Protein complexes play important roles in many cellular processes. Determining the structural aspects of protein interactions will thus further our understanding of these processes, and enhance the potential for intervention in pathologies. Although the structures of many single-chain proteins are known, structures of complexes are rare (of the known biological interactions, where the two protein structures have been solved, only 5% were solved as a complex). My primary research objective is to develop methods for efficient and accurate sampling of docked conformations, including the exploration of backbone flexibility. The improved methods will be used to study biomedically important processes, such as the oligomerization of the capsid protein of HIV. This oligomerization, which involves backbone remodeling, is a crucial step in viral maturation and a long-standing target of therapeutic intervention. I also propose to artificially redesign the sequences of a pair of proteins that do not naturally bind so that they will bind. This result will serve as proof-of-concept for the rational development of protein agonists and antagonists of other proteins. The proposed research involves different methods and concepts from the prediction of transmembrane protein structures which I studied in my doctoral work. Importantly, the protein-design aspects involve experimental work with which I have no prior familiarity. The Baker laboratory has many years of experience in all of the aspects that are new to me. Hence, this fellowship will provide me with the necessary experience for setting up a laboratory for joint computational-experimental study and design of protein interactions.

A combination of different methods is required to unravel the architecture of those macromolecules that are difficult to determine by a single high-resolution method. Small angle X-ray scattering (SAXS) of proteins and complexes in solution can result in structural insights into proteins and complexes in the size range of 30-300 kDa, in particular those that can adopt different functional conformational states. I propose to exploit SAXS for the modeling of large proteins and complexes in combination with other types of information, such as homology-derived restraints and statistical potentials, using the program MODELLER, a widely used program for comparative protein structure modeling. Importantly, I will quantify the ambiguity and accuracy of the resulting models. The method will then be applied to characterize the conformational states of Hsp90, an essential eukaryotic chaperone, throughout the ATP-cycle based on SAXS data and atomic structures of its domains and homologs. Finally, I will explore the theory of electron scattering of vitrified solutions and test it experimentally. This approach may have substantial practical advantages over SAXS because it should require a much smaller volume of less concentrated sample. While my previous research focused on structure at a low-resolution level of ~1 nm, the proposed modeling project will extend my knowledge to atomic representations. Moreover, I will enrich my repertoire of biophysical methods for structural characterization. Both experiences will be indispensable for my future research in the structural characterization of macromolecular assemblies using biophysical methods, in particular by cryo-EM, SAXS, and modeling.

Phagocytosis by specialized phagocytes is a host defense mechanism against pathogens. It is imperative for the cell to efficiently recognize phagosomal content to mediate its further processing for presentation by Class II MHC molecules. A comprehensive understanding of how a cell identifies the substance of a phagosome is still lacking. Signaling molecules attached and clustered in membrane microdomains, such as lipid rafts or the relatively little investigated tetraspanin-webs might well mediate this detection of content. Initial experiments in the Ploegh lab tracked the RFP-tagged version of the tetraspanin CD63 and showed that it is selectively recruited to phagosomes containing C. neoformans and not to phagosomes harboring an equal-sized polystyrene bead. I will investigate if this is a general phenomenon and identify lipid-modified proteins that mediate this effect using azide-tagging of lipids in cells in combination with proteomic analysis of the labeled components. The identified molecules will be tracked with state of the art microscopy as well as knock-down and knock-out technology to elucidate new pathways for phagosomal content recognition. This area of research and the methods are new to me, but a logical continuation of my professional development. Being a chemist by training, I have investigated enzyme kinetics and protein folding using mostly biophysical and biochemical techniques. I am now looking to extend my expertise to the fields of cell biology and immunology in order to be able to combine these approaches in later stages of my career.

