Structural prediction of the beta-domain of metallothionein-3 by molecular dynamics simulation.

The beta-domain of metallothionein-3 (MT3) has been reported to be crucial to the neuron growth inhibitory bioactivity. Little detailed three-dimensional structural information is available to present a reliable basis for elucidation on structure-property-function relationships of this unique protein by experimental techniques. So, molecular dynamics simulation is adopted to study the structure of beta-domain of MT3. In this article, a 3D structural model of beta-domain of MT3 was generated. The molecular simulations provide detailed protein structural information of MT3. As compared with MT2, we found a characteristic conformation formed in the fragment (residue 1-13) at the N-terminus of MT3 owing to the constraint induced by 5TCPCP9, in which Pro7 and Pro9 residues are on the same side of the protein, both facing outward and the two 5-member rings of prolines are arranged almost in parallel, while Thr5 is on the opposite side. Thr5 in MT3 is also found to make the first four residues relatively far from the fragment (residue 23-26) as compared with MT2. The simulated structure of beta-domain of MT3 is looser than that of MT2. The higher energy of MT3 than that of MT2 calculated supports these conclusions. Simulation on the four isomer arising from the cis- or trans-configuration of 6CPCP9 show that the trans-/trans-isomer is energetic favorable. The partially unfolding structure of beta-domain of MT3 is also simulated and the results show the influence of 6CPCP9 sequence on the correct folding of this domain. The correlations between the bioactivity of MT3 and the simulated structure as well as the folding of beta-domain of MT3 are discussed based on our simulation and previous results.

Solution structure and dynamics of human metallothionein-3 (MT-3).

Alzheimer's disease is characterized by progressive loss of neurons accompanied by the formation of intraneural neurofibrillary tangles and extracellular amyloid plaques. Human neuronal growth inhibitory factor, classified as metallothionein-3 (MT-3), was found to be related to the neurotrophic activity promoting cortical neuron survival and dendrite outgrowth in the cell culture studies. We have determined the solution structure of the alpha-domain of human MT-3 (residues 32-68) by multinuclear and multidimensional NMR spectroscopy in combination with the molecular dynamic simulated annealing approach. The human MT-3 shows two metal-thiolate clusters, one in the N-terminus (beta-domain) and one in the C-terminus (alpha-domain). The overall fold of the alpha-domain is similar to that of mouse MT-3. However, human MT-3 has a longer loop in the acidic hexapeptide insertion than that of mouse MT-3. Surprisingly, the backbone dynamics of the protein revealed that the beta-domain exhibits similar internal motion to the alpha-domain, although the N-terminal residues are more flexible. Our results may provide useful information for understanding the structure-function relationship of human MT-3.

Structure and cytotoxicity of a new lanostane-type triterpene glycoside from the sea cucumber Holothuria hilla.

A new triterpene glycoside, hillaside C (1), was isolated from the sea cucumber Holothuria hilla Lesson, which is found in the South China Sea, and its structure has been elucidated by spectral analysis (ESI-MS and NMR) and chemical transformations. Four known compounds, holothuria A, thymine, uracil, and cholesterol, were also obtained. Compound 1 exhibited significant cytotoxicity against eight human tumor cell lines (A-549, MCF-7, IA9, CAKI-1, PC-3, KB, KB-VIN, and HCT-8) with IC50 values in the range of 0.15-3.20 microg/ml.

BmKK4, a novel toxin from the venom of Asian scorpion Buthus martensi Karsch, inhibits potassium currents in rat hippocampal neurons in vitro.

A novel short-chain peptide BmKK4 was isolated from the venom of Asian scorpion Buthus martensi Karsch. It is composed of 30 amino acids including six cysteine residues, and shares less than 25% sequence identity with the known alpha-KTx toxins. The action of BmKK4 on voltage-dependent potassium currents was examined in acutely dissociated hippocampal neurons of rat. BmKK4 (10-100 microM) inhibited both the delayed rectifier and fast transient potassium current in concentration-dependent manners. The inhibition was reversible and voltage-independent. BmKK4 caused a depolarizing shift (about 10 mV) of the steady-state activation curve of the currents, without changing their steady-state inactivation behavior. The unique amino acid sequence and electrophysiological effects suggest that BmKK4 represent a new subfamily of potassium channel toxins.

