Tse-Yu Chung

Effect of sugar positions in ginsenosides and their inhibitory potency on Na + /K + -ATPase activity

AbstractAim:To determine whether ginsenosides with various sugar attachments may act as active components responsible for the cardiac therapeutic effects of ginseng and sanqi (the roots of Panax ginseng and Panax notoginseng) via the same molecular mechanism triggered by cardiac glycosides, such as ouabain and digoxin.Methods:The structural similarity between ginsenosides and ouabain was analyzed. The inhibitory potency of ginsenosides and ouabain on Na+/K+-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of ginsenosides to Na+/K+-ATPase.Results:Ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure, equivalent to the sugar position in cardiac glycosides, and possessed inhibitory potency on Na+/K+-ATPase activity. However, their inhibitory potency was significantly reduced or completely abolished when a monosaccharide was linked to the C-6 or C-20 position of the steroid-like structure; replacement of the monosaccharide with a disaccharide molecule at either of these positions caused the disappearance of the inhibitory potency. Molecular modeling and docking confirmed that the difference in Na+/K+-ATPase inhibitory potency among ginsenosides was due to the steric hindrance of sugar attachment at the C-6 and C-20 positions of the steroid-like structure.Conclusion:The cardiac therapeutic effects of ginseng and sanqi should be at least partly attributed to the effective inhibition of Na+/K+-ATPase by their metabolized ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure.

The effects of macromolecular crowding on the mechanical stability of protein molecules.

Macromolecular crowding, a common phenomenon in the cellular environments, can significantly affect the thermodynamic and kinetic properties of proteins. A single‐molecule method based on atomic force microscopy (AFM) was used to investigate the effects of macromolecular crowding on the forces required to unfold individual protein molecules. It was found that the mechanical stability of ubiquitin molecules was enhanced by macromolecular crowding from added dextran molecules. The average unfolding force increased from 210 pN in the absence of dextran to 234 pN in the presence of 300 g/L dextran at a pulling speed of 0.25 μm/sec. A theoretical model, accounting for the effects of macromolecular crowding on the native and transition states of the protein molecule by applying the scaled‐particle theory, was used to quantitatively explain the crowding‐induced increase in the unfolding force. The experimental results and interpretation presented could have wide implications for the many proteins that experience mechanical stresses and perform mechanical functions in the crowded environment of the cell.

Active ingredients in Chinese medicines promoting blood circulation as Na + /K + -ATPase inhibitors

The positive inotropic effect of cardiac glycosides lies in their reversible inhibition on the membrane-bound Na+/K+-ATPase in human myocardium. Steroid-like compounds containing a core structure similar to cardiac glycosides are found in many Chinese medicines conventionally used for promoting blood circulation. Some of them are demonstrated to be Na+/K+-ATPase inhibitors and thus putatively responsible for their therapeutic effects via the same molecular mechanism as cardiac glycosides. On the other hand, magnesium lithospermate B of danshen is also proposed to exert its cardiac therapeutic effect by effectively inhibiting Na+/K+-ATPase. Theoretical modeling suggests that the number of hydrogen bonds and the strength of hydrophobic interaction between the effective ingredients of various medicines and residues around the binding pocket of Na+/K+-ATPase are crucial for the inhibitory potency of these active ingredients. Ginsenosides, the active ingredients in ginseng and sanqi, substantially inhibit Na+/K+-ATPase when sugar moieties are attached only to the C-3 position of their steroid-like structure, equivalent to the sugar position in cardiac glycosides. Their inhibitory potency is abolished, however, when sugar moieties are linked to C-6 or C-20 position of the steroid nucleus; presumably, these sugar attachments lead to steric hindrance for the entrance of ginsenosides into the binding pocket of Na+/K+-ATPase. Neuroprotective effects of cardiac glycosides, several steroid-like compounds, and magnesium lithospermate B against ischemic stroke have been accordingly observed in a cortical brain slice-based assay model, and cumulative data support that effective inhibitors of Na+/K+-ATPase in the brain could be potential drugs for the treatment of ischemic stroke.

Cloning and expression of pathogenesis-related protein 4 from jelly fig (Ficus awkeotsang Makino) achenes associated with ribonuclease, chitinase and anti-fungal activities

A cDNA fragment (FaPR4) encoding a class I pathogenesis-related protein 4 (PR-4) from Ficus awkeotsang was obtained by PCR cloning. Plant PR-4s were grouped into class I and II, differing by the presence of ChtBD and hinge. The predicted mature FaPR4 comprises N-terminal chitin-binding domain (ChtBD), hinge, Barwin domain and C-terminal extension. FaPR4-C, an N-terminal truncated form of FaPR4, was designed to mimic the structural feature of class II PR-4s. FaPR4 and FaPR4-C were over-expressed in yeast Pichia pastoris, and both recombinants exhibited RNase and anti-fungal activities. To our knowledge, it is the first report that FaPR4, a member of class I PR-4s has RNase activity as class II. FaPR4 possesses better anti-fungal activities toward Fusarium oxysporum and Sclerotium rolfsii than FaPR4-C. Heat-treated FaPR4 remained RNase and anti-fungal activities; while heat-treated FaPR4-C lost those activities. Therefore, ChtBD of FaPR4 may not only contribute to its anti-fungal but also improve the thermal stability of protein. It also implied the correlation of RNase activity with anti-fungal activity of FaPR4-C. Furthermore, FaPR4 was detected to have weak but significant chitinase activity, and its chitinase activity was reduced after heat treatment. The chitinase activity by FaPR4-C was much lower than FaPR4.

