A Quantitative Proteomics Design for Systematic Identification of Protease Cleavage Events

We present here a novel proteomics design for systematic identification of protease cleavage events by quantitative N-terminal proteomics, circumventing the need for time-consuming manual validation. We bypass the singleton detection problem of protease-generated neo-N-terminal peptides by introducing differential isotopic proteome labeling such that these substrate reporter peptides are readily distinguished from all other N-terminal peptides. Our approach was validated using the canonical human caspase-3 protease and further applied to mouse cathepsin D and E substrate processing in a mouse dendritic cell proteome, identifying the largest set of protein protease substrates ever reported and gaining novel insight into substrate specificity differences of these cathepsins.Several protocols for proteome-wide identification of protease processing events were recently published. They all follow strategies in which N-terminal peptides, including neo-N-terminal peptides generated by protease action, are enriched from whole proteome digests before identification (e.g. Refs. 1–4). LC-MS/MS analyses of these peptides often yield hundreds of processing events identified in a single experiment (e.g. Refs. 3–5). The N-terminal COFRADIC1 technology developed in our laboratory (6) has been successful in identifying cleavage events of both canonical (e.g. caspases-3 and -7 (7)) and non-canonical proteases (e.g. HtrA2/Omi (8)). Differential stable isotopic labeling in particular, necessary to univocally distinguish genuine neo-N-terminal peptides, allows analyzing control and protease-treated proteomes in a single run. However, this also introduces the most important bottleneck of the technology: verifying whether the peptide envelope of a neo-N-terminal peptide only carries the isotopic label of the protease-treated sample (see Fig. 1A) often had to be done manually for each identified peptide. This “singleton detection problem” can to some extent be automated by software routines such as ProteinProspector (http://prospector.ucsf.edu/prospector/mshome.htm), the MASCOT Distiller Quantitation Toolbox (www.matrixscience.com/distiller.html), and ICPLQuant (9), although these often need specific or proprietary data formats or can only handle MALDI-MS data (9), and researchers still need to individually check correct calling of a neo-N-terminal peptide (10).Open in a separate windowFig. 1.Manual versus automated annotation of protease cleavage events. A, in a typical setup, a heavy (H) labeled proteome is used for protease treatment, and the light (L) labeled proteome serves as a control. Following mixing and N-terminal COFRADIC sorting, neo-N-terminal peptides generated by the added protease are present as singletons, whereas all other N-terminal peptides are present as couples with (light/heavy) ratios around 1 (0 in log₂ scale). B, a mixture of light and heavy labeled proteins (mixed in a 1:1 ratio) is treated with a protease, and as a result, neo-N-terminal peptides generated by the action of the added protease are now present in light/heavy ratios distributed around 1 (0 in log₂ scale) and are clearly distinct from all other N-terminal peptides that come in ratios around 3 (1.58 in log₂ scale). Both types of peptides are readily quantified, circumventing the need for manual validation.To fully overcome this singleton detection problem, here we present and validate a method for highly automated, software-based quantification and annotation of protein processing events on a proteomics scale based on stable isotopic labeling and positional proteomics. We illustrate its strength by generating the largest set of cathepsin D and E substrates hitherto reported. Furthermore, differences in the specificity profiles of these non-canonical proteases are illustrated by the validation of a cleavage event specific for cathepsin E in filamin-A.     

Drosomycin, an Innate Immunity Peptide of Drosophila melanogaster, Interacts with the Fly Voltage-gated Sodium Channel

