title: Assembly of IMPDH2-based, CTPS-based, and mixed rod/ring structures is dependent on cell type and conditions of induction.
abstract: Inhibition of guanosine triphosphate (GTP) and cytidine triphosphate (CTP) biosynthetic pathways induces cells to assemble rod/ring (RR) structures, also named cytoophidia, which consist of the enzymes cytidine triphosphate synthase (CTPS) and inosine-5'-monophosphate dehydrogenase 2 (IMPDH2). We aim to explore the interaction of CTPS and IMPDH2 in the generation of RR structures. HeLa and COS-7 cells were cultured in normal conditions or in the presence of 6-diazo-5-oxo-L-norleucine (DON), ribavirin, or mycophenolic acid (MPA). Over 90% of DON-treated cells presented RR structures. In HeLa cells, 35% of the RR structures were positive for IMPDH2 alone, 26% were CTPS alone, and 31% were IMPDH2/CTPS mixed, while in COS-7 cells, 42% of RR were IMPDH2 alone, 41% were CTPS alone, and 10% were IMPDH2/CTPS mixed. Ribavirin and MPA treatments induced only IMPDH2-based RR. Cells were also transfected with an N-terminal hemagglutinin (NHA)-tagged CTPS1 construct. Over 95% of NHA-CTPS1 transfected cells with DON treatment presented IMPDH2-based RR and almost 100% presented CTPS1-based RR; when treated with ribavirin, over 94% of transfected cells presented IMPDH2-based RR and 37% presented CTPS1-based RR, whereas 2% of untreated transfected cells presented IMPDH2-based RR and 28% presented CTPS1-based RR. These results may help in understanding the relationship between CTP and GTP biosynthetic pathways, especially concerning the formation of filamentous RR structures.
authors.affiliation: Rheumatology Division, Federal University of Sao Paulo, Sao Paulo SP 04023-062, Brazil; Department of Oral Biology, University of Florida, Gainesville FL 32610-0424, USA. Electronic address: gerson.keppeke@gmail.com.
authors.forename: Gerson Dierley
authors.initials: GD
authors.lastname: Keppeke
authors.affiliation: Department of Oral Biology, University of Florida, Gainesville FL 32610-0424, USA.
authors.forename: S John
authors.initials: SJ
authors.lastname: Calise
authors.affiliation: Department of Oral Biology, University of Florida, Gainesville FL 32610-0424, USA.
authors.forename: Edward K L
authors.initials: EK
authors.lastname: Chan
authors.affiliation: Rheumatology Division, Federal University of Sao Paulo, Sao Paulo SP 04023-062, Brazil; Immunology Division, Fleury Medicine and Health Laboratories, Sao Paulo SP 04102-050, Brazil. Electronic address: luis.andrade@unifesp.br.
authors.forename: Luis Eduardo C
authors.initials: LE
authors.lastname: Andrade
mesh_terms: D000818:Animals; D053898:Biosynthetic Pathways; D019556:COS Cells; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003570:Cytidine Triphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007168:IMP Dehydrogenase / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D007168:IMP Dehydrogenase; C501687:IMPDH2 protein, human; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase
keywords: CTPS; Enzyme aggregation; Enzyme inhibition; IMPDH2; Nucleotide synthesis