Amino Acid Sequence Of Peptide Research Articles

Purification and characterization of antioxidant peptides from yak (Bos grunniens) bone hydrolysates and evaluation of cellular antioxidant activity.

In this study, papain and alcalase were used to generate antioxidant peptides from yak bone protein. The antioxidant activities of hydrolysates in vitro were evaluated by 2,2'-azinobios-(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity, total reducing power, ferrous ion chelating ability and hydroxyl radical scavenging activity. The hydrolysates generated by alcalase possessed the best antioxidant activity among unhydrolyzed protein and samples treated by papain, but the antioxidant activity decreased after simulated gastrointestinal digestion in vitro. The products of simulated gastrointestinal digestion were separated by ultrafiltration and high performance liquid chromatography, and the amino acid sequences of peptides were identified by mass spectrometry. The digestion sites within peptides were predicted by a bioinformatics strategy, and ten peptides were selected for synthesis. Among 10 synthetic peptides, Gly-Phe-Hyp-Gly-Ala-Asp-Gly-Val-Ala, Gly-Gly-Pro-Gln-Gly-Pro-Arg and Gly-Ser-Gln-Gly-Ser-Gln-Gly-Pro-Ala possessed strong antioxidant activities, among which Gly-Phe-Hyp-Gly-Ala-Asp-Gly-Val-Ala had a significant cytoprotective effect in Caco-2 cells under oxidative stress induced by H2O2, which reduced the formation of reactive oxygen species and malondialdehyde, and improved the activity of antioxidant enzymes in cells. These results showed that yak bone peptides exhibited strong antioxidant activity and have a potential value as a new type of natural antioxidant.

Investigating Ugi/Passerini Multicomponent Reactions for the Site-Selective Conjugation of Native Trastuzumab*.

Site-selective modification of proteins has been the object of intense studies over the past decades, especially in the therapeutic field. Prominent results have been obtained with recombinant proteins, for which site-specific conjugation is made possible by the incorporation of particular amino acid residues or peptide sequences. In parallel, methods for the site-selective and site-specific conjugation of native and natural proteins are starting to thrive, allowing the controlled functionalization of various types of amino acid residues. Pursuing the efforts in this field, we planned to develop a new type of site-selective method, aiming at the simultaneous conjugation of two amino acid residues. We reasoned that this should give higher chances of developing a site-selective strategy compared to the great majority of existing methods that solely target a single residue. We opted for the Ugi four-centre three-component reaction to implement this idea, with the aim of conjugating the side-chain amine and carboxylate groups of two neighbouring lysine and aspartate/glutamate. Herein, we show that this strategy can give access to valuable antibody conjugates bearing several different payloads; furthermore, the approach limits the potential conjugation sites to only six on the model antibody trastuzumab.

A Novel Peptide Antibiotic Produced by Streptomyces roseoflavus Strain INA-Ac-5812 With Directed Activity Against Gram-Positive Bacteria.

In this work, we report the isolation and detailed functional characterization for the new non-ribosomally synthesized antibiotic 5812-A/C, which was derived from metabolites of Streptomyces roseoflavus INA-Ac-5812. According to its chemical structure, the studied 5812-A/C preliminary is composed of a cyclic peptide part covalently bounded with an arabinose residue. N-terminal amino acid sequencing of the native peptide has identified its partial structure of Leu-Asp-Gly-Ser-Gly and consisting of a Tyr residue that is supposed to have a two-component peptide nature for the molecule studied. However, the structural analysis of the antibiotic complex derived from S. roseoflavus INA-Ac-5812 is still ongoing. The mechanism of action of 5812-A/C was assessed in comparison with its most related analog, the lipopeptide antibiotic daptomycin, given the presence in both antimicrobials of an L-kynurenine amino acid residue. The inhibitory activity of 5812-A/C against Gram-positive bacteria including methicillin-resistant strain of Staphylococcus aureus was similar to daptomycin. The mechanism of action of 5812-A/C was associated with the disruption of membrane integrity, which differs in comparison with daptomycin and is most similar to the antimicrobial membrane-disturbing peptides. However, 5812-A/C demonstrated a calcium-dependent mode of action. In addition, unlike daptomycin, 5812-A/C was able to penetrate mature biofilms and inhibit the metabolic activity of embedded S. aureus cells. At the same time, 5812-A/C has no hemolytic activity toward erythrocyte, but possessed weak cytotoxic activity represented by heterochromatin condensation in human buccal epithelium cells. The biological properties of the peptide 5812-A/C suggest its classification as a calcium-dependent antibiotic effective against a wide spectrum of Gram-positive pathogenic bacteria.

