Quan

Examining the neuroprotective effects of protocatechuic acid and chrysin on in vitro and in vivo models of Parkinson disease.

Polypharmacology-based strategies using drug combinations with different mechanisms of action are gaining increasing attention as a novel methodology to discover potentially innovative medicines for neurodegenerative disorders. We used this approach to examine the combined neuroprotective effects of two polyphenols, protocatechuic acid (PCA) and chrysin, identified from the fruits of Alpinia oxyphylla. Our results demonstrated synergistic neuroprotective effects, with chrysin enhancing the protective effects of PCA, resulting in greater cell viability and decreased lactate dehydrogenase release from 6-hydroxydopamine-treated PC12 cells. Their combination also significantly attenuated chemically induced dopaminergic neuron loss in both zebrafish and mice. We examined the molecular mechanisms underlying these collective cytoprotective effects through proteomic analysis of treated PC12 cells, resulting in the identification of 12 regulated proteins. Two were further characterized, leading to the determination that pretreatment with PCA and chrysin resulted in (i) increased nuclear factor-erythroid 2-related factor 2 protein expression and transcriptional activity; (ii) modulation of cellular redox status with the upregulated expression of hallmark antioxidant enzymes, including heme oxygenase-1, superoxide dismutase, and catalase; and (iii) decreased levels of malondialdehyde, a known lipid peroxidation product. Treatment with PCA and chrysin also inhibited activation of nuclear factor-κB and expression of inducible nitric oxide synthase. Our findings suggest that natural products, when used in combination, can be effective potential therapeutic agents for treating diseases such as Parkinson disease. A therapy involving both PCA and chrysin exhibits its enhanced neuroprotective effects through a combination of cellular mechanisms: antioxidant cytoprotection and anti-inflammation.

Online two-dimensional porous graphitic carbon/reversed phase liquid chromatography platform applied to shotgun proteomics and glycoproteomics.

A novel fully automatable two-dimensional liquid chromatography (2DLC) platform has been integrated into a modified commercial off-the-shelf LC instrument, incorporating porous graphitic carbon (PGC) separation and conventional low-pH reversed-phase (RP) separation for both proteomics and N-glycomics analyses; the dual-trap column configuration of this platform offers desirable high-throughput analyses with almost no idle time, in addition to a miniaturized setup and simplified operation. The total run time per analysis was only 19 h when using eight PGC fractions for unattended large-scale qualitative and quantitative proteomic analyses; the identification of 2678 nonredundant proteins and 11,984 unique peptides provided one of the most comprehensive proteome data sets for primary cerebellar granule neurons (CGNs). The effect of pH on the PGC column was investigated for the first time to improve the hydrophobic peptide coverage; the performance of the optimized system was first benchmarked using tryptic digests of Saccharomyces cerevisiae cell lysates and then evaluated through duplicate analyses of Macaca fascicularis cerebral cortex lysates using isobaric tags for relative and absolute quantitation (iTRAQ) technology. An additional plug-and-play PGC module functioned in a complementary manner to recover unretained hydrophilic solutes from the low-pH RP column; synchronization of the fractionations between the PGC-RP system and the PGC module facilitated simultaneous analyses of hydrophobic and hydrophilic compounds from a single sample injection event. This methodology was applied to perform, for the first time, detailed glycomics analyses of Macaca fascicularis plasma, resulting in the identification of a total 130 N-glycosylated plasma proteins, 705 N-glycopeptides, and 254 N-glycosylation sites.

Arginine-Facilitated α- and π-Radical Migrations in Glycylarginyltryptophan Radical Cations

We have used model tripeptides GXW (with X being one of the amino acid residues glycine (G), alanine (A), leucine (L), phenylalanine (F), glutamic acid (E), histidine (H), lysine (K), or arginine (R)) to study the effects of the basicity of the amino acid residue on the radical migrations and dissociations of odd-electron molecular peptide radical cations M(·+) in the gas phase. Low-energy collision-induced dissociation (CID) experiments revealed that the interconvertibility of the isomers [G(·)XW](+) (radical centered on the N-terminal α-carbon atom) and [GXW](·+) (radical centered on the π system of the indolyl ring) generally increased upon increasing the proton affinity of residue X. When X was arginine, the most basic amino acid, the two isomers were fully interconvertible and produced almost identical CID spectra despite the different locations of their initial radical sites. The presence of the very basic arginine residue allowed radical migrations to proceed readily among the [G(·)RW](+) and [GRW](·+) isomers prior to their dissociations. Density functional theory calculations revealed that the energy barriers for isomerizations among the α-carbon-centered radical [G(·)RW](+), the π-centered radical [GRW](·+), and the β-carbon-centered radical [GRW(β)(·)](+) (ca. 32-36 kcal mol(-1)) were comparable with those for their dissociations (ca. 32-34 kcal mol(-1)). The arginine residue in these GRW radical cations tightly sequesters the proton, thereby resulting in minimal changes in the chemical environment during the radical migrations, in contrast to the situation for the analogous GGW system, in which the proton is inefficiently stabilized during the course of radical migration.

