides Biomolecules 2011, 1(1), 48-62; doi:10.3390/biom1010048 Received: 29 August 2011; in revised form: 30 November 2011 / Accepted: 5 December 2011 / Published: 14 December 2011 Show/Hide Abstract | Download PDF Full-text (454 KB) Abstract: Glycosylation improves the solubility and stability of proteins, contributes to the structural integrity of protein functional sites, and mediates biomolecular recognition events involved in cell-cell communications and viral infections. The first step toward understanding the molecular mechanisms underlying these carbohydrate functionalities is a detailed characterization of glycan structures. Recently developed glycomic approaches have enabled comprehensive analyses of N-glycosylation profiles in a quantitative manner. However, there are only a few reports describing detailed O-glycosylation profiles primarily because of the lack of a widespread standard method to identify O-glycan structures. Here, we developed an HPLC mapping method for detailed identification of O-glycans including neutral, sialylated, and sulfated oligosaccharides. Furthermore, using this method, we were able to quantitatively identify isomeric products from an in vitro reaction catalyzed by N-acetylglucosamine-6O-sulfotransferases and obtain O-glycosylation profiles of serum IgA as a model glycoprotein.

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Jingyuan Deng, Lirong Tan, Xiaodong Lin, Yao Lu and Long J. Lu Article: Exploring the Optimal Strategy to Predict Essential Genes in Microbes Biomolecules 2012, 2(1), 1-22; doi:10.3390/biom2010001 Received: 11 November 2011; in revised form: 16 December 2011 / Accepted: 19 December 2011 / Published: 27 December 2011 Show/Hide Abstract | Download PDF Full-text (1115 KB) Abstract: Accurately predicting essential genes is important in many aspects of biology, medicine and bioengineering. In previous research, we have developed a machine learning based integrative algorithm to predict essential genes in bacterial species. This algorithm lends itself to two approaches for predicting essential genes: learning the traits from known essential genes in the target organism, or transferring essential gene annotations from a closely related model organism. However, for an understudied microbe, each approach has its potential limitations. The first is constricted by the often small number of known essential genes. The second is limited by the availability of model organisms and by evolutionary distance. In this study, we aim to determine the optimal strategy for predicting essential genes by examining four microbes with well-characterized essential genes. Our results suggest that, unless the known essential genes are few, learning from the known essential genes in the target organism usually outperforms transferring essential gene annotations from a related model organism. In fact, the required number of known essential genes is surprisingly small to make accurate predictions. In prokaryotes, when the number of known essential genes is greater than 2% of total genes, this approach already comes close to its optimal performance. In eukaryotes, achieving the same best performance requires over 4% of total genes, reflecting the increased complexity of eukaryotic organisms. Combining the two approaches resulted in an increased performance when the known essential genes are few. Our investigation thus provides key information on accurately predicting essential genes and will greatly facilitate annotations of microbial genomes.

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Urvashi Rai, Jing Huang, Satish Mishra, Xiangming Li, Takayuki Shiratsuchi and Moriya Tsuji Article: A New Method to Determine Antigen-Specific CD8+ T Cell Activity in Vivo by Hydrodynamic Injection Biomolecules 2012, 2(1), 23-33; doi:10.3390/biom2010023 Received: 25 November 2011; in revised form: 30 December 2011 / Accepted: 1 January 2012 / Published: 5 January 2012 Show/Hide Abstract | Download PDF Full-text (1011 KB) Abstract: Hydrodynamic tail vein (HTV) delivery is a simple and rapid tail vein injection method of a high volume of naked plasmid DNA resulting in high levels of foreign gene expression in organs, especially the liver. Compared to other organs, HTV delivery results in more than a 1000-fold higher transgene expression in liver. After being bitten by malaria-infected mosquitoes, malaria parasites transiently infect the host liver and form the liver stages. The liver stages are known to be the key target for CD8+ T cells that mediate protective anti-malaria immunity in an animal model. Therefore, in this study, we utilized the HTV delivery technique as a tool to determine the in vivo cytotoxic effect of malaria antigen-specific CD8+ T cells. Two weeks after mice were immunized with recombinant adenoviruses expressing malarial antigens, the immunized mice as well as naïve mice were challenged by HTV delivery of naked plasmid DNA co-encoding respective antigen together with luciferase using dual promoters. Three days after the HTV challenge, non-invasive whole-body bioluminescent imaging was performed. The images demonstrate in vivo activity of CD8+ T cells against malaria antigen-expressing cells in liver.

