Search for a Light CP-odd Higgs Boson and Low-Mass Dark Matter at the Belle Experiment.

We report on the first Belle search for a light CP-odd Higgs boson, A^{0}, that decays into low mass dark matter, χ, in final states with a single photon and missing energy. We search for events produced via the dipion transition ϒ(2S)→ϒ(1S)π^{+}π^{-}, followed by the on-shell process ϒ(1S)→γA^{0} with A^{0}→χχ, or by the off-shell process ϒ(1S)→γχχ. Utilizing a data sample of 157.3×10^{6} ϒ(2S) decays, we find no evidence for a signal. We set limits on the branching fractions of such processes in the mass ranges M_{A^{0}}<8.97 GeV/c^{2} and M_{χ}<4.44 GeV/c^{2}. We then use the limits on the off-shell process to set competitive limits on WIMP-nucleon scattering in the WIMP mass range below 5 GeV/c^{2}.

New limits on interactions between weakly interacting massive particles and nucleons obtained with CsI(Tl) crystal detectors.

New limits are presented on the cross section for weakly interacting massive particle (WIMP) nucleon scattering in the KIMS CsI(Tℓ) detector array at the Yangyang Underground Laboratory. The exposure used for these results is 24 524.3 kg·days. Nuclei recoiling from WIMP interactions are identified by a pulse shape discrimination method. A low energy background due to alpha emitters on the crystal surfaces is identified and taken into account in the analysis. The detected numbers of nuclear recoils are consistent with zero and 90% confidence level upper limits on the WIMP interaction rates are set for electron equivalent energies from 3 to 11 keV. The 90% upper limit of the nuclear recoil event rate for 3.6-5.8 keV corresponding to 2-4 keV in NaI(Tℓ) is 0.0098 counts/kg/keV/day, which is below the annual modulation amplitude reported by DAMA. This is incompatible with interpretations that enhance the modulation amplitude such as inelastic dark matter models. We establish the most stringent cross section limits on spin-dependent WIMP-proton elastic scattering for the WIMP masses greater than 20 GeV/c2.

Nucleotide-dependent conformational changes in a protease-associated ATPase HsIU.

BACKGROUND: The bacterial heat shock locus ATPase HslU is an AAA(+) protein that has structures known in many nucleotide-free and -bound states. Nucleotide is required for the formation of the biologically active HslU hexameric assembly. The hexameric HslU ATPase binds the dodecameric HslV peptidase and forms an ATP-dependent HslVU protease. RESULTS: We have characterized four distinct HslU conformational states, going sequentially from open to closed: the empty, SO(4), ATP, and ADP states. The nucleotide binds at a cleft formed by an alpha/beta domain and an alpha-helical domain in HslU. The four HslU states differ by a rotation of the alpha-helical domain. This classification leads to a correction of nucleotide identity in one structure and reveals the ATP hydrolysis-dependent structural changes in the HslVU complex, including a ring rotation and a conformational change of the HslU C terminus. This leads to an amended protein unfolding-coupled translocation mechanism. CONCLUSIONS: The observed nucleotide-dependent conformational changes in HslU and their governing principles provide a framework for the mechanistic understanding of other AAA(+) proteins.

N-terminal processing is essential for release of epithin, a mouse type II membrane serine protease.

Epithin was originally identified as a mouse type II membrane serine protease. Its human orthologue membrane type-serine protease 1 (MT-SP1)/matriptase has been reported to be localized on the plasma membrane. In addition, soluble forms of matriptase were isolated from human breast milk and breast cancer cell-conditioned medium. In this paper, we report a processing mechanism that appears to be required for the release of epithin. CHO-K1 or COS7 cells transfected with single full-length epithin cDNA generated two different-sized proteins in cell lysates, 110 and 92 kDa. The 92-kDa epithin was found to be an N-terminally truncated form of the 110-kDa epithin, and it was the only form detected in the culture medium. The 92-kDa epithin was also found on the cell surface, where it was anchored by the N-terminal fragment. The results of in vivo cell labeling experiments indicate that the 110-kDa epithin is rapidly processed to the 92-kDa epithin. Using site-directed mutagenesis experiments, we identified Gly(149) of the GSVIA sequence in epithin as required for the processing and release of the protein. These results suggest that N-terminal processing of epithin at Gly(149) is a necessary prerequisite step for release of the protein.

