Insect‐Derived Proline‐Rich Antimicrobial Peptides Kill Bacteria by Inhibiting Bacterial Protein Translation at the 70 S Ribosome

Proline‐rich antimicrobial peptides (PrAMPs) have been investigated and optimized by several research groups and companies as promising lead compounds to treat systemic infections caused by Gram‐negative bacteria. PrAMPs, such as apidaecins and oncocins, enter the bacteria and kill them apparently through inhibition of specific targets without a lytic effect on the membranes. Both apidaecins and oncocins were shown to bind with nanomolar dissociation constants to the 70S ribosome. In apidaecins, at least the two C‐terminal residues (Arg17 and Leu18) interact strongly with the 70S ribosome, whereas residues Lys3, Tyr6, Leu7, and Arg11 are the major interaction sites in oncocins. Oncocins inhibited protein biosynthesis very efficiently in vitro with half maximal inhibitory concentrations (IC₅₀ values) of 150 to 240 nmol L ^?1. The chaperone DnaK is most likely not the main target of PrAMPs but it binds them with lower affinity.     

Amino acids of ricin and its polypeptides

Ricin and its corresponding polypeptides (A & B chain) were purified from castor seed. The molecular weight of ricin subunits were 29,000 and 28,000 daltons. The amino acids in ricin determined were Asp45 The22 Ser40 Glu53 Cys4 Gly96 His5 Ile21 Leu33 Lys20 Met4 Phe13 Pro37 Tyr11 Ala45 Val23 Arg20 indicating that ricin contains approximately 516 amino acid residues. The amino acids of the two subunits of ricin A and B chains were Asp23 The12 Ser21 Glu29 Cys2 Gly48 His3 Ile12, Leu17 Lys10 Met2 Phe6 Pro17 Tyr7 Ala35 Val13 Arg13 while in B chain the amino acids were Asp22 The10 Ser19 Glu25 Cys2 Gly47 His1 Ile10, Leu15 Lys11 Met1 Phe7 Pro6 Tyr5 Ala32Val11 Arg10. The total helical content of ricin came around 53.6% which is a new observation.     

Electrospray ionization mass spectrometry and ion mobility analysis of the 20S proteasome complex

Mass spectrometry and gas phase ion mobility [gas phase electrophoretic macromolecule analyzer (GEMMA)] with electrospray ionization were used to characterize the structure of the noncovalent 28-subunit 20S proteasome from Methanosarcina thermophila and rabbit. ESI-MS measurements with a quadrupole time-of-flight analyzer of the 192 kDa alpha7-ring and the intact 690 kDa alpha7beta7beta7alpha7 are consistent with their expected stoichiometries. Collisionally activated dissociation of the 20S gas phase complex yields loss of individual alpha-subunits only, and it is generally consistent with the known alpha7beta7beta7alpha7 architecture. The analysis of the binding of a reversible inhibitor to the 20S proteasome shows the expected stoichiometry of one inhibitor for each beta-subunit. Ion mobility measurements of the alpha7-ring and the alpha7beta7beta7alpha7 complex yield electrophoretic diameters of 10.9 and 15.1 nm, respectively; these dimensions are similar to those measured by crystallographic methods. Sequestration of multiple apo-myoglobin substrates by a lactacystin-inhibited 20S proteasome is demonstrated by GEMMA experiments. This study suggests that many elements of the gas phase structure of large protein complexes are preserved upon desolvation, and that methods such as mass spectrometry and ion mobility analysis can reveal structural details of the solution protein complex.     

