|I01||The Perspective of Mass Spectrometry in Proteomics and protein's post-translational modifications|
Genomics Research Center, Academia Sinica
Recent developments in technology and instrumentation have made mass spectrometry the method of choice for identification of proteins. Current mass spectrometry platform adopting shotgun proteomics approach with collisional activation dissociation (CAD) to facilitate sequence information becomes the major tool for analyzing gel-based spotted samples and total cell lysate. The CAD process severely restricts the detection and location of protein¡¦s post-translational modifications (PTM), such as phosphorylation and glycosylation. New mass spectrometry-based methods involving electron capture dissociation (ECD), electron transfer dissociation (ETD) mechanism result in extensive peptide fragmentation that is indifferent to either peptide sequence or the presence of labile PTMs. A combination of bottom up approach (peptide level) with top down approach (intact protein) to elucidate protein¡¦s PTM will be demonstrated.
Functional proteomic studies of shrimp white spot syndrome virus structural
Institute of Zoology, National Taiwan University
White spot syndrome virus (WSSV), is a large enveloped, double-stranded DNA virus that attacks cultured shrimp and many other crustaceans. Although many WSSV ORFs do not resemble
any known proteins, the entire WSSV genome has been sequenced and shrimp EST database has been established, so a proteomic approach can now be used.
|I03||Mechanism of fibril formation by the gastric cancer-related GISP-like proteins|
Yuan-Chao Loua, Meng-Ru Hoa,b, Wen-Chang Lina, Chinpan Chena
aInstitute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC; bInstitute of Bioinformatic and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan, ROC.
Gastric cancer (GC) is the most prevalent malignant neoplasm and the leading cause of cancer death in many countries. However, the prognosis of GC patients is dismal, emphasizing the necessity for prevention, early detection, and better treatment for GC patients. A novel gene of GISP (GastroIntertinal Secretory Protein) was identified and suggested to have important clinical significance and applications for gastric cancer by our collaborator. Three genes related to GISP that include LITs (lithostathines), PAPs (pancreatitis-associated proteins), and PBCGF (pancreatic beta cell growth factor) were further found based on sequence comparison. These GISP-like proteins all share a common C-type animal lectin motif. Both human LIT and PAP proteins were identified in pathognomonic lesions of Alzheimer¡¦s disease (AD), indicating that they may be involved in AD. Furthermore, both human LIT and PAP were found to form fibrillar aggregates at neutral pH. By contrast, fibrils could not be seen on GISP. To gain insight into the mechanism of fibril formation by GISP-like proteins, we have applied a variety of biophysical experiments for structural studies. In this presentation, NMR solution structure of human PAP and its conformational changes associated with globular to amyloid fibril transformation will be discussed and so do the preliminary studies of other GISP-like proteins. These biophysical studies on GISP-like proteins may provide valuable information for designing and searching the lead compounds for gastric cancer therapeutics as well as for the diseases related to the amyloid fibrils.
|I04||The studies of the low-resolution structural and functional studies for the fidelity of DNA polymerases in solution|
The Genomic Research Center, Academia Sinica, Taiwan, and Department of Chemistry, the Ohio State University, U.S.A
DNA gets damaged by various environmental influences (heat, radiation, oxidative stresses and others). Fortunately, there are repair mechanisms that can correct these defects.
The accuracy (fidelity) of inserting a correct nucleotide (dNTP) into the gap on the damaged site by DNA polymerases is essential for maintaining the genomic stability and preventing cancer-related mutagenesis.
To investigate the molecular mechanism that leads to the nucleotide selection (substrate specificity) of DNA polymerases, we have combined the small-angle scattering (SAS) and stopped-flow fluorescence analyses
for the structure-function studies of the mammalian DNA polymerase £] (Pol £]). Pol £] is a multi-subdomain protein whose subdomains contributed to different roles in the base excision repair (BER) pathway in
the event of DNA damages, and recent medical studies have shown that more than 30% of tumors express variants of Pol £]. Among these mutants, I260Q has tighter DNA binding affinity than the wild type and shown
very poor selection on incorporation of nucleotide (behaves as a mutator).
|I05||FLIM/FRET__ a novel tool for molecular dynamics imaging|
Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan, ROC.
