Effect Of Methionine Oxidation Research Articles

Methionine oxidation selectively enhances T cell reactivity against a melanoma antigen

The impact of the peptide amino acids side-chain modifications on the immunological recognition has been scarcely explored. We investigate here the effect of methionine oxidation on the antigenicity of the melanoma immunodominant peptide 369-YMDGTMSQV-377 (YMD). Using CD8+ Tcell activation assays, we found that the antigenicity of the sulfoxide form is higher when compared to the YMD peptide. This is consistent with free energy computations performed on HLA-A∗02:01/YMD/TCR complex showing that this is lowered upon oxidation, paired with a steep increase in order at atomic level. Oxidized YMD forms were identified at the melanoma cell surface by LC-MS/MS analysis. These results demonstrate that methionine oxidation in the antigenic peptides may generate altered peptide ligands with increased antigenicity, and that this oxidation may occur invivo, opening up the possibility that high-affinity CD8+ Tcells might be naturally primed in the course of melanoma progression, as a result of immunosurveillance.

On the effect of methionine oxidation on the interplay between α-synuclein and synaptic-like vesicles

Human alpha-synuclein (αS) is an intrinsically disordered protein highly expressed in dopaminergic neurons. Its amyloid aggregates are the major component of Lewy bodies, which are considered a hallmark of Parkinson's disease (PD). αS has four different Met, which are particularly sensitive to oxidation, as most of them are found as Met sulfoxide (MetO) in the αS deposits. Consequently, researchers have invested mounting efforts trying to elucidate the molecular mechanisms underlying the links between oxidative stress, αS aggregation and PD. However, it has not been described yet the effect of Met oxidation on the physiological function of αS. Trying to shed light on this aspect, we have here studied a synthetic αS that displayed all its Met replaced by MetO moieties (αS-MetO). Our study has allowed to prove that MetO diminishes the affinity of αS towards anionic micelles (SDS), although the micelle-bound fraction of αS-MetO still adopts an α-helical folding resembling that of the lipid-bound αS. MetO also diminishes the affinity of αS towards synaptic-like vesicles, and its hindering effect is much more pronounced than that displayed on the αS-micelle affinity. Additionally, we have also demonstrated that MetO impairs the physiological function of αS as a catalyst of the clustering and the fusion of synaptic vesicles (SVs). Our findings provide a new understanding on how Met oxidation affects one of the most relevant biological functions attributed to αS that is to bind and cluster SVs along the neurotransmission.

Methionine oxidation accelerates the aggregation and enhances the neurotoxicity of the D178N variant of the human prion protein

The D178N mutation of the prion protein (PrP) results in the hereditary prion disease fatal familial insomnia (FFI). Little is known regarding the effects of methionine oxidation on the pathogenesis of D178N-associated FFI. In the present study, we found that the D178N variant was more susceptible to oxidation than wild-type PrP, as indicated by reverse-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry (MS) analysis. Circular dichroism (CD), differential scanning calorimetry (DSC), thioflavin T (ThT) binding assay studies demonstrated that methionine oxidation decreased the structural stability of the D178N variant, and the oxidized D178N variant exhibited a greater propensity to form β-sheet-rich oligomers and aggregates. Moreover, these aggregates of oxidized D178N PrP were more resistant to proteinase K (PK) digestion. Additionally, using fluorescence confocal microscopy, we detected a high degree of aggregation in D178N-transfected Neuro-2a (N2a) cells after treatment with hydrogen peroxide (H2O2). Furthermore, the oxidation and consequent aggregation of the D178N variant induced greater apoptosis of N2a cells, as monitored using flow cytometry. Collectively, these observations suggest that methionine oxidation accelerates the aggregation and enhances the neurotoxicity of the D178N variant, possibly providing direct evidence to link the pathogenesis of D178N-associated FFI with methionine oxidation.

