Study of the Biological Function and Regulation of CTP Synthase Filamentous Structure

Study of the biological function and regulation of CTP synthase filamentous structure Cytidine triphosphate synthase (CTPsyn), the rate-limiting enzyme for CTP and dCTP production, is recently found to form a unique and conserved cytoplasmic filamentous structure known as cytoophidia. However, the assembly of this structure and its biological functions remain unresolved. dCTP/CTP are the material for DNA/RNA synthesis, and CDP derived from CTP is also involved in phospholipid synthesis. We found that the proto-oncogene Cbl is a critical regulator of cytoophidium structure in the Drosophila follicle cells during endocycle. This disassembly of the cytoophidium structure in mutant resulted in reduction of CTP content, and consequently delayed endoreplication-associated S-phase and defective eggshell integrity. While the E3 ligase activity of Cbl is essential for maintaining cytoophidia, Cbl’s role in this capacity was independent of CTPsyn protein level control or the EGFR signaling. Together, our data link cytoophidia to CTP synthesis for maintaining the nucleotide pool during endoreplication, and further highlight a new role of Cbl in mediating this cellular pathway. This work has been submitted for publication (see preliminary results). Further investigation revealed that starvation in fly induced cytoophidia formation which is similar to that caused by CTPsyn inhibitor, DON treatment. In addition, DON induced cytoophidium in HEp2 cells is sensitive to a STAT3 inhibitor, indicating that stress may serves as an inducer for this structure. Indeed, amino acid depletion conditional medium could induce short filamentous aggregation of CTPsyn in HEp 2 cell as well. Therefore, cytoophidia may involve in more biological functions under stressful conditions. Using the Flag-tagged CTPsyn transgene in fly, we were able to examine this structure at the Electronic Microscopy levels, and to our knowledge this is the first visualization of this structure at the ultra-structure level. Interestingly, the MG132 treatment caused disassembly of cytoophidia both in follicle cells and the germ cells. Given that CTPsyn filaments are also widely observed across organisms, and it may play roles in cell growth through control of DNA and phospholipid synthesis, we propose here to address three major questions related to the assembly of cytoophidia in Drosophila and human cells. 1) What is the composition of cytoophidia? 2) How is the assembly of cytoophidia regulated? 3) What are the biological functions of this subcellular compartment?

Project ID:PA10308-0495
External Project ID:MOST103-2311-B182-004-MY3