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Biomedical subjects

M Nomizu

Publications and source records attributed to M Nomizu.

At least 73 records · Page 4Linked to original sources
PubMed1995

Identification of cell binding sites in the laminin alpha 1 chain carboxyl-terminal globular domain by systematic screening of synthetic peptides.

The laminin alpha 1 chain carboxyl-terminal globular domain has been identified as a site of multiple biological activities. Using a systematic screening for cell binding sites with 113 overlapping synthetic peptide beads that covered this domain, we found 19 potential active sequences. Corresponding synthetic peptides were evaluated for direct cell attachment, spreading, and inhibition of cell spreading to a laminin-1 substrate using several cell lines. Five peptides (AG-10, AG-22, AG-32, AG-56, and AG-73) showed cell attachment activities with cell-type specificities. Cell spreading on AG-10 was inhibited by beta 1 and alpha 6 integrin antibodies and on AG-32 was inhibited by beta 1, alpha 2, and alpha 6 integrin antibodies. In contrast, cell adhesion and spreading on peptide AG-73 were not inhibited by these antibodies. The minimum active sequences of AG-10, AG-32, and AG-73 were determined to be SIYITRF, IAFQRN, and LQVQLSIR, respectively. These sequences are highly conserved among the different species and different laminin alpha chains, suggesting that they play a critical role for biological function and for interaction with cell surface receptors.

Amino Acid Sequence↗
PubMed1995

Structure-activity study of a laminin alpha 1 chain active peptide segment Ile-Lys-Val-Ala-Val (IKVAV).

The IKVAV sequence, one of the most potent active sites of laminin-1, has been shown to promote cell adhesion, neurite outgrowth, and tumor growth. Here we have determined the structural requirements of the IKVAV sequence for cell attachment and neurite outgrowth using various 12-mer synthetic peptide analogs. All-L- and all-D-IKVAV peptides showed cell attachment and neurite outgrowth activities. In contrast, all-L- and all-D-reverse-sequence peptides were not active. Some of the analogs, in which the lysine and isoleucine residues of the IKVAV peptide were substituted with different amino acids, promoted cell attachment, but none of the analog peptides showed neurite outgrowth activity comparable to that of the IKVAV peptide. These results suggest that the lysine and isoleucine residues are critical for the biological functions of the IKVAV peptide.

Amino Acid Sequence↗
PubMed1995

A specific sequence of the laminin alpha 2 chain critical for the initiation of heterotrimer assembly.

Triple-stranded laminin molecules assemble via an alpha-helical coiled-coil structure spanning approximately 600 amino acid residues of each chain. We reported that the C termini of the beta 1 and gamma 1 chains direct the specific dimer and trimer assembly (Utani, A., Nomizu, M., Timpl, R., Roller, P.P., and Yamada, Y. (1994) J. Biol. Chem. 269, 19167-19175). In this study, we focused on the mechanism of trimer formation of the alpha 2 chain utilizing three different approaches. First, competition assays using mutated recombinant alpha 2 chain defined a 25-amino acid sequence at the C terminus of the long arm as an essential site for assembly with beta 1 and gamma 1 chain. Site-specific mutations and synthetic peptides of this site revealed that both positively charged amino acid residues and the alpha-helical structure within this site were critical. Second, overexpression studies of recombinant alpha 2 chain long arm confirmed that the C-terminal end was critical for the trimer assembly within NIH 3T3 cells. Third, circular dichroism spectroscopic examination of the complexes reconstituted in vitro revealed dynamic conformational changes of the alpha 2 and gamma 1 chains in the process of assembly. These studies also revealed that the proper folding of the extreme C terminus of alpha 2 chain was critical for the stability of trimer. From these data, it is concluded that the C terminus of alpha 2 chain long arm is required for the effective initiation of laminin heterotrimer assembly.

3T3 Cells↗
PubMed1995

NP-06: a novel anti-human immunodeficiency virus polypeptide produced by a Streptomyces species.

