Final Report Summary - HEALTH-PROT (Proteins in Health and Disease)
Executive summary:
The HEALTHPROT project enabled creation of a modern center of protein studies with relation to a wide variety of fundamental, medical, industrial and societal aspects. This multifaceted enterprise resulted in publication of 35 papers in various areas of protein structure/function-related research. It also facilitated introduction of modern recruitment procedures for young scientists. The project was based on collaboration of Polish laboratories with nine twinning partners (from UK, Germany, Lithuania, Norway, Italy, France, The Netherlands) and dealt with structure prediction in silico, structure determination by X-ray crystallography, biochemical characterization of protein properties in vitro, functional analyses in vivo using established cell lines in culture, animal-derived primary cells, etc. The project comprised of crystallographic studies of bacterial restriction endonucleases and HTLV, MMLV and XMRV viral reverse transcriptases, development of a number of bioinformatic tools for protein structure prediction, engineering of enzymes acting in RNA modification and DNA repair and degradation, modeling of interactions of single transmembrane helices belonging to proteins from the γ -secretase complex and molecular dynamics simulations of novel structure of presenilin involved in pathogenesis of Alzheimer disease. Within more functional area of research a role of mTOR-CLIP-170 interaction in neurons during their development was elaborated together with identification of several new proteins interacting with mTOR or CLIP-170 in an mTOR-dependent fashion, the involvement of β -catenin in regulation of T-type Ca2+ currents in thalamic neurons and characterization of mechanism and of proteins involved in Store-Operated Calcium Entry (SOCE) in neurons were tackled. Also systematic studies of possible mechanisms by which endocytic proteins may contribute to transcriptional regulation, cell signaling and proliferation were performed that lead to identification of candidate endocytic proteins which affect transcriptional responses in different signaling pathways accompanied by identification of important modulators able to provide fine-tuning and possible integration between different processes. New techniques of culturing airway epithelial cells, sequential and air liquid interface (ALI) were successfully introduced, allowing for application of ciliary proteins immunofluorescence staining, studies of ciliary Notch pathway inhibitors and identification of population-specific set of ciliary genes mutations. The evidences has been accumulated that ATP-binding to MDM2 is a regulatory mechanism in numerous pathways ranging from gene expression involved in the development of cancer cells, to regulation of transcription factors via the PI3/AKT signaling pathway, that MDM2 is involved in the DNA double strand brake response/repair cascade in p53-/- background and that under heat shock conditions (e.g. high fever during an inflammatory process) the overproduction of HSPA6 protein occurs, while under normal physiological temperature its level is almost not detectable. Most of the research problems addressed have direct or indirect relevance to health issues. Their direct societal impact is exemplified by a project-based initiative for foundation of the national parental organization/support group for ciliary disorders.
Project Context and Objectives:
International Institute of Molecular and Cell Biology (IIMCB) in Warsaw is the leading biological research institute in Poland. We study a broad range of proteins involved in cell physiology and pathology. We have at our disposal methods to study proteins by: sequence analyses in silico, structure determination by X-ray crystallography, biochemical characterization of protein properties in vitro, functional analyses in vivo using established cell lines in culture and animal-derived primary cells. Our aim is to expand the range of protein analyses. In particular, we expect our laboratories to introduce novel, state-of-the-art methodologies available in protein research. Moreover, by constant scientific exchange with the leading European centers, we strive to keep abreast with any technological developments in the areas already represented by the Institute. The ultimate goal is to become the main regional centre of reference in modern technologies for protein studies on a research scale and in parallel on a small pre-industrial scale, through reinforcing the S&T potential of our groups.
The HEALTH-PROT project is targeted at continuation and expansion of activities initiated as the FP5 Centre of Excellence in Molecular Bio-Medicine (CEMBM). In the past as the Centre of Excellence we developed an advanced methodology of analysis of complex protein structures with the use of cell and molecular biology techniques, biochemical methods, crystallographic analysis and computer modeling. Our goal is to become a top protein studies Centre in the region by unlocking the potential of all our research groups. This is being achieved mainly by twinning each of the Institute's groups with European groups leading in the field (first objective), through joint research activities, organization of workshops and conferences and participation in consortia within FP7 and European Structural Funds. We create the place for experienced researchers to conduct research at the highest level (second objective), and for junior researchers to obtain the best possible mentoring and a degree based on the top-flight theses. We also intend to reach an ultimate critical mass by completing the organisation of IIMCB's structure (third objective). We aspire to be more innovative towards applications in medicine and biotechnology (fourth objective). In parallel, we popularize science and raise social awareness of the benefits of modern biology and biotechnology (fifth objective). Ultimately, a reinforced S&T potential of our research groups will allow us to become more visible and attractive as a collaborating partner in the European Research Area, for both academia and industry.
Within the HEALTH-PROT project IIMCB has nine twinning partners:
1. University of Cardiff, Great Britain, Ruedi Allemann
2. Institute of Biotechnology, Vilnius, Lithuania, Saulius Klimasauskas
3. Department of Pharmaceutical Sciences, University of Bologna, Italy, Vincenza Andrisano
4. Erasmus Medical Center, Rotterdam, The Netherlands, Casper Hoogenraad
5. Centre for Neuropathology, Ludwig-Maximilians-University of Munich, Germany, Jochen Herms
6. Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway, Harald Stenmark
7. RNA Architecture and Reactivity Unit, Universite Louis Pasteur, CNRS, Strasbourg, France, Roland Marquet
8. Department of Pediatrics and Adolescent Medicine, University of Freiburg; Children and Adolescents University Hospital, Munster, Germany, Heymut Omran
9. Cancer Research of UK Cell Signaling Unit, Edinburgh Cancer Research Centre, University of Edinburgh, Great Britain, Ted Hupp
Project Results:
Matthias Bochtler, Laboratory of Structural Biology
Within the framework of the HEALTH-PROT project, we have collaborated with Cardiff University on endonucleases that are already used or are promising as tools for genetic engineering.
In the first part of the project, we have focused on basic understanding of catalytic types. The group had previously elucidated the structures of PD-(D/E)XK endonucleases and solved the first structures of HNH and phospholipase-like restriction endonucleases. During the realization of the project, work was carried out on three different restriction endonucleases and on the structures of TALENs (?transcriptional activator like effector nucleases). The latter nucleases are of special interest for genetic engineering because of a simple set of design rules which make it possible to generate custom nucleases selective for any arbitrary target sequence. In addition to the work on specific proteins and protein nucleic acid complexes, we have also developed RIBER/DIBER, a tool for crystallographers to rapidly judge crystal content when only diffraction data are available.
ThaI: This nuclease recognizes the CG/CG sequence in methylation sensitive manner and is therefore of interest to analyze epigenetic modifications in eukaryotic genomes. From the perspective of basic science, we wanted to get insight into the recognition of the CG sequence. Interestingly, it turns out that the DNA in the complex is distorted and an amino acid is inserted into the base stack. Similar intercalation has since been observed in the DNA co-crystal structures of Dnmt1 and prokaryotic CG specific methyltransferase. The crystallographic analysis of dinucleotide steps as well as modeling suggest that 5'-pyrimidine-purine-3' steps are easier to unstack than other DNA steps. The intercalation might therefore serve as an indirect means of sequence recognition.
Hpy188I: This nuclease was attractive to us because it cleaves its target sequence with a very unusual stagger (TCN/GA, single nucleotide 3'-overhangs), and because it was predicted to belong to the GIY-YIG family, which also includes transposases and DNA repair enzymes. Despite the widespread use of the GIY-YIG domain for DNA cleavage, a co-crystal structure of a GIY-YIG domain protein with DNA had not been obtained before. Our structure of Hpy188I with DNA, together with an independent structure of another restriction endonuclease of the GIY-YIG type (Eco29kI from Barry Stoddard's group), provide the first glimpses of the catalytic mechanism, and suggest that a tyrosine phenolate acts as the nucleophile in the initiating step of catalysis. Our results have direct implications for GIY YIG nucleases in nucleotide excision repair which among other lesions correct damage from UV exposure in humans.
TALENs: These endonucleases can be designed for any arbitrary target sequence according to simple design rules. Within the HEALTH-PROT project, we have attempted to crystallize a TALEN (in collaboration with Alfred Pingoud). We proposed a prediction for the structural basis of the TALE cipher and ran bioinformatics screens for potential new TALE nucleases. Unfortunately, two crystal structures of TALE proteins were published while the original prediction paper was under review. The manuscript has now been rewritten and is submitted as a review.
RIBER/DIBER: The presence of protein and nucleic acid in a crystallization mixture does not guarantee that both components are present in macromolecular crystals grown in such drops. We have expanded a previously developed tool (DIBER) to DIBER/RIBER. The two programs look for locally strong averages of reflection intensities and unit cell size and use a support vector machine algorithm to predict crystal content based on this information.
Articles submitted:
1. Wojciechowski M, Czapinska H, Bochtler M. CpG depletion and the new bacterial CpG specific DNA methyltransferase M.MpeI.
2. Siwek W, Czapinska H, Bochtler M, Skowronek K, Bujnicki JM. Crystal structure and mechanism of action of the N6-methyladenine dependent type IIM restriction endonuclease R.DpnI.
3. Arpino JA, Czapinska H, Piasecka A, Edwards WR, Barker P, Gajda M, Bochtler M, Jones DD. Structural basis for efficient chromophore communication and energy transfer in a constructed didomain protein scaffold.
Janusz Bujnicki Laboratory of Bioinformatics and Protein Engineering
The activities of the Bujnicki laboratory within the HEALTH-PROT project were focused on two areas: the development of computational methods for prediction and analysis of protein structures and their interactions with nucleic acids, and on experimental analysis of proteins acting on nucleic acids.
Methods development: The main effort of the bioinformatics part of the project was towards methods development. In particular, the postdoctoral researcher employed within HEALTH-PROT, dr Marcin Pawlowski, developed a series of methods for the evaluation of protein structure models. The MQAPmulti program predicts the overall global quality of protein models. It compares structural features generated from a 3D model with those predicted from its primary sequence (secondary structure, solvent accessibility, contact maps), uses statistical potential to estimate the value of pseudo-energy for a single model, uses hydrogen bonds pseudoenergy, and takes into account information from proteins that are evolutionary related to the target protein. We evaluated MQAPmulti method on models generated in the framework of the CASP9 experiment. The correlation between predicted and real global quality of a model (GDT_TS score) was 0.987 i.e. very high. Dr Pawlowski has also developed RecobmineIt, a fully automatic procedure that starts with a number of 3D models generated by other methods, performs local assessment of model quality (by the afore-mentioned MQAPmulti), and constructs hybrid models by splicing best-scored fragments from the original models. RecobmineIt was ranked among the 10 best methods in the CASP 9 experiment. In the course of the HEALTH-PROT project a number of other methods have been also developed:
FILTREST3D (available as a web server and standalone software at: http://filtrest3d.genesilico.pl/) is a computer program for discrimination of structural models using restraints from experimental data (e.g. secondary structure, shape, and intramolecular contacxt determination.
MetalionRNA is a method for predicting positions of metal ions (magnesium, sodium and potassium) for RNA structures defined by a user. It is based on a statistical potential inferred from the analysis of binding sites in experimentally solved RNA structures. It is available as a web server at http://metalionrna.genesilico.pl/.
RIBER is a method for detecting the presence of RNA stems in macromolecular crystals based on diffraction data alone. RIBER complements the previously developed program DIBER intended to search for double-stranded B-DNA and not double-stranded A-RN. The two programs have been implemented as a stand-alone software suite and a web server RIBER/DIBER (available at http://diber.iimcb.gov.pl/) providing an easy way to judge nucleic acid content of a crystal based on a diffraction dataset, before the crystal structure is solved. The new method may help to avoid a laborious phasing procedure when the component or the protein-RNA complex of interest is not present in the crystal.
MetaDisorder is a series of meta-predictors for the prediction of intrinsic disorder in proteins. They are based on third-party primary disorder predictors, and infer the consensus prediction by weighting the accuracy of the input methods. In particular, a meta predictor GSmetaDisorderMD was the top scoring method in the CASP9 benchmark. The Bujnicki group has also developed a disorder predictor GSmetaDisorder3D that uses no third-party disorder predictors, but relies on the analysis of alignments between the query protein sequence and the known protein structures, reported by the protein fold-recognition methods. MetaDisorder server is available at http://iimcb.genesilico.pl/metadisorder/.
Methods application: The Bujnicki group has carried out numerous studies in which they applied software tools for the analysis of structures and functions of proteins and protein-nucleic acid complexes. In line with the topic of the HEALTH-PROT project, the main subject of these analyses were enzymes acting on nucleic acids. Of particular importance for the HEALTH-PROT project were also analyses of the effect of mutations on the stability of protein structures and their potential pathogenic effects in humans.