Several peptide families, including insect antimicrobial peptides, plant protease inhibitors, and ion channel gating modifiers, as well as blockers from scorpions, bear a common CSαβ scaffold. The high structural similarity between two peptides containing this scaffold, drosomycin and a truncated scorpion β-toxin, has prompted us to examine and compare their biological effects. Drosomycin is the most expressed antimicrobial peptide in Drosophila melanogaster immune response. A truncated scorpion β-toxin is capable of binding and inducing conformational alteration of voltage-gated sodium channels. Here, we show that both peptides (i) exhibit anti-fungal activity at micromolar concentrations; (ii) enhance allosterically at nanomolar concentration the activity of LqhαIT, a scorpion alpha toxin that modulates the inactivation of the D. melanogaster voltage-gated sodium channel (DmNa_v1); and (iii) inhibit the facilitating effect of the polyether brevetoxin-2 on DmNa_v1 activation. Thus, the short CSαβ scaffold of drosomycin and the truncated scorpion toxin can maintain more than one bioactivity, and, in light of this new observation, we suggest that the biological role of peptides bearing this scaffold should be carefully examined. As for drosomycin, we discuss the intriguing possibility that it has additional functions in the fly, as implied by its tight interaction with DmNa_v1.The cysteine-stabilized αβ scaffold, CSαβ, contains an α-helix packed against a two-stranded β-sheet stabilized by three spatially conserved disulfide bonds (reviewed in Ref. 1). The CSαβ motif appears in a number of polypeptide families that can exert various biological functions such as: short chain (30–50 residues long) and long chain (60–76 residues long) scorpion toxins that affect voltage-gated ion channels, antimicrobial peptides (of insect and plants) as well as plant protease inhibitors (see Fig. 1) (2, 3).Open in a separate windowFIGURE 1.Diversity of peptides containing the CSαβ motif. Representatives from each of five major groups of peptides containing a CSαβ motif are aligned according to their conserved disulfide bridging and common structural features: two β-strands packed against an α-helix. The featured molecules are from a diverse array of organisms. Scorpion α-toxins: {"type":"entrez-protein","attrs":{"text":"P01484","term_id":"401071","term_text":"P01484"}}P01484 (Aah2 of the North African scorpion Androctonus australis hector), {"type":"entrez-protein","attrs":{"text":"AAB30413","term_id":"546246","term_text":"AAB30413"}}AAB30413 (Ts4 of the Brazilian scorpion Tityus serrulatus); Scorpion β-toxins: {"type":"entrez-protein","attrs":{"text":"P60266","term_id":"1279695545","term_text":"P60266"}}P60266 (Css4 of the Mexican scorpion Centruroides suffusus suffusus), 1BCG_A (Bj-xtrIT of the Israeli black scorpion Hottentota judaica); Scorpion potassium channel blockers: {"type":"entrez-protein","attrs":{"text":"P13487","term_id":"38503412","term_text":"P13487"}}P13487 (charybdotoxin of the Israeli yellow scorpion Leiurus quinquestriatus hebraeus), {"type":"entrez-protein","attrs":{"text":"P0C194","term_id":"93204597","term_text":"P0C194"}}P0C194 (α-KTx 6.11 of the scorpion Opisthacanthus madagascariensis of Madagascar); Insect antimicrobial peptides: {"type":"entrez-protein","attrs":{"text":"NP_523901","term_id":"17737523","term_text":"NP_523901"}}NP_523901 (drosomycin of the fruit fly Drosophila melanogaster), 1I2U_A (heliomicin of the tobacco budworm Heliothis virescens); plant γ-thionins: 1N4N (defensin of the garden petunia Petunia hybrida), {"type":"entrez-protein","attrs":{"text":"AAL85480","term_id":"28624546","term_text":"AAL85480"}}AAL85480 (defensin of peach Prunus persica), {"type":"entrez-protein","attrs":{"text":"AAM62652","term_id":"21553559","term_text":"AAM62652"}}AAM62652 (protease inhibitor II of the thale cress Arabidopsis thaliana).Analysis of the structure-function relationships of several representatives of a subclass of the long chain scorpion toxins family, the scorpion β-toxins (activators of voltage-gated sodium channels (Na_vs)⁵), elucidated their bioactive surfaces including those of the anti-insect excitatory and depressant toxins Bj-xtrIT and LqhIT2 (from Hottentota judaica and Leiurus quinquestriatus hebraeus, respectively (4–6)) and the anti-mammalian β-toxin Css4 (from Centruroides suffusus suffusus (7)). These studies highlighted a conserved pharmacophore positioned on the CSαβ protein core (7). The C-tail, loops, turns, and unstructured stretches that connect to the CSαβ protein core in long chain scorpion toxins constitute a large portion of their exteriors and bear residues that participate in bioactivity (reviewed in Ref. 8). We have recently reported that truncated scorpion β-toxins, lacking the N- and C-terminal regions of the parental peptides but maintaining the CSαβ motif (ΔΔβ-toxins), are able to interact at high affinity with Na_vs (9). Although by themselves, the ΔΔβ-toxins (ΔΔCss4 and ΔΔBj-xtrIT) were nontoxic and did not bind at the receptor sites of the parental toxins, they exhibited an unexpected ability to allosterically facilitate the activity of a scorpion α-toxin (inhibition of Na_v fast inactivation), which binds at receptor site-3 on insect Na_vs (10), and the effect of the marine polyether toxin brevetoxin-2 (PbTx-2, facilitator of Na_v activation), which binds to receptor site-5 (11). However, a short chain potassium channel blocker (charybdotoxin) with a CSαβ structural fold did not exert any of these effects (9). These results indicated that it is not only the CSαβ motif but that specific amino acids at key sites on the protein exterior that can interact with ion channels and either block voltage-gated potassium channels or induce conformational alteration of voltage-gated sodium channels. From a structural viewpoint, the ability of ΔΔBj-xtrIT and ΔΔCss4 to bind to the Na_v, as manifested in modulation of the interaction of receptor site-3 and -5 ligands, suggests that by truncation of the two β-toxins, a masked functional surface was exposed. Because the CSαβ motif appears in several protein families including antimicrobial peptides, potassium channel blockers, and sodium channel gating modifiers (Fig. 1) (2, 3), we explored the possibility that a well characterized CSαβ peptide may exert an additional function known for other peptides bearing this scaffold.For this aim, we tested the ability of a well characterized Drosophila melanogaster anti-fungal peptide drosomycin (DRS) to interact with voltage-gated sodium channels. The solution structure of DRS indicates that this 44-amino acid peptide is cross-linked by four disulfide bonds, of which three render a CSαβ structural fold (Fig. 2) (12). Sequence comparison of the truncated scorpion β-toxin ΔΔCss4 with DRS indicates moderate identity (34%) and similarity (50%), including conservation of six cysteine residues that stabilize the CSαβ motif, which is manifested by a remarkable structural similarity (Fig. 2). Moreover, Lys-3, Asp-11, Asn-12, Glu-13, Gln-21, and Gln-22 of ΔΔCss4, which are involved in the interaction with insect Na_vs, are spatially conserved in DRS (Fig. 2) (9) but not in potassium channel blockers (Fig. 1). In light of the resemblance between the truncated scorpion β-toxin and DRS, we tested whether DRS is able to interact with the D. melanogaster voltage-gated sodium channel DmNa_v1.Open in a separate windowFIGURE 2.Sequence alignment and three-dimensional structures of ΔΔCss4 and DRS. A, schematic diagrams of the Cα model structures of ΔΔCss4 and DRS covered by semitransparent molecular surfaces. The structure of DRS (right panel) is derived from the Protein Data Bank code 1MYN. The ΔΔCss4 model (left panel) is based on the NMR structure of Cn2 (Protein Data Bank code 1Cn2) and is spatially aligned with that of DRS. A was prepared using PyMOL. B, sequences were aligned according to the conserved cysteine residues, and the disulfide bonds formed between cysteine pairs are marked in solid lines. Dashes indicate gaps. Amino acid residues that were identified as part of the interacting surface of ΔΔCss4 with insect Na_vs (9) are shown in sticks according to their chemical nature (blue, positive charge; red, negative charge; green, nonpolar) and are also highlighted in the sequence alignment. Corresponding residues in DRS according to sequence and structural alignments are also shown in sticks.     

