I01 Identification and evolutionary analysis of novel exons and alternative splicing events using cross-species EST-to-genome comparisons in human, mouse and rat

Feng Chi Chen

National Health Research Insititutes, Taiwan.

 

Alternative splicing (AS) is important for evolution and major biological functions in complex organisms. However, the extent of AS in mammals other than human and mouse is largely unknown, making it difficult to study AS evolution in mammals and its biomedical implications.

In this talk, I will describe a cross-species EST-to-genome comparison algorithm (ENACE) that can identify novel exons for EST-scanty species and distinguish conserved and lineage-specific exons. The identified exons represent not only novel exons but also evolutionarily meaningful AS events that are not previously annotated. A genome-wide AS analysis in human, mouse and rat using ENACE reveals a total of 758 novel cassette-on exons and 167 novel retained introns that have no EST evidence from the same species. RT-PCR-sequencing experiments validated ~50 ~80% of the tested exons, indicating high presence of exons predicted by ENACE. ENACE is particularly powerful when applied to closely related species. In addition, our analysis shows that the ENACE-identified AS exons tend not to pass the nonsynonymous-to-synonymous substitution ratio test and not to contain protein domain, implying that such exons may be under positive selection or relaxed negative selection. These AS exons may contribute to considerable inter-species functional divergence. Our analysis further indicates that a large number of exons may have been gained or lost during mammalian evolution. Moreover, a functional analysis shows that inter-species divergence of AS events may be substantial in protein carriers and receptor proteins in mammals. These exons may be of interest to studies of AS evolution.

 

 

I02

Factors differentiating cell fates in a proneural cluster

Chao-Ping Hsu

Institute of Chemistry, Academia Sinica, Taipei, Taiwan

 

The body of an adult fruit fly is covered with evenly spaced external sensory organs composed of four terminally differentiated cells derived from a single sensory organ precursor (SOP) cell. SOP formation involves expression of proneural proteins encoded by achaete (ac) and scute (sc) genes [1-4]. Initially, all cells in a proneural cluster are capable of forming a neural precursor, but only one cell actually forms an SOP. The Notch (N)-Delta (Dl) signaling pathway allows for competition among neighboring cells via lateral inhibition [5,6].

To study the dynamical consequence of such lateral inhibition pathways, we have established numerical models that are capable of reproducing 5 different mutant phenotypes [7]. Most of the models were capable of generating expression patterns for the gene Enhancer of split that were compatible with experimental observations for wild type and two Notch mutants. With these models, our analyses revealed two opposite driving forces that bias the choice between middle cells and peripheral cells in a proneural cluster. Lateral inhibition favors cells that have contact with fewer inhibitory signal-sending neighbors. On the other hand, we found an indirect mechanism that arises from the autonomous regulation of N and the irreversible N-Dl interaction, which decreases the amount of Dl in the peripheral cells and thus suppresses neural fates in these cells. These mechanisms may contribute to a preferential selection of SOPs.

 

 

I03 Investigation of Dimer Interface of Prolyl Dipeptidase DPP-IV

Jai-Hong Cheng1, Gu-Chuan Chen1, Ching-Shu Suen2, Chia-Hui Chien1, Li-Jen Hsu1, Chi-Yuan Chou3, Gu-Gang Chang3, Mingjing Hwang2 and Xin Chen1

1Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan 350, 2Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 115, 3National Yang-Ming UniVersity, Taipei, Taiwan 112, R.O.C.

 

