Research at the NIBN
Engineering affinity scaffolds for cancer imaging and therapy Itay Cohen and Dr. Niv Papo
Research at the NIBN
Ben-Gurion UNIVERSITY OF THE NEGEV Contents
NIBN Overview 5
Cancer Research Group 6 Dr. Eyal Arbely 7 Dr. Roi Gazit 8 Dr. Dan Levy 9 Dr. Niv Papo 10 Prof. Angel Porgador 11 Dr. Barak Rotblat 12 Prof. Varda Shoshan-Barmatz 13
Autoimmune and Metabolic Diseases Group 14 Prof. Amir Aharoni 15 Prof. Angel Porgador 16 Prof. Assaf Rudich 17 Prof. Orian Shirihai 18
Neurodegenerative Diseases Group 19 Dr. Anat Ben-Zvi 20 Prof. Alon Monsonego 21 Prof. Israel Sekler 22 Prof. Varda Shoshan-Barmatz 23
Infectious Diseases Group 24 Dr. Natalie Elia 25 Dr. Eyal Gur 26 Dr. Tomer Hertz 27 Prof. Michael M. Meijler 28
Human Genetic Disorders Group 29 Prof. Ohad Birk 30 Prof. Ruti Parvari 31 Dr. Esti Yeger-Lotem 32
Applied Biotechnology Group 33 Prof. Ohad Medalia 34 Prof. Amir Sagi 35 Dr. Raz Zarivach 36 Dr. Stas Engel 37
Infra-Structure Supporting Group 38 Bioinformatics Core Facility 39 Crystallography Unit 40 Genetics Unit 41 Cytometry, Proteomic and Microscopy Unit 42
Dr. Roee Atlas 43 NIBN Overview
The National Institute for Biotechnology in the Negev Using these approaches, NIBN has already succeeded (NIBN) Ltd. is the first, self-organized, independent to generate commercial outlets in the fields of drug scientific research and development (R&D) body in targets for cancer therapy and infectious diseases, Israel. It has been established through a tri-lateral anti-inflammatory drugs as well as major advances agreement between the government of Israel, in aquaculture technology. These initial commercial Ben-Gurion University of the Negev (BGU) and successes have all been guided by NIBN’s highly philanthropist, Dr. Edgar de Picciotto. In November experienced management team. 2009, the NIBN became a company and implemented a detailed, 8 year applicable-orientated R&D program In December 2014, NIBN members located in several to specifically allow scientific excellence with a BGU buildings will relocate to The Edgar de Picciotto commercial outlet, funded by the NIBN. Family National Institute for Biotechnology in the Negev, a new 5400 sq m building. The building has The missions of the NIBN are to bridge the gap been carefully designed to provide state-of-the- between basic and applied research as well as art scientific infra-structure and to accelerate the forming a major channel between applied science institute’s development and growth, as well as to and industry, resulting in the establishment of a host start-up companies, with the aim of leveraging scientific infrastructure for biotechnology industry in additional commercial activities. the Negev. The strengths of the NIBN are derived from a combination of outstanding scientists, conducting NIBN’s ultimate goals are primarily driven by two multi-disciplinary research with both basic and issues. Firstly, the recruitment of outstanding biotechnological implications, close cooperation scientists as reflected in their academic excellence, with BGU departments, faculties, and research achievements and biotechnological implications of centers, all under the umbrella of a state-of-the-art, their research. Secondly, NIBN’s revered international scientific infrastructure. Such features coalesce to Scientific Advisory Board (SAB) composed of Nobel permit scientific excellence and create the required Prize laureate Prof. Sir Aaron Klug, FRS as well as leverage for the emergence of an internationally- distinguished scientists Prof. Raymond Dwek, FRS, acclaimed and successful biotechnology institute in Prof. Philip Needleman, Prof. Richard Ulevitch, Prof. the Negev and in Israel. Nathan Nelson and Prof. Hermona Soreq. The SAB sets a gold standard of scientific excellence, long- In order to fulfill this ultimate goal, the NIBN term scientific goals and major guidance concerning strongly encourages and funds “cutting-edge”, commercial efforts. creative research in the fields of Cancer Research, Infectious Diseases, Human Genetic Disorders, This booklet presents the current members of the Neurodegenerative Diseases, Autoimmune and NIBN, their selected research projects as well as the Metabolic Diseases and Applied Biotechnology. The Institute's core service facilities. It is our hope that resultant “value through innovation” fuels a plethora the information in this booklet will serve as a major of new biotechnology applications and commercial stepping stone in solidifying the NIBN as a World-Class opportunities. Such business development outlets Research Institute in biotechnology and fostering include: a) the creation of “spin-out” companies commercial opportunities with industry. David Ben- through funding bodies; b) joint collaborative efforts Gurion wrote that “the ultimate test for Israel in our with Big Pharma’; c) exclusive out-licensing of a generation is to prevail, through the power of science technology or product to biotechnology companies. and a pioneering spirit over the wide expanses of the south and the Negev”. At the NIBN, we are translating this vision into reality.
5 Cancer Research Group
An estimated 14 million cancer cases occurred world-wide during 2012 which is expected to increase to about 24 million by 2035. While having a wide diversity of cancer types and characterizations, all cancer types involve several hallmarks include sustaining proliferative signaling, evading growth suppressors and resisting cell death. NIBN’s cancer research group focuses on multi-disciplinary themes including understanding cancer biology through epigenetic modifications (eg. DNA and protein methylation changes), metabolic re-programing, the role of dysregulated cell growth, evading apoptotic cell death and mitochondrial involvement (via voltage-dependent anion channels), the development of appropriate immune-stimulatory strategies by targeting Proliferating Cell Nuclear Antigen (PCNA), the development of innovative animal models to simulate human leukemias and solid cancers, development of innovative diagnostic tools and the rationale design of small molecules, siRNA’s, peptides and protein-based anti-cancer therapeutics including bispecific molecules.
• Dr. Eyal Arbely • Dr. Roi Gazit • Dr. Dan Levy • Dr. Niv Papo • Prof. Angel Porgador • Dr. Barak Rotblat • Prof. Varda Shoshan-Barmatz
6 Expanding the repertoire of ribosomally-synthesized proteins Dr. Eyal Arbely Background The translation mechanism has been evolved over hundreds of millions of years to translate 64 triple-nucleotide codons, each encoding either one of the common 20 amino acids, or the termination of translation. Yet, this machinery is far from exploiting its full potential – proteins with expanded chemistry can be ribosomally synthesized by site-specific incorporation of non-proteinogenic amino acids, using the amber stop codon (UAG) and an orthogonal aminoacyl-tRNA synthetase/tRNA pair. The chemical and structural diversity of such proteins can be expanded even further by exploiting the unique chemistry of bacteriocins - ribosomally synthesized peptides, transformed into biologically active compounds by a series of unique structural and chemical post-translational modifications. Our laboratory is interested in developing and applying different methods for ribosomal expression of chemically and structurally-modified proteins for basic biochemical or biophysical research and as a platform for developing peptide-based, bioactive molecules in Ph.D.: The Hebrew University of general and particularly for novel antibiotics. Jerusalem, Israel Post-doctorate: MRC Center for Current research Protein Engineering and Laboratory of 1. Developing and applying methods for in-vivo Molecular Biology, Cambridge, UK incorporation of non-proteinogenic amino acids: Genetic code engineering allows the site-specific incorporation of Position: Senior Lecturer tailor-made amino acids into recombinant proteins. Equipped with a unique biophysical or chemical property, such amino Department of Chemistry acids may aid in studying the structure and cellular function Faculty of Natural Sciences of proteins. For example, the incorporation of modified amino acids carrying a naturally occurring post-translational E-mail: [email protected] modification such as acetylation or phosphorylation. Using genetic code engineering and directed evolution, we aim Selected publications to develop and apply novel methods for in vivo and in vitro studies of post-translationally modified proteins. Specifically, Arbely E. and Arkin I.T. (2004). Experimental Measurement we are interested in studying the effect of acetylation on the of The Strength of a Cα- H…O Bond in a Lipid Bilayer. J. Am. structure, subcellular localization and DNA binding affinity of Chem. Soc. 126:5362-5363. transcription factors such as NF-κB and p53. In recent years Arkin I.T., Xu H., Jensen M.O., Arbely E., Bennett E.R., Bowers K.J., a direct link was found between acetylation and cellular Chow E., Dror R.O., Eastwood M.P., Flitman-Tene R., Gregersen metabolism. In light of the high frequency of metabolic B.A., Klepeis J.L., Kolossváry I., Shan Y. and Shaw D.E. (2007). disorders associated with diseases ranging from cancer to Mechanism of Na+/H+ Antiporting. Science 317:799-803. obesity, we aim at understanding how cellular metabolism and acetylation level are correlated with acetylation and Arbely E., Rutherford J.T., Sharpe D.T., Ferguson N. and Fersht transcription activity of key transcription factors. A.R. (2009). Downhill versus Barrier-Limited Folding of BBL 1: 2. Bacteriocin engineering and DNA directed peptide synthesis: Energetic and Structural Perturbation Effects upon Protonation Bacteriocins are ribosomally-synthesized and heavily of a Histidine of Unusually Low pKa. J. Mol. Bio. 387:986-992. posttranslationally modified bioactive (usually antimicrobial) peptides. In general, the biosynthesis of bacteriocins Arbely E., Rutherford J.T., Neuweiler H., Sharpe D.T., Ferguson includes a series of chemical and structural modifications of N. and Fersht A.R. (2010). Carboxyl pKa-Values and Acid Denaturation of BBL. J. Mol. Bio. 403:313-327. the C-terminal part of a ribosomally synthesized precursor peptide. Typically, the enzymes that catalyze the biosynthetic Arbely E., Natan E., Brandt T., Allen M.D., Veprintsev D.B., transformations are directed by the N-terminal part of the Robinson C.V., Chin J. W., Joerger A.C. and Fersht A.R. (2011). precursor peptide, which is usually cleaved off, leaving behind Acetylation of Lysine 120 of p53 endows DNA binding the modified and bioactive version of the C-terminal peptide. specificity at effective physiological salt concentration. Proc. Consequently, the biosynthetic enzymes show a high Natl. Acad. Sci. USA. 108:8251-8256. level of promiscuity allowing the biosynthesis of different natural products using the same biosynthetic pathway. Our Arbely E., Torres-Kolbus J., Deiters, A., Chin J.W. (2012). laboratory is studying the biosynthetic pathways of different Photocontrol of tyrosine phosphorylation in mammalian cells bacteriocins aiming at exploiting the high level of promiscuity via genetic encoding of photocaged tyrosine. J. Am. Chem. for the production of DNA encoded libraries of cyclized and Soc. 134:11912-11915. chemically modified peptides.
7 Hematopoietic stem cells during health and disease Dr. Roi Gazit Background Hematopoietic Stem Cells (HSCs), the source of blood and immune cells throughout life, are already clinically-proven in saving thousands of livesin bone marrow transplants. Nevertheless, despite their critical importance in the field of adult stem cells, little is known about the molecular mechanisms underlying HSCs’ functions. Today, bone marrow transplants are still limited by the availability of stem cells and a much deeper understanding is required to generate and expand adequate cells for regenerative medicine. The connection between HSCs, the immune system and cancer, suggest that advanced research of the basic underlying mechanisms will provide new insight and suggest novel treatments by guiding our own adult stem cells.
Current research 1. Elucidating the role of Hlf: Hepatic Leukemia Factor (Hlf) was recently identified as a potent regulator of multipotency and self- renewal of mouse cells in-vitro. Although PAR-bZIP transcription Ph.D.: The Hebrew University of factor is widely recognized in Acute B-cell leukemia with (17:19) Jerusalem, Hadassah Ein-Kerem, Israel translocation of E2A-HLF fusion oncogene, the endogenous role of Hlf is still not yet understood. Characterization of molecular Post-doctorate: Immune Disease mechanism(s), identification of cooperating factors and Institute, Harvard Medical School, USA elucidation of how Hlf enhance self-renewal and multipotency, constitute a significant part of my laboratory’s research efforts. Position: Senior Lecturer The Shraga Segal Department of 2. Reprograming human blood cells back to HSC state: Under normal conditions, mature cells are generated from stem cells Microbiology, Immunology and and following differentiation, they cannot form new stem cells. Genetics However, recent methodological advances have provided ways to reprogram differentiated cells back into stem cells. After Faculty of Health Sciences discovering transcription factors that induce HSC-like activity in mice, we aim to translate such findings into humans. With E-mail: [email protected] the identified and predicted factors, our goal is to validate the ability to reprogram adult human blood cells into multipotent Selected publications and transplantable HSC-like cells. Such cells might eventually be Gazit R., Krizhanovsky V. and Ben-Arie N. (2004). Math1 translated for use into the clinical setting. controls cerebellar granule cell differentiation by regulating multiple components of the Notch signaling pathway. Development 131:903-913. 3. Modulation of HSCs activity within the immune system: The generation of blood cells during normal homeostasis perturbed Gazit R., Gruda R., Elboim M., Arnon T.I., Katz G., Achdout H., upon normal and pathological challenges. HSCs are not only the Hanna J., Qimron U., Landau G., Greenbaum E., Zakay-Rones fundamental source of the hematopoiesis, but also take an active Z., Porgador A. and Mandelboim O. (2006). Lethal influenza role in helping to mount immune responses. Identification of infection in the absence of the natural killer cell receptor HSC’s receptors that directly respond to immune-mediators is still gene Ncr1. Nat. Immunol. 7:517-523. in its infancy and functional studies to understand their responses Gazit R., Weissman I.L. and Rossi D.J. (2008). Hematopoietic to infection are urgently needed. Utilizing specific activators of stem cells and the aging hematopoietic system. Semin. HSC function will pave the way in guiding our own stem cells Hematol. 45:218-224. amd direct the source of all blood and immune cells.
Gur C., Porgador A., Elboim M., Gazit R., Mizrahi S., Stern- Ginossar N., Achdout H., Ghadially H., Dor Y., Nir T., Doviner V., 4. Generate novel Leukemia models: Leukemia is a severe, life- Hershkovitz O., Mendelson M., Naparstek Y. and Mandelboim threateningdisease affecting hundreds of thousands of children, O. (2011). The activating receptor NKp46 is essential for the adults and elderly populations. While significant advancement development of type 1 diabetes. Nat. Immunol. 11:121-128. of treatments have been made that offer complete, or at least partial cure, there are still many types of leukemia that remain Fu W., Ergun A., Lu T., Hill J.A., Haxhinasto S., Fassett M.S., Gazit R., Adoro S., Glimcher L., Chan .S, Kastner P., Rossi D., Collins refractory to therapies. The basic understanding of the human J.J., Mathis D. and Benoist C. (2012). A multiply redundant malignancy source and progression is largely dependent upon genetic switch 'locks in' the transcriptional signature of animal models, which also provide a platform to develop and regulatory T cells. Nat. Immunol. 13:972-980. assess novel therapeutics. Using an advanced lentiviral system for simultaneous overexpression of different transcriptional factors,we Gazit R, Garrison B.S., Rao T.N., Shay T., Costello J., Ericson aim to generate a series of leukemias that closely simulate human J., Kim F., Collins J.J., Regev A., Wagers A.J. and Rossi D.J. Acute Myeloid Leukemia (AML) or Chronic Lymphocytic Leukemia (CLL). (2013). Transcriptome analysis identifies novel regulators of hematopoietic stem and progenitor cells. Stem Cell Report.
