6^th International Conference on Structural Biology August 22-23, 2016 New Orleans, Louisiana, USA

Biography:

R Holland Cheng, Ph D is a Professor of Molecular and Cellular Biology at University of California. Cheng received a master degree  in 1989 and a Ph D degree in 1992 from Purdue University. Cheng has served as a Panel Reviewer for NIH programmed project grant, NIH Microbiology & Infectious Diseases Research Committee, NIH National Center for Research Resources, Wellcome Trust, UK and Earth & Life Sciences Council, Netherlands (2002). Cheng has actively served on the advisory board of the International Microscopy Congress plus regional microscopy societies, American Biographical Institute, and International Virus Assembly Symposium.He is also a regular member of the American Chemical Society,American Society for Microbiology and American Biophysical Society . Cheng has published nearly fifty papers in peer reviewed journals and his research mainly focuses on Proteome imaging of macromolecular systems.

Abstract:

Biography:

Toshiya Senda has completed his PhD from Nagaoka University of Technology (Niigata, Japan) in 1995. He was a research associate in Nagaoka University of Technology (1995-2001) and a senior researcher in Institute of Advanced  Industrial Science  and Technology (2001-2012). Now, he is the director/professor of Structural Biology Research Center of High Energy Accelerator Research Organization in JAPAN. He was awarded the CrSJ (Crystallographic society of Japan) award in 2014 (Structural biology studies of CagA from Helicobacter pylori and histone chaperon CIA/ASF1). 

Abstract:

    Helicobacter pylori    , a gram-negative bacterium  that colonizes the    human gastric mucosa   , is recognized as  a major risk factor for gastric diseases, such as    peptic ulcers    and   gastric carcinomas  . H. pylori delivers an effector protein CagA into gastric epit helial cells and th e EPIYA and CM segments in the C-terminal   intrinsically disordered    (ID) region of CagA (CagA-C) promiscuously interact with cellular  signaling  molecules, such as SHP2 and PAR1b, to deregulate these signaling  proteins. To analyze the structure and function of a large protein complex formed by CagA, SHP2, and PAR1b, we started a  structural biology  study of the CagA-SHP2-PAR1b complex. Since our group already determined the crystal structure of the N-terminal structured region of CagA (CagA-N) (1), we are working on structure and function of CagA-C. Ou  r biochemical analysis suggested that the CBS segment in the CagA-C region interacts with the NBS segment in CagA-N, forming a four-α-helix bundle structure and the CagA-C region adops a lariat-loop like structure. Interestingly, mutations in the NBS and CBS segments caused a reduced biological activity of CagA (humminbird-inducing ability in AGS cells) (1). For further understanding of the  structure-function relationship  of CagA-C, we have confirmed the existence of the lariat-loop like structure. In addition, we have obtained some structural information of the CagA-C region and its interaction with SHP2. We will discuss the structural characteristics of CagA-C and its relationship to the biological activities. 

Biography:

Vesa P Hytonen is a head of the    Protein Dynamics    research group at the University of Tampere. After graduating as a PhD from the University of Jyväskylä, Jyväskylä, Finland at 2005, he conducted postdoctoral training at ETH Zurich, Zürich, Switzerland 2005-2007. He then continued as a postdoctoral researcher at the University of Tampere and established independent res earch group at 2010. His research interests are   mechanobiology  ,  protein engineering  and vaccine research, and    he has authored more than 90 scientific articles. 

Abstract:

   Talin    is cytoplasmic protein essential for cell adhesion. It connects integrin receptors  for   actin   cyto skeleton and acts as a   mechanosensor   by recruiting vinculin molecules as a response to mechanical stress applied on it. We have studied tal in structure and function using extensive   molecular dynamics simulations  ,  small-angle X-ray scattering , cryo-EM and biophysical methods combi ned with cell biology methods. Th ese studies have revealed novel insights into role of talin in integrin activation. We have also been able to develop more detailed picture of about the role of talin rod in mechanosensing and in regulation of cell adhesion dynamics, cell migration and traction force generation.

Biography:

Ren is a staff scientist and lab director at Molecular Foundry, LBNL. He received his BS and MS in  theoretical physics  at Lanzhou University and PhD in material physics at Univ of Science and Tech Beijing. He received his postdoc training at Scripps Research Institute. He started his r esearch lab at UCSF in 2006 and then transfer to LBL in 2010. His research group is supported by DOE, NIH and Instrudy funds. He published more than 70 papers in reputed journals and has been serving as an editorial board member of numerial journals.

Abstract:

Proteins have the unique ability to function specifically and efficiently, which is attained through its three-dimensional (3D) structures and flexibility, as well as necessary conformational changes. However, structural study on proteins that have large-scale flexibility, dynamics, and      heterogeneity      is challenging by current techniques, including     X-ray crystallography    ,    nuclear magnetic resonance    (NMR) spectrum,   small angle scattering   (SAXS) and   electron microsc opy    (EM) single-particle reconstruction. A fundamental approach to study the structure of flexible proteins should be based on the signal from each individual protein molecule itself instead of averaging fr om different protein molecules. EM provide a novel tool to image each individual molecule at atomic resolution level; while   electron tomography   (ET) provide an approach to image a targeted molecule from  a series of tilt angles. Although the signal obtained from an individual molecule has been believed for decades to be too weak to achieve any  3D structure  with a meaningful resolution, we recently re-investigated this possibility carefully and proposed an individual-particle electron tomography (  IPET) approach with a ‘‘focused electron tomography reconstruction’’ (FETR) algorithm to improve the 3D structure resolution via decreasing the reconstructing image size with an iterative refinement process. IPET does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can provide near one nanometer resolution 3D structure from an individual protein molecule. Through the structure determination of each individual molecule, the compar ison of these molecular structures provides a new opportunity to reveal the dynamic character, equilibrium fluctuation, mechanism, aggregation and even structural changes in proteins during a chemical reaction or biological event.

Biography:

Prasanna Kolatkar completed his PhD at the University of Texas at Austin in 1991 in the Department    Chemistry    and   Biochemistry   and post-doctoral studies in the laboratory of Michael Rossmann at Purdue University where he received a Ja ne Coffin Childs Memorial Fund fellowship. Upon moving to Singapore in 1997, he worked at the   Bioinformatics   Center and Institute of Molecular and  Cell Biology  in Singapore before joining the Genome Institute of Singapore in 2001. Subsequently he jo ined QBRI in 2013.  Recently his work has focused on re-engineering of stem cell function through directed mutagenesis of key residues involved in protein-protein interactions.

Abstract:

   Transcription factors    (TFs) are key players in early cell development which direct cells towards specific fates. The Sox family of TFs is composed of 20 members which are responsible for directing a wide variety of fates. The ability of highly similar TFs to carry out such distinct outcomes had been a conundrum to date. We  have studied several   Sox-DNA   (HMG domain) structu res in my laboratory which all display essentially similar structures. The Sox binding motif is only 6-8 bp long and this once again raises the question as to how specificity can be achieved. We discovered that a critical Sox17 residue involved in protein interaction with its partner Oct4 (POU domain) helps to install specificity of binding to its   cognate DNA   and consequently give rise to  endoderm . Subsequently we were able to re-engineer Sox17 through a single resid ue mutant and convert it into a potent pluripotent TF by substituting Sox2 in the standar d induced pluripotent stem cell cocktail composed of 4 TFs. Next we also studied the genomic profiles of native and mutant Sox TFs through a battery of ChIP-seq experiments to    validate the in-vitro results. The integrated application of structural and genomic methods have allowed us to analyze and understand how highly similar TFs can partner each other through key interaction points which give rise to highly specific outcomes. TF function can be altered through modified TF forms as well as novel ligands which could have applications in disease and therapy.

Biography:

Miki Senda has completed her PhD at 2008 from Nagaoka University of Technology. She is an assistant professor of Structural Biology Research Center in High Energy Accelerator Research Organization (KEK). She have several collaborations, in which she has worked as an expert of protein crystallization and crystal quality improvement. She received Oxford Cryosystems Low Temperature Prize at the 63rd Annual meeting of the American Crystallographic Association (ACA) in 2013.

Abstract:

Current     X-ray sources     of    synchrotron radiation    enable us to determi ne the    crystal structures    of proteins even if the crystals were diffracted only to medium or low resolution. However, high-resolution crystal structures ar e still required for the   pharmaceutical   and  biochemical sciences   . When obtained crystals were of poor quality and insufficient for crystal structure determination, post-crystallization treatment could improve the crystal quality. Our experiences of crystal structure analyses of  hist one chaperon  T AF-Iβ, CagA oncoprotein from Helico bacter pylori and GTP sensor PI5P4Kβ showed that crystal soaki ng into cryoprotectants improved crystal-quality (1, 2, 3). Of the three proteins, crystal qualities of CagA and PI5P4Kβ were significantly improved by using more than one cryo-protectant. This method, multi-step soaking method, improved not only the maximum resolution but also success rate of high resolution data collection. Reproducibility of the  crystallization  is another critical problem in determining crystal structure. Anaerobic crystallization and immediate observation method are effective to improve the reproducibility of the crystallization. We would like to report some examples of the crystallization and crystal quality improvement by our strategy. 

