Embryonic stem cells have the capacity to self-renew and differentiate into other cell types. Understanding how this is regulated on the molecular level would enable us to manipulate the process and guide stem cells to generate specific types of cells for safe transplantation. However, complex networks of intracellular cofactors and external signals from the environment all affect the fate of stem cells. Dissecting these molecular interactions in stem cells is a very challenging task and calls for innovative new strategies. We propose to genetically incorporate novel amino acids into proteins directly in stem cells. Through these amino acids we will be able to introduce new chemical or physical properties selectively into target proteins for precise biological study in stem cells.
Nurr1 is a nuclear hormone receptor that has been associated with Parkinson’s disease (PD), which occurs when dopamine (DA) neurons begin to malfunction and die. Overexpression of Nurr1 and other proteins can induce the differentiation of neural stem cells and embryonic stem cells to dopamine (DA) neurons. However, these DA neurons did not survive well in a PD mouse model after transplantation. In addition, it is unclear how Nurr1 regulates the differentiation process and what other cofactors are involved. We propose to genetically introduce a novel amino acid that carries a photocrosslinking group into Nurr1 in stem cells. Upon illumination, molecules interacting with Nurr1 will be permanently linked for identification by mass spectrometry. Using this approach, we aim to identify unknown cofactors that regulate Nurr1 function or are controlled by Nurr1, and to map sites on Nurr1 that can bind agonists. The function of identified cofactors in DA neuron specification and maturation will be tested in mouse and human embryonic stem cells. These cofactors will be varied in combination to search for more efficient ways to induce embryonic stem cells to generate a pure population of DA neurons. The generated DA neurons will be evaluated in a mouse model of PD. Additionally, the identification of the agonist binding site on Nurr1 will facilitate future design and optimization of potent drugs.
Parkinson’s disease (PD) is the second most common human neurodegenerative disorder, and primarily results from the selective and progressive degeneration of ventral midbrain dopamine (DA) neurons. Cell transplantation of DA neurons differentiated from neural stem cells or embryonic stem cells raised great hope for an improved treatment for PD patients. However, DA neurons derived using current protocols do not survive well in mouse PD models, and the details of DA neuron development from stem cells are unclear. Our proposed research will identify unknown cofactors that regulate the differentiation of embryonic stem cells to DA neurons, and determine how agonists activate Nurr1, an essential nuclear hormone receptor for DA neuron specification and maturation. This study may yield new drug targets and inspire novel preventive or therapeutic strategies for PD. These discoveries may be exploited by California’s biotech industry and benefit Californians economically. In addition, we will search for more efficient methods to differentiate human embryonic stem cells into DA neurons, and evaluate their therapeutic effects in PD mouse models. Therefore, the proposed research will also directly benefit California residents suffering from PD.
SYNOPSIS: Complex networks of proteins integrate cellular factors with extrinsic signals from stem cell niches to regulate the pluripotency and self-renewal of stem cells. In this application, the applicant proposes to genetically encode unnatural amino acids (UAAs) into proteins in order to conduct molecular studies of stem cells. The UAAs can possess unique side chains that provide a powerful method for studying the physical, chemical and biological properties of proteins. To genetically encode a desired UAA in stem cells, the PI will generate a new tRNA/synthetase pair that is specific for the UAA, decodes an amber stop codon (TAG), and is orthogonal to endogenous tRNA/synthetase pairs. The codon for the UAA-incorporation site will be mutated to TAG in the target gene.
