Peptide Acylation in Aliphatic Polyesters: a Review of Mechanisms and Inhibition Strategies.

Biodegradable polyesters are widely employed in the development of controlled release systems for peptide drugs. However, one of the challenges in developing a polyester-based delivery system for peptides is the acylation reaction between peptides and polymers. Peptide acylation is an important factor that affects formulation stability and can occur during storage, in vitro release, and after drug administration. This review focuses on the mechanisms and parameters that influence the rate of peptide acylation within polyesters. Furthermore, it discusses reported strategies to minimize the acylation reaction.

pHBMT1, a BAHD-family monolignol acyltransferase, mediates lignin acylation in poplar.

Poplar (Populus) lignin is naturally acylated with p-hydroxybenzoate ester moieties. However, the enzyme(s) involved in the biosynthesis of the monolignol-p-hydroxybenzoates have remained largely unknown. Here, we performed an in vitro screen of the Populus trichocarpa BAHD acyltransferase superfamily (116 genes) using a wheatgerm cell-free translation system and found five enzymes capable of producing monolignol-p-hydroxybenzoates. We then compared the transcript abundance of the five corresponding genes with p-hydroxybenzoate concentrations using naturally occurring unrelated genotypes of P. trichocarpa and revealed a positive correlation between the expression of p-hydroxybenzoyl-CoA monolig-nol transferase (pHBMT1, Potri.001G448000) and p-hydroxybenzoate levels. To test whether pHBMT1 is responsible for the biosynthesis of monolignol-p-hydroxybenzoates, we overexpressed pHBMT1 in hybrid poplar (Populus alba × P. grandidentata) (35S::pHBMT1 and C4H::pHBMT1). Using three complementary analytical methods, we showed that there was an increase in soluble monolignol-p-hydroxybenzoates and cell-wall-bound monolignol-p-hydroxybenzoates in the poplar transgenics. As these pendent groups are ester-linked, saponification releases p-hydroxybenzoate, a precursor to parabens that are used in pharmaceuticals and cosmetics. This identified gene could therefore be used to engineer lignocellulosic biomass with increased value for emerging biorefinery strategies.

Novel Carboxylation Method for Polyetheretherketone (PEEK) Surface Modification Using Friedel-Crafts Acylation.

Recently, polyetheretherketone (PEEK) has shown promising dental applications. Surface treatment is essential for dental applications owing to its poor surface energy and wettability; however, no consensus on an effective treatment method has been achieved. In this study, we attempted to carboxylate PEEK sample surfaces via Friedel-Crafts acylation using succinic anhydride and AlBr3. The possibility of further chemical modifications using carboxyl groups was examined. The samples were subjected to dehydration-condensation reactions with 1H,1H-pentadecafluorooctylamine and N,N'-dicyclohexylcarbodiimide. Furthermore, the sample's surface properties at each reaction stage were evaluated. An absorption band in the 3300-3500 cm-1 wavenumber region was observed. Additionally, peak suggestive of COOH was observed in the sample spectra. Secondary modification diminished the absorption band in 3300-3500 cm-1 and a clear F1s signal was observed. Thus, Friedel-Crafts acylation with succinic anhydride produced carboxyl groups on the PEEK sample surfaces. Further chemical modification of the carboxyl groups by dehydration-condensation reactions is also possible. Thus, a series of reactions can be employed to impart desired chemical structures to PEEK surfaces.

Enzymatic Modification of Cellulose To Unlock Its Exploitation in Advanced Materials.

Nowadays natural biopolymers have a wide variety of uses in various industrial applications, such as food, adhesives and composite materials. Among them, cellulose has attracted the interest of researchers due to its properties: high strength and flexibility, biocompatibility and nontoxicity. Despite that, in many cases its practical use is limited because of poor solubility and/or an unsuitable hydrophilic/hydrophobic balance. In this context, enzymatic modification appears as a powerful strategy to overcome these problems through selective, green and environmentally friendly processes. This minireview discusses the different methods developed for the enzymatic modification of cellulose, emphasizing the type of reaction, the enzymes used (laccases, esterases, lipases, hexokinases, etc.), and the properties and applications of the cellulose derivatives obtained. Considering that cellulose is the most abundant natural polymer on Earth and can be derived from residual lignocellulosic biomass, the impact of its use in bio-based process following the logic of the circular economy is relevant.