Steroid-like compounds in Chinese medicines promote blood circulation via inhibition of Na + /K + -ATPase

AbstractAim:To examine if steroid-like compounds found in many Chinese medicinal products conventionally used for the promotion of blood circulation may act as active components via the same molecular mechanism triggered by cardiac glycosides, such as ouabain.Methods:The inhibitory potency of ouabain and the identified steroid-like compounds on Na+/K+-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of these compounds to Na+/K+-ATPase.Results:All the examined steroid-like compounds displayed more or less inhibition on Na+/K+-ATPase, with bufalin (structurally almost equivalent to ouabain) exhibiting significantly higher inhibitory potency than the others. In the pentacyclic triterpenoids examined, ursolic acid and oleanolic acid were moderate inhibitors of Na+/K+-ATPase, and their inhibitory potency was comparable to that of ginsenoside Rh2. The relatively high inhibitory potency of ursolic acid or oleanolic acid was due to the formation of a hydrogen bond between its carboxyl group and the Ile322 residue in the deep cavity close to two K+ binding sites of Na+/K+-ATPase. Moreover, the drastic difference observed in the inhibitory potency of ouabain, bufalin, ginsenoside Rh2, and pentacyclic triterpenoids is ascribed mainly to the number of hydrogen bonds and partially to the strength of hydrophobic interaction between the compounds and residues around the deep cavity of Na+/K+-ATPase.Conclusion:Steroid-like compounds seem to contribute to therapeutic effects of many cardioactive Chinese medicinal products. Chinese herbs, such as Prunella vulgaris L, rich in ursolic acid, oleanolic acid and their glycoside derivatives may be adequate sources for cardiac therapy via effective inhibition on Na+/K+-ATPase.

Solution Structure of a Phytocystatin from Ananas comosus and Its Molecular Interaction with Papain

The structure of a recombinant pineapple cystatin (AcCYS) was determined by NMR with the RMSD of backbone and heavy atoms of twenty lowest energy structures of 0.56 and 1.11 Å, respectively. It reveals an unstructured N-terminal extension and a compact inhibitory domain comprising a four-stranded antiparallel β-sheet wrapped around a central α-helix. The three structural motifs (G45, Q89XVXG, and W120) putatively responsible for the interaction with papain-like proteases are located in one side of AcCYS. Significant chemical shift perturbations in two loop regions, residues 45 to 48 (GIYD) and residues 89 to 91 (QVV), of AcCYS strongly suggest their involvement in the binding to papain, consistent with studies on other members of the cystatin family. However, the highly conserved W120 appears not to be involved in the binding with papain as no chemical shift perturbation was observed. Chemical shift index analysis further indicates that the length of the α-helix is shortened upon association with papain. Collectively, our data suggest that AcCYS undergoes local secondary structural rearrangements when papain is brought into close contact. A molecular model of AcCYS/papain complex is proposed to illustrate the interaction between AcCYS and papain, indicating a complete blockade of the catalytic triad by AcCYS.

Novel stereoselective bufadienolides reveal new insights into the requirements for Na + , K + -ATPase inhibition by cardiotonic steroids

Cardiotonic steroids (CTS) are clinically important drugs for the treatment of heart failure owing to their potent inhibition of cardiac Na+, K+-ATPase (NKA). Bufadienolides constitute one of the two major classes of CTS, but little is known about how they interact with NKA. We report a remarkable stereoselectivity of NKA inhibition by native 3β-hydroxy bufalin over the 3α-isomer, yet replacing the 3β-hydroxy group with larger polar groups in the same configuration enhances inhibitory potency. Binding of the two 13C-labelled glycosyl diastereomers to NKA were studied by solid-state NMR (SSNMR), which revealed interactions of the glucose group of the 3β- derivative with the inhibitory site, but much weaker interactions of the 3α- derivative with the enzyme. Molecular docking simulations suggest that the polar 3β-groups are closer to the hydrophilic amino acid residues in the entrance of the ligand-binding pocket than those with α-configuration. These first insights into the stereoselective inhibition of NKA by bufadienolides highlight the important role of the hydrophilic moieties at C3 for binding, and may explain why only 3β-hydroxylated bufadienolides are present as a toxic chemical defence in toad venom.