8 Lysine methylation signaling and epigenetics Dr. Dan Levy Background The goal of our research is to understand how intercellular signaling networks regulate oncogenic and cell differentiation processes through epigenetic mechanisms and how dysregulation of these pathways leads to disease. Our work centers on the biology of protein lysine methylation, a post-translational modification that plays a key role in directing cellular and epigenetic programs. Lysine methylation catalyzed by protein lysine methyltransferases (PKMTs) is severely dysregulated in numerous diseases. Given the reversible nature of this chemical modification, targeting lysine methylation holds significant therapeutic promise. Despite the key role lysine methylation serves in modulating a wide range of signaling pathways, the catalytic activity and substrate specificity of the majority of PKMT enzymes remains unknown. Furthermore, Ph.D.: Weizmann Institute of Science, Israel relatively few protein lysine methylation substrates have Post-doctorate: Stanford University, USA been identified and functionally characterized. In our laboratory, we are utilizing a multi-disciplinary Position: Senior Lecturer biochemical, molecular, cellular, proteomic and genomic approaches to address fundamental questions at The Shraga Segal Department of Microbiology, the intersection between chromatin biology and cell Immunology and Genetics signaling. We aim to elucidate the molecular mechanisms, physiological and pathological functions of PKMTs and Faculty of Health Sciences lysine methylation biology. E-mail: [email protected] Current research 1. To identify new methylation events of histone and non-histone proteins and to define the molecular Selected publications mechanisms by which these methyl marks are Levy D., Adamovich Y., Reuven N. and Shaul Y. (2008). Yap1 generated and transduced and to unravel the phosphorylation by c-Abl is a critical step in selective activation biological functions of such methylation events. of pro-apoptotic genes in response to DNA damage. Molecular Cell 29(3):350-361. 2. To define the role of lysine methylation in the Levy D. and Gozani O. (2010). Decoding chromatin goes high tech. modulation of a variety of biological processes and Cell 142(6):844-846. disease models, with a focus on oncogenic and cell differentiation processes. Levy D., Kuo A.J., Chang Y., Schaefer U., Kitson U., Cheung P., Espejo A., Zee B.M., Liu C.L., Tangsombatvisit S., Tennen R.I., Kuo A.Y., Tanjing 3. To further develop peptide and protein microarrays S., Cheung C., Chua K.F., Utz P.J., Shi X., Prinjha R.K., Lee K., Garcia B.A., technologies focusing on protein lysine methylation. Bedford M.T., Tarakhovsky A., Cheng X. and Gozani O. (2011). SETD6 lysine methylation of RelA couples GLP activity at chromatin to tonic repression of NF-kB signaling. Nature Immunology 12(1):29-36. 4. To develop anti-cancer peptide inhibitors to target the enzymatic activity of protein-lysine Chang Y., Levy D., Horton J.R., Peng J., Zhang X., Gozani O. and methyltransferases. Cheng X. (2011). Structural basis of SETD6-mediated regulation of the NF-kB network via methyl-lysine signaling. Nucleic Acids Res.39(15): 6380-6389.
Levy D., Liu C.L., Yang Z., Newman A.M., Alizadeh A.A., Utz P.J.# and Gozani O.# (2011). A proteomic approach for the identification of novel lysine methyltransferase substrates. Epigenetics & Chromatin 4(1):19. #Correspondence authors.
Price J.V, Tangsombatvisit S., Xu G., Yu J., Levy D., Baechler E.C., Gozani O., Varma M., Utz P.J. and Liu C.L. “(2012). On silico” peptide microarrays for high resolution mapping of antibody epitopes and diverse protein-protein interactions. Nature Medicine 18:1434-
9 Utilizing combinatorial approach to target tumor neovasculature for Dr. Niv Papo cancer imaging and therapy
Background Evolution has generated the great diversity of proteins with all different functions required for the processes of life primarily by using the natural twenty amino acids. Despite all efforts to fully understand the nature of proteins, we are still far from being able to create completely new proteins with desired structure and function simply by rational design. By utilizing state-of-the-art yeast surface display (YSD) directed evolution technology, it is now possible to generate protein variants with new functions. Such desired functions could include improvements in protein physico-chemical properties (i.e. expression, solubility and stability) or modifications in substrate affinity and specificity. These optimized properties could be used for a variety of applications including medical diagnostics where issues such as high binding affinities, selectivity, and exceptional thermal, chemical, and protease stability play a major role. In vivo pharmacokinetic characteristics such as serum stability, tissue Ph.D.: Weizmann Institute of Science, penetration, blood clearance and target retention are critically Israel important for therapeutic applications. All these parameters can now be optimized by modern evolution strategies. Post-doctorate: Stanford University, CA, USA Current research Position: Senior Lecturer 1. Analysing protein interaction interfaces: We are developing a YSD Department of Biotechnology compatible method for protein scanning to provide a high-throughput alternative to traditional alanine scanning for mapping of the binding Engineering energy contributions of residues in protein-protein interfaces. To that end, we are using a binomial mutagenesis approach that utilizes yeast- Faculty of Engineering Sciences displayed protein scaffold libraries in which each site within the protein E-mail: [email protected] scaffold is allowed to vary as only the wild-type or a single substitution to alanine. Following YSD-based affinity selection to a specific target, a statistical analysis of the high affinity variant pool is then being used to Selected publications assess the binding contributions of individual side chains in the scaffold. Papo N. *, Braunstein A. *, Eshhar Z. and Shai Y. (2004). Suppression of Human Prostate Tumor Growth in Mice by a 2. Molecular imaging: Cancer treatment is currently shifting towards more Cytolytic D-, L-amino Acid Peptide: Membrane Lysis, Increased personalized approaches which require knowledge about differences in Necrosis, and Inhibition of Prostate-Specific Antigen Secretion. Cancer Res. 64:5779-5786 (*equal contribution). expression patterns of cancer markers. We are using the evolved affinity proteins as in vivo imaging agents to detect and identify these markers. Papo N. and Shai Y. (2005). A Molecular Mechanism for To evaluate these proteins as molecular imaging agents, we are site- Lipopolysaccharide Protection of Gram-Negative Bacteria specifically radiolabeling them and using positron emission tomography from Antimicrobial Peptides. J. Biol. Chem. 280:10378-10387. (PET) imaging to measure their tumor uptake and biodistribution in different cancer models. We are also investigating protein scaffolds as Papo N., Seger D., Makovitzki A., Kalchenko V., Eshhar Z., platforms for integrating cancer imaging and therapy by using a bi- or Degani H. and Shai Y. (2006). Inhibition of Tumor Growth and trifunctional scaffold coupled to effector compounds (such as small- Elimination of Multiple Metastases in Human Prostate and molecule toxins and radioactive isotopes) and radiolabeled probes. Breast Xenografts by Systemic Inoculation of a Host-Defense- Like Lytic Peptide. Cancer Res. 66:5371-5378. 3. Cancer therapy: The therapeutic effect of alternative scaffolds is Kipnis Y.*, Papo N.*, Haran G. and Horovitz A. (2007). Concerted obtained by blocking and antagonizing cancer-related molecular ATP-induced Allosteric Transitions in GroEL Facilitate Release targets. In addition, fusions with cytokines or toxins, which are very of Protein Substrate Domains in an All-or-None Manner. Proc. difficult to produce with antibodies, provide affinity proteins with Natl. Acad. Sci. USA 104:3119-3124 (*equal contribution). effector functions. Cytokine fusions activate the function of cytotoxic Papo N.*, Kipnis Y.*, Haran G. and Horovitz A. (2008). cells at a tumor site, whereas toxin fusions have a direct killing effect. Concerted release of substrate domains from GroEL by ATP Bivalency enhances the affinity of traditional antibodies to surface- is demonstrated with FRET. J. Mol. Biol. 380:717-725 (*equal bound antigens and their Fc region increases their in vivo half-life. Bi- or contribution). oligovalency is achieved in the alternative scaffolds, either by making an oligomer genetically as a head-to-tail fusion protein, by Fc-fusions Papo N., Silverman A.P., Lahti J.L. and Cochran J.R. (2011). or by fusing other oligomerization domains to the protein. Antagonistic VEGF Variants Engineered to Simultaneously Bind to and Inhibit VEGFR2 and αvβ3 Integrin. Proc. Natl. Acad. Sci. USA 108:14067-14072.
10 Recognition of cancer by natural killer cells Prof. Angel Porgador Background The successful eradication of an invading pathogen is dependent upon the coordinated actions of the innate and adaptive immune systems. Belonging to the former, natural killer (NK) cells are able to quickly destroy a wide range of hazardous pathogens such as viruses, tumors, bacteria and parasites. In recent years, it has become evident that NK cells rely on a set of activating receptors, including NKp44, NKp30, and NKp46 (collectively known as natural cytotoxicity receptors, NCRs) and NKG2D to kill their targets.
Current research
1. Characterization of innate immunity responses to tumors and viral infections: The ultimate Ph.D.: Weizmann Institute of Science, Israel goal of our research is to understand how NK cells kill their targets and to identify relevant Post-doctorate: Duke University pathogen-induced ligands that interact with and National Institutes of Health, USA NCRs. Accordingly, we are: a) investigating the in vivo function of NCRs using NKp46 knockout Position: Associate Professor mice challenged with various pathogens and determining mortality, NK distribution, activation The Shraga Segal Department of and trafficking; b) studying the mechanism by Microbiology, Immunology and Genetics which hemagglutinin (HA), an identified NCR ligand, is recognized by NKp46 and NKp44 and Faculty of Health Sciences whether the same mechanism applies to other E-mail: [email protected] viral proteins; c) using the data collected in (a) and (b) to identify novel NCR pathogens and Selected publications pathogen-induced ligands. The results of these studies will serve to develop novel therapeutic Mandelboim O., Lieberman N., Lev M., Paul L., Arnon T., Bushkin Y., Davis D.M., approaches based on NCR recognition of viruses Strominger J.L., Yewdell J.W. and Porgador A. (2001). Recognition of hemagglutinins and tumors. Diseases studied: cancer and on virus-infected cells by NKp46 activates lysis by human NK cells. Nature 409:1055- infectious diseases. 1060. Hershkovitz O., Rosental B., Rosenberg L.A., Navarro-Sanchez M.E., Jivov S., Zilka A., 2. Characterization of the peptidome in the Gershoni-Yahalom O., Brient-Litzler E., Bedouelle H., Ho J.W., Campbell K.S., Rager- blood of disease-bearing hosts: Work in our Zisman B., Despres P. and Porgador A. (2009). NKp44 receptor mediates interaction group seeks to identify peptide patterns among of West Nile and dengue envelope E glycoproteins with Natural Killer cells. J. Immunol. disease-associated blood markers (i.e. the 183(4):2610-2621. blood peptidome and glyco-peptidome) in the **Gur C., Porgador A., Elboim M., Gazit R., Mizrahi S., Stern-Ginossar N., Achdout normal and disease state, to serve as the basis H., Ghadially H., Dor Y., Nir T., Doviner V., Hershkovitz O., Mendelson M., Naparstek for diagnostic kits. At a later stage, these results Y. and Mandelboim O. (2010). The activating receptor NKp46 is essential could lead to the development of peptide-based for the development of type 1 diabetes. Nature Immunol. 11(2):121-128. immunomodulators, inhibitors and other drugs. **AP and OM are equal contributors and co-corresponding authors. Rosental B., Brusilovsky M., Hadad U., Oz D., Appel M.Y., Afergan F., Yossef R., Rosenberg 3. Reactomics approach (with Prof. Raz Jelinek): A L.A., Aharoni A., Cerwenka A., Campbell K.S., Braiman A., and Porgador A. (2011). novel disease diagnostic concept based upon the Proliferating Cell Nuclear Antigen is a novel inhibitory ligand for the natural cytotoxicity screening of interactions of body liquids (blood receptor NKp44. J. Immunol. 187(11):5693-5702. serum/plasma, urine, saliva, tears, amniotic fluid) Jaron-Mendelson M., Yossef R., Appel M.Y., Zilka A., Hadad U., Afergan F., Rosental B., with an array matrix of chromatic biomimetic Engel S., Nedvetzki S., Braiman A. and Porgador A. (2012). Dimerization of NKp46 vesicle detectors comprising lipid entities and Receptor Is Essential for NKp46-Mediated Lysis: Characterization of the Dimerization chromatic reporters. Site by Epitope Mapping. J. Immunol. 188(12):6165-6174. Brusilovsky M., Cordoba M., Rosental B., Hershkovitz O., Andrake M.D., Pecherskaya A., Einarson M.B., Zhou Y., Braiman A., Campbell K.S. and Porgador A. (2013). Genome- Wide siRNA Screen Reveals a New Cellular Partner of NK Cell Receptor KIR2DL4: Heparan Sulfate Directly Modulates KIR2DL4-Mediated Responses. J. Immunol. 191(10):5256-5267.
11 Functions of long noncoding RNA Dr. Barak Rotblat and translation inhibitors in cancer
Background The ‘central dogma’ in biology argues that the genetic information stored in DNA encodes for mRNA, which is translated into proteins that execute functions leading to the manifestation of a trait. Current understanding of biology at the cellular and molecular levels suggests that all stages of the process leading from the expression of protein-coding genes to the manifestation of a trait are highly regulated. In particular, there is high selectivity in terms of which mRNA is translated into a protein in a given cell. However, the vast majority of the genome is transcribed into RNA yet does not encode for proteins. One sub-set of these noncoding RNAs are longer then 200 base pairs and are hence termed long noncoding RNAs (lncRNAs). Although it was previously held that lncRNAs correspond to transcriptional noise, recent studies have Ph.D.: Tel-Aviv University, Israel demonstrated that they function in all aspects of cell biology. Specifically, some lncRNAs regulate the interactions of proteins Post-doctorate: University of British with DNA, RNA or other proteins. Furthermore, lncRNAs interact Colombia, Canada and MRC with polysomes, raising the possibility that they may be involved Toxicology Unit, UK in regulation of proteins synthesis. Because deregulation of cellular signalling pathways, in particular those that regulate translation, Position: Senior Lecturer is a hallmark of many human diseases, such as cancer, we aim to elucidate the biological functions of lncRNAs and proteins that Department of Life Sciences regulate translation in cellular and disease contexts. Faculty of Natural Sciences E-mail: [email protected] Current research 1. lncRNA that support cancer stem cells: The cancer stem cell (CSC) hypothesis argues that in a fraction of the tumor cells Selected publications CSCs are endowed with tumor-initiating capacity, promote tumor growth and are highly resistant to chemotherapy. The Rotblat B., Leprivier G. and Sorensen P.H. (2011). A possible implications of this model are that such cells represent a cell role for long non-coding RNA in modulating signaling population that is responsible for the two most deadly traits pathways. Med Hypotheses 77(6):962-965. of cancer, namely post-treatment relapse and metastasis. Therefore, it is likely that key molecular components of CSCs Rotblat B., Grunewald T.G., Leprivier G., Melino G. and represent attractive drug targets. Accordingly, we study Knight R.A. (2013). Anti-oxidative stress response genes: lncRNAs that are highly expressed in aggressive tumors and bioinformatic analysis of their expression and relevance in in CSCs so as to identify new molecules that promote cancer, multiple cancers. Oncotarget 4(12):2577-2590. which, in turn, correspond to attractive new drug targets. Leprivier G., Remke M., Rotblat B., Dubuc A., Mateo A.R., Kool M., Agnihotri S., El-Naggar A., Yu B., Prakash 2. Negative regulators of translation that promote cancer: To Somasekharan S., Faubert B., Bridon G., Tognon C.E., Mathers support proliferation, cancer cells exhibit increased rates of J., Thomas R., Li A., Barokas A., Kwok B., Bowden M., Smith protein synthesis and hyper-activation of translation-promoting S., Wu X., Korshunov A., Hielscher T., Northcott P.A., Galpin signalling pathways. As such, inhibition of translation is an J.D., Ahern C.A., Wang Y., McCabe M.G., Collins V.P., Jones important aspect of the cellular response to a wide range of R.G., Pollak M., Delattre O., Gleave M.E., Jan E., Pfister S.M., challenges or stresses. Indeed, we have recently demonstrated Proud C.G., Derry W.B., Taylor M.D. and Sorensen P.H. (2013). that cancer cells depend on a negative regulator of translation The eEF2 Kinase Confers Resistance to Nutrient Deprivation by Blocking Translation Elongation. Cell 153(5):1064-1079. to survive the stressful tumor environment. The goal of our studies is to identify coding or noncoding sequences that Rotblat B., Southwell A.L., Ehrnhoefer D.E., Skotte H.N., function as translation inhibitors and which cancer cells exploit Metzler M., Franciosi S., Leprivier G., Somasekharan to enable them to survive stress. Based on our results, we will S.P., Barokas A., Deng Y., Tang T., Mathers J., Cetinbas N., pursue the development of compounds targeting these Daugaard M., Kwok B., Li L., Carnie C.J., Fink D., Nitsch R., translation inhibitors and test them as potential candidates Galpin D.J., Ahern C.A., Melino G., Penninger J.M., Hayden for the treatment of cancer. M.R. and Sorensen P.H. (2014). HACE1 reduces oxidative stress and mutant Huntingtin toxicity by promoting the NRF2 response. Proc. Natl. Acad. Sci. USA 111(8)3032-3037.
12 Novel approaches to cancer therapy Prof. Varda Shoshan-Barmatz Background NIBN Director Many cancer cells undergo re-programming of their metabolism and develop cell survival strategies involving anti-apoptosis defense mechanisms, a hallmark of the majority of cancer types. Found at the outer mitochondrial membrane, the voltage-dependent anion channel (VDAC) assumes a crucial position in the cell serving as a gatekeeper, controlling the metabolic and energy cross-talk between mitochondria and the rest of the cell, and is involved in apoptosis. VDAC is the proposed target for the pro- and anti-apoptotic Bcl2-family of proteins, as well as functioning in the release of apoptotic proteins located in the inter-membranal space. Thus VDAC is considered a key player in cell metabolism and regulation of mitochondria-mediated apoptosis. As such, VDAC1 represents an excellent target to approach in order to impair the re-programmed metabolism of cancer cells and their capability to evade apoptosis.