In preliminary studies, the applicant has solved key technical problems in adapting the unnatural amino acid technology to mammalian cells. He now proposes to validate the method with “proof-of-concept” studies on Nurr1. The Nurr1 protein is an orphan nuclear receptor that is tightly linked to the formation of dopaminergic neurons. Loss of function mutations in Nurr1 are associated with Parkinson's disease in humans. Lack of Nurr1 expression in mice to the loss of federal midbrain dopaminergic neurons. However in the molecular mechanisms of Nurr1action are an enigma. No ligand that it has ever been identified for Nurr1 despite extensive efforts. Nurr1 does not recruit known nuclear receptor co activator proteins. Small molecule inhibitors for Nurr1 have been discovered, but it is unclear whether these molecules interact directly or indirectly with Nurr1. Lastly, Nurr1function has been shown to be cell type specific, suggesting the presence of additional, and as yet unidentified, cell type specific co-regulators. To address this catalog of Nurr1mysteries, the applicant proposes to encode cross-linking, photoactivated unnatural amino acids into strategic positions of the Nurr1protein. There are three specific aims:
Aim 1 is to install an unnatural tRNA, and tRNA synthetase into murine neural stem cells and embryo stem cells. The tRNA/synthetase will be configured to install a photo activated, cross-linking unnatural amino acid. The applicant chooses to start with these murine cell types, because both can be differentiated to dopaminergic neurons using established procedures.
Aim 2 is to identify proteins (and perhaps small molecules) that interact with Nurr1 during the act of differentiation. Towards this end, the cross-linking unnatural amino acid will be incorporated at different positions within the protein. At timed intervals during differentiation the cross-linker will be activated to trap co-regulator proteins and possible small molecule ligands. The trapped proteins/ligands will be identified by mass spectroscopy and other sophisticated physical methods.
The 3rd specific aim is to validate the physiological relevance of new proteins that interact with Nurr1 during dopaminergic formation. Gain of function and loss of function experiments with the genes that encode candidate proteins will be employed.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: In the year 2001, while he was still a graduate student at UC Berkeley, this applicant published a seminal paper in Science entitled "Expanding the Genetic Code of Escherichia coli". The paper described a general method for genetically incorporating unnatural amino acids by embedding a unique codon within the open reading frame of a protein and installing a corresponding tRNA and tRNA synthetase into the geneome. This technology has the potential to change the way live cell imaging and proteomic research is conducted if it can be adapted to mammalian cells. For example, unnatural amino acids with photoactivatable crosslinkers could be used to trap partner proteins, coregulators or small molecule ligands for proteins of interest. For live cell imaging, site-specific incorporation of a single fluorescent amino acid could common problems encountered with fusion proteins incorporating bulky reporters such as GFP.
The study plan is bold and exceptionally innovative. If successful, this work will amount to a technological paradigm shift. The experiments planned are logical and well-described in a very thoughtful way. It remains a risky proposal, although the PI does an excellent job in addressing these issues. One of the clear strengths to this proposal is the PI. He is well-trained with an excellent publication record. Dr. Wang clearly has exceptional training and productivity; he is likely to be a leader in research involving protein technology and structure.
There are a number of attractive features of the PI's approach. As noted by the PI, this method is valuable in investigating molecular interactions because alternative methods such as pulldown or co-immunoprecipitation methods cannot distinguish direct from indirect interactions, and because they may fail to detect weak or transient interactions. Yeast 2-hybrid systems are limited in only detecting protein interactions and provide limited information regarding interacting interfaces. In contrast, the photocrosslinking metho of Dr. Wang is irreversible and allows the detection of weak interactions that can involve proteins, nucleic acid or small molecules. In addition, a variety of modifications, such as fluorescent labeling, posttranslational modification, or photocaging, can be made to proteins at specific sites, allowing for biophysical and molecular studies that are not possible with other methods. For example, the site-specific incorporation of a fluorescent UAA may allow tracking of protein locaization and movement; in contrast, the bulkiness of conventional fluorescent tags on proteins may interfere with the protein's function or binding. Incorporation of fluorescent UAAs that are responsive to the environment, such as pH, may allow one to monitor phosphorylation, acetylation, etc. in the cell. This strategy has never been reported in embryonic stem cells and carries significant promise to the entire field.