Crystal structures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism.

BACKGROUND: The bacterial heat shock locus HslU ATPase and HslV peptidase together form an ATP-dependent HslVU protease. Bacterial HslVU is a homolog of the eukaryotic 26S proteasome. Crystallographic studies of HslVU should provide an understanding of ATP-dependent protein unfolding, translocation, and proteolysis by this and other ATP-dependent proteases. RESULTS: We present a 3.0 A resolution crystal structure of HslVU with an HslU hexamer bound at one end of an HslV dodecamer. The structure shows that the central pores of the ATPase and peptidase are next to each other and aligned. The central pore of HslU consists of a GYVG motif, which is conserved among protease-associated ATPases. The binding of one HslU hexamer to one end of an HslV dodecamer in the 3.0 A resolution structure opens both HslV central pores and induces asymmetric changes in HslV. CONCLUSIONS: Analysis of nucleotide binding induced conformational changes in the current and previous HslU structures suggests a protein unfolding-coupled translocation mechanism. In this mechanism, unfolded polypeptides are threaded through the aligned pores of the ATPase and peptidase and translocated into the peptidase central chamber.

The ATP-dependent CodWX (HslVU) protease in Bacillus subtilis is an N-terminal serine protease.

HslVU is a two-component ATP-dependent protease, consisting of HslV peptidase and HslU ATPase. CodW and CodX, encoded by the cod operon in Bacillus subtilis, display 52% identity in their amino acid sequences to HslV and HslU in Escherichia coli, respectively. Here we show that CodW and CodX can function together as a new type of two-component ATP-dependent protease. Remarkably, CodW uses its N-terminal serine hydroxyl group as the catalytic nucleophile, unlike HslV and certain beta-type subunits of the proteasomes, which have N-terminal threonine functioning as an active site residue. The ATP-dependent proteolytic activity of CodWX is strongly inhibited by serine protease inhibitors, unlike that of HslVU. Replacement of the N-terminal serine of CodW by alanine or even threonine completely abolishes the enzyme activity. These results indicate that CodWX in B.subtilis represents the first N-terminal serine protease among all known proteolytic enzymes.

The HslU ATPase acts as a molecular chaperone in prevention of aggregation of SulA, an inhibitor of cell division in Escherichia coli.

HslVU is an ATP-dependent protease consisting of two multimeric components: the HslU ATPase and the HslV peptidase. SulA, which is an inhibitor of cell division and has high tendency of aggregation, is degraded by HslVU protease. Here we show that HslU plays a role not only as a regulatory component for the HslV-mediated proteolysis but also as a molecular chaperone. Purified HslU prevented aggregation of SulA in a concentration-dependent fashion. This chaperone activity required oligomerization of HslU subunits, which could be achieved by ATP-binding or in the presence of high HslU protein concentrations. hsl mutation reduced the SulA-mediated inhibition of cell growth and this effect could be reversed upon overproduction of HslU, suggesting that HslU promotes the ability of SulA to block cell growth through its chaperone function. Thus, HslU appears to have two antagonistic functions: one as a chaperone for promotion of the ability of SulA in cell growth inhibition by preventing SulA aggregation and the other as the regulatory component for elimination of SulA by supporting the HslV-mediated degradation.

ATP-dependent degradation of SulA, a cell division inhibitor, by the HslVU protease in Escherichia coli.