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Anne Kopp, Mario Hebecker, Eliška Svobodová and Mihály Józsi Review: Factor H: A Complement Regulator in Health and Disease, and a Mediator of Cellular Interactions Biomolecules 2012, 2(1), 46-75; doi:10.3390/biom2010046 Received: 21 December 2011; in revised form: 23 January 2012 / Accepted: 26 January 2012 / Published: 7 February 2012 Show/Hide Abstract | Download PDF Full-text (653 KB) Abstract: Complement is an essential part of innate immunity as it participates in host defense against infections, disposal of cellular debris and apoptotic cells, inflammatory processes and modulation of adaptive immune responses. Several soluble and membrane-bound regulators protect the host from the potentially deleterious effects of uncontrolled and misdirected complement activation. Factor H is a major soluble regulator of the alternative complement pathway, but it can also bind to host cells and tissues, protecting them from complement attack. Interactions of factor H with various endogenous ligands, such as pentraxins, extracellular matrix proteins and DNA are important in limiting local complement-mediated inflammation. Impaired regulatory as well as ligand and cell recognition functions of factor H, caused by mutations or autoantibodies, are associated with the kidney diseases: atypical hemolytic uremic syndrome and dense deposit disease and the eye disorder: age-related macular degeneration. In addition, factor H binds to receptors on host cells and is involved in adhesion, phagocytosis and modulation of cell activation. In this review we discuss current concepts on the physiological and pathophysiological roles of factor H in light of new data and recent developments in our understanding of the versatile roles of factor H as an inhibitor of complement activation and inflammation, as well as a mediator of cellular interactions. A detailed knowledge of the functions of factor H in health and disease is expected to unravel novel therapeutic intervention possibilities and to facilitate the development or improvement of therapies.

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Vesa M. Olkkonen, Olivier Béaslas and Eija Nissilä Review: Oxysterols and Their Cellular Effectors Biomolecules 2012, 2(1), 76-103; doi:10.3390/biom2010076 Received: 29 December 2011; in revised form: 3 February 2012 / Accepted: 7 February 2012 / Published: 15 February 2012 Show/Hide Abstract | Download PDF Full-text (591 KB) Abstract: Oxysterols are oxidized 27-carbon cholesterol derivatives or by-products of cholesterol biosynthesis, with a spectrum of biologic activities. Several oxysterols have cytotoxic and pro-apoptotic activities, the ability to interfere with the lateral domain organization, and packing of membrane lipids. These properties may account for their suggested roles in the pathology of diseases such as atherosclerosis, age-onset macular degeneration and Alzheimer’s disease. Oxysterols also have the capacity to induce inflammatory responses and play roles in cell differentiation processes. The functions of oxysterols as intermediates in the synthesis of bile acids and steroid hormones, and as readily transportable forms of sterol, are well established. Furthermore, their actions as endogenous regulators of gene expression in lipid metabolism via liver X receptors and the Insig (insulin-induced gene) proteins have been investigated in detail. The cytoplasmic oxysterol-binding protein (OSBP) homologues form a group of oxysterol/cholesterol sensors that has recently attracted a lot of attention. However, their mode of action is, as yet, poorly understood. Retinoic acid receptor-related orphan receptors (ROR) α and γ, and Epstein-Barr virus induced gene 2 (EBI2) have been identified as novel oxysterol receptors, revealing new physiologic oxysterol effector mechanisms in development, metabolism, and immunity, and evoking enhanced interest in these compounds in the field of biomedicine.

