Structural and biochemical basis for the inhibition of cell death by APIP, a methionine salvage enzyme Wonchull Kanga,1, Se Hoon Hongb,1, Hye Min Leea, Na Yeon Kima, Yun Chan Lima, Le Thi My Lea, Bitna Limb, Hyun Chul Kima, Tae Yeon Kima, Hiroki Ashidac, Akiho Yokotac, Sang Soo Hahd, Keun Ho Chuna, Yong-Keun Jungb,2, and Jin Kuk Yanga,2 aDepartment of Chemistry, College of Natural Sciences, Soongsil University, Seoul 156-743, Korea; bSchool of Biological Science/Bio-Max Institute, Seoul National University, Seoul 151-742, Korea; cGraduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan; and dDepartment of Chemistry and Research Institute for Basic Sciences, Kyung Hee University, Seoul 130-701, Korea Edited by James A. Wells, University of California, San Francisco, CA, and approved December 4, 2013 (received for review May 10, 2013) APIP, Apaf-1 interacting protein, has been known to inhibit two MTA (5-methylthioadenosine) to methionine through six en- main types of programmed cell death, apoptosis and pyropto- zymatic reaction steps, and MtnB is the third enzyme in the sis, and was recently found to be associated with cancers and pathway and catalyzes the dehydration of MTRu-1-P (5-meth- inflammatory diseases. Distinct from its inhibitory role in cell ylthioribulose-1-phosphate) to DK-MTP-1-P (2,3-diketo-5-meth- death, APIP was also shown to act as a 5-methylthioribulose-1- ylthiopentyl-1-phosphate) (Fig. 1B) (4, 5). In the absence of phosphate dehydratase, or MtnB, in the methionine salvage path- methionine, cells supplemented with MTA exhibit decreased way. Here we report the structural and enzymatic characterization viability when APIP expression is reduced (2, 4). These studies ofhumanAPIPasanMtnBenzymewithaKm of 9.32 μM and a Vmax indicate that APIP is an MtnB enzyme in the methionine salvage of 1.39 μmol min−1 mg−1. The crystal structure was determined at pathway. 2.0-Å resolution, revealing an overall fold similar to members of the The methionine salvage pathway is found in all organisms, zinc-dependent class II aldolase family. APIP/MtnB exists as a tetra- from bacteria to plants and animals (6). The role of this pathway mer in solution and exhibits an assembly with C4 symmetry in the is to recycle MTA, which is a by-product of the polyamine syn- crystal lattice. The pocket-shaped active site is located at the end of thetic process, back to methionine (Fig. 1B). The methionine a long cleft between two adjacent subunits. We propose an enzy- salvage pathway is beneficial as a means of recycling the sulfur matic reaction mechanism involving Glu139* as a catalytic acid/base, present in MTA because assimilation of sulfur is thermody- as supported by enzymatic assay, substrate-docking study, and se- namically costly (6). The metabolic importance of the pathway is quence conservation analysis. We explored the relationship be- underscored in humans because methionine is one of the es- tween two distinct functions of APIP/MtnB, cell death inhibition, sential amino acids needed to be provided through the diet, in and methionine salvage, by measuring the ability of enzymatic which it is one of the most limiting amino acids (6). Recently, the mutants to inhibit cell death, and determined that APIP/MtnB func- methionine salvage pathway attracted medical interest because it tions as a cell death inhibitor independently of its MtnB enzyme was implicated in cell death and inflammation and diseases as- activity for apoptosis induced by either hypoxia or etoposide, but sociated with these processes. For example, metabolites such as dependently for caspase-1-induced pyroptosis. Our results establish MTA and 2-keto-4-methylthiobutyrate (KMTB) have effects of the structural and biochemical groundwork for future mechanistic apoptosis induction (6–9). MTA was also shown to induce cas- studies of the role of APIP/MtnB in modulating cell death and in- pase-1–dependent pyroptosis in the inflammatory response to flammation and in the development of related diseases. Significance squamous carcinoma | systemic inflammatory response syndrome Apaf-1 interacting protein (APIP) inhibits two main types of he programmed death of dangerous cells, either infected or programmed cell death: apoptosis and pyroptosis. In addition, Ttransformed, has critical importance for the survival of the APIP is a 5-methylthioribulose-1-phosphate dehydratase (MtnB) multicellular organism and therefore is also of great medical in the methionine salvage pathway. We verified its enzymatic relevance. APIP, Apaf-1 interacting protein, was initially iden- activity directly through an enzyme assay and determined its tified as an inhibitor of apoptotic cell death induced by hypoxia/ high-resolution structure. Furthermore, we explored the re- ischemia and cytotoxic drugs (1). Recently APIP was also shown lationship between two distinct functions of APIP/MtnB, cell to inhibit pyroptosis, an inflammatory