Abstract
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is well known for its involvement in numerous non-metabolic processes inside mammalian cells. Alternative functions of prokaryotic GAPDH are mainly deduced from its extracellular localization ability to bind to selected host proteins. Data on its participation in intracellular bacterial processes are scarce as there has been to date only one study dealing with this issue. We previously have reported several points of evidence that the GAPDH homolog of Francisella tularensis GapA might also exert additional non-enzymatic functions. Following on from our earlier observations we decided to identify GapA’s interacting partners within the bacterial proteome to explore its new roles at intracellular level. The quantitative proteomics approach based on stable isotope labeling of amino acids in cell culture (SILAC) in combination with affinity purification mass spectrometry enabled us to identify 18 proteins potentially interacting with GapA. Six of those interactions were further confirmed by alternative methods. Half of the identified proteins were involved in non-metabolic processes. Further analysis together with quantitative label-free comparative analysis of proteomes isolated from the wild-type strain strain with deleted gapA gene suggests that GapA is implicated in DNA repair processes. Absence of GapA promotes secretion of its most potent interaction partner the hypothetical protein with peptidase propeptide domain (PepSY) thereby indicating that it impacts on subcellular distribution of some proteins.
Highlights
The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known for its ability to exhibit multiple unrelated functions within a single polypeptide chain (Pancholi and Chhatwal, 2003; Jeffery, 2009)
Several large-scale protein–protein interaction studies performed on Escherichia coli have revealed that bacterial Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), similar to its eukaryotic orthologs or analogs, is able to interact with other intracellular proteins, including metabolic enzymes as well as proteins involved in transcription, protein synthesis, and DNA repair (Butland et al, 2005; Arifuzzaman et al, 2006; Ferreira et al, 2013, 2015)
We used a quantitative proteomic approach based on SILAC metabolic labeling in combination with affinity purification and mass spectrometry to screen for bacterial proteins that bind to the GapA of F. tularensis (Figure 1)
Summary
The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known for its ability to exhibit multiple unrelated functions within a single polypeptide chain (Pancholi and Chhatwal, 2003; Jeffery, 2009). In mammalian cells, it is involved in diverse cellular non-metabolic processes, including gene regulation, DNA repair, cytoskeletal dynamics, cell death, vesicular transport, and many others. Several large-scale protein–protein interaction studies performed on Escherichia coli have revealed that bacterial GAPDH, similar to its eukaryotic orthologs or analogs, is able to interact with other intracellular proteins, including metabolic enzymes as well as proteins involved in transcription, protein synthesis, and DNA repair (Butland et al, 2005; Arifuzzaman et al, 2006; Ferreira et al, 2013, 2015)
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