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

Abstract

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...

Revisiting the methionine salvage pathway and its paralogues

“…It has also close counterparts in eukaryotes (Pirkov et al, 2008). Gene APIP, for example, is the counterpart in H. sapiens where it is involved in the control of the key process of apoptosis (Kang et al, 2014a). Two different types of reactions have been recruited to generate 1,2-dihydroxy-3-keto-5methylthiopentene.…”

APIP, an ERBB3-binding partner, stimulates erbB2-3 heterodimer formation to promote tumorigenesis

“…A recent report showed that APIP exists as a tetrameric complex which is essential for the methionine salvage pathway [32]. In gastric cancer, APIP also forms a tetrameric complex and the inhibition of APIP/APIP interactions blocks its oncogenic activity.…”

Molecular heterogeneity in the novel fusion gene APIP-FGFR2: Diversity of genomic breakpoints in gastric cancer with high-level amplifications at 11p13 and 10q26

“…APIP encodes the APAF1-interacting protein, which has methylthioribulose 1-phosphate dehydratase activity and is involved in the methionine salvage pathway (28). APIP deficiency is associated with cell death and cancer (29,30).…”