Characterization and analysis of antioxidant activity of walnut-derived pentapeptide PW5 via nuclear magnetic resonance spectroscopy

The antioxidative activity of natural products has commonly been studied by free radical scavenging methods. However, the mechanisms by which antioxidation is explored by free radical scavenging methods remain largely unknown. This study analyzed the composition of walnut-derived pentapeptides PW5 with potential biological activity and its oxidation reaction products in 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) test by nuclear magnetic resonance (NMR) spectroscopy. The amino acid sequence of PW5 peptide successfully characterized as Proline-Proline-Lysine-Asparagine-Tryptophan exhibited significant antioxidant activity with lower IC50 value (0.2210 ± 0.0032 mM) compared to glutathione (GSH, 0.2567 ± 0.0023 mM, p < 0.001). Furthermore, we found that the tryptophan residue was the only residue in PW5 with obvious alteration after treatment with ABTS free radicals, which was linked to its potential antioxidant properties. These findings revealed how NMR-characterized structures and oxidation reaction products may be used to explore the antioxidative mechanisms of food-derived peptides as well as other natural products.

Preparation and purification of an immunoregulatory peptide from Stolephorus chinensis of the East Sea of China

Immunomodulatory peptides derived from marine organisms are potential sources of new immunomodulating drugs. This study aimed to investigate the optimal enzymatic hydrolysis conditions of immunoregulatory peptides from Stolephorus chinensis. The relative proliferation rate (RPR) of RAW264.7 macrophages was set as the screening index. The immunoregulatory peptides from S. chinensis was prepared via process optimization, ultrafiltration, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography (RP-HLPC). The amino acid sequence of the immunoregulatory peptide from S. chinensis (IPSC) was identified to be Tyr-Val-Met-Arg-Phe (YVMRF; MW 715.4 Da) using Edman degradation and mass spectrometry. In addition, the macrophages became larger with more pseudopods after IPSC treatment, indictating that IPSC stimulated RAW 264.7 differentiation. IPSC also increased productions of nitric oxide (NO), interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α). Our results provide a theoretical basis for preparing immunomodulatory functional food from S. chinensis in future.

Evaluation of temperature, excipients impact on the primary structure of Ganirelix in an injectable formulation, and comparison with Orgalutran® using MALDI‐TOF/TOF‐MS

Ganirelix is a linear polypeptide consisting of covalently bonded 10 amino acid residues. The amino acid sequence in a peptide determines the properties of the molecule. The slightest change in the primary structure (amino acid sequence) of therapeutic peptides can significantly impact its safety, efficacy, and immunogenicity. Hence, the primary structure analysis of therapeutic peptides is regarded as a critical quality attribute (CQA). A vast array of analytical techniques can be used to capture the primary structure of the peptide. In this study, we applied matrix-assisted laser desorption ionization (MALDI)/tandem time of flight mass spectroscopic (TOF/TOF MS) method to demonstrate the primary structure of Ganirelix in an injectable formulation. The apparent monoisotopic molecular mass of Ganirelix is 1,568.9 Da. The attained primary amino acid sequence of Ganirelix in temperature-stressed generic product matched with the theoretical sequence and showed homology with those of the reference listed drug (RLD).