Current research Ph.D.: Weizmann Institute of Science, Israel Given that mitochondria play a central role in the execution of Post-doctorate: University of Wisconsin- apoptosis and that VDAC1 is the gatekeeper of mitochondrial function and dysfunction, we have generated specific, potent and Madison, USA and University of Toronto, highly effective VDAC1-based cancer therapies that facilitate the Canada death of cancer cells or arrest of cell growth. Four novel strategies towards developing cancer therapies involving VDAC1 are currently Position: Professor, NIBN Director being pursued in my laboratory: Department of Life Sciences 1. Targeting anti-apoptotic proteins using VDAC1-based peptides to minimize the self-defense mechanisms of cancer cells, known Faculty of Natural Sciences to overexpress anti-apoptotic proteins. We have identified VDAC1 sequences involved in interactions between VDAC1 and E-mail: [email protected] anti-apoptotic proteins and demonstrated that VDAC1-based Selected publications peptides prevent the anti-apoptotic proteins’ activity, disturb cell energy homeostasis and lead to apoptotic cell death specifically. Abu-Hamad S., Sivan S. and Shoshan-Barmatz V. (2006). In-vitro, such peptides were shown to be active in a variety of The Voltage-Dependent Anion Channel Down- and over- cancer cell lines regardless of the carried mutations and inhibited expression control Cell's life and death. Proc. Nat. Ac. Sci. USA 386:73-83. glioblastoma tumor growth in-vivo. 2. Arresting cell proliferation by down-regulation of VDAC1 Shoshan-Barmatz V., de Pinto P., Markus Zweckstetter M., Raviv, Z., Keinan N. and Arbel N. (2010). VDAC, a multi- expression – Our results have shown that suppression of VDAC1 functional mitochondrial protein regulating both cell life expression by a single siRNA arrested cell proliferation due to and death. Molecular Aspects of Medicine. 31:227-286 interrupted energy and metabolite supply to the high energy- demanding cancer cells. We have demonstrated proof-of-concept Arbel N., Ben-Hail D. and Shoshan-Barmatz V. (2012). in animal models with cervical and lung cancers. Mediation of the anti-apoptotic activity of BCL-XL upon interaction with VDAC1. J. Biol. Chem. 287(27):23152-23161. 3. We have demonstrated that VDAC1 oligomerization is a general mechanism that is common to many apoptosis inducers, acting Shoshan-Barmatz V., Mizrachi D. and Keinan K. (2013). via different pathways. We have developed a high throughput Oligomerization of the mitochondrial protein VDAC1: From structure to function and cancer therapy. Prog. Mol. Biol. screening (HTS) assay for modulators of VDAC1 oligomerization Transl. Sci. 117:303-334. and have identified pro- and anti-apoptotic drugs acting via modulation of VDAC1 oligomerization. The aim is to develop Prezma T., Shteinfer A., Admoni L., Raviv Z., Sela I., Levi I. and molecules that, a) promote VDAC1 oligomerization andthe Shoshan-Barmatz V. (2013). VDAC1-based peptides: Novel subsequent apoptotic cell death, thereby serving as anti-cancer pro-apoptotic agents and potential therapeutics for B cell therapeutics, and b) inhibitVDAC1 oligomerization thereby chronic lymphocytic leukemia. Cell Death and Disease, e809. doi: 10.1038/cddis.2013.316. PMID:24052077. preventing cell death and allow the rescue of nerve cells in neurodegenerative diseases. Arif, T, Vasilkovsky, L., Refaeli, Y. Konson, A. and Shoshan-Barmatz, V (2014) Silencing VDAC1 expression 4. We have demonstrated an increase in VDAC1 expression levels by siRNA inhibits cancer cell proliferation and tumor following apoptosis induction by various agents, as well as growth in vivo, Molecular Therapy–Nucleic Acids 2014 the correlation between drug efficacy and VDAC1 expression Apr 29;3:e159. doi: 10.1038/mtna.2014.9. level. We propose a new concept according to which several apoptosis-inducing agents and conditions act by up-regulating Weisthal, S., Keinan, N., Ben-Hail, D., Arif, T. and VDAC1 expression in a Ca2+-dependent manner, leading to Shoshan-Barmatz, V. (2014) Ca2+-mediated regulation of VDAC1 expression levels is associated with cell death VDAC1 assembly into high oligomeric structures and thereby to induction Biochim. Biophys. Acta, 1843(10):2270-81 cell death. Further investigations y of this novel mechanism may provide a unique platform for developing a new class of anti- cancer drugs. 13 Autoimmune and Metabolic Diseases Group
Understanding the mechanisms involved in the maintenance of homeostatic immune responses as well as those that have gone awry in disease states, poses a significant challenge. At the NIBN, a robust platform technology called “direct evolution” is being used to generate superior, soluble anti-inflammatory proteins to treat autoimmune diseases such as psoriasis, rheumatoid arthritis (RA), inflammatory bowel disease (IBD) and asthma. Additionally, this group’s efforts include understanding how chronic stress is associated with increased susceptibility to autoimmune disease. Other activities are focused on understanding metabolic dysfunctions related to the role of adipose tissue dysfunction in obesity and associated co-morbidities as well as the causative link suppression of mitochondrial turnover, β-cell dysfunction, apoptosis and the development of type 2 diabetes.
• Prof. Amir Aharoni • Prof. Angel Porgador • Prof. Assaf Rudich • Prof. Orian Shirihai
14 Protein engineering using directed evolution Prof. Amir Aharoni Background Protein engineering has been used extensively in the past twenty years for the study of enzyme structure-function and evolution. Recently, protein engineering using directed evolution has proven to be highly successful, yielding proteins demonstrating increased stability under extreme conditions, increased solubility for expression in heterologous systems, and proteins with novel reaction and substrate specificities. Directed evolution implements an iterative Darwinian optimization process, whereby the fittest variants are selected from a collection of random mutations. Improved variants are identified and isolated by screening or selection for the property of interest. This approach is particularly advantageous in cases in which no prior knowledge of a protein’s mechanism and structure is available.
Current research Ph.D.: Weizmann Institute of Science, 1. Cytosolic sulfotransferases (SULTs) are liver enzymes that detoxify a variety of substrates by transferring sulfate to a variety of acceptor Israel molecules bearing a hydroxyl or an amine group. Sulfation renders Post-doctorate: Weizmann Institute of the product more readily excretable or less pharmacologically Science, Israel and University of British active. The diversity of acceptor compounds for cytosolic SULTs is remarkable, ranging in size, shape and flexibility, from ethanol Columbia, Canada to steroids. These enzymes play important roles in a variety of Position: Associate Professor biological functions, such as modulating the levels of hormones and neurotransmitters. Using directed evolution, we aim to Department of Life Sciences improve the detoxification properties of SULTs by implementing a new high throughput screening methodology that allows for the Faculty of Natural Sciences screening of millions of mutant enzymes in parallel for increases E-mail: [email protected] in catalytic efficiency. Improved mutant enzymes may find ex-vivo biotechnological applications, such as in bioremediation.
2. DNA replication and gene transcription are two ubiquitous Selected publications biological processes in all living organisms. We are currently Fridman Y., Palgi N., Dovrat D., Ben-Aroya S., Hieter P., developing new tools to study these processes using directed Aharoni A. (2010). Subtle Alterations in PCNA-partner evolution methodology. We are focusing on the proliferating Interactions Severely Impair DNA Replication and Repair. cellular nuclear antigen (PCNA) which is a hub protein PLoS Biology. 8(10):e100. orchestrating the DNA replication process in eukaryotic cells. Sadeh A., Baran D., Volokh M., Aharoni A. (2012). We aim to elucidate the importance of PCNA-protein interaction Conserved Motifs in the Msn2-Activating Domain are for DNA replication, repair and cell viability. Another project is Important for Msn2-mediated Yeast Stress Response. J. focused on the study of stress-related transcription factor (TF) in Cell. Sci. 125:3333-3342. yeast. We aim to generate TF mutants that increase the ability of yeast to survive under different stress conditions in order to study Zamir L., Zaretsky M., Fridman Y., Ner-Gaon H., Rubin E., the molecular basis for higher tolerance to different conditions Aharoni A. (2012). Tight co-evolution of PCNA-partner and the linkage between the yeast responses to different stress interaction networks in fungi leads to inter-species conditions. network incompatibility. Proc. Natl. Acad. Sci. USA 109(7):E406-414. 3. Engineering of therapeutic proteins for increased stability and Amar D., Berger I., Amara N., Tafa G., Meijler M., Aharoni A. binding affinity – We are focusing in generating improved soluble (2012). The Transition of Human Estrogen Sulfotransferase receptors for inhibiting pro-inflammatory cytokines involved in from Generalist to Specialist using Directed Enzyme a variety of different autoimmune diseases. Our initial efforts Evolution. J. Mol. Biol. 416(1):21-32. were focused on the development of a unique, soluble IL-17 Zaretsky M., Etzyoni R., Kaye K., Sklair-Tavron L., Aharoni A. receptor therapeutic with improved properties, that was shown (2013). Directed Evolution of a Soluble Human IL-17A e to inhibit psoriasis plaque formation in a mouse model. This Receptor for the Inhibition of Psoriasis Plaque in Mice protein constitutes an important drug candidate for the treatment Model. Chemistry and Biology 20:202-211. of psoriasis in humans.
15 Prof. Angel Porgador Innate immunity and diabetes
Background Natural killer (NK) cells belong to the innate lymphoid cells. Their cytotoxic activity is regulated through the delicate balance between activating and inhibitory signals. NKp46 is a member of the primary activating receptors of NK cells. We previously reported that islets beta cells express ligand(s) to the human NK activation receptor NKp46 and that NKp46 is involved in the development of type 1 diabetes (T1D) autoimmune disease. Subsequently, we hypothesized that blocking of the NKp46 receptor could prevent or hinder T1D development.
Current research Characterization of innate immunity responses to type Ph.D.: Weizmann Institute of Science, Israel 1diabetes. Our research works towards the development of new Post-doctorate: Duke University tools and the identification of novel mechanisms aimed at and National Institutes of Health, USA antagonizing the activity of NKp46 during type I diabetes Position: Associate Professor development. Furthermore, we aim to identify those NKp46 ligand(s) expressed by murine and human islet beta cells. The Shraga Segal Department of Specifically, our goals are to develop blocking anti-NKp46 Microbiology, Immunology and Genetics antibodies or NKp46-derived peptides that prevent NKp46 activity, as well as to investigate mechanisms controlling Faculty of Health Sciences expression of the NKp46 ligand in islet beta cells followed E-mail: [email protected] by ligand characterization.
To date, we have developed monoclonal antibodies Selected publications (mAbs) against murine NKp46. One mAb, termed NCR1.15, Gur C., Porgador A., Elboim M., Gazit R., Mizrahi S., recognizes Ncr1, the mouse homologue of NKp46, and was Stern-Ginossar N., Achdout H., Ghadially H., Dor Y., Nir able to down-regulate the surface expression of NKp46 T., Doviner V., Hershkovitz O., Mendelson M., Naparstek on primary murine NK cells following in vivo antibody Y. and Mandelboim O. (2010). The activating receptor NKp46 is essential for the development of type 1diabetes. injection. Additionally, NCR1.15 treatment led to the Nature Immunol. 11(2):121-128. ** AP and OM are equal down-regulation of cytotoxic activity mediated by NKp46 contributors. but not by other NK receptors. To test the effects of the antibodies on T1D, we followed development of the disease in two models, NOD mice and in mice subjected Jaron-Mendelson M., Yossef R., Appel M.Y., Zilka A., Hadad to low-dose streptozotocin treatment. The results revealed U., Afergan F., Rosental B., Engel S., Nedvetzki S., Braiman A. and Porgador A. (2012). Dimerization of NKp46 Receptor a significantly lower incidence of diabetes in the NCR1.15- Is Essential for NKp46-Mediated Lysis : Characterization of treated group, as comparing to control groups. We are the Dimerization Site by Epitope Mapping. J. Immunol. currently developing humanized monoclonal antibodies 188(12):6165-6174. to human NKp46 that are cross-reactive to murine NKp46. Such antibodies will allow us to directly test the human- suggested prototype in mouse models for suppression or Yossef R., Gur C., Shemesh A., Hadad U., Nedvetzki S., Miletić A., Cerwenka A., Jonjic S., Mandelboim O. and prevention of type 1 diabetes development. Porgador A. Targeting Natural Killer Cell Reactivity by Employing Antibody to NKp46: Implications for Type 1 We are also developing a method to insert human NKp46 Diabetes. Submitted. into hematopoietic stem cells of NCR1-knockout mice with the expectation that functional human NKp46 will be expressed in the developing murine NK cells. Such cells would allow us to test the function of human NKp46- derived peptides in the in vivo blocking of human NKp46 function and allow for better characterization of the effects of this peptide drug-mediated block on the development of induced T1D. 16 The role of adipose tissue Prof. Assaf Rudich dysfunction in obesity and its co-morbidities
Background Over the past 15 years obesity has become the most prevalent preventable risk factor for morbidity and mortality. Although the reasons are complex and still incompletely understood, it is clear that adipose tissue in obesity becomes dysfunctional, and is a major pathogenic contributor to the morbidity that accompanies obesity.
Current research
1. Mechanisms for human adipose tissue dysfunction in obesity: Analysis of human adipose tissues is aimed at unraveling how obesity leads to adipose tissue M.D.: Ben-Gurion University, Israel dysfunction. Utilizing expression, molecular, imaging, functional, and ex-vivo studies of human samples, we are Ph.D.: Ben-Gurion University, Israel identifying the "human adipose tissue stress response" Post-doctorate: Hospital for Sick and unravel its functional significance. The causal Children, Toronto, Canada role of specific pathways and molecular mechanisms identified (such as the role of autophagy, inflammation, Position: Professor and a specific MAP kinase signaling cascade) are further challenged using cellular, ex-vivo and in vivo experimental Department of Clinical Biochemistry models. Functionally we mainly focus on two pathogenic and Pharmacology axes: an adipocyte-macrophage axis that operates within the adipose tissue, and a fat-liver axis that mediates the Faculty of Health Sciences pathogenic role of visceral adiposity. E-mail: [email protected] 2. Investigating potential life-style interventions to alleviate obesity-related morbidities in large Selected publications populations: As part of a larger team of investigators (headed by Prof. Iris Shai), intervention trials are being Nov O., Shapiro H., Ovadia H., Tarnovscki T., Dvir I., Shemesh E., Kovsan J., Shelef I., Carmi Y., Voronov E., Apte R.N., Lewis E., Haim Y., Konrad conducted to identify life-style approaches to limit the D., Bashan N. and Rudich A. (2013). Interleukin-1β regulates fat-liver health burden of the obesity epidemic, and to understand crosstalk in obesity by auto-paracrine modulation of adipose tissue the mechanisms by which such protective effects occur. inflammation and expandability. PloSOne 8(1):e53626. 3. Circulating monocytes as mediators of the environmental Shapiro H., Pecht T., Shaco-Levy R., Harman-Boehm I., Kirshtein impact on adipose tissue in obesity: Circulating B., Kuperman Y., Chen A., Blüher M., Shai I. and Rudich A. (2013). monocytes are the likely precursors of adipose tissue Adipose tissue foam cells are present in human obesity. J. Clin. macrophages that accumulate in obesity. We hypothesize Endocrinol. Metab. 98:1173-1181. that monocytes may constitute a marker of adipose tissue inflammation (and thus, for morbidity-prone Hadad N., Elgazar-Carmon V., Burgazliev O., Solomonov Y., Wueest S., Item F., Konrad D., Rudich A. and Levy R. (2013). Induction of obesity), and are also active players in the pathogenesis cytosolic phospholipase A2α is required for adipose neutrophil of obesity-associated morbidity. Specifically, we entertain infiltration and hepatic insulin resistance early in the course of high the possibility that monocytes may communicate a fat feeding. Diabetes 62:3053-3063. contributing effect of air-borne environmental particles (air pollution) to obesity-associated morbidity. Beck-Haim Y., Tarnovscki T., Bashari D., Rudich A. (2013). A Chromatin Immunoprecipitation (ChIP) protocol for use in whole human adipose tissue. Am. J. Physiol. - Endocrinol. Metab. 305: E1172-E1177.
