Amyloidogenecity and pitrilysin sensitivity of a lysine-free derivative of amyloid beta-peptide cleaved from a recombinant fusion protein

The progressive cerebral deposition of a 40-42 residues amyloid beta-peptide (Abeta) is regarded as a major factor in the onset of the Alzheimer's disease. It has recently been shown that Abeta(1-40) is cleaved by Escherichia coli pitrilysin, a homologue of insulysin, at a specific site. To facilitate the studies on a recognition mechanism of Abeta by pitrilysin, an overproduction system of Abeta(1-40) as a fusion protein with E. coli RNase HI was constructed. This fusion protein was designed such that an Abeta(1-40) derivative, Abeta(1-40)*, in which Lys16 and Lys28 of Abeta(1-40) are simultaneously replaced by Ala, is attached to the C-terminus of E. coli RNase HI and Abeta(1-40)* is separated from RNase HI upon cleavage with lysyl endopeptidase. The fusion protein was overproduced in E. coli in inclusion bodies, solubilized and purified in the presence of guanidine hydrochloride, and cleaved by lysyl endopeptidase. Abeta(1-40)* was purified from the resultant peptide fragments by reverse-phase HPLC. Measurement of the far-UV CD spectra suggests that Abeta(1-40)* is conformationally similar to Abeta(1-40). However, the thioflavin T binding assay suggests that Abeta(1-40)* is more amyloidogenic than Abeta(1-40). Nevertheless, Abeta(1-40)* was cleaved by pitrilysin at the site identical to that in Abeta(1-40).     

Stabilization of E. coli Ribonuclease HI by the 'stability profile of mutant protein' (SPMP)-inspired random and non-random mutagenesis

The change in the structural stability of Escherichia coli ribonuclease HI (RNase HI) due to single amino acid substitutions has been estimated computationally by the stability profile of mutant protein (SPMP) [Ota, M., Kanaya, S. Nishikawa, K., 1995. Desk-top analysis of the structural stability of various point mutations introduced into ribonuclease H. J. Mol. Biol. 248, 733-738]. As well, an effective strategy using random mutagenesis and genetic selection has been developed to obtain E. coli RNase HI mutants with enhanced thermostability [Haruki, M., Noguchi, E., Akasako, A., Oobatake, M., Itaya, M., Kanaya, S., 1994. A novel strategy for stabilization of Escherichia coli ribonuclease HI involving a screen for an intragenic suppressor of carboxyl-terminal deletions. J. Biol. Chem. 269, 26904-26911]. In this study, both methods were combined: random mutations were individually introduced to Lys99-Val101 on the N-terminus of the alpha-helix IV and the preceding beta-turn, where substitutions of other amino acid residues were expected to significantly increase the stability from SPMP, and then followed by genetic selection. Val101 to Ala, Gln, and Arg mutations were selected by genetic selection. The Val101-->Ala mutation increased the thermal stability of E. coli RNase HI by 2.0 degrees C in Tm at pH 5.5, whereas the Val101-->Gln and Val101-->Arg mutations decreased the thermostability. Separately, the Lys99-->Pro and Asn100-->Gly mutations were also introduced directly. The Lys99-->Pro mutation increased the thermostability of E. coli RNase HI by 1.8 degrees C in Tm at pH 5.5, whereas the Asn100-->Gly mutation decreased the thermostability by 17 degrees C. In addition, the Lys99-->Pro mutation altered the dependence of the enzymatic activity on divalent metal ions.     