Angiogenesis Inhibition by a Short 13 Amino Acid Peptide Sequence of Tetrastatin, the α4(IV) NC1 Domain of Collagen IV.

Angiogenesis is defined as the formation of new capillaries by sprouting from the pre-existing microvasculature. It occurs in physiological and pathological processes particularly in tumor growth and metastasis. α1, α2, α3, and α6 NC1 domains from type IV collagen were reported to inhibit tumor angiogenesis. We previously demonstrated that the α4 NC1 domain from type IV collagen, named Tetrastatin, inhibited tumor growth in a mouse melanoma model. The inhibitory activity was located in a 13 amino acid sequence named QS-13. In the present paper, we demonstrate that QS-13 decreases VEGF-induced-angiogenesis in vivo using the Matrigel plug model. Fluorescence molecular tomography allows the measurement of a 65% decrease in Matrigel plug angiogenesis following QS-13 administration. The results are confirmed by CD31 microvessel density analysis on Matrigel plug slices. QS-13 peptide decreases Human Umbilical Vein Endothelial Cells (HUVEC) migration and pseudotube formation in vitro. Relevant QS-13 conformations were obtained from molecular dynamics simulations and docking. A putative interaction of QS-13 with α5β1 integrin was investigated. The interaction was confirmed by affinity chromatography, solid phase assay, and surface plasmon resonance. QS-13 binding site on α5β1 integrin is located in close vicinity to the RGD binding site, as demonstrated by competition assays. Collectively, our results suggest that QS-13 exhibits a mighty anti-angiogenic activity that could be used in cancer treatment and other pathologies with excessive angiogenesis such as hemangioma, psoriasis or diabetes.

Determination of Amino Acid Composition of Ganirelix Acetate in an Injectable Formulation by Pre-column Derivatization with 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate.

Ganirelix is a synthetic decapeptide linked with nine different amino acids. To understand the peptide amino acid sequence or primary structure, the first step is to determine the amino acid composition of the peptide which can be a determining factor for the peptide immunogenicity. Edman degradation is not a suitable analytical technique to identify amino acid sequence present in Ganirelix due to the absence of uncharged N-terminal amino group. To address this challenge, a pre-column derivatization method was developed with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent. In the present work, the Ganirelix active pharmaceutical ingredient present in the injectable formulation was isolated by fraction collection and further purified by flash chromatography. The amino acid composition of Ganirelix is assayed by carrying out acid hydrolysis with 6molL-1 hydrochloric acid solution containing 1% phenol at 100°C for 24h and derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent solution, followed by determination of individual amino acids by reverse-phase chromatography using a C18 column. High resolution was achieved for the nine amino acid mixture. The amino acid composition results of temperature-stressed Ganirelix generic product and reference listed drug are in good agreement with the theoretical molar ratio of label information.

MHCAttnNet: predicting MHC-peptide bindings for MHC alleles classes I and II using an attention-based deep neural model.

MotivationAccurate prediction of binding between a major histocompatibility complex (MHC) allele and a peptide plays a major role in the synthesis of personalized cancer vaccines. The immune system struggles to distinguish between a cancerous and a healthy cell. In a patient suffering from cancer who has a particular MHC allele, only those peptides that bind with the MHC allele with high affinity, help the immune system recognize the cancerous cells.ResultsMHCAttnNet is a deep neural model that uses an attention mechanism to capture the relevant subsequences of the amino acid sequences of peptides and MHC alleles. It then uses this to accurately predict the MHC-peptide binding. MHCAttnNet achieves an AUC-PRC score of 94.18% with 161 class I MHC alleles, which outperforms the state-of-the-art models for this task. MHCAttnNet also achieves a better F1-score in comparison to the state-of-the-art models while covering a larger number of class II MHC alleles. The attention mechanism used by MHCAttnNet provides a heatmap over the amino acids thus indicating the important subsequences present in the amino acid sequence. This approach also allows us to focus on a much smaller number of relevant trigrams corresponding to the amino acid sequence of an MHC allele, from 9251 possible trigrams to about 258. This significantly reduces the number of amino acid subsequences that need to be clinically tested.Availability and implementationThe data and source code are available at https://github.com/gopuvenkat/MHCAttnNet.