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Koji Umezawa, Jinzen Ikebe, Mitsunori Takano, Haruki Nakamura and Junichi Higo Article: Conformational Ensembles of an Intrinsically Disordered Protein pKID with and without a KIX Domain in Explicit Solvent Investigated by All-Atom Multicanonical Molecular Dynamics Biomolecules 2012, 2(1), 104-121; doi:10.3390/biom2010104 Received: 28 December 2011; in revised form: 11 February 2012 / Accepted: 12 February 2012 / Published: 22 February 2012 Show/Hide Abstract | Download PDF Full-text (2526 KB) Abstract: The phosphorylated kinase-inducible activation domain (pKID) adopts a helix–loop–helix structure upon binding to its partner KIX, although it is unstructured in the unbound state. The N-terminal and C-terminal regions of pKID, which adopt helices in the complex, are called, respectively, αA and αB. We performed all-atom multicanonical molecular dynamics simulations of pKID with and without KIX in explicit solvents to generate conformational ensembles. Although the unbound pKID was disordered overall, αA and αB exhibited a nascent helix propensity; the propensity of αA was stronger than that of αB, which agrees with experimental results. In the bound state, the free-energy landscape of αB involved two low free-energy fractions: native-like and non-native fractions. This result suggests that αB folds according to the induced-fit mechanism. The αB-helix direction was well aligned as in the NMR complex structure, although the αA helix exhibited high flexibility. These results also agree quantitatively with experimental observations. We have detected that the αB helix can bind to another site of KIX, to which another protein MLL also binds with the adopting helix. Consequently, MLL can facilitate pKID binding to the pKID-binding site by blocking the MLL-binding site. This also supports experimentally obtained results.

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Alexander Brobeil, Michaela Graf, Moritz Eiber and Monika Wimmer Article: Interaction of PTPIP51 with Tubulin, CGI-99 and Nuf2 During Cell Cycle Progression Biomolecules 2012, 2(1), 122-142; doi:10.3390/biom2010122 Received: 30 December 2011; in revised form: 4 February 2012 / Accepted: 14 February 2012 / Published: 23 February 2012 Show/Hide Abstract | Download PDF Full-text (3499 KB) Abstract: Protein tyrosine phosphatase interacting protein 51 (PTPIP51), also known as regulator of microtubule dynamics protein 3, was identified as an in vitro and in vivo interaction partner of CGI-99 and Nuf-2. PTPIP51 mRNA is expressed in all stages of the cell cycle; it is highly expressed six hours post-nocodazole treatment and minimally expressed one hour post-nocodazole treatment. Recent investigations located PTPIP51 protein at the equatorial plate. This study reports the localization of the PTPIP51/CGI-99 and the PTPIP51/Nuf-2 complex at the equatorial region during mitosis. Moreover, Duolink proximity ligation assays revealed an association of PTPIP51 with the microtubular cytoskeleton and the spindle apparatus. High amounts of phosphorylated PTPIP51 associated with the spindle poles was seen by confocal microscopy. In parallel a strong interaction of PTPIP51 with the epidermal growth factor receptor phosphorylating PTPIP51 at the tyrosine 176 residue was seen. In the M/G1 transition a high level of interaction between PTPIP51 and PTP1B was registered, thus restoring the interaction of PTPIP51 and Raf-1, depleted in mitotic cells. Summarizing these new facts, we conclude that PTPIP51 is necessary for normal mitotic processes, impacting on chromosomal division and control of the MAPK pathway activity.

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Carolina Londono, Cristina Osorio, Vivian Gama and Oscar Alzate Review: Mortalin, Apoptosis, and Neurodegeneration Biomolecules 2012, 2(1), 143-164; doi:10.3390/biom2010143 Received: 31 January 2012; in revised form: 22 February 2012 / Accepted: 23 February 2012 / Published: 1 March 2012 Show/Hide Abstract | Download PDF Full-text (5589 KB) Abstract: Mortalin is a highly conserved heat-shock chaperone usually found in multiple subcellular locations. It has several binding partners and has been implicated in various functions ranging from stress response, control of cell proliferation, and inhibition/prevention of apoptosis. The activity of this protein involves different structural and functional mechanisms, and minor alterations in its expression level may lead to serious biological consequences, including neurodegeneration. In this article we review the most current data associated with mortalin’s binding partners and how these protein-protein interactions may be implicated in apoptosis and neurodegeneration. A complete understanding of the molecular pathways in which mortalin is involved is important for the development of therapeutic strategies for cancer and neurodegenerative diseases.