The prolyl dipeptidase DPP-IV plays diverse and important roles in cellular functions. Its inhibitor has been proven effective in the treatment of human type II diabetes. DPP-IV is a type II membrane-bound prolyl-cleaving dipeptidase. It consists of a short cytoplasmic region of 6 amino acids, a transmembrane domain and an ectodomain. The crystal structure of the ectodomain shows that it is homodimeric with two dimer interfaces consisting of the C-terminal loop and the propeller loop of DPP-IV. Previously, we discovered that single site mutation at the C-terminal loop disrupts dimer formation of DPP-IV (Chien et al., JBC 2004 and Chien et al., Biochemistry, 2006). Monomeric forms of DPP-IV have only a fraction of the enzymatic activities left, while dimeric forms remains enzymatically active like the wild type enzyme. Unlike another monomeric prolyl-cleaving protease called POP, the propeller loop of DPP-IV is much longer and involved in dimerization. In this study, we identified the hydroxyl group of Y248 at the propeller loop is important not only for inter-molecular interaction in dimers, but also for intra-molecular interaction upon formation of monomers. Single site mutation at Y248 (Y248A, Y248T and Y248F) has generated proteins with strikingly different biochemical properties. Quite unexpectedly, we also discovered that the transmembrane domain of DPP-IV is the third dimer interface of DPP-IV. Computer modeling and sequence analysis of the transmembrane domain (TM) has revealed it likely forms alpha helix with the heptad repeats, which is confirmed by the circular dichroism spectrometry. Using bioluminescence resonance energy transfer (BRET) assay, cell transfection and analytical ultracentrifugation, we demonstrated that TM domain indeed forms homodimer in lipid. Site-directed mutagenesis on the TM domain has been carried out to identify key residues involved in dimerization. The series of the study on DPP-IV dimer interface points out that the dimerization might be a folding event of DPP-IV, which has important implication for the biogenesis of DPP-IV and its drug discovery.

 

 

I04 Development of New Methodologies for Carbohydrate Synthesis

Shang-Cheng Hung

Department of Chemistry, National Tsing Hua University, Hsinchu 300, Taiwan

 

Carbohydrates are involved in numerous vital life processes. They are structurally diverse and complex as compared to other biopolymers (proteins and nucleic acids) and are present in micro-heterogeneous forms in nature. Chemical synthesis of carbohydrates, the practical route to procure pure oligosaccharides, is however hampered by two major hurdles, regioselective protection of polyhydroxyls and rapid assembly of glycosidic linkages involving the stereoselective control of £\- or £]-glycosidic bonds. Here, a novel, combinatorial, and highly regioselective method to protect individual hydroxyls of monosaccharide units and install an orthogonal protecting group pattern in a one-pot manner is presented, obviating the necessity to carry out intermittent tedious workups and time-consuming purifications. Hundreds of monosaccharide building blocks have been efficiently prepared. Iterative coupling of these synthons to the assembly of £]-1,6-glucans is demonstrated. Thereby, the combination of one-pot protection method and one-pot glycosylation may offer an efficient protocol to solve the long-standing problem in oligosaccharide synthesis.

References
1. Shie, C.-R.; Tzeng, Z.-H.; Kulkarni, S. S.; Uang, B.-J.; Hsu, C.-Y.; Hung, S.-C.
    Angew. Chem. Int. Ed. 2005, 44, 1665.
2. Wang, C.-C.; Lee, J.-C.; Luo, S.-Y.; Kulkarni, S. S.; Huang, Y.-W.; Lee, C.-C.;
    Chang, K.-L.; Hung, S.-C. Nature 2007, 446, 896.

 

 

I05 Structure-Based Drug Design of a Novel Family of PPARƒ× Partial Agonists

Su-Ying Wu

INational Health Research Institutes, Division of Biotechnology and Pharmaceutical Research
R2-8023, No.35, Keyan Road, Zhunan Town, Miaoli County 350, Taiwan, R.O.C. 886-37-246166 ext 35713, E-mail: suying@nhri.org.tw.

 

Peroxisome proliferator-activated receptor £^(PPAR£^) is well known as the receptor of thiazolidinedione anti-diabetic drugs. Structure-based virtual screening, a method that combines shape-based database search with a docking study and analogue search, has been employed to discover a novel family of PPAR£^ agonists based upon pyrazol-5-ylbenzenesulfonamide. Two analogues in the family show high affinity for, and specificity to, PPAR£^ and act as partial agonists. They also demonstrate glucose-lowering efficacy in vivo. A structural biology study reveals that they both adopt a distinct binding mode and have no H-bonding interactions with PPAR£^. The absence of H-bonding interaction with the protein provides an explanation why both function as partial agonists since most full agonists form conserved H-bonds with the activation function helix (AF-2 helix) which, in turn, enhances the recruitment of co-activators. Moreover, the structural biology and computer docking studies reveal the specificity of the compounds for PPAR£^ could be due to the restricted access to the binding pocket of other PPAR subtypes, i.e. PPAR£\ and PPAR£m, and steric hindrance upon the ligand binding.

