Jie J Zheng, PhD - US grants

The objective of the proposed studies is to use structural and biophysical methods to investigate the molecular mechanisms underlying the specific regulatory and targeting interactions of the Wnt signaling pathway. Wnt signaling plays an important role in embryonic development and in the regulation of cell growth. Inappropriate activation of Wnt signaling has been implicated in cancers and other human diseases. Dishevelled is an important player in the Wnt pathway. It relays the signal from the membrane-bound Wnt receptors to downstream partners. Sequence analysis revealed two new novel protein domains in Dishevelled: the DEP and DIX domains. It is believed that the DEP domain binds to the membrane-bound receptor and that the DIX domain interacts with downstream components. Protein NMR spectroscopy will be used to determine the structures of the two domains and the fundamental chemical nature of the interactions between both domains and their binding partners will be analyzed by structural and biophysical methods. Axin, one of the proteins downstream of Dishevelled, also contains a DIX domain. It has been hypothesized that the Wnt signal passes from Dishevelled to Axin through heterodimerization of the two DIX domains. This hypothesis will be examined by further analysis of the structures of the Axin DIX domain and the DIX heterodimer complex. The applicant suggests that these studies will reveal the first structural and functional information about DEP and DIX domains and, ultimately, may provide insights for the development of pharmaceutical agents that can interfere with specific Wnt signaling events in human disease.

DESCRIPTION (provided by applicant): By linking extracellular matrix (ECM) proteins to the actin cytoskeleton, integrin proteins regulate cell proliferation, migration, and survival. A key player in integrin signaling is focal adhesion kinase (FAK). Activation and localization of FAK to focal adhesions results from the binding of integrins to ECM proteins. Activated FAK relays signal from integrins to downstream components of different pathways. However, inappropriate activation of FAK and inappropriate FAK-regulated signaling has been implicated in cancer and other human diseases. Therefore, the long-term objective is to investigate structures and molecular mechanisms underlying regulatory and targeting interactions of FAK-regulated signaling pathways. This objective will be achieved through the use of biophysical methods. Studies showed that FAK's C-terminal focal adhesion targeting (FAT) domain is necessary and sufficient for the targeting of FAK to focal adhesions, a crucial step in FAK signaling. On the basis of preliminary findings described in this application, it is hypothesized that the FAT domain regulates FAK activation by interacting with FAK's N-terminal FERM domain. Previously, the principal investigator determined the solution structure of the FAT domain by NMR spectroscopy. In proposed studies, this method will be used to determine the chemical nature of interactions between the FAT domain and its binding partners: paxillin, talin, and FAK's FERM domain. Furthermore, the complex molecular interplay among FAK, paxillin, and talin will be evaluated. These studies will provide structural and functional information at the atomic level about the FAT domain and its associated proteins. Also, ligands designed to serve as FAT domain inhibitors, will be tested to determine whether they provide a lead in developing therapies that interfere specifically with abnormal FAK-mediated signaling that contributes to cancer.

[unreadable] DESCRIPTION (provided by applicant): The objective of the proposed studies is to investigate the molecular mechanisms underlying the specific regulatory and targeting interactions of the Wnt signaling pathways by using structural and biophysical methods. Wnt signaling plays an important role in embryonic development and in the regulation of cell growth. Inappropriate activation of Wnt signaling has been implicated in cancers and other human diseases. Dishevelled (Dvl), an important component of the Wnt signaling pathways, relays signals from the membrane-bound Wnt receptor Frizzled (Fz) to downstream partners. Different Wnt signaling pathways diverge at the level of Dvl. However, the mechanism by which Dvl transduces the Wnt signals is unclear, as are the molecular events that regulate pathway specification at the level of Dvl. The proposed structural and biophysical analyses will address these questions by elucidating the fundamental chemical nature of the interactions between Dvl and its binding partners. Dvl is hypothesized to undergo conformational changes upon receiving Wnt signals. Protein NMR spectroscopy will be used to investigate these conformational changes and the mechanism of their regulation by molecular interactions of Dvl and its binding molecules. The Specific Aims are to 1) Determine the binding specificities between the PDZ domains of Dvl proteins and the C-termini of Fz receptors by protein NMR spectroscopy and other physical biochemistry methods; 2) Identify components of the regulatory network that mediates the function of the Dvl proteins; and 3) Ascertain whether Dvl undergoes a conformational change in the transduction of Wnt signaling. The knowledge gained from these investigations will not only help to explain the fundamental chemical nature of the complex molecular interplay at the level of Dvl in Wnt signaling but will also shed light on the maintenance of intracellular signal specificity and the nature of global signaling control. These findings may also be useful in formulating rational approaches to the development of novel pharmaceutical agents that can interfere with specific Wnt signaling events that contribute to human diseases. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cell migration is promoted by the alternating formation and disassembly of focal adhesions (FAs); our long-term goal is to understand the mechanisms that govern these dynamic events. In FA formation, multiple adhesion molecules are concentrated in a small area in which complex protein interactions are highly specific, although some of the binding affinities are relatively weak. Because of the relatively high protein concentration at the FA locus, binding affinity plays a critical role, and phosphorylation-mediated changes in binding affinity can completely alter the balance of interactions within the complex network. Therefore, to elucidate the mechanism that regulates the formation and disassembly of focal adhesions, we must understand in detail the structural and biophysical interactions within FAs and their binding partners and determine how phosphorylation affects these interactions; this is the focus of the project. Specifically, we will study paxillin family proteins and their biding partners in FAs. Paxillin and its related proteins are key players in FAs; as scaffold proteins, they use their N-terminal LD motifs to interact with other FA molecules. One major group of LD binding partners comprises the focal adhesion targeting (FAT) domain of FAK and the FAT-like domains of other FA proteins. Although the FAT-like domains have similar structures, their interactions with the LD motifs of paxillin family proteins differ in many ways. These differences offer an opportune starting point to dissect the dynamics of the complex interactions in FA formation and disassembly. This study will also aid in the design of inhibitors that target specifi binding events in FAs in order to mediate FA dynamics.

Abstract Limbal stem cell deficiency (LSCD) is a major cause, either primary or secondary, of significant visual loss and blindness in many common corneal disorders. Transplantation of autologous limbal stem cells (LSCs) expanded in tissue culture has successfully restored vision and revolutionized the treatment of LSCD. A higher expansion efficiency of the stem/progenitor cell population in culture corresponds to a greater probability of long-term graft survival. The long-term goal of our study is to develop small molecules that can govern LSC self-renewal and differentiation and be used as therapeutic reagents for stem cell-based treatments of LSCD? related disorders. The most efficient expansion method requires feeder cells that provide a proper microenvironment to support the growth of LSCs. Among the external signaling that the feeder cells provide to the LSCs is the Wnt signaling. The central hypothesis of this project is that small molecules can be developed to mimic the Wnt proteins and activate Wnt signaling in the cells. We further propose that these small molecules will increase the efficiency of ex vivo expansion of functional human LSCs. The goal of this application is to use a structure-based drug discovery approach to develop potent Wnt mimics small molecule and to test their ability to increase the efficiency of ex vivo LSC expansion. Because the maintenance of stem cell characteristics in the process of culture expansion is essential for the success of ocular surface reconstruction, the small molecules generated in this project will serve as a platform for the development of novel pharmaceutical reagents for treating other corneal epithelial disorders.