Purification and Primary Structure of a Novel Peptide from the Chinese Scorpion Buthus martensi Karsch.

To purify and characterize peptides from the venom of Chinese scorpion Buthus martensi Karsch, the purification was carried out by gel-filtration, ion exchange and reversed phase HPLC techniques. The purified peptide was reduced by dithioerythritol (DTT), S-alkylated with iodoacetic acid, and subjected to enzymatic cleavages (TPCK-trypsin). The purified fragments from enzymatic cleavage of the peptide were separated by C(18)HPLC, then submitted to the ESI-MS, and Edman degradation for amino acid sequence determination. The mixture was also subjected to tandem mass (MS-MS) analysis. As a result, a novel peptide, named BmK4112, was obtained, with the primary structure being TPYPV NCKTD RDCVM CGLGI SCKNG YCTGQ C, and having three disulfide bonds.

The structural and biological significance of the EAAEAE insert in the alpha-domain of human neuronal growth inhibitory factor.

Human neuronal growth inhibitory factor (hGIF) is able to inhibit the outgrowth of neurons. As compared with the amino acid sequences of metallothionein 1/2, hGIF contains two insertions: a Thr at position 5 and an acidic hexapeptide EAAEAE(55-60) close to the C-terminus. Moreover, all mammalian growth inhibitory factor sequences contain a conserved CPCP(6-9) motif. Previous studies have demonstrated that the TCPCP(5-9) motif is pivotal to its bioactivity, but no specific role has been assigned to the unique EAAEAE(55-60) insert. To investigate the potential structural and biological significance of the EAAEAE(55-60) insert, several mutants were constructed and investigated in detail. Notably, deletion of the acidic insert (the Delta55-60 mutant) reduced the inhibitory activity, whereas the bioactivities of other mutants did not change much. Then, spectroscopic characterization and molecular dynamics simulation were performed to investigate the potential causes of the reduced bioactivity of the Delta55-60 mutant. It was found that the domain-domain interaction mechanism of hGIF was different from that of metallothionein 2. It was also shown that the acidic insert could regulate the interdomain interactions in hGIF, leading to the structural change in the beta-domain, which resulted in the alteration of the solvent accessibility and metal release ability, thus playing an important role in the biological activity of hGIF. Our studies provided useful information on the domain-domain interaction at the molecular level for the first time, and shed new light on the mechanism of the bioactivity of hGIF.

Study on structure-property-reactivity-function relationship of human neuronal growth inhibitory factor (hGIF).

Human metallothionein-3 (hMT3), also named as human neuronal growth inhibitory factor (hGIF), can inhibit the outgrowth of embryonic cortical neurons in the presence of brain extracts. In order to systematically study the structure-property-reactivity-function relationship of hGIF, our laboratory designed a series of mutants and studied their structure, property, reactivity and functions by a series of chemical and biological tools including UV spectroscopy, CD spectroscopy, NMR, chemical reaction and primary neuronal culture assays. In summary, we concluded that the bioactivity of hGIF was regulated by multiple factors, including the (6)CPCP(9) motif, an additional threonine insert at sequence position 5, domain-domain interactions, the structure and stability of the metal-thiolate cluster and the linker. Our studies provide more and more evidences which revealed that the bioactivity of hGIF is mainly related to the essential metal release and its characteristic conformation.

Hillasides A and B, two new cytotoxic triterpene glycosides from the sea cucumber Holothuria hilla Lesson.

Two new triterpene glycosides, hillasides A (1) and B (2), were isolated from the sea cucumber H. hilla Lesson, together with one known glycoside holothuria B (3). Their structures were deduced by extensive spectral analysis and chemical evidences. The presence of conjugated double bonds [22E,24-diene] in the aglycone of 1 is a rare structural feature among sea cucumber glycosides. The two glycosides showed significant cytotoxicity against eight human tumour cell lines (A-549, MCF-7, IA9, CAKI-1, PC-3, KB, KB-VIN and HCT-8) with IC(50) in the range of 0.1-3.8 microg/ml.

