Self-protective responses to norvaline-induced stress in a leucyl-tRNA synthetase editing-deficient yeast strain.

The editing function of aminoacyl-tRNA synthetases (aaRSs) is indispensible for formation of the correct aminoacyl-tRNAs. Editing deficiency may lead to growth inhibition and the pathogenesis of various diseases. Herein, we confirmed that norvaline (Nva) but not isoleucine or valine is the major threat to the editing function of Saccharomyces cerevisiae leucyl-tRNA synthetase (ScLeuRS), both in vitro and in vivo. Nva could be misincorporated into the proteome of the LeuRS editing-deficient yeast strain (D419A/ScΔleuS), potentially resulting in dysfunctional protein folding and growth delay. Furthermore, the exploration of the Nva-induced intracellular stress response mechanism in D419A/ScΔleuS revealed that Hsp70 chaperones were markedly upregulated in response to the potential protein misfolding. Additionally, proline (Pro), glutamate (Glu) and glutamine (Gln), which may accumulate due to the conversion of Nva, collectively contributed to the reduction of reactive oxygen species (ROS) levels in Nva-treated D419A/ScΔleuS cells. In conclusion, our study highlights the significance of the editing function of LeuRS and provides clues for understanding the intracellular stress protective mechanisms that are triggered in aaRS editing-deficient organisms.

Conotoxin αD-GeXXA utilizes a novel strategy to antagonize nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) play essential roles in transmitting acetylcholine-mediated neural signals across synapses and neuromuscular junctions, and are also closely linked to various diseases and clinical conditions. Therefore, novel nAChR-specific compounds have great potential for both neuroscience research and clinical applications. Conotoxins, the peptide neurotoxins produced by cone snails, are a rich reservoir of novel ligands that target receptors, ion channels and transporters in the nervous system. From the venom of Conus generalis, we identified a novel dimeric nAChR-inhibiting αD-conotoxin GeXXA. By solving the crystal structure and performing structure-guided dissection of this toxin, we demonstrated that the monomeric C-terminal domain of αD-GeXXA, GeXXA-CTD, retains inhibitory activity against the α9α10 nAChR subtype. Furthermore, we identified that His7 of the rat α10 nAChR subunit determines the species preference of αD-GeXXA, and is probably part of the binding site of this toxin. These results together suggest that αD-GeXXA cooperatively binds to two inter-subunit interfaces on the top surface of nAChR, thus allosterically disturbing the opening of the receptor. The novel antagonistic mechanism of αD-GeXXA via a new binding site on nAChRs provides a valuable basis for the rational design of new nAChR-targeting compounds.

Conopeptide Vt3.1 preferentially inhibits BK potassium channels containing ß4 subunits via electrostatic interactions.

BK channel ß subunits (ß1-ß4) modulate the function of channels formed by slo1 subunits to produce tissue-specific phenotypes. The molecular mechanism of how the homologous ß subunits differentially alter BK channel functions and the role of different BK channel functions in various physiologic processes remain unclear. By studying channels expressed in Xenopus laevis oocytes, we show a novel disulfide-cross-linked dimer conopeptide, Vt3.1 that preferentially inhibits BK channels containing the ß4 subunit, which is most abundantly expressed in brain and important for neuronal functions. Vt3.1 inhibits the currents by a maximum of 71%, shifts the G-V relation by 45 mV approximately half-saturation concentrations, and alters both open and closed time of single channel activities, indicating that the toxin alters voltage dependence of the channel. Vt3.1 contains basic residues and inhibits voltage-dependent activation by electrostatic interactions with acidic residues in the extracellular loops of the slo1 and ß4 subunits. These results suggest a large interaction surface between the slo1 subunit of BK channels and the ß4 subunit, providing structural insight into the molecular interactions between slo1 and ß4 subunits. The results also suggest that Vt3.1 is an excellent tool for studying ß subunit modulation of BK channels and for understanding the physiological roles of BK channels in neurophysiology.

Critical effect of peptide cyclization on the potency of peptide inhibitors against Dengue virus NS2B-NS3 protease.