HslVU is an ATP-dependent protease consisting of two multimeric components, the HslU ATPase and the HslV peptidase. To gain an insight into the role of HslVU in regulation of cell division, the reconstituted enzyme was incubated with SulA, an inhibitor of cell division in Escherichia coli, or its fusion protein with maltose binding protein (MBP). HslVU degraded both proteins upon incubation with ATP but not with its nonhydrolyzable analog, ATPgammaS, indicating that the degradation of SulA requires ATP hydrolysis. The pulse-chase experiment using an antibody raised against MBP-SulA revealed that the stability of SulA increased in hsl mutants and further increased in lon/hsl double mutants, indicating that SulA is an in vivo substrate of HslVU as well as of protease La (Lon). These results suggest that HslVU in addition to Lon plays an important role in regulation of cell division through degradation of SulA.

Crystallization and preliminary X-ray crystallographic analysis of the protease inhibitor ecotin in complex with chymotrypsin.

Ecotin, a homodimeric protein composed of 142-residue subunits, is a novel protease inhibitor present in the periplasm of Escherichia coli. It shows a broad inhibitory specificity towards a group of serine proteases and binds two molecules of protease to form a tetrameric complex in a distinct chelation mechanism. The ecotin-chymotrypsin complex has been crystallized in the triclinic space group P1 with unit-cell parameters a = 57.29, b = 57.39, c = 79.75 A, alpha = 91.49, beta = 88.63 and gamma = 112.45 degrees. The asymmetric unit contains the whole tetrameric complex, consisting of two molecules of chymotrypsin bound to the ecotin dimer, with a corresponding crystal volume per protein mass (VM) of 2.58 A3 Da-1 and a solvent fraction of 48.9%. The crystals diffract beyond 2.0 A with Cu Kalpha X-rays and are very stable in the X-ray beam. Native X-ray data have been collected from a crystal to approximately 2.0 A Bragg spacing.

LonR9 carrying a single Glu614 to Lys mutation inhibits the ATP-dependent protease La (Lon) by forming mixed oligomeric complexes.

An unusual lon mutation (called lonR9) is dominant over the wild-type gene, which encodes the ATP-dependent protease La (Lon) in Escherichia coli, when present in multicopy plasmids. Here, we cloned and sequenced lonR9, and showed that the mutant gene carries a single point mutation in its open reading frame, which leads to replacement of Glu614 by Lys. The LonR9 protein and its poly-His-tagged form were purified to apparent homogeneity. Both of the purified proteins were capable of inhibiting the ATP-dependent proteolysis and the protein-activated ATP hydrolysis by protease La. Furthermore, the His-tagged LonR9 protein was found to form mixed oligomeric complexes with protease La, upon analysis by chromatography on a metal-chelating column. These results suggest that the phenotypic dominance of the lonR9 mutant is due to the formation of mixed oligomeric complexes between LonR9 and protease La, in which the defective components prevent the function of the wild-type subunits.

Effects of the cys mutations on structure and function of the ATP-dependent HslVU protease in Escherichia coli. The Cys287 to Val mutation in HslU uncouples the ATP-dependent proteolysis by HslvU from ATP hydrolysis.

To define the role of the Cys residues in the ATP-dependent HslVU protease, mutagenesis was performed to replace either Cys261 or Cys287 in HslU with Val and Cys159 in HslV with Ser or Ala. Whereas HslU/C261V could hydrolyze ATP and support the ATP-dependent proteolytic activity of HslV as well as the wild-type HslU, HslU/C287V could not hydrolyze ATP. Nevertheless, HslU/C287V could support the HslV-mediated proteolysis by forming the HslVU complex in the presence of ATP but not its absence, indicating that ATP binding but not its hydrolysis is essential for proteolysis. Whereas treatment of N-ethylmaleimide (NEM) resulted in dissociation of the oligomeric HslU into monomers, the C261V mutation, but not C287V, prevented the NEM effect. These results suggest that Cys261 is involved in oligomerization and that Cys287 is related to the ATPase function of HslU. NEM also dissociated the dodecameric HslV into monomers, and this effect could be prevented by either the C159S or C159A mutation, suggesting the involvement of Cys159 in oligomerization of HslV. Moreover, either mutation abolished both the basal and HslU-activated proteolytic activity of HslV and its ability to activate the HslU ATPase and to form the HslVU complex, indicating that Cys159 is essential for the proteolytic activity of HslV and its interaction with HslU. These results suggest that the Cys residues play an important role in maintaining the structure and function of the HslVU protease.