Electrophoretic analysis of phosphorylation of the yeast 20S proteasome

The 26S proteasome complex, consisting of two multisubunit complexes, a 20S proteasome and a pair of 19S regulatory particles, plays a major role in the nonlysosomal degradation of intracellular proteins. The 20S proteasome was purified from yeast and separated by two-dimensional gel electrophoresis (2-DE). A total of 18 spots separated by 2-DE were identified as the 20S proteasome subunits by peptide mass fingerprinting with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The alpha2-, alpha4- and alpha7-subunits gave multiple spots, which converged into one spot for each subunit when treated with alkaline phosphatase. The difference of pI between phosphorylated and dephosphorylated spots and their reaction against anti-phosphotyrosine antibody suggested that the alpha2- and alpha4-subunits are phosphorylated either at Ser or at Thr residue, and the alpha7-subunit is phosphorylated at Tyr residue(s). These phosphorylated subunits were analyzed by electrospray ionization-quadrupole time of flight-tandem MS (ESI-QTOF-MS/MS) to deduce the phosphorylation sites. The 20S proteasome has three different protease activities: chymotrypsin-like, trypsin-like and peptidylglutamyl peptide-hydrolyzing activities. The phosphatase treatment increased K(m) value for chymotrypsin-like activity of the 20S proteasome, indicating that phosphorylation may play an important role in regulating the proteasome activity.     

SPR imaging biosensor for the 20S proteasome: sensor development and application to measurement of proteasomes in human blood plasma

The 20S proteasome is a multicatalytic enzyme complex responsible for intracellular protein degradation in mammalian cells. Its antigen level or enzymatic activity in blood plasma are potentially useful markers for various malignant and nonmalignant diseases. We have developed a method for highly selective determination of the 20S proteasome using a Surface Plasmon Resonance Imaging (SPRI) technique. It is based on the highly selective interaction between the proteasome’s catalytic β5 subunit and immobilized inhibitors (the synthetic peptide PSI and epoxomicin). Inhibitor concentration and pH were optimized. Analytical responses, linear ranges, accuracy, precision and interferences were investigated. Biosensors based on either PSI and epoxomicin were found to be suitable for quantitative determination of the proteasome, with a precision of ±10% for each, and recoveries of 102% and 113%, respectively, and with little interference by albumin, trypsin, chymotrypsin, cathepsin B and papain. The proteasome also was determined in plasma of healthy subjects and of patients suffering from acute leukemia. Both biosensors gave comparable results (2860 ng·mL-1 on average for control, and 42300 ng·mL-1 on average for leukemia patients).

Synthesis of (S)-N-[methyl-11C]nicotine and its regional distribution in the mouse brain: a potential tracer for visualization of brain nicotinic receptors by positron emission tomography.

A nicotine agonist, 11C-labeled (S)-nicotine, was synthesized by N-methylation of (S)-nornicotine with [11C]-methyl iodide in dimethylformamide-dimethylsulfoxide in order to study nicotinic receptors in the human brain by positron emission tomography. The radiochemical yield of this N-methylation reaction was more than 90% within 5 min. After purification by high performance liquid chromatography the radiochemical purity of the product was more than 99% and the specific radioactivity was 7.4-11.1 GBq/mumol. The regional distribution of (S)-[11C]nicotine in the mouse brain after intravenous injection was compared with that of (R)-[11C]nicotine. After injection of (S)-[11C]nicotine, the regional uptake of radioactivity was in the following order: cortex greater than thalamu approximately hippocampus greater than striatum greater than hypothalamus greater than cerebellum. Moreover, (S)-[11C]nicotine was displaced from the brain by unlabeled (S)-nicotine, but unlabeled (R)-nicotine caused no change in uptake. In contrast, (R)-[11C]nicotine showed a lower brain uptake and lesser regional differences in radioactivity.     

Simultaneous determination of 2‐arachidonoylglycerol, 1‐arachidonoylglycerol and arachidonic acid in mouse brain tissue using liquid chromatography/tandem mass spectrometry

