A Novel Lipase Belonging to the Hormone-sensitive Lipase Family Induced under Starvation to Utilize Stored Triacylglycerol in Mycobacterium tuberculosis

Deb, Chirajyoti; Daniel, Jaiyanth; Sirakova, Tatiana D.; Abomoelak, Bassam; Dubey, Vinod S.; Kolattukudy, Pappachan E.

doi:10.1074/jbc.m505556200

Cited by 245 publications

(286 citation statements)

References 27 publications

(30 reference statements)

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“…To investigate whether this secretion motif is also required for other ESX-5 substrates, we analyzed M. tuberculosis LipY (LipY tub ). LipY tub is a lipase that is secreted via ESX-5, and the only PE protein with a known function (33,34). Although LipY tub contains a large C-terminal domain that exhibits lipase activity, it also has a complete N-terminal PE domain, including the conserved YxxxD/E motif (Fig.…”

Section: Esx-5 Secretion Of M Tuberculosis Lipy Depends On a Similarmentioning

confidence: 99%

General secretion signal for the mycobacterial type VII secretion pathway

Daleke

Ummels

Bawono³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

178

224

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Mycobacterial pathogens use specialized type VII secretion (T7S) systems to transport crucial virulence factors across their unusual cell envelope into infected host cells. These virulence factors lack classical secretion signals and the mechanism of substrate recognition is not well understood. Here we demonstrate that the model T7S substrates PE25/PPE41, which form a heterodimer, are targeted to the T7S pathway ESX-5 by a signal located in the C terminus of PE25. Site-directed mutagenesis of residues within this C terminus resulted in the identification of a highly conserved motif, i.e., YxxxD/E, which is required for secretion. This motif was also essential for the secretion of LipY, another ESX-5 substrate. Pathogenic mycobacteria have several different T7S systems and we identified a PE protein that is secreted by the ESX-1 system, which allowed us to compare substrate recognition of these two T7S systems. Surprisingly, this ESX-1 substrate contained a C-terminal signal functionally equivalent to that of PE25. Exchange of these C-terminal secretion signals between the PE proteins restored secretion, but each PE protein remained secreted via its own ESX secretion system, indicating that an additional signal(s) provides system specificity. Remarkably, the YxxxD/E motif was also present in and required for efficient secretion of the ESX-1 substrates CFP-10 and EspB. Therefore, our data show that the YxxxD/E motif is a general secretion signal that is present in all known mycobacterial T7S substrates or substrate complexes.

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Section: Esx-5 Secretion Of M Tuberculosis Lipy Depends On a Similarmentioning

confidence: 99%

General secretion signal for the mycobacterial type VII secretion pathway

Daleke

Ummels

Bawono³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

178

224

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“…LipY consists of three domains: a typical PE domain at the N terminus followed by a linker domain of unknown function and a C-terminal domain containing the triacylglycerol lipase motif (for a schematic view, see (32), only one orthologue has been reported in a mycobacterial species outside the M. tuberculosis complex (33). This gene is present in M. marinum and codes for a protein with a different domain organization.…”

Section: Homologues Of Lipy Have Evolved Different N-terminal Domainsmentioning

confidence: 99%

“…M. tuberculosis LipY tub is involved in the degradation of triacylglycerols (TAGs) and is the major active lipase under nutrientdeprived conditions. This led to the hypothesis that LipY tub plays a role in fatty acid metabolism during the dormancy and reactivation stages of the M. tuberculosis infection cycle (32). Although the lipolytic activity of LipY tub is expressed by the C-terminal part of the protein (32), the function of its N-terminal PE domain is less clear.…”

mentioning

confidence: 99%

“…This led to the hypothesis that LipY tub plays a role in fatty acid metabolism during the dormancy and reactivation stages of the M. tuberculosis infection cycle (32). Although the lipolytic activity of LipY tub is expressed by the C-terminal part of the protein (32), the function of its N-terminal PE domain is less clear. It has been shown that the PE domain of LipY tub has an inhibiting effect on the lipase activity (33).…”

mentioning

confidence: 99%

“…To date, little is known about the function of the various domains of the PE and PPE proteins. In fact, the M. tuberculosis PE protein LipY (hereafter referred to as LipY tub ) is the only PE protein for which a function has been characterized (32). M. tuberculosis LipY tub is involved in the degradation of triacylglycerols (TAGs) and is the major active lipase under nutrientdeprived conditions.…”

mentioning

confidence: 99%

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Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) Protein Domains Target LipY Lipases of Pathogenic Mycobacteria to the Cell Surface via the ESX-5 Pathway