Mutational analysis of the two ATP-binding sites in ClpB, a heat shock protein with protein-activated ATPase activity in Escherichia coli.

The 93 kDa ClpB (ClpB93) is a heat shock protein and has a protein-activated ATPase activity. To define the role of the two ATP-binding sites in ClpB93, site-directed mutagenesis was performed to replace Lys212 or Lys611 with Thr or Glu. All of the mutant proteins hydrolysed ATP at a higher rate than that seen with ClpB93 at ATP concentrations up to 2 mM. However, ClpB93 carrying mutations in both of the ATP-binding sites could not cleave ATP. Thus any of the two ATP-binding sites seems to be capable of supporting the ATPase activity of ClpB93. The ATPase activities of both ClpB93/K212T and ClpB93/K212E were gradually decreased when ATP concentrations were increased above 2 mM, unlike those of ClpB93, ClpB93/K611T and ClpB93/K611E, which showed a typical saturation curve. Furthermore ADP inhibited ATP hydrolysis by ClpB93/K212T and ClpB93/K212E more effectively than that by the latter proteins, suggesting that the mutations in the first ATP-binding site result in an increase in the affinity of ADP for the second site in ClpB93. In addition, all of the purified ClpB93 and its mutant forms behaved as an oligomer of 400-450 kDa on a Sephacryl S-300 gel-filtration column, whether or not ATP was present. Thus the binding of ATP to either of the two sites seems not to be essential for oligomerization of ClpB93. Although a low-copy plasmid carrying clpB93 could rescue the sensitivity of a clpB-null mutant cell at 52 degreesC, none of the plasmids carrying the mutations in the ATP-binding sites could. Furthermore, incubation at 52 degreesC resulted in a gradual loss of the ATPase activity of ClpB93 carrying the mutations in either of the two ATP-binding sites, but not of the parental ClpB93, indicating that the mutant proteins have a greater tendency to denature at this temperature than the parental ClpB93. These results suggest that both of the ATP-binding sites in ClpB have an important role in maintaining the thermotolerance of the protein and hence in the survival of Escherichia coli at high temperatures.

ATP binding, but not its hydrolysis, is required for assembly and proteolytic activity of the HslVU protease in Escherichia coli.

HslVU is an ATP-dependent protease consisting of two multimeric components: the HslU ATPase and the HslV peptidase. To gain an insight into the role of ATP hydrolysis in protein breakdown, we determined the insulin B-chain-degrading activity and assembly of HslVU in the presence of ATP and its nonhydrolyzable analogs. While beta,gamma-methylene-ATP could not support the proteolytic activity, beta,gamma-imido-ATP supported it to an extent less than 10% of that seen with ATP. Surprisingly, however, HslVU degraded insulin B-chain even more rapidly in the presence of ATPgammaS than with ATP. Furthermore, the ability of ATP and its analogs in supporting the proteolytic activity was closely correlated with their ability in supporting the oligomerization of HslU and the formation of the HslVU complex. However, ADP, which is capable of supporting the HslU oligomerization, could not support the HslVU complex formation or the proteolytic activity, suggesting that the conformation of the ADP-bound HslU oligomer is different from that of ATP-bound form. Thus, it appears that ATP-binding, but not its hydrolysis, is essential for assembly and proteolytic activity of HslVU.

Mutagenesis of two N-terminal Thr and five Ser residues in HslV, the proteolytic component of the ATP-dependent HslVU protease.