Synthesis of piperidine-4-one Derivative Containing Dipeptide: An Acetyl cholinesterase and β-secretase Inhibitor

Background: With the goal of developing Alzheimer's disease therapeutics, we have designed and synthesized novel piperidone fused dipeptide (DPPS) derivatives possessing dual action such as acetylcholinesterase (AChE) and beta-amyloid peptide (Aβ) aggregation inhibition. Designed peptide was synthesized by solid phase peptide synthesis using FMOC chemistry protocol and characterized by mass spectroscopy. Methods: The amino acid sequence in peptide was analyzed by LC-MS-MS. In silico docking analysis was carried out using GLIDE software. The docking score using GLIDE was found to be -7.88 against AChE and -9.74 against BACE1 enzyme. In vitro enzyme inhibition assay was carried out for AChE enzyme and BACE1 enzyme. Results: The IC50 values of AChE inhibition and BACE1 of DPPS were found to be 0.4796 μM/ml and 0.0154 μM/ml, respectively. The correlation of in silico and in vitro results showed that DPPS possessed a greater ability to inhibit BACE1 enzyme.

Identification of Copper-Binding Peptides and Investigation of Functional Properties of Acetes japonicus Proteolysate

In this research, two copper-binding peptides were identified in the proteolysate of Acetes japonicus. Firstly, the examination of the effects of hydrolysis conditions including enzyme type, pH, temperature, enzyme:substrate (E:S) ratio and hydrolysis time on the copper-binding capacity (CBC) was carried out. Secondly, to optimize the hydrolysis, response surface methodology (RSM) was applied via E:S ratio and hydrolysis time for maximizing the CBC of the proteolysate. After that, the amino acid composition and functional properties of the proteolysate was evaluated. Simultaneously, before being tested for CBC, four peptide fractions of 10–30 kDa, 3–10 kDa, 1–3 kDa, and < 1 kDa were recovered from the proteolysate using ultrafiltration. Finally, mass spectrometer / mass spectrometer (MS/MS) was used to determine molecular weight and amino acid sequence of bioactive peptide. The result showed that when using Flavourzyme, pH 6, 60 °C, E:S ratio of 40.33 U/g protein and hydrolysis time of 4.15 h, CBC reached its peak at 2699.72 µgCu2+/g protein. Proteolysate, of which Leu was the major amino acid, the indispensible amino acids comprised of approximately 40% total amino acid content. The proteolysate also exerted great solubility, heat stability, foaming and emulsifying property, oil and water holding capacity (OHC and WHC, respectively). The 1–3 kDa fraction exhibited the highest CBC of 2754.73 ± 91.87 μg Cu2+/g protein, from which two peptides (Asp-Tyr-Met-Leu-Pro-Thr-Asp-Lys-Tyr-Pro-His (1378.6 Da) and Gly-Tyr-Pro-Phe-Asp-Ala-Asp-Ser-Val-Asn-Phe-Pro-Val-His-Gly (1620.7 Da)) were detected. This is the first time that the copper affinity peptides were recovered from the small shrimp that could be utilized as a natural source for copper enhancement.

