Dataset Information

Identification of high molecular weight protein/protein complexes in human cultured cells (HEK293T) LC-MS/MS

ABSTRACT: Phosphoribosyl pyrophosphate synthetase (PRPS) is a highly conserved enzyme that conducts the chokepoint reaction of nucleotide biosynthesis by converting ribose-5-phosphate (R-5-P) to phosphoribosyl pyrophosphate (PRPP). Due to the presence of multiple isoforms and related PRPS-associated proteins in opisthokont species, the precise nature of the PRPS enzyme in cells and tissues remains uncertain. Using proteomics approaches and biochemistry, we demonstrate that these individual components assemble together to form a heterogeneous megadalton complex comprising of PRPS1, PRPS2, and two PRPS associated proteins – PRPSAP1 and PRPSAP2. To validate this and identify other protein/protein complexes that exists in a similar high molecular weight (HMW) range in human (HEK293T) cells, we performed shotgun proteomics on size exclusion chromatography fractions containing HMW proteins/protein complexes. This analysis identified a total of 262 unique proteins, which included ribosomal proteins and CAD confirming enrichment for HMW proteins. This dataset also revealed that among the eight cytosolic enzymes residing in HMW range, two were PRPS isozymes, indicating that the PRPS

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Tom Cunningham

LAB HEAD: Dr. Tom Cunningham

PROVIDER: PXD059156 | Pride | 2025-05-13

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	CunninghamLab_HEK293T_HMWfractions_shotgun_20210820_06_080321.mgf	Mgf
	CunninghamLab_HEK293T_HMWfractions_shotgun_20210820_06_080321.msfView	Msf
	CunninghamLab_HEK293T_HMWfractions_shotgun_20210820_06_080321.raw	Raw

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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

Similar Datasets

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.

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 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.