Annual Report 2004 NCMLS

Transcription

1 Annual Report 2004 NCMLS

2 Postal address PO Box HB Nijmegen The Netherlands Visiting address Geert Grooteplein GA Nijmegen T: +31 (0) F: +31 (0) E: I: Editing: Dr. Margaret Mullally Design: Final Design Photography: Theo Hafmans and Frank Muller Printed by: Thieme MediaCenter, Nijmegen 2 NCMLS

3 One of our prominent achievements of this year was the introduction of the Principal Investigator (PI) model system in July 2004, a system that recognises individual researchers as leaders within defined research themes. Foreword I am pleased to present our annual report It provides an overview of the achievements of the Nijmegen Centre for Molecular Life Sciences in both science and education. The year 2004 was an extremely important and fruitful one. Self evaluation documentation and a site-visit report from an international scientific advisory committee, required for the renewal of the recognition of the NCMLS as a graduate school at the KNAW and VSNU, were prepared and submitted. I am grateful to all who have contributed to this large undertaking. One of our prominent achievements of this year was the introduction of the Principal Investigator (PI) model system in July 2004, a system that recognises individual researchers as leaders within defined research themes. It provides greater freedom to individual researchers, and promises to particularly enhance the career development of talented young researchers within the organisation. Greater emphasis continues to be placed on the closer interactions with the clinical departments of the Radboud University Nijmegen Medical Centre, in addition to intensifying collaborations with both local and international research institutes. The NCMLS graduate school also filed an application for accreditation of a new honours MSc research programme, Molecular Mechanisms of Disease (MMD) at the NVAO (the Dutch-Flemish accreditation organization), and member of the European Consortium for Accreditation. The NVAO accreditation will guarantee the quality of the international research MSc programme in a European context. We hope to attract the most talented students in the field of molecular life sciences and molecular medicine, creating a basis for continuance of excellence within the NCMLS PhD programme. A renewed PhD programme has also been implemented giving impetus to the NCMLS as a recognised graduate school. Our annual report for 2004 highlights some of our younger researchers within the various research themes of our centre and their contribution to the understanding of the molecular and cellular basis of disease. Wishing you enjoyable reading, Carl Figdor Scientific Director NCMLS Annual Report 2004 Carl Figdor Foreword 3

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5 Table of contents Page Foreword 3 Nijmegen Centre for Molecular Life Sciences 7 Research 8 Research Themes 8 Awards 9 Societal Impact 9 Technology Platforms 10 The NCMLS as Graduate School 11 MSc Molecular Mechanisms of Disease 11 International PhD programme 12 Selected Research Highlights NCMLS Theme 1: Infection, immunity and tissue repair 13 Theme 1a Infection and autoimmunity 14 Theme 1b Immune regulation 17 Theme 1c Tissue engineering and pathology 22 Theme 2: Metabolism, transport and motion 25 Theme 2a Energy and redox metabolism 26 Theme 2b Membrane transport and intracellular motility 29 Theme 3: Cell growth and differentiation 31 Theme 3a Functional genomics 32 Theme 3b Neural development 36 Theme 3c Signalling networks 38 Theme 3d Protein structure and design 39 Facts and figures 42 Members of NCMLS 43 Scientific publications Annual Report 2004 Table of contents 5

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7 The NCMLS seeks to achieve greater insights into the complexity of living cells with the purpose of obtaining a multifaceted knowledge of both normal and pathological processes. The NCMLS will pursue its goals Nijmegen in the Centre interests forof Molecular curiositylife driven Sciences research and education. The NCMLS aims to advance innovation in translational research based on the integration of diverse scientific expertise in molecular and medical sciences. Governing Body Prof. Dr. Carel van Os (Director of Research, Radboud University Nijmegen Medical Centre) Prof. Dr. Dirk Ruiter (Dean of Faculty of Radboud University Nijmegen Medical Centre) Prof. Dr. Sjoerd Wendelaar-Bonga (Dean of Faculty of Science, Radboud University Nijmegen) Management Team Prof. Dr. Carl Figdor (Scientific Director, NCMLS) Prof. Dr. Jan van Hest (Faculty of Science) Dr. Margaret Mullally (Assistant Scientific Director, NCMLS) Prof. Dr. Jan Smeitink (Faculty of Radboud University Nijmegen Medical Centre) Research Council Prof. Dr. Gosse Adema (Theme leader) Prof. Dr. Rene Bindels (Sub-theme leader and chair of grants committee) Prof. Dr. Han Brunner (Chair of Science committee, UMCN) Prof. Dr. Carl Figdor (Scientific Director, NCMLS) Prof. Dr. Jan van Hest (Member of Management team) Prof. Dr. Martijn Huynen (CMBI representative) Prof. Dr. John Jansen (DPTE representative) Prof. Dr. Willem Melchers (Chair of Post-doc platform) Prof. Dr. Joost Schalkwijk (Sub-theme leader) Prof. Dr. Jan Smeitink (Member of Management team) Prof. Dr. Henk Stunnenberg (Theme leader) Prof. Dr. Bé Wieringa (Theme leader) Dr. Margaret Mullally (Assistant Scientific Director, NCMLS, member of Management team) Scientific Advisory Committee Prof. Dr. Hans Clevers (chairperson), Hubrecht Laboratory, Utrecht, The Netherlands Prof. Dr. Tony Mikos, Rice University, Houston, US. Prof. Dr. Paola Ricciardi-Castagnoli, University of Milano-Bicocca, Italy Prof. Dr. Rosario Rizzuto, University of Ferrara, Italy. Prof. Dr. Luis. Serrano, EMBL Heidelberg, Germany Prof. Dr. Gert-Jan van Ommen, Leiden University Medical Centre (LUMC), The Netherlands Prof. Dr. John Walker, MRC Dunn, Cambridge, UK. The NCMLS was first recognised as a graduate school in 1995 and again in 2000 by the Royal Netherlands Academy of Arts and Sciences (KNAW). A request for re-accreditation was submitted in 2004 and this is currently under review at the KNAW. Mission: Understanding the cellular basis of disease. The NCMLS seeks to achieve greater insights into the complexity of living cells with the purpose of obtaining a multifaceted knowledge of both normal and pathological processes. The NCMLS will pursue its goals in the interests of curiosity driven research and education. The NCMLS aims to advance innovation in translational research based on the integration of diverse scientific expertise in molecular and medical sciences. The NCMLS brings together researchers in several groups at the Radboud University Nijmegen Medical Centre and the Faculty of Science. There is a particular emphasis on the relationship between fundamental and translational research. The NCMLS seeks to achieve greater insights into the complexity of living cells with the purpose of obtaining a multifaceted knowledge of both normal and pathological processes. The NCMLS will pursue its goals in the interests of curiosity driven research and education. The NCMLS aims to advance innovation in translational research based on the integration of diverse scientific expertise in molecular and medical sciences. Annual Report 2004 Nijmegen Centre for Molecular Life Sciences 7

8 Within the NCMLS, all research and education is linked to the study of molecular life sciences in relation Research to disease. The centre focuses on three thematic research areas, which are further divided into sub-themes. Research Themes Within the NCMLS, all research and education is linked to the study of molecular life sciences in relation to disease. The centre focuses on three thematic research areas, which are further divided into sub-themes. Theme 1: Infection, immunity and tissue repair (Prof. G. Adema) Infection and autoimmunity (Prof. J Schalkwijk), Immune regulation (Prof. G. Adema), and Tissue engineering and pathology (Dr. A. van Kuppevelt). The immune system has the dual task of eliminating pathogens and eradicating incipient tumours, while preventing auto-reactive responses harmful to the host. In maintaining this balance, there is a complex interplay between immune and tissue cells and many stimulatory and inhibitory circuits operate simultaneously. Outcomes are further shaped by genetic and environmental factors. Deregulation of this intricate balance is associated with human diseases, ranging from inflammatory and autoimmune disorders to cancer, infection and transplantation disorders. In each case, prolonged deregulation can initiate a cascade of molecular events ultimately leading to tissue damage and destruction. Tissue engineering is a relatively new field of research aimed at repairing or replacing damaged tissues by implanting smart synthetic biomatrices or stem cells. Immune control is intrinsically involved both in tissue acceptance and in preventing autoimmune attacks on engineered tissues. A multi-disciplinary approach (molecule-mouse-patient) is taken to define the molecular basis of immune regulatory circuits, events that trigger or fuel immune-related disorders and infectious diseases, and tissue pathology & regeneration as well as stem cell behaviour & differentiation. Theme 2: Metabolism, transport and motion (Prof. B. Wieringa) Energy and redox metabolism (Prof. B. Wieringa) and Membrane transport and intracellular motility (Prof. R. Bindels). The study of disease at the molecular level but in the context of the macromolecular world of cellular organelles, the intact cell, or organs and tissues in the entire organism is central to the NCMLS. For example, intrinsic genetic problems or extrinsic factors causing cellular energy deprivation, ion and metabolite and water transport failure, toxic accumulation of intermediates, or ischemia and anoxia caused by cerebro-vascular obstruction are related to a range of diseases, including cancer, neuropathy and myopathy, degenerative disorders like Alzheimer s and Parkinson or ischemic/anoxic organ failure. In addition, conditions such as obesity and type II diabetes or some aspects of ageing, have a direct connection to metabolism and molecular transport and motion. Energy and redox metabolism is also often linked to membrane transport and intracellular motility. Metabolites such as ATP, produced in key pathways like glycolysis and mitochondrial respiratory complexes are consumed as fuel or are needed as, for example, as co-factors for, drugtransporters or the acto-myosin motor and sliding machinery involved in cell movements. Defects in metabolic signalling are often involved in renal disease, cardiomyopathy or brain and muscle disorders due to defects in the production or assembly of ATPases, water channels, or the mitochondrial machinery. Theme 3: Cell growth and differentiation (Prof. H. Stunnenberg) Functional genomics (Prof. H. Stunnenberg), Neural development (Prof. G. Martens), Signalling networks (Prof. J. van Zoelen), and Protein structure and design (Prof. W. de Jong). The fate of all cells lies in the fine balance between growth and differentiation. If this balance is disturbed, uncontrolled growth and deregulated cellular development can lead to disease. Studying the molecular processes that underlie growth and differentiation is pivotal to a basic understanding of the causes of many diseases and malfunctions. Multi-level analysis is used to study the functional blueprint of all cellular decisions, a functional genomics approach is pursued that ranges from deciphering the genome in terms of actively transcribed genes under defined cellular circumstances (such as normal differentiation versus unregulated proliferation) to specific disease-linked genomic studies. Since the single cell cannot be viewed in isolation from its cellular surrounding, decisions within the cell need to be linked to external cues and constraints, and the translation of this approach within cells is at the core of research on signalling networks. In order to understand the molecules that convey the information packaged in the functional genomic blueprint as well as the signals from the cellular outside world, it is also necessary to gain a better understanding of the protein structure and design of these molecules that finally convey the growth and differentiation decisions. Valuable insights can be gained from investigating a specific differentiation programme and neural development is studied as a special case. 8 Research NCMLS

9 Various members of the NCMLS obtain funding through national and international patient-oriented non-profit organizations such as the Kidney Foundation, Dutch Cancer Society, the Diabetic Foundation, or the Rheumatoid Societal impact Various members of the NCMLS obtain funding through national and international patient-oriented non-profit organizations such as the Kidney Foundation, Dutch Cancer Society, the Diabetic Foundation, or the Rheumatoid Arthritis Foundation. In addition, NCMLS members fulfil advisory functions or are board members within these organizations or at the Royal Netherlands Academy of Arts and Sciences (KNAW). Moreover, clinical groups (Prof. J. Berden, Dr. M. Netea, Prof. C. Punt, Prof. T. De Witte, Prof. N. Knoers, Prof. B.J. Kullberg, Prof. J. Smeitink) are in daily interaction with patients or their relatives, inform patient organizations and are involved in public and strategic policy. Some examples of memberships are as follows: Prof. J. Berden is a member of the Scientific Board of the Dutch Diabetes Foundation. Prof. W. van Venrooij is a member of the Standardisation Committee of the International Union of Immunological Societies (WHO). Prof. T. de Witte is treasurer and member of the Board of the European Group for Blood and Marrow Transplantation. Prof. R. Sauerwein is chairman of the Scientific Coordination Committee AMANET (African Malaria Network Trust). Prof. H. Stunnenberg is a member of the Scientific Board of the Dutch Cancer Society. Several examples of translational research are being developed within the NCMLS. The Centre participates in the Dutch Program for Tissue Engineering (DPTE) having received 7million to carry out research in this area (Prof. J. Jansen, Prof. C. Figdor; Dr. A. van Kuppevelt and Dr. P. van der Kraan). The aim of the programme is to culture organs and tissues from living cells such as stem cells and investigate biocompatibility, biodegradability and culture methods. The primary goal is to develop biomaterials that can be used to replace or repair damaged or dysfunctional organs and tissue. Molecular diagnostics for cancer is also being developed (Prof. J. Schalken). The expression of the prostate cancer specific gene DD3/PCA3 is linked to prostate cancer and this can be detected using a specific, highly sensitive assay. As a result, a commercial partner the Canadian biotech company Diagnocure has negotiated a contract with one of the biggest companies in the field of genebased diagnostics (GenProbe). Research on the development of dendritic cell (DC) vaccines is also taking place within NCMLS (Prof. C. Punt, Prof. C. Figdor, Prof. T. de Witte and Prof. G. Adema). Awards Prof. W. van Venrooij received the prestigious Dutch Rheumatology Medal for his research in this field. Dr. F. van Kuppeveld received the Beijerinck Award from the Royal Netherlands Academy of Arts and Sciences for excellence in viral research. Prof. J. van der Meer will be appointed vice-president of the Royal Netherlands Academy of Arts and Sciences as of 1 May Veni: Recipents of the prestigious Veni grants (NWO Dutch Science Organisation) were: Dr. A. Zendman for research on the possibilities of putting a brake on the chronic character of rheumatoid arthritis. Dr. F. Wagener for research on enzymes that steer the immune system. Dr. T. Scheenen for the project The rim of diffusely infiltrating brain tumors: visualizing the invisible with magnetic resonance imaging and spectroscopy. Dr. B. Snel for research on comparative functional genomics: reconstructing systems evolution from functional genomics data to improve function prediction. Dr. S. Spicuglia for the project The fight against malaria. Vidi: Recipient of the prestigious Vidi grant (NWO Dutch Science Organisation) was: Dr. M. Netea for his project on mechanisms of pattern recognition and innate resistance against Candida albicans infection. Annual Report 2004 Societal Impact / Awards 9