Poly(ethylene glycol)-b-poly(1,3-trimethylene carbonate) Amphiphilic Copolymers for Long-Acting Injectables: Synthesis, Non-Acylating Performance and In Vivo Degradation.

This article presents the evaluation of diblock and triblock poly(ethylene glycol)-b-poly(1,3-trimethylene carbonate) amphiphilic copolymers (PEG-PTMCs) as excipients for the formulation of long-acting injectables (LAIs). Copolymers were successfully synthesised through bulk ring-opening polymerisation. The concomitant formation of PTMC homopolymer could not be avoided irrespective of the catalyst amount, but the by-product could easily be removed by gel chromatography. Pure PEG-PTMCs undergo faster erosion in vivo than their corresponding homopolymer. Furthermore, these copolymers show outstanding stability compared to their polyester analogues when formulated with amine-containing reactive drugs, which makes them particularly suitable as LAIs for the sustained release of drugs susceptible to acylation.

Ascorbic acid-induced degradation of liposome-encapsulated acylated and non-acylated anthocyanins of black carrot extract.

BACKGROUND: In the presence of ascorbic acid, the degradation of acylated (sinapic, ferulic and p-coumaric acid derivatives of cyanidin-3-xylosylglucosylgalactoside) and non-acylated anthocyanins of black carrot extract (BCE) encapsulated in liposomes was studied. BCEs (0.2% and 0.4% w/w) were encapsulated in liposomes using different lecithin concentrations (1%, 2% and 4% w/w). RESULTS: The liposomes were prepared with particle diameters of less than 50 nm and zeta potentials of about -21.3 mV for extract-containing liposomes and -27.7 mV for control liposomes. The encapsulation efficiency determined using high-performance liquid chromatography (HPLC) showed that increasing lecithin levels increased the efficiency to 59% at the same extract concentration. The concentrations of total anthocyanins and individual anthocyanins were determined for ascorbic acid (0.1% w/w)-degraded extract and liposomes (containing 0.2% w/w extract). Anthocyanin quantification of both liposomal and extract samples was performed by HPLC using cyanidin-3-O-glucoside chloride as standard. Five anthocyanins in the extract and encapsulated liposomes were quantified during 24 h (0-24 h): cyanidin-3-xylosylglucosylgalactoside 1.0-0.51 and 0.82-0.58 mg g-1 , cyanidin-3-xylosylgalactoside 2.5-1.1 and 2.2-1.7 mg g-1 , cyanidin-3-xylosyl(sinapoylglucosyl)galactoside 0.51-0.14 and 0.35-0.28 mg g-1 , cyanidin-3-xylosyl(feruloylglucosyl)galactoside 1.37-0.41 and 1.06-0.98 mg g-1 , and cyanidin-3-xylosyl(coumaroylglucosyl)galactoside 0.28-0.08 mg g-1 for extract and 0.27-0.26 mg g-1 for liposomes, respectively. CONCLUSIONS: This study demonstrates the potential beneficial effect of liposomal encapsulation on individual, particularly acylated, anthocyanins after addition of ascorbic acid during a storage time of 24 h.

Physical-Chemical Characterization of Octreotide Encapsulated in Commercial Glucose-Star PLGA Microspheres.

Sandostatin LAR (SLAR) is an injectable long-acting release (LAR) microsphere formulation for octreotide based on a biodegradeable glucose star copolymer of d,l-lactic and glycolic acids (PLGA-glu), which is primarily used for the treatment of patients with acromegaly. There currently is no generic SLAR approved in the United States despite expiration of patent coverage. To understand better this important formulation, SLAR was assessed for its composition and physical-chemical properties. Octreotide release kinetics was monitored under physiological conditions over 56 days together with several bioerosion parameters [mass loss, water uptake, pH of release media, polymer molecular weight (Mw), and confocal microscopy after BODIPY uptake]. A significant increase in the amount of released peptide occurred after day 14. After 1 day of incubation in PBST, octreotide was not extractable completely from SLAR during 2 h of the extraction process, but complete extraction was accomplished after 24 h, which suggested that strong and noncovalent PLGA-octreotide interactions occurred beginning in the initial release phase. Leuprolide is considered as a cationic peptide competitor for octreotide-PLGA interactions and its presence in the release medium resulted in more continuous octreotide release from SLAR, which was linearly correlated with the mass loss from the polymer (i.e., an indication of erosion-controlled release). These data strongly suggest that octreotide forms a salt with acid end groups of linear PLGA chains that are either present as impurities in, and/or produced by the degradation of, the PLGA-Glu. This salt is expected to catalyze octreotide acylation and extend peptide release beyond that driven by erosion control. The characterization studies of physicochemical properties of SLAR described here could be useful for the development and regulatory evaluation of generic octreotide microspheres as well as new polymer formulations, in which the polymer strongly interacts with encapsulated peptides.