In mammals, X-chromosome inactivation is a mechanism that ensures equivalent levels of X linked-gene expression between male and female, by silencing randomly one of the two X chromosomes in female cells. The process is established early in development and is initiated by a unique locus, which produces a large non-coding RNA, Xist. The Xist transcript is involved in cis-silencing by coating the entire inactive X chromosome. This induces chromatin changes that include histone modifications and DNA methylation. It has been proposed that LINE repetitive elements, which are particularly enriched on the X chromosome relative to autosomes, could act as “booster” elements to promote efficient inactivation, either by enhancing initial spreading of the inactivation signal, or by facilitating long term maintenance of gene silencing. Recent observations from the host laboratory, showing a correlation between methylation rates of CpG islands of X linked-genes and LINE density, suggest a possible model for the role of LINE elements in maintenance of X inactivation. The project described here aims at directly testing this model by genetically modifying LINE environments and assessing the consequences for gene silencing and CpG methylation. This work will provide further insights in understanding mechanisms of repeat-mediated gene silencing in mammalian genomes and will be a complementary study from my previous research based on the impact of transposons on plant genomes.

The biogenesis and co-transcriptional processing of eukaryotic and viral messenger RNA (mRNA) is regulated in part by direct recruitment of processing factors to the large subunit and C-terminal domain (CTD) of RNA polymerase II (RNAPII). The CTD recruits factors involved in pre-mRNA splicing, 5’- and 3’-processing, pathways essential for efficient mRNA export, stability, and translation. The CTD is comprised of tandem repeats of the heptad consensus sequence YSPTSPS wherein Ser2 and Ser5 are phosphorylated and dephosphorylated in coordination with the transcriptional cycle. The 5’ mRNA cap is the first co-transcriptional modification to occur on the nascent mRNA chain and cap formation occurs through the coordinated activities of three enzymes including RNA triphosphatase, RNA guanylyltransferase and RNA methyltransferase. Recruitment of these enzymes to RNA polymerase II (RNAPII) occurs via direct recognition of the phosphorylated RNAPII CTD. Interestingly, RNA methyltransferase maintains interactions with the phosphorylated CTD beyond early elongation when the CTD is presumably phosphorylated on both Ser2 and Ser5. After each round of transcription, RNAPII is recycled through dephosphorylation of the CTD by phosphatases such as Fcp1. This proposal will focus on utilizing macromolecular crystallography, genetics, and biochemistry to elaborate the mechanistic characterization of these enzymes and their interactions with RNAPII CTD. Consistent with my long-term goal, this study will provide molecular insights into how cellular factors are coordinated and perhaps coupled with transcription to facilitate co-transcriptional processing of mRNA.

During the last decades lung cancer has become the leading cause of cancer deaths in the world. Inactivation of tumor suppressor genes are the events underlying lung tumorigenesis, mainly genetic changes of p16INK4a and p14ARF. Our group has recently unveiled that aberrant expression of Cdc6 is oncogenic by directly repressing the INK4/ARF locus through the RD (4). In the present project, we propose to dissect the silencing mechanism on the INK4/ARF locus in lung cancer as working model. At the molecular level, I will analyze how ectopic Cdc6 and RNAi-RD result in RDINK4/ARF-dependent transcriptional repression, and, ensuing downregulation of the INK4/ARF locus. Moreover, I plan to characterize both the Cdc6 high levels found in NSCLC and endogenous RNAi-RD as internal regulation of the locus using murine models for lung tumorigenesis. In addition, we have a cohort of NSCLC to study whether the silencing of p16INK4/p14ARF by Cdc6 and RNAi-RD drive or contribute to the genomic alterations of p16INK4/p14AF associated to different transformations stages. These valuable tools will allow not only to understand the basis lung tumor biology but also for the development and validation of new tumor intervention strategies as well as for the identification of markers as Cdc6 or endogenous RNAi-RD for early diagnosis. I believe that the proposed studies on analysis of INK4/ARF locus in malignancy represent a cohesive approach to reach my professional goal as an independent researcher. My experience in molecular biology along with the support and help of Dr. Serrano, known as one of the leading researchers in the field, are vital for the successful execution of this ambitious project.