Pecht T., Gutman A., Bashan N. and Rudich A. (2013). Peripheral blood leucocyte sub-classes as potential biomarkers of adipose tissue inflammation and obesity sub-phenotypes in humans. Obes. Rev. (in press).
17 Mitochondrial dynamics in metabolic diseases Prof. Orian Shirihai Background Mitochondria have a very active social life style involving frequent fusion and fission events. Mitochondria that lose their ability to properly respire become excluded from the networking population and will be consumed by the cellular equivalent of a lion, the autophagosome. This forms a pathway of quality control. However, recent studies suggest that arrest of mitochondrial fusion at the cellular level, also termed "fragmentation", is playing a role in the adaptation to excess nutrient environment. Recognizing that excess nutrient environment places mitochondria in a biological conflict of interest may help understanding the link between metabolic and aging associated conditions. We study two disease models in which oxidative damage to mitochondria play a key role in the development of pathology. In diabetes, nutrient-induced oxidative damage has been shown to be a major mediator of endocrine dysfunction and beta cell loss. In bone marrow, oxidative damage induced by iron and heme- intermediates, leads to the development of sideroblastic anemia and myelodysplastic syndrome.
Ph.D.: Technion, Israel Current research Post-doctorate: Harvard University Mitochondrial Dynamics in the beta cell: Mitochondria in β-cells School of Medicine, USA play a key role as integrators of nutrient signals and insulin secretion. One significant manifestation of diabetes is the Position: Associate Professor gradual reduction in mitochondrial capacity to produce signals in response to fuels. The cause of this gradual deterioration is Department of Clinical Biochemistry not yet understood. Our goal is to understand the mechanisms that underlie deterioration of mitochondrial function during the and Pharmacology development of β-cell dysfunction and diabetes. Faculty of Health Sciences We have shown that β-cells respond to the chronic exposure to E-mail: [email protected] high levels of glucose and fatty acids with a drastic reduction in mitochondrial networking through fusion and fission. This Selected publications phenomenon precedes a gradual deterioration of mitochondrial function that is characterized by the generation of a subpopulation Twig G., Elorza A., Molina A.J., Mohamed H., Wikstrom J.D., of mitochondria with reduced membrane potential. Remarkably, Walzer G., Stiles L., Haigh S.E., Katz S., Las G., Alroy J., Wu M., under these conditions, induction of mitochondrial fusion in the Py B.F., Yuan J., Deeney J.T., Corkey B.E. and Shirihai O.S. β-cell prevents the appearance of mitochondria with reduced (2008). Fission and selective fusion govern mitochondrial membrane potential and protects from the detrimental effects of segregation and elimination by autophagy. EMBO J. 27:433- chronic exposure to a nutrient rich environment (see publications 446. list: Molina et al. 2009) Molina A.J., Wikstrom J.D., Stiles L., Las G., Mohamed H., Elorza A., Walzer G., Twig G., Katz S., Corkey B.E. and Shirihai Mitochondrial quality control: By tagging and tracking individual O.S. (2009). Mitochondrial networking protects beta-cells mitochondria in intact β-cells we discovered the existence of a from nutrient-induced apoptosis. Diabetes 58:2303-2315. quality control mechanism that relies on both fusion and fission. Following mitochondrial fission some daughter units depolarize. Las G., Sereda S., Wikstrom J.D., Twig G., and Shirihai O.S. These units display a lower likelihood for subsequent fusion and (2011). Fatty acids suppress autophagic turnover in β-cells. are apparent targets of autophagy (see Twig et al. 2008). Moreover, J. Biol. Chem. 286:42534-42544. this model predicts that the inhibition of mitochondrial dynamics (MtDy) by Gluco-lipo-toxicity (GLT) may have a cumulative effect Liesa M. and Shirihai O.S. (2013). Mitochondrial dynamics and result in an increased portion of dysfunctional units over time. in the regulation of nutrient utilization and energy Such enrichment of dysfunctional mitochondria could explain the expenditure. Cell Metabolism 17:491-506. long lasting effect of GLT, a phenomenon that has been shown to impact animals’ prognosis many months after a high fat diet has Wikstrom J.D., Mahdaviani K., Liesa M., Sereda S.B., Si Y., been discontinued (see Liesa and Shirihai, 2013). Las G., Twig G., Petrovic N., Zingartti C., Graham A., Cinti S., Corkey B.E., Cannon B., Nedergaard J. and Shirihai O.S. Mitochondrial Dynamics in the regulation of Energy efficiency: (2014). Hormone-induced mitochondrial fission is utilized Brown fat is a unique tissue that can reduce its energy efficiency by brown adipocytes as an amplification pathway for in response to hormonal stimulation. Upon stimulation with energy expenditure. EMBO J. 33(5):418-436. adrenergic agonists brown adipocyte mitochondria switch from efficient ATP producing metabolism to heat producing Ferree A., Trudeau K., Zik E., Benador IY1, Gottlieb R.A. and metabolism mediated by uncoupling. We are using this model Shirihai O.S. (2013). MitoTimer probe reveals the impact of to study the role of mitochondrial architecture and dynamics autophagy, fusion, and motility on subcellular distribution in energy efficiency. Our findings indicate that fragmented of young and old mitochondrial protein and on relative mitochondrial network enhances energy expenditure and as mitochondrial protein age. Autophagy 9(11):1887-1896. such may be a mechanism by which we can increase caloric consumption by the brown adipose tissue.
18 Neurodegenerative Diseases Group
With the global phenomenon of aging whereby many countries worldwide will have at least 20% of their population > 65 years old by 2030, the progressive loss of structure or function of neurons, including death of neurons and associated diseases, represents a major clinical burden. Many neurodegenerative diseases including Parkinson’s, Alzheimer’s, and Huntington’s occur as a result of neurodegenerative processes and discovering cellular mechanisms involved in neuronal loss might offer new diagnostic and therapeutic advances. At the NIBN, research groups are focusing on the development of innovative immune and peptide-based therapeutic strategies for Alzheimer’s disease, understanding awry protein-protein interactions in ALS (Lou Gehrig’s disease) as well as utilizing the nematode c. elegans as a model system to unveil mechanisms of neurodegeneration and assessment of naturally-occurring plant extracts to halt cell loss. Furthermore, dissecting the role of the mitochondrial sodium/ calcium exchanger in neuronal diseases such as Parkinson’s, is also being investigated.
· Dr. Anat Ben-Zvi · Prof. Alon Monsonego · Prof. Israel Sekler · Prof. Varda Shoshan-Barmatz
19 Protein folding homeostasis in a multicellular organism Dr. Anat Ben-Zvi Background The long-term health of all metazoan cells is linked to protein quality control. All cells have highly conserved pathways that detect, prevent and resolve protein damage. The absence or malfunction of these pathways can result in developmental arrest, functional decline of diverse cellular machinery and the onset of protein misfolding diseases. When protein folding and clearance is balanced with protein biosynthetic processes, protein homeostasis (proteostasis) is achieved, thereby preventing the accumulation of mis-folded protein and aggregates within cells.
Current research Most protein mis-folding associated diseases exhibit tissue-selective impairment. However, the mechanism for this selectivity and vulnerability remains unknown. Cell-type-specific and tissue-specific regulation of protein expression results in different cellular functions Ph.D.: The Hebrew University of and morphological characteristics. Thus the requirements for and regulation of protein folding may also vary between tissues. We aim Jerusalem, Israel to elucidate how cell-specific differences in protein expression affect Post-doctorate: Northwestern cellular quality control networks. Caenorhabditis elegans (C. elegans) University, Evanston, USA provides an opportunity to study the complex biological networks that determine proteostasis in an intact organism. We employ the Position: Senior Lecturer C. elegans model system to study protein folding in the cell, using a combination of cellular, biochemical and genetic approaches to: Department of Life Sciences 1. Develop a toolbox of folding sensors to monitor proteostasis Faculty of Natural Sciences challenges and determine the genetic and physical interactions with the cellular folding machinery. E-mail: [email protected] 2. Identify cell-specific and cell-nonspecific modifiers of proteostasis in C. elegans and compare the protein folding capacity in Selected publications different cell types. Gidalevitz T*., Ben-Zvi A*., Ho K.H., Brignull H.R. and 3. Evaluate the impact of the expression of various aggregation- Morimoto R.I (2006). Progressive Disruption of Cellular prone proteins on the protein folding capacity in different cell Protein Folding in Models of Polyglutamine Diseases. types. Science 311(5766):1471-1474. 4. Examine the cell non-autonomous regulation of protesostasis Ben-Zvi A., Miller E.A and Morimoto R.I (2009). Collapse during adulthood and how it modulates the onset and of Proteostasis Represents an Early Molecular Event in C. progression of protein mis-folding diseases. elegans Aging. Proc. Natl. Acad. Sci. USA 106(35):14914- 14919.
Bar-Lavan Y., Kosolapov L., Frumkin F. and Ben-Zvi A. (2011). Regulation of cellular protein quality control networks in a multi-cellular organism. FEBS J. 279(4):526- 531.
Shemesh N., Shai N. and Ben-Zvi A. (2013). Germline stem cell arrest inhibits the collapse of somatic proteostasis Early in Caenorhabditis elegans Adulthood. Aging Cell 12(5):814-822.
Karady I., Frumkin A., Dror S., Shemesh N., Shai N. and Ben- Zvi A. (2013). Using Caenorhabditis elegans as a model system to study protein homeostasis in a multicellular organism. JoVE (82):e50840
Feldman N., Kosolapov L. and Ben-Zvi A. (2014). Fluorodeoxyuridine improves Caenorhabditis elegans proteostasis independent of reproduction onset. PLOS ONE 9(1):e85964
20 Novel approaches for the treatment of neurodegenerative Prof. Alon Monsonego and autoimmune diseases
Background Our laboratory is interested in the characterization of key regulatory factors that maintain immune homeostasis and protect against self-originated neurodegenerative and autoimmune diseases. In contrast to current therapeutics that mostly offer an anti-inflammatory approach, our goals for cure and prevention are aimed at immunotherapy, designed to strengthen endogenous beneficial immune reactions.
Current research
1. Neurodegenerative diseases: Our research is focused on characterizing the immune mechanisms, which maintain the neuronal network in health and disease, by studying: a) the differentiation and functional characteristics of Ph.D.: Weizmann Institute of Science, microglia (the brain macrophages) within the brain tissue; b) the role of cytokines and chemokines in regulating glia- Israel neuron interactions; and c) the migration and function of Post-doctorate: Harvard Medical lymphocyte subsets within the brain. School, USA These aspects of the immune system can mediate neuronal Position: Associate Professor recovery by affecting the clearance of toxic forms of amyloid from the brain and by inducing a local milieu The Shraga Segal Department of supportive of neuronal repair. We have developed a unique Microbiology, Immunology and Genetics Alzheimer’s disease (AD) mouse model that lends itself to the development of safe vaccination approaches aimed at Faculty of Health Sciences prevention and therapy of the disease.
E-mail: [email protected] 2. Autoimmunity: Our research is focused on the following aspects: a) characterizing the repertoire of brain-specific Selected publications T cells and their role in aging and the progression of AD; Nemirovsky A., Fisher Y., Baron R., Cohen I.R. and Monsonego b) revealing autoimmune mechanisms associated with A. (2011). Amyloid beta-HSP60 peptide conjugate vaccine chronic stress; and c) the use of 3D-scaffolds to generate treats a mouse model of Alzheimer's disease. Vaccine a transplantable lymphoid-like tissue with immuno- 29(23):4043-4050. regulatory properties. Fisher Y., Nemirovsky A., Baron R. and Monsonego A. (2011). Dendritic cells regulate amyloid-β-specific T-cell entry into These approaches offer a platform for the design of the brain: the role of perivascular amyloid-β. J. Alzheimers Dis. immune-based therapies to a variety of autoimmune 27(1):99-111. disorders, whether they originate from immune deficiency (e.g. cancer, neurodegenerative diseases) or hyper-immune Abutbul S., Shapiro J., Szaingurten-Solodkin I., Levy N., responses (e.g. autoimmune diseases, allergies). Carmy Y., Baron R., Jung S. and Monsonego A. (2012). TGF-β signaling through SMAD2/3 induces the quiescent microglial phenotype within the CNS environment. Glia 60(7):1160-1171.
Fisher Y., Strominger I., Biton S., Nemirovsky A., Baron R. and Monsonego A. (2013). Th1 Polarization of T Cells Injected into the Cerebrospinal Fluid Induces Brain Immunosurveillance. J. Immunol. 192(1):92-102.
Monsonego A., Nemirovsky A. and Harpaz I. (2013). CD4 T cells in immunity and immunotherapy of Alzheimer's disease. Immunology 139(4):438-446.
Harpaz I., Abutbul S., Nemirovsky A., Gal R., Cohen H. and Monsonego A. (2013). Chronic exposure to stress predisposes to higher autoimmune susceptibility in C57BL/6 mice: glucocorticoids as a double-edged sword. Eur. J. Immunol. 43(3):758-769.
21 Ionic control of Ca2+ signaling and metabolism Prof. Israel Sekler Background Ionic gradients control signaling and metabolic activity, with their breakdown being linked to major diseases. The major focus of our lab is to molecular identify key ion transporters and to determine their physiological roles, as well as developing novel opto-molecular strategies for controlling ionic gradients.
Current research Mitochondrial Ca2+ signaling The mitochondrial Na+/Ca2+ exchanger (NCLX) is a key player in mitochondrial and cellular calcium homeostasis. Although its existence was documented some 40 years ago, its molecular identity has remained elusive. By combining molecular silencing, ectopic expression and dominant negative analysis with imaging of mitochondrial and cellular calcium levels, we have identified NCLX as the long-sought exchanger. This finding opens the Ph.D.: Weizmann Institute of Science, Israel door to molecular analysis of the mitochondrial Ca2+ transport Post-doctorate Tenure: Stanford machinery and physiological studies ranging from cardiac University, USA activity to insulin secretion and neuronal activity. Based on an examination of human mutations of NCLX, we are now linking Position: Professor mitochondrial signaling to major human maladies ranging from diabetes to Parkinson’s disease, Alzheimer’s disease and stroke. Department of Physiology and Cell Furthermore, by combing molecular modeling with appropriate Biology screens for potential NCLX agonists and antagonists, we plan to identify novel drugs that might interrupt these major health Faculty of Health Sciences syndromes and presently unmet clinical needs. E-mail: [email protected] Opto-metabolic control Metabolic activity not only controls the life and death of cells but Selected publications is also linked to major diseases when impaired. Although we can readily monitor metabolic activity, we cannot selectivity control Palty R., Ohana E., Hershfinkel M., Volokita M., Elgazar V., Beharier such processes because currently available reagents are devoid O., Silverman W.F., Argaman M. and Sekler I. (2004). Lithium- of selectivity and often act as irreversible metabolic poisons. calcium exchange is mediated by a distinct potassium- independent sodium-calcium exchanger. J. Biol. Chem. 279: In our laboratory, we are focusing on a novel strategy termed 25234-25240. opto-metabolic control. Our aim is to target light-dependent molecular switches into mitochondria, thereby controlling Palty R., Silverman W.F., Hershfinkel M., Caporale T., Sensi S.L., metabolic activity in cells both selectively and in a temporally Parnis J., Nolte C., Fishman D., Shoshan-Barmatz V., Herrmann controlled manner. This strategy will provide global molecular S., Khananshvili D. and Sekler I. (2010). NCLX is an essential component of mitochondrial Na+/Ca2+ exchange. PNAS control of metabolic activities in sub-cellular fractions, cells, 107:436-441. tissues and organs.
Nita I.I., Hershfinkel M., Fishman D., Ozeri E., Rutter G.A., Sensi Heavy metals in health and disease S.L., Khananshvili D., Lewis E.C. and Sekler I. (2012). The Zinc is an essential micronutrient required for growth and mitochondrial Na(+)/Ca(2+) exchanger upregulates glucose dependent Ca(2+) signaling linked to insulin secretion. PLoS development yet is strikingly similar to cadmium, a highly One 7(10):e46649. toxic environmental pollutant. Indeed, due to their similarity, zinc transporters can be hijacked by cadmium, thus gaining Hoch E., Lin W., Chai J., Hershfinkel M., Fu D. and Sekler I. (2012). access into the body. Indeed, the similarity of the two metals is Histidine pairing at the metal transport site of mammalian ZnT so remarkable that there are no chelators that can distinguish transporters controls Zn2+ over Cd2+ selectivity. Proc. Natl. Acad. Sci. U.S.A. 109(19):7202-7207. between the two. By combing molecular modeling with functional assays, we have identified the first mammalian Parnis J., Montana V., Delgado-Martinez I., Matyash V., Parpura transporter and ion binding site that can transport zinc yet V., Kettenmann H., Sekler I. and Nolte C. (2013). Mitochondrial reject cadmium. Our current efforts are focused on identifying exchanger NCLX plays a major role in the intracellular Ca2+ signaling, gliotransmission, and proliferation of astrocytes. J. the functional mechanism(s) of this and other zinc transporters, Neurosci. 33(17):7206-7219. as well as the molecular basis for heavy metal selectivity.