 

 

I06 Lessons from snake venom structure biology research:
Carbohydrates, lipids and proteins as multiple targets for cobra venom actions

Wen-guey Wu

National Synchrotron Radiation Research Center and Institute of Bioinformatics and Structural biology, National Tsing Hua University, Hsinchu, Taiwan

 

Severe tissue necrosis with retarded wound healing is a major symptom of the victims survived from cobra snakebite, but its molecular mechanism remains poorly understood. Cardiotoxins, major components of cobra venoms that belong to the Ly-6 protein family and implicated in tissue damage, are basic polypeptides having lipid- and heparin binding capabilities similar to the cell penetrating peptides. Whereas cardiotoxins are specifically retained on the cell surface via heparan sulfate mediated processes (1), their lipid binding ability appears to be responsible for the membrane insertion, pore formation and cell internalization to further target other intracellular organelles (2). Recent identification of cardiotoxin as non-RGD integrin binding protein (3) also open a new avenue to understand a role of integrin binding in affecting cardiotoxin-induced cytotoxicity with therapeutic potentials as integrin antagonist. Specifically, non-cytotoxic cardiotoxin A5 has been shown to bind to integrin £\v£]3 and inhibit bone resorption. In this talk, we shall present structural biology data to show how cardiotoxins might act toward multiple targets, i.e., glycosaminoglycans in extracellular matrix, glycosphingolipids and integrin receptors in cell membranes, to exhibit its toxicity. Since cobra venoms also consist of other multi-domain proteins such as matrix metalloprotease (MMP) to target multiple molecules on membrane surface, it is suggested that animal toxins may use a complex strategy to explore diverse targets on cell membranes. The significance of multiple targeting by involving multi-domains toxins and/or toxin homologues is finally discussed in terms of snake venom evolution and its potential medical applications.
1. Lee et al., Structural basis of citrate-dependent and heparan sulfate mediated cell
    surface retention of cobra cardiotoxin A3 J. Biol. Chem. 280, 9567-9577 (2005)
2. Wang et al., Glycosphingolipid-facilitated membrane insertion and internalization of
    cobra cardiotoxin. J. Biol. Chem. 281, 656-667 (2006)
3. Wu et al., Non-cytotoxic cobra cardiotoxin A5 binding to integrin and inhibits bone
    resorption J. Biol. Chem.. 281, 7937 (2006)

 

 

I07 Structural study of prostacyclin synthase

Yi-Ching Li1 (§õ©yÀR), Chia-Wang Chiang1 (¦¿¨Î©ô), Hui-Chun Yeh2, Lee-Ho Wang2*,
and Nei-Li Chan1* (¸â°i¥ß)

1Institute of Biochemistry, College of Life Sciences, National Chung Hsing University, Taichung City 402, Taiwan.
2Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center at Houston, 6431 Fannin, Houston, TX 77030, USA.

 

Prostacyclin synthase (PGIS) catalyzes an isomerization of prostaglandin H2 to prostacyclin, a potent mediator of vasodilation and anti-platelet aggregation. Being a member of the cytochrome P450 enzyme superfamily, PGIS shares many spectral features with classical P450 mono-oxygenases, which accept electrons from P450 reductase to activate an oxygen molecue for subsequent substrate hydroxylation. Since the electron-donating P450 reductases play an essential role during catalytic cycle, P450 enzymes are classified into 4 classes (Classes I ¡V IV) according to their redox partners. Among them, Class III P450s, such as PGIS, are unique because they do not require any electron-transfer partner or oxygen as endoperoxides or hydroperoxides are their sole substrates. In addition, the signature mono-oxygenase activity of P450 has not been detected for any Class III enzymes. To provide a better understanding on the structure/function relationship of this unique class of P450 enzymes, we have successfully determined a high resolution crystal structure of substrate-free form of human PGIS recently, representing the first three-dimensional structure of a Class III cytochorme P450. While notable sequence divergence has been recognized between PGIS and other P450s, PGIS exhibits the typical triangular prism-shaped P450 fold with only moderate structural differences. Interestingly, in contrast to other P450 enzymes in which the heme propionates are tightly anchored to the protein matrix, we noticed that the propionate of PGIS heme ring-A forms a water-mediated hydrogen bond to the protein matrix and that of ring-D lacks a defined structure, suggesting a somewhat plasticity of the heme lodging in PGIS. The functional significance of this observation and detailed structural analysis will be presented during the meeting.

