Photodamage attenuation effect by a tetraprenyltoluquinol chromane meroterpenoid isolated from Sargassum muticum.

A tetraprenyltoluquinol meroterpenoid with a chromane moiety, obtained as an epimeric mixture at C3, was isolated for the first time from the dichloromethane fraction of a methanolic extract from the brown alga Sargassum muticum. The structure has been established by means of spectral methods including NMR spectroscopy and MS and comparison with reported data. The compound showed photodamage attenuation on irradiated cells with UVA light, presented protection against intracellular ROS generation in a degree comparable to retinoic acid (9.1-20.6%) and did not display toxicity against human dermal fibroblast cells.

The role of Phe181 in the hexamerization of Helicobacter pylori quinolinate phosphoribosyltransferase.

Quinolinic acid phosphoribosyltransferase (QAPRTase; NadC) catalyzes an indispensable step in NAD biosynthesis, one that is essential for cell survival in prokaryotes, which makes it an attractive target for antibacterial drug therapy. We recently reported the crystal structures of Helicobacter pylori QAPRTase with bound quinolinic acid, nicotinamide mononucleotide, and phthalic acid. The enzyme exists as a hexamer organized as a trimer of dimers, which is essential for full enzymatic activity. The loop between helix alpha7 and strand beta8 contributes significantly to the hydrophobic dimer-dimer interactions. Phe181Pro mutation within the alpha7-beta8 loop disrupts the hexamerization of QAPRTase, and the resultant dimer shows dramatically reduced protein stability and no activity. Our findings thus suggest that compounds able to disrupt its proper oligomerization could potentially function as selective inhibitors of Helicobacter pylori QAPRTase and represent a novel set of antibacterial agents.

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.

Crystallization and preliminary X-ray studies of Trp138Phe/Val185Thr xylose isomerases from Thermotoga neapolitana and Thermoanaerobacterium thermosulfurigenes.

Xylose isomerases from Thermotoga neapolitana (TNXI) and Thermoanaerobacterium thermosulfurigenes (TTXI) share 70.4% sequence identity and are thermostable. The double mutants Trp138Phe/Val185Thr of TNXI and TTXI have higher catalytic efficiencies than TNXI and TTXI, respectively. The Trp138Phe/Val185Thr TNXI and TTXI mutants were overexpressed in Escherichia coli strain BL21(DE3) and purified. Crystals of the two proteins were grown with polyethylene glycol 8000 as the major precipitant by the hanging-drop vapour-diffusion method. Crystals of the TNXI mutant were obtained in the absence of substrate, in complex with glucose and in complex with fructose. Crystals of the TTXI mutant were obtained complexed with glucose. Diffraction data were collected at 1.9, 2.1 and 2.1 A resolution for the fructose-TNXI mutant, glucose-TNXI mutant and substrate-unbound TNXI mutant, respectively. The diffraction data for the glucose-TTXI mutant were collected at 2.0 A resolution. The crystals belong to the orthorhombic space groups C222(1) (TNXI mutant) and P2(1)2(1)2(1) (TTXI mutant). The TNXI and TTXI mutant crystals contain two and four monomers in the asymmetric unit, respectively.

Calreticulin, a calcium-binding molecular chaperone, is required for stress response and fertility in Caenorhabditis elegans.

Calreticulin (CRT), a Ca(2+)-binding protein known to have many cellular functions, including regulation of Ca(2+) homoeostasis and chaperone activity, is essential for heart and brain development during embryogenesis in mice. Here, we report the functional characterization of Caenorhabditis elegans calreticulin (crt-1). A crt-1 null mutant does not result in embryonic lethality but shows temperature-dependent reproduction defects. In C. elegans CRT-1 is expressed in the intestine, pharynx, body-wall muscles, head neurons, coelomocytes, and in sperm. crt-1 males exhibit reduced mating efficiency and defects late in sperm development in addition to defects in oocyte development and/or somatic gonad function in hermaphrodites. Furthermore, crt-1 and itr-1 (inositol triphosphate receptor) together are required for normal behavioral rhythms. crt-1 transcript level is elevated under stress conditions, suggesting that CRT-1 may be important for stress-induced chaperoning function in C. elegans.