Biography:

Jiang obtained his PhD from Yale University and did his postdoctoral training at Rockefeller University. He is currently an Associate Professor in UF and serves as the Faculty Director of Electron Microscopy (EM) at the UF Interdisciplinary Center for Biotechology Research, where EM is used for revealing the structural basis of   biological machineries  . He has published more than 25 papers in reputed journals. His lab focuses on structural and functional studies of intracellular  RNA-binding complexes  and ion channels invovled in membrane excitability and regulated secretion, and has been developing new methods for cryoEM imaging of single molecules.  

Abstract:

The intracellular    innate immune response    takes advantage of the induced filament formation i n order to achieve high sensitivity and   signal amplification  . The RIG-I –MAVS signaling is initiated  by the Rig-I sensing of individual viral RNA (vRNA) molecules, which induce the formation of RIG-I/vRNA dimers. The activated RIG-I dimers together with   poly-ubiquitins   trigger the self-inhibited  MAVS  molecules on the outer surface o f mitochondria to release their CARD domains, interact with each other and f orm fibril structures. We have been working on unde   rstanding the structural basis for the RIG-I –MAVS signaling, both the vRNA-induced RIG-I  dimerization  and the switch of MAVS monomers from the self-inhibited state to the filament form both in vitro and in virus-infected cells. We observed significant heterogeneity in the MAVS filaments. Symmetry analysis of the filaments showed ambiguity in them. In our earlier datasets, a majority of our images agreed better with the C3 symmetry, which led us to prepare filaments under low  ionic strength  to increase electrostatic interaction and filament quality. Sorting of the cryoEM images from these filaments led to a significant portion of the data that showed C1 symmetry, consistent with a group of fil aments formed by renaturization of the denatured CARD domsignal amplification poly-ubiquitins fibril structures signaling  cryoEM images  CARD mutagenesisains. We performed parallel sorting of our dat asets using different algorithms against two different structural models to estimate the heterogeneous composition in the datasets. The two models are not well distinguished by the  mutagenesis  data we have collected. They suggest two different molecular mechanisms and need further experimental examination. A major open question is which one or both filaments are formed inside the virus-infected cells. 

Biography:

Jacinto López-Sagaseta completed his PhD on 2007 and carried out a postdoctoral stage at the University of Chicago. His work has been focused mainly in the characterization of the     endothelial protein     C receptor and its interaction with the    coagulation Factor    VII. During his postdoctoral research, he pursued   structural studies   on antigen recognition by NKT and MAIT cells. He has also  worked on the   structural elucidation   of a  potassium ion channel  bound t  o an inhibitory  ligand . In 2015, Jacinto López-Sagaseta was awarded a Marie Curie European  Grant and joined the Structural Biology Unit of GSK Vaccines  where he is carrying out studies in the context of structural vaccinology. 

Abstract:

Anchored yet exposed to the outside moiety of the bacterial shell, Factor H   binding protein   (fHbp) is one of the main  antigenic  components of Neisseria meningitidis, one of the causative agents of   men ingitis  , an infect ious disease that can cause a fatal outcome or permanent disability within 24 hours of infection. Though there have been described up to three different variants of fHbp, it is fHbp variant 1 (fHbp-1), the subclass showing the highest prevalence amongst MenB strains, and also, one of the actual components of Bexsero, the current licensed vaccine against   serogroup   B Meningococci (MenB). In order to define the structural basis that underlie the recognition of this  highly immunogenic antigen and the broad strain coverage offered by Bexsero, we have determined the crystal structure of a complex between a human Fab and fHbp-1 at a resolution of 2.2 Å. The Fab has been originated from an immunization study that included a recombinant form of fHbp-1, and importantly, it is cross-reactive against all of them. The cross-reactive  epitope  spans along the c-terminal beta barrel of fHbp and encompasses residues that are highly conserved across the different fHbp variants. The hypervariable CDR3 loop of the heavy chain dominates the recognition of the antigen. This crystal structure represents the first evidence, at the atomic level, of the recognition of Neisseria meningitidis fHbp by a human Fab r  aised in an individual upon vaccination, and provides the basis behind the broad strain coverage of the current vaccine against MenB. In addition, the information gathered from this structure will be of high value for future structure-based antigen design. 

Biography:

Chan-Shing Lin has completed his PhD at University of California, Irvine, in 1992. He has published more than 65 papers in reputed journals and has been serving as an Editorial Board Member of Archive of Virology and Frontier journals. He has also consulted biotechnology companies in building the cGMP processes for manufacturing  vaccines  and bio-medicals. 

Abstract:

   Betanodaviruses    cause lethal   nervous necrosis   in more than f orty species of fish. I will present the maps from   cryo-electron microscopy   (cryo-EM), since 2001 when the first map was published, in comparison to  x-ray diffractions . During the low resolution era of 10-20 Å maps, the molecular features of  the  Dragon Grouper Nervous Necrosis Virus  (DGNNV) were intensively studied. Virus-like particles (VLPs) are formed by the single capsid p rotein that was expressed either in E. coli, yeast, or insect cell, while VLPs were not found when 35 amino acids at the N-terminus or four amino acids at C-terminus were deleted. Residues of aspartic acids interacting with cations are found to be important to the VLP stability, whereas the potential disulfide bounds of cysteine residues were not essential for the particle assembly. In the pathway of the DGNNV entry into fish cells, micropinocytosis interacting with heat-shock protein HSP90-like was proposed while the detail interaction of protrusion domain with lipid-bound protein is under investigated using x-ray refractivity. Three domains of the capsid protein (RNA-binding, shell, and protrusion domains) in truncated clones were determined to 4-6 A using x-ray diffraction. The proposed structure of the  Betanodvirus  since 2001 was doubly confirmed, in addition to another cation-binding site at protrusion was found. Recently,  cryoEM  with a direct detection camera to overcome specimen motion and radiation damage issues provides near atomic structures of the T=3 DGNNV VLPs. The structure of the shell domain (52-213^th aa) in weak basic condition was determined to 3.56 Å resolution and an atomic model that was built de novo reveals  protein-protein interactions , calcium-ion bridges, and unique cation interactions. The cation interactions stabilize the particle by holding three subunits in an asymmetric unit of trimer and p atching asymmetric units into an icosahedron and their alterations have led to expansion or disruption of the particle. The cryo-EM structure changes upon immersion in an acidic condition that mimics endocytosis entry pathway suggests that a pH-sensing mechanism for the piscine NNV machinery to deliver its genome.

Biography:

Michael Rossmann graduated from the University of London (B.Sc. General in Physics and Mathematics, 1950; B.Sc. Special 1951 in Physics; M.Sc., 1953) and the University of Glasgow (Ph.D., 1956). He was a postdoctoral fellow with Prof. William Lipscomb at the University of Minnesota (1956-1958) and a Research Associate with Dr. Max Perutz at the MRC Laboratory of    Molecular Biology   , Cambridge, England (1958-1964). Currently he is the Hanley Professor of   Biological Sciences   at Purdue University, where he has worked for the last 52 years. His laboratory utilizes  X-ray crystallography  and electron microscopy to study the structures of animal and bacterial viruses.   

Abstract:

Zika virus belongs to the flavivirus family that includes dengue virus, West Nile virus, yellow fever virus and other human pathogens. The structure of dengue virus was the first flavivirus to be determined. It was accomplished by obtaining a low resolution image of the whole virus by cryoEM and determining the structure of the major capsid protein of the homologous tick-borne encephalitis virus crystallographically. When Zika started to be recognized as a major threat to humanity, cryoEM had advanced to allow us to determine the structure of Zika virus to near-atomic resolution using only cryoEM. The most important difference between dengue and Zika virus was found to be at a glycosylation site which is suspected to be involved in the recognition of molecules on the surface of potential host cells.

Biography:

Liguo Wang completed his PhD at the age of 33 years from Cornell University and his postdoctoral studies from Yale University School of  Medicine . He is an assistant professor in the department of Biological Structure at the University of Washington. He has more than 10 years of expertise in cryo-Electron Microscopy, and developed a method to study structures  of membrane proteins in lipid environments, which make it possible to manipulate the functional states of membrane proteins. He published more than 20 papers in reputed journals. He also served as a reviewer for several high-ranking journals and a reviewer of scientific proposals for a few scientific organizations.