Effect of the N-terminal basic residue on facile Cα–C bond cleavages of aromatic-containing peptide radical cations

Fragmentation of radical cationic peptides [R(G)(n-2)X(G)(7-n)]˙(+) and [R(G)(m-2)XG]˙(+) (X = Phe or Tyr; m = 2-5; n = 2-7) leads selectively to a(n)(+) product ions through in situ C(α)-C peptide backbone cleavage at the aromatic amino acid residues. In contrast, substituting the arginine residue with a less-basic lysine residue, forming [K(G)(n-2)X(G)(7-n)]˙(+) (X = Phe or Tyr; n = 2-7) analogs, generates abundant b-y product ions; no site-selective C(α)-C peptide bond cleavage was observed. Studying the prototypical radical cationic tripeptides [RFG]˙(+) and [KFG]˙(+) using low-energy collision-induced dissociation and density functional theory, we have examined the influence of the basicity of the N-terminal amino acid residue on the competition between the isomerization and dissociation channels, particularly the selective C(α)-C bond cleavage viaβ-hydrogen atom migration. The dissociation barriers for the formation of a(2)(+) ions from [RFG]˙(+) and [KFG]˙(+)via their β-radical isomers are comparable (33.1 and 35.0 kcal mol(-1), respectively); the dissociation barrier for the charge-induced formation of the [b(2)- H]˙(+) radical cation from [RFG]˙(+)via its α-radical isomer (39.8 kcal mol(-1)) was considerably higher than that from [KFG]˙(+) (27.2 kcal mol(-1)). Thus, the basic arginine residue sequesters the mobile proton to promote the charge-remote selective C(α)-C bond cleavage by energetically hindering the competing charge-induced pathways.

Intramolecular hydrogen atom migration along the backbone of cationic and neutral radical tripeptides and subsequent radical-induced dissociations.

Dissociation of peptide radical ions involves competition between charge-induced and radical-induced reactions that can be preceded by isomerization. The isomeric radical cations of the peptide methyl ester [G˙GR-OMe](+) and [GG˙R-OMe](+) provide very similar collision-induced dissociation (CID) spectra, suggesting that isomerization occurs prior to fragmentation. They undergo characteristic radical-induced bond cleavage of the peptide N-terminal amide bond resulting in the y2(+) ion, and of the arginine side-chains Cα-Cβ bond giving protonated allylguanidine {[CH2[double bond, length as m-dash]CHCH2NHC(NH2)2](+), m/z 100}. The absence of a y2(+) fragment ion in the CID of the radical cationic tripeptide [ACH3G˙R](+) and of an m/z 100 ion in the spectrum of [G˙ACH3R](+) (where ACH3 is an α-aminoisobutyric acid residue, which cannot form an α-carbon-centered radical through hydrogen atom transfer) establishes the importance of hydrogen atom migration along the peptide backbone prior to specific radical-induced fragmentations. Herein we use density functional theory (DFT) at the B3LYP/6-31++G(d,p) level to evaluate the barriers for interconversion between the α-carbon-centered radicals and for dissociation. The radical cations [G˙GR](+) and [GG˙R](+) have their radicals located on the α-carbon atoms of the peptide backbone and their charge densities largely sequestered on the guanidine groups of the side-chain of arginine residues. This is in contrast to the isomeric radical cations of [GGG]˙(+), in which the charge resides necessarily on the peptide backbone. The lower charge densities on the backbones of [G˙GR](+) and [GG˙R](+) result in greater structural flexibility, decreasing the barrier for interconversion between these α-carbon-centered radicals to 36.2 kcal mol(-1) (cf. 44.7 kcal mol(-1) for [GGG]˙(+)). The total absence of charge, assessed by examining intramolecular hydrogen atom transfers among the three α-carbon centers of the isomeric neutral α-carbon-centered triglycine radicals [GGG-H]˙, leads to an additional but slight reduction in enthalpy, to approximately 34 kcal mol(-1).