In this post-genome era, visualizing and quantifying protein-protein interactions is a recent trend in biomedical imaging. The ability to see the dynamics behavior of a specific protein
inside the living cells and tissues became possible through the application of fluorescence (Föster) resonant energy transfer (FRET) technique. Applications of the modern photonics technology allow biomedical
analysis at single molecule and nanometer scale with sub-nanosecond time resolution. The integration of fluorescence spectroscopy added a new dimension to light microscopy that enables the investigation of
molecular behavior. Such need, in particular, has been successfully met by the fluorescence resonance energy transfer (FRET), formulated by Förster back in 1948 and reborn again recently due to the advances of
newly developed genetically encoded fluorescent labels and sensors. FRET is a non-radiative quantum-mechanical dipole-dipole interaction process whereby energy from an excited donor/fluorophore is transferred to
an acceptor. FRET occurs when both donor and acceptor have sufficiently large spectral overlap, a favorable dipole-dipole orientation, proximity of 1-10nm and a large enough quantum yield. The dependence of the
coupling efficiency varies with the inverse sixth power of the distance between the acceptor and donor and is designated as the Föster radius, R0 (distance at which the efficiency of energy transfer is 50%).
Upon energy transfer, the fluorescence intensity and the lifetime of the donor reduce as its excited state population is being depleted. FRET measurements in a microscopic object can be conveniently carried out
with fluorescence lifetime imaging microscopy (FLIM).
|I06||Micro- and Nano-Radiology for Life Sciences|
Institute of Physics, Academia Sinica, Taipei 115, Taiwan, ROC
With the rapid development of many biomedical imaging modalities, radiography remains one of the most used imaging methods. On top of its popularity, radiology has also benefited from the new technology capability and recently reached a new level of sophistication and power. With higher coherence of the X-ray source produced by synchrotron, phase difference is added to the absorption as anther, yet much improved, contrast mechanism. Together with the improvement in the detecting system and the capability of fabricate x-ray optic elements with nanometer precision, we can now looking deep inside into matters with unprecedented precision and speed. Sub-£gm resolution radiograph can now be obtained with ease on animals in vivo and fast phenomenon can be observed in a real time fashion. A lateral resolution of 30nm has been demonstrated using nanofabricated x-ray phase zone plate. Phase contrast is implemented in all aspect of the x-ray imaging with great success in revealing sub-cellular structures. The application of this new tool has already helped researches in materials science, biology and medicine. Ongoing developments in the instrumentation and reconstruction algorithms have generated even higher excitement with the recent demonstration of nanometer scale resolution in 3D. It is anticipated that with the new installation of X-ray free electron laser, X-rays can be used to ¡§image¡¨ a single protein molecule with atomic resolution in the near future and to continue the legacy started by Rontgen. Review of this advent of this technology, its application to various domains in science, particularly the relevance to the medicine, nano-science and -technology, and the future potential will be emphasized.
|I07||Medical Imaging Experiments at SPring-8|
SPring-8/JASRI, Hyogo, Japan
SPring-8 is the largest synchrotron radiation facility in the world, with a circumference of 1.4 km.
It is also the one with the highest energy (8 GeV). Thus, it is suitable for high resolution x-ray imaging experiments on biological samples.
SPring-8 has an excellent facility for animal experiments, which makes it possible to work on live animals.
Systems analysis of the extremely halophilic archaeon Halobacterium sp. NRC-1:
Getting biological stories from proteomics data
Rueichi R. Gana, Yu-chieh P. Kaob, Eugene C. Yic,d, Yulun Chiua, Hookeun Leee, Timothy H.Wub, Ruedi Aebersoldc,e, David R. Goodlettf, and Wailap Victor Ngb,g,h
a Institute of Biochemistry, b Institute of Bioinformatics, g Institute of Biotechnology in Medicine,
Mass spectrometry is a valuable technology for revealing the macromolecules at the terminus of the bioinformation. After the completion of the Halobacterium sp. NRC-1 genome sequence, we have initiated several proteome analyses using high-throughput liquid chromatography coupled to electrospray ionization tandem mass spectrometry so as to obtain the essential expression information for making approximate predictions of the biochemical reactions occurred in standard laboratory growth conditions. With the increased number of protein identifications in our recent studies, it becomes necessary to analysis the complex data with more efficient novel techniques. Thus we developed the BMSorter and MSNetwork tools to carry out biomodular analysis which use network information to analyze proteomics data and predict interactions between systems. As an example, an integrated analysis of the enzymes in the amino acid metabolism and citrate cycle systems found up to eight amino acids may be converted to oxaloacetate, fumarate, or oxoglutarate in the citrate cycle for energy production. The analysis also suggested the interconversions of acidic amino acids to meet the high demand of the building blocks for the acidic proteome. The details of such analysis will be discussed in details in the presentation.