Experimental analyses of enzymes acting on nucleic acids: The experimental work in the Bujnicki group has focused on enzymes acting in the context of two subsystems of nucleic acid metabolism: RNA modification and DNA repair and degradation. Major effort within the HEALTH-PROT project was devoted to the development of a labeling methodology with the use of SAM analogues, in collaboration with the prof. Klimasauskas group in the twinning partner institution Vilnius. In the course of a medium-term visit of experienced researchers from the Bujnicki Laboratory to the Klimasauskas laboratory, several RNA methyltransferases have been tested for the ability to catalyze the transfer of extended groups from the cofactor analogs onto the RNA substrate, with retention of the original target specificity of these enzymes. The visit allowed the Bujnicki laboratory to gain the knowledge on labeling RNA by cofactors and to improve the knowledge of the use of HPLC and mass spectrometry for the analysis of nucleic acid modifications. The work on DNA repair enzymes has been done in connection with the bioinformatics analyses (development of the REPAIRtoire database and bioinformatics analyses). One analysis that has been finalized and whose results have been published shortly after the end of the HEALTH-PROT project concerned the structural analysis of a DNA repair enzyme Exonuclease VII (ExoVII), a bacterial nuclease involved in DNA repair and recombination that hydrolyses single-stranded DNA. ExoVII is composed of two subunits: large XseA and small XseB. Thus far, little was known about the molecular structure of ExoVII, the interactions between XseA and XseB, the architecture of the nuclease active site or its mechanism of action. The Bujnicki group used bioinformatics methods to predict the structure of XseA, which revealed four domains: an N-terminal OB-fold domain, a middle putatively catalytic domain, a coiled-coil domain, and a short C-terminal segment. By series of deletion and site-directed mutagenesis experiments on XseA from Escherichia coli they determined that the OB-fold domain is responsible for DNA binding, the coiled-coil domain is involved in binding multiple copies of the XseB subunit, and residues D155, R205, H238, and D241 of the middle domain are important for the catalytic activity but not for DNA binding. Altogether, a model of sequence-structure-function relationships in ExoVII was proposed.
Slawomir Filipek, Laboratory of Biomodeling
Understanding of amyloid formation in Alzheimer's Disease
Alzheimer's disease (AD), the most common form of dementia in the elderly, is characterized by accumulation of the amyloid senile plaques in the brain. Genetic studies show that more than 170 mutations in the PS-1 protein were associated with familial forms of Alzheimer's disease and new mutations are still being identified.
Prof. Andrisano had developed many acetylcholinesterase inhibitors against AD and works on combining them with other drugs against AD. These inhibitors increase concentration of neurotransmitter acetylcholine to help AD patients because they have diminished levels of acetylcholine in brain neurons. Increase of this concentration slow down the pathological changes and the especially loss of mental ability. Unfortunately, these drugs neither stop nor reverse deterioration of brain neurons. They only affect symptoms of this disease. For this reason there are ongoing investigations on complementing of action of these drugs against additional molecular targets, for instance the γ -secretase complex. Because this complex is necessary to proteolize other substrates not only APP so the potential inhibitors should selectively hamper APP proteolysis. Knowledge of the structure of γ -secretase complex and especially interactions between proteins forming this complex would allow designing of selective inhibitors. Analysis of effects of mutations leading to familial form of Alzheimer's disease (FAD) at molecular level would allow to understand mechanisms of overproduction of pathological amyloids.
Drug design is the first step in a long and costly process of searching for novel drugs and the investigations should be focused on biological targets which are the cause of the disease. Multithreading characteristics of AD requires usage of multi-target ligands (MTDL - Multi-Target-Directed Ligands) which can interact with more than one biological target. The cholinergic and amyloid hypotheses functioning to explain onset of development of AD can be employed together to design compounds which can inhibit formation of amyloids and can increase cholinergic transmission.
We also conducted MD simulations of novel structure which is a C-terminal fragment (CTF) of presenilin 1. The structure of this fragment was obtained using NMR techniques in SDS micelles [Sobhanifar et al., PNAS 2010]. Detergent molecules can modify the structure of membrane proteins and especially the location and structure of loops linking the transmembrane helices. Simulations were performed in DPPC (dipalmitoyl-phosphatidylcholine) and DLPC (dilauroyl-phosphatidylcholine) models of lipid bilayers and based on these simulations it was possible to obtain the proper structure of loops on both sides of lipid bilayers. The coarse-grain methods were used to achieve simulation time scale of microseconds such time was needed for the protein structure to adapt to the new environment from SDS micelle to lipid bilayer. In the next step the structure of CTF PS-1 was determined on the atomic level employing the reconstruction procedures from coarse-grain models and by running shorter but more detailed the all-atom simulations.
Realization of scientific tasks in this grant resulted in two papers published in 2012:
1. W. Pulawski, U. Ghoshdastider, V. Andrisano, S. Filipek, Ubiquitous Amyloids, Appl. Biochem. Biotech. (2012) 166, 1626-1643
2. B. Trzaskowski, D. Latek, S. Yuan, U. Ghoshdastider, A. Debinski, S. Filipek, Action of molecular switches in GPCRs - theoretical and experimental studies, Curr. Med. Chem. (2012) 19, 1090-1109
3. M. Naldi, J. Fiori, D. Tedesco, M. Pistolozzi, A. Drake, C. Bertucci, R. Wu, K. Mlynarczyk, S. Filipek, V. Andrisano, Amyloid beta peptide 25-35 self-assembly and its inhibition: a model decapeptide system to gain atomistic and secondary structure details of the Alzheimer's disease process and treatment?, manuscript in preparation.
Jacek Jaworski, Laboratory of Molecular and Cellular Neurobiology
Work of Dr. Jaworski team in frame of HEALTH-PROT grant initially focused on a role of mTOR-CLIP-170 interaction in neurons during their development. An initial finding that started collaboration with Dr. Hoogenraad was that CLIP-170, a microtubule plus end binding protein, a potential mTOR kinase target regulates dendritic arbor morphology. In course of HEALTH-PROT project, in close collaboration with Hoogenraad lab, we showed that:
(i) indeed in neurons CLIP-170 is a substrate of mTOR,
(ii) mTOR dependent dendritogenesis requires presence of CLIP-170,
(iii) mTOR activity regulates protein interactions of CLIP-170 but not its cellular distribution,
(iv) mTOR-dependent cross-talk between microtubules and actin cytoskeleton is needed for proper dendritic development,
(v) CLIP-170 resides in dendritic spines and is key for their proper morphology,
(vi) Dynamic microtubule retraction from spines is important for long-term synaptic depression, a form of synaptic plasticity.
The major findings stemming from these new research directions can be summarized as follows:
(i) p150glued interacts both with CLIP-170 and mTOR and is needed for dendritic arborization. p150glued rescues the effects of CLIP-170 deficiency in neurons.
(ii) β-adaptin interaction with mTOR is needed for initial steps of neuronal endocytosis and regulates interaction of cellular cargo with AP2 complex as well as AP2 complex,
(iii) Both mTORC1 and mTORC2 are needed for proper dendritic arbor development and mTORC2 acts upstream of mTORC1 (Urbanska et al., J. Biol. Chem, under revision),
(iv) mTOR and CLIP-170 are downstream effectors of GSK3. GSK3 blocks mTOR-dependent phosphorylation of CLIP-170.
In frame of HEALTH-PROT grant, newly hired postdoc, Dr. Matylda Macias, has developed pharmacological, animal model of long-term mTOR activity inhibition using rapamycin, in which together with Dr. Swiech, she performed several studies with regard to mTOR activation and +TIPs tissue and subcellular distribution. However, the most appreciable features of mice chronically treated with rapamycin were: a decrease in seizure threshold and hydrocephaly (Macias et al., submitted). Thus, as a major outcome of this part of research we find discovery of an importance of mTOR for water homeostasis in the brain. This topic will be further investigated due to its medical relevance.
Finally, in addition to scientific advancements, several advanced techniques and tools were introduced to the lab thanks to personnel exchange, e.g.:
(i) Fast live-imaging of neurons (visits from Hoogeraad lab)
(ii) Knock-out and transgenic models (visits to van Leuven lab, Leuven, Belgium)
(iii) iPS technology and in vivo analysis of neuroprecursor development (visits to Sala lab, Milano, Italy; Toni lab, Lausanne, Switzerland)
(iv) analysis of endocytic protein interactions (Haucke lab, Berlin, Germany)
(v) Electrophysiological data analysis (visit from Krugers lab, Amsterdam, the Netherlands)
Jacek Kunicki, Laboratory of Neurodegeneration
The project consisted of two major parts both are related to calcium homeostasis and calcium signaling in the brain. In particular, we showed the involvement of β -catenin in regulation of T-type Ca2+ currents in thalamic neurons and we characterized mechanism and proteins involved in Store-Operated Calcium Entry (SOCE) in neurons. The details are described below.
Our previous studies carried out in the Laboratory of Neurodegeneration revealed cell cycle disturbances in immortalized lymphocytes derived from Alzheimer's disease (AD) patients. We decided to check whether the observed cell cycle changes in AD lymphocytes are accompanied by impaired apoptotic response to oxidative stress and to compare AD and Parkinson's disease (PD) lymphocytes. Lymphocytes from sporadic AD and PD patients were from prof. Martin-Requero's laboratory culture collection. Lymphocytes from FAD patients came from the bank at the IIMCB. The vulnerability to oxidative stress was assessed with two independent methods: colorimetric MTT test and JC-1 flow cytometry assay. The preliminary results showed a significant difference in apoptotic response between FAD and SAD lymphocytes. The experiments are now continued in the IIMCB. The data obtained in collaboration with prof. Martin-Requero were included in the PhD thesis of Dr. E. Bialopiotrowicz supervised by Prof. U. Wojda and in the common publication which is currently prepared. Thanks to the collaboration we also broadened our lymphocyte culture bank with cells from Spanish AD and PD patients.
Our group at the Laboratory of Neurodegeneration of IIMCB is studying the role of Calmyrin 2 (CaMy2, CIB2), a novel member of calcium binding proteins Calmyrins. Looking for neuronal CaMy2 function by affinity approach we have discovered a novel CaMy2 interaction partner, Rab5 GTPase the key element in early endosomes. To investigate the potential implication of CaMy2 in endocytosis, we established the collaboration with the expert in the neuronal endocytosis, Prof. Casper Hoogenraad. Our collaborative studies showed that overexpressed CaMy2 colocalizes with Rab5-positive vesicles as well as together with another early endosomal marker, EEA1 protein in rat primary hippocampal neurons in culture. Knock-down of CaMy2 with shRNA greatly affected early endosome localization of Rab5 and EEA1. CaMy2 depletion resulted also in inhibition of AMPA receptor endocytosis known to be mediated by Rab5. Our results obtained in collaboration with Prof. Hoogenraad laboratory indicate a novel mechanism by which Ca2+ signals mediated by CaMy2 regulate Rab5-mediated endocytosis. Now we describe these findings in our common research publication and in the PhD thesis of K. Debowska.
Publications and manuscripts:
Misztal K, Wisniewska MB, Ambrozkiewicz M, Nagalski A, Kuznicki J. WNT protein-independent constitutive nuclear localization of beta-catenin protein and its low degradation rate in thalamic neurons. J Biol Chem. 2011 Sep 9;286(36):31781-8.
Gruszczynska-Biegala J, Pomorski P, Wisniewska MB, Kuznicki J. Differential roles for STIM1 and STIM2 in store-operated calcium entry in rat neurons. PLoS One. 2011 Apr 26;6(4)
Wisniewska MB, Misztal K, Michowski W, Szczot M, Purta E, Lesniak W, Klejman ME, Dabrowski M, Filipkowski RK, Nagalski A, Mozrzymas JW, Kuznicki J. LEF1/beta-catenin complex regulates transcription of the Cav3.1 calcium channel gene (Cacna1g) in thalamic neurons of the adult brain. J Neurosci. 2010 Apr 7;30(14):4957-69.
Wisniewska MB, Nagalski A, Dabrowski M, Misztal K, Kuznicki J. Novel beta-catenin target genes identified in thalamic neurons encode modulators of neuronal excitability. BMC Genomics. 2012, submitted
Debowska K., Blazejczyk M., Jaworski J., Hoogenraad C., Kunicki J, Wojda U. CaMy2 regulates Rab5-mediated clathrin-dependent endocytosis in neurons. Manuscript in preparation
Bialopiotrowicz E, Kuzniewska B, Esteraz N, Martin-Requero A, Kuznicki J, Wojda U. Responses to oxidative stress differentiate lymphocytes from familial and sporadic Alzheimer's disease patients. Manuscript in preparation
Marta Miaczynska Laboratory of Cell Biology
Endosomal proteins in regulation of cell signaling and proliferation.
Within the project, the Laboratory of Cell Biology in collaboration with Prof. Harald Stenmark (Institute for Cancer Research, The Norwegian Radium Hospital, Oslo) and Prof. Marino Zerial (Max Planck Institute, Dresden) performed screens to search for endosomal proteins with novel functions in transcriptional regulation, cell signaling and proliferation. To undertake systematic studies of possible mechanisms by which endocytic proteins may contribute to these processes, we established and performed medium-scale, targeted RNAi screens. We aimed to identify candidate endocytic proteins which affect transcriptional responses in different signalling pathways. While it was not expected that endocytic proteins represented novel core components of the investigated pathways, we wished to identify important modulators able to provide fine-tuning and possible integration between different processes.
Marcin Nowotny, Laboratory of Protein Structure
The aim of our research project within the HEALTH-PROT grant was to perform structural studies of reverse transcriptases (RTs), which catalyze the reverse transcription a multi-step reaction of conversion of single-stranded RNA to double-stranded DNA. In addition to the purely scientific interest in this fascinating process, it is also highly medically relevant, since it is the key step of the proliferation of retroviruses such as human immunodeficiency virus (HIV) and hepatitis B virus (HBV). RTs from both viruses are targets of anti-viral therapy.
