Dataset Information

Affinity purification of PRPS complex in mouse cultured cells (NIH3T3)- LC-MS/MS

ABSTRACT: Phosphoribosyl pyrophosphate synthetase (PRPS) is an enzyme conserved across all forms of life, tracing back to the last universal common ancestor (LUCA). PRPS catalyzes the rate-limiting step in converting ribose-5-phosphate (R5P) to phosphoribosyl pyrophosphate (PRPP), a crucial precursor in the biosynthesis of nucleotides, amino acids, and lipids. While Bacteria and Archaea species typically express a single PRPS enzyme, the presence of multiple PRPS-encoding genes is a hallmark trait of eukaryotes. Given the well-established evolutionary trajectory from opisthokonts to mammals, supported by relatively complete molecular phylogenetic data, we used mammals in our study that harbor five PRPS homologs, as a model system to investigate the evolutionary origins and functional significance of these multiple homologs. In addition to the three isozymes– PRPS1, PRPS2, and PRPS1L1 (testes-specific) – mammals also possess PRPSAP1 and PRPSAP2, which feature unique insertions in the catalytic flexible (CF) loop (referred to as non-homologous regions (NHRs)) compared to the sequences found in PRPS isozymes. In this project, to ascertain whether these evolutionary conserved mammalian PRPS homologs interact with each other, we tagged PRPS1, PRPS2, PRPSAP1, and PRPSAP2 individually with GFP at their C-termini. Immunoprecipitation followed by LC-MS/MS peptide identification from all revealed that all PRPS homologs assemble into a single enzyme complex in mouse (NIH3T3) cells. As a control, we used Flag-NES (Nuclear Export Signal)-GFP tagged cells given that PRPS enzymes localize in the cytoplasm. Employing isogenic cells representing all viable individual or combinatorial assembly states, we dissect the basic organizational principles of the PRPS enzyme complex and characterize the emergent properties responsible for paralog specialization, including new modes of regulation that govern complex assembly and activity in vivo. Collectively, our study demonstrates how evolution has transformed a single PRPS enzyme into a biochemical complex endowed with novel functional and regulatory features that fine-tune mammalian metabolism.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Fibroblast

SUBMITTER: Tom Cunningham

LAB HEAD: Dr. Tom Cunningham

PROVIDER: PXD058829 | Pride | 2025-05-13

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	CunninghamLab_NIH3T3_NESGFP_IP_control1_20210730_04_062121.mgf	Mgf
	CunninghamLab_NIH3T3_NESGFP_IP_control1_20210730_04_062121.msfView	Msf
	CunninghamLab_NIH3T3_NESGFP_IP_control1_20210730_04_062121.raw	Raw
	CunninghamLab_NIH3T3_NESGFP_IP_control2_20220210_13.mgf	Mgf
	CunninghamLab_NIH3T3_NESGFP_IP_control2_20220210_13.msfView	Msf

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Publications

Evolutionary origins and innovations sculpting the mammalian PRPS enzyme complex.

Karki Bibek R BR Macmillan Austin C AC Vicente-Muñoz Sara S Greis Kenneth D KD Romick Lindsey E LE Cunningham J Tom JT

bioRxiv : the preprint server for biology 20241001

The phosphoribosyl pyrophosphate synthetase (PRPS) enzyme conducts a chokepoint reaction connecting central carbon metabolism and nucleotide production pathways, making it essential for life<sup>1,2</sup>. Here, we show that the presence of multiple PRPS-encoding genes is a hallmark trait of eukaryotes, and we trace the evolutionary origins and define the individual functions of each of the five mammalian PRPS homologs - three isozymes (one testis-restricted)<sup>3,4</sup> and two non-enzymatic ...[more]

PMID: 39411161

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Project description:Phosphoribosyl pyrophosphate synthetase (PRPS) is an enzyme conserved across all forms of life, tracing back to the last universal common ancestor (LUCA). PRPS catalyzes the rate-limiting step in converting ribose-5-phosphate (R5P) to phosphoribosyl pyrophosphate (PRPP), a crucial precursor in the biosynthesis of nucleotides, amino acids, and lipids. While Bacteria and Archaea species typically express a single PRPS enzyme, the presence of multiple PRPS-encoding genes is a hallmark trait of eukaryotes. Given the well-established evolutionary trajectory from opisthokonts to mammals, supported by relatively complete molecular phylogenetic data, we used mammals in our study that harbor five PRPS homologs, as a model system to investigate the evolutionary origins and functional significance of these multiple homologs. In addition to the three isozymes– PRPS1, PRPS2, and PRPS1L1 (testes-specific) – mammals also possess PRPSAP1 and PRPSAP2, which feature unique insertions in the catalytic flexible (CF) loop (referred to as non-homologous regions (NHRs)) compared to the sequences found in PRPS isozymes. In this project, to ascertain whether these evolutionary conserved mammalian PRPS homologs interact with each other, we tagged PRPS1, PRPS2, PRPSAP1, and PRPSAP2 individually with GFP at their C-termini. Immunoprecipitation followed by LC-MS/MS peptide identification from all revealed that all PRPS homologs assemble into a single enzyme complex in human (HEK293T) cells. As a control, we used Flag-NES (Nuclear Export Signal)-GFP tagged cells given that PRPS enzymes localize in the cytoplasm. Employing isogenic cells representing all viable individual or combinatorial assembly states, we dissect the basic organizational principles of the PRPS enzyme complex and characterize the emergent properties responsible for paralog specialization, including new modes of regulation that govern complex assembly and activity in vivo. Collectively, our study demonstrates how evolution has transformed a single PRPS enzyme into a biochemical complex endowed with novel functional and regulatory features that fine-tune mammalian metabolism.