Abstract:

Recently, a steadily growing community of researchers has been attracted by the capability of cryo-Electron Microscopy (cryo-EM) to obtain atomic-resolution structures without the need to crystalize their samples. To study membrane protein structures using cryo-EM, membrane proteins are usually extracted from cell membranes and dissolved in detergents. However, both functional and structural studies clearly highlight the importance of a lipid membrane environment to preserve protein integrity and activity. To restore the lipid membrane environment of membrane proteins, I have been developing a platform, called “random spherically constrained” (RSC) single-particle cryo-EM, for both structural and functional studies of membrane proteins. The RSC platform establishes the lipid environment for membrane proteins, and makes it possible, for the first time, to apply desired transmembrane potential to trap voltage-gated ion channels in desired functional states for structural analysis. To confirm the absolute amplitude of the transmembrane potential, the hyperpolarization- activated cyclic nucleotide-gated (HCN2) channel was investigated as a model protein, as it only opens at very negative transmembrane potentials. The results confirmed that a negative potential of -120 mV was successfully established as predicted. The RSC method has been successfully employed to obtain the structures of both the large conductance voltage- and calcium-activated potassium (BK) and HCN2 channels in lipid membranes. This confirmed that the RSC method could be used to manipulate the functional states of other voltage-gated ion channels, which is critical for understanding the mechanism under which the ion channel responds to the transmembrane potential.

Biography:

Jiang obtained his PhD from Yale University and did his postdoctoral training at Rockefeller University. He is currently an Associate Professor in UF and serves as the Faculty Director of   Electron Microscopy   (EM) at the UF Interdisciplinary Center for  Biotechology  Research, where EM is used for revealing the structural basis of biological machineries. He has published more than 25 papers in reputed journals. His lab focuses on structural and functional studies of int racellular  RNA-binding complexes  and ion channels invovled in membrane excitabili ty and regulated secretion, and has been developing new methods for cryoEM imaging of single molecules.

Abstract:

With recent successful development of maximum-likelihood based refinement algorithms, direct electron detectors and motion correction, phase plates, energy filters and automatic data collection and particle selection, preparation of good cryoEM specimens and reliable analysis of conformational and/or compositional heterogeneity in the images of single molecular complexes are becoming the rate-limiting steps in cryoEM studies of biological specimens. ChemiC was developed in my laboratory to facilitate selective enrichment of nanogram amounts of biological complexes to the surfaces of chemically functionalized carbon-films, which, compared to the conventional, physically-treated carbon films, has multiple advantages. It allows affinity-based selection without introducing a significant amount of biomass, retains all attached molecules in a thin layer of vitrified ice that is less than 40 nm thick and has much less ice-scattering noise than conventional 100-150 nm thick ice layers, and enables controlled assembly of specific biological processes or synchronization of biological complexes in a targeted conformational state. This new method has been used for different projects. I will discuss the ChemiC technology and its applications in cryoEM specimen preparation and imaging and will present the recent progress in further development of the technology for different purposes.

Biography:

Kenneth A Taylor has completed his PhD in 1975 from University of California Berkeley on the topic of    Electron Microscopy    and   Electron Diffraction   of Frozen Hydrated Biological Specimens, widely regarded at t he start of the field of  cryo-electron microscopy . He has s  pent 4 years as a Post doctorate at the MRC Laboratory of   Molecular Biology   and 15 years on the faculty at Duke Unive rsity Medical Center. He is currently the Donald L. D. Caspar Professor of  Biological Science . He studies the structure of macromolecular assemblies in muscle, the cyto  skeleton and viruses using cryo-electron microscopy and cryo-electron tomography. 

Abstract:

Many biological systems contain complex mixtures of filaments, such as actin containing and myosin containing filaments and the cytoskeleton. The properties of the interacting partners are keys to understanding how the systems function. In muscle, the filament systems are composed of multiple proteins that modify the basic filament. In the case of actin, the troponin-tropomyosin system provides a mechanism for regulating attachment by the myosin motors and thus muscle contraction. During muscle contraction, myosin motors attach the thin filament in multiple ways producing a heterogeneous system that is difficult to dissect. Many proteins interact with F-actin to form cross-linked bundles. Among these are fimbrin, villin, α-actinin and several glycolytic enzymes and even elevated concentrations of magnesium and polylysine. An im­portant characteristic of these cross-linkers is an ability to form 2-D rafts on positively charged lipid monolayers. Actin rafts should be ideal for structure determination by electron micros­copy (EM) but several problems exist, in particular heterogeneity. All raft cross-linkers produce polymorphic rafts that are either polar or have mixed polarity. Even bundles of defined polarity can have heterogeneous cross linkers. We have developed a strategy for obtaining information from these heterogeneous systems using sub-volume alignment and classification that will be applicable to this type of problem and perhaps many others where there is difficulty obtaining large populations of homogeneous entities, be they filament segments or single particles.

Biography:

Matthew T Swulius has completed his PhD at University of Texas   Health Science   Center at Houston in 2010, where he studied structural and protein compositional changes in the synapse throughout development. He is currently a senior Post-doc in Grant  Jensen’s lab at Caltech, where he has contributed to the field of bacterial  cell biology  by studying the MreB cytoskeleton using electron cryotomography and to eukar  yotic cell division using cryo-focused ion beam milling and correlated light and electron microscopy to image the native structure of the actomyosin ring in fission yeast. 

Abstract:

While electron cryotomography (ECT) is a powerful technique for imaging unique biological structures to molecular resolution, some protein machinery is complex and highly dynamic, making static images difficult to interpret on their own. In such cases, coarse-grained simulations can be employed in order to interpret and test the mechanistic implications of such experimental data. Here we use a combination of ECT and simulation to study the structure and contractile mechanism of the actomyosin ring (AMR) in fission yeast. Because fission yeast are beyond the thickness amenable to cryoEM, we used cryo-focused ion beam milling and cryosectioning to gain access to natively preserved AMRs. ECT revealed bundles of actin filaments running parallel to one another that were “saddling” the leading edge of the septum, and no direct contact between actin and the membrane was observed, refuting the notion that F-actin is connected to the membrane at cytokineteic nodes. After exploring a variety of actomyosin configurations by 3D coarse-grained simulation, we propose a model that best agrees with our and other published experimental data.

Biography:

Youdong Mao has obtained his PhD in Physics from Peking University in 2005 and completed his Postdoctoral training in   Biophysics   at Harvard Medical School in 2012. He has then worked at the Faculty of D ana-Farber Cancer Institute, Harvard Medical School. In 2015, he has joined the Faculty at the School of Physics and Center for  Quantitative Biology  at Peking University. His research interest lies in developing multidisciplinary approaches across the areas of  structural biol ogy , electron microscopy an d high-performance machine learning. 

Abstract:

Machine learning technology represents an intriguing avenue in the methodology development for cryo-EM structure determination. In this presentation, we explore two aspects of machine learning development that help advance cryo-EM data analysis. First, structural heterogeneity in single-particle images presents a major challenge for high-resolution cryo-EM structure determination. Recently we introduce a statistical manifold learning approach for unsupervised single-particle deep classification. When optimized for Intel High-Performance Computing (HPC) processors, our approach implemented in ROME software package, can generate thousands of reference-free class averages within several hours from hundreds of thousands of single-particle cryo-EM images. Deep classification thus assists in computational purification of single-particle datasets for high-resolution reconstruction. Second, particle extraction represents a major practical bottleneck in the structure determination of biological macromolecular complexes by single-particle cryo-EM. We developed a deep learning-based algorithmic framework,  DeepEM , for single-particle recognition from noisy cryo-EM micrographs, enabling automated particle picking, selection and verification in an integrated fashion. Our approach exhibits improved performance and high accuracy when tested on the standard KLH dataset as well as several challenging experimental cryo-EM datasets. 

Biography:

John Miller completed his PhD in   biological sciences   in 1971 from Stanford U niversity, carried out his postdoctoral research in  molecular biology  and physiology and at the University of California,   San Diego, and then accepted a lectureship at Victoria University of Wellington in 1977. He was promoted to full professor in 2008. He teaches courses in mamma lian physiology, cell biology, and development. His research interests are in the mode of action of novel natural products and their development as anticancer agents. He has published more than 113 papers in peer-reviewed journals and has written 4 book chapters.

Abstract:

    Tubulin    , a 50 kDa protein, associates into α, β-heterodimers which polymerize in to the    microtubule   , a maj or cytoskeletal component of all eukaryote cells. Micr otubules consist of 13 protofilaments with the α,β-dimers stacked head-to-tail. Numerous molecules, both    endogenous    and   exogenous  , bind to tubulin and affect it s ability to  polymerize  and  depolymer ize . Insights into the s   tructure of tubulin and the binding sites of different  ligands  were first obtained from       electron crystallography       of Zn- and paclitaxel-stabilize d, antiparallel sheets.      Paclitaxel      is the first known micr otubule-stabilizing agent. More recent studies have used a      T2R complex      consisting of a        stathmin-like domain      bound to two     heterodimers     linked in a head-to- tail fashion. Stathmin, by inducing a     curve    d dimer structure,  prevents its assembly into polymers. This    T2R complex   , often in combination with the enzyme tubu lin tyrosine ligase has allowed detailed structural mapping by    X- ray crystallography    to less than 2.3 Å resolution. There are two known   microtubule-destabilizing sites   on  β-tubulin , the colchicine site and the  vinca alkaloid site, and two sta   bilizing sites, the taxoid site and the laulimalide/peloruside site. Recent    X-ray crystallography    analysis has confirmed the location of the stabilizing sites and shown that ligands that bind the two different sites can bind simultaneously . Adding both types of ligands together can lead to synergistic effects on the dynamicity of the   microtubules  , both in solution and inside cells; however, the mechanisms of stabilization and synergy between  ligands are not fully understood. In collaboration with Eva Nogales at Berkeley, high resolution   cryo-EM   studies are currently underway on the structural association between  peloruside  and tubulin.   

