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A chemical proteomics approach to profiling the ATP-binding proteome of Mycobacterium tuberculosis.

Wolfe LM, Veeraraghavan U, Idicula-Thomas S, Schürer S, Wennerberg K, Reynolds R, Besra GS, Dobos KM - Mol. Cell Proteomics (2013)

Bottom Line: Protein families vital to the survival of the tubercle bacillus during hypoxia emerged from our studies.Specifically, along with members of the DosR regulon, several proteins involved in energy metabolism (Icl/Rv0468 and Mdh/Rv1240) and lipid biosynthesis (UmaA/Rv0469, DesA1/Rv0824c, and DesA2/Rv1094) were found to be differentially abundant in hypoxic versus normal growing cultures.These pathways represent a subset of proteins that may be relevant therapeutic targets for development of novel ATP-competitive antibiotics.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523, USA.

ABSTRACT
Tuberculosis, caused by Mycobacterium tuberculosis, remains one of the leading causes of death worldwide despite extensive research, directly observed therapy using multidrug regimens, and the widespread use of a vaccine. The majority of patients harbor the bacterium in a state of metabolic dormancy. New drugs with novel modes of action are needed to target essential metabolic pathways in M. tuberculosis; ATP-competitive enzyme inhibitors are one such class. Previous screening efforts for ATP-competitive enzyme inhibitors identified several classes of lead compounds that demonstrated potent anti-mycobacterial efficacy as well as tolerable levels of toxicity in cell culture. In this report, a probe-based chemoproteomic approach was used to selectively profile the M. tuberculosis ATP-binding proteome in normally growing and hypoxic M. tuberculosis. From these studies, 122 ATP-binding proteins were identified in either metabolic state, and roughly 60% of these are reported to be essential for survival in vitro. These data are available through ProteomeXchange with identifier PXD000141. Protein families vital to the survival of the tubercle bacillus during hypoxia emerged from our studies. Specifically, along with members of the DosR regulon, several proteins involved in energy metabolism (Icl/Rv0468 and Mdh/Rv1240) and lipid biosynthesis (UmaA/Rv0469, DesA1/Rv0824c, and DesA2/Rv1094) were found to be differentially abundant in hypoxic versus normal growing cultures. These pathways represent a subset of proteins that may be relevant therapeutic targets for development of novel ATP-competitive antibiotics.

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Protein-protein interaction networks of the M. tuberculosis ATPome. The list of protein IDs from our MS analysis was input into the STRING database (STRING version 9.0) to identify known and predicted functional networks. 48% of the proteins in our shotgun analysis were shown to be functionally associated with at least one known interacting partner. Emerging protein clusters are functionally relevant in basic metabolism (i.e. respiratory chain and protein synthesis), cell wall biosynthesis (i.e. fatty acid and peptidoglycan synthesis), and virulence (i.e. lipid synthesis).
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Figure 7: Protein-protein interaction networks of the M. tuberculosis ATPome. The list of protein IDs from our MS analysis was input into the STRING database (STRING version 9.0) to identify known and predicted functional networks. 48% of the proteins in our shotgun analysis were shown to be functionally associated with at least one known interacting partner. Emerging protein clusters are functionally relevant in basic metabolism (i.e. respiratory chain and protein synthesis), cell wall biosynthesis (i.e. fatty acid and peptidoglycan synthesis), and virulence (i.e. lipid synthesis).

Mentions: To find clusters of protein families functionally linked in relevant biochemical pathways, we utilized the list of 81 confidently labeled proteins and expanded our dataset to include nonlabeled proteins that were confidently identified by mass spectrometry (i.e. proteins with total spectral counts across biological replicates >5 with 90% peptide probability) irrespective of ATP labeling (supplemental Table S1). Functional association networks using the web-based Search Tool for the Retrieval of Interacting Genes (STRING version 9.0) (22) were generated from the 81 ATP-binding proteins combined with the 54 unlabeled proteins. Emerging from this data set we visualized clusters of associated protein families (Fig. 7), including members of the polyketide synthase family (Category 1, Lipid Metabolism), ribosomal protein synthesis (Category 2, Information Pathways), and mycolic acid and peptidoglycan synthesis (Category 3, Cell Wall and Processes). Polyketide synthases are large multidomain proteins involved in lipid and mycolic acid biosynthesis. Pks5, Pks12, and Pks13 as well as the phthiocerol dimycoserate synthases PpsABCDE and mycocerosic acid synthase (Mas) work in coordination to synthesize the cell wall-associated and virulence determinant phthiocerol dimycocerosate (40, 41). Within this group of proteins, PpsC was found to bind ATP. PpsC catalyzes the complete reduction of malonyl-CoA in the synthesis of phthiocerol. The localization of the ATP nucleotide probe was not within any of the annotated domains of PpsC. Although PpsC is a nonessential enzyme, its associated protein partners identified in this study do play essential roles (pks12/13) (41).