Crystal structure and functional analysis of the SurE protein identify a novel phosphatase family.

Homologs of the Escherichia coli surE gene are present in many eubacteria and archaea. Despite the evolutionary conservation, little information is available on the structure and function of their gene products. We have determined the crystal structure of the SurE protein from Thermotoga maritima. The structure reveals the dimeric arrangement of the subunits and an active site around a bound metal ion. We also demonstrate that the SurE protein exhibits a divalent metal ion-dependent phosphatase activity that is inhibited by vanadate or tungstate. In the vanadate- and tungstate-complexed structures, the inhibitors bind adjacent to the divalent metal ion. Our structural and functional analyses identify the SurE proteins as a novel family of metal ion-dependent phosphatases.

Assessment of substrate specificity of hepatitis G virus NS3 protease by a genetic method.

The RNA genome of hepatitis G virus (HGV) encodes a large polyprotein that is processed to mature proteins by viral-encoded proteases. The HGV NS3 protease is responsible for the cleavage of the HGV polyprotein at four different locations. No conserved sequence motif has been identified for the cleavage sites of the NS3 protease. To determine the substrate specificity of the NS3 protease, amino acid sequences cleaved by the NS3 protease were obtained from randomized sequence libraries by using a screening method referred to as GASP (Genetic Assay for Site-specific Proteolysis). Based on statistical analyses of the obtained cleavable sequences, a consensus substrate sequence was deduced: Gln-Glu-Thr-Leu-Val downward arrow Ser, with the scissile bond located between Val and Ser. The relevance of this peptide as a cleavable substrate was further supported by molecular modeling of the NS3 protease. Our result would provide an insight on the molecular activity of the NS3 protease and may be useful for the design of substrate-based inhibitors.

Structure-activity analysis of SMAP-29, a sheep leukocytes-derived antimicrobial peptide.

SAMP-29 is a cathelecidin-derived antimicrobial peptide deduced from sheep myeloid mRNA. To elucidate the structural-activity relationship of SMAP-29, several analogues were synthesized and their antibiotic activity was investigated. Compared to parental SMAP-29, SMAP-29(1-17) and [K(22,25,27)]-SMAP-29 retained relatively effective antimicrobial activity (MIC: 1.0-8.0 microM), but resulted in a complete loss of hemolytic activity. Pro-19 --> Ala substitution ([A19]-SMAP-29) in SMAP-29 induced a significant reduction in antibacterial activity. These results suggested that the N-terminal amphipathic alpha-helical region and the C-terminal hydrophobic region of SMAP-29 are responsible for antimicrobial activity and hemolytic activity, respectively, and the central Pro-19 in SMAP-29 plays a critical role in showing improved antibacterial activity. In particular, [K(2,7,13)]-SMAP-29(1-17) showed potent antimicrobial activity under high salt conditions without hemolytic activity. Thus, this short peptide could serve as an attractive candidate for the development of therapeutic antimicrobial drugs. Structural analysis by circular dichroism suggested that SMAP-29 seems to adopt a helix-bend/turn-extended random conformation.

Crystal structure of the MJ0490 gene product of the hyperthermophilic archaebacterium Methanococcus jannaschii, a novel member of the lactate/malate family of dehydrogenases.