Translocation of a Thioether-Bridged Azurin Peptide Fragment via the Sec Pathway in Lactococcus lactis

This study demonstrates for the first time that a thioether-containing peptide, an azurin fragment, can be translocated via the Sec pathway. This methyl-lanthionine was introduced by the nisin modification enzymes. The Sec pathway can therefore be a successful alternative for those cyclized peptides that are inefficiently transported via NisT.Azurin, a cupredoxin produced by Pseudomonas aeruginosa, can selectively enter human cancer cells and induce apoptosis (24) via binding to the tumor suppressor protein p53 (1). The azurin peptide fragment p28, containing amino acids 50 to 77 (LSTAADMQGVVTDGMASGLDKDYLKPDD), still enters human cancer cells and inhibits tumor proliferation (20). Importantly, novel cancer treatments can be based on azurin peptide fragments and derivatives thereof (T. Das Gupta and A. Chakrabarty, 20 March 2008, patent application WO2008033820). Although the pharmacokinetic property of therapeutic peptides is promising, lack of biostability is the major hurdle for their successful application. Consequently, it is very relevant to explore the possibilities for enhancing biostability of these peptides.In our group, we developed a technology to improve the stability of therapeutic peptides by exploiting the nisin synthetase enzymes NisB and NisC for the introduction of thioether bridges. We applied a two-plasmid expression system (7, 8, 12), in which the NisBTC-encoding plasmid is compatible with the substrate-peptide-encoding plasmid. Lactococcus lactis containing this expression system can secrete nonlantibiotic peptides which are dehydrated or stabilized by a thioether ring (8, 16). NisB dehydrates serines and threonines in substrate peptides, NisC couples dehydrated residues stereo- and regioselectively to cysteines, and NisT, the ABC transporter, translocates the modified peptides out of the cell (10, 11, 13, 15). The leader peptide is essential for targeting and modification of the propeptides (23).When transport via NisT is impaired or is less efficient, the Sec pathway of L. lactis is a successful alternative in translocation of dehydrated peptides. When the nisin leader is preceded by a Sec signal peptide or a Tat signal peptide 27 or 44 amino acids long, respectively, modification by NisB and NisC still occurs (12; G. N. Moll, A. Kuipers, R. Rink, A. J. M. Driessen, and O. P. Kuipers, 15 June 2006, patent application WO 2006062398). However, NisC-cyclized prenisin was not translocated via the Sec system (12). This is likely due to the dimensions of fully modified nisin (3), which is too large to fit in the SecY pore (12, 21). Here, we report for the first time that the Sec pathway of L. lactis can translocate a p28 azurin fragment analog with a thioether ring.We previously demonstrated that under culturing conditions, the highly reactive dehydroalanines can spontaneously couple to cysteines, either intra- or extracellularly, whereas the less reactive dehydrobutyrines do not (16). To exclude spontaneous thioether ring formation by dehydroalanines, we mutated serines in positions 51 and 66 to alanines, whereas a single dehydratable threonine was kept in position 52 of the azurin(50-77) peptide fragment. Position 56 was mutated to a cysteine to allow posttranslational introduction of a thioether bridge (Table ​(Table1;1; pNZ8048-derived plasmids).

TABLE 1.

Bacterial strains and plasmids used in this study

Production of Angiotensin-I-Converting-Enzyme-Inhibitory Peptides in Fermented Milks (<Emphasis Type="Italic">Lassi</Emphasis>) Fermented by <Emphasis Type="Italic">Lactobacillus acidophillus</Emphasis> with Consideration of Incubation Period and Simmering Treatment