We have recently enabled photoswitching of a kainate receptor-channel by conjugating it to a tethered ligand. Ligand binding is controlled by a photoisomerizable azobenzene group in the tether, which presents the ligand to its binding site under ultraviolet light, thus opening the ion channel. Visible light removes the ligand from the binding site and results in channel closing. This project aims to extend this approach and develop new photoswitchable glutamate-gated receptors (AMPAR, NMDAR and mGluR). They are multimeric proteins with multiple ligand binding sites, and their operational mechanics are not well understood. Enabling to photoswitch these receptors will open new opportunities to study their gating mechanisms, by allowing to remotely control ligand binding in only one of the receptor subunits, and to distinguish between the events of ligand binding and channel opening. In addition, photoswitchable receptors make it possible to rapidly and reversibly depolarize cells without using invasive electrodes. Fast photoswitching in neurons will allow to generate photo-excitatory post synaptic potentials. In combination with the spatial selectivity of laser scanning microscopes, novel experiments can be designed to study the dynamics of cellular signaling at the intracellular and multicellular levels. The development of these tools is an important step towards the long-term goal of combining remote sensors and actuators of cell activity, in order to rationally interrogate complex cellular systems.

Determining the mechanisms underlying growth and differentiation in mammalian epidermis and its appendages will be important for understanding how this process goes awry in human skin disorders, including cancers. The Wnt signaling pathway is required to instruct skin stem cells (SCs) to make a hair follicle (rather than epidermis). Wnt signaling stabilizes beta-catenin, which in skin SCs acts as a transcriptional co-activator for the DNA binding protein, Tcf3. In the absence of beta-catenin,Tcf3 represses genes. I now propose to 1) elucidate Tcf3’s role in mouse skin SCs, by conditional ablation in vivo and in cultured keratinocytes 2) identify essential direct Tcf3 target genes by microarray,promoter analyses, chromatin immunoprecipitations and other molecular approaches, and 3) explore the functional significance of key Tcf3 targets. My studies should reveal how activated SCs acquire epidermal (Tcf3--) versus early hair follicle (Tcf3++) fates. They should expose whether Tcf3 target genes can be activated by shutting off Tcf3 (epidermal fate) as well as by Wnt-stabilization of beta-catenin (follicle fate) and if so, whether the same Tcf3 target genes are involved in both mechanisms. They should also provide insights as to whether Tcf3/Wnts govern SC activation, SC maintenance/quiescence or epidermal/follicle fate decisions. By means of this proposal, I will not only be able to address these important questions in stem cell biology, but also strengthen my knowledge and background in this field, and acquire the skills and intellectual thinking I will need for a successful academic career in scientific research.

The aim of this project is to investigate the role of 3’-phosphoinositides (PIs) in cancer development using the fruit fly Drosophila melanogaster. Drosophila is a powerful model to study cancer biology, because it mimics most aspects of human cancer development, including uncontrolled cell division, growth and invasion. One of the four 3’-PIs has been strongly correlated to increased cell growth and division and development of human cancer, but its role in metastasis is not known. The correlation between carcinogenesis and the remaining three 3’-PIs has not yet been explored. I will analyse how increase or decrease in levels of the four 3’-PIs affects receptor signalling and the steps of tumour development. This is easily done in Drosophila during larval development, because the larval tissue is transparent and affected cells can be labelled and traced by confocal microscopy. In this way, I will analyse the effects of defective PI metabolism on cell division, growth and metastasis. The genes that affect cancer development will be studied in more detail to determine the underlying molecular mechanisms and their possible involvement in human cancer will be validated in collaboration with geneticists. In this project I will learn the Drosophila model system, tumour biology and confocal microscopy to study cancer development in vivo, whereas I in my previous research have analysed detailed molecular mechanisms using biochemistry and cultured cells. Thus, I will broaden my knowledge significantly. In summary, this project has a great potential to identify novel genes involved in cancer development and will be of great value for my further development as a scientist.

