Tsutomu Nakamura

Role of a BCL9-Related β-Catenin-Binding Protein, B9L, in Tumorigenesis Induced by Aberrant Activation of Wnt Signaling

Wnt signaling plays a crucial role in a number of developmental processes and in tumorigenesis. β-Catenin is stabilized by Wnt signaling and associates with the TCF/LEF family of transcription factors, thereby activating transcription of Wnt target genes. Constitutive activation of β-catenin-TCF–mediated transcription resulting from mutations in adenomatous polyposis coli (APC), β-catenin, or Axin is believed to be a critical step in tumorigenesis among divergent types of cancers. Here we show that the transactivation potential of the β-catenin-TCF complex is enhanced by its interaction with a BCL9-like protein, B9L, in addition to BCL9. We found that B9L is required for enhanced β-catenin-TCF–mediated transcription in colorectal tumor cells and for β-catenin–induced transformation of RK3E cells. Furthermore, expression of B9L was aberrantly elevated in about 43% of colorectal tumors, relative to the corresponding noncancerous tissues. These results suggest that B9L plays an important role in tumorigenesis induced by aberrant activation of Wnt signaling.

Oxidation of archaeal peroxiredoxin involves a hypervalent sulfur intermediate

The oxidation of thiol groups in proteins is a common event in biochemical processes involving disulfide bond formation and in response to an increased level of reactive oxygen species. It has been widely accepted that the oxidation of a cysteine side chain is initiated by the formation of cysteine sulfenic acid (Cys-SOH). Here, we demonstrate a mechanism of thiol oxidation through a hypervalent sulfur intermediate by presenting crystallographic evidence from an archaeal peroxiredoxin (Prx), the thioredoxin peroxidase from Aeropyrum pernix K1 (ApTPx). The reaction of Prx, which is the reduction of a peroxide, depends on the redox active cysteine side chains. Oxidation by hydrogen peroxide converted the active site peroxidatic Cys-50 of ApTPx to a cysteine sulfenic acid derivative, followed by further oxidation to cysteine sulfinic and sulfonic acids. The crystal structure of the cysteine sulfenic acid derivative was refined to 1.77 Å resolution with Rcryst and Rfree values of 18.8% and 22.0%, respectively. The refined structure, together with quantum chemical calculations, revealed that the sulfenic acid derivative is a type of sulfurane, a hypervalent sulfur compound, and that the Sγ atom is covalently linked to the Nδ1 atom of the neighboring His-42. The reaction mechanism is revealed by the hydrogen bond network around the peroxidatic cysteine and the motion of the flexible loop covering the active site and by quantum chemical calculations. This study provides evidence that a hypervalent sulfur compound occupies an important position in biochemical processes.

Crystal structure of peroxiredoxin from Aeropyrum pernix K1 complexed with its substrate, hydrogen peroxide.

Peroxiredoxin (Prx) reduces hydrogen peroxide and alkyl peroxides to water and corresponding alcohols, respectively. The reaction is dependent on a peroxidatic cysteine, whose sulphur atom nucleophilically attacks one of the oxygen atoms of the peroxide substrate. In spite of the many structural studies that have been carried out on this reaction, the tertiary structure of the hydrogen peroxide-bound form of Prx has not been elucidated. In this paper, we report the crystal structure of Prx from Aeropyrum pernix K1 in the peroxide-bound form. The conformation of the polypeptide chain is the same as that in the reduced apo-form. The hydrogen peroxide molecule is in close contact with the peroxidatic Cys50 and the neighbouring Thr47 and Arg126 side chain atoms, as well as with the main chain nitrogen atoms of Val49 and Cys50. Bound peroxide was also observed in the mutant C50S, in which the peroxidatic cysteine was replaced by serine. Therefore, the sulphur atom of the peroxidatic cysteine is not essential for peroxide binding, although it enhances the binding affinity. Hydrogen peroxide binds to the protein so that it fills the active site pocket. This study provides insight into the early stage of the Prx reaction.

Tertiary Structure and Carbohydrate Recognition by the Chitin-Binding Domain of a Hyperthermophilic Chitinase from Pyrococcus furiosus

A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both alpha and beta crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded beta-sheets, was composed of a typical beta-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein-carbohydrate recognition. This is important in the development of technology for the exploitation of biomass.