The lassi, fermented milks product containing angiotensin-I-converting-enzyme (ACE)-inhibitory peptides were produced by using selected Lactobacillus acidophilus NCDC-15 and the incubation period and simmering effect was also optimized for production of ACE-inhibitory peptides. The time–temperature combination for the heat treatment was optimized using RSM. The biological activity was measured in the supernatant of the fermented milk after centrifugation. The lowest IC₅₀ values for the inhibition of angiotensin-converting enzyme (ACE) was found 28.9 ± 0.95 μg protein/ml in the supernatant of milk fermented by L. acidophilus and heated at 78 °C for 10 h. The fractions which showed the highest ACE-inhibitory indexes were further purified by different techniques including solid phase extraction, RP-HPLC and FPLC and the related peptides were identified by LC–MS/MS using the Ultimate 3000 nano HPLC system (Dionex) coupled to a 4000 Q TRAP electro-spray ionization mass spectrometry. The high ACE-inhibitory activity containing fractions of the milk fermented by L. acidophilus contained the sequences of b-casein (b-CN) fragment. The fraction-III showed minimum IC₅₀ value i.e. 14.57 ± 0.72 μg/ml compared with fraction-I and fraction-II. Among these peptides 14 peptides have been identified from the fraction-I of the lassi prepared from L. acidophilus i.e. β-CN f47–56, β-CN f47–57, β-CN f199–209, β-CN f176–182, β-CN f176–183, β-CN f176–184, β-CN f1–7, β-CN f57–68, β-CN f166–175, β-CN f195–206, β-CN f195–207, β-CN f195–209, β-CN f94–106 and β-CN f169–176 showed partially or completely homology to that the milk protein bioactive peptides having ACE inhibitory. The two peptides KVLPVPQK (β-CN f169–176) and YQEPVLGPVRGPFPIIV (β-CN f193–209) have the same sequence as ACE inhibitory peptides (Maeno et al. in J Dairy Sci 79(8):1316–1321, 1996; Yamamoto et al. in J Dairy Sci 77:917–922, 1994b).     

Structural modeling of osteoarthritis ADAMTS4 complex with its cognate inhibitory protein TIMP3 and rational derivation of cyclic peptide inhibitors from the complex interface to target ADAMTS4

The ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) enzyme is a matrix-associated zinc metalloendopeptidase that plays an essential role in the degradation of cartilage aggrecan in arthritic diseases and has been recognized as one of the most primary targets for therapeutic intervention in osteoarthritis (OA). Here, we reported computational modeling of the atomic-level complex structure of ADAMTS4 with its cognate inhibitory protein TIMP3 based on high-resolution crystal template. By systematically examining the modeled complex structure we successfully identified a short inhibitory loop (⁶²EASESLC⁶⁸) in TIMP3 N-terminal inhibitory domain (NID) that directly participates in blocking the enzyme’s active site, which, and its extended versions, were then broken from the full-length protein to serve as the peptide inhibitor candidates of ADAMTS4. Atomistic molecular dynamics simulation, binding energetic analysis, and fluorescence-based assay revealed that the TIMP3-derived linear peptides can only bind weakly to the enzyme (K_d = 74 ± 8 μM), which would incur a considerable entropy penalty due to the high conformational flexibility and intrinsic disorder of these linear peptides. In this respect, we proposed a cyclization strategy to improve enzyme–peptide binding affinity by, instead of traditionally maximizing enthalpy contribution, minimizing entropy cost of the binding, where a disulfide bond was added across the two terminal residues of linear peptides, resulting in a number of TIMP3-derived cyclic peptides. Our studies confirmed that the cyclization, as might be expected, can promote peptide binding capability against ADAMTS4 substantially, with affinity increase by 3-fold, 9-fold and 7-fold for cyclic peptides

,

and

, respectively.     

Endothelial NO and O2− production rates differentially regulate oxidative,nitroxidative, and nitrosative stress in the microcirculation

Endothelial dysfunction causes an imbalance in endothelial NO and O₂⁻ production rates and increased peroxynitrite formation. Peroxynitrite and its decomposition products cause multiple deleterious effects including tyrosine nitration of proteins, superoxide dismutase (SOD) inactivation, and tissue damage. Studies have shown that peroxynitrite formation during endothelial dysfunction is strongly dependent on the NO and O₂⁻ production rates. Previous experimental and modeling studies examining the role of NO and O₂⁻ production imbalance on peroxynitrite formation showed different results in biological and synthetic systems. However, there is a lack of quantitative information about the formation and biological relevance of peroxynitrite under oxidative, nitroxidative, and nitrosative stress conditions in the microcirculation. We developed a computational biotransport model to examine the role of endothelial NO and O₂⁻ production on the complex biochemical NO and O₂⁻ interactions in the microcirculation. We also modeled the effect of variability in SOD expression and activity during oxidative stress. The results showed that peroxynitrite concentration increased with increase in either O₂⁻ to NO or NO to O₂⁻ production rate ratio (Q_O2⁻/Q_NO or Q_NO/Q_O2⁻, respectively). The peroxynitrite concentrations were similar for both production rate ratios, indicating that peroxynitrite-related nitroxidative and nitrosative stresses may be similar in endothelial dysfunction or inducible NO synthase (iNOS)-induced NO production. The endothelial peroxynitrite concentration increased with increase in both Q_O2⁻/Q_NO and Q_NO/Q_O2⁻ ratios at SOD concentrations of 0.1–100 μM. The absence of SOD may not mitigate the extent of peroxynitrite-mediated toxicity, as we predicted an insignificant increase in peroxynitrite levels beyond Q_O2⁻/Q_NO and Q_NO/Q_O2⁻ ratios of 1. The results support the experimental observations of biological systems and show that peroxynitrite formation increases with increase in either NO or O₂⁻ production, and excess NO production from iNOS or from NO donors during oxidative stress conditions does not reduce the extent of peroxynitrite mediated toxicity.     