10 A GMP facility with clean rooms is used for translational research e.g. immunotherapeutic cell therapy, stem cell transplantation and gene therapy. In 2004 a total of 65 patients were treated in NCMLS translational research studies. Technology Platforms Genomics and proteomics The NCMLS groups its research facilities under five well-defined platforms. Animal models Animal models have proven to be of great importance to molecular life scientists. The available disease-related models include those for arthritis, cancer, kidney disease, tissue engineering, heart transplantation, neural disorders, metabolic disorders, osteoporosis, haematopoiesis, fungal and bacterial septicaemia and malaria (P. falciparum). A behavioural testing battery (comprising mice & rats) is also available to investigate the functional consequences of gene environment interactions and for developing transgenic/knock-out models. Molecular imaging Imaging at the molecular level is an essential tool for life scientists. Electron microscopy and other high-resolution instruments such as Atomic Force Microscopy are available within a state-of-the-art facility. Furthermore, confocal laser scanning microscopy, flow cytometry and other fluorescent microscopic techniques are combined to perform dynamic measurements of fluorescent GFP-based tagged proteins (such as FRET & FRAP) and intracellular metabolites. Magnetic resonance facilities for in vivo NMR and MRI of animals and humans (7 Tesla) are also available. Translational research and cellular therapy DNA sequencing as well as micro-array technology for gene expression profiling are now basic tools used by molecular life scientists. In 2004, novel microfluidic-based quantitative PCR became available to perform high throughput quantitative RT-PCR. A state-of-the-art proteomics facility has also been launched with, for example, 2D-electrophoresis, SELDI- TOF and Mass spectrometry (MALDI-TOF, FT-MS,nLC-MS/MS). Bioinformatics The Centre for Molecular and Biomolecular Informatics (CMBI) is the Dutch national centre for computational molecular sciences and is affiliated with the Faculty of Science. This centre is fully integrated within the NCMLS. The CMBI pursues a research programme with topics ranging from computational small-molecule chemistry to bioinformatics. The centre facilities, including a helpdesk databases and software packages are available to external scientists. The CMBI is also involved in organising courses and tutorials to support these scientists, in addition to maintaining www-based servers to give scientists rapid access to commonly used bioinformatics and small-molecule databases and information systems. A GMP facility with clean rooms is used for translational research e.g. immunotherapeutic cell therapy, stem cell transplantation and gene therapy. In 2004 a total of 65 patients were treated in NCMLS translational research studies. 10 Technology Platforms NCMLS

11 The Nijmegen Centre for Molecular Life Science (NCMLS) is the leading graduate school where students The NCMLS follow as Graduate a tailor-made School research programmes shoulder to shoulder with professionals. The Nijmegen Centre for Molecular Life Science (NCMLS) is the leading graduate school where students follow a tailor-made research programmes shoulder to shoulder with professionals. The regulation of cellular processes is crucial for human development, and maintenance of health throughout life. It is clear that cellular malfunction affects common multi-factorial diseases such as diabetes, immune and inflammatory disorders, renal disease, cardiovascular, metabolic and neurodegenerative diseases as well as obesity and certain forms of cancer. In the fight against such diseases, the Nijmegen Centre for Molecular Life Sciences (NCMLS) Graduate School which is part of Radboud University Nijmegen and Radboud University Nijmegen Medical Centre plays a key role. A major goal of the NCMLS is to translate basic knowledge generated from biomedical research into clinical application, in order to improve diagnostics and develop new treatments. All MSc and PhD students are registered junior members of the research school and have the corresponding responsibilities and privileges. Tailor-made tuition Students are guided throughout the practical training period by a supervisor and throughout the entire programme by a mentor, who stimulates them to explore your abilities and develop general research competencies, including reflection. Together with these coaches, students draw up a personal training and supervision plan. Such a broad and interdisciplinary approach to research is particularly important in the international scientific arena. Excellent career prospects There is considerable demand for specialists in fundamental molecular biology and cell biology as well as in its application to the treatment of diseases such as cancer, autoimmune and inflammatory disorders, and metabolic and neurodegenerative disorders. Our MSc enables students to move rapidly into an international PhD programme, giving them a more mature perspective and a broader range of experimental approaches than is possible within standard MSc programmes. They are also prepared for further training as a PhD-level researcher. Graduates are exempted from certain elements of the NCMLS PhD programme. For example, the graduate course is taught during the MSc phase and the main practical project can be incorporated into a PhD project, thus shortening the PhD period. Education is focussed is on the domain of molecular life sciences related to disease and in particular in three main fields related to molecular medicine, cell biology and translational research. Programmes are aligned along the three research themes. The dedicated research programmes within the Graduate school are briefly highlighted as follows: MSc Molecular Mechanisms of Disease The NCMLS offers an exclusive Masters programme in Molecular Mechanisms of Disease, which is taught by top researchers and clinicians. This programme is accredited by the NVAO, the Dutch-Flemish accreditation organisation and a member of ECA, a European consortium for accreditation. A unique and challenging programme This ground-breaking programme translates disease-related basic research in cellular and molecular biology into clinical experimental research in patients. Designed to meet the needs of talented students with the drive, motivation and ambition to push forward their scientific careers, it represents a unique opportunity to develop a research project and build up an international research network. This extremely competitive programme provides a sound balance of theory and practice. We enrol just 30 students per year, each of whom is allocated a personal mentor and research supervisor. This selective approach guarantees excellence, especially during the research training period. According to on of our international partners the prestigious Mayo Clinic in the USA, the programme offers a focused combination of didactic and practical components suited to optimally synthesize current discoveries, conceptual breakthroughs and technological advancement mandatory for the effective education of the new vanguard of multidisciplinary investigators. The MSc programme The programme, which starts each September, is modular in structure, lasts 24 months and is worth 120 European credits. Its main goal is to prepare talented, motivated students for independent research projects, which provide the basis for continuing to take a PhD degree. There is a strong international component and an emphasis on a multidisciplinary approach to answering research questions related to molecular mechanisms of disease. The general modules are taught mainly in the first year, while the focus in the final year is on individual project work leading to a MSc thesis. Translational research and technology platforms Students are exposed to Translational research, which links basic science and the treatment of disease. This is crucial when studying the molecular mechanisms of the following diseases: autoimmune and inflammatory disorders (such as rheumatoid arthritis), renal disease, neurodegenerative diseases, metabolic disorders, microbial infection Annual Report 2004 The NCMLS as Graduate School 11

12 The Nijmegen Centre for Molecular Life Science (NCMLS) is the leading graduate school where students follow a tailor-made research programmes shoulder to shoulder with professionals. (including malarial infections), viral infections and cancer. Students also become familiar with technology platforms which link basic science, technology and disease such as genomics, proteomics, bioinformatics, cellular therapy, tissue engineering and molecular imaging. PhD programme Awards 2004 Winner of best thesis: Edwin Jansen, Dept. of Cell Biology Winner of best student report: Stanleyson Hato, Dept. of Medical Microbiology Master classes and international experience One of the highlights of the programme is a series of Master classes, which are given by leading authorities from renowned international institutes. These offer valuable insights both within and beyond your chosen specialist topics, explaining the current state of the art in important scientific areas. Students participate in exchanges with a number of collaborating institutes and formal NCMLS partners, including the University of Southern Denmark, the University of Münster, the Rudolf Virchow Center for Experimental Biomedicine at Würzburg (both in Germany), the University of Milano-Bicocco in Italy and the Mayo Clinic in the USA. International PhD programme The aim of the NCMLS PhD training programme is to provide PhD students with a multi-faceted education in the field of Molecular Life Sciences. NCMLS PhD students therefore follow an interdisciplinary training programme, containing both compulsory and elective components. The elective components allow a section of the NCMLS training programme to be tailor-made to complement the specific specialisation of the individual student. The training programme encourages both practical and theoretical participation in several NCMLS activities. Entrance requirements The training programme is accessible to PhD students from one of the research groups that are related to the graduate school. Students with a NCMLS M.Sc. degree, with a higher professional degree or with a similar degree from a foreign institute can enter the programme. Medicine, (Medical) Biology, Molecular Mechanisms of Disease, Chemistry, Physics, Mathematics, Computer Science and (Bio) Engineering are suitable preparatory fields of study. Because of the heterogeneity of the knowledge and interest of the students, a diverse education programme, in which many subjects will be taught, is provided. The NCMLS certificate Along side practical training, PhD students will be given the opportunity to broaden their skills through participation of specialised knowledge transfer courses. A certain number of courses are though to be necessary in the development and education of each PhD student as independent researchers. Students that complete a full programme of NCMLS courses will be awarded with the NCMLS certificate. 12 The NCMLS as Graduate School NCMLS

13 Annual Report 2004 Theme 1 Infection, immunity and tissue repair

14 Skin diseases vary from trivial cosmetic problems to life-threatening conditions, as seen in some severe disorders of keratinization and Patrick Zeeuwen cornification. These (Sk)in balance This work was published in: Zeeuwen, PLJM, Vlijmen-Willems, IMJJ van, Olthuis, D, Johanson, HT, Hitomi, K, Hara-Nishimura, I, Powers, James,JCKE, Op Den Camp,HJ, Lemmens, R, & Schalkwijk, J (2004). Evidence that unrestricted legumain activity is involved in disturbed epidermal cornification in cystatin M/E deficient mice. Hum.Mol.Genet 13, Understanding skin disease The role of proteases and their inhibitors in epidermal cornification Skin diseases vary from trivial cosmetic problems to life-threatening conditions, as seen in some severe disorders of keratinization and cornification. These disorders are commonly due to abnormal epidermal differentiation processes, which result in a disturbance of the barrier function of human skin. Recent findings have provided evidence that a disturbed protease-antiprotease balance could cause faulty differentiation processes in the epidermis and hair follicle. Cathepsins were until recently considered to be primarily responsible for intralysosomal protein degradation, mediating housekeeping functions in the cell. Regulation of proteolytic enzyme activity is now well recognized to be essential for cell and tissue homeostasis. Investigations over the last decade have shown that lysosomal cathepsins are involved in a variety of physiological processes such as proenzyme activation, enzyme activation, antigen presentation, hormone maturation, tissue remodeling, and bone matrix resorption. Collectively, cathepsins take part in multiple host systems in both health and disease. Their implication in numerous vital processes and pathologies make them highly attractive targets for drug design. The regulation of activity of cysteine proteases for correct functioning is a fragile balance of many factors, one of the most crucial being their cognate protease inhibitors. Disturbance of this protease-antiprotease balance at a wrong time and location has serious consequences. The importance of regulated proteolysis in epithelia is well demonstrated by the recent identification of the SPINK5 serine proteinase inhibitor as the defective gene in Netherton syndrome, cathepsin C mutations in Papillon-Lefevre syndrome, cathepsin L deficiency in furless mice, targeted ablation of the serine protease Matriptase, and targeted ablation of cathepsin D. Our previous investigations have provided evidence for a novel biochemical pathway that controls transglutaminase-mediated crosslinking and formation of the epidermal stratum corneum. This involves the proteolytic activation of lysosomal proteases that process transglutaminase-3 to its active form. Misregulation of this pathway by unrestrained activity of the asparaginyl endoprotease legumain (Figure), as seen in cystatin M/E deficient mice, leads to abnormal stratum corneum formation, disturbance of skin barrier function and neonatal death. Currently we are generating double knockout mice (cystatin M/E -/- and legumain -/- ). The double knockout will be interesting for a number of reasons including the possibility that cystatin M/E has functions not related to its antiprotease activity. These double knockout mice should also give a definitive answer to the question if the legumaincystatin M/E balance is truly important in the pathway that leads to a correct composition and formation of the stratum corneum. We are also now rescuing the cystatin M/E deficient mice (neonatal lethal) by the introduction of a transgene that drives epidermis-specific cystatin M/E expression on a cystatin M/E null background. By doing this we could eventually study the consequences of cystatin M/E deficiency in other tissues that are affected by unrestricted legumain activity. Figure: Free in situ protease activity of legumain in hair follicles at the level of the infundibulum in cystatin M/E deficient mice. Legumain activity is visualized as a fluorescent precipitate by the hydrolysis of the Suc-Ala-Ala-Asn-NHNapOME substrate. 14 Patrick Zeeuwen Understanding skin disease NCMLS

