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.     

PILRα and PILRβ have a siglec fold and provide the basis of binding to sialic acid

Paired immunoglobulin-like type 2 receptor α (PILRα) and β (PILRβ) belong to the PILR family and are related to innate immune regulation in various species. Despite their high sequence identity, PILRα and PILRβ are shown to have variant sialic acid (SA) binding avidities. To explore the molecular basis of this interaction, we solved the crystal structures of PILRα and PILRβ at resolutions of 1.6 Å and 2.2 Å, respectively. Both molecules adopt a typical siglec fold but use a hydrophobic bond to substitute the siglec-specific disulfide linkage for protein stabilization. We further used HSV-1 glycoprotein B (gB) as a representative molecule to study the PILR–SA interaction. Deploying site-directed mutagenesis, we demonstrated that three residues (Y2, R95, and W108) presented on the surface of PILRα form the SA binding site equivalent to those in siglecs but are arranged in a unique linear mode. PILRβ differs from PILRα in one of these three residues (L108), explaining its inability to engage gB. Mutation of L108 to tryptophan in PILRβ restored the gB-binding capacity. We further solved the structure of this PILRβ mutant complexed with SA, which reveals the atomic details mediating PILR/SA recognition. In comparison with the free PILR structures, amino acid Y2 oriented variantly in the complex structure, thereby disrupting the linear arrangement of PILR residues Y2, R95, and W108. In conclusion, our study provides significant implications for the PILR–SA interaction and paves the way for understanding PILR-related ligand binding.There are two members in the paired immunoglobulin-like type 2 receptor (PILR) family: PILRα and PILRβ (1). Both are expressed as a monomeric transmembrane protein with a single V-set Ig-like (IgV) extracellular domain (2). In the cytoplasmic tail, PILRα bears two immunoreceptor tyrosine-based inhibitory motifs that deliver inhibitory signals by recruiting SHP-1 and SHP-2, whereas PILRβ binds to the DAP-12 molecule bearing a tyrosine-based activation motif (ITAM) for transduction of activating signals (3). Several studies in mice showed that the former is always related to the inhibition of the immune system, whereas the latter plays pivotal roles in activating natural killer (NK) cells and dendritic cells (DCs) and is involved in the mass production of inflammatory factors during infection (4). In addition, a recent report also demonstrated that PILRα could function to regulate neutrophil infiltration via activation of integrins during inflammation (5). Reminiscent of these immune-modulation functions, both receptors are largely expressed on cells of the immune system, especially those of the myeloid lineage such as monocytes, DCs, and macrophages (6, 7). PILRβ is also abundantly expressed on NK cells (6).To exert their regulatory functions, the PILR receptors require engagement of specific ligands via their extracellular domains. Mouse CD99 is the only identified ligand for PILRβ to date (8). However, a set of host molecules, including mouse CD99 (8), PILR-associating neural protein (9), neuronal differentiation and proliferation factor-1 (NPDC1) (7), and collectin-12 (7), can recognize PILRα, implicating important roles of PILRα in diverse processes. In addition to the natural host ligands, PILRα is also hijacked by some viruses, such as HSV-1 (10) and porcine pseudorabies virus (11), for cell entry. The viral surface glycoprotein B (gB) is shown to recognize PILRα and mediate the virus infection (10, 11). Elucidation of the mechanisms underlying these ligand–receptor interactions is important in understanding PILR-involved physiological processes. Current knowledge on these interactions, however, only indicates the involvement of sialic acid (SA) moieties residing on the ligand surface in PILR engagement (7, 8, 12). This character drew parallels between PILRs and siglecs, a family of SA-binding Ig-type lectins (13). Nevertheless, PILRs, unlike siglec molecules, are of low SA-binding avidity and fail to bind to single SA sugars in a glycan microarray (14). The molecular basis of the PILR–SA interaction is an interesting, yet unresolved, issue.In this study, we first solved both PILRα and PILRβ structures, demonstrating that they have siglec-like folds but maintain protein stability by hydrophobic interactions, different from siglecs, which have disulfide bonds. We also developed a Biacore-based assay for quantitative calculations of the PILR–SA interaction based on HSV-1 gB protein. A triresidue motif consisting of Y2, R95, and W108 was identified as a key SA-binding site in PILRα, and a W108L substitution in the motif was shown to be responsible for the inability of PILRβ to interact with gB. We further reported a complex structure of SA bound to a PILRβ L108W mutant protein, thereby presenting the atomic details mediating the PILR–SA interaction.     