Angiotensin II Type I Receptor Agonistic Autoantibody Blockade Improves Cerebral Blood Flow Autoregulation, Blood Brain Barrier Permeability, and Hypertension in the Pre‐Clinical Rat Model of Preeclampsia

BackgroundPreeclampsia (PE) is a pregnancy disorder that is characterized by an increase in blood pressure, angiotensin II type1 receptor agonistic autoantibodies (AT1‐AA), and neurological complications. The pre‐clinical RUPP (reduced uterine perfusion pressure) rat model of PE displays many of the clinical pathologies associated with PE, such as increased blood pressure, AT1‐AAs, blood brain barrier (BBB) permeability, and impaired cerebral blood flow (CBF) autoregulation.HypothesisWe hypothesized that specific inhibition of AT1‐AA, using the epitope binding 7 amino acid peptide sequence (7AA), will improve blood pressure, BBB integrity, and CBF autoregulation in RUPP rats.MethodsSprague Dawley rats, were divided into 3 groups: normal pregnant (NP) (n=19), RUPP (n=19), and RUPP+AT1‐AA inhibition (7AA) (n=21). RUPP surgery was performed on gestational day (GD) 14 and 7AA (2μg/μl) was administered via osmotic minipumps. GD 19, mean arterial pressure (MAP) was determined and BBB permeability was assessed using the in‐vivo imaging system (IVIS). CBF was measured by laser Doppler flowmetry through a 4 mm x 4 mm cranial window under anesthesia. MAP was elevated step‐wise from 100–190 mmHg by infusion of phenylephrine to determine changes in CBF.ResultsMAP was increased in RUPP vs NP (124±3 vs 100±1 mmHg, p&lt;0.05) and decreased in RUPP+7AA vs RUPP (105±2 vs 124±3 mmHg, p&lt;0.05) rats. Using IVIS, Texas Red/FITC green fluorescent imaging was decreased in RUPP+7AA vs RUPP (0.2±0.001 vs 0.6±0.001, p&lt;0.05), indicating improvement in BBB permeability with AT1‐AA inhibition. CBF autoregulation was impaired in RUPP vs NP rats at 140 mmHg (142±7 vs 119±4%, p&lt;0.05) and 160 mmHg (157±10 vs 129±6%, p&lt;0.05) (both pressures are within the normal CBF autoregulatory range). AT1‐AA inhibition prevented impaired CBF at 140mmHg (125±5 RUPP+7AA vs 142±7% RUPP, p&lt;0.05) and 160 mmHg (125±9 RUPP+7AA vs 157±10% RUPP, p&lt;0.05). Furthermore, the increases in CBF at pressures above the autoregulatory range, 180 mmHg (122±10 vs 198±17%, p&lt;0.05) and 190 mmHg (123±11 vs 208±19%, p&lt;0.05), was prevented in RUPP+7AA vs RUPP, respectively.ConclusionAT1‐AA inhibition improved blood pressure, BBB permeability, and CBF autoregulation in the pre‐clinical RUPP rat model of PE, indicating that AT1‐AA inhibition may be a potential therapy used to improve neurological complications during PE.Support or Funding InformationSupported: AHA18CDA34110264

Purification, Characterization and Mechanistic Evaluation of Angiotensin Converting Enzyme Inhibitory Peptides Derived from Zizyphus Jujuba Fruit

The synthetic Angiotensin Converting Enzyme (ACE) inhibitors have side effects and hence demands for natural ACE inhibitors have been rising. The aim of this study is to purify and introduce natural ACE inhibitors extracted from Zizyphus jujuba fruits. Proteins from Zizyphus jujuba were lysed by trypsin, papain and their combination. Acquired peptides were purified and evaluated for ACE inhibitory activity. Peptide fractions with inhibitory activity were sequenced using tandem mass spectrometry. To elucidate the mode of peptide binding to ACE, homology modeling, molecular docking and molecular dynamics simulation were performed. Amino acid sequence of F2 and F4 peptides, which were the most active hydrolysates, were determined to be IER and IGK with the IC50 values of 0.060 and 0.072 mg/ml, respectively. Results obtained by computational analysis revealed that similar to the common ACE competitive inhibitors such as captopril, IER tripeptide binds to the enzyme active site, in vicinity of the zinc binding site, and occupies the S1 and S2’ subsites. Binding occurs through hydrogen bonding with Gln293, Lys522, His524, Tyr531 and also several hydrophobic interactions. Collectively, these findings indicate that IER tripeptide inhibits the rabbit ACE enzyme through a competitive mechanism of inhibition with IC50 values in the millimolar range.