Biography:

K. Ogata has completed his PhD at the age of 27 years from Yokohama City University in 1992 and postdoctoral studies from RIKEN tsukuba institute. In 1997, He became the leader of the regulation of   protein function project  , Kanagawa Academy of Science and Technology (KAST), and from 2001, the professor and chairperson of Department of  Biochemistry , Yokohama City University Graduate School of Medicine. He has published papers in JMB, PNAS, NSMB, and Cell, and had been serving as an editorial board member of BBRC (2009-2010)  

Abstract:

  Transcriptional regulation   is a  fundamental mechanism for  cell proliferation  and differentiation. A ma  jor player in this process is a member of    tanscription factors    (TFs). Activities of TFs are modu lated by chemical modifications such as    phosphorylation    through   cell signaling   pathways directing cell functions.   Molecular mechanism s   of regulation for the functional high-order assembly consisting of multiple TFs and enhancer DNA (enhanceosome) under   cell signaling   are largely unknown. So far, we investigated changes in assembly state of an  enhanceosome  upon a TF’s phosphorylation, using   crystallographical  ,  biophysical , and  molecular dynamical  analyses . Here, we will  illustrate the cas e of Ets1 in  non-phosphorylated  and phosphorylated forms on its target gene enhan cers with or without its partner TFs, Runx1/CBF or Pax5, as an example. A structure-function relationship of a TF-DNA assembly and an effect by phosphorylation will be discussed.

Biography:

Fumio Hirata has completed his PhD from Hokkaido University and postdoctoral studies from State be University of New York, University of Texas, and Rutgers University. He was an associate professor in Kyoto University, and a professor in Institute for Molecular Science. He is currently a professor emeritus of the institute. He has published more than 200 papers in reputed journals and has served as an editorial board member of repute.

Abstract:

There are two    physicochemical    processes which are essential for living bod ies to maintain their life: “  self-organization  ” and “molecular recognition. ”   Protein folding    and formation of  cell-membrane  are typical exam ples of the former process, in which  biomolecules  have to overcome the entropy barrier to organize th  emselves into some characteristic structure. On the other hand, a molecular recognition process concerns whenever a biomolecule performs its function as a “ molecular-machine .” For examples, in order for the enzymatic reaction to occur, substrate molecules should be accommodated first by the protein in its reaction pocket to form so-called an enzyme–substrate (ES) complex. The two processes may not proceed spontaneously if biomolecules and ligand molecules are exisiting by themselves in “   vacuum   ,” because those are not in favor w ith respect to entropy. For instance, the    protein folding    is a process in which a protein folds into a   native conformation  , the state of least  entropy , from the random coil, the s   tate of largest entropy. Then, why do those processes occur spontaneously in our body? It is because there is always “aqueous solution” in the real environment of a living body.

Biography:

Toshiya Senda has completed his PhD from Nagaoka University of Technology (Niigata, Japan) in 1995. He was a research associate in Nagaoka University of Technology (1995-2001) and a senior researcher in Institute of Advanced Industrial Science and Technology (2001-2012). Now, he is the director/professor of Structural Biology Research Center of High Energy Accelerator Research Organization in JAPAN. He was awarded the CrSJ (Crystallographic society of Japan) award in 2014 (Structural biology studies of CagA from Helicobacter pylori and histone chaperon CIA/ASF1).

Abstract:

      GTP       is an energy molecule in the cell and required  in      protein synthesis     . R eduction of GTP concentration results in slow cell growth; inversely, rapidly growing cells have elevated GTP concentration. Because of its vital roles in cell growth, GTP concentration should be monitored and homeostatically regulated in the cell. However, a sensing mech anism of cellular GTP concentration remains elusive. Here, we show that a      lipid kinase     ,     PI5P4Kβ    , ser ves as a    GTP sensor    and GTP  concentration functions as a met  abolic cue via PI5P4Kβ. Our     proteomics     and    biochemical    study revealed that PI5P4Kβ binds GTP  and its enzyme activity is significantly higher with GTP than with ATP; PI5P4Kβ mainly utilizes GTP for phosphorylation of PI5P. Furthermore, the kinetic characters of PI5P4Kβ are suitable to detect the change of cellular GTP concentration. These biochemical characteristics suggested that PI5P4Kβ is a GTP sensor in the cell. However, since PI5P4K β can utilize not only GTP but also    ATP    for the enzyme reaction in the cell, a simple knock out/down experiment is insufficient to analyze the biological function of the GTP-sensing activity of PI5P4Kβ. We therefore took a structural reverse genetic approach. First, we determined crystal structures of  PI5P4Kβ-ATP/GTP complexes and used the   crystal structures   to prepare a PI5P4Kβ mutant that lacks    GTP-sensing activity   without changing  ATP-dependent activity . We then preformed b iological and metabolomic analyses with the PI5P4Kβ mutant, revealing that PI5P4Kβ serves as a GTP-sensor. The GTP-sensing activity of PI5P4Kβ is critical for  metabolic adaptation  and tumorigenesis.  

Biography:

Shigeyuki Yokoyama has obtained his PhD degree from The University of Tokyo in 1981. He was an Associate Professor (1986-1991) and Professor (1991-2012) at The University of Tokyo and presently an Emeritus Professor. He was also appointed as the Director of the    Cellular Signaling    Laboratory (1993-2004), the   Structural Molecular Biology   Laboratory (2004-2006), the  Protein Research  Group (1998-2008) and the Systems and Structural Biology   Center (2008-2013). He is a Distinguished Senior Scientist and directing the Structural Biology Laboratory at RIKEN. He has published more than 800 papers and has been serving a s Editorial Board Members of Nucleic Acids Research etc.

Abstract:

In the      protein synthesis      according to the genetic c ode, each of the twenty     amino acids     is provided as an     amino acyl-tRNA    . We have been studying the mechanisms of the specific aminoacyl-tRNA synt hesis by    crystallography    and    muta gen esis    . Aminoacyl-tRNA synthetases (aaRSs) specifically recognize their substrate amino acid and tRNA species. The 3’-CCA region, conserved in all tRNAs, is single stranded and flexible, of which the terminal adenosine is aminoacylated by the aminoacylation domain of aaRS. The major recognition element, e.g., the anticodon of a tRNA interacts with the site on the tRNA recognition domain, far from the    aminoacylation    catalytic site. Several aaRSs have the editing domain which hydrolyzes incorrectly formed aminoacyl-tRNAs and the terminal adenosine shuttles between the two catalytic sites. For amino acids such as   glutamine   and  selenocysteine , their precursor amino acids are attached to their tRNAs by others’ aaRSs and then enzymatically converted to the correct ones. We found that, in most of these processes of the tRNA and amino acid selection,  the flexibility of the  CCA  region of tRNA is important. On the structural basis, we have engineered the substrate specificities of tyrosiyl, phosphoseryl and pyrrolysyl-tRNA synthetase s for expansion of the genetic code to incorporate various unnatural amino acids site specifically into proteins in mammalian cells and in the  Escherichia coli  protein synthesis in vivo and in vitro. We used the expanded genetic code to produce proteins with epigenetic modifications, drug conjugation or  fluorescence probes  and to photocrosslink protein complexes including membrane protein complexes  in vitro and/or in vivo.

Biography:

Prof. Lei Zhang has completed his PhD at the age of 29 years from Xi’an Jiaotong University and postdoctoral studies from Lawrence Berkeley National Laboratory. He has published more than 30 papers in reputed journals and has been serving as an editorial board member of several journals. His researches focus on the determination of  protein structure  and functions by advanced electron microscpies. 