In addition, the PI has targeted the dopamine system and Nurr1 for his studies. Nurr1 is of interest since it is an orphan nuclear receptor that is highly expressed in postmitotic dopaminergic neurons (DA) and their precursors. A variety of studies have demonstrated that this protein has a critical function in developing dopaminergic neurons. In addition, mutations in Nurr1 are one cause of Parkinson's disease (PD). The information gained from the proposed experiments will help the PI develop an efficient method in order to differentiate human ESCs to DA neurons - and then to evaluate their effect. This goal is important since there is presently suboptimal characterization of Nurr1, and suboptimal ways to differentiate stem cells into DA neurons and allow the continuing growth of these differentiated cells following transplantation. The long-term goal of this study is to develop methods to incorporate UAA into embryonic stem cells (ESCs) in order to allow in vivo investigations of molecular events involving these cells.
The applicant has carefully considered potential pitfalls in his study plan and proposed alternative approaches where possible. As a positive control, he will use the known heterodimerization of Nurr1 with the RXR protein as a model system. One strategic weakness is that the aims 1 and 2 are sequentially linked (aim 2 cannot go forward unless aim 1 is successfu). However that is the nature of high risk/high gain research. Preliminary data inspire confidence that aim one (installation of the tRNA/tRNA synthetase pair) will be successful and that much will be learned from aim two. Time frame is another minor concern. Reviewers were not certain how far into aim 3 (target validation) the applicant will get. There is no way of knowing this until the work from aim 2 starts to unfold.
In specific aim 1, the PI will use methods that are presented in the Preliminary Data and published to genetically introduce UAA containing different photcrosslinking groups into murine NSCs and ESCs. The experiments are logical and well-designed. There are a number of potential problems with the planned experiments that the PI discusses. For example, the UAA may be incorporated into legitimate amber stop codons in other proteins besides the TAG codon that the PI engineers. In addition, inefficiency in incorporation of the UAA will generate truncated proteins that have a dominant negative effect in the planned experiments. Furthermore, the Bpa may interfere with binding because of its bulkiness. This reviewer felt that the PI addressed these issues with appropriate care, although these studies remain risky.
In specific aim 2, the PI, in collaboration with the Evans lab, will carry out transcription profiling of differentiating murine ESCs at selected time points for expression of nuclear receptors, including Nurr1. The results of these experiments (some of which are presented in the Preliminary Data) will guide the timing of expression of UAA-containing Nurr1 and its photocrosslinking. Photocrosslinker UAAs will then be placed at or near various domains of Nurr1 predicted to be involved in interactions of the protein. Following identification of co-regulators, photocrosslinking UAAs can be incorporated into the co-regulators to identify other interacting molecules. Although the placement of the UAAs in Nurr1 is unavoidably a bit like a fishing expedition, the PI is clearly knowledgeable about this protein and appropriately prioritizes the sequence of experiments. A concern that needs to be addressed in this specific aim is whether the incorporation of the UAAs and tRNA/Aminoacyl-tRNA synthetase will interfere with stem cell growth, phenotype, differentiation, etc.
The third aim is highly ambitious but very logical. The applicant assumes that he will identify several Nurr-1 interacting partners, and plans to study their role in DA neuron development by gain and loss of function strategies. The differentiated mouse and human DA neurons will be tested for efficacy in a PD mouse model in collaboration with the Evans lab. Beyond this point, the proposal gets a bit thin on details. The PI hopes to identify more efficient derivation protocols and perhaps improved in vivo survival in animal models of Parkinson's disease. He does not elabborate on these experiments, which are likely to be beyond the 5 year span of this proposal anyway.
If these approaches are successful, the PI plans to investigate interactions with other proteins important in stem cell biology. He outlines some long-term goals involving the application of fluorescent UAAs in imaging studies and the engineering site-specific UAAs that mimic phosphorylation, methylation, etc. to explore the role of posttranslational modification and epigenetics in the function of the protein.