15 During infection, microorganism pathogenassociated molecular patterns (PAMPs) are recognised by Toll-like receptors (TLR), the Mihai Netea major class of signaling Toll-like receptors This work was published in: Netea, MG, Sutmuller, R, Hermann, C, Graaf, CA van der, Meer, JW van der, Krieken, JH van, Hartung, T, Adema, G, & Kullberg, BJ (2004). Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells. J Immunol 172 (6), Understanding the mechanisms of innate immunity From the Th1/Th2 paradigm towards a TLR/Th bias During infection, microorganism pathogenassociated molecular patterns (PAMPs) are recognised by Toll-like receptors (TLR), the major class of signaling receptors, first described in Drosophila. The specificity of TLR recognition has been established for several important PAMPs. While it first seemed that these pathways were aspecific and redundant, recent studies showed that TLRs enable the innate immune system not only to recognize specific PAMPs, but by inducing specific cytokine profiles, TLRs bring specificity to the innate immune system and influence the nature of the adaptive immune response. In response to microbial pathogens, CD4 T cells differentiate into Th1 or Th2 cells. Whereas a Th1-type response is important for the defense against bacterial and fungal pathogens through production of IFN-α, Th2 cells produce IL-4, IL-5 and IL-10, suppressing the host defense against these pathogens. How the nature of infection determines the type of T-cell response is an area of great interest. It now has become apparent that both the initiation of adaptive immune responses and the subsequent T- cell activation and initiation of adaptive immunity are under control of TLRs. There has been strong evidence for an important role of TLRs in driving Th1 responses, whereas the absence of TLR-mediated signals generates Th2 responses. However, recent data, including that from our group, demonstrate specific roles for particular TLRs in the differentiation of Th subsets (including Th2), rather than a model in which the absence of TLR-mediated signals is the driving force of Th2 differentiation. In dendritic cells (DCs), TLR2-mediated signals preferentially induce a Th2 profile, compared with TLR4 activation mainly leading to a Th1 response. Thus, through specific TLR stimulation, DC will process information leading to the polarization of the acquired immune response. By driving specific Th1 or Th2 differentiation, TLRs are therefore a crucial link between innate and acquired immunity. We described an important role for TLR4 in the recognition of Candida albicans, leading to a Th1 response and production of proinflammatory cytokines and neutrophil recruitment. Thus, mice deficient in TLR4 are more susceptible to disseminated candidiasis. In contrast, TLR2-/- knockout mice are more resistant to disseminated Candida infection, and this is associated with increased chemotaxis and enhanced candidacidal capacity of TLR2-/- macrophages. IL- 10 release is severely impaired in the TLR-/- mice. This is accompanied by a 50% decrease in the CD4CD25 regulatory T cell (Treg) population. In vitro studies confirmed that enhanced survival of Treg cells was induced by TLR2 agonists. The deleterious role of Treg cells in the innate immune response during disseminated candidiasis was underscored by the improved resistance to this infection after depletion of Treg cells. In conclusion, C. albicans induces immunosuppression through TLR2-derived signals that mediate increased IL-10 production and survival of Treg cells. This represents a novel mechanism in the pathogenesis of infection, and a step forward in understanding the Th1/Th2 paradigm, in which the pro- versus anti-inflammatory pathways diverge from the moment a microorganism interacts with the leukocytes at the level of the cell membrane, through TLR4 and TLR2. This has important consequences for both our understanding of innate immune system, and for the possible therapeutic use of strategies designed to target specific TLRs. A B Figure: Histology of the kidneys of TLR2 +/+ and TLR2 -/- mice infected with C. albicans. Control TLR2+/+ (A and B) and TLR2 -/- (C and D) mice were infected i.v. with 1 x 105 CFU of C. albicans, and subgroups of five animals were sacrificed to assess the outgrowth of the yeasts in the right kidneys. Serial sections were examined microscopically after staining with periodic acid Schiff and H&E. On day 7 after infection, control TLR2 +/+ mice displayed invasive growth of large amounts of C. albicans both in the renal tissue (A) and pyelum (B), accompanied by moderate inflammatory infiltrates. In contrast, very little growth of Candida was found in the TLR2 -/- mice, which had an almost normal kidney architecture (C), whereas a severe inflammatory infiltrate was present around the pyelum (D). Magnification: x400. Arrows indicate C. albicans microorganisms. C D Annual Report 2004 Mihai Netea Understanding the mechanisms of innate immunity 15

16 Viruses induce a number of alterations in metabolism and structure of their host cell in order to ensure efficient reproduction. Some of Frank van Kuppeveld these alterations can be Pore formation by viruses This work was published in: Campanella, M, Jong, A de, Lanke, K, Melchers, W, Willems, P, Pinton, P, Rizzuto, R, & Kuppeveld, F van (2004). The coxsackievirus 2B protein suppresses apoptotic host cell responses by manipulating intracellular Ca2+ homeostasis. J Biol Chem 279, Understanding virus-host interactions Po(u)re out the calcium; a viral strategy to suppresses apoptotic host cell responses Viruses induce a number of alterations in metabolism and structure of their host cell in order to ensure efficient reproduction. Some of these alterations can be sensed by the host cell and a defensive apoptotic reaction is switched on, aimed at curtailing virus replication. Many viruses have developed countermeasures in order to prevent premature abortion of the viral life cycle. Previously, it has been shown that enteroviruses (small cytolytic RNA viruses that includes poliovirus, coxsackievirus, and ECHOvirus) confer an anti-apoptotic state to infected cells but the underlying molecular mechanism was unknown. We obtained evidence that the enterovirus 2B protein plays a major role in suppressing apoptotic host cell responses by manipulating intracellular Ca 2+ homeostasis. Growing evidence suggests that calcium fluxes between the ER and the mitochondria play a major role in apoptotic signaling. Excessive rises in mitochondrial Ca 2+ can lead to opening of the permeability transition pore and swelling of the mitochondria, leading to the rupture of the mitochondrial outer membrane, and thereby releasing cytochrome c and triggering apoptosis. Accordingly, down-regulation in ER-mitochondrial Ca 2+ signalling was shown to confer protection against a number of apoptotic stimuli. The enterovirus 2B protein is a small protein that localizes at ER and Golgi membranes. All enterovirus 2B proteins contain two hydrophobic regions, of which one is predicted to form a cationic amphipathic α- helix with characteristics typical for the group of membrane-lytic peptides i.e., peptides that build membrane-integral pores by forming multimeric transmembrane bundles (Figure). Previously, we demonstrated homomultimerization reactions of 2B by genetic and biochemical approaches, and in living cells using FRET microscopy. These data strongly suggested that 2B viral protein might reduce the [Ca 2+ ] in ER and Golgi by building pores in the membranes of these organelles. Making use of fluorescent Ca 2+ indicators and organelle-targeted aequorins, genetically encoded Ca 2+ sensors, we now demonstrated that 2B indeed reduces [Ca 2+ ] in ER and Golgi in HeLa cells. This leads to a decrease in the amount of Ca 2+ that can be released from these stores and, as a consequence, in the stimulusinduced rise of [Ca 2+ ] in the mitochondria. Consistent with the above described importance of ER-mitochondrial Ca 2+ signalling, we found that expression of the 2B protein suppressed cell death induced by a number of apoptotic stimuli. 2B viral protein mutants that were unable to reduce the Ca 2+ content of the stores failed to protect against apoptosis. Together, these data support a functional role of the enteroviral protein 2B-induced perturbation of intracellular Ca 2+ signalling in the enteroviral strategy to suppress apoptosis, and provide another physiological example of the regulatory role of Ca 2+ signalling in the modulation of apoptotic cell death. The findings are of major importance for a better understanding of the virus-host interaction and virus-induced cell injury and disease. Figure: pore formation by the enterovirus 2B protein 16 Frank van Kuppeveld Understanding virus-host interactions NCMLS

17 Dendritic cells (DC) are professional antigen presenting cells that play a pivotal role in the induction of immunity. Immature DCs reside Martijn den Brok in peripheral tissues Dendritic cells This work was published in: Brok, MH den, Sutmuller, RP, Voort, R van der, Bennink EJ, Figdor CG, Ruers TJ, & Adema GJ (2004). In situ tumor ablation creates an antigen source for the generation of antitumor immunity. Cancer Res 64(11), Understanding anti-tumour immune response Dendritic cells: bridging the gap to in vivo vaccination Dendritic cells (DC) are professional antigen presenting cells that play a pivotal role in the induction of immunity. Immature DCs reside in peripheral tissues where they take up and process antigens from their surroundings. In a stimulatory environment, like in case of an infection, immature DCs undergo activation, maturation and acquire the capacity to present exogenous antigens in MHC class I. Maturation is accompanied by migration of the DCs to the draining lymph node, where they subsequently present antigens to T lymphocytes to launch the immune response. The possibility of loading DCs with tumour antigens makes them a promising tool to induce immune responses directed against human cancers. Ex-vivo generated, tumour antigen-loaded mature DCs are currently exploited as vaccines in clinical studies. Although tumour-specific responses have been obtained, many questions regarding effective tumour antigens and DC migration remain unanswered. Moreover, ex vivo generation and loading of DCs is time consuming and costly. In vivo loading and maturation of DCs would therefore greatly improve the applicability of DC vaccination and provide a basis to learn more about ex vivo DC vaccine generation. Tumour-destructing techniques, like Radiofrequency ablation (RFA), allow eradication of large tumours. Potentially, in situ tumour destruction can also provide the immune system with an antigen source for the induction of anti-tumour immunity. Antigen presenting cells can take up antigens in the periphery after which they induce specific immune responses. However, virtually nothing is known regarding the induction of immune responses after in situ tumour destruction in either mice or men. We explored the murine B16-OVA melanoma cell line to develop a novel tumour model to explore: 1) the immunological consequences of in situ tumour destruction and 2) the efficacy of a combination approach of tumour destruction and immunostimulation. Applying this model system we demonstrated that following RFA, a weak but detectable immune response develops. Adoptive transfer of splenocytes further indicated that anti-tumour reactivity can be transferred to naïve mice by splenocytes. To augment the response observed, we administered a blocking mab against cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) at the time of tumour destruction. CTLA-4 is believed to guard the threshold for T cell activation and expansion, thus blocking it will result in increased T cell mediated immunity. Interestingly, administration of this antibody strongly enhanced antitumour immunity resulting in long-lasting tumour protection compared to isotype controls. These results illustrate that in situ tumour destruction can provide a useful antigen source for the induction of antitumour immunity, provided that additional immunostimulatory signals are co-administered. In situ tumour destruction in combination with immune modulation creates a unique model to study in situ DC-vaccination, and these results are readily applicable in a clinical setting. Figure: In vivo interventions in the optimal CD8+ T cell anti-tumour response. A) Large amounts of antigens are released from the destructed tumour. Tumour antigens are loaded on DC that start migrating towards the draining lymph nodes. B) Injected TLR ligands can effectively mature the DC. The antigens are processed and presented on MHC molecules to T cells in the lymph node. CTLA-4 mabs will lower the threshold for T cell activation. C) The T cells proliferate and get activated. D) T cells migrate back to the destructed tumour or to micro metastases, where they kill remaining live tumour cells. Annual Report 2004 Martijn den Brok Understanding anti-tumour immune response 17

18 Dendritic cells (DCs) are a group of bone-marrow-derived leukocytes that are considered the guardians of our immune defense system. Alessandra Cambi Their job is to conside- C-type lectin receptors This work was published in: Cambi A, Lange F de, Maarseveen NM van, Nijhuis M, Joosten B, Dijk EM van, Bakker BI de, Fransen JA, Bovee-Geurts PH, Leeuwen FN van, Hulst NF van, & Figdor CG (2004). Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells. J Cell Biol Jan 5 164(1), Understanding pathogen recognition by the immune system DC-SIGN microdomains as docking sites for pathogens Dendritic cells (DCs) are a group of bonemarrow-derived leukocytes that are considered the guardians of our immune defense system. Their job is to constantly sample the tissues for potential intruders like viruses or other pathogens. Therefore these DCs are specialized in uptake, processing, transport and presentation of antigens to T lymphocytes to initiate an immune response. One of the primary interests of our laboratory is to understand how DCs recognize pathogens and how they could be used in the development of novel vaccination strategies against infection and potentially cancer. We have discovered that C-type lectin receptors (CLRs) are constituents of the powerful antigen capture and uptake mechanism of DCs. We found that DC-SIGN, a membranebound CLR highly expressed by DCs, is designed to capture pathogens for intracellular degradation and subsequent antigen loading of MHC molecules. Life-threatening viruses including HIV-1, Ebola, and Hepatitis C virus, but also bacteria and fungi are recognized by DC-SIGN. Interestingly, we observed that expression of DC-SIGN at the surface of DCs was not always sufficient to mediate pathogen uptake. This prompted us to determine the organization of DC-SIGN on the cell membrane at high resolution. We developed a novel whole-mount transmission electron microscopy (TEM) technique that allowed analysis of the DC-SIGN distribution on parts of the surface the cell. During development of DCs from precursor monocytes isolated from peripheral, DC-SIGN becomes organized in well-defined micro-domains, with an average diameter of 200 nm, within the DC plasma membrane (Figure). Further biochemical studies and confocal microscopy indicated that DC-SIGN microdomains reside within cholesterol- and glycosphingolipid-enriched membrane domains also known as lipid rafts. We showed that this typical organization of DC-SIGN in microdomains on the plasma membrane is essential for the binding and internalization of viral particles, suggesting that these multi-molecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host. Our findings demonstrate, for the first time, the importance of relating receptor function with the cell surface organization of membrane receptors. A clustered distribution appeared essential to enhance the interaction as well as the internalization efficiency of DC-SIGN/pathogen complexes. Our data highlight the importance of the plasma membrane as a specialized highly organized microenvironment, where tightly orchestrated interactions among proteins, carbohydrates and lipids occur. It is tempting to speculate that distinct microdomains exist on the cell surface that function as docking and signaling platforms mediating important cellular processes such as phagocytosis, cell migration, or cell mediated killing. A better understanding of the cell surface organization of pathogen uptake receptors like DC-SIGN not only provides insight in the mechanisms exploited by pathogens to escape the immune system but may also help to develop novel strategies to combat life threatening infectious diseases. Figure: (a) DC-SIGN cell surface distribution changes during DC development. High resolution imaging of whole mount samples of dendritic cells by transmission electron microscopy shows that the CLR DC-SIGN is present in distinct nm sized microdomains (colored dots represent different cluster sizes) on differentiated dendritic cells (DC), but still randomly distributed (black. doublets; and single, white dots) on developing DCs that are halfway differentiation. 18 Alessandra Cambi Understanding pathogen recognition by the immune system NCMLS

19 Treatment of patients with metastasized Renal Cell Cancer (RCC) is difficult. The tiumor is resistant against conventional therapies Mischa de Weijert such as chemotherapy Targeted treatments This work was published in: Grabmaier, K, Weijert, MCA de, Verhaegh, GW, Schalken, JA, & Oosterwijk, E (2004). Strict regulation of CAIX(G250/MN) by HIF-1alpha in clear cell renal cell carcinoma. Oncogene 23 (33), Understanding renal cell carcinoma Development and optimisation of monoclonal antibody treatments for renal cell carcinoma Treatment of patients with metastasised Renal Cell Cancer (RCC) is difficult. The tumour is resistant to conventional therapies such as chemotherapy and radiation. With a median survival of approximately one year, it is clear that there is great need for new treatments. The concept of the magic bullets refers to compounds that would seek out malignant cells and destroy them, and having no harmful effects on normal cells. We are investigating such a possibility using a monoclonal antibody treatment that recognises a tumour-associated molecule most abundant on RCC. Patients with metastasised RCC were treated with unmodified antibody or with a radio-labeled antibody, using the antibody as vehicle to guide radioactive substances to tumour cells. In addition, preclinical studies were performed, aimed at optimisation of radioimmunotherapy. In a multicentre phase II study, the safety and efficacy of cg250 in metastatic RCC patients was evaluated in 36 patients. cg250 was given weekly by intravenous infusion for 12 weeks. Ten patients experienced stabilisation of disease and received extended treatment. One patient showed a complete response and significant disease regression was observed during the followup of the treatment in another patient. The median survival after treatment start was 15 months. With a median survival of 15 months after the start of this treatment and two late clinical responses, the antibody treatment appeared to be able to modulate the disease course. Injection of radiolabeled G250 in patients with RCC results in excellent uptake in the tumour sites (Figure). We have performed several trials with iodine-labeled G250 and have now developed new methods to deliver more powerful radionuclides to the tumour. Therapy studies in animals confirmed the improved efficacy of these other radionuclides. We concluded that these are most likely better candidates than conventionally radiolabeled 131I for radioimmunotherapy with cg250 in patients with RCC. A phase I clinical study has been designed which will be paralleled by an identical study with 90Y labeled cg250 at Memorial Sloan-Kettering Cancer Center, New York (MSKCC) to examine whether radioimmunotherapy with this approach can lead to durable responses in RCC patients. Figure: Anterior (left) and posterior (right) images of a patient with metastasized RCC treated with iodinelabeled cg250. Tumour lesions are visible as dark black areas and indicate specific accumulation of cg250. Annual Report 2004 Mischa de Weijer Understanding renal cell carcinoma 19