Effects of Hydrophobic Tail Length Variation on Surfactant-Mediated Protein Stabilization.

Recently, protein therapeutics have gained significant attention as a result of their enhanced selectivity and diminished side effects compared to traditional small-molecule drugs. Despite their advantages, protein formulations typically suffer from stability issues because of aggregation and denaturation during production and storage, often resulting in detrimental immune responses. Surfactants can be used to stabilize and protect proteins in solution by preventing protein adsorption onto interfaces or by forming protective structures in solution. Herein, a detailed structure-activity relationship study is described, demonstrating the role that hydrophobic tail length plays in surfactant-mediated stabilization of the model therapeutic protein IgG. The FM1000 series, originating from a surfactant scaffold that allows for easy structure modulation, was synthesized by a simple 2-step procedure. First, phenylalanine was acylated with a variety of acyl chlorides of differing lengths to yield n-acyl phenylalanine, which was then coupled to Jeffamine M1000, a polyethylene glycol-based amine, to yield the final surfactant. With this FM1000 series, it was observed that the 14 carbon-long tail surfactant (14FM1000) was optimal at preventing IgG aggregation compared to surfactants with tails that were longer or shorter. Using a combination of dynamic surface tensiometry and quartz crystal microbalance with dissipation, it was hypothesized that 14FM1000 was able to prevent IgG adsorption, and therefore aggregation, by adsorbing appreciably onto surfaces quickly. 14FM1000 had the fastest rate of initial adsorption compared to the other surfactants studied. Short-tail surfactants were slow to and did not adsorb appreciably onto surfaces, allowing IgG adsorption. Although long-tail surfactants were also slow to adsorb, allowing IgG to adsorb and aggregate, their equilibrium adsorption was strong. Additionally, 14FM1000 was the most reversibly adsorbed surfactant, likely improving its ability to desorb and adsorb quickly to transient surfaces, therefore protecting the IgG at each new hydrophobic surface and preventing aggregation. By understanding the structure-activity relationship between surfactants and protein stabilization, we move toward more efficient design of future surfactants increasing the stability and utility of important protein therapeutics.

Modulation of Antimicrobial Peptide Conformation and Aggregation by Terminal Lipidation and Surfactants.

The function and properties of peptide-based materials depend not only on the amino acid sequence but also on the molecular conformations. In this paper, we chose a series of peptides Gm(XXKK)nX-NH2 (m = 0, 3; n = 2, 3; X = I, L, and V) as the model molecules and studied the conformation regulation through N-terminus lipidation and their formulation with surfactants. The structural and morphological transition of peptide self-assemblies have also been investigated via transmission electron microscopy, atomic force microscopy, circular dichroism spectroscopy, and small-angle neutron scattering. With the terminal alkylation, the molecular conformation changed from random coil to ß-sheet or α-helix. The antimicrobial activities of alkylated peptide were different. C16-G3(IIKK)3I-NH2 showed antimicrobial activity against Streptococcus mutans, while C16-(IIKK)2I-NH2 and C16-G3(IIKK)2I-NH2 did not kill the bacteria. The surfactant sodium dodecyl sulfonate could rapidly induce the self-assemblies of alkylated peptides (C16-(IIKK)2I-NH2, C16-G3(IIKK)2I-NH2, C16-G3(VVKK)2V-NH2) from nanofibers to micelles, along with the conformation changing from ß-sheet to α-helix. The cationic surfactant hexadecyl trimethyl ammonium bromide made the lipopeptide nanofibers thinner, and nonionic surfactant polyoxyethylene (23) lauryl ether (C12EO23) induced the nanofibers much more intensively. Both the activity and the conformation of the α-helical peptide could be modulated by lipidation. Then, the self-assembled morphologies of alkylated peptides could also be further regulated with surfactants through hydrophobic, electrostatic, and hydrogen-bonding interactions. These results provided useful strategies to regulate the molecular conformations in peptide-based material functionalization.