Dengue virus (DENV) infection is a serious public health threat worldwide that demands effective treatment. In the search for potent virus protease inhibitors, several cone snail venoms were screened against serotype 2 DENV NS2B-NS3 protease, and one conotoxin, MrIA, was identified to have inhibitory activity. The inhibitory activity was attributed to a disulfide bond-mediated loop, from which rational optimization was made to improve the potency and stability. An eight-residue cyclic peptide inhibitor was finally obtained with high potency (inhibitory constant 2.2 µM), stability, and cell permeability. This inhibitor can thus serve as a good lead for DENV drug development. In addition, this work highlights the critical effect of peptide cyclization on the potency of oligopeptide inhibitors against DENV protease, which may advance the design of peptide inhibitors for homologous virus proteases.

A helical conotoxin from Conus imperialis has a novel cysteine framework and defines a new superfamily.

Cone snail venoms are a rich source of peptides, many of which are potent and selective modulators of ion channels and receptors. Here we report the isolation and characterization of two novel conotoxins from the venom of Conus imperialis. These two toxins contain a novel cysteine framework, C-C-C-CC-C, which has not been found in other conotoxins described to date. We name it framework XXIII and designate the two toxins im23a and im23b; cDNAs of these toxins exhibit a novel signal peptide sequence, which defines a new K-superfamily. The disulfide connectivity of im23a has been mapped by chemical mapping of partially reduced intermediates and by NMR structure calculations, both of which establish a I-II, III-IV, V-VI pattern of disulfide bridges. This pattern was also confirmed by synthesis of im23a with orthogonal protection of individual cysteine residues. The solution structure of im23a reveals that im23a adopts a novel helical hairpin fold. A cluster of acidic residues on the surface of the molecule is able to bind calcium. The biological activity of the native and recombinant peptides was tested by injection into mice intracranially and intravenously to assess the effects on the central and peripheral nervous systems, respectively. Intracranial injection of im23a or im23b into mice induced excitatory symptoms; however, the biological target of these new toxins has yet to be identified.

Purification, cDNA cloning, and recombinant expression of chymotrypsin C from porcine pancreas.

Chymotrypsin C is a bifunctional secretory-type serine protease in pancreas; besides proteolytical activity, it also exhibits a calcium-decreasing activity in serum. In this study, we purified activated chymotrypsin C from porcine pancreas, and identified its three active forms. Active chymotrypsin C was found to be different in the length of its 13-residue activation peptide due to carboxydipeptidase (present in the pancreas) degradation or autolysis of the activated chymotrypsin C itself, resulting in the removal of several C-terminus residues from the activation peptide. After limited chymotrypsin C cleavage with endopeptidase Lys C, several purified peptides were partially sequenced, and the entire cDNA sequence for porcine chymotrypsin C was cloned. Recombinant chymotrypsinogen C was successfully expressed in Escherichia coli cells as inclusion bodies. After refolding and activation with trypsin, the comparison of the recombinant chymotrypsin C with the natural form showed that their proteolytic and calcium-decreasing activities were at the same level. The successful expression of chymotrypsin C gene paves the way to further mutagenic structure-function studies.

A novel conotoxin, qc16a, with a unique cysteine framework and folding.

A novel conotoxin, qc16a, was identified from the venom of vermivorous Conus quercinus. qc16a has only 11 amino acid residues, DCQPCGHNVCC, with a unique cysteine pattern. Its disulfide connectivity was determined to be I-IV, II-III. The NMR structure shows that qc16a adopts a ribbon conformation with a simple beta-turn motif formed by residues Gly6, His7 and Asn8. qc16a causes depression symptom in mice when injected intracranially. Point mutation results showed that Asp1, His7 and Asn8 are all essential for the activity of qc16a. Electrophysiologically, qc16a has no strong effect on the whole-cell currents of neurons and the currents of Drosophila Shaker channels, human BK channels and Na(V)1.7 channels. Its specific target still remains to be identified.

Effect of chymotrypsin C and related proteins on pancreatic cancer cell migration.

