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In the human body, all biological components—from individual genes to entire organs—work together to promote normal development and sustain health. This amazing feat of biological teamwork is made possible by an array of intricate and interconnected pathways that facilitate communication among genes, molecules, and cells. Limitations of proteomics technologies often force investigators to treat dynamic systems as either static or as binary options between static states. As with early photography, current approaches to proteomics involve long exposures that capture broadly defined “images” such as “normal vs. diseased”, “the yeast interactome”, or “the nuclear pore complex.” We are largely blind to the dynamics of systems we know are not static but which must be treated as such for the time being because of inadequate tools. Transient interactions, rapid changes in protein activity or location, and post-translational modifications control critical regulatory steps in biology, yet they are like a bird flying through the frame of a carefully composed long exposure: invisible. New strategies complementary to conventional proteomics are necessary to help us determine the rapid, dynamic changes that control physiology. The National Technology Centers for Networks and Pathways (TCNPs) create technologies to measure the dynamics of protein interactions, modifications, translocation, expression, and activity, and to do so with temporal, spatial, and quantitative resolution. The program is intended to bridge the quantitation and interaction aspects of proteomics, breaking out of the artificially static view of complex systems (Sheeley, Breen, and Old, J. Proteome Research (2005) 4,1114). The TCNP program was envisioned specifically to complement conventional proteomics programs at NIH, integrating proteomics with cell biology and biophysics, with an enhanced emphasis on novel approaches not substantially supported in other programs. Five independent centers cooperate in a networked national effort to develop instrumentation, biophysical methods, reagents, and infrastructure for temporal and spatial characterization of complex biochemical pathways and networks of protein interactions. They collaborate with biomedical researchers through several mechanisms, providing a synergistic push-pull between technological advancement and biomedical problem solving. The centers are also tasked with providing broad access to the technologies, methods, and reagents they develop, as well as providing appropriate interdisciplinary academic and peer training for biomedical researchers. The scope of the dynamics problem is very broad. Each center integrates multiple approaches to create a coherent biological, analytical, and informatics strategy, but each focuses on different technologies and systems, with corresponding strengths. Cooperation among the centers allows them to broaden their scope and complement one another. Recompetition of the TCNP ProgramIn the years since the TCNP program was conceived, the need for it has become even clearer. The broader community is beginning to appreciate the value of dynamic approaches. Recognition of the value of research like that supported by the TCNP program shows that new groups will be able to contribute their innovative ideas to the program in the next phase of competition. There is still a significant unmet need for technologies that will enable researchers to use proteomics approaches to identify and validate changes quantitatively across complex, highly dynamic pathways involving transiently interacting proteins. The goal of Phase II is to accelerate the progress of proteomics from static maps to quantitative functional models by the creation, broad application, and dissemination of instrumentation, biophysical methods, reagents, and infrastructure specifically focused on the problem of quantitative temporal and spatial characterization of protein networks and pathways. New approaches to the characterization of these transient interactions will be the focus of the RFA for the second phase of the program. As the technologies developed in the program mature and become sufficiently robust for routine use by non-experts, we anticipate that the impact of TCNP program in the broader biomedical research community will continue to expand substantially. Currently Funded Centers
Implementation Group: Building Blocks, Biological Pathways and Networks Burnham InstituteCenter on Proteolytic Pathways Principal Investigator NIH Science Officer: Salvatore Sechi, PhD (NIDDK) Abstract: Proteolysis is of paramount importance to biological regulation, and its significance is magnified because it is irreversible. Proteolysis regulates the four fundamental aspects of cell behavior: division, death, differentiation and motility. Proteolytic pathways are key to the pathology of virtually every type of human disease including, infection, inflammation, thrombosis, cancer, emphysema, Alzheimer's, etc. The importance and impact of understanding proteolysis "in total" will be immense. Achieving this ultimate goal will be a considerable challenge, and one that will require an ambitious and coordinated effort. Here we propose to form the Center on Proteolytic Pathways (CPP), a National Resource for the study of proteases, their inhibitors, their products, and upstream and downstream regulatory pathways. The strategic goal of the CPP is to develop The Protease Pathway Interrogation