PL FELICIA CHEN-WU MEMORIAL LECTURE :
Mechanotransduction in Endothelial Cells

Shu Chien

Departments of Bioengineering and Medicine, University of California, San Diego,
La Jolla, California, U.S.A.

 

Biophysical factors, such as the shear stress resulting from blood flow and the mechanical strain due to transmural pressure, can modulate functions of endothelial cells (ECs) by activating sequentially the mechano-sensors, signaling pathways, and gene and protein expressions. Laminar or pulsatile shear stress with a significant forward direction causes a transient up-regulation of pro-inflammatory and pro-proliferative genes such as MCP-1, followed by their down-regulation with sustained shearing, which also up-regulates the growth arrest genes and are hence anti-atherogenic. In contrast, the disturbed flow seen at curved regions and branch points of the arterial tree or oscillatory perfusion flow without a significant net forward direction causes the continued up-regulation of MCP-1 and high rates of cell proliferation, and are hence atherogenic. Wound healing of ECs after denudation is enhanced by shear stress with a net forward direction, but not by shear stress without a si gnificant net flow. ECs respond differentially to uniaxial and biaxial stretches. Uniaxial stretch causes a Rho-dependent orientation of stress fibers perpendicular to the direction of stretch, but this is not seen in response to equibiaxial stretch. Uniaxial stretch causes transient JNK activation to inhibit EC proliferation and protects ECs from apoptosis; biaxial stretch causes sustained JNK activation and induces apoptosis. Temporal and spatial variations in shear stress and stretch can cause differential modulations of signal transduction, gene expression, and protein expressions, as well as EC functions. The results on the effects of shearing and stretch indicate that the direction of mechanical forces plays an important role in mechanotransduction and the consequent functional responses of endothelial cells. Interdisciplinary studies at the interface of biophysics, molecular biology, bioengineering and medicine serve to elucidate the mechanisms of mechanotransduction under physiological and pathophysiological conditions and may provide insights into the pathogenesis and treatment of cardiovascular disorders.

 

 

PL1

Sialic Acid-Recognising Proteins and Drug Discovery

M. von Itzstein

Institute for Glycomics, Griffith University, Gold Coast Campus, Queensland, Australia

 

The important roles of carbohydrates and the proteins that recognise them in biological processes are broad in nature [1]. When these roles are associated with a disease a potential drug discovery opportunity can be presented. Various clinically significant pathogens, including viruses, parasites and bacteria utilise carbohydrates and their associated proteins to invade their host, facilitate their lifecycle and as a consequence produce disease [2]. Viruses such as influenza virus, rotavirus, and dengue virus all have essential carbohydrate recognition processes in their replicative cycles that present possible drug discovery targets [2, 3].
We have had a long-term interest in the investigation of a number of these carbohydraterecognising proteins as possible drug discovery targets [see for example 3-5] and some of our most recent work and advances on these target proteins, particularly influenza virus sialidase, and the potential for therapeutic development will be presented.

[1] Carbohydrate-based Drug Discovery, Wong, C-H. (ed), Wiley-VCH, Weinheim (2003).
[2] Carbohydrates in Chemistry and Biology, Ernst, B., Hart, G.W., Sinay, P. (eds), Wiley-VCH, Weinheim (2000).
[3] Wilson, J C, Kiefel, M J and von Itzstein, M (2005). In Handbook of Carbohydrate Engineering, Yarema, K J, (ed), Marcel Dekker, pp 679-740.
[4] Mann, M C, Islam, T, Dyason, J C, Florio, P, Trower, C J, Thomson, R J and von Itzstein, M (2006). Glycoconj. J 23 (1, 2):127 V 133.
[5] Liakatos, A, Kiefel, M J, Fleming, F, Coulson, B and von Itzstein , M (2006). Bioorg. Med. Chem, 14 (3):739-57.

 

 

PL2 A structural view of ion pumping by Ca2+-ATPase

Chikashi Toyoshima

Institute of Molecular and Cellular Biosciences, The University of Tokyo
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan

 

Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (SERCA1a) is an integral membrane protein of 110K and the best characterised member of the P-type (or E1/E2-type) ion translocating ATPases. We solved the structure of this ion pump in 2000 (Nature 405: 647-655) by X-ray crystallography and showed that it consists of 10 transmembrane helices, 3 cytoplasmic domains (A, actuator; N, nucleotide binding; P, phosphorylation) and small lumenal loops. According to the classical E1/E2 theory, transmembrane Ca2+-binding sites have high affinity and face the cytoplasm in E1; in E2, the binding sites have low affinity and face the lumen of sarcoplasmic reticulum (extracellular side). Actual transfer of bound Ca2+ is thought to take place between two phosphorylated intermediates, E1P and E2P. We have determined the crystal structures of this ATPase in 7 different states that nearly cover the entire reaction cycle, and also carried out all-atom molecular dynamics simulations for native structures and some mutants. Thus, we can now describe the mechanism of ion pumping in unprecedented detail and ask fundamental questions. These analyses show that ion pumps use large rearrangements of cytoplasmic domains to move transmembrane gates of ion pathway. In particular, the A-domain is the actuator of the gates and that ATP, phosphate, Ca2+ and Mg2+ are the principal modifiers of the domain interfaces to change the position and orientation of the A-domain. Large domain motions are needed because ion pumps vigorously fluctuate by thermal energy, yet utilises it efficiently for transferring ions across the membrane. In this presentation, structural basis of ion pumping by Ca2+-ATPase is overviewed very briefly.

 

 

PL3 Overview of Research Opportunities at Synchrotron Biomedical Imaging and
Therapy Facilitie

Dean Chapman

Anatomy and Cell Biology, University of Saskatchewan

 

A number of dedicated biomedical research facilities at synchrotrons have or are being built worldwide. These facilities have a strong medical focus with widely varying imaging (and some therapy) capabilities for biological tissues, animal and some human research. This presentation will survey the present and planned facilities with emphasis on the research that is being carried out and the methods being used. Presently the imaging methods can be categorized as absorption and/or phase based with the methods applied in either simple projection or computed tomography modes. The absorption based methods include K-edge subtraction, single and multiple energy imaging. Phase based methods include phase contrast or in-line holography, analyzer based or Diffraction Enhanced Imaging / Multiple Image Radiography.
Radiation therapy methods include Microbeam Radiation Therapy and monochromatic beam therapy (Synchrotron Stereotactic Radiotherapy).
Special emphasis is made on the biomedical research facility being constructed at the Canadian Light Source in Saskatoon, Saskatchewan. The BioMedical Imaging and Therapy (BMIT) facility will provide high intensity, high x-ray energy light for a wide variety of imaging and therapy programs. This $17M facility is now in the detailed design phase with conventional construction underway. Completion of the bulk of the facility and commissioning is envisioned for the summer of 2007.

 

 

PL4 The NS5B Polymerase from HCV: A Target for Drug Design

B. K. Biswala, M. Wanga,b, M.M. Cherneya, L. Chanc, C.G. Yannopoulosc, Darius Bilimoriac, J. Bedardc, M. N. G. Jamesa†

aGroup in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G2H7.
bPresent address; Department of Chemistry and Biochemistry, Arizona State University, Main Campus, P.O. Box 871604, Tempe, AZ 85287-1604, USA.
cViroChem Pharma Inc., 275 Armand Frappier Boulevard, Laval, Quebec, Canada H7V4A7.
Canada Research Chair in Protein Structure and Function.

 

Hepatitis C virus (HCV) chronically infects about 170 million people, ≈ 3% of the worlds population. There is clinical evidence that this chronic infection progresses to cirrhosis then to hepatocellular carcinoma within 10 to 20 years in about 20% and 5% respectively of these infected individuals. Chronic infection by HCV is the leading cause of liver transplantation in USA. To date, there is no vaccine available against HCV. Furthermore, the currently available treatment (a combination of interferon-\ and ribavirin) is expensive and is effective in only 50V60% of patients treated. The development of new effective antiviral compounds for combating this debilitating human pathogen is therefore of paramount importance and is currently an intensive area of pharmaceutical research. The RNA dependent RNA polymerase (NS5B) from Hepatitis C virus (HCV) is a key enzyme in HCV replication. NS5B adopts the classical right-hand fold of polymerases and is constituted of a palm domain, a fingers domain and a thumb domain. The polymerase active site is located in the palm domain. NS5B is a major target for the development of antiviral compounds directed against the HCV. I will discuss the structures of three thiophene-based non-nucleoside inhibitors (NNIs) bound non-covalently to NS5B. Each of the inhibitors binds to NS5B non-competitively to a common binding site in the thumb domain that is ~ 35 Å from the polymerase active site located in the palm domain. The three compounds exhibit IC50s in the range of 340 nM to 470 nM and have common binding features that result in relatively large conformational changes of residues that interact directly with the inhibitors as well as for other residues adjacent to the binding site. Detailed comparisons of the unbound NS5B structure with those having the bound inhibitors present show that residues Pro495 to Arg505 (the N-terminus of the "T" helix) exhibit some of the largest changes. It has been reported that Pro495, Pro496, Val499 and Arg503 are part of the guanosine triphosphate (GTP) specific allosteric binding site located in close proximity to our binding site. It has also been reported that the introduction of mutations to key residues in this region (i.e. Val499Gly) ablate in vivo subgenomic HCV RNA replication. The details of NS5B polymerase/inhibitor binding interactions coupled with the observed, induced conformational changes provide new insights into the design of novel NNIs of HCV.