Michael Witt, Ciliary Proteins Function Project
Work within this project focused on introducing into the laboratory several techniques required for analysis of respiratory epithelium structure and function, including the technically demanding method of culturing respiratory epithelial cells from nasal brushings, known as the in vitro ciliogenesis technique. This technique is an essential technique for analysis of molecular basis of airway disorders, such as cystic fibrosis or primary ciliary dyskinesia (PCD), as well as universal tool for analysis of respiratory epithelium structure and function. In parallel with the chosen method of in vitro ciliogenesis (sequential method of ciliogenesis by Jorissen et al, 1989) several complementary techniques, including collagen isolation from rat tails, coating of the culture flasks with collagen gels and high-speed videomicroscopy of the in vitro differentiated cilia have been successfully implemented into the laboratory. The experience of the team with the culturing the respiratory epithelial cells has been summarized in a review article on the techniques of culturing differentiated respiratory epithelial cells (Bukowy et al, 2011, Journal of Applied Genetics).
Collaboration with the laboratory of Professor Heymut Omran at the Department of Pediatrics and Adolescent Medicine at the University Hospital in M?nster, Germany has allowed to further increase the experience of the team in the method of culturing and analyzing respiratory epithelial cells, as well as introduce into the laboratory the method of immunofluorescence staining of proteins present in cilia, flagella and related structures, in which the laboratory of Professor Omran has a high expertise.
In addition to learning different aspects of the in vitro ciliogenesis technique, collaboration with the Omran's group (reciprocal visits) allowed members of the Witt team to introduce into the lab the method of immunofluorescence staining of proteins present in cilia, flagella and related structures. Both methods were used in the study of the influence of the conserved signaling pathway, Notch pathway, on the direction and degree of the respiratory epithelium differentiation; they are also used in the currently ongoing projects. The whole series of mutations causative for PCD were identified in the Polish population in several genes coding for various ciliary proteins, mainly DNAH5, DNAI1, DNAL2, RSPH9, RSPH4A, RPGR, leading towards a population-specific set of significant diagnostic potential.
Publications and manuscripts:
Bukowy Z, Zietkiewicz E, Witt M. In vitro culturing of ciliary respiratory cells--a model for studies of genetic diseases. J Appl Genet. 2011 Feb;52(1):39-51.
Zietkiewicz E, Nitka B, Voelkel K, Skrzypczak U, Bukowy Z, Rutkiewicz E, Huminska K, Przystalowska H, Pogorzelski A, Witt M. Population specificity of the DNAI1 gene mutation spectrum in primary ciliary dyskinesia (PCD). Respir Res.2010 Dec 8;11:174.
Bukowy-Bieryllo Z, Zietkiewicz E, Loges NT, Wittmer M, Geremek M, Olbrich H, Fliegauf M, Voelkel K, Rutkiewicz E, Rutland J, Morgan LC, Pogorzelski A, Martin J, Haan E, Berger W, Omran H; Witt M. RPGR mutations might cause reduced orientation of respiratory cilia. Pediatric Pulmonology, accepted
Zietkiewicz E, Bukowy-Bieryllo Z, Voelkel K, Klimek B, Dmenska H, Pogorzelski A, Sulikowska-Rowi?ska A, Rutkiewicz E, Witt M. Mutations in radial spoke head genes and ultrastructural cilia defects in East-European cohort of primary ciliary dyskinesia patients PLoS ONE, accepted
Maciej Zylicz, Department of Molecular Biology
The MDM2 protein is an E3-ubiquitin ligase and is a major negative regulator of p53 suppressor protein. However, as it has been shown by us and others, it also possesses intrinsic chaperone like activities towards WT p53 protein and p53 mRNA. Within the Health Prot project, in collaboration with our twinning partners (Prof. Ted Hupp, University of Edinburgh and Prof. Urszula Hibner, CNRS, Montpellier), we have further shown evidences that ATP-binding to MDM2 is a regulatory mechanism in numerous pathways ranging from gene expression involved in the development of cancer cells, to regulation of transcription factors via the PI3/AKT signaling pathway. Dr. Pawel Winiewski, employed within the project, demonstrated that TEK/Tie2 gene expression was significantly affected by MDM2 K454A, ATP-binding deficient mutant, in comparison to MDM2 WT. TEK receptor is involved in the regulation of cell motility, differentiation and angiogenesis, acting predominantly via AKT kinase pathway. Next, he has shown that phosphorylation of AKT is strongly down-regulated by MDM2 K454A and AKT binding to MDM2 is impaired in MDM2 K454A transfected cells. Finally, it has been demonstrated that the ATP-dependent properties of MDM2 regulate activity of two distinct transcription factors: AP-1 and ISGF3 via down-regulation of AKT signaling pathway in H1299 non-small lung cancer cells.
Using the same experimental model, parallel investigations were carried out, focusing on the role of MDM2 on other p53 family members, namely TAp63 and TAp73. Using BAX promoter, we have demonstrated that MDM2 differently modulates the transcriptional activity of both proteins. MDM2 stimulates the transcriptional activity of TAp63, while inhibits the transcriptional activity of TAp73. The preliminary results indicate that MDM2 regulates translation of both TAp63 and TAp73. The results were presented on the 5th Mutant p53 Workshop (Tracz Z, Zylicz A, Zylicz M. (2011) Interactions of p53 mutants with other p53 family members in non-small cell lung cancer cells. In: ?5th Mutant p53 Workshop From bench to bedside across mouse models?, pp. 131, May 22-25, Palazzo Chigi Ariccia, Italy). The preliminary results described above gave prominent impact on successful grant application from the Foundation for Polish Science (Zuzanna Tracz).
Additionally we have shown that MDM2 is also involved in the DNA double strand brake response/repair cascade in p53-/- background. Dr. Bartosz Wawrzynow from the twinning partner laboratory (University of Edinburgh) was employed within the programme for the last six months at IIMCB. He implemented new techniques such as Olympus ScanR live screening platform to measure protein abundance/accumulation and compartment localization upon proteasome inhibition and/or heat shock. Additionally an In-Cell-Western (ICW) assay was established aimed at monitoring the kinetic phosphorylation profile of key protein involved in Homologous Recombination (HR), one of the dominant DNA repair mechanisms in eukaryotic cells.
In collaboration with Marta Maluszek, they have shown that ATP binding by MDM2 is key for its involvement in DNA repair mechanisms. Upon initiation of DNA damage with the use of radiomimetic compound neocarzinostatin (NCS), a pronounced difference in the ubiquitylation and degradation profile between WT and K454A MDM2 occurs. Moreover, the ATP-bound MDM2 is required for efficient homologous recombination and survival rate of cancer cells in response to DNA damage. We have strongly benefited from the Health Prot programme in this avenue of research, as complementary results further strengthening our findings were obtained in the Gray Institute for Radiation Oncology and Biology in Oxford by Marta Matuszek. The preliminary results were presented on the international workshop (Ma?uszek MJ, Zylicz A, Zylicz M. (2011) The role of MDM2 ATP-binding ability in DNA damage response, The MDM2 International Workshop, New York Academy of Sciences, p21, October 16-19, New York, USA) and will serve as a basis for the next grant application.
Molecular chaperones modulate the activity of WT p53 suppressor protein, which is beneficiary for the healthy cell. However, they also stabilize mutants of p53, which in contrast is beneficiary for the cancer cell. One of our projects within the Health Prot programme, focuses on mutant p53 Gain-Of-Function (GOF) mechanism. We have postulated and demonstrated that GOF can be implemented by HSP70/HSPA1 induced aggregation process of mutant p53 forms. Being aggregated they are not able to be ubiquitylated and subsequently degraded, leading to a flux in the cellular homestasis. These preliminary experiments performed on Non Small Lung Cancer Cell lines gave an impact for a new grant proposal, which was obtained from the National Centre of Science. A manuscript of the paper is prepared. Further experiments with using breast cancer model cell lines will be continued. The current data was presented on the international workshops and conferences (Wiech M, Tracz Z, Maluszek M, Pruszko M, Wisniewski P, Zylicz A, Zylicz M. (2011) Hsp70 in mutant p53 gain-of-function phenotypes. In: The Biology of Molecular Chaperones. From basic mechanisms to intervention strategies in disease and aging?, pp. 166, May 19-24, Grundlsee, Austria;
Wiech M, Zylicz A, Zylicz M. (2011) Aggregate prone p53 mutants: the impact of HSP70. In 5th Mutant p53 Workshop From bench to bedside across mouse models, pp. 136, May 22-25, Palazzo Chigi Ariccia, Italy; Tracz Z, Wiech M, Manuszek M, Pruszko M, Zylicz A, Zylicz M. (2011) Stress induced mutant p53 gain-of-function phenotypes. In: 7th NCRI Cancer Conference, November 6-10, Liverpool, UK).
We have also shown that human cells are equipped with numerous functionally distinct subsets of chaperones, some of which seem to be dedicated to (re)folding and some that have evolved to dispose of non-foldable proteins. We investigated the new HSPA6 protein which lacks the generic chaperone-like properties of other HSP70s and may have evolved to maintain specific critical functions under conditions of severe stress. We have shown that under heat shock conditions (e.g. high fever during an inflammatory process) the overproduction of HSPA6 occurs, while under normal physiological temperature its level is almost not detectable. This particular protein is responsible for the transcriptional activity of p53 without need any HSP40 cochaperones. The results are presented in the paper: Hageman J, van Waarde MA, Zylicz A, Walerych D, Kampinga HH. The diverse members of the mammalian HSP70 machine show distinct chaperone-like activities. Biochem J, 2011; 435:127-142.
Potential Impact:
General impacts
EU level
Boosting research recognition. Within three years of HEALTH-PROT implementation IIMCB scientists published 35 publications in recognized journals, visited and hosted distinguished partners from top European centers and with them organized high quality scientific events. The HEALTH-PROT resulted in deepening existing and initiating new promising cooperations. Some of them are described below:
(i) The collaboration between Prof. Michael Witt and his twinning partner Prof. Heymut Omran, currently from Munster, has a long history and a broad range of EU Framework Projects involved: it's been initiated within the Centre of Excellence in Molecular Biomedicine in FP5, continued within FP6 as HEALTH-PROT project, currently being under coverage within FP7 as the project ?Better Experimental Screening and Treatment for Primary Ciliary Dyskinesia, acronym BESTCILIA and PhD programme in Molecular Biology: Studies of nucleic acids and proteins - from basic to applied research; acronym MPD; implemented within Structural Funds IE OP 1.1.2. Programme which is based on a common mentorship of a PhD student involved in a PhD research project in Prof. Witt and Prof. Omran cooperating laboratories.
(ii) Combined efforts of Prof. Kuznicki, Laboratory of Neurodegeneration, IIMCB, with Prof. Jochen Herms, Centre for Neuropathology Ludwig-Maximilians-University of Munich, Germany, towards innovative research resulted in a common patent application. Moreover, these two partners submitted two projects? applications for EU funding: SME-targeted research for developing tools and technologies for high-throughput research project to FP7 and IE OP 1.1.2 PhD programme in Molecular Biology: Studies of nucleic acids and proteins - from basic to applied research (MPD) to EU Structural Funds (546 609 euro). The latter was successful and is now being implemented. This intense and fruitful collaboration extended on Dr. Jaworski Laboratory of Molecular and Cellular Neurobiology, IIMCB. The common interests of Dr. Jaworski and Prof. Herms were expressed in a successfully submitted and currently implemented FP7 Era-Net Neuron project on Imaging Neurogenesis in Neurodegenerative Disease (262 004 euro).
(iii) Similar added value was created from the twinning of Prof. Bujnicki, Laboratory of Bioinformatics and Protein Engineering, IIMCB, with Prof. Saulius Klimasauskas, Laboratory of Biological DNA Modification, Institute of Biotechnology, Vilnius Lithuania. Thanks to scientists? exchange Prof. Klimasauskas entered into collaboration with Prof. Matthias Bochtler, Laboratory of Structural Biology, IIMCB, the outcome of which is a common participation in the TEAM project on Structural biology of methylation and hydroxymethylation funded from EU Structural Funds (488 344 euro).
(iv) Another example of real result bringing cooperation can be a twinning pair of Prof. Zylicz, Department of Molecular Biology, IIMCB and Prof. Ted Hupp, Edinburgh Cancer Research Centre, University of Edinburgh, UK. This long lasting cooperation which stems from the Centre of Excellence project in FP5 was now strengthened and, as a HEALTH-PROT result, evolved in a common participation in two competition based projects: the EU Structural MPD project and the Polish Ministry of Science and Higher Education research grant on Mechanism of oncogenic activities of mutated TP53 (144 722 euro).
All these activities allow for a better recognition and appreciation of IIMCB, and Polish science in general, among Western European reknown partners. In medium and long term it will contribute to a perception of IIMCB and other Polish research institutes as equal scientific partners and will lead to a real integration of Eastern European research institutes into the European Research Area.
Enhancing attractiveness to European researchers. The HEALTH-PROT evoked the idea to create an integrated set of principles on recruitment and approach towards employed researchers. It was materialised in IIMCB official declaration in 2010 to adopt the European Charter for Researchers and the Code of Conduct for the Recruitment of Researchers. As one of the three Polish institutions voluntary committing itself to apply Charter and Code principles, IIMCB?s intention was to increase institute's attractiveness to European researchers by offering them sure and secure favorable working environment, enhancing the quality of research and innovation and boosting international mobility.