Endocannabinoids (ECs), such as anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG), modulate a number of physiological processes, including pain, appetite and emotional state. Levels of ECs are tightly controlled by enzymatic biosynthesis and degradation in vivo. However, there is limited knowledge about the enzymes that terminate signaling of the major brain EC, 2‐AG. Identification and quantification of 2‐AG, 1‐AG and arachidonic acid (AA) is important for studying the enzymatic hydrolysis of 2‐AG. We have developed a sensitive and specific quantification method for simultaneous determination of 2‐AG, 1‐AG and AA from mouse brain and adipose tissues by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using a simple brain sample preparation method. The separations were carried out based on reversed phase chromatography. Optimization of electrospray ionization conditions established the limits of detection (S/N = 3) at 50, 25 and 65 fmol for 2‐AG, 1‐AG and AA, respectively. The methods were selective, precise (%R.S.D. < 10%) and sensitive over a range of 0.02–20, 0.01–10 and 0.05–50 ng/mg tissue for 2‐AG, 1‐AG and AA, respectively. The quantification method was validated with consideration of the matrix effects and the mass spectrometry (MS) responses of the analytes and the deuterium labeled internal standard (IS). The developed methods were applied to study the hydrolysis of 2‐AG from mouse brain extracts containing membrane bound monoacylglycerol lipase (MAGL), and to measure the basal levels of 2‐AG, 1‐AG and AA in mouse brain and adipose tissues. Copyright © 2009 John Wiley & Sons, Ltd.     

Plasma Exosomes of Patients with Breast and Ovarian Tumors Contain an Inactive 20S Proteasome

Exosomes are directly involved in governing of physiological and pathological conditions of an organism through the transfer of information from producing to receiving cells. It can be assumed that exosomes are one of the key players of tumor dissemination since they are very stable and small enough to penetrate from various tissues into biological fluids and then back, thus interacting with tissue target cells. We evaluated the enzymatic activity and the level of 20S proteasome in tissue and exosomes of healthy females (n = 39) and patients with ovarian (n = 50) and breast (n = 108) tumors to reveal the critical role of exosomal cargo in the mediation of different types of metastases. Exosomes from plasma and ascites were isolated and characterized in according to International Society for Extracellular Vesicles guidelines. The level of 20S proteasome in tissue and exosomes was determined using Western blot analysis. Chymotrypsin- and caspase-like (ChTL and CL, respectively) peptidase activities of the proteasomes were determined using fluorogenic Suc-LLVY-AMC and Cbz-LLG-AMC substrates, respectively. We observed increased levels of 20S proteasome in ovarian cancer tissue and luminal B subtype breast cancer tissue as well as in plasma exosomes from cancer patients. Moreover, the level of the 20S proteasome in plasma exosomes and ascites exosomes in patients with ovarian tumors is comparable and higher in ovarian cancer patients with low volume ascites than in patients with moderate and high-volume ascites. We also found increased ChTL and CL activities in breast cancer and ovarian cancer tissues, as well as in peritoneal metastases in ovarian cancer, while proteasomal activity in exosomes from plasma of healthy females and all patients, as well as from ascites of ovarian tumor patients were lower than detection limit of assay. Thus, regardless of the type of tumor metastasis (lymphogenous or peritoneal), the exosomes of cancer patients were characterized by an increased level of 20S proteasome, which do not exhibit enzymatic activity.     

Design and Properties of Novel Proteasome Substrates Containing a Polyglutamine Sequence

Fluorogenic polyglutamine-containing peptides with five and ten glutamine residues in a row, having a FRET pair of EDANS (fluorophore) and Dabcyl (quencher), are characterized using spectral and mass spectrometric methods. The possibility of their hydrolysis by the 20S proteasome is examined. The kinetic parameters (catalytic efficiency) for these substances are determined. The presence of glycine in the substrate significantly decreases the solubility of the substrate and diminishes the efficiency of hydrolysis with the proteasome.     

Substrate specificity of the human proteasome

BACKGROUND: Regulated proteolysis by the proteasome is crucial for a broad array of cellular processes, from control of the cell cycle to production of antigens. RESULTS: The rules governing the N-terminal primary and extended substrate specificity of the human 20S proteasome in the presence or absence of 11S proteasome activators (REGalpha/beta and REGgamma) have been elaborated using activity-based proteomic library tools. CONCLUSIONS: The 11S proteasome activators are shown to be important for both increasing the activity of the 20S proteasome and for altering its cleavage pattern and substrate specificity. These data also establish that the extended substrate specificity is an important factor for proteasomal cleavage. The specificities observed have features in common with major histocompatibility complex (MHC) class I ligands and can be used to improve the prediction of MHC class I restricted cytotoxic T-cell responses.     