Structural basis for targeting avian sarcoma virus Gag polyprotein to the plasma membrane for virus assembly.

For most retroviruses, including HIV-1, binding of the Gag polyprotein to the plasma membrane (PM) is mediated by interactions between Gag's N-terminal myristoylated matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the PM. The Gag protein of avian sarcoma virus (ASV) lacks the N-myristoylation signal but contains structural domains having functions similar to those of HIV-1 Gag. The molecular mechanism by which ASV Gag binds to the PM is incompletely understood. Here, we employed NMR techniques to elucidate the molecular determinants of the membrane-binding domain of ASV MA (MA87) to lipids and liposomes. We report that MA87 binds to the polar head of phosphoinositides such as PI(4,5)P2 We found that MA87 binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups, indicating that the MA87-IP binding is governed by charge-charge interactions. Using a sensitive NMR-based liposome-binding assay, we show that binding of MA87 to liposomes is enhanced by incorporation of PI(4,5)P2 and phosphatidylserine. We also show that membrane binding is mediated by a basic surface formed by Lys-6, Lys-13, Lys-23, and Lys-24. Substitution of these residues to glutamate abolished binding of MA87 to both IPs and liposomes. In an accompanying paper, we further report that mutation of these lysine residues diminishes Gag assembly on the PM and inhibits ASV particle release. These findings provide a molecular basis for ASV Gag binding to the inner leaflet of the PM and advance our understanding of the basic mechanisms of retroviral assembly.

Commelinid Monocotyledon Lignins Are Acylated by p-Coumarate.

Commelinid monocotyledons are a monophyletic clade differentiated from other monocotyledons by the presence of cell wall-bound ferulate and p-coumarate. The Poaceae, or grass family, is a member of this group, and most of the p-coumarate in the cell walls of this family acylates lignin. Here, we isolated and examined lignified cell wall preparations from 10 species of commelinid monocotyledons from nine families other than Poaceae, including species from all four commelinid monocotyledon orders (Poales, Zingiberales, Commelinales, and Arecales). We showed that, as in the Poaceae, lignin-linked p-coumarate occurs exclusively on the hydroxyl group on the γ-carbon of lignin unit side chains, mostly on syringyl units. Although the mechanism of acylation has not been studied directly in these species, it is likely to be similar to that in the Poaceae and involve BAHD acyl-coenzyme A:monolignol transferases.

Proton Oriented-"Smart Depot" for Responsive Release of Ca2+ to Inhibit Peptide Acylation in PLGA Microspheres.

PURPOSE: The purpose of this study was to characterize and detail the mechanism of a smart Ca2+ release depot (Ca3(PO4)2) about its ability for sustainable inhibition on peptide acylation within PLGA microspheres. METHODS: The octreotide acetate release and acylation kinetics were analyzed by RP-HPLC. Changes of Ca2+ concentration and adsorption behavior were determined by a Calcium Colorimetric Assay Kit. The inner pH changes were delineated by a classic pH sensitive probe, Lysosensor yellow/ blue® dextran. Morphological changes of microspheres, adsorption between polymer and additive, transformation of Ca3(PO4)2 were characterized using SEM, FTIR and SSNMR separately. RESULTS: Before and after microspheres formulation, the property and effectiveness of Ca3(PO4)2 were investigated. Compared with a commonly used calcium salt (CaCl2), high encapsulation efficiency (96.56%) of Ca3(PO4)2 guarantees lasting effectiveness. In an increasingly acidic environment that simulated polymer degradation, the poorly water-soluble Ca3(PO4)2 could absorb protons and transform into the more and more soluble CaHPO4 and Ca(H2PO4)2 to produce sufficient Ca2+ according to severity of acylation. The corresponding Ca2+ produce capacity fully met the optimum inhibition requirement since the real-time adsorption sites (water-soluble carboxylic acids) inside the degrading microspheres were rare. A sustained retention of three switchable calcium salts and slow release of Ca2+ were observed during the microsphere incubation. FTIR results confirmed the long-term inhibition effect induced by Ca3(PO4)2 on the adsorption between drug and polymer. CONCLUSIONS: With the presence of the smart Ca2+ depot (Ca3(PO4)2) in the microspheres, a sustainable and long-term inhibition of peptide acylation was achieved.

