To investigate the possible similarity of the proinsulin folding process with insulinlike growth factor I, two swap-like human proinsulin mutant proteins [A 7, A11Ser]-HPI and [A11 Ser, A12Cys]-HPI were prepared. Their in vitro refolding yields, oxidation of free thiol groups, circular dichroism spectra, antibody and receptor binding activities and sensitivity to trypsin digestion were studied and compared with both native HPI and [A 6, A11Ser]-HPI. The results indicate that the shift mutation in the disulfide bond caused more conformational change and a greater decrease in biological activity than the deletion mutation on the proinsulin molecule. However, the shift of the intra-A chain disulfide bond had little effect on the refolding rate of the molecule. In vitro refolding yields of HPI analogues with shift or deletion mutations in the region of the [A6-A11, A7-B7] disulfide bonds were almost as high as that of wild type HPI suggesting that the region of the [A6-A11, A7-B7] disulfide bonds possesses some flexibility as is found in the corresponding region of insulin-like growth factor I.

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We have isolated a temperature-sensitive alanyl-tRNA synthetase mutant from hamster BHK21 cells, designated as is ET12. It has a single nucleotide mutation, converting the 321 st amino acid residue, 321Gly, to Arg. The mutation was localized between two RNA-binding domains of alanyl-tRNA synthetase. Thus far, we have isolated two temperature-sensitive aminoacyl-tRNA synthetase mutants from the BHK21 cell line: is BN250 and is BN269. They are defective in histidyl- and lysyl-tRNA synthetase respectively. Both mutants rapidly undergo apoptosis at the nonpermissive temperature, 39.5°C. is ET12 cells, however, did not undergo apoptosis until 48h after a temperature-shift to 39.5°C, while mutated alanyl-tRNA synthetase of is ET12 cells was lost within 4h. Loss of the mutated alanyl-tRNA synthetase was inhibited by a ubiquitin-dependent proteasome inhibitor, MG132, and by a protein-synthesis inhibitor, cycloheximide. Cell-cycle related proteins were also lost in is ET12 cells at 39.5°C, as shown in is BN250. In contrast, the mutated aminoacyl-tRNA synthetases of is BN250 and is BN269 were stable at 39.5°C. However, the defects of these mutants released EMAPII, an inducer of apoptosis at 39.5°C. No release of EMAPII occurred in is ET12 cells at 39.5°C, consistent with the delay of apoptosis in these cells.

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To address the effects of local structures on structural fluctuations of Escherichia coli dihydrofolate reductase (DHFR), the backbone-fluctuation map was determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) coupled with H/D exchange and pepsin digestion. H/D exchange kinetics was examined at 15°C with 18 identified digestion fragments covering almost the entire amino acid sequence of DHFR. These fragments exhibited significant variations in the first-order rate constant of proton exchange, k_ex (0.47-0.71min^-1), the fraction of deuterium incorporation at the initial stage, D_o (0.20-0.60), the fraction of deuterium incorporation at infinite time, D_??_ (0.75-0.97), and the number of protons protected from exchange, P (0.4-4.7), relative to the corresponding values for the whole DHFR molecule (k_ex=0.51min^-1, D_o=0.41, D_??_=0.85, and P=20.7). H/D exchange was very fast in the fragment comprising residues 5-28 (Met 20 loop), which participates in substrate uptake, and reasonably fast in disordered and hydrophobic fragments, but slow in β-strand-rich fragments. These results indicate that the local structures contribute differently to the fluctuation of the DHFR molecule, and that mass spectrometry coupled with H/D exchange and protease digestion is a useful tool for detecting segmentdependent protein fluctuation.