 

 

I08 Photoacoustic functional and molecular imaging: applications and challenges

Pai-Chi Li

Graduate Institute of Biomedical Electronics and Bioinformatics
Department of Electrical Engineering
National Taiwan University

 

Photoacoustic imaging combines advantages of high optical contrast and low acoustic scattering in biological tissues. Unlike conventional ultrasound that uses an external source to send out acoustic waves, photoacoustic imaging uses transient acoustic waves generated as a result of thermal expansion of tissue heated with short laser pulses. The ultrasonic waves are then detected by an ultrasonic transducer and such signals can be used to reconstruct optical absorption distribution inside the image object. Tissues with different optical properties have different photoacoustic profiles and this enables reconstruction of an acoustic image based on distribution of optical absorption. Currently, this new imaging technology has been explored for breast cancer and prostate cancer. It has also become a promising tool for microcirculation detection, epidermal melanin measurements, and oxygenation monitoring of blood vessel. Despite the widespread interest, photoacoustic imaging has been limited mostly to morphological imaging at the tissue level. In this talk, we will describe the use of gold nanoparticles in developing photoacousitc functional and molecular imaging methods. Specifically, the efficient light absorption of gold nanoparticles and abilities to tune their optical properties will be introduced. Development of advanced photoacoustic imaging technologies for quantitative analysis, measurements of hemodynamic functions, and simultaneous detection of multiple selective targeting will also be presented. New development in combined diagnosis with therapy will also be discussed.

 

 

I09 Orchestrate the multifaceted biophotonics sensing and imaging in vivo: from microscopic, macroscopic to functional

Leu-Wei Lo

Associate Investigator
Division of Medical Engineering Research, National Health Research Institutes
35 Keyan Road, Zhunan Town, Miaoli County 350, Taiwan

 

The panorama of in vivo biophotonics sensing and imaging could be generally divided into three facets: microscopic, macroscopic, and functional; however which should, as a whole, provide integrated information and picture of the target addressed.

With regard to the microscopic biophotonics sensing, the fluorescence nanoparticle-based investigation on blood-brain-barrier (BBB) and hepatic vasculature permeability have been achieved using microdialysis/microlight guide system. Design of a FRET-based microdialysis/microlight guide System for dynamic monitoring of cytokine concentrations in situ is currently undergoing.

With regard to the macroscopic biophotonics imaging, a prototype of small animal optical imaging system has been implemented for dynamic tracking of in vivo biodistributions registered by designed nanocarriers of drugs for efficient theraprutics and/or optical beacons for molecular imaging.

With regard to the functional biophotonics sensing and imaging, the aim is to establish a platform technology for real-time monitoring of clinical important metabolites. Currently, the oxygen-dependent quenching of phosphorescence has been applied to measurements of tissue oxygenation in vivo from minimally-invasive to non-invasive.