22 VDAC1 as a potential target for Prof. Varda Shoshan-Barmatz Alzheimer's disease therapy NIBN Director Background Alzheimer’s disease (AD) is the most common form of dementia in the elderly. AD is characterized by cognitive decline and the occurrence of brain senile plaques and neurofibrillary tangles, as well as associated with the loss of brain synapses and synaptic dysfunction. In consort with these pathologies, increasing evidence also points to structural and functional abnormalities of mitochondria. Clinical findings strongly suggest that early aggregation of the amyloid beta peptide (Aβ) plays a key role in AD pathogenesis. Moreover, Aβ deposits were found both in extracellular and intra-neuronal in the brains of AD patients early in the disease process. Moreover, it has been shown that synaptic damage and mitochondrial dysfunction are early events in AD pathogenesis, with Aβ-induced cytotoxicity having been shown to be preceded by mitochondrial dysfunction and signaling events characteristic of apoptosis in cultured cells. While mitochondrial Ph.D.: Weizmann Institute of Science, Israel dysfunction occurs early in AD and is a prominent feature of the disease, the underlying mechanisms remain poorly understood. Post-doctorate: University of However, it has been recently shown that the voltage-dependent Wisconsin-Madison, USA and anion channel (VDAC), located in the outer mitochondrial membrane University of Toronto, Canada participates in Aβ-induced toxicity. This finding underscores a definitive causative link between awry mitochondrial function and Position: Professor, NIBN Director the development of AD. Department of Life Sciences Current research Faculty of Natural Sciences E-mail: [email protected] VDAC1 functions in both cell metabolism and apoptosis can be modified by Aβ interaction with VDAC1, leading to mitochondrial dysfunction and apoptosis. Using biophysical and biochemical methods, we have demonstrated that Aβ directly interacts with Selected publications VDAC1 and with a peptide corresponding to VDAC1’s N-terminal domain. This peptide completely prevents Aβ entry into PC-12 Shoshan-Barmatz V., Israelson A., Bridiczka D. and and SHSY-5Y cells and resultant cytotoxicity. In addition, silencing Sheu S.S. (2006). The Voltage Dependent Anion Channel VDAC1 expression by a specific siRNA prevented Aβ association (VDAC): Function in Intracellular Signalling, Cell Life and with the cell membrane and intracellular accumulation. Cell Death. Current Pharmaceutical design 12(18):2249- Therefore, we hypothesize that Aβ toxicity involves mitochondrial 2270. impairment mediated via Aβ interaction with both mitochondrial Shoshan-Barmatz V., de Pinto V., Zweckstetter M., Raviv and plasmalemmal (pl)VDAC1. As a VDAC1 N-terminal-based Z., Keinan N. and Arbel N. (2010). VDAC, a multi-functional peptide protects against Aβ-related cytotoxicity and its cell mitochondrial protein regulating both cell life and death. penetration, we hypothesize that Aβ cytotoxicity is mediated via Molecular Aspects of Medicine 31:227-286. its interaction with the VDAC1-N-terminal domain and that Aβ penetrates the cell via pl-VDAC1 that we predict is over-expressed Varda Shoshan-Barmatz (2014). Amyloid-Beta binding in AD brains. However, the exact mechanism by which VDAC1 peptides and use thereof for treating neurodegenerative mediates Aβ cytotoxicity and the sequence of events leading to diseases. U.S. Provisional Patent Application No. such toxicity have yet to be explored. 61/935,363 Our overall goal is to scrutinize the relationship between Aβ interaction with VDAC1 and apoptosis induction and translate these findings into the development of VDAC1-based peptide therapeutics as effective anti-Aβ treatment for AD. Elucidating the role of the VDAC1-Aβ interaction and the involvement of pl-VDAC1 in Aβ cytotoxicity will provide new insights into the mechanisms that control Aβ cytotoxicity in AD pathogenesis and contribute to the development of VDAC1- based pharmacological interventions.
23 Infectious Diseases Group
The development of innovative antibiotics is urgently required to combat the spread of antibiotic-resistant infectious diseases, especially hospital-acquired infections. In direct response to this unmet medical need, groups at the NIBN are developing both synthetic and naturally-occurring molecules to prevent a phenomenon termed Quorum Sensing (bacterial communication). In particular, such molecules are assayed for bioactivity in preventing biofilm formation, a major bacterial resistance mechanism that envelops bacteria rendering them far less susceptible to antibiotics. Other anti-bacterial activities efforts are focused on screening libraries for inhibitory activities targeting bacterial- specific, proteolytic degradation pathways known to be critical for bacterial virulence. Such a strategy is used to specifically derail infectivity associated with Myobacterium tuberculosis infections (via inhibition of a unique pupylation pathway) and E. coli, Klebsiella, Vibrio Cholera and Pseudomonas aeruginosa (via inhibition of Lon protease). Additional activities within the infectious disease group concern host-pathogen co-evolution concerning T and B-cell immunodominance, HLA binding prediction and how all this information can be leveraged for rational vaccine design. Furthermore, research concerning the cell abscission component of cytokinesis, namely the ESCRT machinery, is being adapted in understanding the process of viral budding and infection and how such a process can be arrested with specific inhibitors.
• Dr. Natalie Elia • Dr. Eyal Gur • Dr. Tomer Hertz • Prof. Michael .M. Meijler
24 Cellular dynamics and macromolecular architecture in 3D Dr. Natalie Elia Background Cells are composed of many large, multi-molecular complexes executing distinct cellular functions in a confined space and operating in a dynamic, highly coordinated fashion. Understanding the kinetics and 3D organization of such protein complexes is, therefore, key to understanding cellular mechanisms. Recent advances in fluorescence light microscopy techniques now permit the dissection of the spatiotemporal behavior of proteins in their intact cellular environment. Confocal spinning disk microscopy provides the high imaging speed, high sensitivity and high dynamic range required to measure protein dynamics in cells, while structured illumination microscopy (SIM) is a fluorescence-based super-resolution imaging technique capable of three-dimensional (3D) multi-color imaging at 100 nm lateral and 350 nm axial resolution in any biological sample up to 10 microns in thickness. As such, SIM is one of the most suitable techniques for mapping the spatial organization of protein complexes in their native environment at nanometer scale resolution. The temporal information obtained by spinning disk microscopy and Ph.D.: The Hebrew University of the detailed spatial information obtained by SIM can be integrated to Jerusalem, Israel generate a spatiotemporal map of protein complexes in a given cellular process. Constituting such spatiotemporal maps will facilitate mechanistic Post-doctorate: National Institutes of understanding of different cellular machineries. Health, USA Current research Position: : Senior Lecturer 1. Understanding ESCRT mechanism of action: The ESCRT complex Department of Life Sciences is emerging as one of the major cellular machineries responsible Faculty of Natural Sciences for driving membrane fission in cells. Additionally, a fundamental role for the ESCRT machinery was recently defined in driving the E-mail: [email protected] very late events of cell division that lead to the physical separation of two daughter cells. We aim to elucidate the mechanism of Selected publications action of the ESCRT machinery in driving membrane fission using mammalian cell division as a model system. We hope to Lee H.H., Elia N., Ghirlando R., Lippincott-Schwartz J. do so using advanced quantitative imaging approaches, as well and Hurley J.H. (2008). Midbody targeting of the ESCRT as by developing new tools for arresting the ESCRT pathway. As machinery by a noncanonical coiled coil in CEP55. Science 322(5901):576-580. the ESCRT machinery is involved in numerous cellular functions, including receptor degradation, viral budding and cell division, Rambold A., Kostelecky B., Elia N. and Lippincott-Schwartz our results will have applicative implications including for the J. (2011). Tubular network formation protects mitochondria development of drugs to block uncontrolled cell division, viral from autophagosomal degradation during nutrient infectivity and other cellular processes. starvation. Proc. Natl. Acad. Sci. U.S.A 108(25):10190-10195.
Elia N., Sougrat R., Spurlin T.A., Hurley J.H. and Lippincott- 2. Dissecting the spatiotemporal regulation of cytokinetic Schwartz J. (2011). Dynamics of endosomal sorting abscission: Cell division is one of the most regulated and complex required for transport (ESCRT) machinery during coordinated processes in cell biology. While much is known cytokinesis and its role in abscission. Proc. Natl. Acad. Sci. U.S.A 108(12):4846-4851. Ranked “must read” in Faculty of about the temporal regulation of early division steps (prophase 1000. to anaphase), little is known about the temporal regulation of late division steps. Recent studies indicate that the final steps of Elia N., Fabrikant G., Kozlov M. and Lippincott-Schwartz cell division termed cytokinetic abscission are highly coordinated J. (2012). Computational model of cytokinetic abscission in time and space. However, the regulatory basis of this process, driven by ESCRT III polymerization and remodeling. which terminates cell division giving rise to the formation of Biophysical J. 102(10):2309-2320. Selected for cover. two independent daughter cells, is still unknown. By generating Fridman K., Mader A., Zwerger M., Elia N. and Medalia O. spatiotemporal maps of different cytokinetic proteins at different (2012). Advances in tomography: probing the molecular late stages cytokinesis we aim to elucidate the regulation of architecture of cells. Nat. Rev. Mol. Cell. Bio. 13(11):736-742. cytokinetic abscission and to determine the mechanistic basis for abscission timing. Elia N.*, Ott C. and Lippincott-Schwartz J.* (2013). Incisive imaging and computation for cellular mysteries: lessons from abscission. Cell 155(6):1220-1231. *Corresponding authors
25 Targeting the bacterial Lon protease – a novel approach for the development Dr. Eyal Gur of antibiotic compounds
Background Quality-control systems, consisting of ATP-dependent proteases, chaperones, heat-shock proteins and additional regulatory molecules have evolved to protect cells from the harmful effects of protein unfolding. These networks execute either degradation or refolding of misfolded proteins and assist in the disassembly of protein aggregates. The major enzyme responsible for degrading damaged proteins in bacteria is a hexameric ATP-dependent protease known as Lon. Its importance for proper cellular function is manifested in the phenotypes of Lon mutations, which present various physiological defects. In recent years, it has been established that Lon activity is essential for the virulence of many pathogenic bacteria. Therefore, Lon may serve as a potential target for the development of novel anti-bacterial compounds.
Ph.D.: Tel Aviv University, Israel Current research Post-doctorate: Massachusetts 1. The mechanism of protein degradation by Lon (and other Institute of Technology, USA ATP-dependent proteases) is complex and involves multiple coordinated steps, which act to harvest the energy released from Position: Senior Lecturer ATP hydrolysis to propel protein degradation in a regulated fashion. The multi-step, complex degradation mechanism employed by Department of Life Sciences Lon offers numerous opportunities to inhibit the protease along Faculty of Natural Sciences the substrate processing pathway. Understanding the molecular mechanism of protein degradation by Lon will facilitate the E-mail: [email protected] development of inhibitors of the degradation process. Toward this aim, we use biochemical, genetic and structural approaches to study Lon-mediated proteolysis. 2. Small molecules are defined as non-polymeric organic Selected publications compounds, often isolated from natural sources. Many times small molecules are secondary metabolites that serve biological Gur E. and Sauer R.T. (2008). Recognition of misfolded functions. The advantages of using small molecules are several- proteins by Lon, a AAA+ protease. Genes Dev. 22:2267- 2277. fold. The vast diversity of these compounds (numbering hundreds of thousands of different molecules) and the fact that they have Gur E., Biran D. and Ron E.Z. (2011). Regulated proteolysis evolved to fulfill biological roles, result in a very high probability in Gram negative bacteria - how and when? Nat. Rev. of finding compounds with a desired activity. Moreover, small Microbiol. 9:839-848. molecules can reach most compartments of the human body. Ofer N., Vishkautzan M., Meijler M., Wang Y., Speer A., We have designed a high-throughput screening assay to identify Niederweis M. and Gur E. (2012). Ectoine biosynthesis small molecules that can act as selective Lon inhibitors. Identified in Mycobacterium smegmatis. Appl. Environ. Microbiol. compounds will be further tested for their qualification as anti- 78:7483-7486. bacterial drugs.
Forer N., Korman M., Elharar Y., Vishkautzan M. and Gur 3. Lon is the only ATP-dependent protease known to have a natural E. (2013). The bacterial proteasome and PafA, the Pup protein inhibitor. The T4-bacteriophage protein, PinA, specifically ligase, interact to form a modular protein tagging and inhibits the E. coli Lon protease, apparently by inhibiting degradation machine. Biochemistry 52(50):9029-9035. the activity of the Lon ATPase domain. Understanding the inhibition mechanism of Lon by PinA should facilitate the future Shenkerman Y., Elharar Y., Vishkautzan M. and Gur E. development of novel antibiotic compounds designed to act as (2013). Efficient and simple generation of unmarked gene deletions in Mycobacterium smegmatis. Gene 533:374- specific inhibitors of bacterial Lon proteases. 378.
Ofer N., Forer N., Korman N., Vishkautzan M., Khalaila I. and Gur E. (2013). Allosteric transitions direct protein tagging by PafA, the prokaryotic ubiquitin-like protein (Pup) ligase. J. Biol Chem 288:11287-11293.
26 Computational and experimental approaches to studying Dr. Tomer Hertz immunodominance
Background In the arms race between pathogen and host, the adaptive immune system uses a diverse set of pattern detectors to identify and eliminate pathogens and pathogen infected cells. These detectors bind to short contiguous (T-cells, B-cells) and non-contiguous (B-cells) protein fragments called epitopes. During the course of an infection the immune system focuses its response to a small fraction of the thousands of potential targets. This phenomenon, known as immunodominance, is a fundamental property of the adaptive immune response. Understanding the mechanisms that govern immunodominance is crucial for designing vaccines. Immunodominance is a result of a large number of factors including immunological history, antigen processing and presentation, viral load and kinetics of viral expression, Ph.D.: The Hebrew University of and host genetics. Jerusalem, Israel We are developing computational and experimental tools to study the underlying mechanisms that govern T-cell and B-cell Post-doctorate: Microsoft Research, USA immunodominance in both natural infection and vaccination. The main research objective is to identify both viral and Position: Senior Lecturer host features that define and modulate immunodominance The Shraga Segal Department of hierarchies. The nature of our work is translational, integrating the design and application of computational approaches with Microbiology, Immunology and Genetics clinical and laboratory studies that provide data for validating and refining our computational tools. Faculty of Health Sciences E-mail: [email protected] Current research 1. Using HLA binding predictors to study T-cell immunodominance: Human Leukocyte Antigen alleles Selected publications present short peptides on the surface of cells to cytolytic T-cells. We and others have developed computational tools Hertz T., Nolan D., James I., John M., Gaudieri S., Phillips E., to predict which targets are likely to be presented to cytolytic Huang J.C., Riadi G., Mallal S. and Jojic N. (2011). Mapping the T-cells. We are using these tools to develop computational Landscape of Host-Pathogen Coevolution: HLA Class I Binding tools for predicting which pathogenic targets are likely to and Its Relationship with Evolutionary Conservation in Human and Viral Proteins. Journal of Virology 85:1310-1321. become immunodominant, with applications to vaccine design and analysis of T-cell responses following both Meroz D., Yoon S.W., Ducatez M.F., Fabrizio T.P., Webby R.J., Hertz vaccination and natural infection. T.* and Ben-Tal* N. (2011). Putative amino acid determinants 2. Quantifying the effects of immunological history on of the emergence of the 2009 influenza A (H1N1) virus in the immune responses to natural infection and vaccination: human population. Proceedings of the National Academy of Previous exposure to pathogens and vaccination is one of Sciences of the United States of America 108: 13522–13527. the key factors that bias the response to a novel vaccine Hertz T., Ahmed H., Friedrich D., Horton H., Frahm N., McEl- or infection. We are developing a high-throughput assay rath J., Corey L. and Gilbert P. (2013). HIV-1 Vaccine Induced for profiling immunological history using the technology of T-cell Reponses Cluster in Epitope Hotspots that Differ From antigen microarrays. Those Induced in Natural Infection with HIV-1. PLoS Pathogens 3. Using adjuvants to shift vaccine-induced 9(6):e1003404. immunodominance patterns: Adjuvants are widely used in licensed vaccines to boost the vaccine-induced immune Hertz T., Oshansky-Weilnau, C., Roddam P.L., DeVincenzo responses. We are investigating the effects of different J.P., Caniza M.A., Jojic N., Mallal S., Phillips E., James I., Thomas adjuvants on antibody immunodominance patterns. P., Halloran B. and Corey L. (2013). HLA Targeting Efficiency Correlates with Human T-Cell Response Magnitude and with 4. Computational identification of host specificity Mortality from Influenza A Infection. PNAS 110(33):13492-13497. determinants in influenza infection: Influenza pandemics occur when a novel strain is introduced into the human Keating R., Hertz T., Lukens J.R., Harris T.L., Edwards B.A., Wehenkel population from different animal hosts such as birds and M., McClaren J.L., Brown S.A., Surman S., Hurwitz J., Doherty P.C., swine. The molecular mechanisms that allow an influenza Thomas P.G. and McGargill M.A. (2013). mTOR modulates the anti- virus to adapt to the human host are poorly understood. body response to provide cross-protective immunity to lethal We are developing an approach for computationally influenza infections. Nature immunology 14(12): 1266-1276. elucidating Influenza virus mutations that are essential for the adaptation to the host.