HslVU in E. coli is a new type of ATP-dependent protease consisting of two heat shock proteins: the HslU ATPase and the HslV peptidase that has two repeated Thr residues at its N terminus, like certain beta-type subunit of the 20S proteasomes. To gain an insight into the catalytic mechanism of HslV, site-directed mutagenesis was performed to replace each of the Thr residues with Ser or Val and to delete the first or both Thr. Also each of the five internal Ser residues in HslV were replaced with Ala. The results obtained by the mutational analysis revealed that the N-terminal Thr acts as the active site nucleophile and that certain Ser residues, particularly Ser124 and Ser172, also contribute to the peptide hydrolysis by the HslVU protease. The mutational studies also revealed that both Thr, Ser103, and Ser172, but not Ser124, are involved in the interaction of HslV with HslU and hence in the activation of HslU ATPase as well as in the HslVU complex formation.

Mutational analysis for the role of C-terminal region of ecotin, a dimeric inhibitor of pancreatic serine proteases.

Ecotin is a dimeric molecule that is capable of inhibiting a variety of serine proteases. To clarify the role of the C-terminal region, mutagenesis was performed to delete the C-terminal residues from 130 to 142. The mutant inhibitor behaved as a monomer upon cross-linking analysis followed by gel filtration. The mutation also resulted in a significant increase of the Ki of ecotin on trypsin, chymotrypsin, and elastase (i.e., by 1 to 2 order of magnitude). The mutant ecotin was slightly more sensitive to heating at 100 degrees C than the wild-type ecotin, but became much more sensitive to the heat treatment upon reduction of the intra-chain disulfide bond in its subunits. In addition, treatment with 4 M urea resulted in complete loss of the activity of the mutant ecotin but not that of the wild-type inhibitor. Thus, the C-terminal region of ecotin seems to be required not only for dimerization of the subunits but also for optimal interaction with target proteases and for maintenance in its structural stability, particularly under reducing or denaturing conditions.

Crystal structure analyses of uncomplexed ecotin in two crystal forms: implications for its function and stability.

Ecotin, a homodimeric protein composed of 142 residue subunits, is a novel serine protease inhibitor present in Escherichia coli. Its thermostability and acid stability, as well as broad specificity toward proteases, make it an interesting protein for structural characterization. Its structure in the uncomplexed state, determined for two different crystalline environments, allows a structural comparison of the free inhibitor with that in complex with trypsin. Although there is no gross structural rearrangement of ecotin when binding trypsin, the loops involved in binding trypsin show relatively large shifts in atomic positions. The inherent flexibility of the loops and the highly nonglobular shape are the two features essential for its inhibitory function. An insight into the understanding of the structural basis of thermostability and acid stability of ecotin is also provided by the present structure.

The P1 reactive site methionine residue of ecotin is not crucial for its specificity on target proteases. A potent inhibitor of pancreatic serine proteases from Escherichia coli.

The importance of the P1 reactive site for the specificity of ecotin on target proteases was examined by site-directed mutagenesis. The replacement of Met at the P1 site with Ile, Arg, Glu, or Tyr showed little or no effect on the ability of ecotin to inhibit trypsin. Similar results were obtained for chymotrypsin, except that its replacement with Glu caused about 40% reduction of the inhibitory activity of ecotin. On the other hand, the replacement of the Met residue with Arg, Tyr, or Glu dramatically reduced its ability to inhibit elastase, while that with Ile showed little or no effect. Nevertheless, elastase could be completely inhibited upon incubation with excess amounts of the mutant ecotin containing Arg, Glu, or Tyr. Moreover, all the mutant forms of ecotin could be cleaved at the mutated P1 site upon incubation with trypsin at pH 3.75. In addition, the replacement of a Cys residue in the disulfide bridge with Ser showed little or no effect on the ability of ecotin to inhibit trypsin, chymotrypsin, or elastase. However, the mutant ecotin containing Ser was more sensitive to inactivation by heating at 100 degrees C than the wild-type inhibitor. Furthermore, the wild-type ecotin whose disulfide bond had been reduced and alkylated was also more easily inactivated by heat treatment than the untreated control. These results strongly suggest that the P1 site of ecotin is not crucial for its specificity on target proteases and that the disulfide bridge in ecotin appears to play an important role in maintenance of its structural stability.