Proteomic evidence for genetic epistasis: ClpR4 mutations switch leaf variegation to virescence in Arabidopsis

Chloroplast development in plants is regulated by a series of coordinated biological processes. In this work, a genetic suppressor screen for the leaf variegation phenotype of the thylakoid formation 1 (thf1) mutant combined with a proteomic assay was employed to elucidate this complicated network. We identified a mutation in ClpR4, named clpR4-3, which leads to leaf virescence and also rescues the var2 variegation. Proteomic analysis showed that the chloroplast proteome of clpR4-3 thf1 is dominantly controlled by clpR4-3, providing molecular mechanisms that cause genetic epistasis of clpR4-3 to thf1. Classification of the proteins significantly mis-regulated in the mutants revealed that those functioning in the expression of plastid genes are oppositely regulated while proteins functioning in antioxidative stress, protein folding, and starch metabolism are changed in the same direction between thf1 and clpR4-3. The levels of FtsHs including FtsH2/VAR2, FtsH8, and FtsH5/VAR1 are greatly reduced in thf1 compared with those in the wild type, but are higher in clpR4-3 thf1 than in thf1. Quantitative PCR analysis revealed that FtsH expression in clpR4-3 thf1 is regulated post-transcriptionally. In addition, a number of ribosomal proteins are less expressed in the clpR4-3 proteome, which is in line with the reduced levels of rRNAs in clpR4-3. Furthermore, knocking out PRPL11, one of the most downregulated proteins in the clpR4-3 thf1 proteome, rescues the leaf variegation phenotype of the thf1 and var2 mutants. These results provide insights into molecular mechanisms by which the virescent clpR4-3 mutation suppresses leaf variegation of thf1 and var2.

Arginine-facilitated isomerization: radical-induced dissociation of aliphatic radical cationic glycylarginyl(iso)leucine tripeptides.

The gas phase fragmentations of aliphatic radical cationic glycylglycyl(iso)leucine tripeptides ([G(•)G(L/I)](+)), with well-defined initial locations of the radical centers at their N-terminal α-carbon atoms, are significantly different from those of their basic glycylarginyl(iso)leucine ([G(•)R(L/I)](+)) counterparts; the former lead predominantly to [b(2) - H](•+) fragment ions, whereas the latter result in the formation of characteristic product ions via the losses of (•)CH(CH(3))(2) from [G(•)RL](+) and (•)CH(2)CH(3) from [G(•)RI](+) through C(β)-C(γ) side-chain cleavages of the (iso)leucine residues, making these two peptides distinguishable. The α-carbon-centered radical at the leucine residue is the key intermediate that triggers the subsequent C(β)-C(γ) bond cleavage, as supported by the absence of (•)CH(CH(3))(2) loss from the collision-induced dissociation of [G(•)RL(α-Me)](+), a radical cation for which the α-hydrogen atom of the leucine residue had been substituted by a methyl group. Density functional theory calculations at the B3LYP 6-31++G(d,p) level of theory supported the notion that the highly basic arginine residue could not only increase the energy barriers against charge-induced dissociation pathways but also decrease the energy barriers against hydrogen atom transfers in the GR(L/I) radical cations by ∼10 kcal mol(-1), thereby allowing the intermediate precursors containing α- and γ-carbon-centered radicals at the (iso)leucine residues to be formed more readily prior to promoting subsequent C(β)-C(γ) and C(α)-C(β) bond cleavages. The hydrogen atom transfer barriers for the α- and γ-carbon-centered GR(L/I) radical cations (roughly in the range 29-34 kcal mol(-1)) are comparable with those of the competitive side-chain cleavage processes. The transition structures for the elimination of (•)CH(CH(3))(2) and (•)CH(2)CH(3) from the (iso)leucine side chains possess similar structures, but slightly different dissociation barriers of 31.9 and 34.0 kcal mol(-1), respectively; the energy barriers for the elimination of the alkenes CH(2)═CH(CH(3))(2) and CH(3)CH═CHCH(3) through C(α)-C(β) bond cleavages of γ-carbon-centered radicals at the (iso)leucine side chains are 29.1 and 32.8 kcal mol(-1), respectively.