Anionic effects on the formation of tridentate 4′-chloro-2,2′:6′,2″-terpyridine copper(II) complexes having 1:1 or 1:2 ratio of metal and ligand and their crystal symmetry

A facile synthetic procedure has been used to prepare one five-coordinate and four six-coordinate copper(II) complexes of 4′-chloro-2,2′:6′,2″-terpyridine (tpyCl) ligand with different counterions (, , , , and ) in high yields. They are formulated as [Cu(tpyCl-κ³N,N′,N′′)(SO₄-κO)(H₂O-κO)] · 2H₂O (1), trans-[Cu(tpyCl-κ³N,N′,N″)(NO₃-κO)₂(H₂O-κO)] (2), [Cu(tpyCl-κ³N,N′,N″)₂](BF₄)₂ (3), [Cu(tpyCl-κ³N,N′,N″)₂](PF₆)₂ (4) and [Cu(tpyCl-κ³N,N′,N″)₂](ClO₄)₂ (5) and versatile interactions in supramolecular level including coordinative bonding, O-H?O, O-H?Cl, C-H?F, and C-H?Cl hydrogen bonding, π-π stacking play essential roles in forming different frameworks of 1-5. It is concluded that the difference of coordination abilities of the counterions used and the experimental conditions codominate the resulting complexes with 1:1 or 1:2 ratio of metal and ligand.     

fpr,a Deficient Xer Recombination Site from a Salmonella Plasmid,Fails To Confer Stability by Dimer Resolution: Comparative Studies with the pJHCMW1 mwr Site

Salmonella plasmid pFPTB1 includes a Tn3-like transposon and a Xer recombination site, fpr, which mediates site-specific recombination at efficiencies lower than those required for stabilizing a plasmid by dimer resolution. Mutagenesis and comparative studies with mwr, a site closely related to fpr, indicate that there is an interdependence of the sequences in the XerC binding region and the central region in Xer site-specific recombination sites.Xer site-specific recombination stabilizes many plasmids by resolving dimers created through recombination events (17, 18). Most plasmids'' Xer recombination sites consist of a core recombination site (CRS) that includes two 11-nucleotide binding sites for XerC and XerD, separated by a 6- to 8-nucleotide central region, and a stretch of about 180 bp known as accessory sequences (AS) that bind the architectural proteins PepA and ArgR (Fig. ​(Fig.1A).1A). These elements form a synaptic complex (11, 14) where XerC is activated by interaction with XerD and catalyzes the exchange of the first pair of strands, which results in the formation of a Holliday junction (3, 6) that, in the case of cer (ColE1) or mwr (pJHCMW1), is resolved by Xer-independent processes (Fig. ​(Fig.1B)1B) (2).Open in a separate windowFIG. 1.(A) Comparative diagrams of pFPBT1 and pJHCMW1. The black lines represent regions of homology. The Tn3-like transposons in both plasmids are shown at the correct locations but are not to scale. The gray bar between the plasmid maps identifies the replication (REP) regions, which share 97% homology. The numbers indicate the coordinates in the GenBank database (pJHCMW1, accession number {"type":"entrez-nucleotide","attrs":{"text":"AF479774","term_id":"19698424","term_text":"AF479774"}}AF479774; pFPBT1, accession number {"type":"entrez-nucleotide","attrs":{"text":"AJ634602","term_id":"46019644","term_text":"AJ634602"}}AJ634602). The location and a diagram of the Xer site-specific recombination site are shown below the plasmid diagrams. The different regions of the Xer site-specific recombination site, shown with different colors, are not drawn to scale. ARG, ARG box, ArgR binding site. (B) Schematic representation of dimer resolution mediated by the Xer site-specific recombination reaction. For clarity, the proteins are shown only in the synaptic complex (red rectangle, XerD; green rectangle, XerC; brown oval, PepA; yellow oval, ArgR). Blue lines represent AS, and the CRS is the only region shown with a double line (green and red) representing the two DNA strands. The green line represents the DNA strand exchanged by XerC. Only the Xer-independent pathway of resolution of the Holliday junction (demonstrated for cer and mwr) is shown. (C) Comparison of the nucleotide sequences of mwr, fpr, and cer. The ARG box and different regions of the CRS are individually boxed. The ARG box consensus sequence is shown at the top. Nucleotides mutated in different derivatives are indicated by an arrow (central region) or a star (ARG box). Black dots identify the most conserved nucleotides among several sites (8).The pJHCMW1 plasmid, originally isolated from Klebsiella pneumoniae, includes mwr and the Tn3-like transposon Tn1331, which transposes through a replicative pathway (15, 20, 22). The efficiency of Xer-mediated dimer resolution at mwr when Escherichia coli is cultured in L broth is below the levels needed to stabilize the plasmid; instead, stabilization is mediated by the Tn1331 resolvase acting at the res site (13, 21). However, the efficiency of Xer-mediated dimer resolution at mwr is substantially increased when the cells are cultured in low-osmolarity broth (4, 13). The low levels of dimer resolution observed when cells are cultured in L broth seem to be due to a weak interaction of the substandard mwr ARG box with ArgR, hindering proper formation of the synaptic complex. When the cells are cultured in low-osmolarity growth medium, an increase in the density of negative supercoiling results in an increased stability of the synaptic complex and/or efficiency of catalysis by XerC, leading to a significantly higher efficiency of dimer resolution (23). These characteristics make pJHCMW1 a very unusual plasmid that includes a Xer recombination site that, under certain conditions, is unable to confer stability by resolution of dimers; instead, that function is performed by the cointegrate resolution system of Tn1331. In this study, we report that another plasmid, Salmonella Typhimurium plasmid pFPTB1 (12), includes a Xer recombination site with high homology to mwr (Fig. ​(Fig.1C),1C), from here on called fpr (pFPTB1 Xer recombination site), and a copy of the replicative transposon Tn3-ΔTn1721 (Fig. 1A and C). Plasmid pFPTB1 is the second case reported in which stabilization by dimer resolution is provided by the insertion of a replicative transposon rather than a resident Xer recombination site, suggesting that rather than being exceptional, pJHCMW1 and pFPTB1 may be part of a group of plasmids with these characteristics.The E. coli strains and plasmids used in this study are described in Table ​Table1.1. Plasmid pFPRTT1 was generated by inserting a synthetic DNA fragment with the fpr site sequence (coordinates 2221 to 2520, accession number {"type":"entrez-nucleotide","attrs":{"text":"AJ634602","term_id":"46019644","term_text":"AJ634602"}}AJ634602) from pFPTB1 (12) into the EcoRV site of pUC57 (accession number {"type":"entrez-nucleotide","attrs":{"text":"Y14837","term_id":"2440162","term_text":"Y14837"}}Y14837). Plasmids pTTT1 through pTTT6 were generated by site-directed mutagenesis using the QuikChange II XL kit (Stratagene). Lennox L broth (containing 2% [wt/vol] agar in the case of solid medium) is called high-osmolarity medium (containing 0.5% NaCl; osmolality, 209 mmol/kg); for low-osmolarity growth medium, NaCl was omitted (osmolality, 87 mmol/kg) (13, 23). In vivo resolution assays were carried out as described by Pham et al. (13). Although pFPTB1 has been isolated from S. Typhimurium, we decided it was appropriate to carry out the in vivo experiments with E. coli because it has been shown before that the Xer recombination proteins of S. Typhimurium can substitute for and are highly homologous to the corresponding proteins of E. coli (7), K. pneumoniae Xer recombination proteins share high homology with those of E. coli and can complement mutants, we have observed no difference in levels of resolution with some sites such as dif and cer and minimal differences with mwr (4), and pJHCMW1-like replicons such as pGY1 (Salmonella) (9), pVI678 (E. coli; accession number {"type":"entrez-nucleotide","attrs":{"text":"NC_008597","term_id":"118480566","term_text":"NC_008597"}}NC_008597), and pTKH11 (Klebsiella) (25) are being found in nature across the members of the family Enterobacteriaceae rather than in one specific genus.