Regional level
Sharing experiences with and enhancing participation of INCO countries in FP7. IIMCB has taken actions to share the experiences of participating in the Research Potential programme with Ukrainian scientists and managers from the Institute of Molecular Biology and Genetics (IMBG) in Kiev. IIMCB and IMBG representatives meet every 2 years during Polish-Ukrainian Parnas conferences. Closer bilateral relations between IIMCB and IMBG were established in 2008 thanks to an initiative of IIMCB director Prof. Jacek Kuznicki. During the round table Research organization in the 21st century: experience and achievements of IIMCB, organized by the Ukrainian Ministry of Education and Science Prof. Kuznicki proposed that IIMCB's experience could be used by Ukrainian scientific institutions. In 2009 IIMCB organized a Polish-Ukrainian scientific conference accompanied by the HEALTH-PROT kick-off meeting. The IMBG director, Prof. Elskaya, participated in the IIMCB International Advisory Board meeting and the IMBG managers had meetings with their IIMCB counterparts. One of the results of this cooperation is a common project entitled ?Strengthening cooperation in molecular biomedicine between EU and Ukraine? (FP7-INCO-2011-6, ERA-WIDE), 2011-2014.
National level
Reaching scientific excellence on national level. HEALTH-PROT project contributed to IIMCB excellence on national level: in 2010 IIMCB was ranked number 1 among 36 biological institutions by the Polish Ministry of Science and Higher Education. Prof. Jacek Kuznicki, IIMCB director and HEALTH-PROT coordinator, was in 2011 nominated a member of Science Policy Committee. This body, consisting of 10 experts, assists the Minister of Science and Higher Education among others in: the development of documents relating to the development of science and science and innovation policy and in the development of draft State budget and financial plan setting out the financial resources for science. In 2011 Prof. Michael Witt was elected the Chairman of the Advisory Board for Molecular Genetic Tests and Biobanking, established at the Ministry of Science and Higher Education. The Board is comprised of geneticists, oncologists and laboratory diagnosticians. In 2011 Janusz Bujnicki was elected a member of the Young Scientists Academy, an appendix to the Polish Academy of Sciences. International Institute of Molecular and Cell Biology is a laureate of the Funds for Science 2011 awards in the commercialization of scientific research category.
Learning from the HEALTH-PROT achievements, two neighboring institutes from Warsaw Ochota Campus applied for RegPot funding, of which one with success.
Country-wide initiatives with significant societal impact
During the conference organized within the HEALTH-PROT project a patient family/support group for families with the primary ciliary dyskinesia (PCD) was founded based on IIMCB initiative. The Polish Society for Ciliary Dyskinesia is the first organization gathering these families in Poland with over 60 founding members signing the foundation document. Organization's goals are: to provide support to patients and their carers who have, or are suspected of having, PCD; to bring PCD to the attention of medics who may come across PCD and continue to provide an up to date information service for them and the general public; to promote research to aid diagnosis and treatment of patients with PCD; to ensure patients have access to diagnostic services and on-going care within the framework of current national medical care system.
IIMCB also supports other patient organisations. In 2005, one of our staff members organised the patient association J-elita, bringing together the families of patients who suffer from Crohn's disease and Colitis (J-elita's office is located on the IIMCB premises).
Institutional level
Setting up directions for further development.
(i) The HEALTH-PROT project consolidated Institutes efforts to concentrate on the strategic research problem which is studies of various aspects of protein structure/function. Research performed at IIMCB although basic may lead to better understanding of different mechanisms vital for human life such as: ageing, neurodegeneration, cancer, addictions, etc. Being aware of the market value of scientific results IIMCB founded in 2010 a Technology Transfer Unit Bio&Technology Innovations Platform (http://www.biotechip.pl). This idea originated in the HEALTH-PROT proposal and was appreciated by the EC but funds to this end were cut. IIMCB received funding from Structural Funds and a program of the Ministry of Science and Higher Education to set up a TTU for six bio-tech-med research institutes forming BioCentrum Ochota consortium. The Unit has at its disposal funds to cover stipends for innovative applied research projects, trainings in legal and financial aspects of conducting scientific research such as IPR and commercialization.
(ii) In March 2012 the cooperation agreement between IIMCB and the Max-Planck Society (MPG) was signed. The agreement concerns the establishment of two Max Planck/IIMCB Research Groups, one in IIMCB, other in the Max-Planck Institute of Heart and Lung Research in Bad Nauheim. A research group in IIMCB will be the first in Poland to use the zebrafish as a model of pathomechanisms of various human diseases. Existing significant experience of MPG in this area should greatly facilitate the rapid launch and research progress in this direction.
(iii) Experiences of HEALTH-PROT project allowed to gather expertise and knowledge to further develop innovation potential, measured by medically relevant discoveries, patent applications, and license agreements. Stemming from this was an initiative to create at IIMCB the FishMed Centre to be the first zebrafish research and breeding facility in Poland. A relevant applicaton has been submitted to the FP7. Zebrafish is an excellent organism to study various aspects of human disease because it is fully transparent until adulthood, and its metabolism is similar to that of mammals. The FishMed Centre is designed to twin with six excellent European zebrafish centres. These include the Max Planck Institute for Heart and Lung Research in Bad Nauheim, the Universities of Cambridge, Leiden, Sheffield, and Geneva, and an Austrian IST. The twinning Partners will share their zebrafish models and know-how in joint projects with regard to cancer, neurodegeneration, epigenetics, and tissue regeneration. The zebrafish breeding facility will be organized and a new research group established, headed by a leader selected in an international competition. The costly equipment for the breeding facility will be purchased partly from the grant funds.
(iv) BESTCILIA project within FP7 devoted to primary ciliary dyskinesia (PCD) biology has been developed with weighty input of IIMCB scientists. PCD is a rare disease for which no orphan drug is available and which currently is treated off-label. The main goal of the project is to improve the diagnosis and treatment of PCD patients. This will be achieved through introduction of standardized diagnostic testing for PCD in European countries, where this is currently not available; establishment of a comprehensive PCD registry for systematic data collection on current disease status in order to define the natural course of the disease and to define outcome relevant parameters in a large European-American cohort; setting up an observational trial in PCD for a meta-analysis of existing datasets to describe the clinical phenotype, prognostic factors and predictors of poor outcome; establishment of PCD-specific Health-Related Quality of Life Questionnaires (HRQOLQ); performing a randomized controlled clinical trial on the use of acithromycin in PCD.
Potential impact and exploitation of scientific results
Cooperation between Prof. Jacek Kuznicki and his twinning partner Prof. Jochen Herms resulted in a patent application that has been filed through Ludwig Maximilians Universitat in Munich (Use of [name removed] derivatives, including their pharmacologically useable salts and related structures for the treatment of diseases of the central nervous system, metabolic and cardiac diseases as well as aging and health status itself?). (Kuznicki)
Endonucleases, particularly programmable TALENs, are of interest for genetic manipulation in vivo as well as in vitro. The group has now won new Polish grants to apply some of the knowledge gained from studies of different restriction endonuclease types to TALENs.
In particular, it is planned to search for or develop replacement for the currently used effector nuclease FokI. There has also been an effort to broaden the expertise of the group to include the application of the new nucleases in animal models. As part of the training program of the HEALTH-PROT grant, the PI has learnt P-element mediated transformation of Drosophila melanogaster from Wynand van der Goes van Naters at Cardiff University. He has also had the opportunity to observe lentivirus mediated transgenesis in the mouse during a guest stay in the laboratory of Philippe Ravassard, an expert in mouse transgenesis at Pierre and Marie Curie University and ICM (Paris). The training will help the group to apply basic knowledge on nucleases to animal transgenesis experiments. (Bochtler)
Alzheimer's Disease is a progressive neurodegenerative disease. It affects more than 25 million of people worldwide and this number is strongly increasing nowadays. This is why the search for novel and more effective drugs is extremely important. Novel multi-target-directed drugs are still in their begging phase because of their complex interactions. They can potentially affect different element of amyloid formation cascade in the same time, therefore, could be more efficient. During this grant the new methods of designing of multi-target ligands were developed and they could be used for subsequent studies and analyses. (Filipek)
The work done by the members of Jaworski lab in frame of HEALTH-PROT grant undoubtly will have an important impact on different levels. First, new knowledge regarding mTOR interacting partners as well as effects of mTOR-inhibitors is very relevant clinically as it helps predict adverse effects of mTOR inhibition in clinic. We also got a further insight into novel biological functions of mTOR. In addition, technology introduced to the laboratory moved our research to the next level that resulted in several new collaborations and grant applications. From this perspective, introduction of iPS technology seems to be very important as it already tighten our links to clinicians who are very eager to look into cellular mechanisms in neurons-derived from fibroblasts of real patients (e.g. TSC patients). Currently, with clinicians from several centers around Poland we plan to establish bank of iPS cells derived from patients suffering from various diseases with neurological manifestations. These bank will be of use to whole research community.
Finally, HEALTH-PROT grant allowed hiring specialist in cutting edge techniques in high-resolution microscopy (e.g. EM) and histology which will allow us to research mTOR cellular functions in the brain on subcompartmental level. iPS cell developed in frame of our research will be used as a starting material in several of our studies aiming to:
(i) reveal basic disease mechanisms and
(ii) find new substances preventing/correcting phenotypical outcome of the diseased gene on a cellular level. We also plan to deposit such iPS lines in iPS bank developed by us and our clinical collaborators in the future. (Jaworski)
The collaboration with the twinning partners within the project allowed us to establish at IIMCB a novel platform and methodology for RNAi screens in cultured cells using transcriptional reporter tests or proliferation assays. Since its establishment, this platform has been successfully used in other screens and will be further employed in the future. This is an important development which will be of long-lasting impact. Considering the specific scientific results of the project, identification of novel regulators of AP-1-dependent transcription or proliferation among endosomal proteins is an important discovery of basic research. We are currently continuing the studies of molecular mechanisms of action of the newly identified regulators which will eventually lead to publications. The scientific results of the project belong to fundamental research and have already formed a basis for further research and grant applications (both Polish and international) which were awarded to Dr. Miaczynska. They have already led and will further lead to publications in international peer-reviewed journals. (Miaczynska)
The results obtained within the HEALTH-PROT project are the first structural studies of a monomeric reverse transcriptase in complex with its substrate- RNA/DNA hybrid. Our crystal structure together with SAXS-verified model of the full-length enzyme provide important insights into similarities and differences with extensively studied HIV-1 RT. We were also able to correct the tracing of a substantial part of MMLV RT, which now allows us to draw structure-based conclusions for retroviral RTs in general. We plan to continue these studies, in particular to gain insights into the specific processing of the poly-purine tract an essential but poorly understood step of reverse transcription. HEALTH-PROT project allowed us to initiate studies of reverse transcriptases that we plan to carry on. The information gained during this projects and methods it allowed us to establish such as chemical cross-linking and SAXS will allow us to tackle such important and technically challenging issues as the structural basis of PPT recognition in reverse transcription. (Nowotny)
From the methodology point of view, new techniques and protocols have been successfully used throughout the study, many of which have been introduced to other labs within the IIMCB: a gene expression platform of profile analysis of over 80 genes known to be involved in cancer cells development (SABiosciences, PAHS-033) using RT-PCR (Dr. Pawel Winiewski); Olympus ScanR screening platform to measure protein abundance/accumulation and compartment localization as well as In-Cell-Western assay to monitor kinetics of posttranslational protein modifications (dr. Bartosz Wawrzynow). Considering the specific scientific achievements within the project, it is worth to underline the preliminary data leading to the elucidation of the mechanisms of DNA repair in cancer cells deficient in the TP53 gene as well as the data concerning the mechanisms of gain of new oncogenic functions of mutated p53 in lung cancer. The data provided strong impact for the successful application of the complementary three grant proposals (Alicja Zylicz, Zuzanna Tracz and Milena Wiech). Thus, the continuation of the project is further planned.
The new techniques introduced by employed postdoctoral researchers are commonly used in the laboratory and will be used in the future. One of the scientific aspects of the project concerns the elucidation of drugs resistance during treatment of patients with non-small-lung-cancer. Understanding such fundamental mechanisms would potentially benefit in the verification of current methods of treatment of patients harboring the above type of cancer. It has recently been demonstrated in mouse model that the stress response resulting from gamma-radiation stabilizes mutant p53 and shifts the tumour phenotype to more aggressive, as compared to treatment of Trp53-/- mice. It is tempting to speculate that similar phenomena may occur in human patients who harbour mutated p53 and are treated by gamma-irradiation. If that is the case, then the knowledge of the patient p53 status may be crucial in designing the optimal therapeutic strategies. Our findings elucidate the molecular mechanism of how stress-induced HSP70 stabilizes the mutant p53 protein and suggest new therapeutic approach, whereby HSP70 inhibitors could be used synergistically with gamma-irradiation to treat patients with mutated p53. (Zylicz)
Dissemination activities planned after the end of the project:
The HEALTH-PROT project will be promoted at the conference WIRE 2012, 4-5.06.2012 Krakow, Poland. Prof. Kunicki, HEALTH-PROT coordinator, was invited to moderate one of the panels. Dr. Urszula Bialek-Wyrzykowska, Dorota Libiszowska and Aleksandra Nalecz-Tolak will present the results of the project on a stand.
Around ten publications with the project results are expected to be published by the end of 2012.