Daleke

Cascioferro

Punder

et al. 2011

Journal of Biological Chemistry

123

181

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The type VII secretion system ESX-5 is a major pathway for export of PE and PPE proteins in pathogenic mycobacteria. These mycobacteria-specific protein families are characterized by conserved N-terminal domains of 100 and 180 amino acids, which contain the proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) motifs after which they are named. Here we investigated secretion of the triacylglycerol lipase LipY, which in fast-growing mycobacteria contains a signal sequence, but in slow-growing species appears to have replaced the signal peptide with a PE or PPE domain. Selected LipY homologues were expressed in wild-type Mycobacterium marinum and its corresponding ESX-5 mutant, and localization of the proteins was investigated by immunoblotting and electron microscopy. Our study shows that Mycobacterium tuberculosis PE-LipY (LipY tub ) and M. marinum PPE-LipY (LipY mar ) are both secreted to the bacterial surface in an ESX-5-dependent fashion. After transport, the PE/PPE domains are removed by proteolytic cleavage. In contrast, Mycobacterium gilvum LipY, which has a signal sequence, is not transported to the cell surface. Furthermore, we show that LipY tub and LipY mar require their respective PE and PPE domains for ESX-5-dependent secretion. The role of the PE domain in ESX-5 secretion was confirmed in a whole cell lipase assay, in which wild-type bacteria expressing full-length LipY tub , but not LipY tub lacking its PE domain, were shown to hydrolyze extracellular lipids. In conclusion, both PE and PPE domains contain a signal required for secretion of LipY by the ESX-5 system, and these domains are proteolytically removed upon translocation.Mycobacteria such as Mycobacterium tuberculosis, the etiological agent of tuberculosis, have a highly unusual and complex cell envelope (1). To secrete virulence factors across the cell envelope, these bacteria use specialized protein secretion systems, known as ESX or type VII secretion systems (2-8). Mycobacterial genomes contain up to five genetic loci coding for type VII secretion systems, named ESX-1 to ESX-5 (9, 10). The most well studied system, ESX-1, is responsible for the secretion of 10 substrates, including the important T-cell antigens ESAT-6 and CFP-10, and is required for full virulence of M. tuberculosis (7,(11)(12)(13)(14)(15)(16)(17). Phylogenetic analyses and comparative genomics suggest that the five ESX clusters have evolved by gene duplication and that ESX-5 is the result of the most recent duplication event (9). Interestingly, ESX-5 is restricted to a group of mycobacterial species known as the slow-growing mycobacteria, which include all major pathogens, such as M. tuberculosis, Mycobacterium leprae, and Mycobacterium ulcerans, and the fish pathogen Mycobacterium marinum (9). Four of the ESX loci contain also PE and PPE genes (named after the conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N termini of their respective gene products) (9, 10), and the appearance of ESX-5 predates the huge expansion of these gene families in s...

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Biofragments: An Approach towards Predicting Protein Function Using Biologically Related Fragments and its Application to Mycobacterium tuberculosis CYP126

et al. 2014

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We present a novel fragment-based approach that tackles some of the challenges for chemical biology of predicting protein function. The general approach, which we have termed biofragments, comprises two key stages. First, a biologically relevant fragment library (biofragment library) can be designed and constructed from known sets of substrate-like ligands for a protein class of interest. Second, the library can be screened for binding to a novel putative ligand-binding protein from the same or similar class, and the characterization of hits provides insight into the basis of ligand recognition, selectivity, and function at the substrate level. As a proof-of-concept, we applied the biofragments approach to the functionally uncharacterized Mycobacterium tuberculosis (Mtb) cytochrome P450 isoform, CYP126. This led to the development of a tailored CYP biofragment library with notable 3D characteristics and a significantly higher screening hit rate (14 %) than standard drug-like fragment libraries screened previously against Mtb CYP121 and 125 (4 % and 1 %, respectively). Biofragment hits were identified that make both substrate-like type-I and inhibitor-like type-II interactions with CYP126. A chemical-fingerprint-based substrate model was built from the hits and used to search a virtual TB metabolome, which led to the discovery that CYP126 has a strong preference for the recognition of aromatics and substrate-like type-I binding of chlorophenol moieties within the active site near the heme. Future catalytic analyses will be focused on assessing CYP126 for potential substrate oxidative dehalogenation.

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A Novel Lipase Belonging to the Hormone-sensitive Lipase Family Induced under Starvation to Utilize Stored Triacylglycerol in Mycobacterium tuberculosis

Cited by 245 publications

References 27 publications

General secretion signal for the mycobacterial type VII secretion pathway

General secretion signal for the mycobacterial type VII secretion pathway

Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) Protein Domains Target LipY Lipases of Pathogenic Mycobacteria to the Cell Surface via the ESX-5 Pathway

Biofragments: An Approach towards Predicting Protein Function Using Biologically Related Fragments and its Application to Mycobacterium tuberculosis CYP126

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