TABLE 1.

Strains and plasmids used in this study

In situ alkylation of 4,4′-bipyridine in hydrothermal synthesis of one-dimensional copper(I) bromide compounds

Hydrothermal reaction of CuBr₂, 4,4′-bipyridine and ethanol/methanol generated two copper (I) bromide complexes with in situ alkylated 4,4′-bipyridium, namely [C₁₄H₁₈N₂][Cu₅Br₇] (1) and [C₁₂H₁₄N₂][Cu₄Br₆] (2). The structure of 1 consists of chains and N,N′-diethyl-4,4′-bipyridinium. The underlying structural motif in of 1 is the Cu₅Br capped square pyramid, which is different from the Cu₅Br₂ pentagonal bipyramidal structural motif in various documented anions. The in 1 contains untypical μ₅-bromide, with which five copper atoms forms a capped square pyramid rather than a pentagonal pyramid as predicted by Subramanian and Hoffmann. Compound 2 is isostructural with [C₁₂H₁₄N₂][Cu₄Cl₆] reported by Willett, and consists of chains and N,N′-dimethyl-4,4′-bipyridinium. The chain is composed of alternating Cu₆Br₆ and Cu₂Br₆ units.     

Ruthenium(II) complexes possessing the η-p-cymene ligand

Complexes possessing a soft donor η⁶-arene and hard donor acetylacetonate ligand, [(η⁶-p-cymene)Ru(κ²-O,O-acac-μ-CH)]₂[OTf]₂ (1) (OTf = trifluoromethanesulfonate; acac = acetylacetonate) and {Ar′ = 3,5-(CF₃)-C₆H₃}, were prepared and fully characterized. The lability of the μ-CH linkage for complex 1 and the THF ligand of 2 allow access to the unsaturated cation [(η⁶-p-cymene)Ru(κ²-O,O-acac)]⁺. The reaction of with KTp {Tp = hydridotris(pyrazolyl)borate} produces . The azide complex forms upon reaction of with N₃Ar (Ar = p-tolyl), and reaction of with CHCl₃ at 100 °C yields the chloride-bridged binuclear complex . The details of solid-state structures of [(η⁶-p-cymene)Ru(κ²-O,O-acac-μ-CH)]₂[OTf]₂ (1), and are disclosed.     