Effect of alpha-domain substitution on the structure, property and function of human neuronal growth inhibitory factor.

Human metallothionein-3 (hMT3), also named human neuronal growth inhibitory factor (hGIF), is attractive due to its distinct neuronal growth inhibitory activity, which is not shown by other human MT isoforms. It has been reported that the neuronal growth inhibitory activity arises from the N-terminal beta-domain rather than its C-terminal alpha-domain. However, previous bioassay results have shown that the single beta-domain is less effective at inhibiting the neuron growth than that in intact hMT3 on a molar basis, which suggests that the alpha-domain is indispensable to the neuronal growth inhibitory activity of hMT3. In order to confirm this assumption, we constructed two domain-hybrid mutants, the beta(MT3)-beta(MT3) mutant and the beta(MT3)-alpha(MT1) mutant, and investigated their structural and metal binding properties by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, etc. The results showed that stability of the Cd(3)S(9) cluster of the beta(MT3)-beta(MT3) mutant decreased significantly while the Cd(3)S(9) cluster of the beta(MT3)-alpha(MT1) mutant had a similar stability and solvent accessibility to that of hMT3. Interestingly, the bioassay results showed that the neuronal growth inhibitory activity of the beta(MT3)-beta(MT3) mutant decreased significantly, while the beta(MT3)-alpha(MT1) mutant showed similar inhibitory activity to hMT3. Based on these results, we conclude that the alpha-domain is indispensable and plays an important role in modulating the stability of the metal cluster in the beta-domain by domain-domain interactions, thus influencing the bioactivity of hMT3.

Nobilisides A - C, three new triterpene glycosides from the sea cucumber Holothuria nobilis.

Three new triterpene glycosides, named nobilisides A ( 1), B ( 2) and C ( 3), were isolated from the sea cucumber Holothuria nobilis Selenka. Their structures were deduced by extensive spectral analysis and chemical evidence. Compounds 1 and 3 are non-sulfated monoglycosides while 2 is a sulfated diglycoside. The presence of two conjugated double bonds [22 E,24-diene and 7,9(11)-diene] in the aglycone of 1 is a rare structural feature among sea cucumber glycosides, while 2 and 3 possess the same 22,25-epoxy moiety as their side chains. All three glycosides exhibited significant cytotoxicity against human tumor cell lines.

Mutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3.

Human metallothionein-3 (hMT3), first isolated and identified as a neuronal growth inhibitory factor (GIF), is a metalloprotein expressed predominantly in brain. However, until now, the exact mechanism of the bioactivity of hMT3 is still unknown. In order to study the influence of acid-base catalysis on S-nitrosylation of hMT3, we constructed the E23K mutant of hMT3. During the course of bioassay, we found out unexpectedly that mutation at E23 of hMT3 eliminates the neuronal growth inhibitory activity completely. To the best of our knowledge, it is the first report that other residues, besides the TCPCP motif, in the beta-domain can alter the bioactivity of hMT3. In order to figure out the causes for the loss of bioactivity of the E23K mutant, the biochemical properties were characterized by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, and SNOC reaction. All data demonstrated that stability of the metal-thiolate cluster and overall structure of the E23K mutant were not altered too much. However, the reaction of the E23K mutant with SNOC exhibited biphasic kinetics and the mutant protein released zinc ions much faster than hMT3 in the initial step, while hMT3 exhibited single kinetic process. The 2D [1H-15N] HSQC was also employed to characterize structural changes during the reaction of hMT3 with varying mounts of nitric oxide. It was shown that the resonance of Glu23 disappeared at a molar ratio of NO to protein of 4. Based on these results, we suggest that mutation at Glu23 may alter the NO metabolism and/or affect zinc homeostasis in brain, thus altering the neuronal growth inhibitory activity.

New oleanene-type triterpene saponins from Pueraria peduncularis.

