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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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Primary sequence analysis of ATP-binding peptides and proteins. Each protein sequence was submitted for in silico analysis through InterPro and sorted via Gene Ontology (molecular function). A, approximately 80% of the ATP-binding (i.e. desthiobiotin-labeled) proteins could be mapped to Pfam domains. Binding confidence, high, medium, and low, was empirically determined associated with the quality of labeled peptide spectra (i.e. confident sequence coverage and low signal to noise). B, functions of small molecule binding, transferase, and oxidoreductase activity described the majority of ATPome enzymatic properties.
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Figure 1: Primary sequence analysis of ATP-binding peptides and proteins. Each protein sequence was submitted for in silico analysis through InterPro and sorted via Gene Ontology (molecular function). A, approximately 80% of the ATP-binding (i.e. desthiobiotin-labeled) proteins could be mapped to Pfam domains. Binding confidence, high, medium, and low, was empirically determined associated with the quality of labeled peptide spectra (i.e. confident sequence coverage and low signal to noise). B, functions of small molecule binding, transferase, and oxidoreductase activity described the majority of ATPome enzymatic properties.

Mentions: Over half (59%) of the proteins within the identified M. tuberculosis ATPome harbor essential functions to support growth (23, 24), indicating that the M. tuberculosis ATPome in general is functionally important. A list of all identified proteins and their annotation as essential or nonessential for in vitro growth is provided in supplemental Table S1. Functional annotation of the proteins was conducted to identify the functional domains and domain families that were selectively labeled and enriched via our chemical proteomic techniques. In total, 218 protein domain families (Pfams) were associated with the GO term ATP binding (GO ID:0005524). The amino acid sequence of each identified protein and covalently labeled peptide sequence was subjected to an InterPro pattern search to identify functional domains and associate these regions within the list 218 annotated domain families (Pfams) (25). It was determined that 13 ATP-associated Pfams were represented in the ATPome dataset across all ranges of labeling confidence (low to high, n = 122), and none were represented in proteins identified but not labeled with nucleotide probe (n = 54) (supplemental document S1). Among the ATP-associated Pfams were proteins involved with ATP synthesis (PF00006) and peptidoglycan synthesis (Mur Ligase (PF01225)), as well as protein kinases (PF00069). Overall, ∼80% of the ATPome had peptides that could be mapped to Pfam domains (Fig. 1A). The majority of enzyme functions identified were associated with the following activities: small molecule binding (34%), transferase (17%), and oxidoreductase (16%) (Fig. 1B).


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)

Primary sequence analysis of ATP-binding peptides and proteins. Each protein sequence was submitted for in silico analysis through InterPro and sorted via Gene Ontology (molecular function). A, approximately 80% of the ATP-binding (i.e. desthiobiotin-labeled) proteins could be mapped to Pfam domains. Binding confidence, high, medium, and low, was empirically determined associated with the quality of labeled peptide spectra (i.e. confident sequence coverage and low signal to noise). B, functions of small molecule binding, transferase, and oxidoreductase activity described the majority of ATPome enzymatic properties.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Primary sequence analysis of ATP-binding peptides and proteins. Each protein sequence was submitted for in silico analysis through InterPro and sorted via Gene Ontology (molecular function). A, approximately 80% of the ATP-binding (i.e. desthiobiotin-labeled) proteins could be mapped to Pfam domains. Binding confidence, high, medium, and low, was empirically determined associated with the quality of labeled peptide spectra (i.e. confident sequence coverage and low signal to noise). B, functions of small molecule binding, transferase, and oxidoreductase activity described the majority of ATPome enzymatic properties.
Mentions: Over half (59%) of the proteins within the identified M. tuberculosis ATPome harbor essential functions to support growth (23, 24), indicating that the M. tuberculosis ATPome in general is functionally important. A list of all identified proteins and their annotation as essential or nonessential for in vitro growth is provided in supplemental Table S1. Functional annotation of the proteins was conducted to identify the functional domains and domain families that were selectively labeled and enriched via our chemical proteomic techniques. In total, 218 protein domain families (Pfams) were associated with the GO term ATP binding (GO ID:0005524). The amino acid sequence of each identified protein and covalently labeled peptide sequence was subjected to an InterPro pattern search to identify functional domains and associate these regions within the list 218 annotated domain families (Pfams) (25). It was determined that 13 ATP-associated Pfams were represented in the ATPome dataset across all ranges of labeling confidence (low to high, n = 122), and none were represented in proteins identified but not labeled with nucleotide probe (n = 54) (supplemental document S1). Among the ATP-associated Pfams were proteins involved with ATP synthesis (PF00006) and peptidoglycan synthesis (Mur Ligase (PF01225)), as well as protein kinases (PF00069). Overall, ∼80% of the ATPome had peptides that could be mapped to Pfam domains (Fig. 1A). The majority of enzyme functions identified were associated with the following activities: small molecule binding (34%), transferase (17%), and oxidoreductase (16%) (Fig. 1B).

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