Global Analysis of Myocardial Peptides Containing Cysteines With Irreversible Sulfinic and Sulfonic Acid Post-Translational Modifications

Cysteine (Cys) oxidation is a crucial post-translational modification (PTM) associated with redox signaling and oxidative stress. As Cys is highly reactive to oxidants it forms a range of post-translational modifications, some that are biologically reversible (e.g. disulfides, Cys sulfenic acid) and others (Cys sulfinic [Cys-SO₂H] and sulfonic [Cys-SO₃H] acids) that are considered “irreversible.” We developed an enrichment method to isolate Cys-SO₂H/SO₃H-containing peptides from complex tissue lysates that is compatible with tandem mass spectrometry (MS/MS). The acidity of these post-translational modification (pK_a Cys-SO₃H < 0) creates a unique charge distribution when localized on tryptic peptides at acidic pH that can be utilized for their purification. The method is based on electrostatic repulsion of Cys-SO₂H/SO₃H-containing peptides from cationic resins (i.e. “negative” selection) followed by “positive” selection using hydrophilic interaction liquid chromatography. Modification of strong cation exchange protocols decreased the complexity of initial flowthrough fractions by allowing for hydrophobic retention of neutral peptides. Coupling of strong cation exchange and hydrophilic interaction liquid chromatography allowed for increased enrichment of Cys-SO₂H/SO₃H (up to 80%) from other modified peptides. We identified 181 Cys-SO₂H/SO₃H sites from rat myocardial tissue subjected to physiologically relevant concentrations of H₂O₂ (<100 μm) or to ischemia/reperfusion (I/R) injury via Langendorff perfusion. I/R significantly increased Cys-SO₂H/SO₃H-modified peptides from proteins involved in energy utilization and contractility, as well as those involved in oxidative damage and repair.Cysteine (Cys)¹ is an integral site for protein post-translational modification (PTM) in response to physiological and pathological stimuli. Numerous studies have identified roles for biologically reversible Cys PTM, including disulfides, S-nitrosothiols, and sulfenic acids (Cys-SOH), in the regulation of protein function during redox signaling (reviewed in (1, 2)). Additionally, Cys can be oxidized in pathologies associated with oxidative stress (e.g. neurodegeneration, cancer, and cardiovascular disease (2)). Various redox proteomics methods exist for enrichment of these reversibly oxidized Cys, based on reduction to the thiol and then capture by: 1) alkylation with a chemical tag (e.g. isotope coded affinity tags) (3–6); 2) thiol-disulfide exchange (7–10); or 3) heavy metal ion chelation (11, 12). Oxidative Cys PTMs with predominantly no known means of enzymatic reduction have also been identified. These “over” or “irreversibly” oxidized Cys PTM (sulfinic [Cys-SO₂H] and sulfonic [Cys-SO₃H] acids) are primarily associated with oxidative stress. Only one example of reversible Cys-SO₂H modification has been characterized—in peroxiredoxins (Prx) by the ATP-dependent sulfiredoxin (Srx)(13); however, Srx is not thought to reduce Cys-SO₂H in other proteins, and no mechanism has yet been found for Cys-SO₃H reduction. At basal levels, ∼1–2% of Cys exist as Cys-SO₂H/SO₃H (14), and the RSO₂H modification has functional significance in some proteins (e.g. DJ-1 is activated in Alzheimer''s disease by Cys-SO₂H at Cys-106) (15).Cys-SO₂H/SO₃H are produced via sequential oxidation of Cys-SOH, which itself is formed because of Cys thiol oxidation by reactive oxygen and nitrogen species (ROS/RNS), such as hydrogen peroxide (H₂O₂) or peroxynitrite. This reaction is relatively inefficient and requires three equivalents of oxidant, as well as the protection of the initial Cys-SOH from nucleophilic attack. Therefore, Cys forming these PTM, particularly at biologically relevant concentrations of oxidant, are likely to be highly reactive or located in a unique microenvironment that accommodates their production without prior reduction of the Cys-SOH (e.g. by thiol or amine attack). Such sites may thus be candidates as redox or regulatory sensors (reviewed in (16)). Alternatively, over-oxidation to Cys-SO₂H/SO₃H during elevated oxidative stress may serve as a marker of oxidative damage, and target proteins for degradation.Information on Cys-SO₂H/SO₃H PTM in complex samples has thus far been generated only by amino acid analysis (hydrolyzed lysates) (14) or two-dimensional gel electrophoresis (2-DE), where these PTM cause an acidic shift (17, 18). The former provides no information on specific proteins, whereas the latter relies on the modified population being of sufficient intensity for observation and/or the availability of antibodies against a protein-of-interest. A recent study identified 44 Cys-SO₂H/SO₃H-modified peptides in nonphysiologically H₂O₂ oxidized (440 μm) cells utilizing long column ultra-high pressure liquid chromatography (LC) (19). Global analysis of irreversible Cys-PTM thus requires enrichment that considers: (1) Cys is the second least abundant amino acid in proteins (∼1.5%) (20), and (2) Cys-SO₂H/SO₃H are expected to occupy only 1–2% of these Cys sites, under physiological (and perhaps even pathological) conditions.Specific peptide enrichment by LC followed by bottom-up proteomics is a common approach used successfully for many PTMs (21, 22). Limited studies, however, have explored such techniques for Cys-SO₂H/SO₃H-containing peptides, and none have examined complex lysates—only single purified proteins (23, 24). Given that these PTM are among the most acidic modifications, with an average pK_a of RSO₂H < 2 and RSO₃H ∼−3, it is pertinent to isolate these peptides by exploiting their unique charge distribution. At acidic pH, where nonmodified tryptic peptides will have an average in-solution charge state between one and two (depending on pK_a of acidic residues and the C terminus), Cys-SO₂H/SO₃H-containing peptides will have an added negative charge, and, thus, have average charge distribution ≤ 1. Selection can therefore be performed on either positively or negatively charged resins with the former being a “positive” selection for Cys-SO₂H/SO₃H-containing peptides (retained by the resin), whereas the latter is a “negative” selection (Cys-SO₂H/SO₃H-containing peptides will not be retained by the resin). Both approaches have been used (23, 24) to capture peptides from bovine serum albumin (BSA) oxidized by performic acid – causing scission of disulfide bonds and conversion of Cys to Cys-SO₃H, and methionine (Met) to the sulfone Met(O₂). The studies gave comparative results, with positive selection (24) increasing Cys coverage in comparison to negative selection (23) (60% versus 45%) at the expense of specificity, with more non-Cys peptides observed in the elution.Ultimately, any enrichment approach must be able to purify Cys-SO₂H/SO₃H-containing peptides from cells and tissues under physiological and/or pathological conditions, both of which will generate considerably lower levels of Cys-SO₂H/SO₃H than performic acid. Myocardial ischemia and reperfusion (I/R) injury is characterized by a “burst” of ROS/RNS that is observed upon reperfusion (25, 26). These ROS/RNS overwhelm the natural antioxidant defenses of the heart (27) and lead to oxidative stress that contributes to contractile dysfunction (28–30). Several studies have observed an increase in reversible Cys PTM following I/R (31–36), and an increase in Cys-SO₂H/SO₃H may also contribute to cellular dysfunction that ultimately leads to apoptosis and necrosis that follows prolonged I/R (myocardial infarction). Given the common practice of peptide fractionation with strong cation exchange (SCX) as a first dimension during bottom-up proteomics, we wished to explore its utility in identifying Cys-SO₂H/SO₃H sites in complex samples. Performic oxidized BSA and myocardial protein extracts were utilized to study the interactions occurring at each step of the method, and then the method was applied to myocardial protein extract that had been exposed to a high concentration of a less efficient oxidant (H₂O₂). Finally, the method was used to identify Cys-SO₂H/SO₃H-containing peptides derived from either physiologically relevant concentrations of H₂O₂ (i.e. ≤100 μm, an estimate of the likely pathological H₂O₂ levels (37, 38)) or from rat myocardial tissue subjected to I/R injury.     