The MJ0490 gene, one of the only two genes of Methanococcus jannaschii showing sequence similarity to the lactate/malate family of dehydrogenases, was classified initially as coding for a putative l-lactate dehydrogenase (LDH). It has been re-classified as a malate dehydrogenase (MDH) gene, because it shows significant sequence similarity to MT0188, MDH II from Methanobacterium thermoautotrophicum strain DeltaH. The three-dimensional structure of its gene product has been determined in two crystal forms: a "dimeric" structure in the orthorhombic crystal at 1.9 A resolution and a "tetrameric" structure in the tetragonal crystal at 2.8 A. These structures share a similar subunit fold with other LDHs and MDHs. The tetrameric structure resembles typical tetrameric LDHs. The dimeric structure is equivalent to the P-dimer of tetrameric LDHs, unlike dimeric MDHs, which correspond to the Q-dimer. The structure reveals that the cofactor NADP(H) is bound at the active site, despite the fact that it was not intentionally added during protein purification and crystallization. The preference of NADP(H) over NAD(H) has been supported by activity assays. The cofactor preference is explained by the presence of a glycine residue in the cofactor binding pocket (Gly33), which replaces a conserved aspartate (or glutamate) residue in other NAD-dependent LDHs or MDHs. Preference for NADP(H) is contributed by hydrogen bonds between the oxygen atoms of the monophosphate group and the ribose sugar of adenosine in NADP(H) and the side-chains of Ser9, Arg34, His36, and Ser37. The MDH activity of MJ0490 is made possible by Arg86, which is conserved in MDHs but not in LDHs. The enzymatic assay showed that the MJ0490 protein possesses the fructose-1,6-bisphosphate-activated LDH activity (reduction). Thus the MJ0490 gene product appears to be a novel member of the lactate/malate dehydrogenase family, displaying an LDH scaffold and exhibiting a relaxed substrate and cofactor specificities in NADP(H) and NAD(H)-dependent malate and lactate dehydrogenase reactions.

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.

Crystallization and preliminary X-ray crystallographic analysis of the surE protein from Thermotoga maritima.

The surE protein from Thermotoga maritima is a 247-residue protein of unknown function. Its homologues are well conserved among both the eubacteria and the archaea. It has been overexpressed in soluble form in Escherichia coli. The protein has been crystallized at 296 K using 2-propanol as a precipitant. X-ray diffraction data have been collected to 1.9 A resolution using synchrotron radiation. The crystals belong to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a = b = 115.96, c = 78.60 A, alpha = beta = 90, gamma = 120 degrees. The asymmetric unit contains two monomers of the surE protein, with a corresponding V(M) of 2.72 A(3) Da(-1) and a solvent content of 54.7%.

Crystallization and preliminary X-ray diffraction studies of the guanylate kinase-like domain of PSD-95 protein from rat.

The PSD-95 (postsynaptic density-95) protein, one of the members of the MAGUK (membrane-associated guanylate kinase) family, is composed of three PDZ domains, one SH3 domain and one guanylate kinase-like (GK) domain. The GK domain mediates the scaffolding function of PSD-95 by protein--protein interaction. Here, the GK domain was subcloned, expressed as an intein fusion protein, purified without the intein and then crystallized at room temperature by the hanging-drop vapour-diffusion method using PEG 8000 as a precipitant. The complete native data set was collected to a resolution of 2.35 A using flash-cooling. The crystals belong to the primitive tetragonal space group P4(3) (or P4(1)), with unit-cell parameters a = b = 70.03 (4), c = 37.64 (1) A.

SPIN90 (SH3 protein interacting with Nck, 90 kDa), an adaptor protein that is developmentally regulated during cardiac myocyte differentiation.

In the yeast two-hybrid screening, we have isolated a cDNA clone from a human heart library using Nck Src homology 3 (SH3) domains as bait. The full-length cDNA, which encoded 722 amino acids, was identified as a VIP54-related gene containing an SH3 domain, proline-rich motifs, a serine/threonine-rich region, and a long C-terminal hydrophobic region. We refer to this protein as SPIN90 (SH3 Protein Interacting with Nck, 90 kDa). The amino acid sequence of the SH3 domain has the highest homology with those of Fyn, Yes, and c-Src. SPIN90 was broadly expressed in human tissues; in particular, it was highly expressed in heart, brain, and skeletal muscle, and its expression was developmentally regulated during cardiac myocyte differentiation. SPIN90 is able to bind to the first and third SH3 domains of Nck, in vitro, and is colocalized with Nck at sarcomere Z-discs within cardiac myocytes. Moreover, treatment with antisera raised against SPIN90 disrupted sarcomere structure, suggesting that this protein may play an important role in the maintenance of sarcomere structure and/or in the assembly of myofibrils into sarcomeres.