Blood Compatibility-An Important but Often Forgotten Aspect of the Characterization of Antimicrobial Peptides for Clinical Application.

Acylation of antimicrobial peptides mimics the structure of the natural lipopeptide polymyxin B, and increases antimicrobial and endotoxin-neutralizing activities. In this study, the antimicrobial properties of lactoferrin-based LF11 peptides as well as blood compatibility as a function of acyl chain length were investigated. Beyond the classical hemolysis test, the biocompatibility was determined with human leukocytes and platelets, and the influence of antimicrobial peptides (AMPs) on the plasmatic coagulation and the complement system was investigated. The results of this study show that the acylation of cationic peptides significantly reduces blood tolerance. With increasing acyl chain length, the cytotoxicity of LF11 peptides to human blood cells also increased. This study also shows that acylated cationic antimicrobial peptides are inactivated by the presence of heparin. In addition, it could be shown that the immobilization of LF11 peptides leads to a loss of their antimicrobial properties.

O-GalNAcylation of RANTES Improves Its Properties as a Human Immunodeficiency Virus Type 1 Entry Inhibitor.

Many human proteins have the potential to be developed as therapeutic agents. However, side effects caused by direct administration of natural proteins have significantly slowed expansion of protein therapeutics into the clinic. Post-translational modifications (PTMs) can improve protein properties, but because of significant knowledge gaps, we are considerably limited in our ability to apply PTMs to generate better protein therapeutics. Here, we seek to fill the gaps by studying the PTMs of a small representative chemotactic cytokine, RANTES. RANTES can inhibit HIV-1 infection by competing with it for binding to receptor CCR5 and stimulating CCR5 endocytosis. Unfortunately, RANTES can induce strong signaling, leading to severe inflammatory side effects. We apply a chemical biology approach to explore the potential of post-translationally modified RANTES as safe inhibitors of HIV-1 infection. We synthesized and systematically tested a library of RANTES isoforms for their ability to inhibit inflammatory signaling and prevent HIV-1 infection of primary human cells. Through this research, we revealed that most of the glycosylated variants have decreased inflammation-associated properties and identified one particular glyco variant, a truncated RANTES containing a Galß1-3GalNAc disaccharide α-linked to Ser4, which stands out as having the best overall properties: relatively high HIV-1 inhibition potency but also weak inflammatory properties. Moreover, our results provided a structural basis for the observed changes in the properties of RANTES. Taken together, this work highlights the potential importance of glycosylation as an alternative strategy for developing CCR5 inhibitors to treat HIV-1 infection and, more generally, for reducing or eliminating unwanted properties of therapeutic proteins.

Human Rab small GTPase- and class V myosin-mediated membrane tethering in a chemically defined reconstitution system.

Membrane tethering is a fundamental process essential for the compartmental specificity of intracellular membrane trafficking in eukaryotic cells. Rab-family small GTPases and specific sets of Rab-interacting effector proteins, including coiled-coil tethering proteins and multisubunit tethering complexes, are reported to be responsible for membrane tethering. However, whether and how these key components directly and specifically tether subcellular membranes remains enigmatic. Using chemically defined proteoliposomal systems reconstituted with purified human Rab proteins and synthetic liposomal membranes to study the molecular basis of membrane tethering, we established here that Rab-family GTPases have a highly conserved function to directly mediate membrane tethering, even in the absence of any types of Rab effectors such as the so-called tethering proteins. Moreover, we demonstrate that membrane tethering mediated by endosomal Rab11a is drastically and selectively stimulated by its cognate Rab effectors, class V myosins (Myo5A and Myo5B), in a GTP-dependent manner. Of note, Myo5A and Myo5B exclusively recognized and cooperated with the membrane-anchored form of their cognate Rab11a to support membrane tethering mediated by trans-Rab assemblies on opposing membranes. Our findings support the novel concept that Rab-family proteins provide a bona fide membrane tether to physically and specifically link two distinct lipid bilayers of subcellular membranes. They further indicate that Rab-interacting effector proteins, including class V myosins, can regulate these Rab-mediated membrane-tethering reactions.