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To investigate the role of the A20-B19 disulfide bond in the structure, activity and folding of proinsulin, a human proinsulin (HPI) mutant [A 20, B19Ala]-HPI was prepared. This mutant, together with another proinsulin mutant previously constructed with an A19Tyr deletion, which can also be taken as shifted mutant of the A20-B19 disulfide bond, were studied for their in vitro refolding, oxidation of free thiol groups, circular dichroism spectra, antibody and receptor binding activities and sensitivity to trypsin digestion in comparison with native proinsulin. The results indicate that deletion of the A20-B19 disulfide bond results in a large decrease in the α-helix content of the molecule and higher sensitivity to tryptic digestion. Both the deletion and shift mutations, especially the latter, cause a great decrease in the biological activity of proinsulin analogues. The folding yields of HPI analogues were much lower than that of HPI. And the shift mutant, [ΔA19Tyr]-HPI, was scarcely refolded correctly in vitro and its refolding yield was extremely low. These results suggest that the A20-B19 disulfide bond plays an important role in the structural stabilization and folding of the insulin precursor. By summarizing the refolding studies on proinsulin, a possible folding pathway is proposed.

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Pyridoxine deficiency caused a decrease in the amount of aromatic L-amino acid decarboxylase (AADC) in PC 12 cells to less than 5% of the control. The degree of the enzyme saturation with the coenzyme pyridoxal 5'-phosphate (PLP) was around 90% for both the control and the pyridoxine-deficient cells, contrary to earlier reports by others. Mathematical analysis of the result indicated that the AADC apoenzyme is degraded at least 20-fold faster than the holoenzyme in the cells. To determine the mechanism of the preferential degradation of the apoenzyme, in vitro model studies were carried out. AADC has a flexible loop that covers the active site. This loop was easily leaved by proteases at similar rates for both the holoenzyme and the apoenzyme. However, in the presence of the substrate analog, dopa methyl ester, the holoenzyme was not cleaved by proteases, while the apoenzyme was cleaved similarly. These results indicated that the ligand that forms a Schiff base (aldimine) with PLP is fixed to the active site and stabilizes the flexible loop. The structure of the rat AADC-dopa complex modeled on the crystal structure of pig AADC showed that the flexible loop can fit in the concave surface at the entrance of the active site, its aliphatic and aromatic residues forming hydrophobic interactions with the substrate catechol ring. It was postulated that the flexible loop of the holoenzyme is stabilized in vivo by taking a closed structure that holds the PLP-substrate aldimine, while the apoenzyme cannot bind the substrate and its flexible loop is easily cleaved, leading to the preferential degradation of the apoenzyme.

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It is known that PomA and PomB form a complex that functions as a Na⁺ channel and generates the torque of the Na⁺-driven flagellar motor of Vibrio alginolyticus. It has been suggested that PomA works as a dieter and that the PomA/PomB complex is composed of four PomA and two PomB molecules. PomA does not have any Cys residues and PomB has three Cys residues. Therefore, a mutant PomB (PomB^cl) whose three Cys residues were replaced by Ala was constructed and found to be motile as well. We carried out gel filtration analysis and examined the effect of cross-linking between the Cys residues of PomB on the formation of the PomA/PomB complex. In the presence of dithiothreitol (DTT), the elution profile of the PomA/PomB complex was shifted to a lower apparent molecular mass fraction similar to that of the complex of the wild-type PomA and PomB^cl mutant. Next, to analyze the arrangement of PomA molecules in the complex, we introduced the mutation P172C, which has been shown to cross-link PomA molecules, into tandem PomA dimers (PomA_??_PomA). These mutant dimers showed a dominant-negative effect. DTT could restore the function of PomA_??_P172C and P172C_??_P172C, but not P172C_??_PomA. Interdimer and intradimer cross-linked products were observed; the interdimer cross-linked products could be assembled with PomB. The formation of the interdimer cross-link suggests that the channel complex of the Na⁺-driven flagellar motor is composed of two units of a complex consisting of two PomA and one PomB, and that they might interact with each other via not only PomA but also PomB.

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Cytochrome b₅₆₁ from bovine adrenal chromaffin vesicles contains two hemes b with different midpoint potentials (+150 and +60 mV) and participates in transmembrane electron transport from extravesicular ascorbate to an intravesicular monooxygenase, dopamine β-hydroxylase. Treatment of oxidized cytochrome b₅₆₁ with diethylpyrocarbonate caused a downshift of midpoint potential for the lower component, and this shift was prevented by the presence of ascorbate during the treatment. Present EPR analyses showed that, upon the treatment, the g_Z=3.69 heme species was converted to a non-ascorbate-reducible form, although its g_Z-value showed no appreciable change. The treatment had no effect on the other heme (the g_Z=3.13 species). Raman data indicated that the two heme b centers adopt a six-coordinated low-spin state, in both the reduced and oxidized forms. There was no significant effect of diethylpyrocarbonate-treatment on the Raman spectra of either form, but the reducibility by ascorbate differed significantly between the two hemes upon the treatment. The addition of ferrocyanide enhanced both the reduction rate and final reduction level of the diethylpyrocarbonate-treated cytochrome b₅₆₁ when ascorbate was used as a reductant. This observation suggests that ferrocyanide scavenges monodehy. droascorbate radicals produced by the univalent oxidation of ascorbate and, thereby, increases both the reduction rate and the final reduction level of the heme center on the intravesicular side of the diethylpyrocarbonate-treated cytochrome. These results further clarify the physiological role of this heme center as the electron donor to the monodehydroascorbate radical.

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Several synthetic peptides derived from the C-terminal domain sequence of a hemolytic lectin, CEL-III, were examined as to their action on bacteria and artificial lipid membranes. Peptide P332 (KGVIFAKASVSVKVTASLSK-NH₂), corresponding to the sequence from residue 332, exhibited strong antibacterial activity toward Gram-positive bacteria. Replacement of each Lys in P332 by Ala markedly decreased the activity. However, when all Lys were replaced by Arg, the antibacterial activity increased, indicating the importance of positively charged residues at these positions. Replacement of Val by Leu also led to higher antibacterial activity, especially toward Gram-negative bacteria. The antibacterial activity of these peptides was correlated with their membrane-permeabilizing activity toward the bacterial inner membrane and artificial lipid vesicles, indicating that the antibacterial action is due to perturbation of bacterial cell membranes, leading to enhancement of their permeability. These results also suggest that the hydrophobic region of CEL-III, from which P332 and its analogs were derived, may play some role in the interaction with target cell membranes to trigger hemolysis.

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In order to discover novel invertebrate cytokines from the budding tunicate, Polyandrocarpa misakiensis, we treated the water-insoluble fraction of tunicate homogenates with trypsin. The extracts showed remarkable activities to promote the growth and motility of tunicate cells. The activities were heat-stable and proteinase K-resistant. After anion exchange chromatography, the activities were eluted with detergents such as 0.1% deoxycholic acid. The Fourier transform infrared spectrum indicated large amounts of fatty acids and phospholipids instead of polypeptides in the extracts. Consistently, the activities were extractable with organic solvents such as chloroform. Long chains of n-3 polyunsaturated free fatty acids (FFA), phosphatidylinositol (PI), phosphatidyleholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) were the major components in the lipid-soluble fraction. A cDNA for FFA-releasing enzyme phospholipase A₂ (PLA₂) was cloned. The expression of this gene could be seen in epidermal cells during budding. The recombinant protein, as in the case of the authentic PLA 2, preferred PC and PE as substrates, followed by PS and PI. The resultant FFAs only promoted cell growth, while the remaining lysophospholipids stimulated cell motility. The former contained unsaturated fatty acids (C18:1, C20:5, and C22:6) while the latter did not, suggesting that unsaturated fatty acids are responsible for mitogenic activity in tunicate cells. These results show for the first time that phospholipids and their derivatives are bio-mediators promoting cell growth and cell motility in invertebrates.

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The genomes of more than 100 species have been sequenced, and the biological functions of encoded proteins are now actively being researched. Protein function is based on interactions between proteins and other molecules. One approach to assuming protein function based on genomic sequence is to predict interactions between an encoded protein and other molecules. As a data source for such predictions, knowledge regarding known proteinsmall molecule interactions needs to be compiled. We have, therefore, surveyed interactions between proteins and other molecules in Protein Data Bank (PDB), the protein three-dimensional (3D) structure database. Among 20, 685 entries in PDB (April, 2003), 4, 189 types of small molecules were found to interact with proteins. Biologically relevant small molecules most often found in PDB were metal ions, such as calcium, zinc, and magnesium. Sugars and nucleotides were the next most common. These molecules are known to act as cofactors for enzymes and/or stabilizers of proteins. In each case of interactions between a protein and small molecule, we found preferred amino acid residues at the interaction sites. These preferences can be the basis for predicting protein function from genomic sequence and protein 3D structures. The data pertaining to these small molecules were collected in a database named Het-PDB Navi., which is freely available at http://daisy.nagahama-i-bio.ac.jp/golab/hetpdbnavi.html and linked to the official PDB home page.

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The inhibitory effects of 15 flavonoids on animal fatty acid synthase (FAS, EC 2. 3. 1. 85) were investigated, and 9 of them were found to inhibit FAS with IC₅₀ (the inhibitor concentration inhibiting 50% of the activity of FAS) values ranging from 2 to 112 μM. A structure-activity relationship study showed that the flavonoids containing two hydroxyl groups in the B ring and 5, 7-hydroxyl groups in the A ring in combination with a C-2, 3 double bond were the most inhibitory. Morin (IC₅₀=2.33±0.09 μM) was further investigated kinetically to detail the inhibitory mechanism. The results showed that morin inhibited the overall reaction of FAS competitively with Ac-CoA, noncompetitively with Mal-CoA and in a mixed manner with NADPH. The study indicated that morin bound reversibly to the β-ketoacyl synthase domain of FAS to inhibit the elongation of the saturated acyl groups in fatty acids synthesis.

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Streptomyces griseus trypsin (SGT) is a bacterial trypsin that lacks the conserved disulphide bond surrounding the autolysis loop. We investigated the molecular mechanism by which SGT is stabilized against autolysis. The autolysis loop connects to another surface loop via a salt bridge (Glu146-Arg222), and the Arg222 residue also forms a cation-π interaction with Tyr217. Elimination of these bonds by site-directed mutagenesis showed that the surface salt bridge at Glu146-Arg222 is the main force stabilizing the enzyme against autolysis. The effect of the cation-π interaction at Tyr217-Arg222 is small, however, its presence increases the half-life by about five hours and enhances the protein stability more than three-fold considering the catalytic activity in the presence of the salt bridge. The melting temperature also showed cooperation between the salt bridge and cation-π interaction. These findings show that S. griseus trypsin is stabilized against autolysis through a cooperative network of a salt bridge and cation-π interaction, which compensate for the absence of the conserved C136-C201 disulphide bond.

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CEL-I is a C-type lectin isolated from the Holothuroidea Cucumaria echinata. This lectin shows very high N-acetylgalactosamine-binding specificity. We constructed an artificial gene encoding recombinant CEL-I (rCEL-I) using a combination of synthetic oligonucleotides, and expressed it in Escherichia coli cells. Since the recombinant protein was obtained as inclusion bodies, the latter were solubilized using urea and 2-mercaptoethanol, and the protein was refolded during the purification and dialysis steps. The purified rCEL-I showed comparable hemagglutinating activity to that of native CEL-I at relatively high Ca²⁺-concentrations, whereas it was weaker at lower Ca²⁺-concentrations due to decreased Ca²⁺-binding affinity. rCEL-I exhibited similar carbohydrate-binding specificity to native CEL-I, including strong GaINAc-binding specificity, as examined by hemagglutination inhibition assay. Comparison of the far UV-CD spectra of recombinant and native CEL-I revealed that the two proteins undergo a similar conformational change upon binding of Cat+. Single crystals of rCEL-I were also obtained under the same conditions as those used for the native protein, suggesting that they have similar tertiary structures. Although native CEL-I exhibited strong cytotoxicity toward cultured cells, rCEL-I showed low cytotoxicity. These results indicate that rCEL-I has a tertiary structure and carbohydrate-binding specificity similar to those of native CEL-I. Howeger, there is a subtle difference in the properties between the two proteins probably due to the additional methionine residue at the N-terminus of rCEL-I.

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A peptide fraction isolated from the venom of the Egyptian scorpion Buthus occitanus was proved to have a bradykinin- potentiating activity. In vivo and in vitro modes of action of the isolated bradykinin- potentiating peptide (BPP) on kidneys of guinea pigs were investigated. Animals received five successive i. p. doses of the scorpion BPP (1 μg/g body weight) at one-week intervals. The control animals were i. p. injected with saline solution only. In vivo experiments showed a significant increase in renal tissue PGE₂ content and lipid peroxides of the treated guinea pigs compared to the control animals (p<0.05). Nonsignificant changes were detected in the levels of tissue c-AMP and 5-nucleotidase activity (p>0.05) of the treated animals, while the changes in c-GMP and c-AMP/c-GMP ratio were both significant (p<0.05). In vitro experiments demonstrated enhanced capacity of guinea pig-renal tissue to convert ¹⁴C-linoleic acid to its metabolites, 6-keto-PGF₁α, PGF₂α, PGE₂, TxB₂, PGD₂, and arachidonic acid, in response to the added PBP (1 μg/ml) and bradykinin (1 μg/ml). This enhanced response was abolished upon the addition of 1 μg/ml of BK-inhibitor (D-Arg- [Hyp³, Thi^{5, 6,} Phe⁷]). The capacity for labeled metabolites recovery in BPP treated renal tissue was 19.78%, while it was 13.00% in the basal control. The total increase that evoked by BPP was 62.78%. The results clearly indicate that the isolated BPP induced prostaglandin biosynthesis, which may trigger enhanced glomerular filtration in guinea pigs.

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ALG-2 is a Ca²⁺-1-binding protein that belongs to the penta-EF-hand protein family and associates with several proteins, including annexin VII, annexin XI, and Alix/AIP1, in a Ca²⁺-dependent manner. The yeast two-hybrid system and a biotin-tagged ALG-2 overlay assay were carried out to characterize the interaction between ALG-2 and Alix. The region corresponding to amino acid residues 794 to 827 in the carboxy-terminal proline-rich region of Alix was sufficient to confer the ability to interact directly with ALG-2. This region includes four-tandem PxY repeats. Alanine substitutions indicated that seven proline residues in this region, four in the PxY repeats, and four tyrosine residues in the PxY repeats are crucial for the binding affinity with ALG-2. Endogenous ALG-2 was co-immunoprecipitated in the presence of Ca²⁺ with FLAG-tagged Alix or FLAG-tagged AIixΔEBS, a deletion mutant lacking the endophilin binding consensus sequence, but not with FLAG-tagged AlixΔABS, another mutant lacking the region comprising amino acids 798-841, from the lysates of HEK293 cells transfected with each FLAG-tagged protein expression construct. FLAG-tagged ALG-2 overexpressed in HEK293 cells was also co-immunoprecipitated with Alix in a Ca2+-dependent fashion, whereas FLAG-tagged ALG-2^E47A/E114A, a Ca²⁺-binding deficient mutant of ALG-2, was not detected in the immunoprecipitates of Alix even in the presence of Ca²⁺. Fluorescent microscopic analyses using the carboxy-terminal half of Alix fused with green fluorescent protein (GFP-AIixCT) revealed that endogenous ALG-2 in HeLa cells exhibits a dot-like pattern overlapping with exogenously expressed GFP-AIixCT, and the distribution of GFP-AlixCTΔABS is observed diffusely in the cytoplasm. These results indicate the requirement of ABS in Alix for the efficient accumulation of AlixCT and raise the possibility that ALG-2 participates in membrane trafficking through a Ca²⁺-dependent interaction with Alix.

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Syndecan-2 cooperates with integrin a5pl in cell adhesion to a fibronectin substratum and regulates actin cytoskeletal organization in an expression level-dependent manner; Lewis lung carcinoma-derived P29 cells with high expression form stress fibers, whereas the same tumor-derived low expressers, LM66-H11 cells, form cortex actin [Munesue, S., Kusano, Y., Oguri, K., Itano, N., Yoshitomi, Y., Nakanishi, H., Yamashina, I., and Okayama, M. (2002) Biochem. J. 363, 201-209]. In this study we examined the participation of other cell surface heparan sulfate proteoglycans in this signaling. The two clones expressed syndecan-1, -2 and -4, and glypican-1 at similar levels except for syndecan-2. Treatment of cells with phosphatidylinositol-specific phospholipase C or immobilized anti-syndecan-1 antibodies demonstrated that neither glypican-1 nor syndecan-1 was involved in this signaling, indicating that individual cell surface heparan sulfate proteoglycans have functional specificity. Stimulation with immobilized anti-syndecan-2 or -4 antibodies induced stress fiber formation in P29 cells but not in LM66-Hll cells, despite the similar levels of syndecan-4 expression, suggesting that stress fiber formation required a threshold expression level of syndecan-2 acting downstream of syndecan-4. This was confirmed by cells in which syndecan-2 expression was artificially suppressed by antisense mRNA oligonucleotide treatment or elevated by cDNA transfection. This is the first report demonstrating that syndecan-2 and -4 cooperate in situ in actin cytoskeletal organization.

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The Saccharomyces cerevisiae Na⁺/H⁺ antiporter Nhalp has a two-domain structure consisting of an N-terminal integral membrane region and a C-terminal cytoplasmic region. We previously identified six distinct cytoplasmic domains (C 1-C 6) conserved among yeast species and here we performed detailed structure-function analysis of the C 1 domain (16 residues). Deletion of the C 1 domain causes extensive inhibition of cell-growth under high salinity conditions. Mutants with single residue deletions or various amino acid substitutions affecting the Cl domain were analyzed with respect to salinity-dependent growth and Nhalp localization. The Cl domain was found to consist of two subdomains: (i) The first three N-proximal residues, which in conjunction with the integral membrane region play a crucial role in the targeting of Nhalp to the cytoplasmic membrane, and (ii) the portion between Leu-439 and Thr-449, which is not required for localization, but in which four residues (Gly-440, Arg-441, His-442, and Ile-446) affect salinity-sensitive cell-growth by possibly influencing the antiporter activity. Based on the overall similarity of the two-domain structure of Nhalp to that of mammalian Na⁺/H⁺ antiporters, the functional importance of domains proximal to the membrane region is discussed.

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High mobility group box protein 1, HMGB1, is a major nonhistone chromatin component in higher eukaryotic cells. HMGB1 is thought to be involved in the processes of global nuclear events such as transcription, recombination and repair, but the mechanism of these processes is unclear. Here, we show a concrete example of chromatin structural modulation by HMGB1 in HeLa S 3 cells. A co-immunopurification experiment with Flag-tagged HMGB1 revealed that a portion of HMGB1 in HeLa S 3 cells is included in a large-molecular-weight multiprotein complex. The multiprotein complex including HMGB1 showed ATP hydrolysis and ATP-dependent chromatin structural modulation activities that increased the susceptibility of chromatin to MNase digestion, while HMGB1 alone had no such activity. Thus, HMGB1 in the multiprotein complex is critical for expressing the chromatin structural modulation activity. These results suggest that HMGB1 is involved in chromatin structural modulation in global nuclear events through its interaction with a multiprotein complex in mammalian cells.

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