A change in the regulatory protein profile of a cell drives fate specification. This change is in part controlled at the transcriptional level, but as cells progress to their final fate during embryonic development, regulatory proteins must also be inactivated. One mechanism is removal by ubiquitin-mediated protein degradation which involves the attachment of a multi-ubiquitin chain to target the selected proteins to the 26S proteasome for destruction. These proteins are selected by ubiquitin ligases (E3), and while there are numerous E3 ligases, the best understood are the Cullin based ligases (SCF) which contain Skp1, the scaffold protein Cullin, and the target-binding F-box proteins. There is a rich history demonstrating a role for ubiquitin-mediated protein destruction in cell-cycle regulation, and defects in this process underlie a number of cancers. Furthermore, E3 ligase deficiencies have been linked to neural crest disorders and neurodegenerative diseases, indicating that protein degradation plays a key role throughout embryonic development. While the multistep process of ubiquitination is well understood and targets have been identified, we know less about its function in the regulation of cell fate specification in vertebrate development. To gain a better understanding of the role of protein degradation in neural development, the investigators propose to identify all F-box proteins in Xenopus and use gain- and loss-of-function assays to characterize those expressed in the nervous system throughout development and in the epidermis during neural plate specification. With the completion of these aims, they will then be able to identify F-box binding partners and therefore potential targets whose degradation is required for ectodermal fate specification as well as for progression from neural precursor to differentiated neuron. F-box proteins are the largest class of E3 substrate-interacting proteins and since many bind multiple substrates, they are likely to facilitate the ubiquitination of hundreds of proteins in a variety of cellular and developmental processes. Using bioinformatic tools, thus far the investigators have identified 58 F-box proteins in Xenopus and classified them into three groups based on their protein interaction domain. Furthermore, with in silico genome scanning for REST binding sites, they have identified three F-box proteins that are expressed in the nervous system. The investigators will confirm that they play a role in ubisquitination and that they have a role in neural development by demonstrating interaction with Skp1 of the modular Cullin-based E3 ligase, and with loss-of-function analysis, respectively. The long term goal of this research program, to determine the role of these specific degradation events in the specification and differentiation of the nervous system, is fundamental to understanding the regulation of normal and disordered neural development. NARRATIVE: To transform the fertilized egg into a perfectly formed individual, specific combinations of proteins must be expressed at the right time and place. Loss of expression or misexpression of a single protein can have catastrophic consequences including cyclopia, dwarfism, connective tissue disorders, and limb as well as craniofacial defects. This application will address how the levels of proteins are controlled by degradation to ensure ordered progression through neurogenesis and therefore the proper development of the central nervous system.

Public Health Relevance

To transform the fertilized egg into a perfectly formed individual, specific combinations of proteins must be expressed at the right time and place. Loss of expression or misexpression of a single protein can have catastrophic consequences including cyclopia, dwarfism, connective tissue disorders and limb and craniofacial defects. This proposal will address how the levels of proteins are controlled by degradation to ensure ordered progression through neurogenesis and therefore the proper development of the central nervous system.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Small Research Grants (R03)
Project #
5R03HD060698-02
Application #
8109374
Study Section
Pediatrics Subcommittee (CHHD)
Program Officer
Mukhopadhyay, Mahua
Project Start
2010-07-15
Project End
2013-06-30
Budget Start
2011-07-01
Budget End
2013-06-30
Support Year
2
Fiscal Year
2011
Total Cost
$73,680
Indirect Cost
Name
Georgetown University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
049515844
City
Washington
State
DC
Country
United States
Zip Code
20057
Saritas-Yildirim, Banu; Childers, Christopher P; Elsik, Christine G et al. (2015) Identification of REST targets in the Xenopus tropicalis genome. BMC Genomics 16:380
Saritas-Yildirim, Banu; Pliner, Hannah A; Ochoa, Angelica et al. (2015) Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins. PLoS One 10:e0136929
Saritas-Yildirim, Banu; Silva, Elena M (2014) The role of targeted protein degradation in early neural development. Genesis 52:287-99