Abstract:

      DNA base-pairing       has been used for many years to direct the arrangeme nt of inorganic      nanocrystals      into small grou pings and arrays with tailored optical and electrical properties. The control of      DNA-mediated assem     bly depends crucially on a better understanding of the     three-dimensional (3D) structure     of the    DNA-nanocrystal    hy   bridized building blocks. Existing techniques do not allow for the structural determination of these flexible and heterogeneous samples. Here, we employed    electron tomography    and   negative-staining techniques   to investigat e the   3D structure   of  DNA-nanogold conjugates  that were self-as sembled from a mixture of an 84-base-pair double-stranded DNA (dsDNA) conjugated with two 5-nm nanogold particles for potential substrates in plasmon coupling experiments. We reconstructed 14 electron density maps at a resolution of ~2 nm from each individual dsDNA-nanogold particle using the  individual-particle electron tomography (IPET)  reconstruction method. Using these 3D density maps as a constraint, we projected a standard flexible DNA model onto the observed EM density maps and derived 14 conformations of dsDNA by molecular dynamics simulations. The variation of the conformations was consistent with the variation from liquid solution, but the IPET approach provides the most complete experimental determination of th e flexibility and fluctuation range of these directed nanocrystal assemblies to date. The general features revealed by these experiments can be expected to occur in a broad range of DNA-assembled nanostructures.

Biography:

Irena Roterman-Konieczna has completed his PhD at the age of 35 years from Nicolaus Copernicus Medical Academy Krakow, Poland - postdoctoral studies from Cornell University Ithaca NY USA – Harold Scheraga Group. She is the director of Department of Bioinformatics and Telemedicine at Jagiellonian University – Medical College. She has published more than 25 papers in reputed journals and has been serving as an editorial board member of repute. The field of interst is the protein structure and folding simulation as well as systems biology. General model for protein folding simulation is published by Woodhead Publishing (currently Elsevier). The model for proteome construction is presented in the book published by Springer. She is the Chief Editor of the journal Bio-Algorithms and Med-Systems (de Gruyter).

Abstract:

Fuzzy oil drop model - in contrast to discrete oil drop - introduces the gradual decrease of hydrophobicity from the center of protein molecule (the highest concentration of hydrophobicity) reaching zero level on the surface of protein body. The 3D Gauss function is assumed to represent the idealized hydrophobicity distribution in protein molecule. The real hydrophobicity distribution may differ since it is the result of residues distribution and the pair-wise hydrophobic interaction depending on the intrinsic hydrophobicity of each residue. The difference between idealized distribution and observed one can be measured quantitatively applying the Kullback-Leibler divergence entropy. It measures the distance between observed versus idealized hydrophobicity distribution in the protein as a whole and is able to identify the fragments of high and low similarity. In consequence the localization of high and low stability may be identified since hydrophobic core is assumed to be responsible for tertiary structure stabilization. The directing hydrophbic residues toward the center of protein is the effect of external force field (water environment) during folding process. The balance between external force field (water) and internal force field (non-bonding interaction between toms in protein) is assumed to be the mechanism of protein folding. Local disorder of hydrophobic core (versus idealized one) is quite often related to biological activity (ligand/substrate binding – local hydrophobicity deficiency, protein-protein complexation – local excess of hydrophobicity).

Biography:

Jyh-Yeuan (Eric) Lee completed his PhD from the University of California, Riverside, in   cryo-electron microscopy   (cryo-EM) and  drug resistance  ABC transporters. He has performed his postdoctoral st udies in large-scale membrane protein expression and puri  fication of human ABC transporters and  X-ray crystallography  of the sterol transporter ABCG5/ABCG8 from Texas Tech University Health Sceinces Cent er and the University of Texas Southwestern Medical Center at Dallas. He is currently a postdoctoral research scientist at the McDermott Center in Human Growth and Development at the University of Texas Southwestern Medical Center at Dallas.

Abstract:

   ATP binding cassette    (ABC) transporters play critical roles in maintaining stero l balance in higher   eukaryotes  , as exemplified by  the ABCG5/ABCG8 heterodimer (G5G8) that mediates   sterol   excretion in liver and intestines. Mutations disrupting G5G8 cause sitosterolemia, a disorder characterized by sterol accumulatio n and premature   atherosclerosis  .  Here we use crystallization in  lipid bilayers  to determine the  X-ray structu re  of huma n G5G8 i n a nucleotide-free state at 3.9 Å resolution, generating the first atomic model of an ABC st erol transporter. The structure reveals a new  transmembrane fold  that is present in a large and functionally diverse superfamily of ABC transporters. The transmembrane domains (TMD) are couple d to the nucleotide-binding sites (NBS) by networks of interactions that differ between the active and inactive  ATPases , reflecting the catalytic asymmetry of the transporter. We discovered the TMD  polar network  that may play a role in transmitting signals from ATPase catalysis in the NBS to sterol transport on the TMD. Based on  molecular dynamic simulation  and long-range coevolution analysis, as well as in vivo structure-based functional  mutagenesis , we  propose an updated model for sterol transport mechanism. The G5G8 structure provides a mechanistic framework for understanding sterol transport and the disruptive effects of mutations causing sitosterolemia, and will serve as a new structural template for  homology modelling  to a wide range of transport system that are regulated by ABCG transporters. 

Biography:

Maleki is an expert in food allergy research and has worked in this field for over 18 years. She recieved her Ph.D. from the Univ of Arkansas for Medical Sciences. She has served on the Scientific Advisory Committee to the FDA, the Nataional Peanut Board, and NIH’s Guidelines of the Diagnosis and Management of Food Allergy and as Expert Reviewer for the ($70 million) Canadian AllerGen project. She is a Fellow of the AAAAI and has edited a book and authored over 90 publications, and has had a significant number of invited national and international speaking and reviewer engagements.

Abstract:

Food allergy is on the rise and the prevalence of       peanut allergy       has more than tripled in the U.S. in the last 20 years. Mea nwhile, little is known about why certain proteins in foods are      allergenic      and others are not. Also, it is important to know what happens  to the      allergenicity      of food products after pr ocessing. To assess     processing-induced changes    , the major allergen s were purified from raw (R), and roasted (Ro) peanuts and the structure and     IgE     binding were compared with various    ELISA    and   immunoblots   with  allergic sera ,  circular dic     hroism , mass spectroscopy and e nzymatic digestion. While the structure of the allergens purified following roasting did not show significant changes compared to the raw, the IgE binding and SPT to the roasted samples peanuts were higher. Although allergen structure was found to be more important than linear sequence, it is likely that the roasting-induced chemical modifications are more important for enhanced IgE binding and  immunogenicity  than the structural changes to the major peanut allergens. Mass spectroscopic analysis was utilized to identify specific processing-induced chemical modif ications of the peanut allergens that may contribute to the observed effects. Thermally processed peanut proteins are chemically modified, covalently cross-linked, less soluble, more resistant to digestive enzymes, bind higher levels of IgE, and cause higher skin prick test (SPT) reactivity than raw peanut proteins. This knowledge may be useful in the development of more specific and improved diagnostic, therapeutic and detection tools and potentially lead to development of processes that can result in reduced allergenicity of a food.

Biography:

Tzu-Ching has completed his PhD from University of Nebraska Medical Center in 1999 and postdoctoral studies from Cold Spring Harbor Laboratory in 2003. Since then, he has been working at Academia Sinica, the premier government-funded institution in Taiwan. He is now a Research Fellow with professorship jointly appointed by National Taiwan University. He has published more than 40 papers in reputed journals and has been serving as an advisory board member of competitive journals.

Abstract:

Purpose:   Protein tyrosine phosphatase   N3 (PTPN3) and  mitogen-activated  protein kinase p38 y coordinae to promote Ras-induced oncegenesis. Structural analysis of PTPN3-p38y complex is a challenging task by using traditional  x-ray crystallography . Therefore, chemical cross-linking coupled with mass spectrometry (CX-MS) has been developed as an alternative method to solve this obstac le. Experimental description: Through covalent linkage of two spatially proximate residues within a single or between two  polypeptide  chains, the CX-MS approach provides structural insights into the flexible regions of proteins under any circumstances amenable to analysis. Convention ally, mapping of the protein-protein interaction sites relies on the coupling of lysine residues on the surface. Together with the distance restraint of the selected cross-linker, the segments of interaction surface can be determined. Results: Employing this method, we have mapped the contact interfaces of the PTPN3-p38y complex. Our finding of the solution structure indicated that the  catalytic domain  of PTPN3 interacts with the activation loop of phosphorylated  p38y, illustrating how PTPN3-mediated dephosphorylation of p38y takes place. The CX-MS approach further demonstrated that the PDZ domain of PTPN3 recruits the PDZ-binding motif of p38y, thus stabilizing the active-state complex of PTPN3-p38y. Moreover, the CX-MS analysis defined the autoinhibitory characteristic of the PDZ domain in PTPN3 in the absence of p38y. Conclusions: By combining other approaches such as small-angle x-ray scattering (SAXA) and crystal structure of PTPN3-phospho-p38y peptide, the CX-MS method generates considerable insights into the architecture of the phosphatase-kinase complex assembly.

