Basic Biology II
Human embryonic stem cells (hESCs) have the remarkable capacity of limitless self-renewal. This property is known to be controlled by signaling of a growth factor called Wnt. This proposal investigates the molecular mechanism by which Wnt causes self-renewal. Current conversational wisdom is that Wnt only prolongs the half-life of a protein called β-Catenin. Here we propose the hypothesis that Wnt regulates the stability of a multitude of proteins, all of them characterized by receiving phosphates from an enzyme called Glycogen Synthase Kinase 3 (GSK3). In this view, Wnt would be a general metabolic signal that instructs cells to slow down protein degradation by inhibiting GSK3 activity. How is GSK3 inhibition achieved? This is a key unanswered question in the Wnt signaling field. We propose a new cellular mechanism by which the GSK3 enzyme is sequestered inside intracellular vesicular organelles (called multivesicular bodies) after Wnt signaling. If this GSK3 sequestration hypothesis can be proven, it would constitute an important contribution to stem cell research. Human embryonic stem cells are essential for these investigations because they naturally have very high levels of Wnt signaling. In addition, they have asymmetric mitotic divisions. We will develop methods to investigate why some hESCs differentiate, losing their astonishing self-renewal potential. The experiments proposed will help understand how Wnt signaling maintains the pluripotent state in hESCs. By investigating the hypothesis that Wnt signaling functions as a general regulator of protein stability, we hope to significantly advance the field of human embryonic stem cell research and regenerative medicine.
Statement of Benefit to California:
The State of California benefits from the proposed basic research by strengthening its leadership role in worldwide stem cell research. This project would provide employment for five full-time researchers, who will obtain training in cutting-edge molecular and cell biology research. It has been our experience that trainees leave our laboratory always for better high-tech jobs. Many go into the biotech industry and others into teaching at the college level. Funding this project will benefit California by producing an improved and highly trained workforce. It will also strengthen our already excellent university research system.
EXECUTIVE SUMMARY This proposal focuses on understanding the mechanism by which the Wnt signaling pathway regulates human embryonic stem cell (hESC) pluripotency and self-renewal. The central hypothesis is that Wnt regulates the stability of a number of proteins by inhibiting their phosphorylation by glycogen synthase kinase 3 (GSK3). The applicant proposes a novel cellular mechanism for this inhibition by which Wnt signaling causes GSK3 to be sequestered in intracellular compartments. This sequestration would prevent target protein phosphorylation and subsequent degradation. In Aim 1, the applicant proposes to identify proteins whose stability is regulated by Wnt signaling and investigate potential asymmetric allocation of GSK3-phosphorylated proteins during hESC division. Aim 2 focuses on a specific protein identified in the preliminary studies that has GSK3 phosphorylation sites and may be regulated by Wnt signaling. The applicant proposes to study the regulation of this protein in a model organism and in hESCs. Finally, in Aim 3, the applicant proposes to test the hypothesis that Wnt signaling induces GSK3 sequestration and investigate potential underlying mechanisms. Reviewers found the proposal to be quite innovative, as it is based on a novel hypothesis that Wnt can cause GSK3 to be sequestered away from its substrates. However, they were not convinced of the significance of the proposal for hESC biology. Reviewers agreed that there was little justification for the use of hESCs and that most of the experiments could just as easily be performed in somatic, transformed, or non-human cells. While the proposed research is quite significant for cell biology in general, reviewers did not feel it was likely to have a major impact on the fields of stem cell biology or regenerative medicine. The reviewers found the research plan to be well designed and supported by sound preliminary data. However, they noted that a significant number of experiments involve the model organism rather than hESCs. Reviewers also raised a few concerns about the experimental design. They cautioned that certain methods proposed in hESCs, including uniform expression of epitope-tagged proteins via lentiviral transduction and synchronization of hESC cultures may be technically difficult. Demonstration of facility with these methods in the preliminary data would have been helpful. Reviewers were also concerned that potential pitfalls and alternative strategies are not discussed in the research plan. Reviewers described the principal investigator (PI) as a leader in the field of signal transduction biology with an excellent publication record. They noted that the research team appears adequate to advance the project but no CVs are presented other than the PI’s. Reviewers commented that the PI has only recently started working with hESCs and might benefit from collaboration with an investigator with expertise in this area. Overall, while reviewers appreciated the novelty of this proposal’s central hypothesis and supporting preliminary data, they were not convinced that there is a strong rationale for performing the experiments in hESCs over other model systems. PROGRAMMATIC REVIEW A motion was made to move this application into Tier 1, Recommended for Funding. Reviewers summarized their reviews and discussed whether there is a strong case to conduct the proposed work in hESCs. While reviewers appreciated the scientific questions posed in the application, they agreed that the work would be better suited for simpler model systems. The motion failed.
- Ali Brivanlou