Pancreatic cancer is a malignant cancer with a high mortality rate. The amount of chymotrypsin C in pancreatic cancer cells is only 20% of that found in normal cells. Chymotrypsin C has been reported to be involved in cancer cell apoptosis, but its effect on pancreatic cancer cell migration is unclear. We performed cell migration scratch assays and Transwell experiments, and found that cell migration ability was downregulated in pancreatic cancer Aspc-1 cells that overexpressed chymotrypsin C, whereas the cell migration ability was upregulated in Aspc-1 cells in which chymotrypsin C was suppressed. Two-dimensional fluorescence differential in gel electrophoresis/mass spectrometry method was used to identify the proteins that were differentially expressed in Aspc-1 cells that were transfected with plasmids to induce either overexpression or suppressed expression of chymotrypsin C. Among 26 identified differential proteins, cytokeratin 18 was most obviously correlated with chymotrypsin C expression. Cytokeratin 18 is expressed in developmental tissues in early stages of cancer, and is highly expressed in most carcinomas. We speculated that chymotrypsin C might regulate pancreatic cancer cell migration in relation to cytokeratin 18 expression.

Chemical synthesis and characterization of two α4/7-conotoxins.

α-Conotoxins are small disulfide-constrained peptides that act as potent and selective antagonists on specific subtypes of nicotinic acetylcholine receptors (nAChRs). We previously cloned two α-conotoxins, Mr1.1 from the molluscivorous Conus marmoreus and Lp1.4 from the vermivorous Conus leopardus. Both of them have the typical 4/7-type framework of the subfamily of α-conotoxins that act on neuronal nAChRs. In this work, we chemically synthesized these two toxins and characterized their functional properties. The synthetic Mr1.1 could primarily inhibit acetylcholine (ACh)-evoked currents reversibly in the oocyte-expressed rat α7 nAChR, whereas Lp1.4 was an unexpected specific blocker of the mouse fetal muscle α1ß1γδ receptor. Although their inhibition affinities were relatively low, their unique receptor recognition profiles make them valuable tools for toxin-receptor interaction studies. Mr1.1 could also suppress the inflammatory response to pain in vivo, suggesting that it should be further investigated with respect to its molecular role in analgesia and its mechanism or therapeutic target for the treatment of pain.

A new subfamily of conotoxins belonging to the A-superfamily.

Two novel conotoxins from vermivorous cone snails Conus pulicarius and Conus tessulatus, designated as Pu14.1 and ts14a, were identified by cDNA cloning and peptide purification, respectively. The signal sequence of Pu14.1 is identical to that of α-conotoxins, while its predicted mature peptide, pu14a, shares high sequence similarity with ts14a, with only one residue different in their first intercysteine loop, which contains 10 residues and is rich in proline. Both pu14a and ts14a contain four separate cysteines in framework 14 (C-C-C-C). Peptide pu14a was chemically synthesized, air oxidized, and the connectivity of its two disulfide bonds was determined to be C1-C3, C2-C4, which is the same as found in α-conotoxins. The synthetic pu14a induced a sleeping symptom in mice and was toxic to freshwater goldfish upon intramuscular injection. Using the Xenopus oocyte heterologous expression system, 1µM of pu14a demonstrated to inhibit the rat neuronal α3ß2-containing as well as the mouse neuromuscular α1ß1γδ subtypes of nicotinic acetylcholine receptors, and then rapidly dissociated from the receptors. However, this toxin had no inhibitory effect on potassium channels in mouse superior cervical ganglion neurons. According to the identical signal sequence to α-conotoxins, the unique cysteine framework and molecular target of pu14a, we propose that pu14a and ts14a may represent a novel subfamily in the A-superfamily, designated as α1-conotoxins.

Identification of neuropeptide Y-like conopeptides from the venom of Conus betulinus.

Neuropeptide Y (NPY) is a ubiquitous endocrine neuropeptide found in vertebrate and invertebrate. In our present work, two NPY-like exocrine conopeptides (designated as cono-NPYs) were first identified in the venom of cone snails. Both cono-NPYs showed sequence characteristics of invertebrate NPYs, suggesting that some exocrine venom peptides are probably evolved from the preexisting endocrine peptides during the evolution of cone snails.

Snapshot of the interaction between HIV envelope glycoprotein 120 and protein disulfide isomerase.