Novel Cyclotides and Uncyclotides with Highly Shortened Precursors from Chassalia chartacea and Effects of Methionine Oxidation on Bioactivities

Cyclotides are a new class of plant biologics that display a diverse range of bioactivities with therapeutic potentials. They possess an unusual end-to-end cyclic backbone combined with a cystine knot arrangement, making them exceptionally stable to heat, chemical and enzymatic degradation. Currently, >200 cyclotides have been discovered but only three naturally occurring linear variants (also known as uncyclotides) have been isolated. In this study, we report the discovery of 18 novel peptides, chassatides C1 to C18, composed of 14 new cyclotides and four uncyclotides from Chassalia chartacea (Rubiaceae family). Thus far, this is the largest number of uncyclotides being reported in a single species. Activity testing showed that the uncyclotides not only retain the effectiveness but also are the most potent chassatides in the assays for antimicrobial, cytotoxic, and hemolytic activities. Genetic characterization of novel chassatides revealed that they have the shortest precursors of all known cyclotides hitherto isolated, which represents a new class of cyclotide precursors. This is the first report of cyclotide genes in a second genus, the Chassalia, other than the Hedyotis (Oldenlandia) of the Rubiaceae family. In addition, we also report the characterization of two Met-oxidized derivatives of chassatides C2 and C11. The oxidation of Met residue causes loss of bioactivities, strengthening the importance of the hydrophobic patch for membrane interaction.

Enhanced physical stability of human calcitonin after methionine oxidation

Calcitonin is a blood-calcium-lowering peptide, present in different species, which inhibits the resorption of bone by osteoclasts. Human calcitonin (hCT) is one of the few calcitonin peptides, which contains a methionine residue; this residue is in position 8. Methionines are known to be readily oxidized to sulfoxides both in vivo and in vitro. The current work describes the effect of methionine oxidation on the physical stability of hCT. Aggregation kinetics of human calcitonin were studied at different pH values by intrinsic fluorescence spectroscopy, turbidity at 350 nm, microscopy analyses, Nile Red, and 1,8-ANS fluorescence emission. In all the experiments, methionine oxidation reduced the aggregation rate of human calcitonin. The effect of methionine oxidation was independent of pH. Fluorescence lifetime data also showed that the conformation of hCT in the aggregated state can be influenced by methionine oxidation. A hypothesis for the enhanced physical stability of oxidized hCT is presented and discussed.

Stability of Human Growth Hormone: Influence of Methionine Oxidation on Thermal Folding

Oxidation, particularly of methionine residues, is one of the major chemical degradations of proteins. In a previous publication we studied the conformation of recombinant human growth hormone (r-hGH) selectively oxidized at Met14 and Met125. Conformation of oxidized r-hGH was found not different from that of nonoxidized r-hGH. In this paper, the effect of methionine oxidation on the thermal stability of r-hGH folding was investigated. The thermally induced unfolding process of the oxidized and nonoxidized protein was measured by monitoring the circular dichroism signal at 220 nm. The melting temperatures (T(m)) of the oxidized and nonoxidized r-hGH forms were determined at different pHs and in the presence of salts often used in pharmaceutical formulations. The effect of the location of the oxidized Met residue in the protein and the percentage of oxidation were investigated. Our findings indicate that the monoxidation of the two most accessible methionine residues of r-hGH-Met14 and Met125 - has no effect on the protein conformation. However, oxidation of these residues to form sulfoxides does influence the thermal stability of the protein folding. The presence of the polar oxygen atom on the methionine sulfoxide group thermally destabilizes r-hGH folding. The effect (T(m)) depends upon pH, ionic strength, and the location of the oxidized methionine residues in the protein. The thermal melting of r-hGH and its oxidized products is a highly cooperative process. Methionine oxidation leads to a thermal destabilization of the whole protein folding and is not just a local destabilization.

Effect of Methionine Oxidation on the Structural Properties, Conformational Stability, and Aggregation of Immunoglobulin Light Chain LEN

Light chain amyloidoses arise from the overproduction and abnormal deposition of the immunoglobulin light chain in various organs. LEN is the variable domain of an immunoglobulin light chain originally isolated from the urine of a patient suffering from multiple myeloma, with no sign of renal dysfunction or amyloidosis. LEN was shown to form fibrils in vitro under mildly destabilizing conditions. In this work, we investigated the changes induced by methionine oxidation in the structural properties, conformational stability, and aggregation behavior of immunoglobulin light chain domain LEN. We established that LEN was well-protected from oxidation in its native state, but successful oxidation was achieved in the presence of 4 M GuHCl. Oxidation induced noticeable structural changes in LEN and destabilized this protein. The methionine-oxidized LEN preferred to form amorphous aggregates instead of fibrils. The results indicated that the LEN oxidation may play an important role in amorphous deposition of the protein, but not in its fibrillation.