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Béla Papp, Jean-Philippe Brouland, Atousa Arbabian, Pascal Gélébart, Tünde Kovács, Régis Bobe, Jocelyne Enouf, Nadine Varin-Blank and Ágota Apáti Review: Endoplasmic Reticulum Calcium Pumps and Cancer Cell Differentiation Biomolecules 2012, 2(1), 165-186; doi:10.3390/biom2010165 Received: 28 January 2012; in revised form: 14 February 2012 / Accepted: 17 February 2012 / Published: 5 March 2012 Show/Hide Abstract | Download PDF Full-text (363 KB) Abstract: The endoplasmic reticulum (ER) is a major intracellular calcium storage pool and a multifunctional organelle that accomplishes several calcium-dependent functions involved in many homeostatic and signaling mechanisms. Calcium is accumulated in the ER by Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA)-type calcium pumps. SERCA activity can determine ER calcium content available for intra-ER functions and for calcium release into the cytosol, and can shape the spatiotemporal characteristics of calcium signals. SERCA function therefore constitutes an important nodal point in the regulation of cellular calcium homeostasis and signaling, and can exert important effects on cell growth, differentiation and survival. In several cell types such as cells of hematopoietic origin, mammary, gastric and colonic epithelium, SERCA2 and SERCA3-type calcium pumps are simultaneously expressed, and SERCA3 expression levels undergo significant changes during cell differentiation, activation or immortalization. In addition, SERCA3 expression is decreased or lost in several tumor types when compared to the corresponding normal tissue. These observations indicate that ER calcium homeostasis is remodeled during cell differentiation, and may present defects due to decreased SERCA3 expression in tumors. Modulation of the state of differentiation of the ER reflected by SERCA3 expression constitutes an interesting new aspect of cell differentiation and tumor biology.

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Type of Paper: Article
Title: A New Protein Expression System Using Zebrafish
Authors: Hisayoshi Ishikawa ¹, Eriko Avşar-Ban ¹, Noriko Haiyama ¹, Hideo Miyake ¹, Shin-ichi Akiyama ² and Yutaka Tamaru ¹
Affiliations: ¹ Department of Life Science, Mie University Graduate School of Bioresources, 1577 Kurimamachiya, Tsu, Mie 514-8507, Japan;
E-Mail: ytamaru@bio.mie-u.ac.jp (Y.T.)
² Department of Nephrology, Graduate School of Medicine, Nagoya University, 65 Tsurumai, Syowa, Nagoya 466-8550, Japan
Abstract: In post-genome era, huge amounts of genomic sequencing data are accumulating year by year. Moreover, novel genes encoding predicted open reading frames in organism’s genome are also increasing. Although researchers want to try analyzing them, it is often too difficult to express them by Escherichia coli and so on. Thus, the existing systems for protein expression are not available and suitable especially for membrane-associated proteins. On the other hand, several embryonic lethal genes have so far been found in mammals by knockout technologies. However, even if conditional knockout mice are obtained to analyze such target genes, true function of the genes in body is still unclear. Furthermore, it costs much and takes time to construct them. In order to overcome such technical troubles and complains, we attempt to construct a new protein expression system using zebrafish embryos, especially for membrane-associated proteins. In case of considering protein expression, we first focused on not disturbing development of zebrafish and maintaining the strains. Therefore, since it does not spend too much time expressing target proteins relatively early during development and the expressing cells are easily observed, we chose a gene promoter of zebrafish hatching gland enzyme (zHE1). After screened and optimized the length of the zHE1 promoter, we finally constructed the protein expression vector by the zHE1 promoter (pZex). By using this vector, we succeeded several kinds of membrane- associated proteins such as a tetraspanin CD81 and leucine-rich-repeat-containing G-protein-coupled receptors (LGRs) within 48 hours post fertilization (hpf).

Type of Paper: Article
Title: Recognition of Histone Tail Modifications: Results from a Multi-Scale Study
Authors: R.P. Singh ^1,2, R. Zucca ^1,2,, G. Brysbaert ¹, J. Pelletier^1,2, M.F. Lensink¹, F. Cleri², R. Blossey^1,*
Affiliations: ¹ Interdisciplinary Resarch Institute CNRS USR 3078, USTL, 50 Avenue de Halley, Parc de la Haute Borne, 59658 Villeneuve d’Ascq, France
² IEMN CNRS 8520, USTL, Avenue Poincaré, Cité Scientifique, 59652 Villeneuve d’Ascq, France ; E-Mail: ralf.blossey@iri.univ-lille1.fr (R.B.)
Abstract: The N-terminal tails of histone proteins play a crucial role in transcriptional regulation in eukaryotes; the large variety of post-translational modifications that the different tail residues can undergo has even motivated the proposition of a “histone code”.  However, in most cases the patterns that link histone modifications to defined regulatory events have so far only been established through statistical correlation and a detailed mechanistic understanding of the specific role of these modifications is still lacking. Recently, studies on chromatin remodeling have shown the importance of histone tail states in the initiation and activation of the displacement of nucleosomes along DNA. Spurred by these advances, we present here results from a multi-scale study of histone tail recognition, undertaken with the goal to classify modification patterns by linking functional behavior to quantifiable and physical properties of the motifs. Our study combines i) a docking study of modified histone tails to their readers, ii) a statistical analysis of data from recognition assays and iii) a functional genomics approach to reconstruct the interaction networks of histone tails.