List of Websites:
http://www.iimcb.gov.pl/
Contact details:
Dr Urszula Bialek-Wyrzykowska
International Institute of Molecular and Cell Biology
4 Ks. Trojdena St.
02-109 Warsaw, Poland
Tel. +48 22 59 70 713
Fax +48 22 59 70 715
ulawyrzykowska@iimcb.gov.pl
http://www.iimcb.gov.pl
The HEALTHPROT project enabled creation of a modern center of protein studies with relation to a wide variety of fundamental, medical, industrial and societal aspects. This multifaceted enterprise resulted in publication of 35 papers in various areas of protein structure/function-related research. It also facilitated introduction of modern recruitment procedures for young scientists. The project was based on collaboration of Polish laboratories with nine twinning partners (from UK, Germany, Lithuania, Norway, Italy, France, The Netherlands) and dealt with structure prediction in silico, structure determination by X-ray crystallography, biochemical characterization of protein properties in vitro, functional analyses in vivo using established cell lines in culture, animal-derived primary cells, etc. The project comprised of crystallographic studies of bacterial restriction endonucleases and HTLV, MMLV and XMRV viral reverse transcriptases, development of a number of bioinformatic tools for protein structure prediction, engineering of enzymes acting in RNA modification and DNA repair and degradation, modeling of interactions of single transmembrane helices belonging to proteins from the γ -secretase complex and molecular dynamics simulations of novel structure of presenilin involved in pathogenesis of Alzheimer disease. Within more functional area of research a role of mTOR-CLIP-170 interaction in neurons during their development was elaborated together with identification of several new proteins interacting with mTOR or CLIP-170 in an mTOR-dependent fashion, the involvement of β -catenin in regulation of T-type Ca2+ currents in thalamic neurons and characterization of mechanism and of proteins involved in Store-Operated Calcium Entry (SOCE) in neurons were tackled. Also systematic studies of possible mechanisms by which endocytic proteins may contribute to transcriptional regulation, cell signaling and proliferation were performed that lead to identification of candidate endocytic proteins which affect transcriptional responses in different signaling pathways accompanied by identification of important modulators able to provide fine-tuning and possible integration between different processes. New techniques of culturing airway epithelial cells, sequential and air liquid interface (ALI) were successfully introduced, allowing for application of ciliary proteins immunofluorescence staining, studies of ciliary Notch pathway inhibitors and identification of population-specific set of ciliary genes mutations. The evidences has been accumulated that ATP-binding to MDM2 is a regulatory mechanism in numerous pathways ranging from gene expression involved in the development of cancer cells, to regulation of transcription factors via the PI3/AKT signaling pathway, that MDM2 is involved in the DNA double strand brake response/repair cascade in p53-/- background and that under heat shock conditions (e.g. high fever during an inflammatory process) the overproduction of HSPA6 protein occurs, while under normal physiological temperature its level is almost not detectable. Most of the research problems addressed have direct or indirect relevance to health issues. Their direct societal impact is exemplified by a project-based initiative for foundation of the national parental organization/support group for ciliary disorders.
Project Context and Objectives:
International Institute of Molecular and Cell Biology (IIMCB) in Warsaw is the leading biological research institute in Poland. We study a broad range of proteins involved in cell physiology and pathology. We have at our disposal methods to study proteins by: sequence analyses in silico, structure determination by X-ray crystallography, biochemical characterization of protein properties in vitro, functional analyses in vivo using established cell lines in culture and animal-derived primary cells. Our aim is to expand the range of protein analyses. In particular, we expect our laboratories to introduce novel, state-of-the-art methodologies available in protein research. Moreover, by constant scientific exchange with the leading European centers, we strive to keep abreast with any technological developments in the areas already represented by the Institute. The ultimate goal is to become the main regional centre of reference in modern technologies for protein studies on a research scale and in parallel on a small pre-industrial scale, through reinforcing the S&T potential of our groups.
The HEALTH-PROT project is targeted at continuation and expansion of activities initiated as the FP5 Centre of Excellence in Molecular Bio-Medicine (CEMBM). In the past as the Centre of Excellence we developed an advanced methodology of analysis of complex protein structures with the use of cell and molecular biology techniques, biochemical methods, crystallographic analysis and computer modeling. Our goal is to become a top protein studies Centre in the region by unlocking the potential of all our research groups. This is being achieved mainly by twinning each of the Institute's groups with European groups leading in the field (first objective), through joint research activities, organization of workshops and conferences and participation in consortia within FP7 and European Structural Funds. We create the place for experienced researchers to conduct research at the highest level (second objective), and for junior researchers to obtain the best possible mentoring and a degree based on the top-flight theses. We also intend to reach an ultimate critical mass by completing the organisation of IIMCB's structure (third objective). We aspire to be more innovative towards applications in medicine and biotechnology (fourth objective). In parallel, we popularize science and raise social awareness of the benefits of modern biology and biotechnology (fifth objective). Ultimately, a reinforced S&T potential of our research groups will allow us to become more visible and attractive as a collaborating partner in the European Research Area, for both academia and industry.
Within the HEALTH-PROT project IIMCB has nine twinning partners:
1. University of Cardiff, Great Britain, Ruedi Allemann
2. Institute of Biotechnology, Vilnius, Lithuania, Saulius Klimasauskas
3. Department of Pharmaceutical Sciences, University of Bologna, Italy, Vincenza Andrisano
4. Erasmus Medical Center, Rotterdam, The Netherlands, Casper Hoogenraad
5. Centre for Neuropathology, Ludwig-Maximilians-University of Munich, Germany, Jochen Herms
6. Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway, Harald Stenmark
7. RNA Architecture and Reactivity Unit, Universite Louis Pasteur, CNRS, Strasbourg, France, Roland Marquet
8. Department of Pediatrics and Adolescent Medicine, University of Freiburg; Children and Adolescents University Hospital, Munster, Germany, Heymut Omran
9. Cancer Research of UK Cell Signaling Unit, Edinburgh Cancer Research Centre, University of Edinburgh, Great Britain, Ted Hupp
Project Results:
Matthias Bochtler, Laboratory of Structural Biology
Within the framework of the HEALTH-PROT project, we have collaborated with Cardiff University on endonucleases that are already used or are promising as tools for genetic engineering.
In the first part of the project, we have focused on basic understanding of catalytic types. The group had previously elucidated the structures of PD-(D/E)XK endonucleases and solved the first structures of HNH and phospholipase-like restriction endonucleases. During the realization of the project, work was carried out on three different restriction endonucleases and on the structures of TALENs (?transcriptional activator like effector nucleases). The latter nucleases are of special interest for genetic engineering because of a simple set of design rules which make it possible to generate custom nucleases selective for any arbitrary target sequence. In addition to the work on specific proteins and protein nucleic acid complexes, we have also developed RIBER/DIBER, a tool for crystallographers to rapidly judge crystal content when only diffraction data are available.
ThaI: This nuclease recognizes the CG/CG sequence in methylation sensitive manner and is therefore of interest to analyze epigenetic modifications in eukaryotic genomes. From the perspective of basic science, we wanted to get insight into the recognition of the CG sequence. Interestingly, it turns out that the DNA in the complex is distorted and an amino acid is inserted into the base stack. Similar intercalation has since been observed in the DNA co-crystal structures of Dnmt1 and prokaryotic CG specific methyltransferase. The crystallographic analysis of dinucleotide steps as well as modeling suggest that 5'-pyrimidine-purine-3' steps are easier to unstack than other DNA steps. The intercalation might therefore serve as an indirect means of sequence recognition.
Hpy188I: This nuclease was attractive to us because it cleaves its target sequence with a very unusual stagger (TCN/GA, single nucleotide 3'-overhangs), and because it was predicted to belong to the GIY-YIG family, which also includes transposases and DNA repair enzymes. Despite the widespread use of the GIY-YIG domain for DNA cleavage, a co-crystal structure of a GIY-YIG domain protein with DNA had not been obtained before. Our structure of Hpy188I with DNA, together with an independent structure of another restriction endonuclease of the GIY-YIG type (Eco29kI from Barry Stoddard's group), provide the first glimpses of the catalytic mechanism, and suggest that a tyrosine phenolate acts as the nucleophile in the initiating step of catalysis. Our results have direct implications for GIY YIG nucleases in nucleotide excision repair which among other lesions correct damage from UV exposure in humans.
TALENs: These endonucleases can be designed for any arbitrary target sequence according to simple design rules. Within the HEALTH-PROT project, we have attempted to crystallize a TALEN (in collaboration with Alfred Pingoud). We proposed a prediction for the structural basis of the TALE cipher and ran bioinformatics screens for potential new TALE nucleases. Unfortunately, two crystal structures of TALE proteins were published while the original prediction paper was under review. The manuscript has now been rewritten and is submitted as a review.
RIBER/DIBER: The presence of protein and nucleic acid in a crystallization mixture does not guarantee that both components are present in macromolecular crystals grown in such drops. We have expanded a previously developed tool (DIBER) to DIBER/RIBER. The two programs look for locally strong averages of reflection intensities and unit cell size and use a support vector machine algorithm to predict crystal content based on this information.
Articles submitted:
1. Wojciechowski M, Czapinska H, Bochtler M. CpG depletion and the new bacterial CpG specific DNA methyltransferase M.MpeI.
2. Siwek W, Czapinska H, Bochtler M, Skowronek K, Bujnicki JM. Crystal structure and mechanism of action of the N6-methyladenine dependent type IIM restriction endonuclease R.DpnI.
3. Arpino JA, Czapinska H, Piasecka A, Edwards WR, Barker P, Gajda M, Bochtler M, Jones DD. Structural basis for efficient chromophore communication and energy transfer in a constructed didomain protein scaffold.
Janusz Bujnicki Laboratory of Bioinformatics and Protein Engineering
The activities of the Bujnicki laboratory within the HEALTH-PROT project were focused on two areas: the development of computational methods for prediction and analysis of protein structures and their interactions with nucleic acids, and on experimental analysis of proteins acting on nucleic acids.
Methods development: The main effort of the bioinformatics part of the project was towards methods development. In particular, the postdoctoral researcher employed within HEALTH-PROT, dr Marcin Pawlowski, developed a series of methods for the evaluation of protein structure models. The MQAPmulti program predicts the overall global quality of protein models. It compares structural features generated from a 3D model with those predicted from its primary sequence (secondary structure, solvent accessibility, contact maps), uses statistical potential to estimate the value of pseudo-energy for a single model, uses hydrogen bonds pseudoenergy, and takes into account information from proteins that are evolutionary related to the target protein. We evaluated MQAPmulti method on models generated in the framework of the CASP9 experiment. The correlation between predicted and real global quality of a model (GDT_TS score) was 0.987 i.e. very high. Dr Pawlowski has also developed RecobmineIt, a fully automatic procedure that starts with a number of 3D models generated by other methods, performs local assessment of model quality (by the afore-mentioned MQAPmulti), and constructs hybrid models by splicing best-scored fragments from the original models. RecobmineIt was ranked among the 10 best methods in the CASP 9 experiment. In the course of the HEALTH-PROT project a number of other methods have been also developed:
FILTREST3D (available as a web server and standalone software at: http://filtrest3d.genesilico.pl/) is a computer program for discrimination of structural models using restraints from experimental data (e.g. secondary structure, shape, and intramolecular contacxt determination.
MetalionRNA is a method for predicting positions of metal ions (magnesium, sodium and potassium) for RNA structures defined by a user. It is based on a statistical potential inferred from the analysis of binding sites in experimentally solved RNA structures. It is available as a web server at http://metalionrna.genesilico.pl/.
RIBER is a method for detecting the presence of RNA stems in macromolecular crystals based on diffraction data alone. RIBER complements the previously developed program DIBER intended to search for double-stranded B-DNA and not double-stranded A-RN. The two programs have been implemented as a stand-alone software suite and a web server RIBER/DIBER (available at http://diber.iimcb.gov.pl/) providing an easy way to judge nucleic acid content of a crystal based on a diffraction dataset, before the crystal structure is solved. The new method may help to avoid a laborious phasing procedure when the component or the protein-RNA complex of interest is not present in the crystal.
MetaDisorder is a series of meta-predictors for the prediction of intrinsic disorder in proteins. They are based on third-party primary disorder predictors, and infer the consensus prediction by weighting the accuracy of the input methods. In particular, a meta predictor GSmetaDisorderMD was the top scoring method in the CASP9 benchmark. The Bujnicki group has also developed a disorder predictor GSmetaDisorder3D that uses no third-party disorder predictors, but relies on the analysis of alignments between the query protein sequence and the known protein structures, reported by the protein fold-recognition methods. MetaDisorder server is available at http://iimcb.genesilico.pl/metadisorder/.
Methods application: The Bujnicki group has carried out numerous studies in which they applied software tools for the analysis of structures and functions of proteins and protein-nucleic acid complexes. In line with the topic of the HEALTH-PROT project, the main subject of these analyses were enzymes acting on nucleic acids. Of particular importance for the HEALTH-PROT project were also analyses of the effect of mutations on the stability of protein structures and their potential pathogenic effects in humans.
