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.

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.

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.

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.

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.

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.

Isolation and cloning of a conotoxin with a novel cysteine pattern from Conus caracteristicus.

A new conotoxin, ca16a, containing 8 cysteine residues was purified, sequenced, and cloned from a worm-hunting snail, Conus caracteristicus. This conotoxin is an extremely hydrophilic peptide comprising 34 residues, with 4 acidic and 4 basic residues. It is rich in polar Gly, Ser, and Thr residues and includes a hydroxylated Pro residue. The cysteine arrangement pattern of ca16a (-C-C-CC-C-CC-C-, designated as framework #16) is distinct from that of other known conotoxins. Furthermore, the signal peptide sequence of this conotoxin does not share any homology with those of other conotoxins. Leu residues account for almost 50% of its 20-residue signal peptide. The unique cysteine framework and signal peptide sequence of ca16a suggest that it belongs to a new conotoxin superfamily.

Two different groups of signal sequence in M-superfamily conotoxins.

M-superfamily conotoxins can be divided into four branches (M-1, M-2, M-3 and M-4) according to the number of amino acid residues in the third Cys loop. In general, it is widely accepted that the conotoxin signal peptides of each superfamily are strictly conserved. Recently, we cloned six cDNAs of novel M-superfamily conotoxins from Conus leopardus, Conus marmoreus and Conus quercinus, belonging to either M-1 or M-3 branch. These conotoxins, judging from the putative peptide sequences deducted from cDNAs, are rich in acidic residues and share highly conserved signal and pro-peptide region. However, they are quite different from the reported conotoxins of M-2 and M-4 branches even in their signal peptides, which in general are considered highly conserved for each superfamily of conotoxins. The signal sequences of M-1 and M-3 conotoxins composed of 24 residues start with MLKMGVVL-, while those of M-2 and M-4 conotoxins composed of 25 residues start with MMSKLGVL-. It is another example that different types of signal peptides can exist within a superfamily besides the I-conotoxin superfamily. In addition to the different disulfide connectivity of M-1 conotoxins from that of M-4 or M-2 conotoxins, the sequence alignment, preferential Cys codon usage and phylogenetic tree analysis suggest that M-1 and M-3 conotoxins have much closer relationship, being different from the conotoxins of other two branches (M-4 and M-2) of M-superfamily.

Biological characteristics of dengue virus and potential targets for drug design.

Dengue infection is a major cause of morbidity in tropical and subtropical regions, bringing nearly 40% of the world population at risk and causing more than 20,000 deaths per year. But there is neither a vaccine for dengue disease nor antiviral drugs to treat the infection. In recent years, dengue infection has been particularly prevalent in India, Southeast Asia, Brazil, and Guangdong Province, China. In this article, we present a brief summary of the biological characteristics of dengue virus and associated flaviviruses, and outline the progress on studies of vaccines and drugs based on potential targets of the dengue virus.

Cloning and isolation of a Conus cysteine-rich protein homologous to Tex31 but without proteolytic activity.

We cloned and isolated a cysteine-rich protein, designated Mr30, from Conus marmoreus. Mr30 belongs to the cysteine-rich secretory protein family that is highly homologous to Tex31 previously obtained from Conus textile and reported as a protease responsible for processing of pro-conotoxins. Mr30, purified by a procedure similar to that of Tex31, indeed showed low proteolytic activity. However, further investigations revealed that the detected protease activity actually resulted from a trace amount of protease(s) contamination rather than from Mr30 itself. This finding led us to rethink the role of conus cysteine-rich secretory proteins: they were probably not responsible for the processing of pro-conotoxins as previously deduced, but their real biological functions remained to be clarified.

Molecular cloning, expression and characterization of protein disulfide isomerase from Conus marmoreus.