This is very much a "methods" grant that will be driven by Dr. Wang (although one suspects that it will be the Evans lab that really carries out specific aim 3). One must raise concerns as to how invested Dr. Wang is in the stem cell field. Is this proposal just an opportunity to test his method? Despite these concerns, the proposal is likely to generate new powerful protein tools that can be applied to stem cell research, and provide support for a young innovative investigator who is likely to make important contributions to the field. Dr. Wang's position at the Salk and his close relationship with outstanding scientists involved in stem cell work make it likely that Dr. Wang's work will have an important impact on stem cell research.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The applicant has had a brilliant career so far. He received his bachelor degree in chemistry from Beijing University - widely considered to be the top university in all of China. He trained with Peter Schultz at UC Berkeley and received his Ph.D. in bioorganic chemistry in 2002. He did postdoctoral studies under Dr. Roger Tsien at UCSD from 2002-2005, where he was a Merck Fellow of the Damon Runyon Cancer Research Foundation. Following his three-year interval of postdoctoral research, the applicant was appointed to the faculty of the Salk Institute. Dr. Wang has been highly effective in every stage of training. His publication record is outstanding both in terms of quantity and in quality: Dr. Wang lists over 20 papers published since 2000, many with him as senior author, many in outstanding journals. He was named by the MIT Technology Review TR100 as one of the world’s top young innovators. He was awarded the Amersham Biosciences and Science Grand Prize for Young Scientists by AAAS and the Grand Prize of Collegiate Inventors Competition by National Inventors Hall of Fame. As an investigator at the Salk Institute, he has been awarded a Beckman Young Investigator grant and a Searle Scholar award. He is being mentored by Ron Evans and Fred Gage, who are both distinguished investigators involved in investigations of stem cells. Finally, he has written a thoughtful plan for career development. His qualifications are simply outstanding.
Dr. Wang clearly has exceptional training and productivity. He is likely to be a leader in research involving protein technology and structure. His position at the Salk and his close relationship with outstanding scientists involved in stem cell work make it likely that Dr. Wang's work will have an important impact on stem cell research.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The director of the Center for Chemical Biology and Proteomics at The Salk Institute has written a strong letter support for Dr. Wang. Strategic alliances with the biology and chemistry departments at the University of California San Diego provide Dr. Wang with access to a cadre of top-tier graduate students. The scientific environment and core facilities at the Salk Institute are outstanding. The Wang lab is 400 sq. feet and part of the Skirball Center for Chemical Biology and Proteomics at the Salk Institute. There is a large amount of shared and Core equipment and facilities and there are plans to build a CIRM-funded stem cell core laboratory. The only concern from the perspective of career development is that the Director's ladder provides no insights into long term career prospects for Dr Wang at the Salk Institute. What will happen to Dr. Wang, if/when he is promoted to the rank of associate Professor at the end of his initial five-year appointment? Is there true academic tenure at the end of the tunnel? Rolling tenure as per EMBO labs and Cold Spring Harbor? How does it work?
This is clearly an exceptional environment for investigators entering into the stem cell field. There are the resources at Salk as well as extensive collaborations with other institutions in San Diego. There are plans to recruit individuals in the stem cell field, including two new faculty members and several research assistants in the area of reprogramming and epigenetics. The applicant is supported by senior and reputable scientists in the stem cell field.
DISCUSSION: One reviewer described this as the most exciting grant that he had read in a decade. This as a creative new approach to studying protein interactions that could potentially change the way science is done, as did the cre/lox technology earlier. The technology of introducing unnatural amino acids into proteins will allow proteins to be labelled fluorescently or to be covalently linked to neighboring proteins for biochemical analysis. The protein used in this study, Nurr 1, is a good candidate molecule for testing with this method. It is an orphan receptor that has links to Parkinson's Disease. The PI will try to identify Nurr1-interacting proteins.
The PI was described as outstanding, the research environment and institutional support as excellent. The only reservations expressed about this proposal were that this is a methods grant rather than a stem cell grant, and there was some concern that PI might not be committed to stem cell research. However, collaborations with Rusty Gage and Evans are reassuring that the research will have an impact on the field.