Platform (PPIP) technology. This platform technology will be comprised of four foundation technologies: 1.) Activity-based Protease Profiling; 2.) Protease Activity Imaging Technology; 3.) Product Terminal Isotope Coding; and 4.) the in silico environment called Protease Map. A key and unique feature of this technology platform is its focus squarely on measuring activity, as opposed to just abundance. The CPP will contain a Training component to ensure that a new cadre of scientists is trained to tackle protease biology with the most modem of techniques and most cutting-edge technology. An Outreach component is planned to ensure that the CPP achieves high visibility, and ultimately numerous connections with the scientific research community. Carnegie-Mellon University/University of PittsburghFluorescent Probes and Imaging for Networks and Pathways Principal Investigator NIH Science Officer: Laurie Nadler, PhD (NIMH) Abstract: Mellon Pitts Corporation proposes to form a nationally visible and responsive center focused on fluorescent probe and imaging technologies for investigating pathways and networks in real time and at high resolution in living cells. The proposed Center will be formed by combining the experience and infrastructure of two already existing Centers in Pittsburgh. The research component of the Center will create a powerful toolbox of intracellular fluorescent labels and biosensors that can be used to study many, if not all, the proteins in pathways and networks of living cells. The strong fluorescent probe development program blends genetics, protein structure, nucleic acid structure and fluorescent dye chemistry. The probes we propose to generate will be genetically expressible so that exogenous macromolecules will not have to be transported into the living cells to be studied. Four “Driving Biology Projects” are to be part of the overall Center program in addition to the Technology Development Core. These projects are located at the University of Pittsburgh, Carnegie Mellon, Stanford, and Berkeley. Their responsibility is to assist the new center in identifying the optimal technologies for development and for providing crucial feedback regarding the utility of the technologies. Imaging and informatics technologies are integrated with the new fluorescent probe technologies so that these combined tools will help cell biologists obtain large amounts of spatial and temporal information about pathways in living cells. Included in the proposal are programs for making the fluorescent probe and imaging technologies robust so that they can be provided to a wide range of cell biologists in academic research, pharmaceutical drug discovery, and biotechnology and to disseminate these technologies and provide training. The Johns Hopkins UniversityNetworks and Pathways of Lysine Modification Principal Investigator NIH Science Officer: Adam Felsenfeld, PhD (NHGRI) Abstract: Protein modification on histone lysines is critical for controlling gene expression, which itself controls the incredibly variable and plastic expression of the proteome in diverse cell types. Modifications on lysine are chemically diverse and include acetylation, methylation, ubiquitylation, and sumoylation. Increasingly, acetyl- and methyl-lysines are being discovered in a host of other proteins, only some of which directly control gene expression. Ubiquitylation controls the life and death of most proteins, as well as many other diverse protein functions. Remarkably, the pathways regulating the interplay between these diverse modifications on lysines have been very little studied. The network of these modification pathways is very poorly understood indeed; many lysine-modifying proteins are encoded by multi-gene families, with redundant activities, and have multiple substrates. Thus genetic and computational approaches for decrypting such redundancy are needed to dissect the complex networks defined by these signaling pathways. This proposal combines such techniques with a new and innovative proteomics technology, protein microarrays, and a new affinity ligand technology for identifying novel acetyltransferases. These newer approaches will be complemented in this Technology Center for Networks and Pathways by a heavy emphasis on the development and application of innovative mass spectrometry technologies, including sensitive technologies for quantifying dynamics of lysine modification in cells. Dissecting how lysine modifications change in response to biological stimuli is in its infancy largely because sensitive and robust experimental techniques for quantifying protein modification are needed and will be developed here. A unique instrumentation system aimed at profiling lysine modification in single cells will be developed, which will open a whole new world of single-cell profiling to examine the epigenetic changes that occur in individual cells as they develop, as they age, or as cancer progresses. Diverse Driving Biological Projects centered on lysine acetylation, methylation, and ubiquitylation, as well as Training and Technology Dissemination efforts, are extensively integrated with the Technology Development components of the proposal. Because lysine modification is intertwined with human health, aging, and disease at many levels, these technologies could have far-reaching effects. Rockefeller UniversityNew Tools for Exploring the Dynamic Interactome Principal Investigator NIH Science Officer: Christine Colvis, PhD (NIDA) Abstract: The genomic