Two new oleanane-type triterpene saponins named pedunsaponins B (2) and C (3) were isolated from the roots of Pueraria peduncularis. Their structures were determined to be 3-O-(6-O-methyl)-beta-glucuronopyranosyl-3beta,15alpha-dihydroxyolean-12-en-16-one (2), and 3-O-beta-glucopyranosyl-(1 --> 3)-beta-glucuronopyranosyl-3beta,15alpha-dihydroxyoleana-12-en-16-one (3), on the basis of spectroscopic evidence.

A new triterpenoid from Entodon okamurae Broth.

One new triterpenoid, entokamurol (1), was isolated from Entodon okamurae Broth, together with other nine compounds, namely dryocrassol (2), chrysophamol (3), physcion (4), 10-nonacosamnol (5), n-hexadecanol (6), phthalic acid isodibutyl ester (7), curcumol (8), beta-sitosterol (9) and daucosterol (10). Their structures were elucidated on the basis of extensive NMR (DEPT, DQF-COSY, HMQC, HMBC and NOESY), IR and MS studies. All the compounds were isolated and identified from the genus of Entodon for the first time, and it is also the first report of a guaiane-type sesquiterpenoid and compounds with anthraquinone skeleton in mosses.

BmTx3B, a novel scorpion toxin from Buthus martensi Karsch, inhibits delayed rectifier potassium current in rat hippocampal neurons.

AIM: To examine the effect of BmTx3B, a novel short-chain peptide isolated from the venom of Asian scorpion Buthus martensi Karsch, on voltage-gated potassium channels. METHODS: Two types of voltage-dependent potassium currents were recorded from dissociated hippocampal neurons of neonatal rat in whole-cell voltage-clamp mode, and separated based upon their kinetic properties. RESULTS: BmTx3B (10-100 micromol/L) selectively inhibited the delayed rectifier potassium current (I(K)), without affecting the fast transient potassium current (I(A)). The inhibition of the peptide on I(K) was reversible, concentration-dependent and voltage-independent. BmTx3B did not affect the steady-state activation and inactivation kinetics of the current. CONCLUSION: The short-chain scorpion peptide BmTx3B selectively blocked the delayed rectifier potassium channel.

Intercedensides A-C, three new cytotoxic triterpene glycosides from the sea cucumber Mensamaria intercedens Lampert.

Three new triterpene glycosides, intercedensides A (1), B (2), and C (3), were isolated from the sea cucumber Mensamria intercedens Lampert, which is found in the South China Sea, and their structures have been elucidated by spectroscopic analysis (NMR and ESIMS) and chemical transformations. Intercedensides A (1) and C (3) have a conjugated double bond (22E,24-diene) in the side chain of the aglycon. Intercedenside B (2) has two beta-D-xylose and two sulfate groups in the carbohydrate chain. All three glycosides showed significant cytotoxicity against 10 human tumor cell lines with ED(50) in the range 0.6-4.0 microg/mL. Intercedenside A (1) exhibited significant in vivo antineoplastic activity against mouse Lewis lung cancer and mouse S180 sarcoma. On the basis of these initially promising results, intercedensides A-C merit further study as potential anticancer agents.

Isolation and structure of the cytotoxic cycloheptapeptide phakellistatin 13.

A new cyclic heptapeptide phakellistatin 13 (1) had been isolated from the sponge Phakellia fusca Thiele, collected at Yongxing Island of China. Its structure was elucidated as cyclo-(Pro1-Trp-Leu-Thr-Pro2-Gly-Phe) on the basis of MS, UV, IR, and high-field NMR (600 MHz) analysis. The compound was significantly cytotoxic against the human hepatoma BEL-7404 cell line with an ED(50) < 10(-2) microg/mL.

Lingshuiol, a novel polyhydroxyl compound with strongly cytotoxic activity from the marine dinoflagellate Amphidinium sp.

A novel polyhydroxy compound with a linear carbon-chain, lingshuiol (1), had been isolated from the cultured marine dinoflagellate Amphidinium sp. Its structure was elucidated by extensive analysis of 2D NMR spectral data. Lingshuiol possessed a powerful cytotoxic activity against A-549 and HL-60 cells in vitro with the IC(50) of 0.21 and 0.23 microM, respectively.