Biography:

Mar. 2014, Ph. D, Ritsumeikan University Apr. 2014 - Mar. 2015, Postdoctoral Fellow, Ritsumeikan University Apr. 2015 - present, Assistant Professor, Ritsumeikan University During the Ph. D course, I worked with Prof. Takeshi Kikuchi and tried to apply a  course grained simulation  for analyzing folding mechanism of proteins. Since 2014, I have been working with Prof. Fumio Hirata and trying to apply 3D-RISM theory to the drug screening and to develop new methods based on 3D-RISM theory. 

Abstract:

  Cyclodextrin   is a cyclic molecule formed by s ix to eight  glucopyranose  units and i nclude a small  hydrophobic  molecule into a cavity. Cyclodextrins are widely used as a additive of foods and   drugs , because cyclodextrins have  low toxicity and  its physicochemical characteristics can be varied by the substitution  of hydroxyl group. Although   rational design   of cyclodextrin derivatives are desired to develop a new pharmaceutical products based on inclusion ability of the cycrodextrins, designing of a functional Cyclodextrin derivative have been practiced in an empirical way so far. In this study, we tried to dev elop a procedure based on  MM/3D-RISM  method to screen drug can didate cyclodextrin derivative and assessed the capability of our procedure on the test system, for instance, 2-Hydroxypropyl-β-Cyclodextrin and small molecules. To accomplish this objective, we tried to get appropriate ensemble of the complex state and isolated state, and, after that, predict the  binding free energy . Our procedure reproduce reasonable correlation between the experimental and calculated binding free energy and we conclude MM/3D-RISM method have an ability to distinguish the tightly bind compounds from the candidate molecules if we get an appropriate ensemble. Now we are applying this procedure to screen a functional cyclodextrin derivative.   We are also trying to improve our procedure.

Biography:

Ihosvany Camps holds a Bachelor degree in    Physics    from the Faculty of Physics, University of Havana (Cuba, 1995), Master degree in Physics from the Faculty of Physics, University of Havana (Cuba, 1996) and a PhD in Physics from the Institute of Physics, Federal Fluminense  University (Brazil, 2001). He has experience in   Condensed Matter Physics   and  Computational Modelling . Currently, has as m  ain research lines the study of electronic properties of  nanostructures  and the molecular modelling of organic and inorganic systems including the modelling of drugs ( rat ional drug design , fragment-based drug design, and de novo des ign), molecular docking, and studies on polymorphism of pharmaceutical solids. 

Abstract:

The   molecular docking   is the most used technique to  theoretically study  ligand-receptor interactions . The goal of the tal k is to present the evolution and fundamental aspects behind the molecular docking together with other  computational modelling  methods capable of giving better information about the ligand-protein interactions than simple docking and ligands with  better     binding energy    . In the first part of the talk, the types of docking (rigid-rigid, rigid-flexible and flexible-flexible) together with its different methods of im plementation (mainly    Monte Carlo   ,   Genetic Algorism   an  d Ant Colony Optimization) are analyzed side-by-side with other techniques like full complex optimization,   quantum mechanic   polarized ligand docking and fragment molecular orbital. The use and meaning of evaluation fun ctions (score functions) is discussed together with the softwares were they are implemented and they possible relationship with experimental variables. The second part of the talk is dedicated to different methods used to generate new ligands with better interaction (binding) energy to specific targets. Such techniques include   fragment-based drug design   and  de novo design . These techniques generate libraries with thousands of molecules that should be filtered using  criteria likes the Tanimoto coefficient of similarity toge ther with ADME (adsorption, distribution, metabolism, excretion) descriptors in order to obtain better drug candidates. We acknowledge financial support of FAPEMIG.  

Biography:

Sunyoung Kim has completed her PhD at the University of Michigan and the University of Padova, Italy and Post-doctoral studies at the University of Minnesota. She is a Member of the Louisiana Cancer Research Consortium and Founder of a spin-out company to personalize medicine with structural biomarkers. She leads a collaborative, multilab nanomotor research program. She has published more than 25 papers and serves as an Editorial Board Member of the Journal of Biological Chemistry. In addition, she has performed a variety of administrative roles at the departmental, institutional and state levels, as well as held elected positions for national scientific societies.

Abstract:

  ATP hydrolysis   requires that a proton f rom the water  nucleophile  must be abstr acted and transferred in order to create a hydroxide capable of attacking the substrate. In crystallographic capture of ATP hydrolysis, a two-water cluster is found in the active site of two diffe rent kinesin isoforms. These data suggest that a proton is shared between the  lytic water , positioned for gamma-phosphate attack, and the second water that serves as a  general base. The unusual short distance between the two  orthosteric water molecules , observed by crystallography, is confirmed by solvent kinetic isotope experiments. The positive kinetic isotope e ffect (KIE) confirms proton abstraction from water commits kinesin to catalysis and its    pH-dependence    verifies that switch   salt-bridge   residues direct  chemotransduction . Additionally, a classical descr iption for this proton transfer is  refuted by the  KIE  magnitude, tempe rature-independent Arrhenius pre-exponential fa ctor rati os, and activation energy differences. Taken together, we conclude that the first step in kinesin catalysis has a tunneling component, a quantum mechanical event by which a particle transfers through a reaction barrier. This first detection of tunneling in an ATPase is of consequence for two reasons. First,  proton tunneling  is likely widespread in biomolecules, rather than solely a characteristic of metalloenzymes. Second, energy barrier penetration by proton tunneling is an alternate explanation to classical transition-state stabilization theory for the fast reactivities of motor proteins. 

Biography:

Mohammed A. Mansour has completed his PhD at the age of 28 years from Nagoya University graduate school of      Medicine     , Japan in the field of     Biochemistry     of    cancer    and   cell signaling  . He has b een ass igned the position of lecturer of Biochemistry and Cancer Biology in  Tanta University faculty of Science, Egypt since October 2015. He has published up to 10 papers in highly reputed journals including oncotarget, FEBS J, Exp Cell Res and Tumor Biol. He is interested in understanding the molecular   pathogenesis   of human solid, hard -to-cure cancers. During his PhD project, he focused on the identification of alterations of  tumor  suppressor genes and oncogenes in colon cancer, followed by elucidation of their detailed biological and biochemical functions. This detailed work res ulted in 3 first author publications, and two co-author papers from collaborative studies.  His research thus focuses on taking clinically relevant targets, understanding their molecular function, and determining how to modulate them in the laboratory and in in vivo models.

Abstract:

  Histone deacetylases   (HDACs) pla y critical roles in  apoptosis  and contribute to t he proliferation of  cancer  cells. AR-42 is a novel Class I and II  HDAC inhibitor that shows cytotoxicity ag  ainst multiple human cancer cell lines. Recently, it was reported that AR-42 inhibits the activity of HDAC5 in  hepatocellular carcinoma  (HCC). However, the binding mode of action and molecular docking of this inhibitor is not reported. This study aims to evaluate the possible therapeutic efficacy of this inhibitor by testing its    dock ing    with different targets of HDACs. HDAC5 was found to be upregulated in HCC tissues compared to adjacent normal tissues, and this was correlated with reduced patient survival. Treatment with AR-42 decreased HCC cell growth and increased caspase-dependent apoptosis, and this was rescued by HDAC5 overexpression. We demonstrated that AR-42 can inhibit t he   deacetylation   ac tivity of HDAC5 by assessing the docking using   Swiss dock   software. SwissDock is a web service to predict the molecular interactions that may occur between a target protein and a small  molecule. Then, we visualized our docking results using  UCSF Chimera , a highly extensible, interactive molecular visualizatio n and analysis system. Chimera can read molecular structures and associated data in a large number of formats, display t he structures in a variety of representations, and generate highquality images and animations. Taken together, these results demonstrate for the first time that AR-42 targets HDAC5 and induces its inhibition in human   hepatocellular carcinoma  . AR-42 therefore shows potential as a new  drug candidate  for HCC therapy.  

Biography:

Shengli Zhang has completed his PhD at the age of 28 years from Lanzhou University. He is the director of Physics Department at Xi’an Jiaotong University, a top ten university in China. He has published more than 100 papers in reputed journals. His current researches focus on the determination of protein structures and functions by using molecular dynamics simulaitons and electron miscroscopy.

Abstract:

Cholesteryl ester transfer protein (CETP) decreases the level of atheroprotective high-density lipoprotein cholesterol (HDL-C) while elevating the level of atherogenic low-density lipoprotein cholesterol (LDL-C). Several CETP inhibitors have been evaluated in large-scale clinical trials for treating cardiovascular diseases (CVDs) within the last decade. Although the structure of mutated CETP has been revealed through electron microscopy (EM) and the X-ray crystallography, the structure of native CETP in physiological conditions and the detailed mechanism of how CE transfers from HDL to LDL at atomic resolution remains unclear. Here, we employed all-atom molecular dynamics simulations to study the native CETP structure and CE transfer mechanism. We propose the structure of native CETP, which is more difference in some aspects with the crystal structure. The simulation results showed that the tunnel is sufficiently large to mediate the transfer of a CE molecule through CETP with a predicted transfer rate comparable to physiological measurements. Analyses of the interactions and energies between the CE and CETP tunnel during transfer indicated several residues that might regulate CETP function during CE transfer. This study provides insight into the CE transfer mechanism for future development of CETP inhibitors.

