Functional Identification of APIP as Human mtnB, a Key Enzyme in the Methionine Salvage Pathway

Mary, Camille; Duek, Paula D.; Salleron, Lisa; Tienz, Petra; Bumann, Dirk; Bairoch, Amos Marc; Lane, Lydie

doi:10.1371/journal.pone.0052877

Cited by 25 publications

(30 citation statements)

References 54 publications

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“…For complementation of the pta mutant, we cloned pta together with its native promoter (intergenic region upstream of the ackA-pta operon) on a medium copy number plasmid (pA15 ori). All strains carried plasmid pNF106 (SC101 ori) for doxycycline/anhydrotetracyclineinducible expression of gfp (39 Shigella Infections. HeLa cells, CaCo-2 cells, and HUVECs were infected with polylysine-treated Shigella.…”

Section: Methodsmentioning

confidence: 99%

Shigella reroutes host cell central metabolism to obtain high-flux nutrient supply for vigorous intracellular growth

Kentner

Martano

Callon

et al. 2014

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

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Shigella flexneri proliferate in infected human epithelial cells at exceptionally high rates. This vigorous growth has important consequences for rapid progression to life-threatening bloody diarrhea, but the underlying metabolic mechanisms remain poorly understood. Here, we used metabolomics, proteomics, and genetic experiments to determine host and Shigella metabolism during infection in a cell culture model. The data suggest that infected host cells maintain largely normal fluxes through glycolytic pathways, but the entire output of these pathways is captured by Shigella, most likely in the form of pyruvate. This striking strategy provides Shigella with an abundant favorable energy source, while preserving host cell ATP generation, energy charge maintenance, and survival, despite ongoing vigorous exploitation. Shigella uses a simple three-step pathway to metabolize pyruvate at high rates with acetate as an excreted waste product. The crucial role of this pathway for Shigella intracellular growth suggests targets for antimicrobial chemotherapy of this devastating disease.infectious diseases | host-pathogen interactions

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Section: Methodsmentioning

confidence: 99%

Shigella reroutes host cell central metabolism to obtain high-flux nutrient supply for vigorous intracellular growth

Kentner

Martano

Callon

et al. 2014

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

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“…Distinct from its inhibitory role in the programmed cell death, APIP was recently shown to act as an enzyme in the methionine salvage pathway (2,4). The amino acid sequence of human APIP exhibits 23-26% identity to the previously characterized Bacillus and yeast 5-methylthioribulose-1-phosphate dehydratase (MtnB) (4).…”

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confidence: 99%

“…The methionine salvage pathway converts MTA (5-methylthioadenosine) to methionine through six enzymatic reaction steps, and MtnB is the third enzyme in the pathway and catalyzes the dehydration of MTRu-1-P (5-methylthioribulose-1-phosphate) to DK-MTP-1-P (2,3-diketo-5-methylthiopentyl-1-phosphate) (Fig. 1B) (4,5). In the absence of methionine, cells supplemented with MTA exhibit decreased viability when APIP expression is reduced (2,4).…”

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confidence: 99%

Structural and biochemical basis for the inhibition of cell death by APIP, a methionine salvage enzyme