A facile preparation method for new two-component supramolecular hydrogels and their performances in adsorption,catalysis, and stimuli-response

In this study, we prepared a novel multifunctional two-component supramolecular hydrogel (T-G hydrogel) via two organic molecules in ethanol/water mixed solvents. In addition, we prepared gold nanoparticle/T-G (AuNPs/T-G) composite hydrogels using T-G hydrogel as a template for stabilizing AuNPs by adding HAuCl₄ and NaBH₄ during the heating and cooling process of T-G hydrogels. The morphology and microstructure of the as-prepared hydrogels were characterized using SEM, TEM, XRD, and FT-IR. The hydrogels prepared by solutions that contained different ethanol/water volume ratios exhibited different microstructures, such as sheets, strips, and rods. The obtained T-G hydrogels exhibited a sensitive response to pH changes in the process of sol–gel transformation and showed good adsorption properties for model organic dyes. In the presence of NaBH₄, the obtained AuNP/T-G composite hydrogels exhibited the excellent catalytic performance for 4-nitrophenol (4-NP) degradation. Thus, the current research provides new clues in developing new multifunctional two-component supramolecular gel materials and exhibits potential applications for wastewater treatment.

New two-component supramolecular hydrogels were prepared via a self-assembly process, demonstrating potential applications in adsorption and catalysis as well as sensor materials.     

Risk-based,response-adapted therapy for early-stage extranodal nasal-type NK/T-cell lymphoma in the modern chemotherapy era: A China Lymphoma Collaborative Group study

We aimed to determine the survival benefits of chemotherapy (CT) added to radiotherapy (RT) in different risk groups of patients with early-stage extranodal nasal-type NK/T-cell lymphoma (ENKTCL), and to investigate the risk of postponing RT based on induction CT responses. A total of 1360 patients who received RT with or without new-regimen CT from 20 institutions were retrospectively reviewed. The patients had received RT alone, RT followed by CT (RT + CT), or CT followed by RT (CT + RT). The patients were stratified into different risk groups using the nomogram-revised risk index (NRI). A comparative study was performed using propensity score-matched (PSM) analysis. Adding new-regimen CT to RT (vs RT alone) significantly improved overall survival (OS, 73.2% vs 60.9%, P < .001) and progression-free survival (PFS, 63.5% vs 54.2%, P < .001) for intermediate-risk/high-risk patients, but not for low-risk patients. For intermediate-risk/high-risk patients, RT + CT and CT + RT resulted in non-significantly different OS (77.7% vs 72.4%; P = .290) and PFS (67.1% vs 63.1%; P = .592). For patients with complete response (CR) after induction CT, initiation of RT within or beyond three cycles of CT resulted in similar OS (78.2% vs 81.7%, P = .915) and PFS (68.2% vs 69.9%, P = .519). For patients without CR, early RT resulted in better PFS (63.4% vs 47.6%, P = .019) than late RT. Risk-based, response-adapted therapy involving early RT combined with CT is a viable, effective strategy for intermediate-risk/high-risk early-stage patients with ENKTCL in the modern treatment era.     

Mitochondrial genome variation in male LHON patients with the m.11778G > A mutation

Metabolic Brain Disease - Leber hereditary optic neuropathy (LHON) is a mitochondrial disorder with symptoms limited to a single tissue, optic nerve, resulting in vision loss. In the majority of...     

Characteristic and prognosis of acute large vessel occlusion in anterior and posterior circulation after endovascular treatment: the ANGEL registry real world experience

There were limited studies comparing the anterior (AC) and posterior (PC) circulation acute ischemic strokes (AIS). Our study aimed to evaluate distinct features of AC and PC strokes regarding clinical, vascular risk, pathogenesis and outcome factors after endovascular procedures. This multicenter prospective study registered 873 patients with acute large occlusion of anterior circulation stroke (ACS) and posterior circulation stroke (PCS). Patients who underwent endovascular procedures were included in this study. The differences in ACS and PCS regarding baseline characteristics, post-operative intracranial hemorrhage and outcomes were evaluated. A total of 741 patients were included in the data analysis. Intravenous thrombolysis (31.5%), atrial fibrillation (22.7%) and stent thrombectomy (82.4%) were more frequently observed in ACS patients. While higher NIHSS score, hypertension (67.6%) and balloon angioplasty (20.7%) were more prevalent in PCS patients. Symptomatic intracranial hemorrhage was more common in ACS (7.4% vs 2.8%). However, a 3-month follow-up outcomes were better in ACS with higher functional independence and low mortality rate than PCS (46.8% vs 30.3% and 16.4% vs 33.8%, respectively, P?<?0.01). In this large prospective study, there were significant differences in the pathogenesis of stroke and treatment procedure between ACS and PCS which influence the clinical outcome. These findings could lead to a tailored clinical procedures and treatment strategies to improve the prognosis in both groups.