form of cell death, in- death inhibition and methionine salvage, and determined that duced by Salmonella infection (2). Thus, APIP has been impli- it functions as a cell death inhibitor independently of its MtnB cated in two major types of programmed cell death: apoptosis enzyme activity for apoptosis, but dependently for caspase-1– and pyroptosis. In apoptosis, APIP inhibits the mitochondrial induced pyroptosis. Our results provide groundwork for stud- pathway involving caspase-9 but not the receptor pathway in- ies of the role of APIP/MtnB in development of cancers and volving caspase-8 (1, 3). In pyroptosis, APIP’s inhibitory function inflammatory diseases. was recently revealed in a functional genetic screen for the SNP associated with increased caspase-1–mediated cell death in re- Author contributions: Y.-K.J. and J.K.Y. designed research; W.K., S.H.H., H.M.L., N.Y.K., Salmonella Y.C.L., L.T.M.L., B.L., H.C.K., T.Y.K., H.A., A.Y., S.S.H., and K.H.C. performed research; W.K., sponse to infection (2) and subsequently confirmed by S.H.H., H.A., A.Y., Y.-K.J., and J.K.Y. analyzed data; and Y.-K.J. and J.K.Y. wrote the paper. APIP cell viability assays (2, 4). Intriguingly, other SNPs near were The authors declare no conflict of interest. found in patients suffering from systemic inflammatory response This article is a PNAS Direct Submission. syndrome (2), which further implicates APIP in inflammation. Data deposition: The atomic coordinates and structure factors have been deposited in the Distinct from its inhibitory role in the programmed cell death, Protein Data Bank, www.pdb.org (PDB ID code 4M6R). APIP was recently shown to act as an enzyme in the methionine 1W.K. and S.H.H. contributed equally to this work. salvage pathway (2, 4). The amino acid sequence of human 2 To whom correspondence may be addressed. E-mail: [email protected] or jinkukyang@ APIP exhibits 23–26% identity to the previously characterized ssu.ac.kr. Bacillus and yeast 5-methylthioribulose-1-phosphate dehy- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. dratase (MtnB) (4). The methionine salvage pathway converts 1073/pnas.1308768111/-/DCSupplemental. E54–E61 | PNAS | Published online December 23, 2013 www.pnas.org/cgi/doi/10.1073/pnas.1308768111 Downloaded by guest on September 29, 2021 PNAS PLUS Fig. 1. APIP as an MtnB enzyme in the methionine salvage pathway. (A) Initial reaction rate was plotted at seven different concentrations of the substrate MTRu-1-P for Michaelis-Menten kinetic analysis. Data represent mean values with SE from three independent measurements. (B) Methionine salvage pathway BIOCHEMISTRY characterized in Homo sapiens and Saccharomyces cerevisiae converts MTA to methionine (Met) through the common six enzymatic reactions. Dashed line represents B. subtilis methionine salvage reaction steps distinct from H. sapiens and S. cerevisiae. Gray colored enzymatic steps and metabolites represent biochemical links that are not conceptually part of the methionine salvage pathway. AdoMet, S-adenosyl-l-methionine; dAdoMet, decarboxylated AdoMet; DHK-MTPene, 1,2-dihydroxy-3-keto-5-methylthiopentene; HK-MTPenyl-1-P, 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate; Met, l-methionine; MTOB, 4-methylthio-2-oxobutyrate; MTR, 5-methylthioribose. bacterial infection (2). In addition, the 5-methylthioadenosine (4), which was supported by cell viability assays from two in- phosphorylase (MTAP, which catalyzes the first step) is a tumor dependent studies, as noted above (2, 4). We tested the MtnB suppressor implicated in a various human cancers (6, 10), and aci- enzyme activity of human APIP in an in vitro enzyme assay. The reducton dioxygenase 1 (ADI1, also called MtnD, which catalyzes preparation of MTRu-1-P, the substrate of MtnB, was not suc- the fifth step) has a similar role in prostate cancer (11, 12). Hu- cessful because of the difficulties in purification after synthesis. So man APIP/MtnB, which is the focus of the present study, is an- MTRu-1-P was indirectly supplied to the assay system by addition other example of a methionine salvage enzyme that is implicated of MtnA enzyme, which precedes the MtnB reaction in the in cell death and inflammation. APIP/MtnB was recently repor- methionine salvage pathway, and its substrate, MTR-1-P (meth- B ted to be up-regulated in squamous carcinoma cells from tongue ylthioribose-1-phosphate) (Fig. 1 ). The concentration of and larynx (13) and down-regulated in the cells and tumors of MTRu-1-P was calculated from the equilibration constant be- non–small-cell lung carcinoma (14). In addition, APIP/MtnB is tween MTR-1-P and MTRu-1-P. More details are described in Materials and Methods implicated in inflammatory conditions that likely involve caspase- . Product formation was measured by – monitoring the increase in absorbance at 280 nm for the re- 1 dependent pyroptosis, such as systemic inflammatory response Bacillus syndrome (2).