From an extract of a Streptomyces culture, we identified and purified a novel compound, NP-06, which is active against human immunodeficiency virus (HIV) in vitro. Analyses indicate that NP-06 is a hydrophobic 21-mer oligopeptide, N terminally cyclized through the side chain of Asp-9, containing two intramolecular cystine linkages with a molecular weight of 2,163.4. The 50% inhibitory concentrations were 2.8 and 1.3 microM when NP-06 was tested for in vitro anti-HIV-1 activity in ATH8 cells and phytohemagglutinin-activated peripheral blood mononuclear cells, respectively, NP-06 appears to block the early stage of HIV-1 infection, most likely at the stage of virus-cell fusion.

Amino Acid Sequence↗
PubMed1994

Assembly of synthetic laminin peptides into a triple-stranded coiled-coil structure.

Laminin, a large multidomain glycoprotein specific to basement membranes, is a heterotrimer with alpha, beta, and gamma chains held together in an alpha-helical coiled-coil structure. Synthetic peptides comprising two 51-mers (B1 and B2) from the beta 1 and gamma 1 subunits and a 55-mer (M) from alpha 2 were used to study the molecular mechanisms in laminin chain assembly. Using the synthetic peptides in various mixing experiments, the heterotrimer (B1-B2-M) was preferentially produced. The thermal stability of the heterotrimer increased dramatically (by approximately 20 degrees C) over that of the B1-B2 heterodimer as measured by circular dichroism (CD) spectroscopy. The B1-B1 homodimer (Tm = 60 degrees C) showed higher thermal stability when compared to B1-B2 and B2-B2 dimers. However, the B1 + B2 mixture produced principally the B1-B2 heterodimer. These results suggested that the preferential formations of heterodimer was regulated by kinetic interactions between each chain. The B2 and M peptides have many hydrophobic isoleucine residues which were replaced by leucines. These substitutions were predicted to favor an alpha-helical conformation and a higher propensity for zipper formation. B2L and ML, in which all isoleucine residues were replaced by leucine, showed significantly increased alpha-helicities. While B2L was able to form heterodimers and heterotrimers similar to B2, ML was not able to participate in heterotrimer formation as efficiently as the M peptide. The thermal stability of B1-B2L was comparable to that of B1-B2, but B2L and/or ML containing trimers showed lower thermal stability than B1-B2-M. These results suggest that the isoleucine residues in the alpha 2 and gamma 1 chains are critical for stabilizing the heteromeric triple-stranded coiled-coil structure.

Amino Acid Sequence↗
PubMed1994

D-Val22 containing human big endothelin-1 analog, [D-Val22]Big ET-1[16-38], inhibits the endothelin converting enzyme.

Endothelin converting enzyme (ECE) is essential for generation of the biological effects of endothelin-1 (ET-1) from a precursor, big endothelin-1 (Big ET-1). We synthesized four analogs of human Big ET-1[16-38], substituted with single D-amino acids at P1, P2, P1' and P2' positions. ECE activity was determined using an ET-1 specific radioimmunoassay system. None of the D-amino acid containing Big ET-1 analogs were apparently cleaved by ECE, however, one of the synthetic peptides, [D-Val22]Big ET-1[16-38], strongly inhibited the ECE activity. Furthermore, when this D-Val22 containing peptide was preadministered to rat striatum, it was found to inhibit the dopamine release induced by Big ET-1. This result suggests that the D-Val22 containing peptide inhibits the ECE activity in vivo. The D-Val22 containing inhibitor offers hope of developing more potent and highly specific ECE inhibitors of therapeutic significance.

Amino Acid Sequence↗
PubMed1994

The short amino acid sequence Pro-His-Ser-Arg-Asn in human fibronectin enhances cell-adhesive function.