Polyproline-rich peptides associated with Torpedo californica acetylcholinesterase tetramers

Acetylcholinesterase (AChE) terminates cholinergic neurotransmission by hydrolyzing acetylcholine. The collagen-tailed AChE tetramer is a product of 2 genes, ACHE and ColQ. The AChE tetramer consists of 4 identical AChE subunits and one polyproline-rich peptide, whose function is to hold the 4 AChE subunits together. Our goal was to determine the amino acid sequence of the polyproline-rich peptide(s) in Torpedo californica AChE (TcAChE) tetramers to aid in the analysis of images that will be acquired by cryo-EM. Collagen-tailed AChE was solubilized from Torpedo californica electric organ, converted to 300 kDa tetramers by digestion with trypsin, and purified by affinity chromatography. Polyproline-rich peptides were released by denaturing the TcAChE tetramers in a boiling water bath, and reducing disulfide bonds with dithiothreitol. Carbamidomethylated peptides were separated from TcAChE protein on a spin filter before they were analyzed by liquid chromatography tandem mass spectrometry on a high resolution Orbitrap Fusion Lumos mass spectrometer. Of the 64 identified collagen-tail (ColQ) peptides, 60 were from the polyproline-rich region near the N-terminus of ColQ. The most abundant proline-rich peptides were SVNKCCLLTPPPPPMFPPPFFTETNILQE, at 40% of total mass-spectral signal intensity, and SVNKCCLLTPPPPPMFPPPFFTETNILQEVDLNNLPLEIKPTEPSCK, at 27% of total intensity. The high abundance of these 2 peptides makes them candidates for the principal form of the polyproline-rich peptide in the trypsin-treated TcAChE tetramers.

Brevinin-2GHk from Sylvirana guentheri and the Design of Truncated Analogs Exhibiting the Enhancement of Antimicrobial Activity.

Brevinins are an important antimicrobial peptide (AMP) family discovered in the skin secretions of Ranidae frogs. The members demonstrate a typical C-terminal ranabox, as well as a diverse range of other structural characteristics. In this study, we identified a novel brevinin-2 peptide from the skin secretion of Sylvirana guentheri, via cloning transcripts, and identifying the expressed mature peptide, in the skin secretion. The confirmed amino acid sequence of the mature peptide was designated brevinin-2GHk (BR2GK). Moreover, as a previous study had demonstrated that the N-terminus of brevinin-2 is responsible for exerting antimicrobial activity, we also designed a series of truncated derivatives of BR2GK. The results show that the truncated derivatives exhibit significantly improved antimicrobial activity and cytotoxicity compared to the parent peptide, except a Pro14 substituted analog. The circular dichroism (CD) analysis of this analog revealed that it did not fold into a helical conformation in the presence of either lipopolysaccharides (LPS) or TFE, indicating that position 14 is involved in the formation of the α-helix. Furthermore, three more analogs with the substitutions of Ala, Lys and Arg at the position 14, respectively, revealed the influence on the membrane disruption potency on bacteria and mammalian cells by the structural changes at this position. Overall, the N-terminal 25-mer truncates demonstrated the potent antimicrobial activity with low cytotoxicity.

A Halotolerant Endo-1,4-β-Xylanase from Aspergillus clavatus with Potential Application for Agroindustrial Residues Saccharification.