 

 

I10 Protein based room temperature molecular magnet synthesis and its application on silicon substrates

Chia-Ching Chang1,2,3, Pei-Hsin Chen1, Kien Wen Sun4, Lou-Sing Kan5

1: Department of Biological Science and Technology, National Chiao-Tung University.
2: Institute of Physics, Academia Sinica.
3: National Nano Device laboratories
4: Institute of Molecular Science and Department of Applied Chemistry, National Chiao-Tung University.
5: Institute of Chemistry, Academia Sinica

 

Metallothionein-2 (MT-2) is a cysteine-rich protein that binds seven divalent transition metal ions avidly via its metal-thiol linkages. A magnetic metallothionein-2 containing Mn and Cd (Mn,Cd-MT-2) has been synthesized by protein refolding process. There are two Mn and five Cd atoms per molecule. No trace of Fe was detected by atomic absorption spectroscopy. The Curie temperature Tc is around 430K. The uniform size distribution, tested by dynamic light scattering, indicated that each Mn,Cd-MT-2 molecule is a single molecular magnet. The molecules are then placed into nanopores prepared on silicon (0 0 1) surfaces using electron beam lithography and reactive ion-etching techniques. We have observed the self-assemble growth of the MT molecules on the patterned Si surface such that the MT molecules have grown into rod or ring type three dimensional nanostructures, depending on the patterned nanostructures on the surface. This engineered molecule shows molecular magnetization and is biocompatible with conventional semiconductors. These features make Mn,Cd-MT-2 a good candidate for biological applications and sensing sources of new nanodevices. Using molecular self-assembly and topographical patterning of the semiconductor substrate, we can close the gap between bio-molecules and nanoelectronics built into the semiconductor chip.

 

 

I11 Crystal structure of Helicobacter pylori an aliphatic amidase AmiE and formamidase AmiF reveals a conserved catalytic triad in the nitrilase superfamily

Wen-Ching Wang, and Chiu-Lien Hung, Cheng-Yu Chen, and Yu-Wen Hua

Institute of Molecular and Cellular Biology and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan

 

Helicobacter pylori AmiE and AmiF are two aliphatic amidases and belong to members of the nitrilase superfamily. The crystal structure of AmiF was solved to 1.75 Å resolution using single-wavelength anomalous dispersion methods. The structure consists of a homohexamer related by 3-fold symmetry in which each subunit has an £\-£]-£]-£\ four-layer architecture characteristic of the nitrilase superfamily. One exterior a layer faces the solvent, whereas the other one associates with that of the neighbor subunit, forming a tight £\-£]-£]-£\-£\-£]-£]-£\ dimer. The apo and liganded crystal structures of an inactive mutant C166S were also determined to 2.50 and 2.30 Å, respectively. These structures reveal a small formamide-binding pocket that includes C166, E60, and K133 catalytic residues, in which C166 acts as a nucleophile. Analysis of the liganded AmiF and N-carbamoyl D-amino acid amidohydrolase binding pockets reveals a common Cys-Glu-Lys triad, another conserved glutamate, and different subsets of ligand-binding residues. The crystal structure of AmiE was also solved to 2.38-Å resolution with molecular replacement methods. The AmiE structure also reveals the homologous architecture and conserved catalytic residues (C165, E59, and K133). Molecular dynamic simulations show that the conserved triad has minimal fluctuations, catalyzing the hydrolysis of a specific nitrile or amide in the nitrilase superfamily efficiently.

 

 

I12 A Machine at Work: Structures of the Pre-fusion and Post-fusion Forms

Hsien-Sheng Yin

The former research associate in Howard Hughes Medical Institute; Assistant Professor, Institute of Bioinformatics and Structural Biology & Department of Life Science, National Tsing Hua University, Taiwan.

 

The paramyxoviridae are enveloped viruses that include mumps virus, parainfluenza virus 5 and human parainfluenza viruses 1-4 (hPIV).Many members of this viral family are significant human and animal pathogens, and newly emergent deadly paramyxoviruses (Nipah and Hendra viruses) have been identified. Like other enveloped viruses such as influenza virus and HIV, require fusion of the viral and cellular membranes to enter the cell. Two viral glycoproteins are key to this process: a variable attachment protein (HN, H or G) and a more conserved fusion (F) protein. F is though to drive membrane fusion by coupling irreversible protein refolding to membrane juxtaposition, by initially folding into a metastable form that subsequently undergoes discrete conformational changes to a low energy state. We previously reported the structure of the uncleaved, secreted hPIV3 F0 ectodomain (solF), truncated before the transmembrane domain. Unexpectedly, this structure contains the postfusion 6-helix bundle (HB), indicating that F protein cleavage is not required to attain the postfusion and that the F transmembrane domain and/or the cytoplasmic tail are important for the folding to, or stability of, the prefusion metastable.

