Met Residues Research Articles

Influence of Met/Leu amino acid changes on catalytic properties and oxidative and thermal stability of yeast D-amino acid oxidase

The oxidative stability of enzymes is mostly dependent on the stability of the Cys and Met residues. Three single point mutants with Met/Leu substitutions in D-amino acid oxidase (DAAO, EC 1.4.3.3) from the yeast Trigonopsis variabilis (TvDAAO) are prepared and characterized. The positions for the amino acid residue substitutions are selected based on multiple alignment of different DAAO amino acid sequences and analysis of the three-dimensional structure of TvDAAO. It is shown that the substrate specificity profile ischanged for all of the mutants. The KM values for the small and bulky D-amino acidsare increased and decreased, respectively. One of the Met/Leu substitutions results in a two- to threefold increase in thermal stability as compared to the wild-type enzyme. A method for the determination of TvDAAO stability in the presence of hydrogen peroxide is developed and the oxidative stability of wild-type and mutant TvDAAOs is studied. It is shown that none of thethree mutations changes the oxidative stability of the enzymes.

Spectroscopic and Theoretical Study of Cu(I) Binding to His111 in the Human Prion Protein Fragment 106-115.

The ability of the cellular prion protein (PrPC) to bind copper in vivo points to a physiological role for PrPC in copper transport. Six copper binding sites have been identified in the nonstructured N-terminal region of human PrPC. Among these sites, the His111 site is unique in that it contains a MKHM motif that would confer interesting CuI and CuII binding properties. We have evaluated CuI coordination to the PrP(106–115) fragment of the human PrP protein, using NMR and X-ray absorption spectroscopies and electronic structure calculations. We find that Met109 and Met112 play an important role in anchoring this metal ion. CuI coordination to His111 is pH-dependent: at pH >8, 2N1O1S species are formed with one Met ligand; in the range of pH 5–8, both methionine (Met) residues bind to CuI, forming a 1N1O2S species, where N is from His111 and O is from a backbone carbonyl or a water molecule; at pH <5, only the two Met residues remain coordinated. Thus, even upon drastic changes in the chemical environment, such as those occurring during endocytosis of PrPC (decreased pH and a reducing potential), the two Met residues in the MKHM motif enable PrPC to maintain the bound CuI ions, consistent with a copper transport function for this protein. We also find that the physiologically relevant CuI-1N1O2S species activates dioxygen via an inner-sphere mechanism, likely involving the formation of a copper(II) superoxide complex. In this process, the Met residues are partially oxidized to sulfoxide; this ability to scavenge superoxide may play a role in the proposed antioxidant properties of PrPC. This study provides further insight into the CuI coordination properties of His111 in human PrPC and the molecular mechanism of oxygen activation by this site.

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

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

Structure–activity relationship of antioxidant dipeptides: Dominant role of Tyr, Trp, Cys and Met residues

Antioxidant activities of amino acids and synthetic dipeptides were studied to elucidate the structure–activity relationship of antioxidant dipeptides. Results showed that Tyr- and Trp-containing dipeptides showed the highest radical scavenging activities in both ABTS and ORAC assays with Trolox equivalent (TE) values ranging from 0.69 to 4.97 µmol TE/µmol peptide, followed by Cys- and Met-containing dipeptides. With respect to their reducing power and Fe2+ chelating abilities, only Cys-containing dipeptides showed moderate reducing power, and none of them displayed Fe2+chelating ability. It indicated that antioxidant dipeptides mainly acted as radical scavengers and the presence of Tyr, Trp, Cys or Met residue with electron/hydrogen donating ability was the driving force for dipeptides to scavenge radicals. Additionally, Tyr- and Trp-containing dipeptides with Tyr/Trp residue at the N-terminus showed stronger antioxidant activities than that at the C-terminus, and the neighboring residue also affected their activities by steric effect, hydrophobicity and hydrogen bonding among others.

Abstract 17135: Distribution of Anti-oxidative Activity During Remodeling of High Density Lipoprotein: Studies With CSL112