Synergistic sites in the central cell-adhesive domain of fibronectin (FN) substantially enhance cell adhesion mediated by the alpha 5 beta 1 integrin receptor for fibronectin. We characterized a critical minimal sequence needed for synergistic activity using site-directed mutagenesis and homology scanning using intramolecular chimeras. The minimal cell-binding domain of FN consisting of the 9th and 10th type III FN repeat was expressed in an Escherichia coli expression system. This protein retained high biological activity when assayed using a competitive inhibition assay for FN-mediated adhesion of baby hamster kidney or HT-1080 cells. In contrast, a construct consisting of the 8th and 10th repeat displayed very low biological activity. By replacing various portions of the 8th repeat with homologous 9th repeat segments, we mapped the synergistic region to the center of the 9th repeat. When a very short peptide sequence, Pro-His-Ser-Arg-Asn (PHSRN), from the 9th repeat was substituted for the homologous pentapeptide site in the 8th repeat sequence, the recombinant protein showed markedly enhanced activity. Further mutagenesis analysis suggested that the arginine residue of this pentapeptide sequence is important for function. We also identified a weaker adjacent synergy region other than the PHSRN region. Epitope mapping of an anti-FN monoclonal antibody that inhibits FN-mediated adhesion identified the same critical regions. A synthetic peptide containing the PHSRN sequence showed neither competitive inhibitory activity in solution nor synergy with a soluble RGD-containing peptide. However, when the same synthetic peptide was positioned via a covalent bond at the corresponding site of the normally inactive 8th repeat, it mediated an enhancement of adhesive activity. These results identify a pentapeptide site that synergistically enhances the cell-adhesive activity of the FN RGD sequence.

Amino Acid Sequence↗
PubMed1994

Apoptosis in human fibrosarcoma cells is induced by a multimeric synthetic Tyr-Ile-Gly-Ser-Arg (YIGSR)-containing polypeptide from laminin.

The YIGSR (Tyr-Ile-Gly-Ser-Arg) peptide, derived from the laminin beta 1 chain, decreases tumor metastasis and growth in experimental animals. The mechanism responsible for this inhibition is not known. We now report that a 16-mer branched form of YIGSR, synthesized by the multimeric antigen peptide system, induced the apoptosis of HT-1080 cells in vitro at 30 micrograms/ml (approximately 3 microM). Tumor cells treated with this peptide showed the expected morphological changes associated with apoptosis, acridine orange staining of nuclei, increased numbers of 3'-OH ends of DNA in nuclei, a DNA ladder pattern on agarose gels, and increased transforming growth factor beta 1 mRNA by Northern blot. The specificity of this peptide was confirmed by inhibition of apoptosis with a neutralizing antibody to the peptide. In addition, the branched 16-mer peptides of scrambled sequence did not induce apoptosis. Our in vitro results suggest that apoptosis may play a role in the antimetastatic and antitumor effects associated with the YIGSR peptide.

Amino Acid Sequence↗
PubMed1994

Laminin chain assembly. Specific sequences at the C terminus of the long arm are required for the formation of specific double- and triple-stranded coiled-coil structures.

The assembly of laminin chains into double- and triple-stranded structures was studied with recombinant proteins derived from the C-terminal alpha-helical region of the long arm of laminin. Both affinity assays and structural studies demonstrated chain-specific assembly of the B1, B2, and A chains, while associations between homotypic and homologous chains could not be observed. These results suggest that chain selection is controlled by the C-terminal region. Deletion mapping in the B1e and B2e chains identified two sites important for dimer and trimer formation. These two sites were separated by 23 amino acids in the B1e chain, whereas they were adjacent in the B2e chain. The Ae and Am chains contained only one site for trimerization. Site-directed mutagenesis revealed that charged amino acid residues within these sites were essential for association. These results suggest that distinct sites within the C-terminal alpha-helical region of the laminin long arm are critical for the chain-specific assembly of these macro-molecules.