Antibacterial, antitumor and hemolytic activities of alpha-helical antibiotic peptide, P18 and its analogs.

The alpha-helical antibiotic peptide (P18: KWKLFKKIPKFLHLAKKF-NH2) designed from the cecropin A(1-8)-magainin 2 (1-12) hybrid displayed strong bactericidal and tumoricidal activity without inducing hemolysis. The effect of the Pro9 residue at central position of P18 on cell selectivity was investigated by Pro9 --> Leu or Pro9 --> Ser substitution. Either substitution markedly reduced the antibacterial activity of P18 and increased hemolysis, although it did not significantly affect cytotoxicity against human transformed tumor and normal fibroblast cells. These results suggest that a proline kink in alpha-helical antibiotic peptide P18 serves as a hinge region to facilitate ion channel formation on bacterial cell membranes and thus plays an important role in providing high selectivity against bacterial cells. Furthermore, to investigate the structure-antibiotic activity relationships of P18, a series of N- or C-terminal deletion and substitution analogs of P18 were synthesized. The C-terminal region of P18 was related to its antibiotic activity and alpha-helical conformation on lipid membranes rather than N-terminal one. Higher alpha-helicity of the peptides was involved in the hemolytic and antitumor activity rather than antibacterial activity. Except for [L9]-P18 and [S9]-P18, all the designed peptides containing a Pro residue showed potent antibacterial activity, although they did not induce a cytolytic effect against human erythrocyte and normal fibroblast cells at the concentration required to kill bacteria. In particular, P18 and some analogs (N-1, N-2, N-3, N-3L and N-4L) with potent bactericidal and tumoricidal activity and little or no normal cell toxicity may serve as an attractive candidate for the development of novel anti-infective or antitumor agents.

Crystallization and preliminary X-ray crystallographic studies of response regulator for cyanobacterial phytochrome, Rcp1.

The key response-regulator gene of light regulation, rcp1, from Synechocystis sp. has been overexpressed, purified and subsequently crystallized using ammonium sulfate as a precipitant in forms suitable for X-ray crystallographic studies. A native data set was collected to a resolution of 2.5 A at cryogenic temperature. The crystals belong to the hexagonal space group P6(3), with unit-cell parameters a = b = 89.04 (5), c = 60.29 (3) A. The Matthews parameter suggests that Rcp1 crystallizes with two molecules per asymmetric unit.

Nucleoside diphosphate kinase from the hyperthermophilic archaeon Methanococcus jannaschii: overexpression, crystallization and preliminary X-ray crystallographic analysis.

Nucleoside diphosphate (NDP) kinase is a key enzyme in maintaining cellular pools of all nucleoside triphosphates. NDP kinase from the hyperthermophilic archaebacterium Methanococcus jannaschii has been overexpressed in Escherichia coli and crystallized at 297 K using polyethylene glycol 4000 as precipitant. The crystal is hexagonal, belonging to the space group P6(3), with unit-cell parameters a = b = 72.89, c = 100.87 A. The asymmetric unit contains two subunits of NDP kinase, with a corresponding crystal volume per protein mass (V(M)) of 2.38 A(3) Da(-1) and a solvent content of 48.3%. Native X-ray diffraction data to 2.30 A resolution have been collected using synchrotron X-rays.

CRAMP analogues having potent antibiotic activity against bacterial, fungal, and tumor cells without hemolytic activity.