Neurodegenerative diseases are characterized by progressive loss of neurons and synapses. Deregulation of Ca+2 is suggested to be involved in early stages of the disease and trigger its pathology. Ca+2 has been implicated in synaptic plasticity, therefore, the neurodegenerative process may initiate at synapses. Synaptic Ca+2 levels are regulated by voltage-gated Ca+2 channels and glutamate receptors, both implicated in neurodegeneration. Cdk5, a serine/threonine kinase, phosphorylates CASK, a protein that binds N-type voltage-gated Ca+2 channel and alter its activity. Cdk5 also regulates the function of NMDA receptors. We suggest an hypothesis whereby dysregulated cdk5 alters the activity of NMDA receptors and voltage gated Ca+2 channels and by thus modify synaptic Ca+2 levels, leading to synaptic dysfunction and eventually neurodegeneration. We will examine the role of cdk5 in regulating its synaptic substrates, maintaining synaptic Ca+2 homeostasis, and employ mice with altered cdk5 activity to determine if dysregulated cdk5 can induce changes in synaptic Ca+2 levels and in different aspects of synaptic function early before any observed signs of the disease. This proposal will provide new insight on early changes that occur in synapses and on the molecular process which leads to neurodegeneration. This fellowship will provide me the opportunity to conduct research in an area that differs from my previous research experience. I have never worked on synapses, Ca+2 signaling or neurodegeneration. I will be exposed to a variety of ideas and techniques, which will help me to establish my own laboratory and investigate molecular processes that lead to neurodegeneration.

The proposed project aims to delineate the mechanisms by which the salvador/warts/hippo (sav/wts/hpo) pathway restricts organ size during development and disease. When activity of the sav/wts/hpo pathway is reduced, cells grow and proliferate excessively and are resistant to programmed cell death. Defining the mechanisms by which this pathway functions is important, as several components of this pathway have been implicated in the genesis of human cancer. This study will employ a multi-faceted approach to investigate the mechanisms by which the sav/wts/hpo pathway restricts cell growth and number. We will confirm whether candidate downstream targets of the sav/wts/hpo pathway identified by microarray analysis, are genuine transcriptional targets of this pathway. We will then use Drosophila genetics and cell biology to examine whether these novel target genes regulate cell growth downstream of this pathway. Additionally, we will use bioinformatics to identify gene regulatory elements that are used by the sav/wts/hpo pathway to control the expression of known sav/wts/hpo target genes, as well as target genes that have been newly-identified in this study. The CDA is critical for the establishment of my independent research program and will allow me to assemble a team to perform the experiments outlined here. Ultimately I aim to establish a laboratory that makes important insights into the mechanisms that control cell growth and proliferation, and hence, organ size, during development. These studies should have implications for diseases such as cancer, which arise in part due to excessive cell growth and proliferation and insensitivity to programmed cell death.

In order to understand the evolution of genes and their function it is important to identify and quantify the evolutionary forces at work. To this end, neutral substitution rates of genes are estimated, which has so far mainly been done by estimating substitution rates at synonymous sites, i.e. at codon sites at that nucleotide changes do not change the encoded amino acid. However, there is accumulating evidence that synonymous sites might evolve under constraints also in primates. So far it was not possible to approach this question in a quantitative manner, because appropriate models that take into account the different mutation rates in the hypermutable dinucleotide CG (CpG) are missing. During this fellowship we intend to develop a likelihood method to quantify selection on synonymous and on non-synonymous sites at the same time. We will design this method to also accommodate CpGs as mutational hotspots. The neutral substitution rate will be inferred using intronic or flanking sequences. In addition we will estimate the strength of selection at synonymous sites by contrasting between-species divergence with human diversity data. These measures of constraint on synonymous sites will help to unravel the nature of constraints on synonymous sites and also improve the inference of selection at non-synonymous sites. Hence, I will supplement my skills in data analysis with mathematical and statistical knowledge. In the dawn of the post-genomic era with all the diverse large-scale functional datasets, sound statistical skills will be indispensable for successful research.