Amino Acid Sequence↗
PubMed1994

Lymphocytes and promonocytes attach to the synthetic [Tyr5,12, Lys7]- polyphemusin II peptide.

The [Tyr5,12,Lys7]-polyphemusin II peptide (T22) has been shown to inhibit HIV-1 replication in lymphocytes. The mechanism of T22 inhibition of HIV-1 replication is not known but may involve T22 competition with HIV-1 for attachment sites on the plasma membrane of targeted cells. Here we find that three human immunocyte cell lines (H9, Jurkat, and U-937) attach to T22. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), has been shown to activate intracellular protein kinase C and to stimulate lymphocyte attachment to various substrates through specific cell surface receptors. Here we find that TPA treatment enhances attachment of the immunocytes to T22 by three- to four-fold. These data demonstrate that T22 binds to immunocyte cell surfaces and support the hypothesis that T22 may inhibit HIV-1 replication by competing with the virus for a common cell surface receptor(s).

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Cyclic peptide inhibitors of phosphatidylinositol 3-kinase p85 SH2 domain binding.

Cyclic hexameric peptides based on the amino acid sequence "Gly-Xxx-Val-Pro-Met-Leu", where Xxx is either phosphotyrosyl (pTyr) residue or a hydrolytically stable pTyr mimetic, were examined for their ability to bind to the C-terminal SH2 domain of the p85 phosphoinositol 3-kinase (PI 3-kinase). The cyclic peptides retained significant binding affinity relative to their linear counterparts. Potency varied depending on Xxx in the order: phosphonomethyl phenylalanine (Pmp, ID50 = 5.2 microM) < phosphonodifluoromethyl phenylalanine (F2Pmp, ID50 = 2.2 microM) < pTyr (ID50 = 1.0 microM), with Xxx = Tyr being inactive (ID50 > 500 M). Greatly reduced potency was observed when Xxx was of the unnatural D-configuration. The cyclic peptides represent conformationally constrained ligands which should be useful in the development of p85 SH2 domain-directed inhibitors.

Amino Acid Sequence↗
PubMed1994

Nonhydrolyzable phosphotyrosyl mimetics for the preparation of phosphatase-resistant SH2 domain inhibitors.

Src homology 2 (SH2) domains participate in protein tyrosine kinase (PTK)-mediated cellular signal transduction through their ability to bind with high affinity to phosphotyrosyl (pTyr)-bearing protein sequences. Although peptides containing pTyr competitively inhibit the binding between phosphoproteins and cognate SH2 proteins in a sequence-specific manner, such peptides are rapidly dephosphorylated by cellular phosphatases. We now describe our efforts to develop SH2 inhibitory peptides containing phosphatase-resistant pTyr surrogates. The parent compound, (phosphonomethyl)phenylalanine (Pmp), is a phosphonate-based mimetic of pTyr in which the phosphate ester oxygen (> COPO3H2) has been replaced by a methylene unit (> CCX2PO3H2, X2 = H2). Pmp analogues bearing fluorine (X2 = H, F or X2 = F2) or hydroxyl (X2 = H, OH) substituents on the phosphonate alpha-methylene carbon have been prepared and incorporated into peptides for use as SH2 domain inhibitors. In an assay using the C-terminal SH2 domain of phosphatidylinositol (PI) 3-kinase, peptides having a GXVPML sequence [where X = pTyr, Pmp, hydroxy-Pmp (HPmp), monofluoro-Pmp (FPmp), and difluoro-Pmp (F2Pmp)] exhibited binding potency in the order HPmp < Pmp < FPmp < F2Pmp = pTyr. Distinct peptide sequences which bind selectively with Src and Grb2 SH2 domains were also prepared with pTyr and F2Pmp. The F2Pmp peptides bound with high (0.2- to 5-fold) relative affinity, compared to analogous pTyr peptides. We conclude that peptides containing F2Pmp bind to SH2 domains with high affinity and specificity and, being resistant to cellular phosphatases, should provide a generally useful tool for disrupting SH2 domain-mediated signaling pathways in intact cells.

