Annual Report 2004 NCMLS

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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

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