The use of non-potable water (such as seawater) is an attractive alternative for water intensive processes such as biomass pretreatment and saccharification steps in the production of biochemicals and biofuels. Identification and application of halotolerant enzymes compatible with high-salt conditions may reduce the energy needed for non-potable water treatment and decrease waste treatment costs. Here we present the biochemical properties of a halotolerant endo-1,4-β-xylanase produced by Aspergillus clavatus in submerged fermentation, using paper sludge (XPS) and sugarcane bagasse (XSCB), and its potential application in the hydrolysis of agroindustrial residues. The peptide mass fingerprint and amino acid sequencing of the XPS and XSCB enzymes showed primary structure similarities with an endo-1,4-β-xylanase from Aspergillus clavatus (XYNA_ASPCL). Both enzyme preparations presented good thermal stability at 50°C and were stable over a wide range of pH and Vmax up to 2450U/mg for XPS. XPS and XSCB were almost fully stable even after 24h of incubation in the presence of up to 3M NaCl, and their activity were not affected by 500mM NaCl. Both enzyme preparations were capable of hydrolyzing paper sludge and sugarcane bagasse to release reducing sugars. These characteristics make this xylanase attractive to be used in the hydrolysis of biomass, particularly with brackish water or seawater.

Characterization and antifungal activity of a plant peptide expressed in the interaction between Capsicum annuum fruits and the anthracnose fungus.

Plant defensins are low molecular weight basic peptides ranging from 5 to 7 kDa, with capacity of inhibiting various pathogens, including fungi. They are present in different tissues of plants, including floral parts and fruits of Capsicum sp. The IIF48 extract, present in immature fruits of Capsicum annuum inoculated with C. gloeosporioides, was able to inhibit up to 100% growth ‘in vitro’ of the fungus Colletotrichum gloeosporioides. The main objective of this work was the purification and antifungal activity characterization of a defense-related plant defensin-like isolated of the IIF48 immature fruits extract. The IIF48 extract was subjected to HPLC purification and 13 fractions were obtained, followed by a tricine gel electrophoresis to obtain the protein profile. The different fractions were submitted to a growth inhibition assay against C. gloeosporioides fungus. Fraction 7 (F7) was the most active causing 73% inhibition. Because of the higher F7 activity and the presence of only a peptide of approximately 5 kDa this fraction was subjected to N-terminal sequencing. F7 fraction was carried out plasma membrane permeabilization assays, induction of intracellular ROS production analysis and investigated mitochondrial membrane potential. The F7 fraction showed significant inhibitory activity on the tested fungus, besides promoting membrane permeabilization, induction of endogenous ROS production in Colletotrichum cells and impairing mitochondrial functionality. The first 18 amino acid sequence of the F7 fraction peptide suggests homology to plant-like defensin and was named IIFF7Ca. We also concluded that IIFF7Ca peptide has an effective antimicrobial action against the fungus C. gloeosporioides.

Identification and Characterization of a New Carboxylesterase 2 Isozyme, mfCES2C, in the Small Intestine of Cynomolgus Monkeys.

In contrast to a single human carboxylesterase 2 (CES2) isozyme (hCE2), three CES2 genes have been identified in cynomolgus monkeys: mfCES2A, mfCES2B, and mfCES2C . Although mfCES2A protein is expressed in several organs, mfCES2B is a pseudogene and the phenotype of the mfCES2C gene has not yet been clarified in tissues. In previous studies, we detected an unidentified esterase in the region of CES2 mobility upon nondenaturing PAGE analysis of monkey intestinal microsomes, which showed immunoreactivity for anti-mfCES2A antibody. The aim of the present study was to identify this unidentified esterase from monkey small intestine. The esterase was separated on nondenaturing PAGE gel and digested in-gel with trypsin. The amino acid sequences of fragmented peptides were analyzed by tandem mass spectrometry. The unidentified esterase was shown to be identical to mfCES2C (XP_015298642.1, predicted from the genome sequence data). mfCES2C consists of 559 amino acid residues and shows approximately 90% homology with mfCES2A (561 amino acid residues). In contrast to the ubiquitous expression of mfCES2A, mfCES2C is only expressed in the small intestine, kidney, and skin. The hydrolytic properties of recombinant mfCES2C, expressed in HEK293 cells, with respect to p-nitrophenyl derivatives, 4-methylumbelliferyl acetate, and irinotecan were similar to those of recombinant mfCES2A. However, mfCES2C showed a hydrolase activity for O-n-valeryl propranolol higher than mfCES2A. It is concluded that the previously unidentified monkey intestinal CES2 is mfCES2C, which shows different hydrolytic properties to mfCES2A, depending on the substrate. SIGNIFICANCE STATEMENT: In the present research, we determined that mfCES2C, a novel monkey CES2 isozyme, is expressed in the small intestine and kidney of the cynomolgus monkey. Interestingly, mfCES2C showed a relatively wide substrate specificity for ester-containing compounds. These findings may, in early stages of drug development, support the use of in vitro-to-in vivo extrapolation for the intestinal hydrolysis of ester drugs in the cynomolgus monkey.