Amino Acid Sequence↗
PubMed1993

Multimeric forms of Tyr-Ile-Gly-Ser-Arg (YIGSR) peptide enhance the inhibition of tumor growth and metastasis.

The Tyr-Ile-Gly-Ser-Arg (YIGSR) peptide derived from the laminin B1 chain has been shown to decrease tumor growth and metastasis. Utilizing the multimeric antigen peptide system assembled on a branched lysine core, we synthesized several sizes of multimeric YIGSR, (CH3CO-Tyr-Ile-Gly-Ser-Arg-Gly)16-Lys8-Lys4-Lys2 -Lys-Gly [(Ac-YIGSRG)16 K8K4K2KG] (designated Ac-Y16), (Ac-YIGSRG)8K4K2KG (Ac-Y8), and (Ac-YIGSRG)4K2KG (Ac-Y4), and related peptides, Ac-(YIGSRG)4-NH2 (Ac-Y4L) and Ac-YIGSR-NH2 (Ac-Y1) and evaluated their biological activities in inhibiting tumor growth and metastasis. Coinjection of 0.2 mg/mouse of Ac-Y16 i.v. with B16-F10 mouse melanoma cells inhibited lung colony formation by 97%, whereas 0.2 mg/mouse of Ac-Y1 inhibited by 50%. The larger the peptide (Ac-Y16 > Ac-Y8 > Ac-Y4 > Ac-Y1), the more inhibitory effect there was on lung metastasis. Ac-Y16 also inhibited the growth of s.c.-injected B16-F10 tumors. These data demonstrate that the multimeric YIGSR peptides strongly enhanced the activity of YIGSR in inhibiting tumor growth and metastasis and suggest that these compounds are potentially useful for clinical applications.

Amino Acid Sequence↗
PubMed1993

Quantitative structure-activity relationship (QSAR) study of elastase substrates and inhibitors.

One hundred Suc-X-Y-Ala-pNA peptides: (Suc: succinyl, pNA: p-nitroanilide, X, Y: Gly, Ala, Val, Leu, Ile, Phe, Pro, alpha-aminobutyric acid, norvaline, norleucine) were synthesized and their reaction constants with porcine pancreatic elastase (Km, kcat and kcat/Km) were determined. These reaction constants were quantitatively analyzed using the Free-Wilson/Fujita-Ban method. The contribution of amino acid side chains to the reaction constants Km, kcat and kcat/Km, expressed logarithmically, was found to be additive. On the other hand, 19 elastase inhibitors of the general formula CF3CO-X-Y-Ala-pNA (X,Y: ten amino acids) were synthesized, and their inhibition constants were compared with the Michaelis constant for the corresponding substrates and analyzed using free-energy-related substituent constants. In the analysis of amino acid side chains in the Y position, the Ki value of the inhibitor was generally correlated to the Km value of the substrate, which corresponded to the inhibitor, thus confirming the validity of the equation. log(1/Ki) = 1.271 log(1/Km) + 4.831 This study may serve as a prototypical approach to unraveling structure-activity relationships of peptide substrates and inhibitors of medicinal or agricultural importance.

Amino Acid Sequence↗
PubMed1993

In vitro anti-human immunodeficiency virus (HIV) activities of transition state mimetic HIV protease inhibitors containing allophenylnorstatine.