Experimental analyses of enzymes acting on nucleic acids: The experimental work in the Bujnicki group has focused on enzymes acting in the context of two subsystems of nucleic acid metabolism: RNA modification and DNA repair and degradation. Major effort within the HEALTH-PROT project was devoted to the development of a labeling methodology with the use of SAM analogues, in collaboration with the prof. Klimasauskas group in the twinning partner institution Vilnius. In the course of a medium-term visit of experienced researchers from the Bujnicki Laboratory to the Klimasauskas laboratory, several RNA methyltransferases have been tested for the ability to catalyze the transfer of extended groups from the cofactor analogs onto the RNA substrate, with retention of the original target specificity of these enzymes. The visit allowed the Bujnicki laboratory to gain the knowledge on labeling RNA by cofactors and to improve the knowledge of the use of HPLC and mass spectrometry for the analysis of nucleic acid modifications. The work on DNA repair enzymes has been done in connection with the bioinformatics analyses (development of the REPAIRtoire database and bioinformatics analyses). One analysis that has been finalized and whose results have been published shortly after the end of the HEALTH-PROT project concerned the structural analysis of a DNA repair enzyme Exonuclease VII (ExoVII), a bacterial nuclease involved in DNA repair and recombination that hydrolyses single-stranded DNA. ExoVII is composed of two subunits: large XseA and small XseB. Thus far, little was known about the molecular structure of ExoVII, the interactions between XseA and XseB, the architecture of the nuclease active site or its mechanism of action. The Bujnicki group used bioinformatics methods to predict the structure of XseA, which revealed four domains: an N-terminal OB-fold domain, a middle putatively catalytic domain, a coiled-coil domain, and a short C-terminal segment. By series of deletion and site-directed mutagenesis experiments on XseA from Escherichia coli they determined that the OB-fold domain is responsible for DNA binding, the coiled-coil domain is involved in binding multiple copies of the XseB subunit, and residues D155, R205, H238, and D241 of the middle domain are important for the catalytic activity but not for DNA binding. Altogether, a model of sequence-structure-function relationships in ExoVII was proposed.
Slawomir Filipek, Laboratory of Biomodeling
Understanding of amyloid formation in Alzheimer's Disease
Alzheimer's disease (AD), the most common form of dementia in the elderly, is characterized by accumulation of the amyloid senile plaques in the brain. Genetic studies show that more than 170 mutations in the PS-1 protein were associated with familial forms of Alzheimer's disease and new mutations are still being identified.
Prof. Andrisano had developed many acetylcholinesterase inhibitors against AD and works on combining them with other drugs against AD. These inhibitors increase concentration of neurotransmitter acetylcholine to help AD patients because they have diminished levels of acetylcholine in brain neurons. Increase of this concentration slow down the pathological changes and the especially loss of mental ability. Unfortunately, these drugs neither stop nor reverse deterioration of brain neurons. They only affect symptoms of this disease. For this reason there are ongoing investigations on complementing of action of these drugs against additional molecular targets, for instance the γ -secretase complex. Because this complex is necessary to proteolize other substrates not only APP so the potential inhibitors should selectively hamper APP proteolysis. Knowledge of the structure of γ -secretase complex and especially interactions between proteins forming this complex would allow designing of selective inhibitors. Analysis of effects of mutations leading to familial form of Alzheimer's disease (FAD) at molecular level would allow to understand mechanisms of overproduction of pathological amyloids.
Drug design is the first step in a long and costly process of searching for novel drugs and the investigations should be focused on biological targets which are the cause of the disease. Multithreading characteristics of AD requires usage of multi-target ligands (MTDL - Multi-Target-Directed Ligands) which can interact with more than one biological target. The cholinergic and amyloid hypotheses functioning to explain onset of development of AD can be employed together to design compounds which can inhibit formation of amyloids and can increase cholinergic transmission.
We also conducted MD simulations of novel structure which is a C-terminal fragment (CTF) of presenilin 1. The structure of this fragment was obtained using NMR techniques in SDS micelles [Sobhanifar et al., PNAS 2010]. Detergent molecules can modify the structure of membrane proteins and especially the location and structure of loops linking the transmembrane helices. Simulations were performed in DPPC (dipalmitoyl-phosphatidylcholine) and DLPC (dilauroyl-phosphatidylcholine) models of lipid bilayers and based on these simulations it was possible to obtain the proper structure of loops on both sides of lipid bilayers. The coarse-grain methods were used to achieve simulation time scale of microseconds such time was needed for the protein structure to adapt to the new environment from SDS micelle to lipid bilayer. In the next step the structure of CTF PS-1 was determined on the atomic level employing the reconstruction procedures from coarse-grain models and by running shorter but more detailed the all-atom simulations.
Realization of scientific tasks in this grant resulted in two papers published in 2012:
1. W. Pulawski, U. Ghoshdastider, V. Andrisano, S. Filipek, Ubiquitous Amyloids, Appl. Biochem. Biotech. (2012) 166, 1626-1643
2. B. Trzaskowski, D. Latek, S. Yuan, U. Ghoshdastider, A. Debinski, S. Filipek, Action of molecular switches in GPCRs - theoretical and experimental studies, Curr. Med. Chem. (2012) 19, 1090-1109
3. M. Naldi, J. Fiori, D. Tedesco, M. Pistolozzi, A. Drake, C. Bertucci, R. Wu, K. Mlynarczyk, S. Filipek, V. Andrisano, Amyloid beta peptide 25-35 self-assembly and its inhibition: a model decapeptide system to gain atomistic and secondary structure details of the Alzheimer's disease process and treatment?, manuscript in preparation.
Jacek Jaworski, Laboratory of Molecular and Cellular Neurobiology
Work of Dr. Jaworski team in frame of HEALTH-PROT grant initially focused on a role of mTOR-CLIP-170 interaction in neurons during their development. An initial finding that started collaboration with Dr. Hoogenraad was that CLIP-170, a microtubule plus end binding protein, a potential mTOR kinase target regulates dendritic arbor morphology. In course of HEALTH-PROT project, in close collaboration with Hoogenraad lab, we showed that:
(i) indeed in neurons CLIP-170 is a substrate of mTOR,
(ii) mTOR dependent dendritogenesis requires presence of CLIP-170,
(iii) mTOR activity regulates protein interactions of CLIP-170 but not its cellular distribution,
(iv) mTOR-dependent cross-talk between microtubules and actin cytoskeleton is needed for proper dendritic development,
(v) CLIP-170 resides in dendritic spines and is key for their proper morphology,
(vi) Dynamic microtubule retraction from spines is important for long-term synaptic depression, a form of synaptic plasticity.
The major findings stemming from these new research directions can be summarized as follows:
(i) p150glued interacts both with CLIP-170 and mTOR and is needed for dendritic arborization. p150glued rescues the effects of CLIP-170 deficiency in neurons.
(ii) β-adaptin interaction with mTOR is needed for initial steps of neuronal endocytosis and regulates interaction of cellular cargo with AP2 complex as well as AP2 complex,
(iii) Both mTORC1 and mTORC2 are needed for proper dendritic arbor development and mTORC2 acts upstream of mTORC1 (Urbanska et al., J. Biol. Chem, under revision),
(iv) mTOR and CLIP-170 are downstream effectors of GSK3. GSK3 blocks mTOR-dependent phosphorylation of CLIP-170.
In frame of HEALTH-PROT grant, newly hired postdoc, Dr. Matylda Macias, has developed pharmacological, animal model of long-term mTOR activity inhibition using rapamycin, in which together with Dr. Swiech, she performed several studies with regard to mTOR activation and +TIPs tissue and subcellular distribution. However, the most appreciable features of mice chronically treated with rapamycin were: a decrease in seizure threshold and hydrocephaly (Macias et al., submitted). Thus, as a major outcome of this part of research we find discovery of an importance of mTOR for water homeostasis in the brain. This topic will be further investigated due to its medical relevance.
Finally, in addition to scientific advancements, several advanced techniques and tools were introduced to the lab thanks to personnel exchange, e.g.:
(i) Fast live-imaging of neurons (visits from Hoogeraad lab)
(ii) Knock-out and transgenic models (visits to van Leuven lab, Leuven, Belgium)
(iii) iPS technology and in vivo analysis of neuroprecursor development (visits to Sala lab, Milano, Italy; Toni lab, Lausanne, Switzerland)
(iv) analysis of endocytic protein interactions (Haucke lab, Berlin, Germany)
(v) Electrophysiological data analysis (visit from Krugers lab, Amsterdam, the Netherlands)
Jacek Kunicki, Laboratory of Neurodegeneration
The project consisted of two major parts both are related to calcium homeostasis and calcium signaling in the brain. In particular, we showed the involvement of β -catenin in regulation of T-type Ca2+ currents in thalamic neurons and we characterized mechanism and proteins involved in Store-Operated Calcium Entry (SOCE) in neurons. The details are described below.
Our previous studies carried out in the Laboratory of Neurodegeneration revealed cell cycle disturbances in immortalized lymphocytes derived from Alzheimer's disease (AD) patients. We decided to check whether the observed cell cycle changes in AD lymphocytes are accompanied by impaired apoptotic response to oxidative stress and to compare AD and Parkinson's disease (PD) lymphocytes. Lymphocytes from sporadic AD and PD patients were from prof. Martin-Requero's laboratory culture collection. Lymphocytes from FAD patients came from the bank at the IIMCB. The vulnerability to oxidative stress was assessed with two independent methods: colorimetric MTT test and JC-1 flow cytometry assay. The preliminary results showed a significant difference in apoptotic response between FAD and SAD lymphocytes. The experiments are now continued in the IIMCB. The data obtained in collaboration with prof. Martin-Requero were included in the PhD thesis of Dr. E. Bialopiotrowicz supervised by Prof. U. Wojda and in the common publication which is currently prepared. Thanks to the collaboration we also broadened our lymphocyte culture bank with cells from Spanish AD and PD patients.
Our group at the Laboratory of Neurodegeneration of IIMCB is studying the role of Calmyrin 2 (CaMy2, CIB2), a novel member of calcium binding proteins Calmyrins. Looking for neuronal CaMy2 function by affinity approach we have discovered a novel CaMy2 interaction partner, Rab5 GTPase the key element in early endosomes. To investigate the potential implication of CaMy2 in endocytosis, we established the collaboration with the expert in the neuronal endocytosis, Prof. Casper Hoogenraad. Our collaborative studies showed that overexpressed CaMy2 colocalizes with Rab5-positive vesicles as well as together with another early endosomal marker, EEA1 protein in rat primary hippocampal neurons in culture. Knock-down of CaMy2 with shRNA greatly affected early endosome localization of Rab5 and EEA1. CaMy2 depletion resulted also in inhibition of AMPA receptor endocytosis known to be mediated by Rab5. Our results obtained in collaboration with Prof. Hoogenraad laboratory indicate a novel mechanism by which Ca2+ signals mediated by CaMy2 regulate Rab5-mediated endocytosis. Now we describe these findings in our common research publication and in the PhD thesis of K. Debowska.
Publications and manuscripts:
Misztal K, Wisniewska MB, Ambrozkiewicz M, Nagalski A, Kuznicki J. WNT protein-independent constitutive nuclear localization of beta-catenin protein and its low degradation rate in thalamic neurons. J Biol Chem. 2011 Sep 9;286(36):31781-8.
Gruszczynska-Biegala J, Pomorski P, Wisniewska MB, Kuznicki J. Differential roles for STIM1 and STIM2 in store-operated calcium entry in rat neurons. PLoS One. 2011 Apr 26;6(4)
Wisniewska MB, Misztal K, Michowski W, Szczot M, Purta E, Lesniak W, Klejman ME, Dabrowski M, Filipkowski RK, Nagalski A, Mozrzymas JW, Kuznicki J. LEF1/beta-catenin complex regulates transcription of the Cav3.1 calcium channel gene (Cacna1g) in thalamic neurons of the adult brain. J Neurosci. 2010 Apr 7;30(14):4957-69.
Wisniewska MB, Nagalski A, Dabrowski M, Misztal K, Kuznicki J. Novel beta-catenin target genes identified in thalamic neurons encode modulators of neuronal excitability. BMC Genomics. 2012, submitted
Debowska K., Blazejczyk M., Jaworski J., Hoogenraad C., Kunicki J, Wojda U. CaMy2 regulates Rab5-mediated clathrin-dependent endocytosis in neurons. Manuscript in preparation
Bialopiotrowicz E, Kuzniewska B, Esteraz N, Martin-Requero A, Kuznicki J, Wojda U. Responses to oxidative stress differentiate lymphocytes from familial and sporadic Alzheimer's disease patients. Manuscript in preparation
Marta Miaczynska Laboratory of Cell Biology
Endosomal proteins in regulation of cell signaling and proliferation.
Within the project, the Laboratory of Cell Biology in collaboration with Prof. Harald Stenmark (Institute for Cancer Research, The Norwegian Radium Hospital, Oslo) and Prof. Marino Zerial (Max Planck Institute, Dresden) performed screens to search for endosomal proteins with novel functions in transcriptional regulation, cell signaling and proliferation. To undertake systematic studies of possible mechanisms by which endocytic proteins may contribute to these processes, we established and performed medium-scale, targeted RNAi screens. We aimed to identify candidate endocytic proteins which affect transcriptional responses in different signalling pathways. While it was not expected that endocytic proteins represented novel core components of the investigated pathways, we wished to identify important modulators able to provide fine-tuning and possible integration between different processes.
Marcin Nowotny, Laboratory of Protein Structure
The aim of our research project within the HEALTH-PROT grant was to perform structural studies of reverse transcriptases (RTs), which catalyze the reverse transcription a multi-step reaction of conversion of single-stranded RNA to double-stranded DNA. In addition to the purely scientific interest in this fascinating process, it is also highly medically relevant, since it is the key step of the proliferation of retroviruses such as human immunodeficiency virus (HIV) and hepatitis B virus (HBV). RTs from both viruses are targets of anti-viral therapy.