revolution has been empowered by technologies that have determined a vast pool of genetic information. While nucleic acids encode this information, it is the proteins that act on it. Proteins are incredibly diverse in their abundance and their properties, making them highly versatile for the dynamic tasks at hand but at the same time exceptionally difficult to analyze. It is for these reasons that the proteomic revolution still lags behind the genomic revolution. Indeed, the comprehensive analysis of the dynamic properties of proteins in cells is still largely beyond current capabilities. Here, we seek to revolutionize proteomics by synergistically combining improvements in established techniques with new approaches. We will overcome major bottlenecks in 3 key areas of proteomics technology. First, we will reform the production stage for generating intact macromolecular complexes, so that we will be able to freeze a tagged macromolecular complex in place, within moments of visualizing its position in the cell, and then isolate it together with all its components and neighbors. Second, we will optimize the analysis of each complex such that its macromolecular composition, structure, and dynamics will be quantified and analyzed. Third, we will develop software to integrate our data and represent in unprecedented detail the actions of the macromolecular players in many dynamic subcellular assemblies. We will seek to make these techniques rapid, robust, and routine by beta testing them in 4 experimental systems. These systems focus on aspects of the genetic information pathway, because (i) this is core to eukaryotes, and (ii) it will allow us to develop techniques to analyze the interactions of all 3 information-carrying biological macromolecules (DMA, RNA, and proteins). First, we will walk along great stretches of chromatin, determining the normal flux of structural proteins and regulatory factors that together comprise dynamic segments of the genome. Second, we will follow the course of RNA after transcription, as it is processed, packaged, and exported from the nucleus; we will enumerate the proteins that dance attendance on each kind of RNA molecule during its maturation. Finally, we will expose how two pathogenic human viruses (HIV and CMV) subvert their host's genetic information pathway and supplant it with their own. By creating a National Center for Dynamic Interactome Research, we will be coupling an established mass spectrometry resource, cell biology laboratories, a systems biology resource, and a computational biology center. As part of the larger NIH roadmap, the Center's aim will be to create new and useful tools to elucidate the dynamics of macromolecular interactions. In summary, the present proposal seeks the support to advance our methods into totally new areas, and to spread these methods amongst the biomedical community. The Center will enable the community to assemble the kinds of detailed, dynamic representations of the interactions in the cell that will help elucidate the principles underlying all cellular processes, thus bridging the gaps between functional genomics, proteomics, and systems biology. University of Connecticut School of Medicine and DentistryPolarity in Networks and Pathways Principal Investigator NIH Science Officer: Joseph Breen, PhD (NIAID) Abstract: Existing proteomics technologies can provide a wealth of information about the biochemistry operating in cells. And systems biology tools are being developed to analyze and model these data. However, they fail to address the fundamental questions of how the spatial organization of molecules in cells is established and how it is utilized to control cell function. To answer these questions, we will need new tools and new theoretical frameworks that specifically include consideration of cell morphology and dynamic spatial molecular distributions. This proposal aims to establish a Technology Center for Networks and Pathways (TCNP) that will integrate microscope technologies for making quantitative in vivo live cell measurements with new physical formulations and computational tools that will produce spatially realistic quantitative models of intracellular dynamics. The model predictions will then be validated with new measurements as well as novel intracellular manipulation technologies also to be developed in our proposed TCNP. These new technologies will be developed and disseminated by the Center for Cell Analysis and Modeling (CCAM) at the University of Connecticut Health Center (UCHC). The technology will be disseminated throughout the research community via training programs, Web-based instructional material, a repository of molecular probes, and a database of data and models. The proposed work builds on a firm foundation. CCAM is the home of the Virtual Cell, a computational environment for cell biological modeling, and also hosts a variety of projects in biophotonics and live cell microscopic imaging methods as well as a state-of-the-art user facility for nonlinear, confocal, and widefield microscopy. CCAM is the scientific home of an extraordinary confluence of expertise in physics, chemistry, software engineering, and experimental cell biology that is unique for a medical school and is ideal for the concerted multi-pronged effort that is planned for the TCNP. |
This page last reviewed: March 19, 2008
Division of Program Coordination, Planning, and Strategic Initiatives • National Institutes of Health • Bethesda, Maryland 20892