We have previously shown that reconstituted HDL has the ability to inactivate phospholipid hydroperoxides (PLOOH) in oxidized LDL by virtue of redox-active Met residues present in apolipoprotein A-I (apoA-I), with the level of activity being proportional to the amount of apoA-I protein added. CSL112 is human apoA-I, reconstituted with phosphatidylcholine to form HDL particles suitable for infusion. Infusing CSL112 into human subjects or adding it to human plasma ex vivo, causes remodeling of endogenous HDL. Similar remodeling occurs upon incubation of CSL112 with purified HDL3 and results in accumulation of three HDL species: enlarged HDL (HDL2), a smaller, dense species (HDL3c-like), and lipid-poor apoA-I (pre-β1 HDL). Here we examine the distribution of anti-oxidant activity upon remodeling of native plasma HDL induced by CSL112. We incubated CSL112 with human plasma HDL3, isolated the three populations of remodeled HDL particles by density gradient ultracentrifugation and compared (on a total protein basis) their ability to inactivate PLOOH derived from oxidized LDL as well as their apoA-I content of unoxidized and oxidized Met residues. Both parent CSL112 and HDL3 displayed anti-oxidative activity, inactivating 22±4 and 47±9% of PLOOH, respectively (n=4). Upon remodeling, this activity was mainly found in enlarged HDL2 and smaller HDL3c-like species (PLOOH inactivation, 46±3 and 47±9%, respectively) and was low (23±17%) in lipid-poor apoA-I. In parallel, apoA-I Met residues were oxidized in HDL; oxidation was several fold lower in the lipid-poor apoA-I (12±3%) compared to larger species (62±1% and 69±2% in HDL2 and HDL3c-like particles), consistent with a limited accessibility of apoA-I Met to PLOOH molecules in the lipid-poor particles. These studies further confirm the unequal distribution of HDL function among HDL subclasses and emphasize the important role of HDL remodeling in the production and maintenance of HDL functionality. The antioxidant properties of CSL112 add to its potential as a promising therapy for reducing the high risk of early recurrent atherothrombotic events following acute MI (AMI). A Phase IIb trial (AEGIS-I; NCT02108262) of CSL112 in AMI patients is ongoing.

Point Mutation Ile137-Met Near Surface Conferred Psychrophilic Behaviour and Improved Catalytic Efficiency to Bacillus Lipase of 1.4 Subfamily.

Bacillus lipolytic enzymes of subfamily 1.4 are industrially attractive because of its alkaline optimum pH and broad substrate specificity. The activity and stability of these enzymes for a limited temperature range (30-50 °C) need attention for its industrial application. In the present study, Bacillus subtilis LipJ was rationally designed for low-temperature adaptation. Small amino acids with lower volume and without side chain branches have high occurrence among psychrophilic proteins. Met residue is reported to be preferred for cold adaptation as it is thermolabile in nature and undergoes oxidation at high temperature. Therefore, the Ile137 residue, three residues downstream the catalytic residue Asp133, was substituted by Met. Biochemical study demonstrated that variant Ile137Met was optimally active at 20 °C whereas parent enzyme was most active at 37 °C. The variant retained 70-80% relative activity at 10 °C where parent enzyme demonstrated low activity. Ile137Met was observed to be unstable at and above 30 °C. Kinetic study demonstrated increased K m and k cat values for variant referring improved catalytic efficiency but poor substrate affinity. Homolog modelling predicted lowered number of weak interactions by substituted Met137 as molecular basis of increased flexibility of variant. Hence, increased structure flexibility might be responsible for poor substrate affinity but increased molecular motion for higher catalysis at cold.

Development of a catalytically stable and efficient lipase through an increase in hydrophobicity of the oxyanion residue

In-silico and empirical site-directed substitutions of oxyanion Q114 of the wildtype T1 lipase with that of hydrophobic Leu and Met residues were carried out to afford the Q114L and Q114M lipases, respectively. Using the esterification production of menthyl butyrate as a reaction model, evaluation on the catalytic efficiency of the three lipases was performed. It was found that Leu evidently improved the catalytic activity of the Q114L lipase, achieving the highest conversions of menthyl butyrate under varying experimental conditions that may be attributable to its lower total energy when compared with both the T1 and Q114M lipases. The diminishing catalytic activity of T1 lipase observable following substitution with Met (Q114M lipase) may be due to formation of three additional cavities within the vicinity of the mutation, lesser density of the protein core and susceptibility to high temperature, particularly under prolonged reaction time. Therefore, it can be concluded that the substitution of Leu into the oxyanion-114 had rendered favorable structural changes that enhanced the catalytic activity of the T1 lipase, envisaging that such approach may also be of applied value for improving the catalytic activity of other bacterial lipases within the I.5 family.

Methyl Relaxation Measurements Reveal Patterns of Fast Dynamics in a Viral RNA-Directed RNA Polymerase.