Biography:

Seth Pincus received his M.D. from New York University. Following a    Pediatrics    residency at the University of Utah, he trained in the   Immunology   Branch of NCI. His first faculty appointment was at the University of Utah, f ollowed by stints at the NIAID Rocky Mountain Labs, at Montana State University where he Chaired the Department of   Microbiology  , and from there to his current position in the Departments of  Microbiology  and Pediatrics, at LSU School of Medicine, New Orleans. He has published over 100 peer-re veiwed papers, textbook chapters, and  invited articles. He has served on or chaired multiple NIH study sections and editorial boards.

Abstract:

The     HIV envelope protein     (Env) is the sole    virus    protein expressed on th e surface of    virions    and infected cells. Consequently,  the development of   anti-Env antibodies   (Abs) for therapeut ic applications is the subject of intense investigation. Anti-Env Abs can be used to neutralize cell-free virus and kill HIV-infected cells. After almost two decades with little progress, the introduction of recombinant DNA techniques has lead to a spate of highly effec tive and broadly reactive neutralizing Abs in the past ten years. HIV Env consists of two, non-covalently linked   glycoproteins  , the transmembrane  anchor gp41 , and the re ceptor-binding surface protein gp120. Neutralizing sites have been  ide  ntified on both gp120 and gp41. Working with anti-HIV immunotoxins, we have also mapped the targets of these cytoxic agents. Interestingly, there was little correlation between neutralization and cell killing activities. In an effort to increase  cytotoxic activity , we have made double variable domain (DVD) Abs that bind to structures on gp120 and gp41 that are the most effective targets for anti-HIV immunotoxins. Neither Ab, alone or in combination, neutralized HIV well. We were initially disappointed that the DVD’s offered no improve  ment in cytotoxicity over a mixture of the two Abs, but then bemused to discover that the DVDs were highly effective at neutralizing HIV infection. Activity was both broad and potent. I will discuss mechanisms whereby two weakly neutralizing Abs become more potent when combined into a single molecule.

Biography:

Elizabeth J Goldsmith received her Ph. D. in    Pysical Chemistry    at UCLA, worked with Dave Eisenberg, Max Perutz, and Robert Fletterick , and has been at UT Southwestern for 27 years, in the Dept of   Biophyscs  . Her publications concern  structural biology  and biochemistry    of protein kinases and other conformationally regulated proteins. 

Abstract:

    MAP     kinase modules, a cascade of three kinase enzymes,  a    MAP3K   ,    M AP2K     and   MAPK  , pr opagate diverse extracellular signals to downstream  effectors. The two double phosphorylation reactions catalyzed by the modules occur in a precise order that maximizes the number of reaction steps between signal and final activation. Activation is associated with the final Thr phosphorylation of the    MAPK   . The observed order of phosphorylation events suggests an excursion from the   Ser/Thr kinase activity   of the  MAP3K  into   Tyr kinase activity   of  the central   dual specificity   MAP2K and back to  Ser/Thr kinase activity .   The reaction order, and thus the tempo of  MAPK activation  changes in cancer mutants of the MAP2K.  

Biography:

Kikuchi has completed his PhD from Osaka City University and postdoctoral studies from Cornell University. Now, he is a professor of Department of  Bioinformatics  in Ritsumeikan University. Currently, he is interested in developing a new technique to predict 3D structural properties of a protein. 

Abstract:

Extracting  3D structural  information from a protein amino acid sequence is one of the  significant problems in molecular bioinformatics. In particular, informa tion on protein folding mechanism must be able to be decoded from the sequence of a protein if all information of the 3D structure is included in it. However, extracting the informa tion on protein folding from its sequence is very difficult and the only limited information can be obtained by standard bioinfomatics techniques such as multiple sequence alignment or even more sophisticated techniques. Extracting 3D structural information from a protein amino acid sequence is one of the significant problems in molecular bioinformatics. In particular, information on protein folding mechanism must be able to be decoded from the sequence of a protein if all information of the  3D structure  is included in it. However, extracting the information on  protein folding  from its sequence is very difficult and the only limited information can be obtained by standard  bioinfomatics  techniques such as multiple sequence alignment or even more sophisticated techniques. Our recent s tudies have demonstrated that the analyses of protein sequences based on the inter-residue average distance statistics with standard bioinformatics techniques and evolutionary analyses provide the information on protein folding nuclei. In the present study, we try to show the analyses of sequences of several protein families with specific 3D properties. We focus on the following properties; (1) similar partial 3D structures beyond a super family, (2) folding mechanisms of a 3D structure ubiquitously exists, (3) folding mechanisms of proteins with very highly homologous sequences but different 3D structures. In the present work, we take (1) E-to-H helix unit in globin family, (2) beta-Trefoil protein family, and (3) immunoglobulin binding proteins related proteins. We use the average distance map method and F value analysis which predict the compact regions and highly contact residues along the sequence of a given protein. Such information provides significant parts for protein folding along the sequence and also we compare such segments with the conservative hydrophobic residues and then we try to deduce the folding mechanism of a protein. In this way, we analyze the proteins in the categories above. We will present the possible folding mechanisms of these proteins based on the present analyses.

Biography:

Barry Hurlburt received his PhD in biochemistry from the University of Virginia and postdoctoral studies at Stanford University. In 1990 he became Assistant Professor at the University of Arkansas for Medical Sciences. He was promoted and tenured in 1997. In 2001 he moved to the USDA in New Orleans and is focused on peanut and tree nut allergies. He has published more than 50 papers in international journals and has recieved numerous extramural grants to support his research.

Abstract:

Rationale: Many    PR-10    proteins are allergens when inhaled or ingested. One proposed function of these pr oteins is delivering   bio-active   compounds to wounds  and/or the developing plant. We examined   ligand   binding to seven known PR-10 allergens. Ligand binding could well affect IgE binding. Methods: We generated pure, recombinant Ara h 8.01, Ara h 8.02, Cor a  1.02, Cor a 1.04, Que a 1.02, Que a 1.03 and Bet v 1.01 from peanut, hazelnut, white oak and birch respectively. Twenty three putative ligands were tested for binding using a fluorescence assay. Results: All of the proteins bound   apigenin  ,  daidzein , genistein, querceti n and resveratrol. Que a 1.03 bound the widest array of ligands including several fatty acids. Preliminary   structural studies   show changes in protein structure with ligand binding. Conclusions: Our results support the  theory that these PR-10 allergens’ function in vivo is as a delivery vehicle for   bio-active compounds  . Now that we have identified  biologically-relevant ligands  we will test the possibility that binding them to PR-10 proteins may influence allergenic potential.  

Biography:

Olga I Podgornaya is the Head of the Group of    Non-coding DNA    in the Institute of Cytology (INC RAS), professor at StPetersburg State University (SPbSU) and FEFU, Vladivostok. She was sent after graduation from SPbSU to the newly developed Far East RAS Center in Vladivostok. In 10 year s she became a staff member in INC RAS. There she turned to   gene engineering   and  DNA-binding proteins . When the iron curtain fell, Dr.Po  dgornaya worked in USA and United Kingdom, visited Canada, Germany, France. Grants from DOE USA (1993-2005) and Wellcome Trust (UK, 1992-1996) helped to survive during dark time for Russian science. She leads of the group of young researchers interested in eucaryotic  chromatin structure  and bioinformatics.  

Abstract:

     Transposable elements      (TE) constitute no less than 48% of  the human assembled     genome     and their positions  determined. Alu repeats (SINE) are located mostly to the     gene-rich regions    , while LINEs enrich facultative    heterochromatin   . TE positions in both mouse and human genomes are very s  imilar and cause the synteny. The comparison of the    transcription    intensity is done by   bioinformatics   tools in 32 human and mouse homologous tissues for ~12000 orthol ogous genes using   transcriptome   databases. Human shows a greater fraction of tissue-specific genes and a greater ratio of the total expression of tissue-specific genes to housekeeping genes in each tissue studied, which suggests a generall y higher level of evolutionary  cell differentiation  (specialization). It could be  supposed that TE positions fixed for ~65Myr (rodent  and primate separation) and SINEs and LINEs attraction to the potentially activeinactive parts of the genome may be responsible for the chromatin landscape shaping. The substitution of four mouse SINEs with Alu-repeat correlate with the enhanced transcription. The large regions of classic heterochromatin enriched with  tandem repeats  (TR) poorly investigated though it is the mostly variable part of the genome. The TR or TE substitution in the mammalian genome could go on through sperm-mediated gene transfer followed by homologous recombination. The scheme suggested may remove two of the contradictions of the Modern evolutionary synthetic theory: (1  ) gene mutations are not necessary for the progressive evolution but repetitive elements substitution; (2) ancient mammalians could have litters with the number of offspring altered and inbred mating was possible for the descendant altered. 