人红细胞20S蛋白酶体的蛋白质组学表征及不同来源20S蛋白酶体异质性的研究

通过整合差速离心和非变性聚丙烯酰胺凝胶电泳(native-PAGE)技术,建立了能有效分离20S蛋白酶体(20S core particle,CP)的方法.与传统纯化方法比较,此方法具有经济、快速的特点,并且能够对不同组织细胞来源的CP进行分离.利用本方法对人红细胞来源的CP亚基进行了2-DE分离和MALDI-TOF/TOF MS鉴定.结果显示,可鉴定出33个具有不同相对分子量和等电点的蛋白点,此数量远远多于CP亚基的14种.此外,利用非变性/变性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(native/SDS-PAGE)技术,比较了来源于酵母、小鼠肝脏、人红细胞、人胰腺癌细胞系SW1990和PANC-1的CP及其亚基在电泳行为方面的差异,进行了蛋白酶体异质性初探.     

Substrate binding and sequence preference of the proteasome revealed by active-site-directed affinity probes

Background: The proteasome is a multicatalytic protease complex responsible for most cytosolic protein breakdown. The complex has several distinct proteolytic activities that are defined by the preference of each for the carboxyterminal (P1) amino acid residue. Although mutational studies in yeast have begun to define substrate specificities of individual catalytically active β subunits, little is known about the principles that govern substrate hydrolysis by the proteasome.Results: A series of tripeptide and tetrapeptide vinyl sulfones were used to study substrate binding and specificity of the proteasome. Removal of the aromatic amino-terminal cap of the potent tripeptide vinyl sulfone proteasome inhibitor 4-hydroxy-3-iodo-2-nitrophenyl-leucinyl-leucinyl-leucine vinyl sulfone resulted in the complete loss of binding and inhibition. Addition of a fourth amino acid (P4) to the tri-leucine core sequence fully restored inhibitory potency. 1251-labeled peptide vinyl sulfones were also used to examine inhibitor binding and to determine the correlation of subunit modification with inhibition of peptidase activity. Changing the amino acid in the P4 position resulted in dramatically different profiles of β-subunit modification.Conclusions: The P4 position, distal to the site of hydrolysis, is important in defining substrate processing by the proteasome. We observed direct correlations between subunit modification and inhibition of distinct proteolytic activities, allowing the assignment of activities to individual β subunits. The ability of tetrapeptides, but not tripeptide vinyl sulfones, to act as substrates for the proteasome suggests there could be a minimal length requirement for hydrolysis by the proteasome. These studies indicate that it is possible to generate inhibitors that are largely specific for individual β subunits of the proteasome by modulation of the P4 and carboxy-terminal vinyl sulfone moieties.     

A molecular approach to rationally constructing specific fluorogenic substrates for the detection of acetylcholinesterase activity in live cells,mice brains and tissues

Acetylcholinesterase (AChE) is an extremely critical hydrolase tightly associated with neurological diseases. Currently, developing specific substrates for imaging AChE activity still remains a great challenge due to the interference from butyrylcholinesterase (BChE) and carboxylesterase (CE). Herein, we propose an approach to designing specific substrates for AChE detection by combining dimethylcarbamate choline with a self-immolative scaffold. The representative P10 can effectively eliminate the interference from CE and BChE. The high specificity of P10 has been proved via imaging AChE activity in cells. Moreover, P10 can also be used to successfully map AChE activity in different regions of a normal mouse brain, which may provide important data for AChE evaluation in clinical studies. Such a rational and effective approach can also provide a solid basis for designing probes with different properties to study AChE in biosystems and another way to design specific substrates for other enzymes.

In this work, a new approach was developed for designing the representative P10 with high selectivity and sensitivity for imaging AChE activity in the cells and normal mouse brain.     