27 Chemical biology of bacterial communication Prof. Michael M. Meijler Background Cell-to-cell communication is used by single-cell organisms to coordinate their behavior and function in such a way that they can adapt to changing environments and possibly compete with multicellular organisms. This phenomenon has been termed “quorum sensing” (QS). Examples of QS-controlled behaviors are biofilm formation, virulence factor expression, antibiotic production and bioluminescence. These processes are beneficial to a bacterial population only when they are carried out in a coordinated fashion.
Current research Quorum Sensing An important focus of my group’s research is the study of bacterial intra- and interspecies signaling. Cell-to-cell communication is used by single-cell organisms to coordinate their behavior and function Ph.D.: Weizmann Institute of Science, in such a way that they can adapt to changing environments and possibly compete with multicellular organisms. This phenomenon Israel has been termed “quorum sensing” (QS). Examples of QS-controlled Post-doctorate: The Scripps Research behaviors are biofilm formation, virulence factor expression, Institute, USA antibiotic production and bioluminescence. These processes are beneficial to a bacterial population only when they are carried out in Position: Associate Professor a coordinated fashion. Quorum sensing systems exist in both gram- positive and -negative bacteria and a variety of oligopeptides and Department of Chemistry N-acyl-homoserine lactones have been identified as QS molecules. Faculty of Natural Sciences However, many QS systems have not been characterized fully, thus we will attempt to clarify the role of various QS molecules in bacterial E-mail: [email protected] signaling (in species such as Pseudomonas aeruginosa, Salmonella typhimurium, Helicobacter pylori). Through the synthesis and evaluation of QS molecules and potential antagonists we will develop Selected publications methodologies to study a wide variety of newly discovered and undiscovered QS molecules. Currently, as part of two different studies Amara N., Mashiach R., Amara D., Krief P., Spieser S.A., to design QS antagonists of P. aeruginosa, we have synthesized several Bottomley M.J., Aharoni A. and Meijler M.M. (2009). highly active covalent and non-covalent QS inhibitors. Covalent Inhibition of Bacterial Quorum Sensing. J. Am. Chem. Soc. 131:10610-10619.
Dubinsky L., Jarosz L.M., Amara N., Krief P., Kravchenko Bacterial-Eukaryotic Interkingdom Signaling V., Krom B.P. and Meijler M.M. (2009). Synthesis and Recent reports have shown that several QS molecules can also have validation of a probe to identify quorum sensing a direct effect on eukaryotes. Diverse eukaryotes have been found receptors. Chem. Commun. 47:7378-7380. to react strongly to the presence of these compounds. My group currently examines the hypothesis that diverse eukaryotic species Rayo J., Amara N., Krief P. and Meijler M.M. (2011). Live have developed mechanisms to react to the presence of specific cell labeling of native intracellular bacterial receptors bacterial QS molecules in a receptor-mediated fashion. We perform using aniline-catalyzed oxime ligation. J. Am. Chem. Soc. 133:7469-7475. focused experiments designed to provide greater insight into the primary molecular mechanism of QS molecule induced effects on Ganin H., Danin-Poleg Y., Kashi Y. and Meijler M.M. mammals, fungi and nematodes. Identification of specific QSM (2012). Vibrio cholerae Autoinducer CAI-1 Interferes with receptors in eukaryotes will allow us to further understand the Pseudomonas aeruginosa Quorum Sensing and Inhibits complex mechanisms of coexistence and the evolution of coexistence its Growth. ACS Chem. Biol. 7:659-665. between prokaryotes and eukaryotes. Ultimately insights obtained from these experiments could lead to: a) new approaches for the Dubinsky L., Delago A., Amara N., Krief P., Rayo J., Zor treatment of P. aeruginosa infections, most notably in the clinical T., Kravchenko V.V. and Meijler M.M. (2013). Species setting of cystic fibrosis, as well as to potential new drugs for the selective diazirine positioning in tag-free photoactive quorum sensing probes. Chem. Commun. 49:5826-5828. treatment of autoimmune diseases; b) an increased understanding of the general principles that guide the evolution of symbiotic Mandabi A., Ganin H., Rayo J. and Meijler M.M. (2013). relationships between competing species; c) the development of Karrikins from Plant Smoke Modulate Bacterial Quorum an integrated platform that will enable the discovery of unknown Sensing. Chem. Commun. 50(40): 5322-5325. receptors for small hydrophobic bioactive compounds.
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Human Genetic Disorders Group
The study of the genetic basis of diseases in defined inbred communities offers a unique opportunity for identifying and characterizing disease-associated genes which are direct causes of clinical disease. The Genetics & Bioinformatics groups' research is focused on integrating diverse data types for personalized diagnosis, utilizing protein structure data, understanding and predicting the behavior of complex biological networks and interpretation of high throughput experiments, including expression profiles and next-generation sequencing. It conducts functional genomics and proteomics studies in order to identify new drug targets, to discover novel molecular pathways of disease, to validate mechanisms of actions of drugs, and to develop diagnostic tools for diseases, with a special focus on genetic diseases of the Bedouins population in the Negev.
• Prof. Ohad Birk • Prof. Ruti Parvari • Dr. Esti Yeger-Lotem
29 Identification and characterization of genes associated with human Prof. Ohad Birk diseases: Unraveling of novel drug targets and developing tools for diagnosis of genetic diseases
Background Genetic studies of unique inbred consanguineous populations in southern Israel enable unraveling of the molecular basis of hereditary diseases. The laboratory’s research focuses on the identification and characterization of genes associated with human diseases. The research is of importance to three areas: 1. Medicine: Allowing for molecular diagnosis of hereditary human diseases, enabling carrier detection and prenatal diagnosis. 2. Science: Discovery of the molecular basis for human diseases and of normal molecular developmental pathways. 3. Biotechnology: Discovery of novel drug targets for hereditary M.D.: Tel Aviv University, Israel disorders, as well as developing diagnostic tools for genetic diseases. Ph.D.: Weizmann Institute of Science, Israel Post-doctorate: National Institutes of Current research Health, USA Studies being performed rely on: Residency in Pediatrics: Sheba Medical 1. Linkage analysis studies of large inbred families, mostly from Center, Israel Bedouin communities of southern Israel 2. Molecular analysis of chromosomal aberrations associated with Fellowship in Clinical Human Genetics: specific human disorders Soroka Medical Center and NIH 3. Microarray analysis of disease vs. normal tissues Position: Head Genetics Institute, Soroka 4. Functional genomics analysis (in vitro and in vivo) of disease- University Medical Center associated genes uncovered in 1-3 above The Shraga Segal Department of Once disease-associated genes are identified, study of these genes Microbiology, Immunology and Genetics is performed – this icludes biochemical and structural analysis of the encoded proteins, cellular studies of mutated cells and the Faculty of Health Sciences generation and analysis of animal models for the diseases. We have thus far identified the molecular basis of 15 human diseases, E-mail: [email protected] including myopia, short stature, a variant of seborrheic dermatitis and psoriasis and other rare and common diseases, including 6 severe neurodegenrative diseases in the Bedouin and the Jewish population. The findings are of both scientific and medical interest and are immediately implemented in massive carrier testing and prenatal diagnosis. Selected publications Birk O.S., Casiano D.E., Wassif C.A., Cogliati T., Zhao L., Zhao Y., Grinberg A., Huang S., Kreidberg J.A., Parker K.L., Porter F.D. and Westphal H. Barel O., Shalev S.A., Ofir R., Cohen A., Zlotogora J., Shorer Z., Galia Mazor, (2000). The LIM homeobox gene Lhx9 is essential for mouse gonad Finer G., Khateeb S., Zilberberg N. and Birk O.S. (2008). Maternally formation. Nature 403(6772):909-913. inherited Birk Barel mental retardation dysmorphism syndrome caused by a mutation in the genomically imprinted potassium channel KCNK9. Birnbaum R.Y., Zvulunov A., Hallel-Halevy D., Cagnano E., Finer G., Ofir R., Am. J. Hum. Genet. 83:1-7. Geiger D., Silberstein E., Feferman Y. and Birk O.S. (2006). Seborrhea-like dermatitis with psoriasiform elements caused by a mutation in ZNF750, Gefen A., Cohen R. and Birk O.S. (2010). Syndrome to gene (S2G): in- encoding a putative C2H2 zinc finger protein. Nature Genetics 38:749- silico identification of candidate genes for human diseases. Hum. Mutat. 751. 31(3):229-236.
Narkis G., Ofir R., Manor E., Landau D., Elbedour K. and Birk O.S. (2007). Agamy O., Ben Zeev B., Lev D., Marcus B., Fine D., Su D., Narkis G., Ofir R., Lethal congenital contractural syndrome type 2 (LCCS2) is caused by a Hoffmann C., Leshinsky-Silver E., Flusser H., Sivan S., Söll D., Lerman-Sagie T. mutation in ERBB3 (Her3), a modulator of the phosphatidylinositol-3- and Birk O.S. (2010). Mutations disrupting selenocysteine formation cause kinase/Akt pathway. Am. J. Hum. Genet. 81:589-595. progressive cerebello-cerebral atrophy. Am. J. Hum. Genet. 87(4):538-544.
30 Prof. Ruti Parvari Identification and elucidation of the functions of genes associated with human genetic diseases
Background The research conducted in my laboratory aims to identify the mutations causing human diseases using genetic approaches. This is enabled by the simple pattern of inheritance of diseases caused by mutations in single genes in the highly consanguineous families being treated at the Soroka University Medical Center.
Current research We use exome sequencing and microarray genotyping techniques to identify the mutated genes in various disease Ph.D.: Weizmann Institute of Science, states. The identification of the mutation is followed by Israel functional studies to understand the mechanism of action of the normal compared to the mutated gene. These studies Post-doctorate: Johns Hopkins are carried out in cell cultures as well as animal models. University, USA The diseases presently being studied in the laboratory are: laterality defects (situs inversus and heterotaxia), primary Position: Associate Professor ciliary dyskinesia, cardiomyopathies, male infertility, insensitivity to pain and various hormonal and metabolic The Shraga Segal Department of disorders. Microbiology, Immunology and Genetics
Faculty of Health Sciences The identification of the genetic factors, their function and E-mail: [email protected] the pathways in which they act are expected to contribute to a better understanding of the pathogenesis of the related diseases, ultimately leading to improved diagnosis, treatment and prevention. The elucidation of the pathways leading to the diseases promises the identification of novel drug targets for the benefit of a large patient population.
Selected publications Philip M., Arbellle J.E., Segev Y. and Parvari R. (1998). Male hypogonadism due to a mutation in the gene for the beta- subunit of the follicle stimulating hormone. New England J. Med. 24:1729-1732. Cox J.J., Sheynin J., Shorer Z., Reimann F., Nicholas A.K., Zubovic L., Baralle M., Wraige E., Manor E., Levy J., Woods C.G. and Parvari Parvari R., Hershkovitz E., Grossman N., Gorodischer R. (2010). Congenital insensitivity to pain: novel SCN9A missense R., Loeys B., Zecic, A., Mortier G., Gregory S., Sharony and in-frame deletion mutations. Hum Mutat. 31(9):E1670-E1686. R., Kambouris M., Sakati N., Meyer B.F., Al Aqeel A.I., Al Humaidan A.K., Al Zanhrani F., Al Swaid A., Al Othman J., Mazor M., Alkrinawi S., Chalifa-Caspi V., Manor E., Sheffield V.C., Diaz G.A., Weiner R., Khan K.T., Gordon R. and Gelb B.D.; HRD/ Aviram M. and Parvari R. (2011). Primary Ciliary Dyskinesia Autosomal Recessive Kenny-Caffey SyndromeConsortium. Caused by Homozygous Mutation in DNAL1, Encoding Dynein (2002). Mutation of TBCE causes hypoparathyroidism- Light Chain 1. Am. J. Hum. Genet. 88(5):599-607. retardation-dysmorphism and autosomal recessive Kenny- Caffey syndrome. Nature Genetics 32(3):448-452. Muhammad E., Reish O., Ohno Y., Scheetz T., DeLuca A., Searby C., Regev M., Benyamini L., Fellig Y., Kihara A., Sheffield V.C. and Levy-Litan V., Hershkovitz E., Avizov L., Leventhal N., Parvari R. (2013). Congenital myopathy is caused by mutation Bercovich D., Chalifa-Caspi V., Manor E., Buriakovsky S., of HACD1. Hum Mol Genet. 22(25):5229-5236. Hadad Y., Goding J. and Parvari R. (2010). Autosomal- recessive hypophosphatemic rickets is associated with an inactivation mutation in the ENPP1 gene. Am. J. Hum. Genet. 86(2):273-278.
31 Computational systems biology of human disease Dr. Esti Yeger-Lotem Background A comprehensive understanding of the molecular basis of incurable human diseases is essential for opening new avenues for treatment. In an effort to elucidate their molecular basis, human diseases are increasingly studied using high-throughput approaches, offering unprecedented genomic, transcriptomic and proteomic views into their etiology. However, independent analyses of the resulting data typically enable only a limited understanding of disease processes. For instance, mRNA profiling assays identify transcriptional changes that occur during disease, but do not reveal the cellular pathways that lead to these changes. On the other hand, integrative analysis has great potential to reveal a much broader view of disease processes, which, in turn, could improve diagnostics and accelerate the search for a cure. We are developing computational approaches to meaningfully integrate diverse large-scale molecular data. By applying these approaches to top-quality disease data we aim to gain a broad Ph.D.: Technion, Israel Institute of Technology insight into disease mechanisms.
Post-doctorate: Whitehead Institute for Current research Biomedical Research, UK & Massachusetts Institute of Technology, USA 1. Network biology of human disease: Molecular interaction networks have proven to be a leading methodology for Position: Senior Lecturer elucidating complex cellular processes from diverse, large- scale molecular data. We are developing network optimization Department of Clinical Biochemistry and techniques to both distill and meaningfully integrate diverse Pharmacology molecular data in order to reveal the underlying disease Faculty of Health Sciences processes. 2. Illuminating the molecular basis of Parkinson’s disease: E-mail: [email protected] The cellular pathways leading to neuronal cell death in this common neurodegenerative disorder (1% of the population Selected publications over the age of 50) are not fully understood. To reveal these Yeger-Lotem E. and Margalit H. (2003). Detection of pathways we are applying network approaches to state-of- regulatory circuits by integrating the cellular networks of the-art molecular data about the disease. protein-protein interactions and transcription regulation. Nucleic Acids Research 31:6053-6061. 3. The toxic determinants of protein overexpression: Protein overexpression is associated with various human diseases Yeger-Lotem E., Sattath S., Itzkovitz S., Kashtan N., Milo R., including neurodegenerative disorders and cancer. We are Pinter R.Y., Alon U. and Margalit H. (2004). Network motifs in characterizing the set of proteins whose overexpression is toxic the integrated cellular network of transcription regulation using a wide range of bioinformatic techniques. and protein-protein interaction. Proceedings of the National Academy of Sciences USA (PNAS) 101:5934-5939.
Yeger-Lotem E., Riva L., Su L.J., Gitler A., Cashikar A., King O.D., Auluck P.K., Geddie M.L., Valastyan J.S., Karger D.R., Lindquist S. and Fraenkel E. (2009). Bridging the gap between high- throughput genetic and transcriptional data reveals cellular pathways responding to alpha-synuclein toxicity. Nature Genetics 41:316-323.