20 Apoptosis is of crucial importance for cells of the immune system as it is required for selection and maintenance of cellular homeostasis. Joop Jansen Deregulation of apopo- Mitochondrial integrity This work was published in: Graaf, AO de, Heuvel, LP van den, Dijkman, HB, Abreu, RA de, Birkenkamp, KU, Witte, T de, Reijden, BA van der, Smeitink, JA, & Jansen, JH (2004). Bcl-2 prevents loss of mitochondria in CCCP-induced apoptosis. Exp Cell Res 299 (2) Understanding regulation of the intrinsic pathway in apoptosis Bcl-2 prevents loss of mitochondria in CCCP-induced apoptosis Apoptosis is of crucial importance for cells of the immune system as it is required for selection and maintenance of cellular homeostasis. Deregulation of apopotosis plays a major role in carcinogensis of several types of tumours. It is activated either through an extrinsic or an intrinsic pathway. The extrinsic pathway is engaged through death receptors (members of the TNF receptor superfamily) at the cell surface that can cause caspase-activation by the formation of death-inducing signaling complexes. The intrinsic pathway of apoptosis is mediated through mitochondria. Upon several intracellular signals, mitochondria transmit cell death signals to the cytosol, leading to the activation of caspases and the execution of apoptotic degradation and cell death. A number of changes occur in mitochondria following apoptotic signals, such as loss of mitochondrial membrane potential, generation of reactive oxygen species (ROS), opening of the permeability transition (PT) pore and release of apoptogenic factors like cytochrome c and SMAC (second mitochondria-derived activator of caspase) into the cytosol. Once released in the cytosol, these factors activate caspases, which cleave several cellular substrates contributing to the degradation of the cell. Bcl-2 family proteins regulate apoptosis at the level of mitochondria. In several types of cancer, the genes encoding bcl-2 family members are mutated. Many proteins of the bcl-2 family loicalize to the mitochondria, but their mode of action remains poorly defined. Bcl-2 is a anti-apoptotic protein that was identified as the critical gene that is deregulated by the t(14;18) chromosomal translocation in non-hodgkin s lymphoma. To examine the mechanism of Bcl-2 function, we investigated the effects of bcl-2 overexpression on apoptosis induced by the protonophore carbonyl cyanide m- chlorophenyl hydrazone (CCCP). This compound induces apoptosis by rendering the mitochondrial inner membrane permeable for protons and causing dissipation of the proton gradient across the inner mitochondrial membrane. We show that Bcl-2 protects against CCCP-induced apoptosis, however without preventing the decrease in mitochondrial membrane potential or the alterations in cellular ATP content. Furthermore, the anti-apoptotic effect of Bcl- 2 takes place upstream of caspase activation and nuclear changes associated with apoptosis, since these were markedly inhibited in cells overexpressing Bcl-2. Exposure to CCCP induced a strong decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, with apparent swelling and loss of cristae in parental cells. Bcl-2 clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. Interestingly, in cells that were not exposed to CCCP, a higher number of intact mitochondria was observed in Bcl-2 overexpressing cells compared to parental and vector transduced cells, as shown by electron microscopy. This indicates that Bcl-2 functions to preserve mitochondrial integrity, also in the absence of pro-apoptotic signals and suggests that bcl-2 prevents apoptosis by preserving mitochondrial integrity. Figure: Electron microscopic analysis shows mitochondrial swelling and severe disruption of mitochondrial structures in CCCP-treated cells. (A) untreated Jurkat-parental cell, showing elongated mitochondria with normal cristae structure. (B) apoptotic Jurkatparental cell treated with 25 mm CCCP for 8 h with condensed and fragmented nucleus (indicated by N) and cytoplasmic vacuoles. Only a few mitochondria are present and they appear swollen and have lost cristae (indicated by an asterisk). (C) Detail of a disrupted mitochondrion with an inclusion and some remaining cristae (MI) in a CCCP-treated Jurkat-parental cell. (D) Non-apoptotic Jurkat-parental cell treated with CCCP for 8 h, showing myelin-like figures (indicated by arrowheads). (E) untreated Jurkat-Bcl-2 cell, showing elongated mitochondria with normal cristae structure. (F) Jurkat-Bcl-2 cell treated with CCCP for 8 h. Many normal mitochondria (M) are still present, but disrupted mitochondria that are swollen and have lost crista 20 Joop Jansen Understanding regulation of the intrinsic pathway in apoptosis NCMLS

21 An ageing population requires that materials be developed that can replace missing or damaged body parts on basis of physiological John Jansen principles. Develop- Bone repair This work was published in: Ruhe, PQ, Kroese-Deutman, HC, Wolke, JG, Spauwen, PH, & Jansen, JA (2004). Bone inductive properties of rhbmp-2 loaded porous calcium phosphate cement implants in cranial defects in rabbits. Biomaterials 25 (11), Understanding bone disease Bone morphogenetic protein 2 enhances tissue formation An ageing population requires that materials be developed that can replace missing or damaged body parts on basis of physiological principles. Development of implant materials that show an improved (smart) tissue response are of primary interest. For example, in surgical disciplines where bone has to be repaired, augmented or improved, bone substitutes are essential. Natural bone is not, however, always adequate. For example, frequently these socalled bone grafts resorb after implantation. Furthermore, they cannot be used for joint and tooth replacement, and recently worries have been raised about the transfer of infectious diseases. Therefore, interest has dramatically increased in the use of synthetic materials for replacement of lost or damaged bone tissue. The generic name of these tissue alternatives is biomaterials. A lot of prerequisites have been postulated for the ideal scaffold biomaterial for bone tissue engineering with regard to mechanical properties, biocompatibility, biodegradability, absence of allergic reactions, disease transmission, delivery of the osteoinductive factor, gross architecture, shaping, clinical handling, and regulatory issues. However, none of the currently available scaffold materials meets all of the formulated demands. In viewof this,we have focused on the use of calcium phosphate (Ca-P) cements. Besides the clinical advantage that this cement can be injected directly into a bone defect, it shows a fast deposition of new bone at the cement surface and is considered to be extremely biocompatible. white rabbits. The implantation period was either 2 or 10 weeks. Histological analysis of retrieved specimens revealed evident bone formation in the rhbmp-2 loaded Ca-P cement discs (pore fill: 18±6%) after 10 weeks of implantation. Bone formation occurred only in rhbmp-2 loaded porous Ca- P cement discs. Degradation of the Ca-P cement could not be confirmed after 10 weeks of implantation. The scaffold maintained its shape and stability during this time period. This demonstrates that using calcium phosphate (Ca-P) cements, an osteoconductive and macroporous structure can be induced during the setting reaction. Furthermore, the osteoinductive properties of recombinant human bone morphogenetic protein-2 (rhbmp-2)- loaded porous Ca-P cement was positively evaluated. Therefore Ca-P cement is a very suitable and promising carrier material for ectopic bone engineering. Porous Ca-P cement discs were made and loaded with rhbmp-2 in vitro and implanted subcutaneously in the back of New Zealand Figure: Light micrograph showing extensive bone formation in rhbmp-2 loaded porous CaP cement. BM = bone marrow, OB = osteoblast, OC = osteocyte. Annual Report 2004 John Jansen Understanding bone disease 21

22 Cancer diagnosis is mainly based on morphologic findings at pathological examination. The complex morphological changes of Iris Nagtegaal neoplastic cells toge- Expression profiling This work was published in: Nagtegaal ID, Gaspar CG, Peltenburg LT, Marijnen CA, Kapiteijn E, Velde CJ van de, Fodde R, & Krieken JH van (2004). Radiation induces different changes in expression profiles of normal rectal tissue compared with rectal carcinoma. Virchows Arch Epub Dec 16. Understanding rectal cancer Radiotherapy influences expression profiles in rectal cancer without affecting clinical features Cancer diagnosis is mainly based on morphologic findings at pathological examination. The complex morphological changes of neoplastic cells together with specific stromal reactions are the final result of a series of genetic alterations. However, the latter are in general poorly associated with morphology and behavior of carcinoma. DNA microarrays are aimed at global expression profiles rather than single genes and may provide additional insight in the relation between morphology and gene expression. Rectal cancer can be divided into two main histomorphological subtypes differing in prognosis as well as biological behaviour, presumably as a result of differences in underlying gene expression profile. Mucinous carcinomas are morphologically different from the more common, not otherwise specified (n.o.s.) adenocarcinoma, and are characterised by a substantial amount of mucin being retained within the tumour stroma. Studies of the histogenesis of this particular tumour type have lead to the hypothesis of a separate genetic pathway along which these tumours develop. The relative absence of p53 mutations, the high frequencies of K-ras mutations and c-myc amplification, and the strong MUC2 expression support this hypothesis. Recently, we showed that, in the framework of a randomized trial on preoperative radiotherapy in combination with total mesorectal excision, short-term preoperative radiotherapy increased the number of mucinous lesions in rectal carcinoma patients. Changes in cancer phenotype can occur in the course of disease, though it is not clear how this is reflected by changes in prognosis and gene expression profiles. We hypothesize, based on pre-radiation samples of the patients, that the increase in mucinous carcinomas due to radiation is caused by a morphological switch. We evaluated a large cohort (1449) of carcinomas from rectal cancer patients belonging to a randomised radiotherapy trial for the presence and amount of mucinous component. Results were compared with data from the pre-irradiation biopsies and revealed the presence of two distinct classes of mucinous carcinomas in the radiotherapy group, namely uninduced and induced mucinous carcinomas. Clinical data (including follow up), conventional pathological parameters and immuno-histochemical data from these patient and their tumours were compared. Gene expression profiles of the different groups of mucinous carcinomas and adenocarcinomas were analyzed using the Affymetrix Human Cancer Chips. Clinical and pathological characteristics of induced mucinous carcinomas were more comparable with the common adenocarcinomas rather than with those of the original mucinous carcinomas. Prognosis of induced mucinous carcinomas was significantly better than original mucinous carcinomas (91.2% versus 39.3% recurrence free interval, p = 0.02). Surprisingly, notwithstanding the latter difference in prognosis, the expression profile of radiation-induced mucinous carcinomas was very closely related with that of their uninduced counterparts. We show that within a time interval of 5 days, the morphology of a subset of rectal adenocarcinomas can change into mucinous carcinoma and that this change is related to a change in gene expression profile but not to the clinical features. Studies are in progress to find the mechanism behind this finding. A B C D E F G H I Figure: Histopathological features in mucinous carcinoma. (A-D) Patterns of walls of mucinous lakes: acellular (A), monolayer (B), multi-layer (C), and irregular (D). (E-G) Contents of the lumina of mucin lakes: single cells (E), clumps of cells (F), and signet ring cells (G). (H, I) Two types of growth pattern can be distinguished, circumscribed (H) and diffuse infiltrating growth (I). 22 Iris Nagtegaal Understanding rectal cancer NCMLS

23 Scientists are familiar with two types of sequences: the nucleotide sequence in DNA/RNA and the amino acid sequence in Gerdy ten Dam proteins. Now, a third Heparan sulphate This work was published in: Dam, GB ten, Westerlo, EM van de, Smetsers, TF, Willemse, M, Muijen, GN van, Merry, CL, Gallagher, JT, Kim, YS, & Kuppevelt, TH van (2004). Detection of 2- O-sulfated iduronate and N-acetylglucosamine units in heparan sulfate by an antibody selected against acharan sulfate (IdoA2S-GlcNAc)n. J Biol Chem 279 (37), Understanding saccharides in cell biology Heparan sulphate saccharide sequences in (patho) biology: a third level of bioinformation Scientists are familiar with two types of sequences: the nucleotide sequence in DNA/RNA and the amino acid sequence in proteins. Now, a third type of sequence is emerging: the monosaccharide sequence in polysaccharides. This is especially exemplified by the heparan sulphates (HS), a ubiquitous class of long, unbranched polysaccharides present at cell surfaces and extracellular matrices. HS bind and modulate a myriad of proteins including growth factors and their receptors, cytokines, adhesion proteins and proteases. They have been coined master regulators of molecular interaction. HS chains can be up to 150 disaccharides in length, and due to selective modifications of the basic polysaccharide structure (e.g. N- deacetylation, N- and O-sulfation, epimerisation) an enormous potential for sequence variability exists (Figure). Up to now 20 different disaccharides have been identified in HS. A HS chain of 100 disaccharides can therefore, in theory, have ( ) different sequences reflecting the potential for structural heterogeneity. This makes HS highly information-dense molecules. Within the monosaccharide sequences of HS lie structures for specific interaction with proteins, providing a third level of regulatory biomolecular information. This information, however, has been largely unexplored due to the complexity of HS and the lack of appropriate tools to study them. Antibodies are obvious tools to study the location and function of specific HS domains. However, HS are poorly immunogenic and raising antibodies has therefore been problematic. To circumvent this we adopted the antibody phage display technology since this system allows the generation of (human) antibodies against self-antigens. As a source of HS we choose acharan sulphate from the giant African snail since, in contrast to the heterogeneous HS from mammalians, it has a homogenous structure consisting of the repeating disaccharide α- D-N-acetylglucosaminyl-2-O-sulfo-α-Liduronic acid (GlcNAc-IdoA2S). Acharan sulphate is an anti-tumour agent and demonstrates anti-angiogenic activities. A unique antibody was selected that identified a HS oligosaccharide epitope containing N-acetylated glucosamine and 2-O-sulphated iduronic acid residues. An immunohistochemical survey using various rat organs revealed a distinct distribution of the epitope, which was primarily present in the basal laminae of most (but not all) blood vessels and of some epithelia, including human skin. No staining was observed in the HS-rich tumour matrix of metastatic melanoma, suggesting a specific HS constitution of tumours. Sequence-specific antibodies to polysaccharides like HS present valuable tools to study the realm of saccharides in cell biology. They will be instrumental in elucidating the new frontier in molecular life science: glycobiology. Figure: Biosynthesis of heparan sulphates. A number of enzymes including glycosyltransferases, sulfotransferases and an epimerase create specific monosaccharide sequences forming domain structures, which selectively bind and modulate proteins. Annual Report 2004 Gerdy ten Dam Understanding saccharides in cell biology 23