胰凝乳蛋白酶显色底物的合成及动力学

胰凝乳蛋白酶是一类重要的蛋白水解酶,在其纯化及应用时常常需要进行酶活性的测定;现在一般应用的底物存在溶解度不好、不稳定、灵敏度低或需要特殊的仪器等问题。本文设计合成了一类以对硝基苯胺为显色基团的底物,通过动力学测定研究了氨基酸结构对专一性的影响,该类显色底物可以较好地用于胰凝乳蛋白酶活性测定。     

Conformational energy calculation on the neurotensin c-terminal pentapeptide Arg–Pro–Tyr–Ile–Leu OH

The conformational behavior of the C-terminal neurotensin pentapeptide, Arg–Pro–Tyr–Ile–Leu OH [NT(9–13)], was investigated using empirical energy calculations. A special aim was to display the specific contribution of each residue to induce conformations able to interact with biological receptors. Restrictions were then introduced in intramolecular interactions involving the Arg side chain and the terminal COOH group. The stablest conformations include in the order of decreasing stability: a distorted helical form for the C-terminal tetrapeptide, a (Pro₂–Tyr₃) β turn I, an α helix, an extended form, and a (Tyr₂–Ile₃) β turn III, which are energetically rather close (ΔE < 3 kcal/mol). The NT(9–13) peptide appears then as a rather flexible molcule with a noteworthy ability of adaptation to a substrate. Extended forms would be in agreement with a zipper model of interactions with receptors, whereas folded forms involving helices and β, γ turns would support a lock and key model. The specific contribution of side chains, specially those of Tyr and Arg residues as well as the key position of the Pro residue emerge clearly from this study.     

Solid-state glycation of beta-lactoglobulin by lactose and galactose: localization of the modified amino acids using mass spectrometric techniques

The Maillard reaction is commonly encountered during food processing or storage, and also in human nutrition, hence there is a need for analytical methodologies to identify and characterize the modified proteins. This paper reports specific methods using mass spectrometric techniques to localize protein modifications induced by lactose and galactose on beta-lactoglobulin (beta-Lg) under solid-state glycation conditions. The extent of glycation was first determined by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The specific identification of lactose-modified amino acid residues was realized using both NanoESI-MS, NanoESI-MS/MS (neutral loss scanning modes) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) (with and without guanidination of lysine residues) on unfractionated digests. The results indicated that, after 8.25 h of incubation, the lysine residues were the main targets of lactose-induced modification. In addition to the 15 lysine residues, Leu1 (NH2 terminal) and the Arg124 were also found to be modified, thus leading to a total of 17 different modified amino acid residues (versus 15 found by LC/ESI-MS measurement). In a second set of experiments, different strategies consisting of constant neutral loss and precursor ion scanning were compared to characterize galactose-induced modifications. Owing to the high level of beta-Lg glycation, the combined use of these different strategies appeared to be necessary for determining the galactose-modified sites after 8.25 h of incubation. Thus, among the 22 galactose adducts deduced from the LC/ESI-MS measurement, apart from the N-terminal and classical lysine residues, we also observed a few arginine residues (Arg40, Arg124 and Arg148) that were modified, and also dialkylations on specific lysine residues (Lys47, Lys75).     

Essential of Proline and Valine Residues in the Peptide Derived from Lactoferrin for Angiotensin Converting Enzyme Inhibition

We synthesized Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu, the peptide contained in lactoferrin (Lf), to identify the angiotensin converting enzyme (ACE) inhibition. In an attempt to know the structure‐activity relationship of this peptide, we replaced Pro (the third amino acid residues from N‐terminal) or Val (the fourth amino acid residues from N‐terminal) with Ala (neutral amino acid), Glu (acidic amino acid) or Lys (basic amino acid) to produce six peptides. From the in vitro ACE inhibition (IC₅₀) of these synthesized peptides, the original peptide (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu) showed higher ACE inhibition than the replaced six peptides. Thus, replacement of Pro at the third amino acid residues or Val at the fourth position with Ala, Glu or Lys revealed the ACE inhibition to be lower than the original form of Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu. Otherwise, we added one peptide at the C‐terminal of Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu and found both products with an addition of Val (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu‐Val) or Ile (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu‐Ile) showing a lower ACE inhibition than the original one. The ACE inhibitions produced by both replaced peptides were without significance. Also, deletion of the last peptide at the C‐terminal (Leu‐Arg‐Pro‐Val‐Ala‐Ala) failed to produce a marked change of ACE inhibition as compared to the original one. These results suggest that Pro and Val are essential in the peptide for inhibition of ACE activity.     

