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

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.The 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 exhibits 23–26% identity to the previously characterized Bacillus and yeast 5-methylthioribulose-1-phosphate dehydratase (MtnB) (4). 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). These studies indicate that APIP is an MtnB enzyme in the methionine salvage pathway.Open in a separate windowFig. 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 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.The methionine salvage pathway is found in all organisms, from bacteria to plants and animals (6). The role of this pathway is to recycle MTA, which is a by-product of the polyamine synthetic process, back to methionine (Fig. 1B). The methionine salvage pathway is beneficial as a means of recycling the sulfur present in MTA because assimilation of sulfur is thermodynamically costly (6). The metabolic importance of the pathway is underscored in humans because methionine is one of the essential amino acids needed to be provided through the diet, in which it is one of the most limiting amino acids (6). Recently, the methionine salvage pathway attracted medical interest because it was implicated in cell death and inflammation and diseases associated with these processes. For example, metabolites such as MTA and 2-keto-4-methylthiobutyrate (KMTB) have effects of apoptosis induction (6–9). MTA was also shown to induce caspase-1–dependent pyroptosis in the inflammatory response to bacterial infection (2). In addition, the 5-methylthioadenosine phosphorylase (MTAP, which catalyzes the first step) is a tumor suppressor implicated in a various human cancers (6, 10), and aci-reducton dioxygenase 1 (ADI1, also called MtnD, which catalyzes the fifth step) has a similar role in prostate cancer (11, 12). Human APIP/MtnB, which is the focus of the present study, is another example of a methionine salvage enzyme that is implicated in cell death and inflammation. APIP/MtnB was recently reported to be up-regulated in squamous carcinoma cells from tongue and larynx (13) and down-regulated in the cells and tumors of non–small-cell lung carcinoma (14). In addition, APIP/MtnB is implicated in inflammatory conditions that likely involve caspase-1–dependent pyroptosis, such as systemic inflammatory response syndrome (2).Studies of APIP/MtnB to date have focused mainly on its functional role either in cell death or in methionine salvage. To gain a better understanding of APIP/MtnB at a molecular and biochemical level, we carried out a structural and biochemical characterization in this study. The MtnB enzyme activity of APIP was verified by an in vitro enzyme assay. In addition, the crystal structure was determined at 2.0-Å resolution, which revealed details of the active site architecture and led to a proposed catalytic mechanism. Furthermore, we explored the relationship between two distinct functions of APIP/MtnB, cell death inhibition, and methionine salvage, by testing its enzymatic mutants derived from the crystal structure for their ability to inhibit two main types of programmed cell death: pyroptosis and apoptosis.     

A new Centiloid method for 18F-florbetaben and 18F-flutemetamol PET without conversion to PiB

European Journal of Nuclear Medicine and Molecular Imaging - We developed a new method to directly calculate Centiloid (CL) units of 18F-florbetaben (FBB) and 18F-flutemetamol (FMM) without...     

Short-term dual antiplatelet therapy (DAPT) followed by P2Y12 monotherapy versus traditional DAPT in patients undergoing percutaneous coronary intervention: meta-analysis and viewpoint

Journal of Thrombosis and Thrombolysis - The optimal duration dual antiplatelet therapy (DAPT) after percutaneous coronary intervention (PCI) is subject to debate. A short-duration DAPT (one month...     

Clinical features and treatment outcomes of limited-stage mantle cell lymphoma: Consortium for Improving Survival of Lymphoma report

Annals of Hematology - Limited-stage (Ann Arbor stage I or II) mantle cell lymphoma (MCL) is an extremely rare disease. Thus, there is little data on the clinical features and treatment outcomes of...     

Tumor Treated by Endoscopy

Background

This study was conducted to examine the clinical usefulness and efficacy of endoscopic curettage on benign bone tumor.

Methods

Thirty-two patients (20 men and 12 women) with benign bone tumor were included in the study. The patients were aged between five and 76 years; the mean follow-up period was 27.05 months (range, 9.6 to 39.9 months). The primary sites include simple bone cyst (9 cases), fibrous dysplasia (6 cases), enchondroma (5 cases), non-ossifying fibroma (4 cases), bone infarct (3 cases), aneurysmal bone cyst (1 case), chondroblastoma (1 case), osteoblastoma (1 case), intraosseous lipoma (1 case), and Brodie abscess (1 case). A plain radiography was performed to assess the radiological recovery. Radiological outcomes, including local recurrence and bone union, were evaluated as excellent, good, poor, and recurred.

Results

In our series, there were 27 cases (84.4%) of good or better outcomes, six cases (18.8%) of complications (4 local recurrence, 1 wound infection, and 1 pathologic fracture).

Conclusions

Our results showed that endoscopic curettage and bone graft had a lower rate of recurrence and a higher cure rate in cases of benign bone tumor. It can, therefore, be concluded that endoscopic curettage and bone graft might be good treatment modalities for benign bone tumors.     

Tissue-Specific Antioxidant Responses in Pale Chub (Zacco platypus) Exposed to Copper and Benzo[a]pyrene

In this study, antioxidant responses including lipid peroxidation (LPO), superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST), were evaluated in the liver, gill and muscle tissues of pale chub (Zacco platypus) exposed to copper (Cu) and benzo[a]pyrene (BaP). Cu exposure induced significant antioxidant responses in Z. platypus, particularly in the liver, whereas BaP exposure had a negligible effect. Following Cu exposure, both SOD and CAT activity increased in a concentration-dependent manner, showing significant correlations with malondialdehyde (MDA) levels as a measure of LPO (r = 0.646 and 0.663, respectively). SOD, CAT and GST mRNA levels were also enhanced following Cu exposure, except at 20 μg L^?1, although significant correlations with antioxidant enzyme activities were not found. The results of this study suggest that combined information on SOD and CAT activities together with LPO levels in the liver could be a useful indicator for assessing oxidative stress in freshwater fish.     

Chemically induced Salmonella enteritidis ghosts as a novel vaccine candidate against virulent challenge in a rat model

Salmonella enteritidis ghosts (SEGs), non-living empty bacterial cell envelopes were generated by using the minimum inhibitory concentration (MIC) of sodium hydroxide (NaOH) and investigated as a vaccine candidate in rats. To determine the immunogenicity and protective efficacy of SEG vaccine, rats were divided into four groups: group A (non-vaccinated control), group B (orally vaccinated), group C (intramuscularly vaccinated) and group D (intramuscularly vaccinated with complete Freund's adjuvant). Vaccination of rats with SEGs induced significant immune responses before and after virulent challenge. Rats vaccinated with SEGs showed significant increases in serum IgG antibodies after challenging with virulent S. enteritidis on week 8 and week 10 (P < 0.01). During the vaccination period, groups B, C and D showed significantly higher serum bactericidal activity (SBA) compared to group A (P < 0.01). Most importantly, bacterial loads in vaccinated groups were significantly lower than in the non-vaccinated group (P < 0.01). In conclusion, these results show that the chemically induced SEGs as a vaccine candidate against virulent challenge.