Acceleration of matrix metalloproteinase-1 production and activation of platelet-derived growth factor receptor beta in human coronary smooth muscle cells by oxidized LDL and 4-hydroxynonenal

Increases in matrix metalloproteinases (MMPs) at atherosclerotic lesions are involved in the migration of smooth muscle cells (SMCs) into the intima and to the rupture of plaques, being implicated in the progression of atherosclerosis. The present study examined the mechanisms underlying the production of MMP-1, interstitial collagenase-1, induced by oxidized low-density lipoprotein (oxLDL) and 4-hydroxynonenal (4-HNE), factors proposed to play a pivotal role in atherogenesis, in human coronary SMCs. oxLDL promoted the production of MMP-1 with the preceding phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Immunoprecipitation of platelet-derived growth factor receptor beta (PDGFR-beta) revealed that oxLDL induced tyrosine phosphorylation of the receptor. Inhibition of the activation of PDGFR-beta and ERK1/2 resulted in a suppression of the production of MMP-1. Consistently, 4-HNE also elicited the production of MMP-1 with the preceding phosphorylation of PDGFR-beta and ERK1/2. The 4-HNE-induced production of MMP-1 was prevented when the activation of PDGFR-beta and ERK1/2 was inhibited. The present results suggest that the activation of PDGFR-beta and ERK1/2 is involved in the production of MMP-1 in oxLDL- and 4-HNE-stimulated human coronary SMCs.     

Total synthesis of two isoflavone C-glycosides: genistein and orobol 8-C-beta-D-glucopyranosides

Genistein and orobol 8-C-beta-D-glucopyranosides (1 and 3) were firstly synthesized in overall yields of 39% and 41% from 2,4-di-O-benzylphloroacetophenone (4), as follows: (1) the formation of the chalcone (6, 7) by aldol condensation of the benzyl-protected C-glycosylphloroacetophenone (5), a key intermediate of the total synthesis of 1 and 3 and synthesized by a C-glycosylation method involving the O-->C glycoside rearrangement of 4 in 96% yield; (2) the formation of isoflavones (10, 11 and 12, 13) by the formation of acetals by oxidative rearrangement of the protected chalcones (8 and 9) using Tl(NO3)3, followed by acid-catalyzed cyclization; (3) a final debenzylation by hydrogenolysis.     

Ca2+-Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis: the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca²⁺ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca²⁺ at 80°C. This maturation process was completed within 30 min at 80°C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin*) but forms an inactive complex with T. kodakaraensis mat-subtilisin*, at lower temperatures. At 80°C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca²⁺ but was activated upon incubation with Ca²⁺ at 80°C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin*, indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only ~5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin* and noncompetitively inhibited its activity with a K_i of 25 ± 3.0 nM at 20°C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.     

Expression screening of 17q12-21 amplicon reveals GRB7 as an ERBB2-dependent oncogene

Gene amplification is a major genetic alteration in human cancers. Amplicons, amplified genomic regions, are believed to contain "driver" genes responsible for tumorigenesis. However, the significance of co-amplified genes has not been extensively studied. We have established an integrated analysis system of amplicons using retrovirus-mediated gene transfer coupled with a human full-length cDNA set. Applying this system to 17q12-21 amplicon observed in breast cancer, we identified GRB7 as a context-dependent oncogene, which modulates the ERBB2 signaling pathway through enhanced phosphorylation of ERBB2 and Akt. Our work provides an insight into the biological significance of gene amplification in human cancers.     

Amounts of Sr and Ca Eluted from Deciduous Enamel to Artificial Saliva Related to Dental Caries

This study was performed to elucidate the relationship between dental caries and the levels of Sr and Ca eluted from enamel, and to examine whether these elements are useful as factors to assess caries risk. The available 103 (Sr) and 108 (Ca) samples were obtained among 111 collected deciduous teeth. The healthy regions of enamel were decalcified in artificial saliva at pH 6.2 and 5.5. The eluted levels of these elements from enamel were determined using atomic absorption spectrophotometry. Sr and Ca levels were not affected by the sex nor tooth type. Sr levels of the caries-experienced tooth (CE) group were 2.6-fold (pH 6.2) and 2.2-fold (pH 5.5) higher than those of the sound tooth (ST) group, respectively. Furthermore, the Sr levels were significantly higher in the teeth with treated than in those with untreated caries. Only at pH 6.2 was a significant difference found in Ca levels between the ST and CE groups. In the ST group, at pH 5.5, both the Sr and Ca levels significantly increased when the children had six or more carious teeth. The Sr and Ca elution levels were significantly inhibited in the teeth receiving fluoride application every 3 or 4 months compared to those that were not. These findings indicate that Sr can be an indicator of the acid resistance of teeth, and a useful factor to assess future caries risk.     