Folding analysis of hormonal polypeptide calcitonins and the oxidized calcitonins using electrospray ionization mass spectrometry combined with H/D exchange

Conformational change of calcitonins has been examined by measuring the rate of hydrogen/deuterium (H/D) exchange in amino acids. Time dependent m/z shift caused by H/D exchange was monitored by electrospray ionization quadrupole ion trap mass spectrometry (ESI-QIT MS). The rate constants of H/D exchange were calculated from apparent first-order kinetics. The time course of H/D exchange exhibited two phases of faster and slower exchange. The smaller rate constant (k2) estimated from the slower H/D exchange was correlated with an alpha-helix content that reflected the folding state. The order of k2 values obtained for human calcitonin (hCT), porcine calcitonin (pCT), salmon calcitonin (sCT), and elcatonin (ECT) was hCT > pCT approximately sCT > ECT. Although the amino acid sequence of sCT is similar to that of ECT, their k2 values were considerably different. The results suggest that ECT is relatively rigid on the N-terminal side cyclic structure in the folded state. Further, the effect of methionine oxidation on k2 has been examined. In the oxidized pCT that possesses similar biological activity with the intact pCT, the k2 values obtained were nearly equal. The k2 of hCT increased via methionine oxidation, and the biological activity was weakened by oxidation. This suggested that methionine oxidation of hCT produced unfolding in the secondary structure and that oxidative unfolding of hCT led to the loss of biological activity. The results indicate that the H/D exchange rate constant may be used as an informative parameter to elucidate the relationship between the folded state and biological activity of polypeptides like calcitonins with secondary structure.

In vitro methionine oxidation of recombinant human leptin.

To investigate the role and importance of the four methionines in recombinant human leptin, and the effect of methionine oxidation in leptin structural stability and biological activity. Oxidized leptin derivatives were prepared in the presence of H2O2 and t-butylhydroperoxide, separated by RP-HPLC, and characterized by peptide mapping and LC/MS. Their biophysical and biological properties were studied. Six major species of oxidized leptins were detected: two mono-oxidized, one di-oxidized, two tri-oxidized, and one tetra-oxidized. Further oxidation at cystine disulfide was also detected. Kinetic analysis indicated that oxidation at Met1 and Met69 proceeded first and independently. In 48 mM t-butylhydroperoxide, the pseudo first-order rate constants, k1 and k69, were 1.5 x 10(-3) and 2.3 x 10(-4) min-1. No change in the secondary or tertiary structure was detected for Met1 mono-oxidized and Met1, Met69 di-oxidized leptins. The Met1 mono-oxidized leptin retained full potency as compared to native leptin. A slight decrease of thermostability and a significant loss of the in vitro bioactivity were observed for Met1, Met69 di-oxidized leptin. Both Met55 and Met137 were not oxidized in t-butylhydroperoxide but only in H2O2. They appeared to be much less accessible to oxidation and might interact with the hydrophobic core structure of the leptin molecule. The oxidation of leptin occurred in the order of Met1 > Met69 >> Met55 approximately Met137, and the importance for maintaining leptin structural integrity was Met55 approximately Met137 >> Met69 approximately Met1. Met69, but not Met1, plays a critical role in the protein stability and activity.

Effect of methionine oxidation and deletion of amino-terminal residues on the conformation of parathyroid hormone. Circular dichroism studies.

Circular dichroism (CD) studies of parathyroid hormone (PTH), its oxidized forms, and some fragments of the hormone are described. The CD spectrum of native PTH (84 amino acids) and the active fragment, 1-34 PTH, suggests that most of the secondary structure resides in the amino-terminal segment of this hormone. Oxidation of the methionine residue at position 18 has a small impact on secondary structure, whereas oxidation of the methionine at position 8 produces substantial changes. Oxidation of both methionines produces secondary structure changes that are greater than the sum of those seen upon oxidation of the individual methionines. The CD spectrum for the 3-34 fragment of PTH is identical to that of the 1-34 fragment, and that of the 7-34 fragment is only slightly different. The spectra of the 13-34 and 19-34 fragments are markedly altered from that of the 1-34 peptide, and those of the 9-84 and 19-84 fragments of native PTH are significantly different from the intact hormone. Computer-assisted estimates of secondary structure content, and difference spectra, were utilized to evaluate the secondary structure content of the peptides. These results suggest that residues 6-12 are important in formation of helical secondary structure and that a reverse turn may be important for the folding of PTH into a conformation with high affinity for receptors. Residues 1 and 2 appear to make no contribution to the secondary structure and may be directly involved in activation of receptors.