Online coupling of hydrophilic interaction/strong cation exchange/reversed-phase liquid chromatography with porous graphitic carbon liquid chromatography for simultaneous proteomics and N-glycomics analysis

In this study we developed a fully automated three-dimensional (3D) liquid chromatography methodology-comprising hydrophilic interaction separation as the first dimension, strong cation exchange fractionation as the second dimension, and low-pH reversed-phase (RP) separation as the third dimension-in conjunction downstream with additional complementary porous graphitic carbon separation, to capture non-retained hydrophilic analytes, for both shotgun proteomics and N-glycomics analyses. The performance of the 3D system alone was benchmarked through the analysis of the total lysate of Saccharomyces cerevisiae, leading to improved hydrophilic peptide coverage, from which we identified 19% and 24% more proteins and peptides, respectively, relative to those identified from a two-dimensional hydrophilic interaction liquid chromatography and low-pH RP chromatography (HILIC-RP) system over the same mass spectrometric acquisition time; consequently, the 3D platform also provided enhanced proteome and protein coverage. When we applied the integrated technology to analyses of the total lysate of primary cerebellar granule neurons, we characterized a total of 2201 proteins and 16,937 unique peptides for this primary cell line, providing one of its most comprehensive datasets. Our new integrated technology also exhibited excellent performance in the first N-glycomics analysis of cynomolgus monkey plasma; we successfully identified 122 proposed N-glycans and 135 N-glycosylation sites from 122 N-glycoproteins, and confirmed the presence of 38 N-glycolylneuraminic acid-containing N-glycans, a rare occurrence in human plasma, through tandem mass spectrometry for the first time.

Discovery, Synthesis, and Functional Characterization of a Novel Neuroprotective Natural Product from the Fruit of Alpinia oxyphylla for use in Parkinson’s Disease Through LC/MS-Based Multivariate Data Analysis-Guided Fractionation

Herein we report the discovery of a novel lead compound, oxyphylla A [(R)-4-(2-hydroxy-5-methylphenyl)-5-methylhexanoic acid] (from the fruit of Alpinia oxyphylla), which functions as a neuroprotective agent against Parkinsons disease. To identify a shortlist of candidates from the extract of A. oxyphylla, we employed an integrated strategy combining liquid chromatography/mass spectrometry, bioactivity-guided fractionation, and chemometric analysis. The neuroprotective effects of the shortlisted candidates were validated prior to scaling up the finalized list of potential neuroprotective constituents for more detailed chemical and biological characterization. Oxyphylla A has promising neuroprotective effects: (i) it ameliorates in vitro chemical-induced primary neuronal cell damage and (ii) alleviates chemical-induced dopaminergic neuron loss and behavioral impairment in both zebrafish and mice in vivo. Quantitative proteomics analyses of oxyphylla A-treated primary cerebellar granule neurons that had been intoxicated with 1-methyl-4-phenylpyridinium revealed that oxyphylla A activates nuclear factor-erythroid 2-related factor 2 (NRF2)-a master redox switch-and triggers a cascade of antioxidative responses. These observations were verified independently through western blot analyses. Our integrated metabolomics, chemometrics, and pharmacological strategy led to the efficient discovery of novel bioactive ingredients from A. oxyphylla while avoiding the nontargeting, labor-intensive steps usually required for identification of bioactive compounds. Our successful development of a synthetic route toward oxyphylla A should lead to its availability on a large scale for further functional development and pathological studies.

Comparative and quantitative proteomics reveal the adaptive strategies of oyster larvae to ocean acidification.

Decreasing pH due to anthropogenic CO2 inputs, called ocean acidification (OA), can make coastal environments unfavorable for oysters. This is a serious socioeconomical issue for China which supplies >70% of the worlds edible oysters. Here, we present an iTRAQ‐based protein profiling approach for the detection and quantification of proteome changes under OA in the early life stage of a commercially important oyster, Crassostrea hongkongensis. Availability of complete genome sequence for the pacific oyster (Crassostrea gigas) enabled us to confidently quantify over 1500 proteins in larval oysters. Over 7% of the proteome was altered in response to OA at pHNBS 7.6. Analysis of differentially expressed proteins and their associated functional pathways showed an upregulation of proteins involved in calcification, metabolic processes, and oxidative stress, each of which may be important in physiological adaptation of this species to OA. The downregulation of cytoskeletal and signal transduction proteins, on the other hand, might have impaired cellular dynamics and organelle development under OA. However, there were no significant detrimental effects in developmental processes such as metamorphic success. Implications of the differentially expressed proteins and metabolic pathways in the development of OA resistance in oyster larvae are discussed. The MS proteomics data have been deposited to the ProteomeXchange with identifiers PXD002138 (http://proteomecentral.proteomexchange.org/dataset/PXD002138).