Transition state mimetic tripeptide human immunodeficiency virus (HIV) protease inhibitors containing allophenylnorstatine [(2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] were synthesized and tested for activity against HIV in vitro. Two compounds, KNI-227 and KNI-272, which were highly potent against HIV protease with little inhibition of other aspartic proteases, showed the most potent activity against the infectivity and cytopathic effect of a wide spectrum of HIV strains. As tested in target CD4+ ATH8 cells, the 50% inhibitory concentrations of KNI-227 against HIV type 1 LAI (HIV-1LAI), HIV-1RF, HIV-1MN, and HIV-2ROD were 0.1, 0.02, 0.03, and 0.1 microM, respectively, while those of KNI-272 were 0.1, 0.02, 0.04, and 0.1 microM, respectively. Both agents completely blocked the replication of 3'-azido-2',3'-dideoxythymidine-sensitive and -insensitive clinical HIV-1 isolates at 0.08 microM as tested in target phytohemagglutinin-activated peripheral blood mononuclear cells. The ratios of 50% cytotoxic concentrations to 50% inhibitory concentrations for KNI-227 and KNI-272 were approximately 2,500 and > 4,000, respectively, as assessed in peripheral blood mononuclear cells. Both compounds blocked the posttranslational cleavage of the p55 precursor protein to generate the mature p24 Gag protein in stably HIV-1-infected cells. The n-octanol-water partition coefficients of KNI-227 and KNI-272 were high, with log Po/w values of 3.79 and 3.56, respectively. Degradation of KNI-227 and KNI-272 in the presence of pepsin (1 mg/ml, pH 2.2) at 37 degrees C for 24 h was negligible. Current data warrant further careful investigations toward possible clinical application of these two novel compounds.

Antiviral Agents↗
PubMed1992

The all-D-configuration segment containing the IKVAV sequence of laminin A chain has similar activities to the all-L-peptide in vitro and in vivo.

Laminin is a basement membrane glycoprotein that has diverse biological activities. A sequence on the A chain containing IKVAV (Ile-Lys-Val-Ala-Val) has been shown to promote neurite outgrowth, cell adhesion, and tumor growth and metastasis. Here we have determined the structural requirements of this synthetic peptide for biological activity. Twelve-amino acid-long all-L- (LAM-L) and all-D-peptide (LAM-D) segments as well as an alternating D- and L-amino acid-containing peptide (LAM-DL), which included the IKVAV sequence (residues 2097-2108), were synthesized. Circular dichroism spectral analysis revealed a mirror image conformation of LAM-D and LAM-L with mainly beta-sheet and to a minor extent alpha-helical structure. LAM-DL did not exhibit any significant ordered conformational features. LAM-D and LAM-L showed similar cell attachment activities for rat pheochromocytoma cells (PC12), whereas LAM-DL was inactive. A peptide analog with randomized IKVAV sequence (LAM-RM) was also inactive. A similar trend was observed in competition experiments of the four peptides with laminin in analogous cell attachment assays. In in vivo experiments, both LAM-D and LAM-L were capable of increasing tumor growth when subcutaneously injected into mice with murine melanoma cells B16F10. Results indicate that the conformational status of the IKVAV domain is a contributing factor in determining the biological activity but that there is no strict requirement for a specific chirality. There is a likely sequence specificity to the IKVAV region.

Amino Acid Sequence↗
PubMed1992

Synthesis and conformation of the trimeric coiled-coil segment of laminin.

A disulfide linked 95-mer parallel hetero-trimeric active site segment of laminin was designed and synthesised. The three subunits, A (32-mer), B1 (30-mer) and B2 (33-mer), were prepared by Boc-based solid-phase peptide synthesis involving a two-step trimethylsilyl bromide-thioanisole and HF deprotection procedure. The interlinking of the three subunits was accomplished by the stepwise selective formation of two disulfide bridges using air-oxidation and thallium (III) trifluoroacetate oxidation. The conformations of the synthetic peptides were studied by circular dichroism (CD) spectroscopy, showing that the hetero-dimer, B1-B2, one of the homo-dimers, B1-B1, and the trimer are 30 to 40% in the alpha-helical conformation in aqueous buffer. Variable temperature CD studies demonstrated that the trimer is considerably more stable (melting temperature (Tm) = 61 degrees) than the hetero-dimer, B1-B2 (Tm = 36 degrees).

Amino Acid Sequence↗