Dual mechanisms involved in development of diverse biological activities of islet-activating protein, pertussis toxin, as revealed by chemical modification of lysine residues in the toxin molecule

Islet-activating protein (IAP), pertussis toxin, is an oligomeric protein composed of an A-protomer and a B-oligomer. There seem to be at least two molecular mechanisms by which IAP exerts its various effects in vivo and in vitro. On the one hand, some of the effects were not significantly affected by acetamidination of the epsilon-amino groups of the lysine residues in the molecule. These include the activities in vitro (1) catalyzing ADP-ribosylation of one of the membrane proteins directly, (2) enhancing membrane adenylate cyclase activity in C6 cells, (3) reversing receptor-mediated inhibition of insulin or glycerol release from pancreatic islets or adipocytes, respectively, and the activities in vivo (4) inhibiting epinephrine-induced hyperglycemia, (5) potentiating glucose-induced hyperinsulinemia, (6) reducing hypertension and increasing the heart rate in genetically hypertensive rats. These activities are concluded to develop as a result of ADP-ribosylation catalyzed by the A-protomer which is rendered accessible to its intramembrane substrate thanks to the associated B-oligomer moiety. Thus, neither the enzymic activity of the A-protomer nor the transporting activity of the B-oligomer needs free amino groups of the lysine residues in the IAP molecule. On the other hand, additional effects of IAP, such as (1) mitogenic, (2) lymphocytosis-promoting, (3) histamine-sensitizing, (4) adjuvant and (5) vascular permeability increasing, were markedly suppressed by acetamidination of the intrapeptide lysine residues. The free epsilon-amino group of lysine would play an indispensable role in the firm (or divalent) attachment of the B-oligomer of IAP to the cell surface that is responsible for development of these activities.

Kinetic and crystallographic analyses of the catalytic domain of chitinase from Pyrococcus furiosus- the role of conserved residues in the active site.

The hyperthermostable chitinase from the hyperthermophilic archaeon Pyrococcusu2003furiosus has a unique multidomain structure containing two chitin‐binding domains and two catalytic domains, and exhibits strong crystalline chitin hydrolyzing activity at high temperature. In order to investigate the structure–function relationship of this chitinase, we analyzed one of the catalytic domains (AD2) using mutational and kinetic approaches, and determined the crystal structure of AD2 complexed with chito‐oligosaccharide substrate. Kinetic studies showed that, among the acidic residues in the signature sequence of familyu200318 chitinases (DXDXE motif), the second Asp (D2) and Glu (E) residues play critical roles in the catalysis of archaeal chitinase. Crystallographic analyses showed that the side‐chain of the catalytic proton‐donating E residue is restrained into the favorable conformer for proton donation by a hydrogen bond interaction with the adjacent D2 residue. The comparison of active site conformations of familyu200318 chitinases provides a new criterion for the subclassification of familyu200318 chitinase based on the conformational change of the D2 residue.

Structure of the catalytic domain of the hyperthermophilic chitinase from Pyrococcus furiosus

The crystal structure of the catalytic domain of a chitinase from the hyperthermophilic archaeon Pyrococcus furiosus (AD2(PF-ChiA)) has been determined at 1.5 A resolution. This is the first structure of the catalytic domain of an archaeal chitinase. The overall structure of AD2(PF-ChiA) is a TIM-barrel fold with a tunnel-like active site that is a common feature of family 18 chitinases. Although the catalytic residues (Asp522, Asp524 and Glu526) are conserved, comparison of the conserved residues and structures with those of other homologous chitinases indicates that the catalytic mechanism of PF-ChiA is different from that of family 18 chitinases.

Crystal structure of thioredoxin peroxidase from aerobic hyperthermophilic archaeon Aeropyrum pernix K1.