The overall objective of this proposal is to address key issues in transcriptional control in the yeast Schizosaccharomyces pombe. My focus is on a multiprotein complex, named SAGA, originally discovered in the yeast Saccharomyces cerevisiae, which controls transcription initiation by RNA polymerase II. SAGA is conserved throughout eukaryotes. The proposed experiments are to identify SAGA and its functions in the distantly related fission yeast S. pombe, in which chromatin organization and transcriptional regulation are remarkably different from that in S. cerevisiae and directly relevant to higher eukaryotes. This proposal contains two sets of experiments to address this issue. The first set addresses the roles of SAGA in key cellular processes, such as cell-cell separation and establishment of a specific cell mating identity. In the second set, a thorough biochemical analysis of SAGA in S. pombe will provide a necessary characterization of the complex in this model system. These experiments may help elucidate conserved as well as yet-unidentified specific mechanisms of transcription regulation among eukaryotes. During my Ph.D., I became fascinated by how tissue-specific gene expression is controlled by specific transcriptional mechanisms in mammals. As these mechanisms remain poorly understood, I decided to gain skills into fundamental transcription mechanisms using yeast and eventually to translate this knowledge back into mammals. My research background consists mainly of work in mice and this postdoctoral fellowship will give me the opportunity to learn how to answer new, but related biological questions, using a different model system.

The hepatitis C virus (HCV) core protein plays an important role in virus-host cell interactions. Importantly, transgenic mice expressing core accumulate intracellular lipids, a condition called steatosis, and develop hepatocellular carcinoma. The group of M. Ott has recently shown that a fraction of core localizes to the mitochondrial outer membrane via a C-terminal targeting motif. The goal of this fellowship is to study the role of mitochondrial core protein in HCV pathogenesis. We will generate core proteins mutant in the mitochondrial localization motif and and study their effect on apoptosis and lipid droplet formation in vitro. We will also introduce these mutations into transgenic mice who conditionally express core and study development of steatosis and hepatocellular carcinoma in vivo. Special focus lies on a potential physical and functional interaction of core with the recently identified sorting protein PACS-2 that integrates ER-mitochondria communication and regulates lipid transfer and apoptosis. My previous research was performed in yeast: I described an altruistic aspect of cell death during aging and characterized new cell death pathways and executors of apoptosis in yeast. This fellowship will allow me to move into the new field of molecular virology. I will acquire new expertise by working with primary mammalian cells, virally infected cells, and transgenic mice. This will importantly extend and complement my current training focussed on yeast cell biology and genetics. In the future, I would like to become an independent researcher who continues to investigate molecular mechanisms of diseases with direct implications for diagnosis and treatment.

The innate immune system uses a wide variety of microbial recognition receptors to identify potential pathogens. The main players in innate immunity are phagocytes such as neutrophils, macrophages, and dendritic cells (DC). All these cell types can express on the cell membrane Dectin-1, a C-type lectin-like receptor that recognizes different b-glucans and can mediate phagocytosis of intact yeasts, which contain b-glucans on their cell walls. In an attempt to explore the signaling pathways triggered by this receptor, the host laboratory has observed the specific involvement of Syk in Dectin-1 mediated signaling for synthesis of IL-10 and IL-2 in DCs, and the association of this kinase to the cytoplasmic tail of Dectin through the phosphorylated membrane proximal tyrosine. Dectin-1 also collaborates with other receptors such as TLR2 present on DCs to generate different responses. The purpose of this project is to determine whether ligand engagement can affect the conformation and/or oligomerization state of Dectin-1 and define the signaling pathways that get activated in response to different Dectin-1 ligands and to co-engagement of Dectin-1 with TLR2. Results from this study will lead to a better understanding of how different innate receptors expressed on DCs participate in the transmission of information to generate an adaptive immune response. The work in innate immune response represents a complete change in the field of work at the intellectual and technical level, but, at the same time, I can take advantage of my background for development of this project. This training will be very important to learn to develop a complete project by myself in the near future.