 

 

I13 A systems biology approach to understanding endoderm specification in zebrafish

Tzu-Min Chan, Hua-Ling Chen, Wen-Fang Tseng, Chung-Hao Chao, Te-Hsuan Jang, Yu-I Lin, Hong-I Hsu, Chang-Ben Huang, Chiou-Hwa Yuh

Associate Investigator
Division of Molecular and Genomic Medicine, National Health Research Institute, 35, Keyan Road, Zhunan Town, Miaoli County 350, Taiwan, ROC.

 

The genomic program for development operates mainly by the regulated expression of genes encoding transcription factors and the signaling pathways. Complex genetic regulatory networks that contain all the interactions between those genes control developmental fate. In the post-genomic era, the most urgent topic in studying complex biological systems is functional genomics.
In the vertebrate development, the process of gastrulation leads to three germ layers: ectoderm, endoderm and mesoderm. The endoderm originates from the most marginal blastomeres of blastula stage embryos in zebrafish. Previous evidences demonstrated seven signaling pathways antagonize each other specify the cell lineage. How those signals regulate the transcription factors in turns to specify the cell fate is not well understood. To decipher the underlying mechanism of embryo specification and differentiation, we start to analyze the most important transcription factors in early development by Morpholino antisense oligonucleotide to knockdown the specific genes¡¦ expression. We had found Sox32, Foxa2, Gata5, Gata6, Sox17 and Otx2 regulated many transcription factors from Q-PCR analysis and microarray data. The interrelationship between those transcription factors established subcircuits that ensure the stable expression domain by ¡§AND¡¨ logic between two or more transcription factors, by positive feedback loop to lockdown the territory, and to turn off the genes¡¦ expression by repression system at later stage. This architecture of endoderm networks is common in the entire vertebrate, and the genes involved in the endoderm formation are also evolutionary conserved. Further analysis on the regulation of those transcription factors, we used cross species comparison on the genome sequence of those genes revealed three evolutional-conserved regions. We also searched for the transcription factor binding sites on those conserved non-coding region and identified many important node for the networks. We have generated several GFP reporter constructs and found those three DNA fragments have the function as enhancer. Surprisingly, we found one repressor that is not evolutionary conserved. The individual regulatory modules from those transcription factors have been studied in details by microinjection, co-inject with Morpholino antisense oligonucleotide and mutational analysis. We will also examine the transcription factors¡¦ binding site genome-wide by Chromatin-Immunoprecipitation coupled with Microarray analysis (ChIP-chip).
This is the first systematic approach toward transcriptional regulation in zebrafish development. Study the networks of relationship and interaction between those evolutionary conserved transcription factors can help us identify the kernel of the GRNs. By comparison of different species GRNs, we hope to solve the mystery of developmental specification and their relationship to evolutional changes.

 

 

I14 Construction of Whole Genomic and Proteomic Tree Based on
DNA and Protein Probes

Ping-Chiang Lyu1, 2, Chi-Ching Li2, Wei-Cheng Lo2 and Szu-Ming Lai1, 2

1Department of Life Sciences; 2Institute of Bioinformatics and Structural Biology,
National Tsing Hua University, Hsinchu 30013, Taiwan

 

The classification of microorganisms is difficult because they have various morphological and environmental distributing properties. Since 1970, taxonomy systems have been developed based on some stable and standard molecular biomarkers; for instance, sequence similarity of SSU RNA (small subunit ribosomal RNA) is the first and still wildly used biomarker nowadays for prokaryotes. However, it has been supposed insufficient to classify all kinds of organisms by using one or only a few biomarkers. After the year 2000, the development of genome sequencing techniques has been so rapid that it is now possible to analyze the evolutionary relationships of organisms on the scale of whole genomes.