|I09||How winged helix/forkhead proteins using conserved residues to recognize diverse DNA sequence|
Chwan-Deng Hsiao1, Kuang-Lei Tsai1,2, Cheng-Yang Huang1, Chia-Hao Chang3, Yuh-Ju Sun2, and Woei-Jer Chuang3
1Institute of Molecular Biology, Academia Sinica, Taipei, 115, Taiwan, 2Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, 300, Taiwan, and 3Department of Biochemistry, National Cheng Kung University, College of Medicine, Tainan 701, Taiwan
Winged helix/forkhead proteins have shown to have similar binding specificity to the core sequence. In addition, these proteins have conserved amino acid sequences in the putative recognition helix. This raises an intriguing question as how proteins use conserved residues to recognize distinct core sequences. To date, more than 200 winged helix/forkhead proteins have been identified but only two forkhead protein/DNA complexes have been reported. Prior to this study, little is known about how winged helix/forkhead proteins recognize diverse DNA sequence. We report here a 3-D crystal structure of human FOXK1a bound to a 16-base pair DNA duplex containing promoter sequence. This complex structure provides a new insight into that how the DNA-binding specificities of winged helix/forkhead proteins may be regulated by their less conserved regions. The present structural study also offers the first view of a cooperative binding of ILF to DNA that contains the core sequence. The cooperativity of ILF can arise through DNA conformability in the absence of strong protein-protein interactions. The structural evidence present here was also consistent with our biochemical data.
|I10||How Do Two Membrane Proteins Talk to Each Other: Signal Relay From A Photosensory Receptor, NpSRII to Its Partner Protein, NpHtrII|
Chii-Shen Yang* and John L. Spudich#
*Department of Biochemical Science and Technology, National Taiwan University
The Natronobacterium pharaonis HtrII (NpHtrII), transducer interacts with its cognate photoactive sensory rhodopsin receptor, SRII, to mediate phototaxis responses. HtrII was shown to have two transmembrane (TM) helices and forms a homodimer. To investigate how the photo-activated SRII relays activation signal to HtrII, a series of single cysteines were substituted into an engineered cysteine-less HtrII at 38 positions in its two transmembrane domains. Oxidative disulfide cross-linking efficiencies of the monocysteine mutants were measured with or without photoactivation of SRII. The results reveal conformationally active regions in the TM2 of HtrII and a face along the length of TM2 that becomes more available for cross-linking upon receptor photoactivation. One TM2 mutants, G83F, showed loss of phototaxis responses, which indicates Gly83 is critical for maintaining the proper conformation of HtrII for signal relay from the photoactivated receptor. Furtmore, the cytoplasmic loops of SRII and the membrane- proximal cytoplasmic domain of its bound transducer HtrII were examined in the dark and in the light activated state by fluorescent probes and cysteine crosslinking. Light decreased the accessibility of E-F loop position 154 in the SRII-HtrII complex, but not in free SRII, consistent with HtrII proximity, which was confirmed by tryptophans placed within a 5-residue region identified in the HtrII membrane-proximal domain that exhibited fluorescence resonance energy transfer (FRET) to an fluorescent acceptor at position 154 in SRII. The energy transfer was eliminated in the signaling deficient HtrII mutant G83F without loss of affinity for SRII. Finally, the presence of SRII and HtrII reciprocally inhibit homodimer disulfide cross-linking reactions in their membrane-proximal domains, showing that each interferes with the others self-interaction in this region. A mechanism is thus proposed in which photoactivation alters the SRII-HtrII interaction in the membrane-proximal region during the signal relay process.
Single molecule dynamics of Triplex DNA:
Direct visualization and measurement.
Chia-Sheng Chang1*, Chia-Ching Chang1,2,3*, Po-Yen Lin1, Yen-Fu Chen1, and Lou-Sing Kan4*
1Institute of Physics, Academia Sinica. 2Department of Biological Sciences and Technology, National Chiao Tung University. 3National Nano Device Laboratories. 4Institute of Chemistry, Academia Sinica
Atomic force microscopy (AFM) has been recognized as a powerful tool to both image and manipulate a single molecule in either air or solution environment. Meanwhile, fluorescence images given rise from a pair of molecular beacons are regularly used to study the dynamic behaviors of a DNA¡¦s formation/dissociation. In this study, we have combined these two techniques to directly monitor and measure the dissociation dynamics of a DNA triplex in the single molecule level. During a DNA pulling process by AFM, single-molecule fluorescence images reveal the physical distance between the Hoogsteen and Watson strands of DNA triplex while the force curve of AFM denotes the dissociation mode of the triplex DNA, which takes a two-state process.
|I12||Positrom Emission Tomography for Biomolecular Imaging|
National Yang-Ming University Medical School National PET/Cyclotron Center Taipei Veterans General Hospital, Taiwan
Biomolecular imaging is defined as the characterization and measurement of biological processes in living animals, model systems, and humans at
the cellular and molecular level using imaging detectors. The goal of biomolecular imaging is to understand the dynamics, kinetics and the grade of misregulation of
biological and biochemical processes in vivo, in order to obtain relevant information for the diagnosis and treatment of a disease and for predictions about the efficacy of a therapy.