24 24 NCMLS

25 Annual Report 2004 Theme 2 Metabolism, transport and motion

26 Mitochondrial disorders are diseases with a defect present in the mitochondrion but they commonly refer to diseases that are caused Marieke Coenen by disturbances in the Oxidative phosphorylation This work was published in: Coenen, MJ, Antonicka, H, Ugalde, C, Sasarman, F, Rossi, R, Heister, JG, Newbold, RF, Trijbels, FJ, Heuvel, LP van den, Shoubridge, EA, & Smeitink, JA (2004). Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency. N Engl J Med 351 (20), Understanding Mitochondrial Disorders Nuclear genes and mitochondrial translation: a new class of genetic disease Mitochondrial disorders are diseases with a defect present in the mitochondrion but they commonly refer to diseases that are caused by disturbances in the oxidative phosphorylation (OXPHOS) system. This system present in the mitochondrial inner membrane consists of five multi subunit complexes of which the individual subunits, with the exception of complex II, are encoded either by mitochondrial (mt) or nuclear (n) DNA. Most research has been focussed on the structural buildings blocks of the OXPHOS system in patients with a single enzyme deficiency. This has lead to the identification of many mutations in nuclear as well as mitochondrial encoded structural building blocks of the OXPHOS system. The cause of diseases associated with a deficit in more than one complex and of nuclear origin has been unclear, and the prevalence of such mitochondrial disorders is similar to that of the aggregate of disorders caused by the deficiency of a single complex. We determined an association between the mutation of a nuclear gene encoding a protein that mediates translation of mitochondrial DNA (mtdna) and the occurrence of early fatal hepato-encephalopathy in two siblings, each with a deficit in more than one complex of the respiratory chain. We used the fibroblasts of a patient with a combined deficiency of complex I, III and IV. Firstly, we excluded the presence of mutations in the mtdna. Secondly we performed microcell mediated chromosome transfer to investigate which chromosome harboured the gene, which might be the cause of the deficiency seen in the patient (Figure 1). We have shown that chromosome three should harbour the mutated gene. Two genes involved in the functioning of the OXPHOS system were present in this region. Sequence analysis of these genes has led to the identification of a mutation in EFG1. The protein encoded by this gene is involved in the formation of protein from RNA on the mitochondrial ribosome. Visualisation of the mitochondrial translation products showed a decrease in overall mitochondrial translation. This is the first time that a mutated nuclear gene has been identified in a patient with a combined deficiency of complex I, III and IV of the OXPHOS system. Synthesis of mitochondrial proteins requires a number of initiation, elongation, and termination (or release) factors, all of which are encoded by nuclear genes. The gene EFG1 mutated in the patients encodes just one protein of the whole elongation machinery. The elongation factor, EFG together with GTP controls the translocation of trnas and mrna on the ribosome (Figure 2). With the detection of the mutation in EFG1, we are a step closer to understanding of mitochondrial functioning. The main question for the future will be whether we will be able to develop a rational successful treatment for patients with a mitochondrial disorder. In order to achieve this we will first have to gain a good insight into all the genes involved in mitochondrial function/dysfunction. Figure 1: Chromosome transfer Mouse cell lines that contain one human chromosome (red) are induced to form micronuclei. Each micronucleus contains a few chromosomes, some with and some without the human chromosome. The micronuclei are isolated and fused with the patient cell line. After selection, only the patient cell lines that contain the donor human chromosome survive. (Yellow mitochondria, normal; blue mitochondria, OXPHOS enzyme complex deficient). Figure 2: Translocation of peptidyl trna from A to P site. Ribosome moves one codon to the right. And the now uncharged trna moves from the P to the E site. 26 Marieke Coenen Understanding Mitochondrial Disorder NCMLS

27 An increasing number of inheritable neurodegenerative diseases have been linked to the pathological expansion of unstable simple Bé Wieringa sequence motifs (also Myotonic Dystrophy This work was published in: Spada, AR La, Richards, RI, & Wieringa, B (2004). Dynamic mutations on the move in Banff. Nat Genet 36 (7), Understanding Neurodegenerative Disorders DNA instability and macromolecular aggregation expand problems in Myotonic Dystrophy An increasing number of inheritable neurodegenerative diseases have been linked to the pathological expansion of unstable simple sequence motifs in the human genome. In normal human DNA, these motifs occur at several locations and are commonly polymorphic. In disease states, one specific motif can become pathogenic once its length exceeds the normal range. Individuals belonging to particular disease families are at risk because their motif repeat size typically exceeds a threshold length and has become intergenerationally or somatically unstable. Currently little is known about the precise mechanism of pathogenesis of DNA instability. Genetic and cell biological studies have now revealed that instability may be coupled to defective DNA repair or recombination. Pathogenic features generally occur at the RNA or protein level in tissues where the gene is expressed. The location of the mutation can differ and can be in (i) protein coding segments, yielding expanded tracts with single amino acid repeats such as polyq in Huntington s or spinocerebellar ataxias, or polya in infantile spasms syndrome; or (ii) in non-translated gene segments, yielding premrnas with expansions in their UTRs or introns. However, the nature of the affected gene products differs vastly between disorders. Against this background it is surprising that expansion disorders share a number of typical features. This point has now become more clear by intensive study of several archetypal forms of expansion disorders, including myotonic dystrophy type 1 (DM1), the most common form of neuromuscular dystrophy in adults. One of the main problems of DM1 is that the muscle symptoms of weakness and myotonia and also other core features like the cognitive disturbances and cardiac conduction defects progressively worsen throughout life, frequently ending in premature death. Our studies revealed that DM1 is caused by abnormal expansion of a (CTG) n repeat in the myotonic dystrophy protein kinase (DMPK) gene on human chromosome 19. This DNA expansion occurs over generations in DM1 families and during ageing in all tissues in individual DM1 patients. In tissues where the gene is expressed, like muscle and brain, (CUG) n expanded transcripts from the DMPK gene may become trapped in cell nuclei, forming abnormal RNA protein aggregates. This process may be the main contributor to DM1 pathogenesis, presumably by compromising global transcription or splicing of specific mrnas. A current hypothesis is that the formation of RNA protein aggregates in DM1 just as in the disorders with expanded polyq or polya products - may challenge the ubiquitin proteasome machinery for removal of abnormally folded and aggregated proteins and thereby exerts global cell energy stress leading to cell loss. Other studies involving animal and cell models, however, revealed that corruption of the normal expression profile of the DMPK protein itself, may contribute to the progressive disease phenotype. Thus, the complex DM1 phenotype may be best explained as a mixture of gene specific and common (i.e., ribonucleoprotein aggregation) problems. Future DM1 research may help to reveal a better picture of the primary cytotoxic principles that are common to all expansion disorders. Figure: Expanding repeat disorders: Intra-nuclear or cytosolic protein-protein or protein-rna aggregates trigger the ubiquitin-proteasome machinery and cause cell stress. Annual Report 2004 Bé Wieringa Understanding Neurodegenerative Disorders 27

28 The advent of genomics data has led to descriptions of the cell as networks, providing an abstract presentation in which the nodes of the Vera van Noort networks are e.g. pro- Gene co-expression networks This work was published in: Noort, V van, Snel, B, & Huynen, MH (2004) The yeast coexpression network has a small-world, scale-free architecture and can be explained by a simple model. EMBO reports 5, Understanding cellular networks Removing the mystery of cellular networks using Occam s Razor The advent of genomics data has led to descriptions of the cell as networks, providing an abstract presentation in which the nodes of the networks are e.g. proteins that are connected to each other when they physically interact. Such cellular networks show surprising similarities with networks such as the World Wide Web, social networks, airport traffic networks and many others. Firstly, they are so-called scale-free networks. This means that there is no typical number of connections per node; rather the distribution of the number of connections per node follows a power law. In other words, there are many nodes with few connections and a small number of nodes (the so-called hubs) with many connections. In case of the www, many pages link to the hub Google but very few links lead to personal home-pages. Secondly, these networks have a small-world architecture. Any two nodes can be connected to each other via very few links, while indirect neighbours also tend to link to each other. Biological networks, like the metabolic network and the protein-protein interaction network, also tend to show scale-free and small-world behaviour. This network structure has been invoked to explain features of biological systems, such as the tolerance of yeast to most single-gene deletions. For this reason, some researchers have attributed the global network properties of biological systems to natural selection. However, it is very well possible that such networks are merely the byproduct of selection at other levels, or even a natural consequence of mechanisms of genome evolution. Applying Occam s razor ( one should not increase, beyond what is necessary, the number of entities required to explain anything ), also called the principle of parsimony or law of economy, we set out to test whether selection is really required to explain the structure of biological networks. We have taken the gene co-expression network in yeast as our model system. This scale-free, small-world network consists of genes that are connected when they are expressed under similar cell conditions. Within this network we observed a correlation between co-expression of paralogs and their sequence identity. Previous models of network evolution could neither account for the combined scale-free and small-world properties nor for the correlation between co-expression and sequence similarity. We build a simple, neutralist s model (Figure) consisting of (1) co-duplication of genes with their Transcription Factor Binding Sites (TFBSs), (2) deletion and duplication of individual TFBSs and (3) gene loss. We let the system evolve for 100 generations without selection at any level. Afterwards, we constructed the co-expression network by connecting those genes that share multiple TFBSs. Our model could reproduce the scalefree and small-world network architecture as well as the homology relations between coregulated genes, hence natural selection is not needed to obtain this network architecture. The special properties of the network arise simply from the types of genetic evolutionary events to which we are most accustomed: gene duplication, point mutation and gene loss. This does not exclude the possibility of selection at the network level or that the network architecture is in some way or another exploited by the cell, but it does call for a more sober view in interpreting network architectures in terms of selections and the benefits for the cell. Figure: A simple model of gene duplication and the loss and gain of transcription factor binding sites. 28 Vera van Noort Understanding cellular networks NCMLS

29 For terrestrial animals like we are, it is essential to be able to regulate our body water homeostasis depending on our needs. The Fabrizio de Mattia kidney is the main Aquaporins This work was published in: Mattia, F de, Savelkoul, PJ, Bichet, DG, Kamsteeg, EJ, Konings, IB, Marr, N, Arthus, MF, Lonergan, M, Os, CH van, Sluijs, P van der, Robertson, G, & Deen, PM (2004). A novel mechanism in recessive nephrogenic diabetes insipidus: wild-type aquaporin-2 rescues the apical membrane expression of intracellularly retained AQP2-P262L. Hum Mol Genet 13 (24), Understanding water homeostasis The mis-sorting signal in the Aquaporin-2 water channel determines recessive NDI For terrestrial animals like we are, it is essential to be able to regulate our body water homeostasis depending on our needs. The kidney is the main organ to regulate this water balance. Per day, our blood volume of 6 litres is filtered 30 times in the kidney, resulting in the formation of 180 litres of pro-urine. 90% of this pro-urine volume is constitutively reabsorbed in a transcellular fashion in the early renal nephron, and which is facilitated by the water-selective Aquaporin-1 (AQP1) channels. Modulation of renal water reabsorption occurs through the anti-diuretic hormone vasopressin (AVP), which is released by the pituitary in states of hypernatremia or hypovolemia and which induces water reabsorption in renal collecting duct cells by binding to its vasopressin V2-receptor, elevation of intracellular camp levels and insertion of homotetrameric Aquaporin-2 (AQP2) water channels in the apical plasma membrane. In congenital nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unresponsive to AVP, this mechanism is impaired, resulting in a severe loss of water, dehydration and mental retardation later in life if not diagnosed soon after birth. NDI is caused by mutations in the gene encoding V2R, which comprises the X-linked form, or in AQP2, which results in the autosomal recessive and dominant forms of NDI. In contrast to recessive NDI, we have found that AQP2 mutants in dominant NDI are all functional water channels, but are sorted to other subcellular organelles than wt-aqp2 (1-4). Since AQP2 is expressed as a homotetramer and these AQP2 mutants are properly folded in the ER, they form heterotetramers with wt-aqp2 and missort the wt/mutant AQP2 complexes. The resulting lack of sufficient levels of wt-aqp2 in the apical membrane of collecting duct cells provides an explanation for dominant NDI. In contrast to mutants in recessive NDI, AQP2 mutations found in dominant NDI are all located in the C-terminal tail and underscore the importance of this part of the protein in AQP2 sorting. In our study, patients of two families with recessive NDI appeared compound heterozygotes for AQP2-A190T or AQP2-R187C mutants, together with AQP2-P262L. Since mutations in the AQP2 C-tail, where P262 resides, usually cause dominant NDI, the underlying cell biological mechanism was investigated. Upon expression in oocytes, AQP2-P262L was a properly-folded and functional aquaporin in contrast to the classical mutants, AQP2-R187C and AQP2- A190T. Expressed in polarized cells, AQP2- P262L was retained in intracellular vesicles and did not localize to the endoplasmic reticulum. Upon co-expression, however, AQP2-P262L interacted with wt-aqp2, but not with AQP2-R187C, resulting in a rescued apical membrane expression of AQP2- P262L. In conclusion, our study reveals a novel cellular phenotype in recessive NDI in that AQP2-P262L acts as a mutant in dominant NDI, except for that its missorting is overruled by apical sorting of wt-aqp2. Our findings demonstrate for the first time that the recessive inheritance of a disease involving a channel can be due to two cell-biological mechanisms. Figure: Missorting of wt-aqp2 to the basolateral membrane explains dominant NDI. To increase water uptake from pro-urine in hypernatremia, the anti-diuretic hormone (ADH) induces the translocation of AQP2 water channels from intracellular vesicles to the apical membrane of renal collecting duct cells (green; middle section). In autosomal dominant NDI (dndi), patients are unable to concentrate their urine due to an AQP2 mutation in one allele. Here, confocal analysis of polarized cells shows that such a mutant in dndi (AQP2-insA) translocates to the basolateral membrane with ADH, as it co-localizes with E-Cadherin (blue). Upon coexpression (right), AQP2-insA (red) missorts wild-type AQP2 (green) to the basolateral membrane, thereby providing an explanation for dndi in this family. Annual Report 2004 Fabrizio de Mattia Understanding water homeostasis 29