CRAMP-18 (GEKLKKIGQKIKNFFQKL) is the antibacterial sequence derived from CRMAP, a member of cathelicidin-derived antimicrobial peptides. To develop the novel antibiotic peptides useful as therapeutic drugs requires strong antibiotic activity against bacterial and fungal cells without hemolytic effect. To this goal, the analogues were designed to increase only net positively charge by Lys-substitution of positions 2, 9, 13, or 16 at the hydrophilic helix face of CRAMP-18 without any change at the hydrophobic helix face. In particular, Lys-substitution (K(2)-CRAMP-18) of position 2 in CRAMP-18 induced the enhanced antibiotic activity without any increase in hemolysis. Thus, this peptide may provide a useful template for the design novel antibiotic peptides for the treatment of infectious diseases. Additional CD spectra studies suggested that the alpha-helical structure of the peptides plays an important role in killing bacterial and fungal cells, but the increase of alpha-helical content is less connected with the enhanced antibiotic activity.

Calreticulin couples calcium release and calcium influx in integrin-mediated calcium signaling.

The engagement of integrin alpha7 in E63 skeletal muscle cells by laminin or anti-alpha7 antibodies triggered transient elevations in the intracellular free Ca(2+) concentration that resulted from both inositol triphosphate-evoked Ca(2+) release from intracellular stores and extracellular Ca(2+) influx through voltage-gated, L-type Ca(2+) channels. The extracellular domain of integrin alpha7 was found to associate with both ectocalreticulin and dihydropyridine receptor on the cell surface. Calreticulin appears to also associate with cytoplasmic domain of integrin alpha7 in a manner highly dependent on the cytosolic Ca(2+) concentration. It appeared that intracellular Ca(2+) release was a prerequisite for Ca(2+) influx and that calreticulin associated with the integrin cytoplasmic domain mediated the coupling of between the Ca(2+) release and Ca(2+) influx. These findings suggest that calreticulin serves as a cytosolic activator of integrin and a signal transducer between integrins and Ca(2+) channels on the cell surface.

Crystallization and preliminary x-ray crystallographic analysis of NAD+-dependent DNA ligase from Thermus filiformis.

A highly thermostable DNA ligase from Thermus filiformis has been crystallized at room temperature using methoxypolyethylene glycol 5000 as a precipitant. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 90.63, b = 117.80, c = 98. 65 A, beta = 115.56 degrees. Two molecules of DNA ligase are present in the asymmetric unit, giving a crystal volume per protein mass (V(m)) of 3.1 A(3) Da(-1) and a solvent content of 61%. A native data set extending to 3.0 A resolution has been collected at 100 K using synchrotron X-rays.

Caspase-mediated cleavage of p130cas in etoposide-induced apoptotic Rat-1 cells.

Apoptosis causes characteristic morphological changes in cells, including membrane blebbing, cell detachment from the extracellular matrix, and loss of cell-cell contacts. We investigated the changes in focal adhesion proteins during etoposide-induced apoptosis in Rat-1 cells and found that during apoptosis, p130cas (Crk-associated substrate [Cas]) is cleaved by caspase-3. Sequence analysis showed that Cas contains 10 DXXD consensus sites preferred by caspase-3. We identified two of these sites (DVPD(416)G and DSPD(748)G) in vitro, and point mutations substituting the Asp of DVPD(416)G and DSPD(748)G with Glu blocked caspase-3-mediated cleavage. Cleavage at DVPD(416)G generated a 74-kDa fragment, which was in turn cleaved at DSPD(748)G, yielding 47- and 31-kDa fragments. Immunofluorescence microscopy revealed well-developed focal adhesion sites in control cells that dramatically declined in number in etoposide-treated cells. Cas cleavage correlated temporally with the onset of apoptosis and coincided with the loss of p125FAK (focal adhesion kinase [FAK]) from focal adhesion sites and the attenuation of Cas-paxillin interactions. Considering that Cas associates with FAK, paxillin, and other molecules involved in the integrin signaling pathway, these results suggest that caspase-mediated cleavage of Cas contributes to the disassembly of focal adhesion complexes and interrupts survival signals from the extracellular matrix.