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RN1-00577-1: Genetic Encoding Novel Amino Acids in Embryonic Stem Cells for Molecular Understanding of Differentiation to Dopamine Neurons
Recommendation: Recommended for funding
Scientific Score: 96
First Year Funds Requested: $528,343
Total Funds Requested: $2,626,937
Public Abstract (provided by applicant)
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.
Statement of Benefit to California (provided by applicant)
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.
Review
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.
In preliminary studies, the applicant has solved key technical problems in adapting the unnatural amino acid technology to mammalian cells. S/he now proposes to validate the method with “proof-of-concept” studies on an orphan nuclear hormone receptor that is tightly linked to the formation of dopaminergic neurons. Loss-of-function mutations in this receptor are associated with Parkinson’s disease (PD) in humans. Lack of receptor expression in mice leads to the loss of midbrain dopaminergic neurons. However, the molecular mechanisms of action of this receptor are an enigma. No ligand has ever been identified for the receptor despite extensive efforts, and it does not recruit known nuclear receptor co-activator proteins. Small molecule inhibitors for the receptor have been discovered, but it is unclear whether these molecules interact directly or indirectly with the receptor. Lastly, receptor function has been shown to be cell-type specific, suggesting the presence of additional, and as yet unidentified, cell-type specific co-regulators. The applicant proposes to utilize the unnatural amino acid technology to address this catalog of mysteries.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: As a graduate student, this applicant published a seminal paper describing a general method for genetically incorporating unnatural amino acids into the genome. 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 address common problems encountered with fusion proteins incorporating bulky reporters such as green fluorescent protein.
The study plan is bold and exceptionally innovative. If successful, one reviewer felt that 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 Principal Investigator (PI) does an excellent job in addressing these issues. One of the clear strengths to this proposal is the PI, who is well-trained with an excellent publication record.
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 method described is irreversible and allows the detection of weak interactions that can involve proteins, nucleic acids, 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 localization 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 an important orphan receptor for these studies. The protein 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 this receptor are one cause of Parkinson’s disease (PD). The information gained from the proposed experiments will help the PI develop an efficient method to differentiate human ESCs to DA neurons – and then to evaluate their effect. This goal is important since there is presently suboptimal characterization of this receptor, 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 the study plan and proposed alternative approaches where possible. Preliminary data inspire confidence that aim one 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 three the applicant will get. There is no way of knowing this until the work from aim two starts to unfold.
This is very much a “methods” grant that will be driven by the PI (although one suspects that the PI’s collaborator will carry out specific aim three). One must raise concerns as to how invested the PI is in the stem cell field. Is this proposal just an opportunity to test this 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. The applicant’s close relationship with outstanding scientists involved in stem cell work make it likely that this 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. After receiving a bachelor degree in chemistry from a top foreign university, the applicant received a Ph.D. in bioorganic chemistry in 2002 at a top institution in California. S/he did postdoctoral studies from 2002-2005, and was a Merck Fellow of the Damon Runyon Cancer Research Foundation. Following a three-year interval of postdoctoral research, the applicant was appointed to the faculty of the current institution.
The candidate has been highly effective in every stage of training. His/her publication record is outstanding both in terms of quantity and in quality: the CV lists over 20 papers published since 2000, many with the applicant as senior author, many in outstanding journals. The applicant was named by the MIT Technology Review TR100 as one of the world’s top young innovators. S/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 independent investigator, the applicant has been awarded a Beckman Young Investigator grant and a Searle Scholar award. The candidate is being mentored by two distinguished investigators involved in investigations of stem cells. Finally, the applicant has written a thoughtful plan for career development. His/her qualifications are simply outstanding.
The candidate has clearly had exceptional training and productivity. S/he is likely to be a leader in research involving protein technology and structure.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The director of the applicant’s institution has written a strong support letter support. Strategic alliances with the biology and chemistry departments at nearby institutions provide the applicant with access to a cadre of top-tier graduate students. The scientific environment and core facilities at the institution are outstanding. The applicant’s lab is 400 sq. feet and the institution has a large amount of shared and core equipment and facilities. 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 letter provides no insights into long term career prospects for the applicant. What will happen to the applicant, if/when s/he is promoted to the rank of Associate Professor at the end of the initial five-year appointment?
This is clearly an exceptional environment for investigators entering into the stem cell field. There are the resources of the institution as well as extensive collaborations with other institutions in the area. 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 s/he had read in a decade. S/he commented that 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 could allow proteins to be labeled fluorescently or to be covalently linked to neighboring proteins for biochemical analysis. The hormone receptor to be studied is a good candidate molecule for testing with this method. It is an orphan receptor that has been linked to Parkinson’s disease. The PI will try to identify receptor-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 important figures in stem cell research in California are reassuring that the research will have an impact on the field.
The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:
- None