30 Renal organic anion transporters play a critical role in the tubular elimination of a wide variety of drugs and toxicants, and in particular of their Frans Russel negatively Detoxification This work was published in: Smeets, PHE, Aubel RAMH van, Wouterse AC, Heuvel JJMW van den, Russel FGM (2004). Contribution of multidrug resistance protein 2 (MRP2/ABCC2) to the renal excretion of p-aminohippurate (PAH) and identification of MRP4 (ABCC4) as a novel PAH transporter. J Am Soc Nephrol 15, Understanding muti-drug resistance Role of MRP2 and MRP4 in the renal excretion of xenobiotics Renal organic anion transporters play a critical role in the tubular elimination of a wide variety of drugs and toxicants, and in particular of their negatively charged glucuronide, sulfate, glycine and glutathione conjugates. During the past decade, significant progress has been made in the molecular identification and characterisation of these transporters. It is now appreciated that the renal organic anion system is a complex organization of over 15 plasma membrane proteins, many of which are isoforms that use ATP and transmembrane ion gradients to drive active drug secretion from blood into urine. The large number of different transporters with overlapping substrate specificity emphasizes the importance of renal organic anion excretion for the body. A major challenge is to elucidate the relative contribution of individual transporters to the overall clearance of specific anionic drugs. In this paper we studied the role of multidrug resistance protein 2 (MRP2/ABCC2) and 4 (MRP4/ABCC4) in the apical efflux of p-aminohippurate (PAH), the classical substrate used in the characterization of organic anion transport in renal proximal tubular cells. Whereas basolateral transporters for PAH uptake from blood into the cell have been well characterized, there is still little knowledge on the apical urinary efflux transporters. MRP2 is localized to the apical membrane and has recently been shown by us and others to mediate ATP-dependent PAH transport, but its contribution to urinary PAH excretion is not known. In this study we have shown that renal excretion of PAH in isolated perfused kidneys from wildtype and Mrp2-deficient (TR-) rats is not different. Uptake of [ 14 C]PAH in Sf9 membrane vesicles expressing human MRP2 exhibited a low affinity for PAH (~5 mm). Human MRP4, which has recently been localized by us to the apical membrane (Figure 1), had a much higher affinity for PAH (K m = 160 ± 50 µm). Real-time PCR and Western blot analysis showed that expression of MRP4 in human kidney is approximately 5-fold higher as compared with MRP2. In this report we have shown that MRP4 is a novel PAH transporter, which has higher affinity for PAH and is expressed higher in kidney than MRP2, and may therefore be more important in renal PAH excretion. Its specific role relative to other apical organic anion transporters remains to be elucidated. To study this, multiple knock-out or knockdown models of the genes encoding these proteins may be required because of the high redundancy within this group of transporters. Figure: Immunofluorescence microscopic analyses of multidrug resistance protein 2 (MRP2) and 4 (MRP4) in a proximal tubule of human kidney. Double-labeling with pab hm4-p3 and monoclonal antibody M2III-6 was used for detection of MRP4 and MRP2, respectively. Superimposition demonstrates colocalization of MRP4 with MRP2 in the brush border membrane. 30 Frans Russel Understanding muti-drug resistance NCMLS

31 Annual Report 2004 Theme 3 Cell growth and differentiation

32 The programme of gene expression in an individual cell of a multicellular organism is not wholly specified by the DNA sequence itself. It Huiqing Zhou is epigenetic informa- Chromatin re-modelling This work was published in: Høiby, T, Mitsiou DJ, Zhou H, Erdjument-Bromage H, Tempst P & Stunnenberg HG (2004). Cleavage and proteasome-mediated degradation of the basal transcription factor TFIIA. EMBO J 23, Understanding gene regulation From single transcription factor analysis to global ChIP-on-chip profiling Gene expression in an individual cell of a multicellular organism is not wholly specified by the DNA sequence itself. It is epigenetic information layered upon it which provides the filter that dictates how, when and where the genetic information should be expressed in developing organisms. Despite the availability of several completely sequenced genomes, little is known about how genes interact and regulate each other within a given cell type, to specify identity and function. A realistic model of cellular regulation based on current knowledge points to multiple interacting networks operating at the epigenetic, transcriptional, posttranscriptional, translational and posttranslational levels, with feedback between the various levels. Characterisation of the epigenetic code which is the combinatorial sum of DNA methylations, histone modifications and histone variant can be used to determine whether a chromatin conformation is transcriptionally poised or inactive. To this end, it is also necessary to identify the full complement of enzymes that modify the histones. We set out to characterise the epigenome using high through-put technologies such as advanced mass spectrometry applied to histone post-translational modifications and complex walking. The challenge is to decipher the epigenetic code and to unravel its meaning. This requires a comprehensive documentation of coding combinations, the hierarchies and cross-signaling within the code. Of the currently known histone tail modifications, lysine acetylation has been studied most extensively. Acetylation of histones is a reversible and highly dynamic process catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Although considerable progress has been made in deciphering the role of the HDAC-containing complexes in regulation of gene expression, the histone substrate specificity and therefore the mechanistic role of the various HDAC-complexes in establishing a histone code remained largely unknown. Our findings provide the first evidence that distinct HDAC-containing co-repressor complexes play different roles in the regulation of chromatin structure and transcription. At a greater level of genome specificity, the establishment of patterns of gene expression for particular cell types depends on the binding of transcriptional factors and co-factors to the chromatin. It remains unclear whether interaction of the transcriptional machinery with chromatin is cause or consequence of local chromatin environment. Therefore the identification of the ensemble of histone marks and factors, where they bind along the genome, in what complexes and how they dynamically affect or respond to local chromatin context are all important questions. We have established high throughput ChIP-on-chip to profile histone marks and transcription factor at a global genome wide level. Profiling of the acetylation modification of histone H3 on lysine 9 (brown peak) throughout the human X-chromosome reveals that this mark coincides with the transcription start site of active genes. In many cases, the peak of histone modification is offset with the presumed start of transcription (such as ZNF185 gene) suggesting that the gene annotation maybe incorrect. Our group has also made progress in elucidation of a hitherto enigmatic feature of the general transcription factor, TFIIA: its proteolytic cleavage. The determination and characterisation of the cleavage site and the observation that cleavage leads to subsequent degradation to fine-tune the amount of TFIIA in the cell implies that TFIIA cleavage is a highly regulated, cell type-dependent event. It also leads to the discovery of MLL protease Taspase1 as the enzyme responsible for TFIIA cleavage. These studies and unpublished data suggest that cleavage by Taspase1 regulates the levels of TFIIA, MLL and other substrates in the cell and plays a critical role in transcription and development. Figure: ChIP-on-chip profiling of the histone H3 lysine 9 acetylation on a 200kb fragment covering the Q-arm of human chromosome X. A 50-mer oligonucleotide tile path microarray at 100 bp reolution was designed to cover the entire unique part of the human X-chromosome. The top track shows the relative enrichment (log2chip/input); the line indicates 95% confidence value. The lower track shows the currently annotated position and direction of transcription of genes. 32 Huiqing Zhou Understanding gene regulation NCMLS

33 Limb malformations are among the most common human birth defects. Normal limb development relies on the careful orchestration of the interplay Ernie Bongers be- Small patella syndrome This work was published in: Bongers EM, Duijf PH, Beersum SE van, Schoots J, Kampen A van, Burckhardt A, Hamel BC, Losan F, Hoefsloot LH, Yntema HG, Knoers NV & Bokhoven H van (2004). Mutations in the human TBX4 gene cause small patella syndrome. Am J Hum Genet 74, Understanding limb development Mutations in the human TBX4 gene cause abnormal limb development Limb malformations are among the most common human birth defects. Normal limb development relies on the careful orchestration of the interplay between transcription factors, signalling molecules and structural proteins. Mutations in single genes that give rise to limb malformations provide more insight into these complex molecular networks. Several key players in limb development have been identified already by studying rare genetic human disorders that comprise limb malformations. Here, we have found that a gene from the T-Box family of transcription factors, TBX4, is mutated in a syndrome with abnormal development of lower limb structures in small patella syndrome. This result is of considerable interest, since it reveals that specific molecular pathways are involved in the development programmes of the arm and leg. Small patella syndrome (SPS) is an autosomal dominant skeletal dysplasia characterised by patellar aplasia or hypoplasia and anomalies of the pelvis and feet (Figure). Linkage analysis identified an SPS critical region of 5.6-cM on chromosome 17q22 by haplotype analysis. A Putative loss-of-function mutations were found in a positional gene encoding T-box protein 4 (TBX4) in 5 families with SPS. The five heterozygous mutations included one nonsense-, two missense-, one frameshift-, and one splice-site mutation, which were all absent in 100 control chromosomes. Several mutations predict truncated proteins or no protein at all, indicating that TBX4 gene dosage is critical for normal hind limb development. The two amino acid substitutions affect highly conserved residues in the T-box (G248V) and at the C-terminal end (Q531R) and very likely disrupt crucial functions of the TBX4 protein. Several mutations are de novo, which is compelling evidence for being causative for SPS in these patients. B TBX4 belongs to the T-box gene family, encoding transcription factors characterized by a strongly conserved DNA-binding motif (T-box domain). Mutations in several T-box genes are associated with human developmental disorders, including TBX1 in DiGeorge syndrome, TBX22 in isolated cleft palate, TBX3 in ulnarmammary syndrome, and TBX5 mutations in Holt-Oram syndrome. Mutations in both TBX3 and TBX5 give rise to disrupted upper limb development. Critical stage-dependent roles of Tbx4 in lower limb initiation and outgrowth were shown by misexpression studies of dominant-negative Tbx4 in chicken. A legless phenotype was seen after early disruption of development, and truncated legs and hypomorphic distal structures during Tbx4 misexpression at later developmental stages. The human SPS phenotype suggests that human TBX4 mutations do not affect early steps in limb development, such as limb bud initiation, but do have a profound effect at later stages. The relative mild limb phenotype and the absence of other developmental anomalies in SPS suggest a higher degree of redundancy for TBX4 in comparison to TBX3 and TBX5. The present identification of heterozygous TBX4 mutations in SPS patients, together with the similar skeletal phenotype of animals lacking Tbx4 establish the importance of TBX4 in the developmental pathways of the lower limbs and the pelvis in humans. Figure: Pedigree structure of a family with dominant Small Patella Syndrome. Note the small patella in panel B. Annual Report 2004 Ernie Bongers Understanding limb development 33

34 Recent developments in DNA microarray technologies, pioneered by the NCMLS microarray group have Lisenka Vissers resulted in a robust Chromosomal micro-deletions/-duplications This work was published in: Vissers, LE, Ravenswaaij, CM van, Admiraal, R, Hurst, JA, Vries, BB de, Janssen, IM, Vliet, WA van der, Huys, EH, Jong, PJ, Hamel, BC, Schoenmakers, EF, Brunner, HG, Veltman, JA, & Kessel, AG van (2004). Mutations in a new member of the chromodomain gene family cause CHARGE syndrome. Nat Genet 36 (9), Understanding charge syndrome New technology leads to gene identification for congenital disorder DNA microarray technologies, pioneered by the NCMLS microarray group, have resulted in a robust high resolution ( Kb) screening method for the identification of submicroscopic deletions and/or duplications throughout the entire human genome. These so-called micro-deletions and microduplications are a frequent cause of human congenital and acquired disorders, including mental retardation, malformation syndromes and cancer. The identification of such genomic alterations is instrumental in the search for genomic regions harboring causative genes. Recently, we applied our novel approach to the identification of a gene underlying CHARGE syndrome. CHARGE syndrome denotes a non-random occurrence of ocular coloboma, heart defects, choanal atresia, retarded growth and development, genital hypoplasia, ear anomalies and deafness. Most patients with CHARGE syndrome are sporadic and its cause was, until recently, unknown. We hypothesized that CHARGE syndrome might be due to a genomic microdeletion and/or a mutation in a gene affecting early embryonic development. We used microarray-based Comparative Genomic Hybridization (array CGH) to screen patients with CHARGE syndrome for submicroscopic DNA copy number alterations. By doing so, two de novo overlapping micro-deletions in chromosome 8q12 were identified. A 2.3 Mb region of deletion overlap was defined using a tiling resolution microarray. Subsequent sequence analysis of genes residing within this critical region revealed mutations in the CHD7 gene in 10 of the 17 CHARGE patients tested without micro-deletions. The CHD7 gene encodes a novel member of the chromodomain family of proteins, a family that plays an essential role in early embryonic development affecting multiple organ systems. We showed for the first time that high resolution genome-wide screening by array CGH is an effective approach for the identification of disease genes. This approach is of special relevance for sporadic malformation syndromes that cannot be tackled by other mapping approaches because of reproductive lethality. Since the identification of the CHD7 gene, mutation analysis and micro-deletion screening have been implemented into our routine DNA diagnostic services. In addition, we are studying the genomic mechanisms underlying recurrent genomic alterations, including the role of so-called large-scale copy number variations (LCVs) in the development of multi-factorial diseases. a b Figure: Two individuals with CHARGE syndrome, detailed genomic view of 8q12 and organization of CHD7. (a) Transcript map of the deleted 8q12 genomic region. The shortest region of deletion overlap two individuals is shown. Genes in green were screened for mutations. Cen, centromeric; Tel, telomeric. (b) Genomic structure of CHD7 indicating the positions of seven nonsense mutations (circles), two mis-sense mutations (squares) and one intron-exon boundary mutation (triangle). The corresponding functional CHD7 domains are marked (colored bars). 34 Lisenka Vissers Understanding charge syndrome NCMLS