Michael Witt, Ciliary Proteins Function Project
Work within this project focused on introducing into the laboratory several techniques required for analysis of respiratory epithelium structure and function, including the technically demanding method of culturing respiratory epithelial cells from nasal brushings, known as the in vitro ciliogenesis technique. This technique is an essential technique for analysis of molecular basis of airway disorders, such as cystic fibrosis or primary ciliary dyskinesia (PCD), as well as universal tool for analysis of respiratory epithelium structure and function. In parallel with the chosen method of in vitro ciliogenesis (sequential method of ciliogenesis by Jorissen et al, 1989) several complementary techniques, including collagen isolation from rat tails, coating of the culture flasks with collagen gels and high-speed videomicroscopy of the in vitro differentiated cilia have been successfully implemented into the laboratory. The experience of the team with the culturing the respiratory epithelial cells has been summarized in a review article on the techniques of culturing differentiated respiratory epithelial cells (Bukowy et al, 2011, Journal of Applied Genetics).
Collaboration with the laboratory of Professor Heymut Omran at the Department of Pediatrics and Adolescent Medicine at the University Hospital in M?nster, Germany has allowed to further increase the experience of the team in the method of culturing and analyzing respiratory epithelial cells, as well as introduce into the laboratory the method of immunofluorescence staining of proteins present in cilia, flagella and related structures, in which the laboratory of Professor Omran has a high expertise.
In addition to learning different aspects of the in vitro ciliogenesis technique, collaboration with the Omran's group (reciprocal visits) allowed members of the Witt team to introduce into the lab the method of immunofluorescence staining of proteins present in cilia, flagella and related structures. Both methods were used in the study of the influence of the conserved signaling pathway, Notch pathway, on the direction and degree of the respiratory epithelium differentiation; they are also used in the currently ongoing projects. The whole series of mutations causative for PCD were identified in the Polish population in several genes coding for various ciliary proteins, mainly DNAH5, DNAI1, DNAL2, RSPH9, RSPH4A, RPGR, leading towards a population-specific set of significant diagnostic potential.
Publications and manuscripts:
Bukowy Z, Zietkiewicz E, Witt M. In vitro culturing of ciliary respiratory cells--a model for studies of genetic diseases. J Appl Genet. 2011 Feb;52(1):39-51.
Zietkiewicz E, Nitka B, Voelkel K, Skrzypczak U, Bukowy Z, Rutkiewicz E, Huminska K, Przystalowska H, Pogorzelski A, Witt M. Population specificity of the DNAI1 gene mutation spectrum in primary ciliary dyskinesia (PCD). Respir Res.2010 Dec 8;11:174.
Bukowy-Bieryllo Z, Zietkiewicz E, Loges NT, Wittmer M, Geremek M, Olbrich H, Fliegauf M, Voelkel K, Rutkiewicz E, Rutland J, Morgan LC, Pogorzelski A, Martin J, Haan E, Berger W, Omran H; Witt M. RPGR mutations might cause reduced orientation of respiratory cilia. Pediatric Pulmonology, accepted
Zietkiewicz E, Bukowy-Bieryllo Z, Voelkel K, Klimek B, Dmenska H, Pogorzelski A, Sulikowska-Rowi?ska A, Rutkiewicz E, Witt M. Mutations in radial spoke head genes and ultrastructural cilia defects in East-European cohort of primary ciliary dyskinesia patients PLoS ONE, accepted
Maciej Zylicz, Department of Molecular Biology
The MDM2 protein is an E3-ubiquitin ligase and is a major negative regulator of p53 suppressor protein. However, as it has been shown by us and others, it also possesses intrinsic chaperone like activities towards WT p53 protein and p53 mRNA. Within the Health Prot project, in collaboration with our twinning partners (Prof. Ted Hupp, University of Edinburgh and Prof. Urszula Hibner, CNRS, Montpellier), we have further shown evidences that ATP-binding to MDM2 is a regulatory mechanism in numerous pathways ranging from gene expression involved in the development of cancer cells, to regulation of transcription factors via the PI3/AKT signaling pathway. Dr. Pawel Winiewski, employed within the project, demonstrated that TEK/Tie2 gene expression was significantly affected by MDM2 K454A, ATP-binding deficient mutant, in comparison to MDM2 WT. TEK receptor is involved in the regulation of cell motility, differentiation and angiogenesis, acting predominantly via AKT kinase pathway. Next, he has shown that phosphorylation of AKT is strongly down-regulated by MDM2 K454A and AKT binding to MDM2 is impaired in MDM2 K454A transfected cells. Finally, it has been demonstrated that the ATP-dependent properties of MDM2 regulate activity of two distinct transcription factors: AP-1 and ISGF3 via down-regulation of AKT signaling pathway in H1299 non-small lung cancer cells.
Using the same experimental model, parallel investigations were carried out, focusing on the role of MDM2 on other p53 family members, namely TAp63 and TAp73. Using BAX promoter, we have demonstrated that MDM2 differently modulates the transcriptional activity of both proteins. MDM2 stimulates the transcriptional activity of TAp63, while inhibits the transcriptional activity of TAp73. The preliminary results indicate that MDM2 regulates translation of both TAp63 and TAp73. The results were presented on the 5th Mutant p53 Workshop (Tracz Z, Zylicz A, Zylicz M. (2011) Interactions of p53 mutants with other p53 family members in non-small cell lung cancer cells. In: ?5th Mutant p53 Workshop From bench to bedside across mouse models?, pp. 131, May 22-25, Palazzo Chigi Ariccia, Italy). The preliminary results described above gave prominent impact on successful grant application from the Foundation for Polish Science (Zuzanna Tracz).
Additionally we have shown that MDM2 is also involved in the DNA double strand brake response/repair cascade in p53-/- background. Dr. Bartosz Wawrzynow from the twinning partner laboratory (University of Edinburgh) was employed within the programme for the last six months at IIMCB. He implemented new techniques such as Olympus ScanR live screening platform to measure protein abundance/accumulation and compartment localization upon proteasome inhibition and/or heat shock. Additionally an In-Cell-Western (ICW) assay was established aimed at monitoring the kinetic phosphorylation profile of key protein involved in Homologous Recombination (HR), one of the dominant DNA repair mechanisms in eukaryotic cells.
In collaboration with Marta Maluszek, they have shown that ATP binding by MDM2 is key for its involvement in DNA repair mechanisms. Upon initiation of DNA damage with the use of radiomimetic compound neocarzinostatin (NCS), a pronounced difference in the ubiquitylation and degradation profile between WT and K454A MDM2 occurs. Moreover, the ATP-bound MDM2 is required for efficient homologous recombination and survival rate of cancer cells in response to DNA damage. We have strongly benefited from the Health Prot programme in this avenue of research, as complementary results further strengthening our findings were obtained in the Gray Institute for Radiation Oncology and Biology in Oxford by Marta Matuszek. The preliminary results were presented on the international workshop (Ma?uszek MJ, Zylicz A, Zylicz M. (2011) The role of MDM2 ATP-binding ability in DNA damage response, The MDM2 International Workshop, New York Academy of Sciences, p21, October 16-19, New York, USA) and will serve as a basis for the next grant application.
Molecular chaperones modulate the activity of WT p53 suppressor protein, which is beneficiary for the healthy cell. However, they also stabilize mutants of p53, which in contrast is beneficiary for the cancer cell. One of our projects within the Health Prot programme, focuses on mutant p53 Gain-Of-Function (GOF) mechanism. We have postulated and demonstrated that GOF can be implemented by HSP70/HSPA1 induced aggregation process of mutant p53 forms. Being aggregated they are not able to be ubiquitylated and subsequently degraded, leading to a flux in the cellular homestasis. These preliminary experiments performed on Non Small Lung Cancer Cell lines gave an impact for a new grant proposal, which was obtained from the National Centre of Science. A manuscript of the paper is prepared. Further experiments with using breast cancer model cell lines will be continued. The current data was presented on the international workshops and conferences (Wiech M, Tracz Z, Maluszek M, Pruszko M, Wisniewski P, Zylicz A, Zylicz M. (2011) Hsp70 in mutant p53 gain-of-function phenotypes. In: The Biology of Molecular Chaperones. From basic mechanisms to intervention strategies in disease and aging?, pp. 166, May 19-24, Grundlsee, Austria;
Wiech M, Zylicz A, Zylicz M. (2011) Aggregate prone p53 mutants: the impact of HSP70. In 5th Mutant p53 Workshop From bench to bedside across mouse models, pp. 136, May 22-25, Palazzo Chigi Ariccia, Italy; Tracz Z, Wiech M, Manuszek M, Pruszko M, Zylicz A, Zylicz M. (2011) Stress induced mutant p53 gain-of-function phenotypes. In: 7th NCRI Cancer Conference, November 6-10, Liverpool, UK).
We have also shown that human cells are equipped with numerous functionally distinct subsets of chaperones, some of which seem to be dedicated to (re)folding and some that have evolved to dispose of non-foldable proteins. We investigated the new HSPA6 protein which lacks the generic chaperone-like properties of other HSP70s and may have evolved to maintain specific critical functions under conditions of severe stress. We have shown that under heat shock conditions (e.g. high fever during an inflammatory process) the overproduction of HSPA6 occurs, while under normal physiological temperature its level is almost not detectable. This particular protein is responsible for the transcriptional activity of p53 without need any HSP40 cochaperones. The results are presented in the paper: Hageman J, van Waarde MA, Zylicz A, Walerych D, Kampinga HH. The diverse members of the mammalian HSP70 machine show distinct chaperone-like activities. Biochem J, 2011; 435:127-142.
Potential Impact:
General impacts
EU level
Boosting research recognition. Within three years of HEALTH-PROT implementation IIMCB scientists published 35 publications in recognized journals, visited and hosted distinguished partners from top European centers and with them organized high quality scientific events. The HEALTH-PROT resulted in deepening existing and initiating new promising cooperations. Some of them are described below:
(i) The collaboration between Prof. Michael Witt and his twinning partner Prof. Heymut Omran, currently from Munster, has a long history and a broad range of EU Framework Projects involved: it's been initiated within the Centre of Excellence in Molecular Biomedicine in FP5, continued within FP6 as HEALTH-PROT project, currently being under coverage within FP7 as the project ?Better Experimental Screening and Treatment for Primary Ciliary Dyskinesia, acronym BESTCILIA and PhD programme in Molecular Biology: Studies of nucleic acids and proteins - from basic to applied research; acronym MPD; implemented within Structural Funds IE OP 1.1.2. Programme which is based on a common mentorship of a PhD student involved in a PhD research project in Prof. Witt and Prof. Omran cooperating laboratories.
(ii) Combined efforts of Prof. Kuznicki, Laboratory of Neurodegeneration, IIMCB, with Prof. Jochen Herms, Centre for Neuropathology Ludwig-Maximilians-University of Munich, Germany, towards innovative research resulted in a common patent application. Moreover, these two partners submitted two projects? applications for EU funding: SME-targeted research for developing tools and technologies for high-throughput research project to FP7 and IE OP 1.1.2 PhD programme in Molecular Biology: Studies of nucleic acids and proteins - from basic to applied research (MPD) to EU Structural Funds (546 609 euro). The latter was successful and is now being implemented. This intense and fruitful collaboration extended on Dr. Jaworski Laboratory of Molecular and Cellular Neurobiology, IIMCB. The common interests of Dr. Jaworski and Prof. Herms were expressed in a successfully submitted and currently implemented FP7 Era-Net Neuron project on Imaging Neurogenesis in Neurodegenerative Disease (262 004 euro).
(iii) Similar added value was created from the twinning of Prof. Bujnicki, Laboratory of Bioinformatics and Protein Engineering, IIMCB, with Prof. Saulius Klimasauskas, Laboratory of Biological DNA Modification, Institute of Biotechnology, Vilnius Lithuania. Thanks to scientists? exchange Prof. Klimasauskas entered into collaboration with Prof. Matthias Bochtler, Laboratory of Structural Biology, IIMCB, the outcome of which is a common participation in the TEAM project on Structural biology of methylation and hydroxymethylation funded from EU Structural Funds (488 344 euro).
(iv) Another example of real result bringing cooperation can be a twinning pair of Prof. Zylicz, Department of Molecular Biology, IIMCB and Prof. Ted Hupp, Edinburgh Cancer Research Centre, University of Edinburgh, UK. This long lasting cooperation which stems from the Centre of Excellence project in FP5 was now strengthened and, as a HEALTH-PROT result, evolved in a common participation in two competition based projects: the EU Structural MPD project and the Polish Ministry of Science and Higher Education research grant on Mechanism of oncogenic activities of mutated TP53 (144 722 euro).
All these activities allow for a better recognition and appreciation of IIMCB, and Polish science in general, among Western European reknown partners. In medium and long term it will contribute to a perception of IIMCB and other Polish research institutes as equal scientific partners and will lead to a real integration of Eastern European research institutes into the European Research Area.
Enhancing attractiveness to European researchers. The HEALTH-PROT evoked the idea to create an integrated set of principles on recruitment and approach towards employed researchers. It was materialised in IIMCB official declaration in 2010 to adopt the European Charter for Researchers and the Code of Conduct for the Recruitment of Researchers. As one of the three Polish institutions voluntary committing itself to apply Charter and Code principles, IIMCB?s intention was to increase institute's attractiveness to European researchers by offering them sure and secure favorable working environment, enhancing the quality of research and innovation and boosting international mobility.