The oxidative folding of disulfide-rich conotoxins is essential for their biological functions. In vivo, disulfide bond formation is mainly catalyzed by protein disulfide isomerase. To elucidate the physiologic roles of protein disulfide isomerase in the folding of conotoxins, we have cloned a novel full-length protein disulfide isomerase from Conus marmoreus. Its ORF encodes a 500 amino acid protein that shares sequence homology with protein disulfide isomerases from other species, and 70% homology with human protein disulfide isomerase. Enzymatic analyses of recombinant C. marmoreus protein disulfide isomerase showed that it shared functional similarities with human protein disulfide isomerase. Using conotoxins tx3a and sTx3.1 as substrate, we analyzed the oxidase and isomerase activities of the C. marmoreus protein disulfide isomerase and found that it was much more efficient than glutathione in catalyzing oxidative folding and disulfide isomerization of conotoxins. We further demonstrated that macromolecular crowding had little effect on the protein disulfide isomerase-catalyzed oxidative folding and disulfide isomerization of conotoxins. On the basis of these data, we propose that the C. marmoreus protein disulfide isomerase plays a key role during in vivo folding of conotoxins.

From the identification of gene organization of alpha conotoxins to the cloning of novel toxins.

In the venoms of cone snails, alpha conotoxins are competitive antagonists of nicotinic acetylcholine receptors. Eleven novel cDNA and eight partial gene sequences (including two pseudogenes) of alpha conotoxins were identified from five species of cone snail. As expected, every cDNA encodes a precursor of prepropeptide. In all the partial genes of alpha conotoxins identified, there is a long intron inserted at a fixed position in the pro-region, dividing the encoding region into two exons. The mutation rate in exon I (encoding the signal peptide and a part of pro-region) is much lower than that in exon II (encoding the other part of pro-region, the mature peptide and 3' untranslational region). Interestingly, the sequences at the 5' and 3' end of introns are highly conserved. In addition, in the identified introns exist long dinucleotide (e.g. "GT", "CA") or trinucleotide ("CAT") repeats. In the special case of Pu 1.1, there are five almost identical repeats of a 150 bp sequence in the long intron. Taking advantage of the conserved 3' end sequence of intron, 16 alpha conotoxins, as well as a pseudogene and three kappa A conotoxins, were identified from their genomic DNAs. Based on the comparison of these cDNA and gene sequences, a hypothesis of the alpha conotoxin evolution was proposed.

Solution structure of an M-1 conotoxin with a novel disulfide linkage.

The M-superfamily of conotoxins has a typical Cys framework (-CC-C-C-CC-), and is one of the eight major superfamilies found in the venom of the cone snail. Depending on the number of residues located in the last Cys loop (between Cys4 and Cys5), the M-superfamily family can be divided into four branches, namely M-1, -2, -3 and -4. Recently, two M-1 branch conotoxins (mr3e and tx3a) have been reported to possess a new disulfide bond arrangement between Cys1 and Cys5, Cys2 and Cys4, and Cys3 and Cys6, which is different from those seen in the M-2 and M-4 branches. Here we report the 3D structure of mr3e determined by 2D (1)H NMR in aqueous solution. Twenty converged structures of this peptide were obtained on the basis of 190 distance constraints obtained from NOE connectivities, as well as six varphi dihedral angle, three hydrogen bond, and three disulfide bond constraints. The rmsd values about the averaged coordinates of the backbone atoms were 0.43 +/- 0.19 A. Although mr3e has the same Cys arrangement as M-2 and M-4 conotoxins, it adopts a distinctive backbone conformation with the overall molecule resembling a 'flying bird'. Thus, different disulfide linkages may be employed by conotoxins with the same Cys framework to result in a more diversified backbone scaffold.

Characterization of novel M-superfamily conotoxins with new disulfide linkage.