Sensitive Western-Blot Analysis of Azide-Tagged Protein Post Translational Modifications Using Thermoresponsive Polymer Self-Assembly.

Western-blot (WB) is a powerful analytical technique for protein identification in complex biological samples and has been widely used in biological studies for decades. Detection specificity and sensitivity of WB largely relies on quality of the antibodies and performance of the conjugated HRP. However, the application of WB analysis for the detection of protein post-translational modifications (PTMs) is hampered by the low abundance of protein PTMs and by the limited availability of antibodies that specifically differentiate various kinds of PTMs from their protein substrates. Therefore, new recognition mechanisms and signal amplification strategies for WB analysis of protein PTMs is in high demand. In this work, we prepared a soluble polymer that detects various azide-tagged PTM proteins in WB analysis using triarylphosphine and HRP modified thermoresponsive polymer. Specific and efficient detection of azide-tagged PTM protein is achieved via the bioorthogonal reaction between azide and triarylphosphine. More importantly, the chemiluminiscent signal in the WB analysis is largely amplified by the temperature induced self-assembly of numerous thermoresponsive polymer chains carrying multiple HRPs. As a result, approximately 100 times more sensitive detection than commercial antibodies is achieved by this method using standard PTM proteins. Though, this new reagent does not directly detect native PTMs in cell, tissue or blood samples, it still has important application potential in protein PTM studies, considering the wide availability of azide-tagging techniques to a variety of PTMs.

Role of Acyl Chain Composition of Phosphatidylcholine in Tafazzin-Mediated Remodeling of Cardiolipin in Liposomes.

Remodeling of the acyl chain compositions of cardiolipin (CL) species by the transacylase tafazzin is an important process for maintaining optimal mitochondrial functions. The results of mechanistic studies on the tafazzin-mediated transacylation from phosphatidylcholine (PC) to monolyso-CL (MLCL) in artificial lipid membranes are controversial. The present study investigated the role of the acyl chain composition of PC in the Saccharomyces cerevisiae tafazzin-mediated remodeling of CL by examining the structural factors responsible for the superior acyl donor ability of dipalmitoleoyl (16:1) PC over dipalmitoyl (16:0) PC. To this end, we synthesized systematic derivatives of dipalmitoleoyl PC; for example, the location of the cis double bond was migrated from the Δ9-position toward either end of the acyl chains (the Δ5- or Δ13-position), the cis double bond in the sn-1 or sn-2 position or both, was changed to a trans form, and palmitoleoyl and palmitoyl groups were exchanged in the sn-1 and sn-2 positions, maintaining similar PC fluidities. Analyses of the tafazzin-mediated transacylation from these PCs to sn-2'-MLCL(18:1-18:1/18:1-OH) in the liposomal membrane revealed that tafazzin strictly discriminates the molecular configuration of the acyl chains of PCs, including their glycerol positions (sn-1 or sn-2); however, the effects of PC fluidity on the reaction may not be neglected. On the basis of the findings described herein, we discuss the relevance of the so-called thermodynamic remodeling hypothesis that presumes no acyl selectivity of tafazzin.

Preferential hydrolysis of truncated oxidized glycerophospholipids by lysosomal phospholipase A2.