Combinatorial Peptide Library for Probing the Selectivity of the s-1 Subsite of Proteases

A combinatorial tetrapeptide library, Suc-Ala-Phe-Arg-AA₁-OR, in which R = p-formamidobenzyl ester and AA₁ = 17 of the 20 natural occurring amino acids, has been synthesized chemically and separated by a reverse phase HPLC. The library was used to study the s-1 subsite specificity of various proteases. The preferred substrate at the s-1 subsite of chymotrypsin is in the order of Trp > Tyr > Phe > Met > Leu. This agreed with the reported data that the favored substrate at the s-1 subsite for chymotrypsin-catalyzed hydrolysis is an aromatic amino acid residue. The hydrophobic amino acid residues at this subsite can be hydrolysized after a longer incubating time. This procedure of selective hydrolysis of a peptide library was used to probe the selectivity of s-1 subsites of four proteases isolated from Bacillus stearothermophilus, subtilisin Carlsberg, subtilisin BPN' and an engineered protease subtilisin 8397. The protease from Bacillus stearothermophilus favored the substrate with residue Lys, and Arg at the s-1 subsite as a trypsin-like protease. The relative reactivities of amino acid residues in the protease-catalyzed hydrolysis of the library can be used as a fingerprint to identify the protease in a protease family.     

Proteasome function in antigen presentation: immunoproteasome complexes, Peptide production, and interactions with viral proteins

Proteasomes are the major nonlysosomal protein degradation machinery in eukaryotic cells and they are largely responsible for the processing of antigens for presentation by the MHC class I pathway. This review concentrates on recent developments in the area of antigen processing. Specialized proteasomes called immunoproteasomes and an 11S regulator of proteasomes (PA28) are induced by interferon-gamma, but it is not entirely clear why changes in proteasome structure are beneficial for antigen presentation. Different proteasome complexes have distinct subcellular distributions and subtle differences in cleavage specificity. Thus it is likely that the efficiency of production of MHC class I binding peptides varies in different locations. Immunoproteasome subunits are enriched at the ER where TAP transports peptides for association with newly synthesized MHC class I molecules. There is recent evidence to suggest that antigen presentation from viral expression vectors, or from peptides that are either delivered by bacterial toxins or derived from signal peptides, require proteasome activity for generation of the correct C-terminus of the epitope. The correct N-terminus may be generated by recently identified ER associated aminopeptidases. A number of viral protein interactions with proteasome subunits have been reported and such interactions may interfere with host anti-viral defenses and also contribute to mechanisms of cell transformation.     

Chaperone activities of the 26S and 20S proteasome

The accumulation of misfolded or damaged proteins causes the failure of normal cell structure and functions necessary for growth and viability. To abort this adverse development, defective proteins must be rapidly repaired by molecular chaperones or destroyed by energy-dependent cytoplasmic proteases. A balance among these processes ultimately maintains cellular homeostasis. In eukaryotes, the 26S proteasome, a protease/chaperone complex, is a central component in the protein triage decision process. The 26S proteasome generally acts as a ubiquitination system, though it also selectively degrades structurally abnormal proteins in an ubiquitin-independent manner. In either case, all substrate proteins must undergo structural changes and stabilization necessary for their rapid degradation. It has, therefore, often been suggested that several chaperone functions are closely related to the stimulation of proteasomal degradation. This review summarizes recent discoveries pertaining to chaperone activities in the proteasomal degradation pathway, and to their regulation of protein breakdown mediated by the proteasome.