Synthesis and structures of copper and gold complexes of the P,N ligands RNC(Bu)C(H)RPPh2 (R = SiMe3, H)

The reaction of the chelating P,N ligand RNC(Bu^t)CH(R)PPh₂ (R = SiMe₃) (1) with CuCl and CuCl₂ (probably by way of reduction to Cu(I) by the phosphine ligand) or Cu(NCCH₃)₄ClO₄ yielded the dimeric 1:1 complex [Cu{PPh₂CH(R)C(Bu^t)NR}Cl]₂ (2) or the monomeric 2:1 complex [Cu{PPh₂CH(R)C(Bu^t)NR}₂]ClO₄ (3), respectively. The presence of trace amounts of water during the reaction resulted in the successive cleavage of the two trimethylsilyl groups of the ligand and the formation of the monomeric chelate complexes [Cu{PPh₂CH(R)C(Bu^t)NH}₂]ClO₄ (4) and [Cu{PPh₂CH₂C(Bu^t)NH}₂]ClO₄ (5). Oxidation of 5 by atmospheric oxygen led to small quantities of the blue Cu(II) complex [Cu{(O)PPh₂CH₂C(Bu^t)NH}₂](ClO₄)₂ (6). The dimeric gold complexes [Au{PPh₂CH₂C(Bu^t)NH}]₂X₂ (X = BF₄, ClO₄) (7) were similarly obtained from the previously described Au{PPh₂CH(R)C(Bu^t)NR}Cl by replacing the covalently bound chlorine with the weakly coordinating anions in the presence of small quantities of water. The solution and solid state structures (except 5) of all complexes were determined by NMR spectroscopy and X-ray crystallography.     

Water-soluble N-carboxymethylchitosan derivatives: Preparation,characteristics and its application

In this work, only N-substituted chitosan derivatives (water-soluble N-carboxymethylchitosan derivatives: N-CMC) with different degrees of substitution were obtained by reaction of a fully deacetylated chitosan (derived from deacetylation of chitosan using decrystallized method) with monochloroacetic acid at pH 8 and temperature of 90 °C. The structure of N-carboxymethylchitosan and chitosan was characterized by IR, ¹H, ¹³C and ¹H–¹³C NMR-HSQC spectra. In the IR spectrum of the N-carboxymethylchitosan, the appearance of peak at 1742 cm^?1 was assigned for CO group of NHCH₂COOH of substituted chitosan. In the ¹H NMR spectra, the peaks at about 3.81÷4.06 ppm, assigned for CH₂ groups of NHCH₂ and N(CH₂)₂, were the major feature, while in the ¹H–¹³C NMR-HSQC spectra, signals of CH₂ confirmed the presence of these two different substituted CH₂ groups. The degree of substitution (DS) of N-monosubstitution (DS_N-mono) decreased from 0.47 to 0.03 meanwhile that of N,N-disubstitution (DS_N,N-di) increased from 0.52 to 0.96 since the mass ratio of chitosan/monochloroacetic acid changing from 1/1 to 1/4. The N-carboxymethylchitosan derivatives have been used for adsorption Cu(II) ion from aqueous solution. The results shown that the optimum conditions for adsorption Cu(II) ion in nitrate solution were pH 6.5, temperature of 30 °C, for 60–90 min and the substituted chitosan derivative having DS_N-mono of 0.16 and DS_N,N-di of 0.81 had maximum adsorption capacity of 192 mg Cu(II) per gram of N-CMC.