Introduction. The peroxiredoxin (Prx) constitutes a family of antioxidant proteins that act as peroxidases that reduce hydrogen peroxide and alkyl peroxide to water and the corresponding alcohol, respectively. Prxs participate in the antioxidative mechanism called the “thioredoxin system.” The typical 2-Cys Prxs have two conserved redox-active cysteines: the peroxidatic cysteine and the resolving cysteine. The peroxidase reaction results in an intersubunit disulfide bond between these redox-active cysteines concomitant with the reduction of peroxide substrate. The tertiary structures for Prxs from several sources have been reported, distinct quaternary structures, for example, monomer, dimer, and toroid-shaped decamer, being observed depending on the subtype and redox state. However, the tertiary structure of a hyperthermophilic Prx has not been reported to date. We previously identified the genes for thioredoxin peroxidase, thioredoxin, and thioredoxin reductase in the genome of Aeropyrum pernix K1, which lives at the highest temperature among the aerobic organisms whose genome sequences are available. We presented the first evidence of a set of proteins involved in the thioredoxin system in archaea by confirming the activity. Mutational analyses of thioredoxin peroxidase from A. pernix K1 (ApTPx) showed that peroxidatic Cys50 and resolving Cys213 form an intersubunit disulfide bond upon oxidation and that another cysteine residue (Cys207) is not essential for the peroxidase function. Although we had proposed a ring structure consisting of eight subunits, we found a fivefold axis and five twofold axes in a crystal of ApTPx (C207S mutant) in a later study. This suggested that the overall structure of ApTPx consists of decameric ring, which is homologous to Prxs from human, Salmonella typhimurium, and Crithidia fasciculate. In this article, we describe the tertiary structure of a selenomethionine derivative of the C207S mutant of ApTPx (SeC207S) in the reduced form determined at 2.0-Å resolution. We also discuss the characteristic features of this hyperthermostable Prx protein.

Crystal structure of the cambialistic superoxide dismutase from Aeropyrum pernix K1 - insights into the enzyme mechanism and stability

Aeropyrumu2003pernix K1, an aerobic hyperthermophilic archaeon, produces a cambialistic superoxide dismutase that is active in the presence of either of Mn or Fe. The crystal structures of the superoxide dismutase from A.u2003pernix in the apo, Mn‐bound and Fe‐bound forms were determined at resolutions of 1.56, 1.35 and 1.48u2003Å, respectively. The overall structure consisted of a compact homotetramer. Analytical ultracentrifugation was used to confirm the tetrameric association in solution. In the Mn‐bound form, the metal was in trigonal bipyramidal coordination with five ligands: four side chain atoms and a water oxygen. One aspartate and two histidine side chains ligated to the central metal on the equatorial plane. In the Fe‐bound form, an additional water molecule was observed between the two histidines on the equatorial plane and the metal was in octahedral coordination with six ligands. The additional water occupied the postulated superoxide binding site. The thermal stability of the enzyme was compared with superoxide dismutase from Thermusu2003thermophilus, a thermophilic bacterium, which contained fewer ion pairs. In aqueous solution, the stabilities of the two enzymes were almost identical but, when the solution contained ethylene glycol or ethanol, the A.u2003pernix enzyme had significantly higher thermal stability than the enzyme from T.u2003thermophilus. This suggests that dominant ion pairs make A.u2003pernix superoxide dismutase tolerant to organic media.

Expression from engineered Escherichia coli chromosome and crystallographic study of archaeal N,N′‐diacetylchitobiose deacetylase

In order to develop a structure‐based understanding of the chitinolytic pathway in hyperthermophilic Pyrococcus species, we performed crystallographic studies on N,N′‐diacetylchitobiose deacetylases (Dacs) from Pyrococcus horikoshii (Ph‐Dac) and Pyrococcus furiosus (Pf‐Dac). Neither Ph‐Dac nor Pf‐Dac was expressed in the soluble fraction of Escherichia coli harboring the expression plasmid. However, insertion of the target genes into the chromosome of E. coli yielded the soluble recombinant protein. The purified Pyrococcus Dacs were active and thermostable up to 85 °C. The crystal structures of Ph‐Dac and Pf‐Dac were determined at resolutions of 2.0 Å and 1.54 Å, respectively. The Pyrococcus Dac forms a hexamer composed of two trimers. These Dacs are characterized by an intermolecular cleft, which is formed by two polypeptides in the trimeric assembly. In Ph‐Dac, catalytic Zn situated at the end of the cleft is coordinated by three side chain ligands from His44, Asp47, and His155, and by a phosphate ion derived from the crystallization reservoir solution. We considered that the bound phosphate mimicked the tetrahedral oxyanion, which is an intermediate of hydrolysis of the N‐acetyl group, and proposed an appropriate reaction mechanism. In the proposed mechanism, the Nε atom of His264 (from the adjacent polypeptide in the Ph‐Dac sequence) is directly involved in the stabilization of the oxyanion intermediate. Mutation analysis also indicated that His264 was essential to the catalysis. These factors give the archaeal Dacs an unprecedented active site architecture a Zn‐dependent deacetylases.