A noncanonical binding site of linezolid revealed via molecular dynamics simulations.

Linezolid, an antibiotic of oxazolidinone family, is a translation inhibitor. The mechanism of its action that consists in preventing the binding of aminoacyl-tRNA to the A-site of the large subunit of a ribosome was embraced on the basis of the X-ray structural analysis of the linezolid complexes with vacant bacterial ribosomes. However, the known structures of the linezolid complexes with bacterial ribosomes poorly explain the linezolid selectivity in suppression of protein biosynthesis, depending on the amino acid sequence of the nascent peptide. In the present study the most probable structure of the linezolid complex with a E.coli ribosome in the A,A/P,P-state that is in line with the results of biochemical studies of linezolid action has been obtained by molecular dynamics simulation methods.

Quantitative Mass Spectrometric Analysis of Autoantibodies as a Paradigm Shift in Autoimmune Serology.

Affecting ~7–9% of individuals worldwide (1), autoimmunity is a relatively common condition that can cause substantial morbidity and mortality (2). However, there are considerable challenges in finding robust and accurate biomarkers for this heterogeneous group of diseases. Serum autoantibodies have served as archetypal diagnostic biomarkers for autoimmune diseases over decades (3). As pathologic species, they can be used to monitor disease activity and treatment responses (4). Most diagnostic laboratory tests for autoantibodies utilize conventional assays such as the solid-phase enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA). All of these assays quantitate amounts of autoantibodies in the bodily fluid but fail to delineate their molecular composition. Multiplex assays have emerged for autoantibody high-throughput screening that enable rapid identification of subsets of patients to facilitate diagnostic and predictive medicine (5). This is particularly important since multiple autoantibodies are often responsible for autoimmune disease (6). However, such conventional assays cannot unravel clonal evolution and dynamic autoimmune responses. Frustratingly, prediction of disease onset and flares with these biomarkers remains suboptimal. Mass spectrometry (MS) is an analytical technique which can identify proteins by determining the amino acid sequence of peptides derived from each protein. MS can also measure changes in relative abundance of specific proteins as a consequence of treatment, and with appropriate standards, quantify absolute abundance. MS has been used previously to analyze specific antibodies or the repertoire of antibodies in order to better understand the dynamics of humoral immune responses in vaccinated animals (7). This technology has been used to characterize autoantibodies in diseases such as systemic lupus erythematosus (SLE) and Sjogren's syndrome (SS) by identifying their immunoglobulin variable region (IgV) subfamily usage and mutational profiles at a molecular level (8). Despite conventional immunoassays determining stability in autoantibody profiles, MS-based quantitative proteomics has been used to uncover the dynamic changes in molecular signatures and levels of autoantibodies as the disease progresses (9). Subtle nuances in the molecular profile of patient autoantibodies can be identified, paving the way for new diagnostic biomarkers that can anticipate the onset or severity of disease before conventional biomarkers or immunoassays (10). This exciting technology hence offers a unique opportunity to identify pathogenic “rogue” and/or protective clonotypes that characterize autoimmune diseases. By deconstructing these clonotypes by quantitative proteomics and establishing a database of clonotypes with their corresponding pathogenicity, this would possibly facilitate identification of at-risk patients for deterioration, or predict response to targeted therapy.