A chemical proteomics approach to profiling the ATP-binding proteome of Mycobacterium tuberculosis.

Wolfe LM, Veeraraghavan U, Idicula-Thomas S, Schürer S, Wennerberg K, Reynolds R, Besra GS, Dobos KM - Mol. Cell Proteomics (2013)

Protein-protein interaction networks of the M. tuberculosis ATPome. The list of protein IDs from our MS analysis was input into the STRING database (STRING version 9.0) to identify known and predicted functional networks. 48% of the proteins in our shotgun analysis were shown to be functionally associated with at least one known interacting partner. Emerging protein clusters are functionally relevant in basic metabolism (i.e. respiratory chain and protein synthesis), cell wall biosynthesis (i.e. fatty acid and peptidoglycan synthesis), and virulence (i.e. lipid synthesis).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675820&req=5

Figure 7: Protein-protein interaction networks of the M. tuberculosis ATPome. The list of protein IDs from our MS analysis was input into the STRING database (STRING version 9.0) to identify known and predicted functional networks. 48% of the proteins in our shotgun analysis were shown to be functionally associated with at least one known interacting partner. Emerging protein clusters are functionally relevant in basic metabolism (i.e. respiratory chain and protein synthesis), cell wall biosynthesis (i.e. fatty acid and peptidoglycan synthesis), and virulence (i.e. lipid synthesis).
Mentions: To find clusters of protein families functionally linked in relevant biochemical pathways, we utilized the list of 81 confidently labeled proteins and expanded our dataset to include nonlabeled proteins that were confidently identified by mass spectrometry (i.e. proteins with total spectral counts across biological replicates >5 with 90% peptide probability) irrespective of ATP labeling (supplemental Table S1). Functional association networks using the web-based Search Tool for the Retrieval of Interacting Genes (STRING version 9.0) (22) were generated from the 81 ATP-binding proteins combined with the 54 unlabeled proteins. Emerging from this data set we visualized clusters of associated protein families (Fig. 7), including members of the polyketide synthase family (Category 1, Lipid Metabolism), ribosomal protein synthesis (Category 2, Information Pathways), and mycolic acid and peptidoglycan synthesis (Category 3, Cell Wall and Processes). Polyketide synthases are large multidomain proteins involved in lipid and mycolic acid biosynthesis. Pks5, Pks12, and Pks13 as well as the phthiocerol dimycoserate synthases PpsABCDE and mycocerosic acid synthase (Mas) work in coordination to synthesize the cell wall-associated and virulence determinant phthiocerol dimycocerosate (40, 41). Within this group of proteins, PpsC was found to bind ATP. PpsC catalyzes the complete reduction of malonyl-CoA in the synthesis of phthiocerol. The localization of the ATP nucleotide probe was not within any of the annotated domains of PpsC. Although PpsC is a nonessential enzyme, its associated protein partners identified in this study do play essential roles (pks12/13) (41).

Bottom Line: Protein families vital to the survival of the tubercle bacillus during hypoxia emerged from our studies.Specifically, along with members of the DosR regulon, several proteins involved in energy metabolism (Icl/Rv0468 and Mdh/Rv1240) and lipid biosynthesis (UmaA/Rv0469, DesA1/Rv0824c, and DesA2/Rv1094) were found to be differentially abundant in hypoxic versus normal growing cultures.These pathways represent a subset of proteins that may be relevant therapeutic targets for development of novel ATP-competitive antibiotics.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523, USA.

ABSTRACT
Tuberculosis, caused by Mycobacterium tuberculosis, remains one of the leading causes of death worldwide despite extensive research, directly observed therapy using multidrug regimens, and the widespread use of a vaccine. The majority of patients harbor the bacterium in a state of metabolic dormancy. New drugs with novel modes of action are needed to target essential metabolic pathways in M. tuberculosis; ATP-competitive enzyme inhibitors are one such class. Previous screening efforts for ATP-competitive enzyme inhibitors identified several classes of lead compounds that demonstrated potent anti-mycobacterial efficacy as well as tolerable levels of toxicity in cell culture. In this report, a probe-based chemoproteomic approach was used to selectively profile the M. tuberculosis ATP-binding proteome in normally growing and hypoxic M. tuberculosis. From these studies, 122 ATP-binding proteins were identified in either metabolic state, and roughly 60% of these are reported to be essential for survival in vitro. These data are available through ProteomeXchange with identifier PXD000141. Protein families vital to the survival of the tubercle bacillus during hypoxia emerged from our studies. Specifically, along with members of the DosR regulon, several proteins involved in energy metabolism (Icl/Rv0468 and Mdh/Rv1240) and lipid biosynthesis (UmaA/Rv0469, DesA1/Rv0824c, and DesA2/Rv1094) were found to be differentially abundant in hypoxic versus normal growing cultures. These pathways represent a subset of proteins that may be relevant therapeutic targets for development of novel ATP-competitive antibiotics.

Show MeSH
Related in: MedlinePlus