Biography:

Thomas (alias Tom) Scior received his PhD as a German Pharmacist at the University of Tubingen (1994), did his postdoctoral training at CNRS, Strasbourg, France and became a professor and pioneered the computational molecular laboratory at the     Pharmacy     Department at Free Public University Benemérita Universidad Autónoma de Puebla, Mexico, (www.buap.mx); E-mail: tscior@gmail.com, thomas.scior @correo.buap.mx (deprecated), language skills (7), research skills (   Computational Medicinal chemistry   ,   computational molecular simulations   by  docking , SAR, QSAR, VS,  MD, QM, bioinformati cs, very low homology protein structure modeling and analogy models). CV METRICS > 80 undergraduate pharmacy courses; > 10 graduate students; 48 scientific publication  s; > 1000 scientific citations w/o auto-citations; so far predictive models were confirmed experimentally in one trial – one hit cycles: side directed mutagenesis and antidote against tetanus or botulinum for all vertebrates under international patent application (EP15186045.9, 2015). Memberships: ACS, AFM, AOG (Apotheker Ohne Grenzen), DPhG.

Abstract:

The tenet of   homology modeling   relies on the tendency that the divergence of structure directly reflect s the variation in amino acid  sequence identities . Hence the higher the sequenc e identity (homology) between a target and a template structure the more reliable the pred icted target structure should be. This has been a useful paradigm to generate motifs, folds and domains of the proteins that have not been crystallized. Albeit, the study of dynamic  protein-protein interactions  (PPIs) that underpins natural phenomena – gating ions through cell membrane channels in the present case – requires the identification of functional interfaces. Here we demonstrate  how a hitherto unknown PPI can be studied even under extremely low homology conditions (far distant phylogenic relationships) by an analogy  protein modeling . This approach can overcome the problem of structural uncertain ties due to random sequence similarities and can lead to additional insights where three-dimensional data is missing. It proposes – what we call – a common epi-homology feature which is neither structure-bound nor related to protein activity. Eventually, it was the distinction between reversibility and irreversibility of P PIs. The finding enabled us to identify two adjacent amino acids at the postulated reversible interface between two subunits (alpha and beta-1) of the skeletal muscle voltage-gated Na+ channel (Nav1.4). A single side-directed mutagenesis study combined with subsequent electro  physiological characterizations provided the proof of concept and validation. The outcome was a double mutant (T109A, N110A, called TANA for short) that caused the highest loss-of-function effect in the literature (“Identification of Nav β1 residues involved in the modulation of the sodium channel Nav1.4”; Islas AA, Sánchez-Solano A, Scior T, Millan-PerezPeña L, Salinas-Stefanon EM; PLoS One. 8(12): e81995; 2013; “Predicting a double mutant in the twilight zone of low homology modeling for the skeletal muscle voltage-gated sodium channel subunit beta-1 (Nav1.4 β1)”; Scior T, Paiz-Candia B, Islas ÁA, Sánchez-Solano A, Millan-Perez Peña L, Mancilla-Simbro C, Salinas-Stefanon EM; Comput Struct Biotechnol J. 13: 229-240; 2015).

Biography:

Angel A. Islas received a BSc in Biology from the University of Puebla (BUAP), Mexico in 2007, a MRes in Medical and Molecular Biosciences with strand in Neurosciences awarded by the University of Newcastle Upon Tyne, United Kingdom in 2009 and a PhD in Physiological Sciences from BUAP in 2013. In 2014-2015, he carried out postdoctoral studies on Computational Biophysics at King’s College London in the United Kingdom. His postgraduate studies have been funded by the Mexican Council of Science and Technology (CONACyT). He was appointed research advisor in 2015 at the BUAP in Puebla, Mexico where he is currently collaborating with the department of Pharmacy and the laboratory of Biophysics at the Institute of Physiology. By combining electrophysiology with computational methods, his published work has contributed to the understanding of the modulation of the Na+ channel and the mechanism of action of K+ and Na+ channel inhibitors.

Abstract:

The tenet of homology modeling relies on the tendency that the divergence of structure directly reflects the variation in amino acid sequence identities. Hence the higher the sequence identity (homology) between a target and a template structure the more reliable the predicted target structure should be. This has been a useful paradigm to generate motifs, folds and domains of the proteins that have not been crystallized. Albeit, the study of dynamic protein-protein interactions (PPIs) that underpins natural phenomena – gating ions through cell membrane channels in the present case – requires the identification of functional interfaces. Here we demonstrate how a hitherto unknown PPI can be studied even under extremely low homology conditions (far distant phylogenic relationships) by an analogy protein modeling. This approach can overcome the problem of structural uncertainties due to random sequence similarities and can lead to additional insights where three-dimensional data is missing. It proposes – what we call – a common epi-homology feature which is neither structure-bound nor related to protein activity. Eventually, it was the distinction between reversibility and irreversibility of PPIs. The finding enabled us to identify two adjacent amino acids at the postulated reversible interface between two subunits (alpha and beta-1) of the skeletal muscle voltage-gated Na+ channel (Nav1.4). A single side-directed mutagenesis study combined with subsequent electrophysiological characterizations provided the proof of concept and validation. The outcome was a double mutant (T109A, N110A, called TANA for short) that caused the highest loss-of-function effect in the literature (“Identification of Nav β1 residues involved in the modulation of the sodium channel Nav1.4”; Islas AA, Sánchez-Solano A, Scior T, Millan-PerezPeña L, Salinas-Stefanon EM; PLoS One. 8(12): e81995; 2013; “Predicting a double mutant in the twilight zone of low homology modeling for the skeletal muscle voltage-gated sodium channel subunit beta-1 (Nav1.4 β1)”; Scior T, Paiz-Candia B, Islas ÁA, Sánchez-Solano A, Millan-Perez Peña L, Mancilla-Simbro C, Salinas-Stefanon EM; Comput Struct Biotechnol J. 13: 229-240; 2015).

Biography:

Eduardo M Salinas-Stefanon received his MD from the University of Puebla (BUAP), Mexico in 1980, he received his PhD in Physiological Sciences in 1996 awarded by University of Colima, Mexico. He carried out his postdoctoral studies under the supervision of Dr W. Giles at the University of Calgary, Canada in Pharmacology and Electrophysiology and with Dr P.H. Backx at the Centre for Cardiovascular Research, University of Toronto, Canada. He became a Full Professor in Physiology in 1984 at the Medical Faculty (BUAP), Mexico. He co-founded the Institute of Physiology BUAP in 1995, becoming head of the Department of Cardiac Biophysics. He became a member of the National Research System in 1993. His main research field includes cardiac physiology (pharmacology and biophysics) and protein-protein interactions of the subunits of the Na+ channel, including molecular dynamics of cardiac drugs. He is member of the Biophysical Society and SOBLA (Latin American Biophysical Society) since the 90s.

Abstract:

The tenet of homology modeling relies on the tendency that the divergence of structure directly reflects the variation in amino acid sequence identities. Hence the higher the sequence identity (homology) between a target and a template structure the more reliable the predicted target structure should be. This has been a useful paradigm to generate motifs, folds and domains of the proteins that have not been crystallized. Albeit, the study of dynamic protein-protein interactions (PPIs) that underpins natural phenomena – gating ions through cell membrane channels in the present case – requires the identification of functional interfaces. Here we demonstrate how a hitherto unknown PPI can be studied even under extremely low homology conditions (far distant phylogenic relationships) by an analogy protein modeling. This approach can overcome the problem of structural uncertainties due to random sequence similarities and can lead to additional insights where three-dimensional data is missing. It proposes – what we call – a common epi-homology feature which is neither structure-bound nor related to protein activity. Eventually, it was the distinction between reversibility and irreversibility of PPIs. The finding enabled us to identify two adjacent amino acids at the postulated reversible interface between two subunits (alpha and beta-1) of the skeletal muscle voltage-gated Na+ channel (Nav1.4). A single side-directed mutagenesis study combined with subsequent electrophysiological characterizations provided the proof of concept and validation. The outcome was a double mutant (T109A, N110A, called TANA for short) that caused the highest loss-of-function effect in the literature (“Identification of Nav β1 residues involved in the modulation of the sodium channel Nav1.4”; Islas AA, Sánchez-Solano A, Scior T, Millan-PerezPeña L, Salinas-Stefanon EM; PLoS One. 8(12): e81995; 2013; “Predicting a double mutant in the twilight zone of low homology modeling for the skeletal muscle voltage-gated sodium channel subunit beta-1 (Nav1.4 β1)”; Scior T, Paiz-Candia B, Islas ÁA, Sánchez-Solano A, Millan-Perez Peña L, Mancilla-Simbro C, Salinas-Stefanon EM; Comput Struct Biotechnol J. 13: 229-240; 2015).