Molecular dynamics (MD) simulations combined with biochemical studies have suggested the presence of long-range networks of functionally relevant conformational flexibility on the nanosecond time scale in single-subunit RNA polymerases in many RNA viruses. However, experimental verification of these dynamics at a sufficient level of detail has been lacking. Here we describe the fast, picosecond to nanosecond dynamics of an archetypal viral RNA-directed RNA polymerase (RdRp), the 75 kDa P2 protein from cystovirus ϕ12, using analyses of (1)H-(1)H dipole-dipole cross-correlated relaxation at the methyl positions of Ile (δ1), Leu, Val, and Met residues. Our results, which represent the most detailed experimental characterization of fast dynamics in a viral RdRp until date, reveal a highly connected dynamic network as predicted by MD simulations of related systems. Our results suggest that the entry portals for template RNA and substrate NTPs are relatively disordered, while conserved motifs involved in metal binding, nucleotide selection, and catalysis display greater rigidity. Perturbations at the active site through metal binding or functional mutation affect dynamics not only in the immediate vicinity but also at remote regions. Comparison with the limited experimental and extensive functional and in silico results available for homologous systems suggests conservation of the overall pattern of dynamics in viral RdRps.

OP46 - Superoxide-mediated post-translational modification of tyrosine residues

Superoxide is a byproduct of aerobic respiration. Its in vivo formation is associated with many inflammatory diseases, oxidative stress and aging. Regardless of the rapid spontaneous disproportion of superoxide to give hydrogen peroxide and oxygen, aerobic organisms cannot survive without further catalyzing the rate of this reaction by superoxide dismutase. Therefore, chemical reactions, which contribute to the detrimental effects of superoxide, have to be fast enough to compete with the catalytic dismutation process. The reaction of superoxide with tyrosyl radicals fulfills this criterion and we have shown in a series of publications (see below) that radical addition reactions to tyrosine residues may represent a possible mechanism for superoxide toxicity. Tyr residues are major sites of radical attack on proteins and they also form at active sites of pivotal enzymes (such as cyclo-oxygenase or ribonucleotide reductase) during turnover. This presentation will focus on the mechanisms of the reaction of superoxide with tyrosyl radicals in peptides and proteins in a wide range of model systems. We will present that addition of superoxide to tyrosyl radicals result in the formation of novel Tyr-hydroperoxide species, which can further react with 1) Met residues via intramolecular oxygen transfer to form Met sulfoxide or 2) Cys residues to generate the corresponding disulfide species. These reactions produce a modified bicyclic Tyr monoxide species, which favorably undergo a Michael addition reaction with thiols (such as glutathione) to form novel Tyr-Cys cross-linked adducts.

Turning Escherichia coli into a Frataxin-Dependent Organism.

Fe-S bound proteins are ubiquitous and contribute to most basic cellular processes. A defect in the ISC components catalyzing Fe-S cluster biogenesis leads to drastic phenotypes in both eukaryotes and prokaryotes. In this context, the Frataxin protein (FXN) stands out as an exception. In eukaryotes, a defect in FXN results in severe defects in Fe-S cluster biogenesis, and in humans, this is associated with Friedreich’s ataxia, a neurodegenerative disease. In contrast, prokaryotes deficient in the FXN homolog CyaY are fully viable, despite the clear involvement of CyaY in ISC-catalyzed Fe-S cluster formation. The molecular basis of the differing importance in the contribution of FXN remains enigmatic. Here, we have demonstrated that a single mutation in the scaffold protein IscU rendered E. coli viability strictly dependent upon a functional CyaY. Remarkably, this mutation changed an Ile residue, conserved in prokaryotes at position 108, into a Met residue, conserved in eukaryotes. We found that in the double mutant IscUIM ΔcyaY, the ISC pathway was completely abolished, becoming equivalent to the ΔiscU deletion strain and recapitulating the drastic phenotype caused by FXN deletion in eukaryotes. Biochemical analyses of the “eukaryotic-like” IscUIM scaffold revealed that it exhibited a reduced capacity to form Fe-S clusters. Finally, bioinformatic studies of prokaryotic IscU proteins allowed us to trace back the source of FXN-dependency as it occurs in present-day eukaryotes. We propose an evolutionary scenario in which the current mitochondrial Isu proteins originated from the IscUIM version present in the ancestor of the Rickettsiae. Subsequent acquisition of SUF, the second Fe-S cluster biogenesis system, in bacteria, was accompanied by diminished contribution of CyaY in prokaryotic Fe-S cluster biogenesis, and increased tolerance to change in the amino acid present at the 108th position of the scaffold.