 

 

PL5 Electron Cryo-microscopy of Biological Nano-Machines

Wah Chiu

Baylor College of Medicine, Houston, TX 77010, USA

 

A biological nanomachine is made up of multiple molecular components to perform specific biological functions. A biological nanomachine can assume different sizes and shapes depending on its functional states. Frozen-hydrated preparation of biochemically purified biological nanomachines can be imaged in an electron cryomicroscope. Image reconstruction methods have been developed to reconstruct 3-D structures from single nanomachine images at subnanometer resolution. Mining of salient structure features within the molecular components of a large nanomachine is a daunting task. Computational and visualization tools have been developed to extract features such as alpha helices and beta sheets of protein subunits with a high degree of reliability. 3-D structure at subnanometer resolution can be combined with sequenced-based structure prediction methods to derive pseudo atomic models of molecular components of a nanomachine. Examples will be used to demonstrate our experimental and computational procedures for determining protein folds and the mechanism of structural changes during different physiological states of biological nanomachines.

 

 

PL6 Modeling the structures of proteins and macromolecular assemblies

Andrej Sali

California Institute for Quantitative Biomedical Research, Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, CA, USA.

 

The structures of most protein domains will eventually be characterized by structural genomics, which aims to determine representative structures in most protein fold families by experiment, allowing the remaining protein sequences to be modeled with useful accuracy by computational methods. In the case of assemblies, however, the structure is usually obtained by a number of experimental methods of varying accuracy and resolution (eg, X-ray crystallography of the subunits, low-resolution electron microscopy of the assembly, and chemical cross-linking). Therefore, there is a need for a computational framework that can take into account all available information about the structure of an assembly and calculate at the appropriate resolution all models that are consistent with the given input. To this end, it is useful to express structure determination as an optimization problem. The three components of this approach are (i) representation of an assembly; (ii) a scoring function consisting of individual spatial restraints; and (iii) optimization of the scoring function to obtain the models. This approach will be illustrated by the structural characterization of several protein assemblies.

1. Sali A., Glaeser R., Earnest T., Baumeister W. From words to literature in structural proteomics. Nature, 2003, 422, 216-225.
2. Alber, F., Kim, M., Sali, A. Structural characterization of assemblies from overall shape and subcomplex compositions. Structure 13, 435-445, 2005.
3. M. Topf, A. Sali. Combining Electron Microscopy and Comparative Protein Structure Modeling. Current Opinion in Structural Biology 15, 578-585, 2005.

 

 

PL7 A rational drug design approach: antibodies targeting IgE for treating asthma and allergy.

Tse Wen Chang

The Genomics Research Center, Academia Sinica, Taipei 115, Taiwan

 

The development of anti-IgE antibodies for prophylactic and therapeutic applications in allergic diseases has taken nearly two decades. More than 20 Phase II and III clinical trials have been done on adult and pediatric asthma, seasonal and perennial allergic rhinitis, sensitivity to peanuts, and several other indications. Anti-IgE has been approved in the U.S.A., European Union, and several other regions for treating patients with severe asthma. More than 70,000 difficult-to-treat asthma patients have used asthma with a response rate of 90%.

Therapeutic anti-IgE monoclonal antibodies are the results of rational drug design, taking into consideration the structural features of IgE and membrane-bound IgE (mIgE). Unlike ordinary antibodies specific for human IgE, a therapeutic anti-IgE, while capable of binding tightly to free IgE and mIgE, does not bind to IgE bound by the high-affinity IgE.Fc receptors (Fc`RI) on mast cells and basophils and by the low-affinity IgE.Fc receptors (Fc`RII, or CD23) on B cells, granulocytes, and many other cell types. The therapeutic anti-IgE possesses multiple potential pharmacological effects and is efficacious in treating allergic patients in whom IgE mediates the major allergic responses. Studies on the unique structural mIgE C`mX domain and on the regulatory effects of anti-C`mX monoclonal antibodies will be an area for future investigation of the potential of long-term IgE regulation.