Near-UV Circular Dichroism and UV Resonance Raman Spectra of Individual Tryptophan Residues in Human Hemoglobin and Their Changes upon the Quaternary Structure Transition

The aromatic residues such as tryptophan (Trp) and tyrosine (Tyr) in human adult hemoglobin (Hb A) are known to contribute to near-UV circular dichroism (CD) and UV resonance Raman (RR) spectral changes upon the R → T quaternary structure transition. In Hb A, there are three Trp residues per αβ dimer: at α14, β15, and β37. To evaluate their individual contributions to the R → T spectral changes, we produced three mutant hemoglobins in E. coli; rHb (α14Trp→Leu), rHb (β15Trp→Leu), and rHb (β37Trp→His). Near-UV CD and UVRR spectra of these mutant Hbs were compared with those of Hb A under solvent conditions where mutant rHbs exhibited significant cooperativity in oxygen binding. Near-UV CD and UVRR spectra for individual Trp residues were extracted by the difference calculations between Hb A and the mutants. α14 and β15Trp exhibited negative CD bands in both oxy- and deoxy-Hb A, whereas β37Trp showed positive CD bands in oxy-Hb A but decreased intensity in deoxy-form. These differences in CD spectra among the three Trp residues in Hb A were ascribed to surrounding hydrophobicity by examining the spectral changes of a model compound of Trp, N-acetyl-l-Trp ethyl ester, in various solvents. Intensity enhancement of Trp UVRR bands upon the R → T transition was ascribed mostly to the hydrogen-bond formation of β37Trp in deoxy-Hb A because similar UVRR spectral changes were detected with N-acetyl-l-Trp ethyl ester upon addition of a hydrogen-bond acceptor.     

NMR and CD analysis of an intermediate state in the thermal unfolding process of mouse lipocalin-type prostaglandin D synthase

We previously reported that the thermal unfolding of mouse lipocalin-type prostaglandin D synthase (L-PGDS) is a completely reversible process under acidic conditions and follows a three-state pathway, including an intermediate state (I) between native state (N) and unfolded state. In the present study, we investigated the intermediate state of mouse C65A L-PGDS and clarified the local conformational changes in the upper and bottom regions by using NMR and CD spectroscopy. The (1)H-(15)N HSQC measurements revealed that the backbone conformation was disrupted in the upper region of the β-barrel at 45°C, which is around the T(m) value for the N ? I transition, but that the signals of the residues located at the bottom region of L-PGDS remained at 54°C, where the maximum accumulation of the intermediate state was found. (1)H-NMR and CD measurements showed that the T(m) values obtained by monitoring Trp54 at the upper region and Trp43 at the bottom region of the β-barrel were 41.4 and 47.5°C, respectively, suggesting that the conformational change in the upper region occurred at a lower temperature than that in the bottom region. These findings demonstrate that the backbone conformation of the bottom region is still maintained in the intermediate state.     

A new prenylflavonoid isolated from propolis collected in the Solomon Islands

The new prenylflavonoid, solophenol A (1), together with three known compounds, bonannione A (2), sophoraflavanone A (3) and (2S)-5,7-dihydroxy-4'-methoxy-8-prenylflavanone (4), were isolated from propolis collected from Malaita Island in The Solomon Islands. The structure of each compound was determined by spectroscopic methods, including mass spectrometry and 2D NMR. Compound 1 exhibited potent 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity.