Biomedical subjects

Kunihiro Kitamura

Publications and source records attributed to Kunihiro Kitamura.

14 recordsLinked to original sources

PubMed2006

Quantitative evaluation of each catalytic subsite of cathepsin B for inhibitory activity based on inhibitory activity-binding mode relationship of epoxysuccinyl inhibitors by X-ray crystal structure analyses of complexes.

To quantitatively estimate the inhibitory effect of each substrate-binding subsite of cathepsin B (CB), a series of epoxysuccinyl derivatives with different functional groups bound to both carbon atoms of the epoxy ring were synthesized, and the relationship between their inhibitory activities and binding modes at CB subsites was evaluated by the X-ray crystal structure analyses of eight complexes. With the common reaction in which the epoxy ring of inhibitor was opened to form a covalent bond with the SgammaH group of the active center Cys29, the observed binding modes of the substituents of inhibitors at the binding subsites of CB enabled the quantitative assessment of the inhibitory effect of each subsite. Although the single blockage of S1' or S2' subsite exerts only the inhibitory effect of IC50 = approximately 24 microM (k2 = approximately 1250 M(-1) s(-1)) or approximately 15 microM (k2 = approximately 1800 M(-1) s(-1)), respectively, the synchronous block of both subsites leads to IC50 = approximately 23 nM (k2 = 153,000 - 185,000 M(-1) s(-1)), under the condition that (i) the inhibitor possesses a P1' hydrophobic residue such as Ile and a P2' hydrophobic residue such as Ala, Ile or Pro, and (ii) the C-terminal carboxyl group of a P2' residue is able to form paired hydrogen bonds with the imidazole NH of His110 and the imidazole N of His111 of CB. The inhibitor of a Pn' > or = 3' substituent was not potentiated by collision with the occluding loop. On the other hand, it was suggested that the inhibitory effects of Sn subsites are independent of those of Sn' subsites, and the simultaneous blockage of the funnel-like arrangement of S2 and S3 subsites leads to the inhibition of IC50 = approximately 40 nM (k2 = approximately 66,600 M(-1) s(-1)) regardless of the lack of Pn' substituents. Here we present a systematic X-ray structure-based evaluation of structure-inhibitory activity relationship of each binding subsite of CB, and the results provide the structural basis for designing a more potent CB-specific inhibitor.

Animals↗

PubMed2006

Effect of N-terminal region of eIF4E and Ser65-phosphorylation of 4E-BP1 on interaction between eIF4E and 4E-BP1 fragment peptide.

To clarify the contribution of N-terminal region of eukaryotic initiation factor 4E (eIF4E) to the interaction with 4E-BP and to investigate the effect of 4E-BP phosphorylation on the interaction with eIF4E, the interaction profiles of the Ser65-unphosphorylated and phosphorylated peptides (Thr37-Thr70 fragment of 4E-BP1) with full-length and N-terminal 33 residues-deleted eIF4Es were investigated by fluorescence and SPR methods. The effect of N-terminal region of eIF4E on the interaction with 4E-BP1 peptides was shown to be dependent on the interaction state, that is, the steady-state fluorescence and kinetic-state SRP analyses showed the positive and negative contributions of the N-terminal region to the interaction with the peptide, respectively, despite its unphosphorylated or phosphorylated state. The comparison of the association constants of the peptide with those of full-length 4E-BP1 indicated the importance of N-terminal (1-36) and/or C-terminal (71-118) sequence of 4E-BP1 for the interaction, although the MD simulations suggested that the alpha-helical region (Arg56-Cys62) of 4E-BP1 peptide is sufficient for keeping the interaction. The MD simulations also indicated that a charge-dependent rigid hydration shell formed around the phosphate group makes the molecular conformation rigid, and single Ser65 phosphorylation is insufficient for releasing 4E-PB1 peptide from eIF4E.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Binding mode of novel 1-substituted quinazoline derivatives to poly(ADP-ribose) polymerase-catalytic domain, revealed by X-ray crystal structure analysis of complexes.

In order to clarify the role of the 1-substituent of quinazoline derivatives in their inhibitory activity against poly(ADP-ribose) polymerase (PARP), two novel inhibitors, 1 [8-hydroxy-1-(3-morpholinopropyl)-quinazoline-2,4(1H,3H)-dione] and 2 [8-hydroxy-1-(3-phenoxypropyl)-quinazoline-2,4(1H,3H)-dione], were synthesized and subjected to X-ray crystal analysis in complex with the PARP C-terminal catalytic domain (PARP-CD), which requires NAD+ coenzyme for biological function. The nicotinamide-mimicking part of the quinazoline skeleton of 1 and 2 were both located at the nicotinamide subsite of the NAD+-binding pocket in the same manner as previously reported inhibitors: three hydrogen bonds [(Gly-863)NH-O12, (Gly-863)O-HN3 and (Ser-904)O(gamma)-O12] and stacking interaction between the Tyr-907 phenol and the quinazoline ring. On the other hand, the N-morpholinoprop-3-yl moiety introduced at the 1-position of the quinazoline ring in 1 bridged the large gap between the donor site and the acceptor site through a (Met-890)NH-O20(morpholine) hydrogen bond, where the donor and the acceptor sites are classified as the binding sites of NAD+ and the ADP moiety of the poly(ADP-ribose) chain, respectively. In contrast, the N-phenoxyprop-3-yl moiety in 2 formed hydrophobic interactions close to the adenosine-binding site of NAD+, unlike the hydrogen bond such as in 1. As the inhibitory activities of 1 and 2 for PARP were much more potent than those of the unsubstituted nicotinamide analogues, these results suggest that the occupation of the proximal region of the ADP phosphate-and adenosine-binding subsite of the donor site or that of the gap between the donor and the acceptor site by the 1-substituent of quinazoline may increase the inhibitory activity considerably. The nearly equal inhibitory activities of 1 and 2, despite of their different binding modes at the active site, indicate that this 1-substituent is promising in improving the bioavailability of the inhibitor without compromising its inhibitory activity.

Animals↗

PubMed2005

Structural basis for mRNA Cap-Binding regulation of eukaryotic initiation factor 4E by 4E-binding protein, studied by spectroscopic, X-ray crystal structural, and molecular dynamics simulation methods.

Taking advantage of the Trp73 residue located close to the 4E-BP binding site of eIF4E, the interaction between the 4E-BP isoform and eIF4E was investigated by the Trp fluorescence titration method. Although no significant difference was observed among the association constants of three 4E-BP isoforms, the binding preference of 4E-BP2 over 4E-BP1 and -BP3 was shown, probably due to the effect of a 4E-BP2-specific LDRR (60-63) sequence for the binding with eIF4E. By contrast, surface plasmon resonance (SPR) analyses showed the binding preference of 4E-BP1, although the difference among the isoforms was also not significant. This inconsistency with fluorescence analysis likely resulted from the different observation points of the interaction, i.e., local and overall interactions observed by the fluorescence and SPR methods, respectively. To clarify the structural basis for these spectroscopic results, the crystal structure of the ternary complex of m7GpppA-eIF4E-4E-BP1 fragment (Thr36-Thr70) was analyzed by the X-ray diffraction method. Crystal structure analysis at 2.1 A resolution revealed that the 4E-BP1 fragment, assigned to the Pro47-Pro66 peptide moiety, adopted a reverse L-shaped conformation involving the beta sheet and alpha-helical structures and was located at the root of the handle of the temple-bell-shaped eIF4E through hydrophilic and hydrophobic interactions. Based on the observed binding mode, possible interactions with the three 4E-BP isoforms have been discussed. On the other hand, since the crystal structural comparison with the previously determined m7GpppA-eIF4E-4E binary complex showed that the docking of the 4E-BP1 fragment does not significantly affect the overall tertiary structure and cap-binding scaffold of eIF4E, the dynamic regulation of the cap-binding of eIF4E by 4E-BP1 was investigated by molecular dynamics (MD) simulations. Consequently, the simulation suggested that (i) the helical region of the 4E-BP1 peptide is important for the binding with eIF4E, (ii) the existence of a cap structure stabilizes the binding of eIF4E with 4E-BP, (iii) the binding of 4E-BP stabilizes the cap-binding pocket of eIF4E, and (iv) the phosphorylation of Ser67 alone does not induce the separation of 4E-BP from eIF4E, but increases the structural rigidity of 4E-BP. These results provide the structural basis for the mRNA cap-binding regulation of eIF4E by 4E-BP.

Adaptor Proteins, Signal Transducing↗

PubMed2005

A detailed unfolding pathway of a (beta/alpha)8-barrel protein as studied by molecular dynamics simulations.

The (beta/alpha)(8)-barrel is the most common protein fold. Similar structural properties for folding intermediates of (beta/alpha)(8)-barrel proteins involved in tryptophan biosynthesis have been reported in a number of experimental studies; these intermediates have the last two beta-strands and three alpha-helices partially unfolded, with other regions of the polypeptide chain native-like in conformation. To investigate the detailed folding/unfolding pathways of these (beta/alpha)(8)-barrel proteins, temperature-induced unfolding simulations of N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia coli were carried out using a special-purpose parallel computer system. Unfolding simulations at five different temperatures showed a sequential unfolding pathway comprised of several events. Early events in unfolding involved disruption of the last two strands and three helices, producing an intermediate ensemble similar to those detected in experimental studies. Then, denaturation of the first two betaalpha units and separation of the sixth strand from the fifth took place independently. The remaining central betaalphabetaalphabeta module persisted the longest during all simulations, suggesting an important role for this module as the incipient folding scaffold. Our simulations also predicted the presence of a nucleation site, onto which several hydrophobic residues condensed forming the foundation for the central betaalphabetaalphabeta module.

Aldose-Ketose Isomerases↗

PubMed2003

Structural features of human initiation factor 4E, studied by X-ray crystal analyses and molecular dynamics simulations.

The structural features of human eIF4E were investigated by X-ray crystal analyses of its cap analog (m(7)GTP and m(7)GpppA) complexes and molecular dynamics (MD) simulations of cap-free and cap-bound eIF4Es, as well as the cap-bound Ser209-phosphorylated eIF4E. Crystal structure analyses at 2.0 A resolution revealed that the molecule forms a temple-bell-shaped surface of eight antiparallel beta-structures, three alpha-helices and ten loop structures, where the N-terminal region corresponds to the handle of the bell. This concave backbone provides a scaffold for the mRNA cap-recognition pocket consisting of three receiving parts for the 5'-terminal m(7)G base, the triphosphate, and the second nucleotide. The m(7)G base is sandwiched between the two aromatic side-chains of Trp102 and Trp56. The two (m(7)G)NH-O (Glu103 carboxy group) hydrogen bonds stabilize the stacking interaction. The basic residues of Arg157 and Lys162 and water molecules construct a binding pocket for the triphosphate moiety, where a universal hydrogen-bonding network is formed. The flexible C-terminal loop region unobserved in the m(7)GTP complex was clearly observed in the m(7)GpppA complex, as a result of the fixation of this loop by the interaction with the adenosine moiety, indicating the function of this loop as a receiving pocket for the second nucleotide. On the other hand, MD simulation in an aqueous solution system revealed that the cap-binding pocket, especially its C-terminal loop structure, is flexible in the cap-free eIF4E, and the entrance of the cap-binding pocket becomes narrow, although the depth is relatively unchanged. SDS-PAGE analyses showed that this structural instability is highly related to the fast degradation of cap-free eIF4E, compared with cap-bound or 4E-BP/cap-bound eIF4E, indicating the conferment of structural stability of eIF4E by the binary or ternary complex formation. MD simulation of m(7)GpppA-bound Ser209-phosphorylated eIF4E showed that the size of the cap-binding entrance is dependent on the ionization state in the Ser209 phosphorylation, which is associated with the regulatory function through the switching on/off of eIF4E phosphorylation.

Amino Acid Sequence↗

PubMed2003

Development of hardware accelerator for molecular dynamics simulations: a computation board that calculates nonbonded interactions in cooperation with fast multipole method.

Evaluation of long-range Coulombic interactions still represents a bottleneck in the molecular dynamics (MD) simulations of biological macromolecules. Despite the advent of sophisticated fast algorithms, such as the fast multipole method (FMM), accurate simulations still demand a great amount of computation time due to the accuracy/speed trade-off inherently involved in these algorithms. Unless higher order multipole expansions, which are extremely expensive to evaluate, are employed, a large amount of the execution time is still spent in directly calculating particle-particle interactions within the nearby region of each particle. To reduce this execution time for pair interactions, we developed a computation unit (board), called MD-Engine II, that calculates nonbonded pairwise interactions using a specially designed hardware. Four custom arithmetic-processors and a processor for memory manipulation ("particle processor") are mounted on the computation board. The arithmetic processors are responsible for calculation of the pair interactions. The particle processor plays a central role in realizing efficient cooperation with the FMM. The results of a series of 50-ps MD simulations of a protein-water system (50,764 atoms) indicated that a more stringent setting of accuracy in FMM computation, compared with those previously reported, was required for accurate simulations over long time periods. Such a level of accuracy was efficiently achieved using the cooperative calculations of the FMM and MD-Engine II. On an Alpha 21264 PC, the FMM computation at a moderate but tolerable level of accuracy was accelerated by a factor of 16.0 using three boards. At a high level of accuracy, the cooperative calculation achieved a 22.7-fold acceleration over the corresponding conventional FMM calculation. In the cooperative calculations of the FMM and MD-Engine II, it was possible to achieve more accurate computation at a comparable execution time by incorporating larger nearby regions.

Algorithms↗

PubMed2003

Targeting of MIST to Src-family kinases via SKAP55-SLAP-130 adaptor complex in mast cells.

MIST (mast cell immunoreceptor signal transducer; also termed Clnk) is an adaptor protein structurally related to SLP-76-family hematopoietic cell-specific adaptor proteins. We demonstrate here that two major MIST-associated phosphoproteins expressed in mast cell lines are SLAP-130 and SKAP55, adaptors known to interact with the Src-homology (SH) 2 domain of Src-family protein tyrosine kinases (PTKs). MIST directly associated with SLAP-130 via its SH2 domain, and collaboration of SLAP-130 with SKAP55 was required for the recruitment of MIST to Lyn. Furthermore, MIST was preferentially recruited to Fyn rather than Lyn, which is regulated by higher affinity binding of SLAP-130 and SKAP55 with the Fyn-SH2 domain than the Lyn-SH2 domain. Our results suggest that the MIST-SLAP-130-SKAP55 adaptor complex functions downstream of high-affinity IgE receptor-associated Src-PTKs in mast cells.

Adaptor Proteins, Signal Transducing↗

PubMed2003

A novel homocysteine-responsive gene, smap8, modulates mitogenesis in rat vascular smooth muscle cells.

We isolated the cDNA of a gene, designated smooth muscle-associated protein 8 (smap8), during a search for new genes expressed in human aortic smooth muscle cells. The full-length smap8 cDNA is 3241 bp long and contains an open reading frame of 1113 bp encoding an approximately 45 kDa soluble protein identical to NDRG4 protein. Smap8 mRNA was expressed predominantly in the brain and heart, and moderately in vascular smooth muscle cells. Expression of smap8 mRNA was induced within 3-12 h by treatment with 10 mm homocysteine in rat aortic smooth muscle cells (A10 cells). Expression of exogenous smap8 markedly reduced both the proliferation and migration rates of rat A10 cells, however, PDGF-induced proliferation was significantly enhanced in smap8-expressed cells compared with mock-transfected cells. To ascertain the involvement of smap8 in mitogenesis, we tested the effects of stimulation of smap8, MEK1/2 or ERK1/2, which is known as a proliferation relating intermediate, by various growth factors and cytokines. PDGF was the most prominent in promoting phosphorylation of the smap8 protein. PDGF-dependent phosphorylation of smap8 was induced prior to ERK1/2 activation, and was repressed by staurosporine, a general inhibitor of serine/threonine kinases. Furthermore, activation of both MEK1/2 and ERK1/2 was markedly enhanced in these cells. Smap8 might therefore regulate the potentiation of ERK1/2 signalling induced by PDGF treatment. Our results imply that smap8 is involved in the regulation of mitogenic signalling in vascular smooth muscle cells, possibly in response to a homocysteine-induced injury.

Amino Acid Sequence↗

PubMed2002

A novel parallel algorithm for large-scale Fock matrix construction with small locally distributed memory architectures: RT parallel algorithm.

We developed a novel parallel algorithm for large-scale Fock matrix calculation with small locally distributed memory architectures, and named it the "RT parallel algorithm." The RT parallel algorithm actively involves the concept of integral screening, which is indispensable for reduction of computing times with large-scale biological molecules. The primary characteristic of this algorithm is parallel efficiency, which is achieved by well-balanced reduction of both communicating and computing volume. Only the density matrix data necessary for Fock matrix calculations are communicated, and the data once communicated are reutilized for calculations as many times as possible. The RT parallel algorithm is a scalable method because required memory volume does not depend on the number of basis functions. This algorithm automatically includes a partial summing technique that is indispensable for maintaining computing accuracy, and can also include some conventional methods to reduce calculation times. In our analysis, the RT parallel algorithm had better performance than other methods for massively parallel processors. The RT parallel algorithm is most suitable for massively parallel and distributed Fock matrix calculations for large-scale biological molecules with more than thousands of basis functions.

Algorithms↗

PubMed2002

Identification of a novel smooth muscle associated protein, smap2, upregulated during neointima formation in a rat carotid endarterectomy model.

Proliferation of aortic smooth muscle cells is an important event in vascular lesion formation. To identify new genes that are involved in neointima formation, we constructed an aortic 3'-directed cDNA library. The novel cDNA of a gene designated smooth muscle associated protein 2 (smap2) was isolated. The full-length cDNA of smap2 is 2914 base pairs long and contains an open reading frame of 1338 base pairs. Dot blot analysis revealed that smap2 was expressed particularly in aorta. The deduced amino acid sequence of smap2 contains two thyroglobulin type-1 domains, two EF-hand calcium-binding domains and putative signal peptide. Furthermore, we demonstrated that smap2 mRNA was upregulated during neointima formation in a rat carotid endarterectomy model. These findings suggest that smap2 might be involved in the progression of atherosclerosis in aorta.

Amino Acid Sequence↗

PubMed2002

Structural basis for development of cathepsin B-specific noncovalent-type inhibitor: crystal structure of cathepsin B-E64c complex.

In order to elucidate the substrate specificity of the Sn subsites (n=1-3) of cathepsin B, its crystal structure inhibited by E64c [(+)-(2S,3S)-3-(1-[N-(3-methylbutyl)amino]-leucylcarbonyl)oxirane-2-carboxylic acid] was analyzed by the X-ray diffraction method. Iterative manual rebuilding and convenient conjugate refinement of structure decreased R- and free R-factors to 19.7% and to 23.9%, respectively, where 130 water molecules were included for the refinement using 14,759 independent reflections from 10 to 2.3 A resolution. The epoxy carbonyl carbon of E64c was covalently bonded to the Cys(29) S(gamma) atom and the remaining parts were located at Sn subsites (n=1-3). The substrate specificity of these subsites was characterized based on their interactions with the inhibitor. Base on these structural data, we developed a novel cathepsin B-specific noncovalent-type inhibitor, which may bind to S2'-S3. The molecular design of possessing structural elements of both CA074 and E64c, assisted by energy minimization and molecular dynamics (MD) simulation, may lead to a new lead noncovalent-type inhibitor.

Animals↗

PubMed2002

Crystal structures of 7-methylguanosine 5'-triphosphate (m(7)GTP)- and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)-bound human full-length eukaryotic initiation factor 4E: biological importance of the C-terminal flexible region.

The crystal structures of the full-length human eukaryotic initiation factor (eIF) 4E complexed with two mRNA cap analogues [7-methylguanosine 5'-triphosphate (m(7)GTP) and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)] were determined at 2.0 A resolution (where 1 A=0.1 nm). The flexibility of the C-terminal loop region of eIF4E complexed with m(7)GTP was significantly reduced when complexed with m(7)GpppA, suggesting the importance of the second nucleotide in the mRNA cap structure for the biological function of eIF4E, especially the fixation and orientation of the C-terminal loop region, including the eIF4E phosphorylation residue. The present results provide the structural basis for the biological function of both N- and C-terminal mobile regions of eIF4E in translation initiation, especially the regulatory function through the switch-on/off of eIF4E-binding protein-eIF4E phosphorylation.

Amino Acid Sequence↗

PubMed2002

Molecular characterization of mammalian dicarbonyl/L-xylulose reductase and its localization in kidney.

In this report, we first cloned a cDNA for a protein that is highly expressed in mouse kidney and then isolated its counterparts in human, rat hamster, and guinea pig by polymerase chain reaction-based cloning. The cDNAs of the five species encoded polypeptides of 244 amino acids, which shared more than 85% identity with each other and showed high identity with a human sperm 34-kDa protein, P34H, as well as a murine lung-specific carbonyl reductase of the short-chain dehydrogenase/reductase superfamily. In particular, the human protein is identical to P34H, except for one amino acid substitution. The purified recombinant proteins of the five species were about 100-kDa homotetramers with NADPH-linked reductase activity for alpha-dicarbonyl compounds, catalyzed the oxidoreduction between xylitol and l-xylulose, and were inhibited competitively by n-butyric acid. Therefore, the proteins are designated as dicarbonyl/l-xylulose reductases (DCXRs). The substrate specificity and kinetic constants of DCXRs for dicarbonyl compounds and sugars are similar to those of mammalian diacetyl reductase and l-xylulose reductase, respectively, and the identity of the DCXRs with these two enzymes was demonstrated by their co-purification from hamster and guinea pig livers and by protein sequencing of the hepatic enzymes. Both DCXR and its mRNA are highly expressed in kidney and liver of human and rodent tissues, and the protein was localized primarily to the inner membranes of the proximal renal tubules in murine kidneys. The results imply that P34H and diacetyl reductase (EC ) are identical to l-xylulose reductase (EC ), which is involved in the uronate cycle of glucose metabolism, and the unique localization of the enzyme in kidney suggests that it has a role other than in general carbohydrate metabolism.

Acetoin Dehydrogenase↗