Barshir R., Basha O., Eluk A., Smoly I.Y., Lan A. and Yeger-Lotem E. (2013). The TissueNet database of human tissue protein- protein interactions. Nucleic Acids Research 41:D841-844.
Basha O., Tirman S., Eluk A. and Yeger-Lotem E. (2013). ResponseNet2.0: Revealing signaling and regulatory pathways connecting your proteins and genes-now with human data. Nucleic Acids Research 41:W198-203.
Lan A., Ziv-Ukelson M. and Yeger-Lotem E. (2013). A context- sensitive framework for the analysis of human signalling pathways in molecular interaction networks. ISMB 2013 and Bioinformatics 29:i210-216. 32 Applied Biotechnology Group
Unraveling the structure-function relationships of proteins and molecular complexes is pivotal for an improved understanding of cellular processes. NIBN’s applied biotechnology group comprises of prominent scientists and state-of-the-art infrastructure in the fields of microscopy, protein crystallography, protein engineering as well as nano-technology, which has been further strengthened through NIBN’s establishment of the Aaron Klug Integrated Centre for Biomolecular Structure and Function. Current efforts include electron tomography (3D electron microscopy) of cells and organelles, understanding magnetosome biology with the intent to generate tailor-made magnetite particles for various nano- and bio-technological applications, understanding the structural basis of ligand receptor interactions important in drug discovery, optimizing yields of recombinant protein expression with site-specifically incorporated unnatural amino acids and functional genomics and proteomics related to reproduction and calcium biomineralization.
• Prof. Ohad Medalia • Prof. Amir Sagi • Dr. Raz Zarivach • Dr. Stas Engel
33 Applied uses of electron tomography
Background Prof. Ohad Medalia Improvements in electron microscopy, in conjunction with advances in computer-controlled systems, now enable biological specimens to be investigated at sub-critical dose exposure, thereby minimizing beam-induced radiation damage to negligible levels. The development of cryo-electron microscopy ensures “close-to- life” preparation by vitrification, even for biological samples as large as intact organelles and cells. Since neither fixation nor staining is required, vitrification maintains the integrity of a cell and causes almost no artifacts. Thus, vitrification allows cells to be arrested in various functional states. Indeed, electron tomography of ice- embedded specimens is the only methodology that allows retrieval of 3-D structural information from large polymorphic structures, such as intact cells and organelles, at a resolution of 4-6 nm. Although tomograms of intact cells cannot be enhanced using averaging procedures, they exhibit a sufficient signal-to-noise ratio to allow segmentation and comparison of macromolecular complexes in situ. As such, cryo-electron tomography can be a useful tool in medical research and drug discovery processes. Ph.D.: Weizmann Institute of Science, Israel Current research Post-doctorate: Max-Planck-Institute 1. Structural analysis of the nuclear lamina: Forming a boundary for Biochemistry, Germany between the nucleus and the cytoplasm, the nuclear envelope consists of two concentric membranes (outer and inner) Position: Associate Professor connected at nuclear pores and an underlying lamina, which is a network-like scaffold structure providing mechanical Department of Life Sciences, BGU stability for the nucleus, cell and tissue. As the main structural Faculty of Natural Sciences constituents of the nuclear lamina, lamins are also present in the nucleoplasm. These proteins are members of the intermediate Department of Biochemistry, Zurich filament (IF) protein superfamily and are probably the ancestors University of all IF proteins. Point mutations in lamin proteins cause a set of diseases recently detected in the elderly. As such, we are presently E-mail: [email protected] addressing the structure of the nuclear lamina.
Selected publications 2. Structural analysis of integrin-mediated cell adhesion: Cell Fridmann K., Mader A., Zwerger M., Elia N. and Medalia O. adhesions play an important role in the organization, growth, (2012). Advances in tomography: probing the molecular maturation and function of living cells. The interaction of cells architecture of cells. Nat Rev. Mol. Cell Biol. 13:736-742. with the extracellular matrix also plays an essential role in a variety of disease states, including tumor formation and metastasis Maimon T., Elad N., Dahan I. and Medalia O. (2012). inflammation and repair of wounded tissues. However, the The human nuclear pore complex as revealed by cryo- electron tomography. Structure 20:998-1006. structure of the machinery involved remains unknown. By understanding the structure of these macromolecular assemblies Elad N., Volberg T., Patla I., Hirschfeld-Warneken V., we will be able to suggest new approaches for inhibiting cell Grashoff C., Spatz J.P., Fässler R., Geiger B. and Medalia O. adhesion. (2013). The role of integrin-linked kinase in the molecular architecture of focal adhesions. J. Cell Sci. 126:4099-4107. 3. Electron tomography and drug discovery: Individual proteins Harapin J., Eibauer M. and Medalia O. (2013). Structural and macromolecular complexes can be detected in 3-D by analysis of supramolecular assemblies by cryo-electron electron tomography. By using labeled drug candidates (lead tomography. Structure 21:1522-1530. compounds), the specificity as well as the affinities of a drug inside a cell can be resolved in situ. For example, drug-antigen interactions can be detected and visualized in living cells at early stages in the drug development process and at relatively low cost. We are developing strategies for labeling drugs in order to resolve their network of interactions within cells.
34 Genes and gene products in comparative and applied Prof. Amir Sagi endocrinology: Regulation of sexual differentiation, reproduction, calcium mobilization and growth in marine and freshwater invertebrates
Background Crustacean models are employed in our laboratory for the study of genes and gene products related to processes of sexual differentiation and skeletal biomineralization. In particular, we study the endocrine regulation of sexual differentiation, gonad maturation, growth, molt and the related processes of calcium mobilization and biomineralization. Control of the above events will enable the development of biotechnological tools for; crop improvement via monosex culturing, soft shell-based products and human food Ph.D.: The Hebrew University of additives and drugs. Jerusalem, Israel Current research Post-doctorate: Woods Hole Marine 1. Gonad maturation, vitellogenesis and lipoprotein synthesis. Biological Laboratory and University The vitellogenin gene, its expression pattern and bioinformatic of Connecticut, USA entities. Position: Professor 2. Regulation of growth, molt and calcium mobilization. The role of ecdysteroids and eyestalk neuropeptides in the regulation of Department of Life Sciences events related to the molt cycle, exoskeleton, gastrolith formation Faculty of Natural Sciences and gene expression in the above target organs. 3. Functional genomics of growth regulators and skeletal proteins E-mail: [email protected] related to gender and biomineralization. 4. Sex-determination, a search for sex-specific genomic, transcriptomic and proteomic markers to asses the role of the Selected publications androgenic gland and its secretion. Shechter A., Glazer L., Cheled S., Mor E., Weil S., Berman A., 5. Sexual plasticity, including the regulatory role of the androgenic Bentov S., Aflalo E.D., Khalaila I. and Sagi A. (2008). A gastrolith gland in sex-differentiation and intersexuality of crustaceans. protein serving a dual role in the formation of an amorphous Development of biotechnologies for the production of monosex mineral containing extracellular matrix. Proc. Nat. Acad. Sci. USA. crustacean populations. 106(20):7129-7134. Ventura T., Rosen O. and Sagi A. (2011). From the discovery of 6. Food additives and drugs based on natural products derived from the crustacean androgenic gland to the insulin-like hormone in edible crustaceans. Both cellular and extracellular components six decades. Gen. Comp. Endocrinol. 173(3):381-388. Including are being tested. cover art. Ventura T. and Sagi A. (2012). The insulin-like androgenic gland hormone in crustaceans: from a single gene silencing to a wide array of sexual manipulation-based biotechnologies. Biotechnology Advances 30:1543-1550. Sagi A., Manor R. and Ventura T. (2013). Gene Silencing in Crustaceans: From Basic Research to Biotechnologies. Genes 4:620-645. Ventura T., Manor R., Aflalo E.D., Chalifa-Caspi V., Weil S., Sharabi O. and Sagi A. (2013). Post-embryonic transcriptomes of the prawn Macrobrachium rosenbergii: multigenic succession through metamorphosis. PLoS ONE 8(1):e55322. Rosen O., Weil S., Manor R., Roth Z., Khalaila I. and Sagi A. (2013). A crayfish insulin-like binding protein: Another piece in the androgenic gland insulin-like hormone puzzle is revealed. Journal of Biological Chemistry 288:22289-22298.
35 Crystallographic studies of biological macromolecules Dr. Raz Zarivach Background Structural biology aims to understand the chemistry, interactions and basic biological functions governed by the three-dimensional structure of macromolecules. Knowledge of the 3-D structure of a protein can provide enormous insight into the function of that protein, facilitating elucidation of its biochemical function and its interactions with other proteins, RNA, DNA, or membranes in the cell. Similarly, protein-ligand interactions are crucial in many biological processes with implications for drug targeting and gene expression.
X-ray crystallography is the most prolific technique for the structural analysis of proteins and protein complexes and remains the 'gold standard' in terms of accuracy. Using X-ray crystallography, we can now determine high-resolution structures, up to atomic or even electronic details, enabling a full understanding of macromolecules and their interactions. Crystallography is the key methodology for macromolecule-ligand interaction research in structure-based Ph.D.: Weizmann Institute of Science, drug design, either shedding light on the molecular details of such Israel interactions or for use in ligand-screening of large ligand libraries. Post-doctorate: University of British Columbia, Canada Current research
Position: Senior Lecturer 1. Magnetotactic bacteria are a phylogenetically and morphologically diverse group of microorganisms that share an Department of Life Sciences ability to create magnetosomes. Magnetosomes are biomineral Faculty of Natural Sciences organelles that sense geomagnetic fields and aid the bacteria in aligning themselves accordingly. The magnetosome organelle E-mail: [email protected] is comprised of 30-50 nm aligned iron oxide magnetite crystals, surrounded by a lipid bilayer membrane vesicle. There are several types of magnetosome-forming proteins, all encoded by genes Selected publications within a genomic island common to magnetotactic bacteria. These proteins include a set of incorporated membrane proteins Zeytuni N., Ozyamak E., Ben-Harush K., Davidov G., Levin M., Gat Y., Moyal T., Brik A., Komeili A. and Zarivach that facilitate vesicle formation, vesicle localization and iron R. (2011). Self-recognition mechanism of MamA, a transport and a set of proteins that control magnetite formation magnetosome-associated TPR-containing protein, and size. A large number of the proteins involved in magnetosome promotes complex assembly. Proc. Natl. Acad. Sci. USA. formation are of unknown function. Magnetite crystals formed by 108(33):E480-E487. magnetotactic bacteria have a high potential for applications in nanotechnology and biotechnology, which require specifically Zeytuni N., Baran D., Davidov G. and Zarivach R. designed particle surfaces of distinct shape and size. Abiomimetic (2012). Inter-phylum structural conservation of the magnetosome-associated TPR-containing protein, approach, utilizing purified proteins, possibly mutated forms, MamA. J. Struct. Biol. 180(3):479-487. enables design and control of the magnetite crystals, for uses such as protein tags and fluorophores. For commercial use, magnetite Zeytuni N. and Zarivach R. (2012). Structural and crystals with a permanent stable magnetic dipole moment at functional discussion of the tetra-trico-peptide repeat, room temperature and with a specific size can be designed. a protein interaction module. Structure 20(3):397-405. 2. As a member of the Faculty of Natural Sciences at BGU, the Guttman C., Davidov G., Shaked H., Kolusheva S., Bitton laboratory serves as a core facility and is involved in protein R., Ganguly A., Miller J.F., Chill J.H. and Zarivach R. (2013). structure determination and structural studies of other proteins. Characterization of the N-terminal domain of BteA: A Collaborative research is performed with the laboratories of Bordetella Type III secreted cytotoxic effector. PLoS ONE 8(1):e55650. Dr. Amir Aharoni, which addresses directed evolution, Prof. Varda Shoshan-Barmatz, which studies membrane proteins Guttman C., Davidov G., Yahalom A., Shaked H., Kolusheva and ion channels and Prof. Amir Sagi, which investigatess S., Bitton R., Barber-Zucker S., Chill J.H. and Zarivach R. biomineralization. (2013). BtcA, a Class IA Type III Chaperone, Interacts with the BteA N-Terminal Domain through a Globular/Non- Globular Mechanism. PLoS ONE 8(12):e81557.
36 Interactions between bio-molecules as a target for pharmacological Dr. Stas Engel intervention
Background Protein-protein interactions (PPI) play an essential role in virtually all cellular processes. In biological systems, information is conveyed by the action of small-molecule messengers (Ca2+, cAMP, neurotransmitters, etc.) and via PPI. While mimetics of the former are widely used to intervene in the functions of biological systems, we have only a rudimentary capacity to manipulate PPI. Increasingly, PPI are considered to be potential targets for the development of selective tools to modulate the functions of biological systems. Targeting PPI using drugs may represent a new strategy to combat diseases that have remained intractable through conventional therapies.
Current research
1. Functional mimetics of the intracellular domain (ICD) of Ph.D.: Ben-Gurion University, Israel G protein coupled receptors (GPCR) as a tool for studying the structural basis of GPCR interactions with intracellular Post-doctorate: The National Institutes molecular targets and for drug discovery: We are developing of Health, USA soluble mimetics of the ICD to solve the problem of receptor availability for structural studies and thus enable the direct study Position: Senior Lecturer of GPCR interactions with intracellular effector molecules such as G proteins, arrestins, etc. In particular, soluble mimetics will allow Department of Clinical Biochemistry NMR spectroscopy and crystallography, which have only had and Pharmacology limited use to date, to be performed. Furthermore, ICD mimetics could provide a novel and suitable platform for developing Faculty of Health Sciences compounds that interfere with the formation of GPCR complexes E-mail: [email protected] with intracellular effector molecules. Such compounds will aid GPCR signal transduction research and may provide substantial therapeutic benefit. Selected publications Tikhonova I.G., Sum C.S., Neumann S., Engel S., Raaka B.M., 2. Combinatorial in vitro selection methods to identify active Costanzi S. and Gershengorn M.C. (2008). Discovery of novel compounds targeting protein-protein interactions, in agonists and antagonists of the free fatty acid receptor particular the formation of GPCR/G protein complexes: (FFAR1) using virtual screening. Journal of Medicinal Despite recent progress in the discovery of small-molecules Chemistry 51(3): 625–633. that bind to protein-protein contact surfaces, the logistics of Engel S., Skoumbourdis A., Childress J., Neumann S., targeting small-molecule compounds to large protein-protein Deschamps J.R., Thomas C.J., Colson A.O., Stefano C. and interfaces remains a challenge. When definitive structural data Gershengorn M.C. (2008). A Virtual Screen for Diverse are unavailable, statistical approaches of bulk selection, such as Ligands: Discovery of Selective G Protein-Coupled Receptor in vitro display methods, are instrumental in the discovery of new Antagonists. Journal of the American Chemical Society active compounds. We will use combinatorial in vitro selection 130(15):5115-5123. methods to identify active peptides that target the formation of specific GPCR/G protein complexes. Active peptides targeting Tikhonova I.G., Best R.B., Engel S., Hummer G., Gershengorn M.C. and Costanzi S. (2008). Atomistic insights into rhodopsin the ICD/G protein interfaces may represent a starting point for activation from a dynamic model. Journal of the American the development of a new class of signaling, pathway-specific Chemical Society 130(31):10141-10149. therapeutics to treat GPCR-related disorders.
Mendelson M., Yossef R., Appel M.Y., Zilka A., Hadad U., 3. Molecular modeling in structure-based drug discovery: It Afergan F., Rosental B., Engel S., Braiman A., and Porgador A. is a known fact that the binding pockets (cavities) observed (2012). Dimerization of NKp46 receptor is essential for NKp46- in the structure of protein/ligand complexes are frequently mediated lysis: characterization of the dimerization site by undetectable in the absence of the ligand. We are developing epitope mapping. Journal of Immunology 188(12):6165- 6174. novel computational approaches to predict the location of “hot- spots” on the surface of proteins, which have a propensity to form Kuttner Y.Y. and Engel S. (2012). Protein hot spots - the islands interactions with small molecules. This approach will potentially of stability. Journal of Molecular Biology 415(2):419-428. facilitate the rational design of ligands that bind at protein- protein interfaces. Kuttner Y.Y., Nagar T. and Engel S. (2013). Surface dynamics in allosteric regulation of protein-protein interactions: Modulation of calmodulin functions by Ca2+. PLOS Computational Biology 9(4): e1003028. 37 Infra-Structure Supporting Group
The Core Facilities group is focused on providing expertise in consort with state-of-the-art equipment to advance all research projects in an optimal manner. NIBN’s core services include: the Genetics Unit, the Proteomics Unit, the Bioinformatics Unit, the Crystallography Unit and the Microscopy Unit. These units provide advanced services including: identifying novel proteins and their interactions with macromolecules (proteins, RNA and DNA), DNA microarrays analysis, DNA sequencing, cells sorting, high resolution microscopy, Cell-sorting Unit, Center for Protein Crystallization, robotic high-throughput screening and bio- informatics units.