Type of Paper: Article
Title: Prediction of Cyclin Proteins using machine learning methods
Author: Krishna Kumar Kandaswamy
Affiliation: Institute for Neuro- and Bioinformatics, Building: 64, 2F, Room 27, Ratzeburger Allee 160, 23538 Lübeck, Germany;
E-Mail: Krishna@inb.uni-luebeck.de
Abstract: Cyclins are active during the cell cycle and facilitate cyclin-dependent kinases to phosphorylate different substrates. Identification of cyclins from protein sequence is more challenging due to the poor sequence identity which often falls below the twilight zone. We reported a Random Forest based approach, for the prediction of cyclin proteins from sequence using sequence derived properties. Our approach can be easily extended to recognizing other specific functional properties and should be a useful tool for the high-throughput and large-scale analysis of proteomic and genomic data.

Type of Paper: Review
Title: Plant Leucine-Rich Repeat (LRR)-Containing Proteins with Non-LRR Islands Interrupting LRRs
Authors: Norio Matsushima, Tomoko Mikami, Hiroki Miyashita and Keiko Yamada
Affiliation: Division of Biophysics, School of Health Sciences, Sapporo Medical University, Sapporo, Hokkaido 060-8556, Japan;
E-Mail: matusima@sapmed.ac.jp (N.M.)
Abstract: LRRs (leucine rich repeats) are present in over 50,000 proteins. Non-LRR, island regions (IRs) interrupting LRRs are widely distributed. We developed a method to identify IRs (LRR@IRpred). This review describes LRR@IR proteins from various plant species, identified by LRR@IRpred. A great number of LRR-RLKs and LRR-RLPs, that contain a single transmembrane-spanning region, have LRRs intersected by a single IR in which the number of repeat units in the preceding LRR block (N1) is greater than the number of the following block (N2); N1 " N2. Examples include; the A. thaliana TMK1 homologs have 13 LRRs with N1 = 10 and N2 = 3; and A. thaliana BRI1-related proteins have 22 LRRs with N1 = 17 and N2 = 5. The homologs of A. thaliana BRI1, A. thaliana RPP27, and tomato Cf have variable N1, while N2 is highly conservative; N1 = 14 - 33 and N2 = 4. The IRs in the homologs of TMK1, BRI1 and BRI1-related proteins contains cysteine clusters that likely form a cap structure. The rule of N1 " N2 would play a novel, significant role in ligand-interaction and/or dimerization of the LRR-RLKs and the LRR-RLPs. The structure and evolution of plant LRR@IR proteins also are discussed.

Type of Paper: Article
Title: Ursolic Acid, a Natural Pentacyclic Triterpenoid, Inhibits Na⁺/K⁺-ATPase Activity and Prevents TNF-a-induced Gene Expression by Blocking Amino Acid Transport and De Novo Protein Synthesis
Authors: Tomonobu Yokomichi¹, Kyoko Morimoto¹, Nana Oshima², Yuriko Yamada¹, Liwei Fu³, Shigeru Taketani¹, Masayoshi Ando⁴, and Takao Kataoka^1,2,*
Affiliations: ¹Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
²Center for Biological Resources and Informatics, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
³Graduate School of Science and Technology, Niigata University, Igarashi 2-8501, Nishi-ku, Niigata 950-2181, Japan
⁴Department of Chemistry and Chemical Engineering, Niigata University, 2-8050 Ikarashi, Nishi-ku, Niigata 950-2181, Japan;
E-Mail: takao.kataoka@kit.ac.jp (T.K.)
Abstract: Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-a, induce the expression of a wide variety of genes, including intercellular adhesion molecule-1 (ICAM-1). Ursolic acid (3b-hydroxy-urs-12-en-28-oic acid), a natural pentacyclic triterpenoid, was found to inhibit the cell-surface ICAM-1 expression induced by TNF-a in human lung carcinoma A549 cells. In response to TNF-a, ursolic acid diminished ICAM-1 protein expression almost completely and ICAM-1 mRNA expression partially. Ursolic acid prevented amino acid uptake as well as de novo protein synthesis, but did not obviously affect nucleoside uptake and the subsequent DNA/RNA syntheses. This inhibitory profile of ursolic acid was similar to that of the Na⁺/K⁺-ATPase inhibitor, ouabain. Consistent with this notion, ursolic acid was found to inhibit the activity of Na⁺/K⁺-ATPase. Thus, our present study reveals a novel molecular mechanism in which ursolic acid inhibits Na⁺/K⁺-ATPase activity and prevents the TNF-a-induced gene expression by blocking amino acid transport and de novo protein synthesis.
Keywords: ICAM-1; Na⁺/K⁺-ATPase; protein synthesis; ursolic acid; TNF-a