Kang

Hong

Lee

et al. 2013

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

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APIP, Apaf-1 interacting protein, has been known to inhibit two main types of programmed cell death, apoptosis and pyroptosis, and was recently found to be associated with cancers and inflammatory diseases. Distinct from its inhibitory role in cell death, APIP was also shown to act as a 5-methylthioribulose-1-phosphate dehydratase, or MtnB, in the methionine salvage pathway. Here we report the structural and enzymatic characterization of human APIP as an MtnB enzyme with a K m of 9.32 μM and a V max of 1.39 μmol min −1 mg −1 . The crystal structure was determined at 2.0-Å resolution, revealing an overall fold similar to members of the zinc-dependent class II aldolase family. APIP/MtnB exists as a tetramer in solution and exhibits an assembly with C4 symmetry in the crystal lattice. The pocket-shaped active site is located at the end of a long cleft between two adjacent subunits. We propose an enzymatic reaction mechanism involving Glu139* as a catalytic acid/base, as supported by enzymatic assay, substrate-docking study, and sequence conservation analysis. We explored the relationship between two distinct functions of APIP/MtnB, cell death inhibition, and methionine salvage, by measuring the ability of enzymatic mutants to inhibit cell death, and determined that APIP/MtnB functions as a cell death inhibitor independently of its MtnB enzyme activity for apoptosis induced by either hypoxia or etoposide, but dependently for caspase-1-induced pyroptosis. Our results establish the structural and biochemical groundwork for future mechanistic studies of the role of APIP/MtnB in modulating cell death and inflammation and in the development of related diseases.squamous carcinoma | systemic inflammatory response syndrome T he programmed death of dangerous cells, either infected or transformed, has critical importance for the survival of the multicellular organism and therefore is also of great medical relevance. APIP, Apaf-1 interacting protein, was initially identified as an inhibitor of apoptotic cell death induced by hypoxia/ ischemia and cytotoxic drugs (1). Recently APIP was also shown to inhibit pyroptosis, an inflammatory form of cell death, induced by Salmonella infection (2). Thus, APIP has been implicated in two major types of programmed cell death: apoptosis and pyroptosis. In apoptosis, APIP inhibits the mitochondrial pathway involving caspase-9 but not the receptor pathway involving caspase-8 (1, 3). In pyroptosis, APIP's inhibitory function was recently revealed in a functional genetic screen for the SNP associated with increased caspase-1-mediated cell death in response to Salmonella infection (2) and subsequently confirmed by cell viability assays (2, 4). Intriguingly, other SNPs near APIP were found in patients suffering from systemic inflammatory response syndrome (2), which further implicates APIP in inflammation.Distinct from its inhibitory role in the programmed cell death, APIP was recently shown to act as an enzyme in the methionine salvage pathway (2, 4). The amino acid sequence of human APIP exhib...

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“…Unexpectedly, the mechanism for this effect appeared to occur through APIP's previously unexplored role as a methionine salvage enzyme, 12 which has since been confirmed by two other labs. 13,14 This example demonstrates how cellular GWAS of a complex trait (pyroptosis) can lead to unexpected connections among cell biological pathways and also connections to risk and outcomes of related diseases.…”

Section: Expanding the Cell Biologist's Toolbox To Cellular Gwasmentioning

confidence: 99%

The marriage of quantitative genetics and cell biology

Ko¹,

Jaslow²

2014

BioArchitecture

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icrotubules play a central role in many essential cellular processes, including chromosome segregation, intracellular transport, and cell polarity. As these dynamic polymers are crucial components of eukaryotic cellular architecture, we were surprised by our recent discovery that a common human genetic difference leads to variation in microtubule stability in cells from different people. A single nucleotide polymorphism (SNP) near the TUBB6 gene, encoding class V β-tubulin, is associated with the expression level of this protein, which reduces microtubule stability at higher levels of expression. We discuss the novel cellular GWAS (genome-wide association study) platform that led to this discovery of natural, common variation in microtubule stability and the implications this finding may have for human health and disease, including cancer and neurological disorders. Furthermore, our generalizable approach provides a gateway for cell biologists to help interpret the functional consequences of human genetic variation.

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Functional Identification of APIP as Human mtnB, a Key Enzyme in the Methionine Salvage Pathway

Cited by 25 publications

References 54 publications

Shigella reroutes host cell central metabolism to obtain high-flux nutrient supply for vigorous intracellular growth

Shigella reroutes host cell central metabolism to obtain high-flux nutrient supply for vigorous intracellular growth

Structural and biochemical basis for the inhibition of cell death by APIP, a methionine salvage enzyme

The marriage of quantitative genetics and cell biology

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