The M-superfamily with the typical Cys framework (-CC-C-C-CC-) is one of the seven major superfamilies of conotoxins found in the venom of cone snails. Based on the number of residues in the last Cys loop (between C4 and C5), M-superfamily conotoxins can be provisionally categorized into four branches (M-1, M-2, M-3, M-4) [Corpuz GP, Jacobsen RB, Jimenez EC, Watkins M, Walker C, Colledge C, Garrett JE, McDougal O, Li W, Gray WR, et al. (2005) Biochemistry44, 8176-8186]. Here we report the purification of seven M-superfamily conotoxins from Conus marmoreus (five are novel and two are known as mr3a and mr3b) and one known M-1 toxin tx3a from Conus textile. In addition, six novel cDNA sequences of M-superfamily conotoxins have been identified from C. marmoreus, Conus leopardus and Conus quercinus. Most of the above novel conotoxins belong to M-1 and M-2 and only one to M-3. The disulfide analyses of two M-1 conotoxins, mr3e and tx3a, revealed that they possess a new disulfide bond arrangement (C1-C5, C2-C4, C3-C6) which is different from those of the M-4 branch (C1-C4, C2-C5, C3-C6) and M-2 branch (C1-C6, C2-C4, C3-C5). This newly characterized disulfide connectivity was confirmed by comparing the HPLC profiles of native mr3e and its two regioselectively folded isoforms. This is the first report of three different patterns of disulfide connectivity in conotoxins with the same cysteine framework.

Template-assisted rational design of peptide inhibitors of furin using the lysine fragment of the mung bean trypsin inhibitor.

Highly active, small-molecule furin inhibitors are attractive drug candidates to fend off bacterial exotoxins and viral infection. Based on the 22-residue, active Lys fragment of the mung bean trypsin inhibitor, a series of furin inhibitors were designed and synthesized, and their inhibitory activity towards furin and kexin was evaluated using enzyme kinetic analysis. The most potent inhibitor, containing 16 amino acid residues with a Ki value of 2.45x10(-9) m for furin and of 5.60x10(-7) m for kexin, was designed with three incremental approaches. First, two nonessential Cys residues in the Lys fragment were deleted via a Cys-to-Ser mutation to minimize peptide misfolding. Second, residues in the reactive site of the inhibitor were replaced by the consensus substrate recognition sequence of furin, namely, Arg at P1, Lys at P2, Arg at P4 and Arg at P6. In addition, the P7 residue Asp was substituted with Ala to avoid possible electrostatic interference with furin inhibition. Finally, the extra N-terminal and C-terminal residues beyond the doubly conjugated disulfide loops were further truncated. However, all resultant synthetic peptides were found to be temporary inhibitors of furin and kexin during a prolonged incubation, with the scissile peptide bond between P1 and P1' being cleaved to different extents by the enzymes. To enhance proteolytic resistance, the P1' residue Ser was mutated to D-Ser or N-methyl-Ser. The N-methyl-Ser mutant gave rise to a Ki value of 4.70x10(-8) m for furin, and retained over 80% inhibitory activity even after a 3 h incubation with the enzyme. By contrast, the d-Ser mutant was resistant to cleavage, although its inhibitory activity against furin drastically decreased. Our findings identify a useful template for the design of potent, specific and stable peptide inhibitors of furin, shedding light on the molecular determinants that dictate the inhibition of furin and kexin.

Two novel O-superfamily conotoxins from Conus vexillum.

O-superfamily conotoxins include several families that have diverse pharmacological activity on Na+, K+ or Ca2+ channels. These superfamily toxins have been mainly found in fish-hunting and mollusk-hunting Conus species. Here, we reported two novel O-superfamily conotoxins, vx6a and vx6b, purified from a worm-hunting cone snail, Conus vexillum. Though their cysteine framework and signal peptides share high similarity with those of other members of O-superfamily, the mature vx6a and vx6b both have a low sequence homology with others. To test the biological function of vx6a, the toxin was chemically synthesized and then tested on the locust dorsal unpaired median (DUM) neuron system which containing various ion channels. Although no any activity on ion channels was found on the DUM neuron system, vx6a could clearly elicit a series of symptoms in mouse via intracranial injection, such as quivering, climbing, scratching, barrel rolling and paralysis of limbs at different dose.