The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein 120 (gp120) binds to cell surface receptors and mediates HIV entry. Previous studies suggest the cell surface protein disulfide isomerase (PDI) might interact with disulfide bond(s) of gp120 and thus facilitate HIV-1 entry. In the present study, a kinetic trapping approach was used to capture the disulfide cross-linking intermediate between gp120 and PDI. Active site mutant PDIs were prepared in which the C-terminal cysteine at the active site was replaced by a serine. The active site mutant PDIs were able to covalently cross-link with gp120 through a mixed disulfide bond in vitro. The cross-linking efficiency was enhanced by CD4 protein (primary receptor of HIV-1) and was inhibited both by bacitracin (a PDI inhibitor) and by catalytically inactive PDI. The present results suggested the cell surface PDI might play a role in HIV entry in vivo.

Novel conopeptides in a form of disulfide-crosslinked dimer.

In our present work, seven conotoxins and conopeptides were cloned from four cone snail species based on the M-superfamily signal peptides. Among them, two conopeptides, Vt3.1 and Vt3.2, showed unusual sequence characteristics. Both of them contained two cysteines that are separated by just one non-cysteine residue. In vitro, the chemically synthesized Vt3.1 formed dimers with different intermolecular disulfide linkages. Only the dimer with crossed disulfides showed bioactivity when injected into the intraventricular region of mice brains. Therefore, Vt3.1 and Vt3.2 represent a new group of conopeptides that form disulfide-crosslinked dimers in vitro and probably in vivo.

Two short D-Phe-containing cysteine-free conopeptides from Conus marmoreus.

The cysteine-free conopeptides are naturally occurring components of the venom of cone snails and have been relatively less investigated than the cysteine-containing conopeptides. In this work, we used thiol-exchange chromatography to isolate cysteine-free conopeptides from the venom of Conus marmoreus. The full-length previously reported conomarphin and two novel shortened forms of it were found in the cysteine-free conopeptide fraction. The two shortened conomarphins also contain a D-Phe and a hydroxylated proline residue, suggesting complex post-translational modifications of these conopeptides. In particular, the shorter conomarphin fragment shares significant sequence similarity with conophans. This work demonstrated that thiol-exchange chromatography is a useful approach to isolate cysteine-free conopeptides from venom cocktail. The newly identified conopeptides further revealed the complexity of post-translational processing and the high diversity of conotoxins.

Small peptides derived from the Lys active fragment of the mung bean trypsin inhibitor are fully active against trypsin.

The Bowman-Birk protease inhibitors have recently attracted attention for their potential as cancer preventive and suppressing agents. They contain two canonical binding loops, both consisting of nine highly conserved residues capable of inhibiting corresponding serine proteases. In this study, we cloned the cDNA of the mung bean trypsin inhibitor, one of the most studied Bowman-Birk protease inhibitors. A modified peptide, Lys33GP, with 33 residues derived from the long chain of the Lys active fragment of mung bean trypsin inhibitor, was successfully expressed in Escherichia coli as a glutathione-S-transferase fusion protein. The recombinant product was obtained with a high yield, and exhibited potent inhibitory activity. Meanwhile, a shorter peptide composed of only 16 residues (the Lys16 peptide), corresponding to the active core of the fragment, was synthesized. Both the recombinant and the synthesized peptides had the same inhibitory activity toward trypsin at a molar ratio of 1 : 1, implying that the Lys16 peptide with two disulfide bonds is possibly the essential structural unit for inhibitory activity. Using site-directed mutagenesis, the P(1) position Lys was replaced by Phe, and the resulting mutant, Lys33K/F, was determined to have potent chymotrypsin inhibitory activity. Both Lys33GP and the Lys33K/F mutant may be potential pharmaceutical agents for the prevention of oncogenesis.

Characterization of a novel alpha4/4-conotoxin, Qc1.2, from vermivorous Conus quercinus.

As part of continuing studies of the identification of gene organization and cloning of novel alpha-conotoxins, the first alpha4/4-conotoxin identified in a vermivorous Conus species, designated Qc1.2, was originally obtained by cDNA and genomic DNA cloning from Conus quercinus collected in the South China Sea. The predicted mature toxin of Qc1.2 contains 14 amino acid residues with two disulfide bonds (I-III, II-IV connectivity) in a native globular configuration. The mature peptide of Qc1.2 is supposed to contain an N-terminal post-translationally processed pyroglutamate residue and a free carboxyl C-terminus. This peptide was chemically synthesized and refolded for further characterization of its functional properties. The synthetic Qc1.2 has two interconvertible conformations in aqueous solution, which may be due to the cis-trans isomerization of the two successive Pro residues in its first Cys loop. Using the Xenopus oocyte heterologous expression system, Qc1.2 was shown to selectively inhibit both rat neuronal alpha3beta2 and alpha3beta4 subtypes of nicotinic acetylcholine receptors with low potency. A block of about 63% and 37% of the ACh-evoked currents was observed, respectively, and the toxin dissociated rapidly from the receptors. Compared with other characterized alpha-conotoxin members, the unusual structural features in Qc1.2 that confer to its receptor recognition profile are addressed.