Type of Paper: Article
Title: Inositol Phosphates Induce the Membrane Docking of Lipid-Binding Domains
Authors: Robert V. Stahelin^1,2,*, Jordan L. Scott², Emmanuel Adu-Gyamfi², Katherine E. Ward², Diana Murray³ and Tatiana G. Kutateladze⁴
Affiliations: ¹ Department of Biochemistry & Molecular Biology, Indiana University School of Medicine-South Bend, South Bend, IN 46617, USA; E-Mail: rstaheli@iupui.edu
² Department of Chemistry & Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
³ Department of Pharmacology, Columbia University, New York, NY 11032, USA
⁴ Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, 80045, USA.
Abstract: At least 11 lipid-binding domains have been identified to date some of which bind phosphoinositides (PIs) with high affinity and specificity.  These include ENTH, FYVE, PX, and PH domains, which harbor a pocket that can coordinate different PIs.  PI binding to these lipid-binding domains has been shown to induce the membrane penetration as well as recruit parent effector proteins to cellular membranes.  Generally, affinity of soluble inositol phosphates for ENTH, FYVE, PX, and PH domains is lower than PI embedded membrane affinity due to lack of hydrophobic interactions between the domains and the membranes. However, the role of inositol phosphates in the membrane docking and cellular regulation has not been rigorously explored.  This study demonstrates that inositol phosphates induce the membrane docking of ENTH, FYVE, PX and PH domains. Additionally, inositol phosphate coordination by these domains can recapitulate the biological activity of these domains.  In closing, this study suggests the role of inositol phosphates be considered in the cellular regulation of ENTH, FYVE, PX, and PH domain containing proteins. (M_abstract).
Keywords: Lipid-binding; lipid-binding domains; inositol phosphates; membrane penetration; phosphoinositides

Type of Paper: Article
Title: Rational Design of Membrane Active Antimicrobials Guided by X-rays
Author: David Gidalevitz
Affiliation: Center for Molecular Study of Condensed Soft Matter (μCoSM), and Division of Physics, BCPS Department, Illinois Institute of Technology, Chicago, Illinois 60616, USA; E-Mail: gidalevitz@iit.edu (D.G.)
Abstract: Multi-drug resistant bacteria pose an increasing threat to public health. Antimicrobial peptides (AMPs) have emerged as a promising candidate in the fight against persistent bacteria, because AMPs kill pathogens via a mechanism remarkably different from that of conventional antibiotics. However, the therapeutic potential of the peptides is diminished by their susceptibility to proteolysis, high production costs, and a strong dependence of their activity on pH, salt concentration, and temperature. Biochemists face the paramount challenge to de novo design of small molecules, which would act via a similar mechanism as AMPs, yet would avert their disadvantages. The rational design of the synthetic antimicrobials requires the utmost understanding of structure-activity relationships (SAR) of AMPs. A crucial step in antimicrobial activity of the peptides is their interactions with lipids in bacterial membranes. AMPs bind to, disorder, and locally bend a bacterial lipid membrane to form pores and depolarize membrane potential that ultimately results in the pathogen’s death. Synchrotron X-ray scattering techniques offer insights into the aforementioned steps in the activity of AMPs with a sub-nm spatial and sub-minute time resolutions. Here we review how synchrotron X-ray scattering methods have contributed to the understanding of SARs of AMPs and aided to the rational design of their synthetic counterparts.