Disease is a biological process in which molecular errors cause failure of the normal, well-regulated function of cells. Molecular imaging provides the means to examine
individual organ systems for these molecular errors of disease. Within the general contex of nuclear medicine, positron emission tomography (PET) offers advantages of high sensitivity,
use of physiological elements and accurate quantification. In recent years it has been shown that PET is capable of obtaining in vivo metabolic images of small animals with excellent
image resolution and high sensitivity.
|I13||Biological pathway and gene network construction on cancer systems biology|
Department of Life Science, National Taiwan University
Systems biology is a new field in biology that aims at system-level understanding of biological systems. Recent progress in the field of molecular biology enables us
to obtain huge amounts of data. Furthermore, with the advent of high-throughput proteomics and microarray technologies, the study of systems biology has become possible. The hope of the rapid
translation of 'genes to drugs' has foundered on the reality that disease biology is complex, and that drug development must be driven by insights into biological responses. Systems biology
aims to describe and to understand the operation of complex biological systems and ultimately to develop predictive models of human disease.
|I14||Network characteristics of essential genes in E. coli|
Institute of Bioinformatics, National Yang-Ming University, Taipei, Taiwan
Recently a single-gene deletion library for all of the predicted genes of E. coli has been established and simultaneously 303 genes are identified as essential genes in LB medium. Elucidating the essentiality of these essential genes is a key to understand the system level organization of living cells. Here we present our analysis to address this issue from the perspectives of protein-protein interaction and metabolic networks. We have performed a comparison between essential and non-essential genes within an interaction network of E. coli and found that essential genes have significantly more links than the non-essential genes, validating earlier findings in budding yeast. Furthermore, other topological features such as clustering-coefficients, characteristic path lengths, diameter, and betweenness centrality are investigated to find their correlation with essential genes. Based on the known metabolic reactions of E. coli collected from the KEGG (Kyoto Encyclopedia of Genes and Genomes) and EcoCyc databases, a simple metabolic simulator is developed to reconstruct and navigate all the possible pathways and participated metabolites. In-silico gene deletion is performed to obtain the variation of metabolite distributions and examine the effect of missing links. Our results indicate that the average number of affected metabolites and reactions by deletion of essential genes are significantly higher than non-essential genes.
|I15||Discovery and Mechanistic Study of Novel Inhibitors against Hepatitis C Virus Helicase|
Ji-Wang Chern,,† Chien-Shu Chen,† Chun-Tang Chiou,†Grace Shiahuy Chen,‡ and Ding-Shinn Chen¡±
†School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan, ‡Department of Applied Chemistry, Providence University, Shalu, Taiwan, ¡±Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan, and Faculty of Life Science, National Yang-Ming University, Taipei, Taiwan
Patients with persistent Hepatitis C virus (HCV) infection are at risk of developing liver cirrhosis and hepatocellular carcinoma. The current recommended HCV
treatments use interferon monotherapy or a combination of interferon and ribavirin, which have limitations due to unsatisfactory response rates and adverse side effects. Therefore,
there is a great need to develop more effective agents for the treatment of HCV infection. The HCV NS3 is a bifunctional enzyme, of which the one-third of N-terminus possesses serine
protease activity and the remaining two-thirds of C-terminal region has RNA helicase/NTPase activity. The crystal structures of HCV NS3 helicase have been revealed and show similar
global Y-shaped conformation consisting of three domains. It was our intention to find potential HCV helicase inhibitors by blocking the closing of the interface between domains 1
(ATPase domain) and 2 (RNA binding domain).
|I16||Development of Functionalized Nanoparticles as Affinity Probes for Target Proteins Enrichment and Identification|
Department of Chemistry, National Tsing Hua University, Taiwan
Biomolecule-conjugated nanoparticles (NPs) have been demonstrated to have promising applications in bioanalysis. In this talk, I will focus on our recently progress in the development of functionalized nanoparticles and their applications in separation of target proteins. Due to its large surface area to volume ratio and homogeneity in aqueous solution, nanoparticle was demonstrated as a good carrier of affinity probe for target protein separation. The surface plasma resonance (SPR) was used to investigated the interactions between carbohydrate conjugated NPs and target proteins. The results showed that the ligand affinity with target protein was significantly enhanced when it was assembled on the nanoparticle. The MALDI-TOF MS analysis was applied to identify the protein binding epitope for the ligand. This approach was further extended to identify the biomarker in human serum.