The ternary structure of the double-headed arrowhead protease inhibitor API-A complexed with two trypsins reveals a novel reactive site conformation.

The double-headed arrowhead protease inhibitors API-A and -B from the tubers of Sagittaria sagittifolia (Linn) feature two distinct reactive sites, unlike other members of their family. Although the two inhibitors have been extensively characterized, the identities of the two P1 residues in both API-A and -B remain controversial. The crystal structure of a ternary complex at 2.48 A resolution revealed that the two trypsins bind on opposite sides of API-A and are 34 A apart. The overall fold of API-A belongs to the beta-trefoil fold and resembles that of the soybean Kunitz-type trypsin inhibitors. The two P1 residues were unambiguously assigned as Leu(87) and Lys(145), and their identities were further confirmed by site-directed mutagenesis. Reactive site 1, composed of residues P5 Met(83) to P5' Ala(92), adopts a novel conformation with the Leu(87) completely embedded in the S1 pocket even though it is an unfavorable P1 residue for trypsin. Reactive site 2, consisting of residues P5 Cys(141) to P5' Glu(150), binds trypsin in the classic mode by employing a two-disulfide-bonded loop. Analysis of the two binding interfaces sheds light on atomic details of the inhibitor specificity and also promises potential improvements in enzyme activity by engineering of the reactive sites.

New conotoxins define the novel I3-superfamily.

We purified two novel conotoxins, designated as ca11a and ca11b, from the venom of Conus caracteristicus. Based on the amino acid sequence of mature ca11a, we cloned its full-length cDNA. Based on the signal peptide of ca11a, several ca11a-like conotoxins were cloned from C. caracteristicus and C. pulicarius. These novel conotoxins have an I-superfamily cysteine pattern but with a novel signal peptide sequence, suggesting they belong to a new branch of I-superfamily, designated as I(3)-superfamily. Additionally, two O-superfamily conotoxins were also cloned based on the signal peptide of ca11a, suggesting a possible evolutionary relationship between O- and I-superfamilies.

Recombinant expression of a chitosanase and its application in chitosan oligosaccharide production.

Recently, considerable attention has been focused on chitosan oligosaccharides (COSs) due to their various biological activities. COSs can be prepared by enzymatic degradation of chitosan, which is the deacetylation product of chitin, one of the most abundant biopolymers in nature. In the current study, we recombinantly expressed a chitosanase and used it for COS preparation. A bacillus-derived GH8 family chitosanase with a 6xHis tag fused at its N-terminal was expressed in the Escherichia coli strain BL21(DE3) as a soluble and active form. Its expression level could be as high as 500 mg/L. Enzymatic activity could reach approximately 140,000 U/L under our assay conditions. The recombinant chitosanase could be purified essentially to homogeneity by immobilized metal-ion affinity chromatography. The enzyme could efficiently convert chitosan into monomer-free COS: 1g of enzyme could hydrolyze about 100 kg of chitosan. Our present work has provided a cheap chitosanase for large-scale COS production in industry.

Structure-function relationship of bifunctional scorpion toxin BmBKTx1.

As the first identified scorpion toxin active on both big conductance Ca2+-activated K+ channels (BK) and small conductance Ca2+-activated K+ channels (SK), BmBKTx1 has been proposed to have two separate functional faces for two targets. To investigate this hypothesis, two double mutants, K21A-Y30A and R9A-K11A, together with wild-type toxin were expressed in Escherichia coli. The recombinant toxins were tested on cockroach BK and rat SK2 channel for functional assay. Mutant K21A-Y30A had a dramatic loss of function on BK but retained its function on SK. Mutant R9A-K11A did not lose function on BK or SK. These data support the two functional-face hypothesis and indicate that the BK face is on the C-terminal beta-sheet.