We have developed a whole genome clustering approach based on the frequency of biologically meaningful probes. We compared bacteria, archaea and fungi to build the genomic and proteomic based tree by an unsupervised clustering method. Our results showed that, the genomic tree grouped together microorganisms with similar GC contents, and the proteomic tree clustered bacteria, archaea and fungi into two branches, where the latter two share the same node. Furthermore, the proteomic tree agreed well with the traditional phylogeny at the basal branches while the distal classifications seemed to reflect phenotypic features, such as the parasitism, thermophilicity, methanogenesis and photosynthesis, better than traditional SSU RNA-based classifications.

 

 

I15 The First Phase Report of a Structural Genomics Study of the Plant Pathogen Xanthomonas campestris

Shan-Ho Chou1,5,* Ko-Hsin Chin5, Zhi-Le Tu1, Chao-Yu Yang1, Tso-Ning Li1, Shu-Ju Liao1, Yi-Che Su1, Wei-Ting Kuo1, Cheng-Jen Liao1, Yi-Chun Teng1, Jhong-Nan Chen1, Wen-Ting Lo1, Tzu-Huei Kao1, Yu-Sheng Fang1, Ping-Jiang Lyu2, Peter Tsai3, Andrew H.J. Wang4

1Institute of Biochemistry, National Chung-Hsing University, Taichung, 40227, Taiwan
2Dept. of Life Science, National Tsing-Hwa University, Shin-Chu, Taiwan
3Dept. of Life Science, National Yang-Ming University, Pei-Tou, Taiwan
4Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei, Taiwan
5National Chung Hsing University Biotechnology Center, National Chung-Hsing University, Taichung, 40227, Taiwan

 

Structural genomics is paving a way to understand the intricate interaction pathways among proteins in a whole organism, and is emerging as a powerful approach for functional annotation of genes it encodes. We have been studying the structural genomics of Xanthomonas campestris pv. campestris, a gram-negative bacterium that is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. Due to its academic and industrial values, we have endeavored to identify and characterize the structures and functions of proteins encoded in Xcc by using X-ray crystallography or high resolution NMR techniques.
Because each protein has its own characteristics in terms of protein stability or crystallization capability, a high throughput approach for determining each protein structure in any organism turns out to be impractical. It is necessary to fine tune the conditions for expression level, protein solubility, and tendency to crystallization etc. So far, we have successfully applied the LIC (ligation-independent cloning) strategy to facilitate the gene cloning procedure, and used a high magnetic field to improve protein crystallization.
Until now, approximately 500 target genes of this bacterium have been successfully constructed in expression vectors. Among them, about 50% gene products are soluble, and 200 proteins have been purified. By screening crystallization condition using a robot system, we obtained about 100 crystals, of which ~30% were found to be suitable for structure determination. So far, fifteen structures have been solved using this structural genomics approach, many of which provide clues to biochemical functions that can be experimentally confirmed. Several novel fold or subfamilies of well-known folds have been identified.

 

 

I16 Morphology of £]-amyloid Peptide 1-42 Fibril

Yi-Cheng Chen

Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan

 

Alzheimer¡¦s disease (AD) is a member of amyloid disease. AD patients show two major pathological hallmarks in their brains, amyloid senile plaques (SPs) and neurofibrillary tangles (NFTs). The major components found in SPs are two small, hydrophobic peptide-amyloid £]ƒnspinning residues 1-40 (A£]1-40) and 1-42 (A£]1-42). The formation of A£] aggregation is the main cause for the neuron toxicity and therefore Alzheimer¡¦s disease. A£] Fibrillogensis has been proposed to be a two-step mechanism involving an initial slow, lag and nucleated period and followed by a rapid fibril propagation and aggregation stage. Investigation of fibrillary morphology of A£] peptide has been extensively studied. In a recent study in our laboratory, we discovered an unusual form of aggregated A£]1-42 with a biscuit-like morphology in acidic solution by atomic force microscopy. This fibrillary aggregate has a diameter of 2£gm and a thickness of 20nm. A comparison with usual rod-like A£] fibril, further analyses of aggregation mechanism indicates that the morphology of A£] in nucleation state might play a determined role on the final morphology of A£] fibril.