35 Transcription initiation represents the primary and most regulated step of eukaryotic gene expression. In humans, a set of approximately Zainab Jallow two thousand transcrip- TATA binding protein This work was published in: Jallow, Z, Jacobi UG, Weeks, DL, Dawid, IB & Veenstra GJC (2004). Specialized and redundant roles of TBP and a vertebrate-specific TBP paralog in embryonic gene regulation in Xenopus. Proc. Natl. Acad. Sci. USA 101, (Zainab Jallow and Rike Jacobi contributed equally to this paper). Understanding initiation of transcription Gene-selectivity of the general transcription machinery Transcription initiation represents the primary and most regulated step of eukaryotic gene expression. In humans, a set of approximately two thousand transcription factors integrate cellular and intercellular signals upon a set of general transcription factors (GTFs) which function to recruit RNA polymerase II to the promoters of proteinencoding genes. TATA binding protein (TBP) is a key component of the general transcription machinery. TBP can bind to promoters directly or via TBP-associated factors (TAFs), and can recruit other GTFs and RNA polymerase II to the promoter. Our interest in TBP was first triggered when we found that translation of maternal TBP mrna is highly regulated during embryonic development of Xenopus, our vertebrate model system. Accumulation of TBP during blastula stages coincides with and contributes to the onset of embryonic transcription. In the course of our experiments to assess the role of TBP in the global regulation of transcription, we blocked TBP accumulation by antisense knockdown. The unexpected result was that much of the early embryonic transcription did not depend on TBP. Part of the TBP-independent transcription could be attributed to a TBP paralog known as TBP-like factor (TLF). This factor is not found in yeast but is specific to metazoans. Recently we identified and characterized another TBP family member, TBP2, which is specific for vertebrates. Using in vitro assays we found that TBP2 can bind to the TATA box, as expected on basis of sequence and structure information (Figure). Moreover, TBP2 can support basal transcription in vitro, similar to TBP. We discovered that TBP2 is highly abundant in oocytes, but is expressed at lower levels in embryos. TBP exhibits a different expression pattern; TBP cannot be detected in oocytes, but is highly abundant in embryos from late blastula stages onwards. Using chromatin immunoprecipitation (ChIP) we found that TBP2 is recruited to promoters in Xenopus oocytes in the absence of detectable TBP recruitment, suggesting that TBP2 represents an oocyte TBP replacement factor. The low TBP2 levels in embryos prompted us to examine the extent to which TBP2 functions during development. Using ChIP assays we determined that TBP2 is recruited to promoters in embryos. We knocked down TBP2 expression with antisense oligonucleotides and found that TBP2 is essential for gastrulation and for embryonic transcription of a subset of genes. We also addressed the question as to what extent TBP and TBP2 might be functionally redundant in embryos. TBP2 protein is much less abundant than TBP in embryos, and as it turns out, moderate overexpression of TBP2 partially rescues an antisense knockdown of TBP and restores transcription of many TBP-dependent genes. Therefore, in embryos TBP and TBP2 are coexpressed and exhibit partial redundancy for a subset of TBP-dependent genes, even though both factors are also required and play gene-selective roles in embryonic transcription initiation. Our further research will include analysis of protein-protein interactions of TBP family members to identify potentially novel mechanisms of transcription initiation, and developmental pathway analysis to determine the biological function of a variable transcription machinery. Figure: The core domain of the general transcription factor TBP (red) bound to DNA (grey). The core domain of vertebrate-specific TBP2 is identical to that of TBP except for nine residues (blue) which could be important for protein-protein interactions. Annual Report 2004 Zainab Jallow Understanding initiation of transcription 35

36 Interference with a tumour s blood supply is an effective way to halt tumour progression, and even induce tumour regression. It has William Leenders also become possible VEGF inhibition This work was published in: Leenders, WP, Kusters, B, Verrijp, K, Maass, C, Wesseling, P, Heerschap, A, Ruiter, D, Ryan, A, & Waal, R de (2004). Antiangiogenic therapy of cerebral melanoma metastases results in sustained tumor progression via vessel co-option. Clin Cancer Res 10 (18 Pt 1), Understanding tumour vascularisation Pitfalls of anti-angiogenic therapy Interference with a tumour s blood supply is an effective way to halt tumour progression, and even induce tumour regression. It has also become possible to efficiently interfere with vascularisation. Most of the compounds that have been developed with this aim, interfere with signaling by Vascular Endothelial Growth Factor-A (VEG-A), the most potent angiogenic factor known. An example that was recently highlighted is Avastin, a humanized antibody against VEGF-A (Genentech). This is the first angiogenesis inhibitor that, in combination with chemotherapy, showed improved survival in a phase III clinical trial involving patients with advanced colorectal cancer. Despite this encouraging result, the view that anti-angiogenic therapies can be used to turn cancer into a chronic disease is too optimistic. The models currently used consist of tumours that are implanted subcutaneously in the flanks of animals, where virtually no pre-existent vessels are present. This obviously results in angiogenesisdependent outgrowth of tumours, and from this point of view the anti-tumoural effect of angiogenesis-inhibition is not surprising. However, are these models clinically relevant? Here it is important to realise that the aim of anti-angiogenic therapy is to halt progression of otherwise untreatable cancers, mostly multiple metastases which grow in tissues with a dense pre-existent vasculature. We have shown that angiogenesis might just not be a prerequisite for tumour growth in these situations, because of the ability of tumour cells to exploit the pre-existent vasculature, a process which is now referred to as vessel co-option. Co-option of pre-existent vessels has now been shown in liver, lung and brain, tissues that are prone to metastasis. To investigate whether vessel-co-option might explain the disappointing results of clinical trials, we used an animal model which may be more clinically relevant than subcutaneous tumours. VEGF-A-expressing tumour cells were injected in the internal carotid artery of mice, a method that reproducibly leads to the development of highly angiogenic tumours in the brain parenchyma. One of the hallmarks of these angiogenic tumours is the absence of the bloodbrain barrier in the leaky tumour vessels, enabling visualization of the tumours by in vivo MRI using Gd-DTPA, a low Mw contrast agent that, after extravasation, accumulates in the tumour interstitia and generates an increased MR signal. We treated mice carrying such tumours with ZD6474 (AstraZeneca), an orally available inhibitor of VEGF signal transduction. Although ZD6474 inhibited angiogenesis, this did not result in tumour regression, but rather to progression via a vessel co-opting phenotype. Importantly, VEGF-A inhibition resulted in a restored blood-brain barrier in tumour-associated vessels, and consequently tumours could not be detected anymore in Gd-DTPA enhanced MRI (Figure). These results have two profound implications. Firstly, Gd-DTPA-enhanced MRI scans of brain tumours after anti-angiogenic therapy may falsely suggest tumour regression. Secondly, when applying combined antiangiogenic therapy and chemotherapy of brain tumours, the order of administration may be crucial to the outcome: anti-angiogenic therapies may close down the bloodbrain barrier and thus prevent effective tumour-uptake of cytotoxic compounds. placebo ZD6474 Pre - contrast Post - contrast CD34 blood vesselstaining Figure: Effects of VEGF-inhibition on tumour phenotype. CD34 staining show the typical phenotype of an angiogenic tumour with irregular and dilated vessels which are also leaky (notice contrast enhancement in MRI). Treatment with the VEGFR2 inhibitor ZD6474 results in MR-invisible tumours that are able to progress by co-opting pre-existent vessels. 36 William Leenders Understanding tumour vascularisation NCMLS

37 Protein secretion plays a fundamental role in a number of biological processes. An important step in protein secretion concerns the Gerard Martens transport of secretory p24 cargo receptors This work was published in: Bouw, G, Huizen, R van, Jansen, EJR & Martens, GJM (2004). A cell-specific transgenic approach in Xenopus reveals the importance of a functional p24 system for a secretory cell. Mol. Biol. Cell 15, Understanding protein transport Cell-type specific transgene expression and the role of p24 in ER-to-Golgi protein cargo transport Protein secretion plays a fundamental role in a number of biological processes. An important step in protein secretion concerns the transport of secretory cargo proteins through the early secretory pathway. This step involves cargo selection, transport vesicle formation, quality control to recycle misfolded cargo, and cycling of the COPI- and COPII-coated vesicles between the endoplasmic reticulum (ER) and Golgi. One of the major constituents of the transport vesicles is the p24 family of type I transmembrane proteins that can be subdivided into p24α, - β, -γ and -δ subfamilies. A number of functional models have been proposed for the elusive mechanism of action of p24, (including a role as cargo receptor, membrane organiser, or regulator of vesicle budding, as well as in the ER quality control system). However, defining the role of p24 has turned out to be difficult. For instance, deletion of all p24 proteins resulted in viable yeast, whereas genetic ablation of a single p24 family member caused early lethality in mice. We therefore investigated the role of the p24 system in a well-defined secretory cell using a transgenic approach, namely by targeting p24 transgene expression specifically to the Xenopus intermediate pituitary melanotrope cell (Figure). This cell is involved in background adaptation of the animal and produces high levels of its major secretory cargo, the prohormone proopiomelanocortin (POMC). For transgenic overexpression, we selected one of the Xenopus melanotrope p24 proteins that was coexpressed with POMC, the p24δ 2 protein, and fused it to the N-terminus of GFP. The transgene product effectively displaced not only the p24δ proteins but also other endogenous p24 members, whereby the degree of displacement depended on the level of the fusion protein, resulting in a melanotrope cell p24 system that consisted predominantly of the p24δ 2 GFP protein. Despite the severely distorted p24 machinery, the subcellular structures as well as the level of POMC synthesis were normal in these cells. However, an aberrant POMC processing product and less POMC-derived peptides were produced, and the number and pigment content of the skin melanophores was reduced, impairing the ability of the transgenic animal to fully adapt to a black background. Together, our results are most consistent with a role for p24 in the transport of newly synthesized secretory cargo proteins through the early stages of the secretory pathway or in the processing of secretory cargo by recruiting the proper components of the biosynthetic machinery into ER-to- Golgi cargo transport carriers. Furthermore, our transgenic approach in a physiological context has shown that distortion of the complex p24 system results in an affected profile of prohormone-derived bioactive peptides with the eventual consequence at the level of the target cell receiving the secretory signals. Current and future studies include transgenic experiments with the three other upregulated p24 proteins (p24α 3, -β 1 and - γ 3 ). We hope that the possibility of stable, non-mosaic Xenopus transgenesis combined with the experimental advantages of this lower vertebrate will enable us to elucidate the role of proteins of unknown function, in particular the long-sought role of p24 in the early secretory pathway. Figure: Various levels of p24δ 2 -GFP transgene expression specific in the intermediate pituitary of living stage-45 Xenopus embryos. Bar equals 0.4 mm. Annual Report 2004 Gerard Martens Understanding protein transport 37

38 Studies on phosphotyrosine-based signaling pathways that direct growth and development often address the actions of protein tyrosine kinases, not in Rick Wansink the Protein tyrosine phosphatases This work was published in: Wansink, DG, Peters, W, Schaafsma, I, Sutmuller, RPM, Oerlemans, F, Adema, G, Wieringa, B, Zee, CEEM van der & Hendriks, W (2004). Mild Impairment of Motor Nerve Repair in Mice Lacking PTP-BL Tyrosine Phosphatase Activity. Physiol Genomics 19, Understanding signals that control growth and development Studies on protein tyrosine phosphatases Studies on phosphotyrosine-based signaling pathways that direct growth and development often address the actions of protein tyrosine kinases, not in the least because mutant variants of these enzymes have been implicated in many disease states, including cancer. Hence, timely termination of phosphotyrosine-based signals is of equal importance, a process mediated by the protein tyrosine phosphatase (PTP) enzyme family. Our interest is studying PTP signaling at the molecular and cellular level, and also in mouse models. All PTPs contain one or two copies of the well-conserved phosphotyrosine-specific phosphatase domain and their catalytic activity exceeds that of tyrosine kinases by several hundred fold. Hence, there is a clear need for tight regulation and confinement of PTP activity in vivo, and clear phenotypic consequences for the organism may be expected following mutation of PTP genes. Therefore we were slightly disappointed to learn that the PTP-BL P mice created (that express a truncated version of PTP-BL and consequently lack the enzymatic activity of this largest cytoplasmic PTP) appeared fully normal at first glance. To identify cellular processes in these mice that require the catalytic activity of PTP-BL, several different experiments were performed. Our novel sciatic nerve repair assay, that had proven itself in the phenotypic analysis of mice deficient in LAR PTP activity disclosed at least one. Mouse PTP-BL is a 270-kDa cell-cortical enzyme that consists of a KIND domain, a FERM domain, five PDZ domains, and a single PTP domain. KIND, FERM and PDZ domains are all protein interaction motifs and indeed multiple PTP-BL-associating proteins have been identified, leading to proposed roles for PTP-BL in microfilament dynamics, cytokinesis, apoptosis, and neurite outgrowth. These all could be put to the test in mice lacking the PTP-BL PTP moiety. PTP-BL P mice appeared viable and fertile and, although PTP-BL was expressed in most hematopoietic cell lineages, no alterations of thymocyte development were detected. Sciatic nerve lesioning revealed that sensory nerve recovery was unaltered in these mice. In contrast, a mild but significant impairment of motor nerve repair was observed. These findings exclude an essential role as a phosphotyrosine phosphatase for PTP-BL and are in line with a role as scaffolding or anchoring molecule. Figure: PTP-BL immunostaining is visible in small (arrowheads) and large (arrows) dorsal root ganglia sensory neurons (A) and in most motor neurons in the ventral horn of the spinal cord (B, arrows). Bar = 50 mm. 38 Rick Wansink Understanding signals that control growth and development NCMLS

