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.     

Appendiceal Skip Inflammation and Ulcerative Colitis

In recent decades, the appearance of inflammation near the appendix in patients with distal ulcerative colitis (UC) has been highlighted. Many epidemiological studies have confirmed the link between appendectomy and decreased incidence of UC. However, the clinical significance of appendiceal orifice inflammation (AOI) or peri-appendiceal red patch (PARP) as a “skip lesion” in UC has not been well elucidated. In this review, we summarized the literature regarding AOI/PARP and the role of this lesion in relation to UC. Since the appendiceal “skip area” in UC was first described in 1958, several reports using histologic examination of colectomy specimens and colonoscopy have been published. AOI/PARP has been more frequently associated with distal, mild UC than extensive, severe disease. Although it is still controversial, AOI/PARP seems to have little prognostic implication in the disease course of UC, including remission, relapse and proximal disease extension. However, some case reports have raised the possibility of a relationship between AOI/PARP and appendiceal neoplasms such as appendiceal cystadenoma and appendiceal adenocarcinoma. In addition, some investigators have treated UC patients who were resistant to conventional medical therapy with appendectomy and have reported inconsistent therapeutic effect. Further research may lead to the discovery of etiologic and pathogenic roles of appendiceal inflammation in UC.     

Effect of Mineral Trioxide Aggregate Surface Treatments on Morphology and Bond Strength to Composite Resin

Introduction

The aim of this study was to evaluate the micromorphologic changes that accompany different surface treatments on mineral trioxide aggregate (MTA) and their effect on the bond strength to the composite resin with 4 adhesive systems.

Methods

Three types of MTA cement, ProRoot MTA (WMTA) (Dentsply, Tulsa, OK), MTA Angelus (AMTA) (Angelus, Londrina, PR, Brazil), and Endocem MTA (EMTA) (Maruchi, Wonju, Korea), were prepared and stored for a week to encourage setting. Surface treatment was performed using phosphoric acid or self-etch primer, and an untreated MTA surface was prepared as a control. The surface changes were observed using scanning electron microscopy. MTA surfaces were bonded with 4 adhesive systems, including Scotchbond Multipurpose (3M ESPE, St Paul, MN), Single Bond 2 (3M ESPE), Clearfil SE BOND (Kuraray, Osaka, Japan), and AdheSE One F (Ivoclar Vivadent, Schaan, Liechtenstein), to evaluate the adhesive effectiveness of MTA followed by composite resin restoration. The shear bond strength of the polymerized specimens was tested.

Results

For WMTA and AMTA, untreated surfaces showed an irregular crystalline plate with clusters of globular aggregate particles. For EMTA, the untreated surface presented a reticular matrix with acicular crystals. After surface treatment, superficial crystalline structures were eroded regardless of the MTA cement and adhesive system used. WMTA bonded significantly more strongly than AMTA and EMTA, regardless of the adhesive system used. In the WMTA and AMTA groups, AdheSE One F showed the highest bond strength to the composite. For EMTA, no significant differences were found across adhesive systems.

Conclusions

Acidic treatment of the MTA surface affected the micromorphology and the bond strength to the composite. Within the limitations of this study, using a 1-step self-etch adhesive system might result in a strong bond to WMTA when the composite resin restoration is required over MTA cement.     

Diagnostic utility of the soluble triggering receptor expressed on myeloid cells-1 in bronchoalveolar lavage fluid from patients with bilateral lung infiltrates

Background  

Differential diagnosis of patients with bilateral lung infiltrates remains a difficult problem for intensive care clinicians. Here we evaluate the diagnostic role of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) in bronchoalveolar lavage (BAL) specimens from patients with bilateral lung infiltrates.     

Clinical significance of healthcare-associated infections in community-onset Escherichia coli bacteraemia

BACKGROUND: The increasing antimicrobial resistance of Escherichia coli infection is of great concern, even for community-onset infections. METHODS: We conducted a retrospective cohort study of patients with E. coli bacteraemia who visited the emergency department of the Samsung Medical Center from February 2002 to December 2005 to identify the risk factors for mortality and association between healthcare-associated (HCA) infection and mortality. We classified community-onset E. coli bacteraemia into community-acquired (CA) and HCA infections. RESULTS: A total of 508 patients with E. coli bacteraemia were enrolled (mean age, 61.8 +/- 14.3 years; male/female, 191:317). The HCA E. coli bacteraemia had significantly higher severity of illness and higher antimicrobial resistance rate than CA bacteraemia. The overall 30-day mortality rate was 13.6% (69/508) and the mortality of HCA infections was significantly higher than that of CA infections (26.4% versus 9.6%, P < 0.001). In multivariate analysis, high Charlson's co-morbidity index (OR 4.84, 95% CI 2.14-10.95, P < 0.001), high Pitt bacteraemia score (OR 32.03, 95% CI 13.08-74.43, P < 0.001), presentation with acute renal failure (OR 4.11, 95% CI 1.90-8.89, P < 0.001) and HCA bacteraemia (OR 2.34, 95% CI 1.09-5.01, P = 0.030) were found to be the significant risk factors for 30-day mortality in E. coli bacteraemia. CONCLUSIONS: The mortality rate of HCA E. coli bacteraemia was higher than twice that of CA bacteraemia and HCA bacteraemia was one of the significant risk factors for mortality, even after adjusting for a large number of potential confounders.     

The Patterns of Limb Length,Height, Weight and Body Mass Index Changes after Total Knee Arthroplasty

The objective of this retrospective review of 466 patients was to document changes in limb length, leg length discrepancy (LLD), height, weight, and body mass index (BMI) 1 year after TKA and the patterns of height, weight, and BMI during 5 years. To determine change patterns over 5 years, the data of 291 patients were analyzed and compared with those of age and gender-matched normal subjects. Limb length, height, and weight increased, BMI remained unchanged, and LLD decreased 1 year after TKA. The bilateral group had a greater height increase and lower rate of LLD. Preoperative mechanical tibiofemoral angle was related to limb length increase, and patients with a smaller preoperative BMI showed more weight gain. During the 5 years, weight and BMI at 1 year were maintained, but height diminished, while the healthy population showed a decreasing trend in weight.