Truncated oxidized glycerophospholipids (ox-PLs) are bioactive lipids resulting from oxidative stress. The catabolic pathways for truncated ox-PLs are not fully understood. Lysosomal phospholipase A2 (LPLA2) with phospholipase A and transacylase activities is a key enzyme in phospholipid homeostasis. The present study assessed whether LPLA2 could hydrolyze truncated ox-PLs. Incubation of LPLA2 with liposomes consisting of 1,2-O-octadecenyl-sn-glycero-3-phosphocholine (DODPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or truncated oxidized phosphatidylcholine (ox-PC)/N-acetylsphingosine (NAS) under acidic conditions resulted in the preferential deacylation at the sn-1 position of the truncated ox-PCs. Additionally, the release of free fatty acid from the truncated ox-PCs preferentially occurred compared with the NAS-acylation. Incubation of LPLA2 with the liposomes consisting of DODPC/DOPC/truncated ox-PC/NAS resulted in the same preferential fatty acid release from the truncated ox-PC. The cationic amphiphilic drug, amiodarone, did not inhibit such fatty acid release, indicating that truncated ox-PCs partition from the lipid membrane into the aqueous phase and react with free LPLA2. Consistent with this mechanism, the hydrolysis of some truncated ox-PCs, but not DOPC, by LPLA2 was detected at neutral pH. Additionally, LPLA2-overexpressed Chinese hamster ovary cells efficiently catabolized truncated ox-PC and were protected from growth inhibition. These findings support the existence of a novel catabolic pathway for truncated ox-PLs via LPLA2.

A Porphyromonas gingivalis Periplasmic Novel Exopeptidase, Acylpeptidyl Oligopeptidase, Releases N-Acylated Di- and Tripeptides from Oligopeptides.

Exopeptidases, including dipeptidyl- and tripeptidylpeptidase, are crucial for the growth of Porphyromonas gingivalis, a periodontopathic asaccharolytic bacterium that incorporates amino acids mainly as di- and tripeptides. In this study, we identified a novel exopeptidase, designated acylpeptidyl oligopeptidase (AOP), composed of 759 amino acid residues with active Ser(615) and encoded by PGN_1349 in P. gingivalis ATCC 33277. AOP is currently listed as an unassigned S9 family peptidase or prolyl oligopeptidase. Recombinant AOP did not hydrolyze a Pro-Xaa bond. In addition, although sequence similarities to human and archaea-type acylaminoacyl peptidase sequences were observed, its enzymatic properties were apparently distinct from those, because AOP scarcely released an N-acyl-amino acid as compared with di- and tripeptides, especially with N-terminal modification. The kcat/Km value against benzyloxycarbonyl-Val-Lys-Met-4-methycoumaryl-7-amide, the most potent substrate, was 123.3 ± 17.3 µm(-1) s(-1), optimal pH was 7-8.5, and the activity was decreased with increased NaCl concentrations. AOP existed predominantly in the periplasmic fraction as a monomer, whereas equilibrium between monomers and oligomers was observed with a recombinant molecule, suggesting a tendency of oligomerization mediated by the N-terminal region (Met(16)-Glu(101)). Three-dimensional modeling revealed the three domain structures (residues Met(16)-Ala(126), which has no similar homologue with known structure; residues Leu(127)-Met(495) (ß-propeller domain); and residues Ala(496)-Phe(736) (α/ß-hydrolase domain)) and further indicated the hydrophobic S1 site of AOP in accord with its hydrophobic P1 preference. AOP orthologues are widely distributed in bacteria, archaea, and eukaryotes, suggesting its importance for processing of nutritional and/or bioactive oligopeptides.

Maize Tricin-Oligolignol Metabolites and Their Implications for Monocot Lignification.

Lignin is an abundant aromatic plant cell wall polymer consisting of phenylpropanoid units in which the aromatic rings display various degrees of methoxylation. Tricin [5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one], a flavone, was recently established as a true monomer in grass lignins. To elucidate the incorporation pathways of tricin into grass lignin, the metabolites of maize (Zea mays) were extracted from lignifying tissues and profiled using the recently developed 'candidate substrate product pair' algorithm applied to ultra-high-performance liquid chromatography and Fourier transform-ion cyclotron resonance-mass spectrometry. Twelve tricin-containing products (each with up to eight isomers), including those derived from the various monolignol acetate and p-coumarate conjugates, were observed and authenticated by comparisons with a set of synthetic tricin-oligolignol dimeric and trimeric compounds. The identification of such compounds helps establish that tricin is an important monomer in the lignification of monocots, acting as a nucleation site for starting lignin chains. The array of tricin-containing products provides further evidence for the combinatorial coupling model of general lignification and supports evolving paradigms for the unique nature of lignification in monocots.