Binding orientation and specificity of calmodulin to rat olfactory cyclic nucleotide-gated ion channel.

Calmodulin (CaM), the primary intracellular Ca2+ receptor, regulates a large number of key enzymes and controls a wide spectrum of important biological responses. Recognition between CaM and its target sequence in rat olfactory cyclic nucleotide-gated ion channel (OLFp) was investigated by circular dichroism (CD), fluorescence, and NMR spectroscopy. Fluorescence data showed the OLFp tightly bound to CaM with a dissociation constant of 12 nM in a 1:1 stoichiometry. Far-UV CD data showed that approximately 60% of OLFp residues formed α-helical structures when associated with CaM. NMR data showed that most of the 15N–1H HSQC cross-peaks of the 15N-labeled CaM not only shifted but also split into two sets of peaks upon association with the OLFp. Our data indicated that the two distinct CaM/OLFp complexes existed simultaneously with stable structures that were not interexchangeable within the NMR time scale. In light of the palindromic sequence of OLFp (FQRIVRLVGVIRDW) for CaM targeting, we proposed that the helical OLFp with C2 symmetry may bind to CaM in two orientations. This hypothesis is supported by the observation that only one set of 15N–1H HSQC cross-peaks of the 15N-labeled CaM was detected upon association with OLFp-M13 chimeric peptide (OLFMp), a mutated OLFp lacking the palindromic feature. The binding specificity of OLFMp to CaM was restored when the palindromic feature was destroyed. Binding modes of CaM/OLFp and CaM/OLFMp simulated by molecular docking were in accord with their distinct patterns observed in HSQC spectra. Our studies suggest that the palindromic residues in OLFp are crucial for the orientation-specific recognition by CaM.

Inhibition of Na+/K+-ATPase by Antcins, Unique Steroid-Like Compounds in Antrodia camphorate

The inhibition of Na(+)/K(+) -ATPase by versatile steroid-like compounds contributes to the putative therapeutic effects of many Chinese medicinal cardiac products via the same molecular mechanism triggered by cardiac glycosides. Five major steroid-like compounds, antcin A, B, C, H, and K were isolated from Niuchangchih (Antrodia camphorata), a unique Taiwan mushroom, and all inhibited Na(+)/K(+) -ATPase. Antcin A exhibited significantly higher inhibitory potency than the other four antcins, though weaker than ginsenoside Rh2 . In contrast, cortisone (an analogous steroid with anti-inflammatory effects stronger than antcin A) showed no detectable inhibitory potency. Molecular modeling has shown that antcins bind to Na(+)/K(+) -ATPase with the steroidal skeleton structurally upside-down in comparison with ginsenoside Rh2 . The inhibitory potency of antcin A is attributed to steroidal hydrophobic interaction within the binding pocket and the formation of three hydrogen bonds between its carboxyl group and two cationic residues around the cavity entrance of Na(+)/K(+) -ATPase. The presence of an additional carbonyl or hydroxyl group at C7 of the other four antcins leads to severe repulsion in the hydrophobic pocket, and thus significantly reduces inhibitory potency. It is proposed that antcin A is a bi-functional compound that exerts anti-inflammatory effects and that enhances blood circulation via two different molecular mechanisms.

Magnesium lithospermate B extracted from Salvia miltiorrhiza elevats intracellular Ca2+ level in SH-SY5Y cells.

AbstractAim:To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na+/K+-ATPase, leads to the elevation of intracellular Ca2+ level as observed in cells treated with cardiac glycosides.Methods:Viability of SH-SY5Y neuroblastoma cells treated with various concentrations of ouabain or MLB was measured. Intracellular Ca2+ levels were visualized using Fluo4-AM (fluorescent dye) when cells were treated with ouabain or MLB in the presence or absence of KB-R7943 (Na+/Ca2+ exchanger inhibitor) and 2-APB (IP3 receptor antagonist). Molecular modeling was conducted for the docking of ouabain or MLB to Na+/K+-ATPase. Changes of cell body and dendrite morphology were monitored under a microscope.Results:severe toxicity was observed in cells treated with ouabain of concentration higher than 1 μmol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca2+ levels were substantially elevated by MLB (1 μmol/L) and ouabain (1 μmol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 μmol/L) or 2-APB (100 μmol/L). Equivalent interaction with the binding cavity of Na+/K+-ATPase was simulated for ouabain and MLB by forming five hydrogen bonds, respectively. Treatment of ouabain (1 μmol/L), but not MLB (1 μmol/L), induced dendritic shrink of SH-SY5Y cells.Conclusion:Comparable to ouabain, MLB leads to the elevation of intracellular Ca2+ level presumably via the same mechanism by inhibiting Na+/K+-ATPase. The elevated Ca2+ levels seem to be supplied by Ca2+ influx through the reversed mode of the Na+/Ca2+ exchanger and intracellular release from endoplasmic reticulum.