• Dr. Vered Caspi • Dr. Anat Shahar • Dr. Micha Volokita • Dr. Alon Zilka
38 Core Facilities Bioinformatics Core Facility Headed by Dr. Vered Caspi
Ph.D.: Weizmann Institute of Science, Israel Post-Doctorate: Weizmann Institute of Science, Israel Position: Research Associate Bioinformatics Core Facility, Head E-mail: [email protected] Mission and services The NIBN Bioinformatics Core Facility was established in September 2003 with the aim of providing scientists with opportunities to significantly advance their research with cutting edge bioinformatics resources and methodologies. To date, the Core Facility provides data analysis services, consultation and training to scientists all over Israel from both academia and industry. Our main areas of expertise include the analysis and re-analysis of data obtained from genomic technologies (e.g. Next Generation Sequencing, Mass spec proteomics profiling and DNA microarrays), as well as in mining biological databases, bioinformatics programming and biostatistics. In addition, assistance is provided in designing appropriate experiments using genomic technologies and in writing relevant sections in grant proposals. Our team includes bioinformaticians and programmers with strong backgrounds in biology, bioinformatics and statistics, with much prior expertise in analyzing high-throughput genomic datasets. Our efforts are supported by the necessary hardware infrastructure, including cutting-edge commercial and publicly available software. Details of our main areas of expertise and the services we provide are shown below. Next Generation Sequencing (NGS) data analysis • de novo sequence assembly and annotation of novel genomes and transcriptomes • Gene expression profiling (RNA-Seq) • Protein-DNA and protein-RNA interaction analysis (Chip-Seq) • Micro-RNA discovery and profiling • Comparative bacterial genome analyses and metagenomics Mass Spectrometry Proteomics Profiling • Construction of a reference proteome • Differential protein expression analysis • Pathway and gene ontology enrichment analyses DNA microarray data Analysis and meta-analysis • Differential gene expression analysis • Genetic studies using SNP and CNV arrays Selected publications Amir E.D., Bartal O., Morad E., Nagar T., Sheynin J., Parvari R. and Chalifa-Caspi V. (2010). KinSNP software for homozygosity mapping of disease genes using SNP microarrays. Hum Genomics 4(6):394-401.
Grafi G., Chalifa-Caspi V., Nagar T., Plaschkes I., Barak S. and Ransbotyn V. (2011). Plant response to stress meets dedifferentiation. Planta, 233(3):433-438.
Mazor M., Alkrinawi S., Chalifa-Caspi V., Manor E., Sheffield V.C., Aviram M. and Parvari R. (2011). Primary ciliary dyskinesia caused by homozygous mutation in DNAL1, encoding dynein light chain 1. Am. J. Hum. Genet. 88(5):599-607.
Ventura T., Manor R., Aflalo E.D., Chalifa-Caspi V., Weil S., Sharabi O. and Sagi A. (2013). Post-embryonic transcriptomes of the prawn Macrobrachium rosenbergii: multigenic succession through metamorphosis. PLoS One 8(1):e55322.
Bakshi S., Chalifa-Caspi V., Plaschkes I., Perevozkin I., Gurevich M. and Schwartz R. (2013). Gene expression analysis reveals functional pathways of glatiramer acetate activation. Expert Opin. Ther. Targets 17(4):351-362.
Toker L., Bersudsky Y., Plaschkes I., Chalifa-Caspi V., Berry G.T., Buccafusca R., Moechars D., Belmaker R.H. and Agam G. (2014). Inositol-Related Gene Knockouts Mimic Lithium's Effect on Mitochondrial Function. Neuropsychopharmacology 39(2):319-328.
39 Core Facilities Crystallography Unit Headed by Dr. Anat Shahar
Ph.D.: Technion, Israel Institute of Technology Position: Research Associate Macromolecular Crystallography Research Center (MCRC), Head E-mail: [email protected]
Background
Macromolecular Crystallography uses X-ray radiation to determine the three dimensional structure of proteins and nucleic acids. X-ray crystallography remains the gold standard technique achieving results in the high- resolution range, up to atomic details. The knowledge of molecular structures at atomic level resolution. facilitates research into protein-ligand and protein-protein interactions, is a pre-requisite for structure-based functional studies and rational drug design as well as for understanding various biochemical processes and biological systems. The Macromolecular Crystallography Research Center (MCRC) was established in order to address a variety of such biological questions.
Structure determination of biological macromolecules by X-ray crystallography is a linear process, which involves several steps including cloning, expression, purification and crystallization of the target molecules as well as data collection and model building. However, producing high-quality and well diffracting crystals remains the major bottleneck in this process. In order to overcome this challenge, the MCRC is fully equipped with the appropriate reagents and robotics for providing the services below:
Services • Crystallization setup experiments using various commercial kits to screen for appropriate crystallization conditions • Optimization of crystal growth conditions • Diffraction analysis using either a home source diffractometer or the European Synchrotron Radiation Facility (ESRF) in Grenoble, France • 3D structure determination Equipment 1. Rigaku RU-H3RHB X-Ray diffractometer composed of: – Rotating-anode X-ray generator (Rigaku) – Confocal Max-FluxTM (CMF) beam conditioning optics (Osmic Inc.) – MAR345 image plate detector (Marresearch) 2. Marresearch MarμX X-ray system composed of: – GeniX-3D Cu Microbeam X-ray generator (Xenocs) – MAR345 image plate detector (Marresearch) – Low temperature system (Oxford cryosystems)
Crystallization system including: • Rock maker (Formulatrix) software for designing, automatic set-up and tracking of crystallization experiments • Formultor (Formulatrix) liquid handler • NT8 Drop Setter (Formulatrix) automated dispenser • Rock Imager (Formulatrix) automated imaging system • Rock Maker Web (Formulatrix) for following after images of the crystallization plates. The website can be reached from both inside and outside Ben-Gurion University.
40 Core Facilities
Genetics Unit Headed by Dr. Micha Volokita
Ph.D.: The Hebrew University of Jerusalem, Israel Post-Doctorate: Michigan State University, USA Position: Research Associate DNA microarray and sequencing laboratory, Head E-mail: [email protected]
Background The NIBN core laboratory for DNA microarrays and DNA sequencing provides a number of services that rely on high-priced instruments not usually at the daily disposal of a standard level research laboratory.
Services: • DNA Sequencing • Genotyping by analysis of DNA fragments • Whole genome expression analyses • Whole genome methylation mapping • Whole genome mapping of cis-regulatory elements • Single nucleotide polymorphisms (SNPs) genotyping • Detection of unknown genetic mutations and SNPs • Resequencing • Tiling arrays
Equipment • ABI Prism 3100 genetic analyzer • Affymetrix DNA microarray platform, which includes a GeneChip Scanner 3000 7G, a Fluidics Station 450, a Hybridization Oven 640, and a powerful computer workstation with dual Xeon processors loaded with GeneChip Operating Software (GCOS) • Transgenomics WAVE system instruments: A denaturing high pressure liquid chromatography (dHPLC) machine • 2100 Electrophoresis Bioanalyzer
Selected publications Narkis G., Ofir R., Landau D., Manor E., Volokita M., Hershkowitz R., Elbedour K. and Birk O.S. (2007). Lethal Contractural Syndrome Type 3 (LCCS3) Is Caused by a Mutation in PIP5K1C, Which Encodes PIPKI gamma of the Phophatidylinsitol Pathway. Am J Hum Genet. 81:530-539. Najami N., Janda T., Barriah W., Kayam G., Tal M., Guy M. and Volokita M. (2008). Ascorbate Peroxidase Gene Family in Tomato: Its Identification and Characterization. Molec Genet Genomics. 279:171-82. Volokita M, Rosilio-Brami T and Rivkin N, Zik M. (2011) Combining comparative sequence and genomic data to ascertain phylogenetic relationships and explore the evolution of the large GDSL-lipase family in land plants. Mol Biol Evol. 28(1):551-65.
41 Core Facilities
Cytometry, Proteomic and Microscopy Unit Headed by Dr. Alon Zilka
Ph.D.: Ben-Gurion University of the Negev, Israel Post-Doctorate: Ben-Gurion University of the Negev, Israel Position: Research Associate Cytometry, Proteomic and Microscopy Unit, Head E-mail: [email protected]
Background
The service unit is equipped with high end instruments to help identify inter-molecular interactions between proteins, peptides, nucleic acids and other small molecules. The unit contains a confocal microscope and a high throughput Fluorescent/confocal microscope for quality/large scale image acquisition and analysis. Flow cell analyzers and a high speed sorter enable the analysis and sorting of cell sub populations. A Robot and a high speed plate reader provide a high throughput workbench connected to a powerful fluorescence/ luminescence/absorbance analyzer. The unit contains the following instruments: • A laser scanning confocal microscope FV1000 equipped with 405, 488 and 543nm lasers (Olympus). • Proteon - A protein interaction array system (Surface Plasmon Resonance) for measuring inter-molecular affinity in a high throughput mode (Biorad). • Monolith - A Micro Scale Thermophoresis instrument for measuring inter-molecular affinity using capillaries to minimize the amounts of materials needed (Nanotemper). • Operetta - A high throughput fluorescent/confocal microscope for large scale image acquisition and statistical image analysis (Perkin Elmer) • Synergy - A high speed cell sorter containing 2 independent sorting modules with 375, 488 and 561/594nm lasers on module 1 and 405, 488 and 640nm lasers on module 2. Each module has 6 fluorescent channels and can sort up to 4 sub populations (iCyt) • Two Flow cytometers analyzers, one with 488 and 633nm lasers and 4 fluorescent channels (Calibur) and another with 405 and 640nm lasers and 5 fluorescent channels an EV channel and a sample auto loader (Eclipse) (BD and iCyt, respectively). • A high throughput freedom Evo Robot system connected to a high speed sophisticated plate reader (M1000) (Tecan)
Selected publications
Zilka A., Garlapati S., Dahan E., Yalosky V. and Shapira M. (2001). Developmental regulation of Heat Shock Protein 83 in Leishmania; 3’ processing and mRNA stability control transcript abundance and translation is directed by a determinant in the 3’- untranslated region. Journal of Biological Chemistry. 276:47922-47929. Zilka A., Landau G., Hershkovitz O., Bloushtain N., Bar-Ilan A., Benchetrit F., Fima E., van Kuppevelt T.H., Gallagher J.T., Elgavish S. and Porgador A. (2005). Characterization of the Heparin/Heparan Sulfate Binding Site of the Natural Cytotoxicity Receptor NKp46. Biochemistry. 44:14477-14485. Hershkovitz O., Jivov S., Bloushtain N., Zilka A., Landau G., Bar-Ilan A., Glicklis R., van Kuppevelt T. H. and Porgador A. (2007). Characterization of the recognition of tumor cells bythe natural cytotoxicity receptor, NKp44. Biochemistry. 46:7426-7436. Cagnanol E., Hershkovitz O., Zilka A., Bar-Ilan A., Sion-Vardy1 N., Mandelboim O., Benharroch D. and Porgador A. (2008). Expression of ligands to NKp46 in benign and malignant melanocyte. Journal of Investigative Dermatology. 128:972-979. Zilka A, Mendelson M, Rosental B, Hershkovitz O and Porgador A. (2010). Generating NK cell receptor-Fc chimera proteins from 293T cells and considerations of appropriate glycosylation. Methods Mol Biol. 612:275-283.
42 Dr. Roee Atlas Transforming basic biological Deputy Director research into therapy for Patients Professional background: Prior to his appointment at NIBN, Dr. Roee Atlas held various positions in the biotech industry. As such, he gained considerable expertise in managing the development of pharmaceuticals/biologicals from the initial phases to the preclinical and clinical stages. Specifically, he led tissue regeneration product development in an early- stage company developing an autologous cell therapy technology. Prior to that, Dr. Atlas led a seed company centered on the development of small molecules for use as anesthesia/pain indications. While at Omrix Bio-pharmaceuticals in Rehovot, he headed the tissue regeneration unit of the R&D department. Leading a multidisciplinary team consisting of scientists, engineers, and regulatory personnel, Dr. Atlas focused on the development of therapeutic products based on cells, proteins and polymer combinations to treat bone, blood vessel and muscle pathologies.
Functions of the NIBN Deputy Director: Ph.D.:Weizmann Institute of Science, Israel The NIBN is a unique institute that provides scientists with both the environment and tools to explore the applied potential of their basic research. To realize this Post-doctorate: Columbia Medical potential, it is essential that projects be developed in an industry-oriented School, New York City manner. Accordingly, a major role of the Deputy Director is to facilitate efforts by leading NIBN scientists to transfer basic academic research onto an applied E-mail: [email protected] path, culminating in a commercially viable technology or therapeutic product. Implementation of those strategies leading to an industry-oriented research Selected publications program with the concomitant scientific leverage necessary for commercial interest is of the utmost importance for the NIBN. To generate this traction, NIBN Caspi M., Atlas R., Kantor A., Sapir T. and Reiner O. (2000). Interaction between LIS1 and doublecortin, two lissencephaly supports many outsourcing activities often beyond the scope of an academic gene products. Hum. Mol. Genet. 9:2205-2213. setting that serve to complement and propel basic R&D efforts into commercial- valued candidate technologies or therapeutic products. Atlas R., Behar L., Elliott E. and Ginzburg I. (2004). The insulin- like growth factor mRNA binding-protein IMP-1 and the Ras- regulatory protein G3BP associate with tau mRNA and HuD NIBN places significant emphasis on early dialogue with regulatory and clinical protein in differentiated P19 neuronal cells. J. Neurochemistry experts to understand unmet medical needs, the fastest route to clinical testing, 89:613-626. and the most reliable path for the development of clinically safe and reliable Elliott E*., Atlas R*., Lange A. and Ginzburg I. (2005). BDNF induces products. Under the guidance of the Deputy Director, NIBN’s multi-pronged a rapid dephosphorylation of tau protein in differentiated P19 scientific approach relies on an experienced management team to fuel external neurons. European Journal of Neuroscience 22:1081-1089. interest in NIBN’s projects in the form of collaborations, out-licensing and spin-off (*authors contributed equally to the study). opportunities. Atlas R., Behar L., Sapoznik S. and Ginzburg I. (2007). A dynamic association with Polysomes during P19 neuronal differentiation Major Activities of the Deputy Director: and a UTR-dependent translation regulation of the tau mRNA by the tau mRNA-associated proteins IMP1, HuD, and G3BP1. • Implementation of appropriate strategies to translate basic Journal of Neuroscience Research 85:173-183. research into projects bearing significant leverage, ultimately creating therapeutic product candidates of commercial value to Lesman A., Koffler J., Atlas R., Blinder Y.J., Kam Z. and Levenberg the NIBN. S. (2011). Engineering vessel-like networks within multicellular fibrin-based constructs. Biomaterials 32(31):7856-7869. • Strengthening NIBN-funded projects through in-depth understanding of the underlying science and commercial Patent Applications: competition. “A Fibrin based therapeutic preparation and use thereof”, Application # 207586 Roee Atlas, Israel Nur, Lily Bar, Roberto • Engaging appropriate VC’s, investment bodies, SMEs and big Meidler, Dharanajh Sridevi, Charito Buenusceco, Anthony Kim. Pharma for potential collaborations, business opportunities, and 12/2010 commercialization of NIBN applied technologies.
“Device for Administrating fluids and uses thereof”, Application # • Maintenance, expansion, and strengthening of the NIBN patent 207715 Moti Meron, Israel Nur, Roee Atlas. 12/2010 portfolio. “Device for spraying fluids in proximity to a surface”, Application • Creating spin-off companies, licensing of technologies and # 213375 Roee Atlas, Moti Meron, Assaf Gershonovitch, Amatzia providing services based on NIBN state-of-the-art facilities. Gantz. 6/2011 “Anesthetic Neutralization methods”, Application # 61748453 • Promoting collaborations with research institutes, governmental Sagi Polani, Asher Polani, Roee Atlas 03/2013 bodies and non-profit organizations to accelerate development of NIBN technologies. “Cytotoxic methods using Peroxynitrite”, Application # 61738410 Sagi Polani, Asher Polani, Roee Atlas 12/2012 • Pursue relevant grant opportunities to support on-going projects.
43 The National Institute for Biotechnology in the Negev Ltd.
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