Ribosomal protein S19 (RPS19) is a 16-kDa protein found mainly as a component of the ribosomal 40 S subunit. Its mutations are responsible for Diamond Blackfan anemia, a congenital disease characterized by defective erythroid progenitor maturation. Dysregulation of RPS19 has therefore been implicated in this defective erythropoiesis, although the link between them is still unclear. Two not mutually exclusive hypotheses have been proposed: altered protein synthesis and loss of unknown functions not directly connected with the structural role of RPS19 in the ribosome. A role in rRNA processing has been surmised for the yeast ortholog, whereas the extracellular RPS19 dimer has a monocyte chemotactic activity. Three proteins are known to interact with RPS19: FGF2, complement component 5 receptor 1, and a nucleolar protein called RPS19-binding protein. We have used a yeast two-hybrid approach to identify a fourth protein: the serine-threonine kinase PIM1. The present study describes our use of proteomics strategies to look for proteins interacting with RPS19 to determine its functions. Proteins were isolated by affinity purification with a GST-RPS19 recombinant protein and identified using LCMS/MS analysis coupled to bioinformatics tools. We identified 159 proteins from the following Gene Ontology categories: NTPases (ATPases and GTPases; five proteins), hydrolases/helicases (19 proteins), isomerases (two proteins), kinases (three proteins), splicing factors (five proteins), structural constituents of ribosome (29 proteins), transcription factors (11 proteins), transferases (five proteins), transporters (nine proteins), DNA/RNA-binding protein species (53 proteins), other (one dehydrogenase protein, one ligase protein, one peptidase protein, one receptor protein, and one translation elongation factor), and 13 proteins of still unknown function. Proteomics results were validated by affinity purification and Western blotting. These interactions were further confirmed by co-immunoprecipitation using a monoclonal RPS19 antibody. Many interactors are nucleolar proteins and thus are expected to take part in the RPS19 interactome; however, some proteins suggest additional functional roles for RPS19.

Orru, S., Aspesi, A., Armiraglio, M., Caterino, M., Loreni, F., Ruoppolo, M., et al. (2007). Analysis of the ribosomal protein S19 interactome. MOLECULAR & CELLULAR PROTEOMICS, 6(3), 382-393 [10.1074/mcp.M600156-MCP200].

Analysis of the ribosomal protein S19 interactome

LORENI, FABRIZIO;
2007-01-01

Abstract

Ribosomal protein S19 (RPS19) is a 16-kDa protein found mainly as a component of the ribosomal 40 S subunit. Its mutations are responsible for Diamond Blackfan anemia, a congenital disease characterized by defective erythroid progenitor maturation. Dysregulation of RPS19 has therefore been implicated in this defective erythropoiesis, although the link between them is still unclear. Two not mutually exclusive hypotheses have been proposed: altered protein synthesis and loss of unknown functions not directly connected with the structural role of RPS19 in the ribosome. A role in rRNA processing has been surmised for the yeast ortholog, whereas the extracellular RPS19 dimer has a monocyte chemotactic activity. Three proteins are known to interact with RPS19: FGF2, complement component 5 receptor 1, and a nucleolar protein called RPS19-binding protein. We have used a yeast two-hybrid approach to identify a fourth protein: the serine-threonine kinase PIM1. The present study describes our use of proteomics strategies to look for proteins interacting with RPS19 to determine its functions. Proteins were isolated by affinity purification with a GST-RPS19 recombinant protein and identified using LCMS/MS analysis coupled to bioinformatics tools. We identified 159 proteins from the following Gene Ontology categories: NTPases (ATPases and GTPases; five proteins), hydrolases/helicases (19 proteins), isomerases (two proteins), kinases (three proteins), splicing factors (five proteins), structural constituents of ribosome (29 proteins), transcription factors (11 proteins), transferases (five proteins), transporters (nine proteins), DNA/RNA-binding protein species (53 proteins), other (one dehydrogenase protein, one ligase protein, one peptidase protein, one receptor protein, and one translation elongation factor), and 13 proteins of still unknown function. Proteomics results were validated by affinity purification and Western blotting. These interactions were further confirmed by co-immunoprecipitation using a monoclonal RPS19 antibody. Many interactors are nucleolar proteins and thus are expected to take part in the RPS19 interactome; however, some proteins suggest additional functional roles for RPS19.
2007
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore BIO/11 - BIOLOGIA MOLECOLARE
English
Con Impact Factor ISI
adenosine triphosphatase; DNA binding protein; exportin 1; guanosine triphosphatase; helicase; hydrolase; initiation factor; intein; isomerase; ligase; oxidoreductase; peptidase; phosphotransferase; protein S19; receptor protein; ribosome protein; RNA binding protein; transcription factor; transferase; Y box binding protein 1; article; bioinformatics; controlled study; enzyme activity; functional proteomics; human; human cell; immunoprecipitation; liquid chromatography; mass spectrometry; priority journal; protein analysis; protein determination; protein function; protein isolation; protein processing; protein protein interaction; protein purification; translation regulation; validation process; Western blotting; Glutathione Transferase; Humans; K562 Cells; Protein Interaction Mapping; Proteomics; Recombinant Fusion Proteins; Ribosomal Proteins
Orru, S., Aspesi, A., Armiraglio, M., Caterino, M., Loreni, F., Ruoppolo, M., et al. (2007). Analysis of the ribosomal protein S19 interactome. MOLECULAR & CELLULAR PROTEOMICS, 6(3), 382-393 [10.1074/mcp.M600156-MCP200].
Orru, S; Aspesi, A; Armiraglio, M; Caterino, M; Loreni, F; Ruoppolo, M; Santoro, C; Dianzani, I
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/30749
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