Regional level
Sharing experiences with and enhancing participation of INCO countries in FP7. IIMCB has taken actions to share the experiences of participating in the Research Potential programme with Ukrainian scientists and managers from the Institute of Molecular Biology and Genetics (IMBG) in Kiev. IIMCB and IMBG representatives meet every 2 years during Polish-Ukrainian Parnas conferences. Closer bilateral relations between IIMCB and IMBG were established in 2008 thanks to an initiative of IIMCB director Prof. Jacek Kuznicki. During the round table Research organization in the 21st century: experience and achievements of IIMCB, organized by the Ukrainian Ministry of Education and Science Prof. Kuznicki proposed that IIMCB's experience could be used by Ukrainian scientific institutions. In 2009 IIMCB organized a Polish-Ukrainian scientific conference accompanied by the HEALTH-PROT kick-off meeting. The IMBG director, Prof. Elskaya, participated in the IIMCB International Advisory Board meeting and the IMBG managers had meetings with their IIMCB counterparts. One of the results of this cooperation is a common project entitled ?Strengthening cooperation in molecular biomedicine between EU and Ukraine? (FP7-INCO-2011-6, ERA-WIDE), 2011-2014.
National level
Reaching scientific excellence on national level. HEALTH-PROT project contributed to IIMCB excellence on national level: in 2010 IIMCB was ranked number 1 among 36 biological institutions by the Polish Ministry of Science and Higher Education. Prof. Jacek Kuznicki, IIMCB director and HEALTH-PROT coordinator, was in 2011 nominated a member of Science Policy Committee. This body, consisting of 10 experts, assists the Minister of Science and Higher Education among others in: the development of documents relating to the development of science and science and innovation policy and in the development of draft State budget and financial plan setting out the financial resources for science. In 2011 Prof. Michael Witt was elected the Chairman of the Advisory Board for Molecular Genetic Tests and Biobanking, established at the Ministry of Science and Higher Education. The Board is comprised of geneticists, oncologists and laboratory diagnosticians. In 2011 Janusz Bujnicki was elected a member of the Young Scientists Academy, an appendix to the Polish Academy of Sciences. International Institute of Molecular and Cell Biology is a laureate of the Funds for Science 2011 awards in the commercialization of scientific research category.
Learning from the HEALTH-PROT achievements, two neighboring institutes from Warsaw Ochota Campus applied for RegPot funding, of which one with success.
Country-wide initiatives with significant societal impact
During the conference organized within the HEALTH-PROT project a patient family/support group for families with the primary ciliary dyskinesia (PCD) was founded based on IIMCB initiative. The Polish Society for Ciliary Dyskinesia is the first organization gathering these families in Poland with over 60 founding members signing the foundation document. Organization's goals are: to provide support to patients and their carers who have, or are suspected of having, PCD; to bring PCD to the attention of medics who may come across PCD and continue to provide an up to date information service for them and the general public; to promote research to aid diagnosis and treatment of patients with PCD; to ensure patients have access to diagnostic services and on-going care within the framework of current national medical care system.
IIMCB also supports other patient organisations. In 2005, one of our staff members organised the patient association J-elita, bringing together the families of patients who suffer from Crohn's disease and Colitis (J-elita's office is located on the IIMCB premises).
Institutional level
Setting up directions for further development.
(i) The HEALTH-PROT project consolidated Institutes efforts to concentrate on the strategic research problem which is studies of various aspects of protein structure/function. Research performed at IIMCB although basic may lead to better understanding of different mechanisms vital for human life such as: ageing, neurodegeneration, cancer, addictions, etc. Being aware of the market value of scientific results IIMCB founded in 2010 a Technology Transfer Unit Bio&Technology Innovations Platform (http://www.biotechip.pl). This idea originated in the HEALTH-PROT proposal and was appreciated by the EC but funds to this end were cut. IIMCB received funding from Structural Funds and a program of the Ministry of Science and Higher Education to set up a TTU for six bio-tech-med research institutes forming BioCentrum Ochota consortium. The Unit has at its disposal funds to cover stipends for innovative applied research projects, trainings in legal and financial aspects of conducting scientific research such as IPR and commercialization.
(ii) In March 2012 the cooperation agreement between IIMCB and the Max-Planck Society (MPG) was signed. The agreement concerns the establishment of two Max Planck/IIMCB Research Groups, one in IIMCB, other in the Max-Planck Institute of Heart and Lung Research in Bad Nauheim. A research group in IIMCB will be the first in Poland to use the zebrafish as a model of pathomechanisms of various human diseases. Existing significant experience of MPG in this area should greatly facilitate the rapid launch and research progress in this direction.
(iii) Experiences of HEALTH-PROT project allowed to gather expertise and knowledge to further develop innovation potential, measured by medically relevant discoveries, patent applications, and license agreements. Stemming from this was an initiative to create at IIMCB the FishMed Centre to be the first zebrafish research and breeding facility in Poland. A relevant applicaton has been submitted to the FP7. Zebrafish is an excellent organism to study various aspects of human disease because it is fully transparent until adulthood, and its metabolism is similar to that of mammals. The FishMed Centre is designed to twin with six excellent European zebrafish centres. These include the Max Planck Institute for Heart and Lung Research in Bad Nauheim, the Universities of Cambridge, Leiden, Sheffield, and Geneva, and an Austrian IST. The twinning Partners will share their zebrafish models and know-how in joint projects with regard to cancer, neurodegeneration, epigenetics, and tissue regeneration. The zebrafish breeding facility will be organized and a new research group established, headed by a leader selected in an international competition. The costly equipment for the breeding facility will be purchased partly from the grant funds.
(iv) BESTCILIA project within FP7 devoted to primary ciliary dyskinesia (PCD) biology has been developed with weighty input of IIMCB scientists. PCD is a rare disease for which no orphan drug is available and which currently is treated off-label. The main goal of the project is to improve the diagnosis and treatment of PCD patients. This will be achieved through introduction of standardized diagnostic testing for PCD in European countries, where this is currently not available; establishment of a comprehensive PCD registry for systematic data collection on current disease status in order to define the natural course of the disease and to define outcome relevant parameters in a large European-American cohort; setting up an observational trial in PCD for a meta-analysis of existing datasets to describe the clinical phenotype, prognostic factors and predictors of poor outcome; establishment of PCD-specific Health-Related Quality of Life Questionnaires (HRQOLQ); performing a randomized controlled clinical trial on the use of acithromycin in PCD.
Potential impact and exploitation of scientific results
Cooperation between Prof. Jacek Kuznicki and his twinning partner Prof. Jochen Herms resulted in a patent application that has been filed through Ludwig Maximilians Universitat in Munich (Use of [name removed] derivatives, including their pharmacologically useable salts and related structures for the treatment of diseases of the central nervous system, metabolic and cardiac diseases as well as aging and health status itself?). (Kuznicki)
Endonucleases, particularly programmable TALENs, are of interest for genetic manipulation in vivo as well as in vitro. The group has now won new Polish grants to apply some of the knowledge gained from studies of different restriction endonuclease types to TALENs.
In particular, it is planned to search for or develop replacement for the currently used effector nuclease FokI. There has also been an effort to broaden the expertise of the group to include the application of the new nucleases in animal models. As part of the training program of the HEALTH-PROT grant, the PI has learnt P-element mediated transformation of Drosophila melanogaster from Wynand van der Goes van Naters at Cardiff University. He has also had the opportunity to observe lentivirus mediated transgenesis in the mouse during a guest stay in the laboratory of Philippe Ravassard, an expert in mouse transgenesis at Pierre and Marie Curie University and ICM (Paris). The training will help the group to apply basic knowledge on nucleases to animal transgenesis experiments. (Bochtler)
Alzheimer's Disease is a progressive neurodegenerative disease. It affects more than 25 million of people worldwide and this number is strongly increasing nowadays. This is why the search for novel and more effective drugs is extremely important. Novel multi-target-directed drugs are still in their begging phase because of their complex interactions. They can potentially affect different element of amyloid formation cascade in the same time, therefore, could be more efficient. During this grant the new methods of designing of multi-target ligands were developed and they could be used for subsequent studies and analyses. (Filipek)
The work done by the members of Jaworski lab in frame of HEALTH-PROT grant undoubtly will have an important impact on different levels. First, new knowledge regarding mTOR interacting partners as well as effects of mTOR-inhibitors is very relevant clinically as it helps predict adverse effects of mTOR inhibition in clinic. We also got a further insight into novel biological functions of mTOR. In addition, technology introduced to the laboratory moved our research to the next level that resulted in several new collaborations and grant applications. From this perspective, introduction of iPS technology seems to be very important as it already tighten our links to clinicians who are very eager to look into cellular mechanisms in neurons-derived from fibroblasts of real patients (e.g. TSC patients). Currently, with clinicians from several centers around Poland we plan to establish bank of iPS cells derived from patients suffering from various diseases with neurological manifestations. These bank will be of use to whole research community.
Finally, HEALTH-PROT grant allowed hiring specialist in cutting edge techniques in high-resolution microscopy (e.g. EM) and histology which will allow us to research mTOR cellular functions in the brain on subcompartmental level. iPS cell developed in frame of our research will be used as a starting material in several of our studies aiming to:
(i) reveal basic disease mechanisms and
(ii) find new substances preventing/correcting phenotypical outcome of the diseased gene on a cellular level. We also plan to deposit such iPS lines in iPS bank developed by us and our clinical collaborators in the future. (Jaworski)
The collaboration with the twinning partners within the project allowed us to establish at IIMCB a novel platform and methodology for RNAi screens in cultured cells using transcriptional reporter tests or proliferation assays. Since its establishment, this platform has been successfully used in other screens and will be further employed in the future. This is an important development which will be of long-lasting impact. Considering the specific scientific results of the project, identification of novel regulators of AP-1-dependent transcription or proliferation among endosomal proteins is an important discovery of basic research. We are currently continuing the studies of molecular mechanisms of action of the newly identified regulators which will eventually lead to publications. The scientific results of the project belong to fundamental research and have already formed a basis for further research and grant applications (both Polish and international) which were awarded to Dr. Miaczynska. They have already led and will further lead to publications in international peer-reviewed journals. (Miaczynska)
The results obtained within the HEALTH-PROT project are the first structural studies of a monomeric reverse transcriptase in complex with its substrate- RNA/DNA hybrid. Our crystal structure together with SAXS-verified model of the full-length enzyme provide important insights into similarities and differences with extensively studied HIV-1 RT. We were also able to correct the tracing of a substantial part of MMLV RT, which now allows us to draw structure-based conclusions for retroviral RTs in general. We plan to continue these studies, in particular to gain insights into the specific processing of the poly-purine tract an essential but poorly understood step of reverse transcription. HEALTH-PROT project allowed us to initiate studies of reverse transcriptases that we plan to carry on. The information gained during this projects and methods it allowed us to establish such as chemical cross-linking and SAXS will allow us to tackle such important and technically challenging issues as the structural basis of PPT recognition in reverse transcription. (Nowotny)
From the methodology point of view, new techniques and protocols have been successfully used throughout the study, many of which have been introduced to other labs within the IIMCB: a gene expression platform of profile analysis of over 80 genes known to be involved in cancer cells development (SABiosciences, PAHS-033) using RT-PCR (Dr. Pawel Winiewski); Olympus ScanR screening platform to measure protein abundance/accumulation and compartment localization as well as In-Cell-Western assay to monitor kinetics of posttranslational protein modifications (dr. Bartosz Wawrzynow). Considering the specific scientific achievements within the project, it is worth to underline the preliminary data leading to the elucidation of the mechanisms of DNA repair in cancer cells deficient in the TP53 gene as well as the data concerning the mechanisms of gain of new oncogenic functions of mutated p53 in lung cancer. The data provided strong impact for the successful application of the complementary three grant proposals (Alicja Zylicz, Zuzanna Tracz and Milena Wiech). Thus, the continuation of the project is further planned.
The new techniques introduced by employed postdoctoral researchers are commonly used in the laboratory and will be used in the future. One of the scientific aspects of the project concerns the elucidation of drugs resistance during treatment of patients with non-small-lung-cancer. Understanding such fundamental mechanisms would potentially benefit in the verification of current methods of treatment of patients harboring the above type of cancer. It has recently been demonstrated in mouse model that the stress response resulting from gamma-radiation stabilizes mutant p53 and shifts the tumour phenotype to more aggressive, as compared to treatment of Trp53-/- mice. It is tempting to speculate that similar phenomena may occur in human patients who harbour mutated p53 and are treated by gamma-irradiation. If that is the case, then the knowledge of the patient p53 status may be crucial in designing the optimal therapeutic strategies. Our findings elucidate the molecular mechanism of how stress-induced HSP70 stabilizes the mutant p53 protein and suggest new therapeutic approach, whereby HSP70 inhibitors could be used synergistically with gamma-irradiation to treat patients with mutated p53. (Zylicz)
Dissemination activities planned after the end of the project:
The HEALTH-PROT project will be promoted at the conference WIRE 2012, 4-5.06.2012 Krakow, Poland. Prof. Kunicki, HEALTH-PROT coordinator, was invited to moderate one of the panels. Dr. Urszula Bialek-Wyrzykowska, Dorota Libiszowska and Aleksandra Nalecz-Tolak will present the results of the project on a stand.
Around ten publications with the project results are expected to be published by the end of 2012.
List of Websites:
http://www.iimcb.gov.pl/
Contact details:
Dr Urszula Bialek-Wyrzykowska
International Institute of Molecular and Cell Biology
4 Ks. Trojdena St.
02-109 Warsaw, Poland
Tel. +48 22 59 70 713
Fax +48 22 59 70 715
ulawyrzykowska@iimcb.gov.pl
http://www.iimcb.gov.pl