39 Cells are continuously subjected to all sorts of stress. This ranges from increased temperatures and changes in ph to the wear and tear of Guido Kappé normal ageing. Such Heat shock proteins This work was published in: Kappé, G, Aquilina, JA, Wunderink, L, Kamps, B, Robinson, CV, Garate, T, Boelens, WC & Jong, WW de (2004). Tsp36, a tapeworm small heat-shock protein with a duplicated a-crystallin domain, forms dimers and tetramers with good chaperone-like activity. Proteins 57, Understanding molecular chaperones and protein folding Learning about chaperoning from a tapeworm stress protein Cells are continuously subjected to all sorts of stress. This ranges from increased temperatures and changes in ph to the wear and tear of normal ageing. Such insults cause protein damage, which affects their normal structures and functions. Stress often results in unfolding and aggregation of the affected proteins, which is a great threat to cellular viability. To cope with these deleterious effects, the cell is provided with an intricate defense system consisting of the so-called heat-shock proteins. These proteins are molecular chaperones that interact with unfolded and damaged proteins. The chaperoning process results in either refolding of the protein or transfer to the cellular protein breakdown system, the proteasome. A special group of chaperones is formed by the small heat-shock proteins (shsps). These shsps are characterized by their small subunit size (15-30 kda) and by the presence of a conserved structural region, the α-crystallin domain (ACD). The shsps occur in virtually all organisms and cell types, and are especially effective in binding unfolding proteins and keeping them in a soluble form. Other chaperones then may refold the bound protein or transfer it to the proteasome. shsps are especially important in maintaining cytoskeletal integrity, and protect the cell against apoptosis. In humans, shsps are found around the protein aggregates that occur in neurodegenerative diseases like Alzheimer s and in some types of muscular degeneration. Increased levels of shsps are present in certain tumours and associated with poor prognosis. The working mechanisms of shsps are poorly understood, but the conserved ACD is considered to play a central role. We therefore became interested in a unique type of shsp with a duplicated ACD, which only occurs in flatworms. We expressed and purified this protein, called Tsp36, from the tapeworm Taenia saginata. It turned out that Tsp36 occurs in two complex forms, as dimers in a reducing environment, and as tetramers under non-reducing conditions. In vitro experiments showed that the tetrameric form has greater chaperoning capacity than the dimeric form. This is the first example of a chaperone that regulates its activity depending on the reduction state in the cell. An exciting finding was that Tsp36 could be crystallized and its three-dimensional structure determined by X- ray diffraction studies, in collaboration with Robin Stamler and Christine Slingsby (Birkbeck College, London). Although the structures of two distantly related shsps, from a bacterium and a plant, were already available, this is the first known crystal structure of an animal shsp, and thus of greater relevance for understanding the structure-function relation of human shsps. From the Tsp36 sequence and structure it appears indeed that animal shsps have a different manner of subunit interaction, suggesting that substrate binding and chaperoning mechanisms differ from that in non-animal shsps. The Tsp36 oligomer shows how shsps can bind irregular structure and secondary structural elements. Knowing the structure of an animal shsp now facilitates the design and interpretation of future experiments to unravel the various interactions involved in the functioning of shsps, and to understand the effects of posttranslational modifications and specific disease-causing mutants of human shsps. Figure: The structure of dimeric Tsp36 with the two ACDs (purple) and a conserved disease-related arginine (blue) indicated in one of the subunits. Annual Report 2004 Guido Kappé Understanding molecular chaperones and protein folding 39

40 Since the early 1900s enzyme kinetics has been studied in bulk, aqueous solution. Only recently it has been proven possible to Alan Rowan monitor in real-time the Enzyme kinetics This work was published in Velonia, K, Flomenbom, O, Loos, D, Masuo, S, Cotlet, M, Engelborghs, Y, Hofkens, J, Rowan, AE, Klafter, J, Nolte, RJM, Schryver, FC de (2004). Single enzyme kinetics of CALB catalyzed hydrolysis. Epub 23 Dec Understanding the dynamic behavior of enzymes Single enzyme studies on a virus-enzyme hybrid Since the early 1900s enzyme kinetics has been studied in bulk, aqueous solution. Only recently it has been proven possible to monitor in real-time the dynamic behavior of a single enzyme on a surface, using confocal fluorescence microscopy (CFM) and highly sensitive photo detectors. We recently developed a novel approach, in which a lipase was immobilized on a glass substrate, and parked in the focus of a confocal microscope (Figure 1). A pro-fluorescent substrate is added, which upon enzymatic hydrolysis is converted into a fluorescent product, whose emission is recorded using the microscope. We applied this procedure to investigate a virus particle (PVX, potato virus X), which had been genetically engineered in such a way, that it contains enzyme moieties (CALB, lipase B of Candida Antarctica) linked at its periphery (Figure 1). The real-time enzymatic activity of the PVX- CALB hybrid particle can be seen in Figure 2. The traces indicate on-off behavior of the enzyme as observed before. The enzyme activity is completely blocked upon addition of a specific inhibitor (Paraoxon, Figure 2 bottom left), proving that the events are the result of lipase action. Often, enzyme activity is higher at higher substrate concentrations, eventually reaching a maximum (Michaelis-Menten kinetics). The reverse appears to be true for the present system: at higher substrate concentrations, the activity drops, probably as a result of product inhibition. Based on these results, we conclude that the virus particles decorated with enzymes have been constructed and remain active. This approach offers opportunities for the construction and measuring of defined cascade catalytic systems operating like the metabolons in nature. Figure 1: (Top) Experimental setup. (Bottom left) Impression of the pvx-calb particle. (Bottom right) CFM image of an active particle. Figure 2: (Top) Representative time trace, showing peaks of fluorescence at the location of the virus. (Bottom left) Addition of an inhibitor (paraoxon) blocks the activity completely. (Bottom right) Activity as a function of substrate concentration. 40 Alan Rowan Understanding the dynamic behavior of enzymes NCMLS

41 Facts and figures Members of NCMLS Scientific publications 2004 Annual Report 2004

42 Research: 345 PhD students: 172 Support staff: 93 Facts and figures PhD theses: Scientific publications: Patents 25 (21 UMC + 4 NWI) 390 (298 UMC + 92 NWI) 4 (3 UMC + 1 NWI) Our people as: Research: 345 PhD students: 172 Support staff: 93 Our output as: PhD theses: Scientific publications: Patents 25 (21 UMC + 4 NWI) 390 (298 UMC + 92 NWI) 4 (3 UMC + 1 NWI) 42 Facts and figures NCMLS

43 Members of NCMLS I: Infection, Immunity and II: Metabolism, transport III: Cell growth and Tissue Repair and motion differentiation PI PI PI Adema* Wieringa* Stunnenberg* Figdor Huynen Cremers Jansen Smeitink Geurts van Kessel Schalkwijk* Bindels* Martens* van den Berg Deen Schalken van Venrooij Russel van Zoelen* van Kuppevelt* de Jong* van Krieken van Bokhoven PI- Edu / Tech. platform PI- Edu / Tech. platform PI- Edu / Tech. platform Sauerwein Willems Vriend Torensma de Pont van der Kraan Fransen Aspiring PI Aspiring PI Aspiring PI van Kuppeveld van den Heuvel Franke van de Loo Nijtmans Roepman Melchers Hoenderop Logie Netea Knoers Lohrum Pruijn Drenth Veenstra Dolstra Masereeuw Wesseling Joosten de Waal van Leeuwen Hendriks Oosterwijk van Leeuwen van der Reijden van der Vlag Affiliated PI Affiliated PI Affiliated PI Berden Blom Brunner Galama Heerschap Gielen Kullberg Smits Olijve van der Meer Wevers Theuvenet Hoogerbrugge Jenks van Delft Hilbrands Scheenen van Hest Punt Nolte Raymakers Rowan de Witte Rutjes Jansen Speller Lubsen * subtheme leader Our people from Faculty of Science Individual researchers from the local research community form close collaborations with the research school, without being full members. These are NCMLS Affiliated Principal Investigators, contributing more than 50% of their total research to other institutes (e.g. IMM or CNCN) or to clinical practice. Some junior staff within the Affiliated Principal Investigators groups are however full members of the NCMLS. Annual Report 2004 Members of NCMLS 43

44 Scientific publications Aarbiou, J., Schadewijk, A. van, Stolk, J., Sont, J.K., Boer, W.I., Rabe, K.F., Krieken, JH van, Mauad, T., & Hiemstra, P.S. (2004). Human neutrophil defensins and secretory leukocyte proteinase inhibitor in squamous metaplastic epithelium of bronchial airways. Inflamm Res 53 (6), Aarbiou, J., Verhoosel, RM, Wetering, S. van, Boer, W.I., Krieken, JH van, Litvinov, S.V., Rabe, K.F., & Hiemstra, P.S. (2004). Neutrophil defensins enhance lung epithelial wound closure and mucin gene expression in vitro. Am J Respir Cell Mol Biol 30 (2), Abbink, EJ, Wal, PS van der, Sweep, CG, Smits, P., & Tack, CJ (2004). Compared to glibenclamide, repaglinide treatment results in a more rapid fall in glucose level and beta-cell secretion after glucose stimulation. Diabetes Metab Res Rev 20 (6), Alieke, GV, Bont, N. de, Netea, M.G., Demacker, PN, Meer, JW Van der, Stalenhoef, AF, & Kullberg, B.J. (2004). Apolipoprotein-E-deficient mice exhibit an increased susceptibility to disseminated candidiasis. Med Mycol 42 (4), Al-Shali, K, Cao, H, Knoers, N, Hermus, AR, Tack, CJ, & Hegele, RA (2004). A single-base mutation in the peroxisome proliferator-activated receptor gamma4 promoter associated with altered in vitro expression and partial lipodystrophy. J Clin Endocrinol Metab 89 (11), Artz, MA, Boots, J.M., Ligtenberg, G., Roodnat, J.I., Christiaans, M.H., Vos, P.F., Moons, P, Borm, G, & Hilbrands, L.B. (2004). Conversion from cyclosporine to tacrolimus improves quality-of-life indices, renal graft function and cardiovascular risk profile. Am J Transplant 4 (6), Artz, MA, Borm, G, & Hilbrands, L.B. (2004). Letter: Conversion from cyclosporine to tacrolimus improves quality of life indices, renal graft function and cardiovascular risk profile. Am J Transplant 4 (12), Artz, MA, Hilbrands, L.B., Borm, G, Assmann, KJ, & Wetzels, JF (2004). Blockade of the renin-angiotensin system increases graft survival in patients with chronic allograft nephropathy. Nephrol Dial Transplant 19 (11), Aruga, J, Ogura, H, Shutoh, F, Ogawa, M., Franke, B., Nagao, S, & Mikoshiba, K. (2004). Locomotor and oculomotor impairment associated with cerebellar dysgenesis in Zic3-deficient (Bent tail) mutant mice. Eur J Neurosci 20 (8), Assen, S. van, Bosma, F., Staals, LM, Kullberg, B.J., Melchers, WJ, Lammens, M, Kornips, FH, Vos, P.E., & Fikkers, B.G. (2004). Acute disseminated encephalomyelitis associated with Borrelia burgdorferi. J Neurol 251 (5), Assmann, B.E., Robinson, RO, Surtees, RA, Brautigam, C, Heales, SJ, Wevers, R.A., Zschocke, J., Hyland, K., Sharma, R, & Hoffmann, G.F. (2004). Infantile Parkinsonism-dystonia and elevated dopamine metabolites in CSF. Neurology 62 (10), Bakel, H van, Huynen, M., & Wijmenga, C. (2004). Prokaryotic diversity of the Saccharomyces cerevisiae Atx1p-mediated copper pathway. Bioinformatics 20 (16), Balkom, BW van, Graat, MP, Raak, M van, Hofman, E., Sluijs, P. van der, & Deen, PM (2004). Role of cytoplasmic termini in sorting and shuttling of the aquaporin-2 water channel. Am J Physiol Cell Physiol 286 (2), C Barratt-Boyes, SM, & Figdor, C.G. (2004). Current issues in delivering DCs for immunotherapy. Cytotherapy 6 (2), Bartels-Stringer, M., Terlunen, L, Siero, H, Russel, FG, Smits, P., & Kramers, C. (2004). Preserved vascular reactivity of rat renal arteries after cold storage. Cryobiology 48 (1), Barth, P.G., Majoie, CB, Gootjes, J, Wanders, RJ, Waterham, H.R., Knaap, M.S. van der, Klerk, JB de, Smeitink, J, & Poll-The, B.T. (2004). Neuroimaging of peroxisome biogenesis disorders (Zellweger spectrum) with prolonged survival. Neurology 62 (3), Bartholdi, D, Zumsteg, D, Verrips, A., Wevers, R.A., Sistermans, E, Hess, K, & Jung, HH (2004). Spinal phenotype of cerebrotendinous xanthomatosis a pitfall in the diagnosis of multiple sclerosis. J Neurol 251 (1), Bartsch, G, Fitzpatrick, J.M., Schalken, J.A., Isaacs, J, Nordling, J., & Roehrborn, C.G. (2004). Consensus statement: the role of prostate-specific antigen in managing the patient with benign prostatic hyperplasia. BJU Int 93 Suppl 1, Bekkers, RL, Massuger, LF, Bulten, J., & Melchers, WJ (2004). Epidemiological and clinical aspects of human papillomavirus detection in the prevention of cervical cancer. Rev Med Virol 14 (2), Benito, MJ, Murphy, E, Murphy, E.P., Berg, W.B. van den, FitzGerald, O, & Bresnihan, B. (2004). Increased synovial tissue NF-kappa B1 expression at sites adjacent to the cartilage-pannus junction in rheumatoid arthritis. Arthritis Rheum 50 (6), Berg, W.B. van den (2004). Mechanisms of action of diacerein, the first inhibitor of interleukin-1 in osteoarthritis. Presse Medicale 33 (9), S10-S Berglin, E., Padyukov, L., Sundin, U., Hallmans, G., Stenlund, H., van Venrooij, W.J., Klareskog, L. and Rantapää-Dahlqvist, S. (2004) A combination of autoantibodies to cyclic citrullinated peptide (CCP) and HLA-DRB1 locus antigens is strongly associated with future onset of rheumatoid arthritis. Arhritis Res. Ther. 6:R303-R Berk, LC van den, Ham, M.A. van, Lindert, MM te, Walma, T., Aelen, J., Vuister, G.W., & Hendriks, WJ (2004). The interaction of PTP-BL PDZ domains with RIL: an enigmatic role for the RIL LIM domain. Mol Biol Rep 31 (4), Bernabe, D Beltran-Valero, Voit, T., Longman, C, Steinbrecher, A., Straub, V., Yuva, Y, Herrmann, R., Sperner, J, Korenke, C, Diesen, C, Dobyns, W.B., 25 Brunner, H.G., Bokhoven, H van, Brockington, M., & Muntoni, F. (2004). Mutations in the FKRP gene can cause muscle-eye-brain disease and Walker- Warburg syndrome. J Med Genet 41 (5), e Bernsen, M.R., Diepstra, JH, Mil, P van, Punt, CJ, Figdor, C.G., Muijen, GN van, Adema, G.J., & Ruiter, D.J. (2004). 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