Hypochlorous Acid Generated by Neutrophils Inactivates ADAMTS13: AN OXIDATIVE MECHANISM FOR REGULATING ADAMTS13 PROTEOLYTIC ACTIVITY DURING INFLAMMATION

ADAMTS13 is a plasma metalloproteinase that cleaves large multimeric forms of von Willebrand factor (VWF) to smaller, less adhesive forms. ADAMTS13 activity is reduced in systemic inflammatory syndromes, but the cause is unknown. Here, we examined whether neutrophil-derived oxidants can regulate ADAMTS13 activity. We exposed ADAMTS13 to hypochlorous acid (HOCl), produced by a myeloperoxidase-H2O2-Cl(-) system, and determined its residual proteolytic activity using both a VWF A2 peptide substrate and multimeric plasma VWF. Treatment with 25 nm myeloperoxidase plus 50 μm H2O2 reduced ADAMTS13 activity by >85%. Using mass spectrometry, we demonstrated that Met(249), Met(331), and Met(496) in important functional domains of ADAMTS13 were oxidized to methionine sulfoxide in an HOCl concentration-dependent manner. The loss of enzyme activity correlated with the extent of oxidation of these residues. These Met residues were also oxidized in ADAMTS13 exposed to activated human neutrophils, accompanied by reduced enzyme activity. ADAMTS13 treated with either neutrophil elastase or plasmin was inhibited to a lesser extent, especially in the presence of plasma. These observations suggest that oxidation could be an important mechanism for ADAMTS13 inactivation during inflammation and contribute to the prothrombotic tendency associated with inflammation.

Membranous adenylyl cyclase 1 activation is regulated by oxidation of N- and C-terminal methionine residues in calmodulin

Membranous adenylyl cyclase 1 (AC1) is associated with memory and learning. AC1 is activated by the eukaryotic Ca2+-sensor calmodulin (CaM), which contains nine methionine residues (Met) important for CaM-target interactions. During ageing, Met residues are oxidized to (S)- and (R)-methionine sulfoxide (MetSO) by reactive oxygen species arising from an age-related oxidative stress. We examined how oxidation by H2O2 of Met in CaM regulates CaM activation of AC1. We employed a series of thirteen mutant CaM proteins never assessed before in a single study, where leucine is substituted for Met, in order to analyze the effects of oxidation of specific Met. CaM activation of AC1 is regulated by oxidation of all of the C-terminal Met in CaM, and by two N-terminal Met, M36 and M51. CaM with all Met oxidized is unable to activate AC1. Activity is fully restored by the combined catalytic activities of methionine sulfoxide reductases A and B (MsrA and B), which catalyze reduction of the (S)- and (R)-MetSO stereoisomers. A small change in secondary structure is observed in wild-type CaM upon oxidation of all nine Met, but no significant secondary structure changes occur in the mutant proteins when Met residues are oxidized by H2O2, suggesting that localized polarity, flexibility and structural changes promote the functional changes accompanying oxidation. The results signify that AC1 catalytic activity can be delicately adjusted by mediating CaM activation of AC1 by reversible Met oxidation in CaM. The results are important for memory, learning and possible therapeutic routes for regulating AC1.

A Cisplatin Derivative that Inhibits Collagen Fibril‐Formation in vitro

Using an in vitro random screening of small-molecule compounds, we discovered cis-diamminedichloroplatinum(II) (cisplatin), an anticancer agent, as a potential inhibitor of collagen fibril-formation. The inhibitory effect was found only when cisplatin was dissolved in dimethylsulphoxide (DMSO), indicating that the active species were cisplatin derivatives formed in the DMSO solution. The cisplatin derivatives inhibited the formation of collagen fibrils in vitro without affecting the triple-helical conformation of the collagen molecules. Incubation with the cisplatin solution in DMSO also inhibited in situ deposition of collagen fibrils in a human umbilical vein endothelial cell (HUVEC) culture. In addition, the derivatization of cisplatin in DMSO abolished the cytotoxicity of the original compound. The platinum complex was further revealed to interact with specific sites on the collagen triple helix, and the binding sites were suggested to contain His and/or Met residues. Mass spectrometry analysis of the cisplatin solution in DMSO and a structure-activity relationship study strongly suggested that the active compound is [Pt(NH3 )2 (Cl)(DMSO)](+) . This platinum complex will be useful for investigating molecular mechanisms of collagen self-assembly and for drug development for the treatment of fibrotic diseases.

Structure of mouse Clr-g, a CTL ligand for NK receptor NKR-P1F

Natural killer (NK) cells are large granular lymphocytes with innate immune reaction against tumor cells or cells affected by viral infection. They have a variety of receptors on their surface which mediate contact with cells tested by NK cells. Using X-ray crystallography, we determined a structure of an extracellular part of mouse C-type lectin related protein g (Clr-g, [1], PDB code 3RS1), a ligand for NK receptor NKR-P1F. The ligand and the receptor are both of C-type lectin like fold and this is the first determined structure of a CTL ligand of an NK receptor. The protein was produced in E. coli. The rod-like crystals appeared by spontaneous crystallization of the pure protein (2.5 mg/ml) on tube walls. Diffraction data of the vitrified crystal were measured at synchrotron BessyII of HZB in Berlin and were processed up to 1.95 Å. The protein was found in the form of dimers similar to that of CD69. The N-terminus of the chain (residues Met, Asn, Lys) is in the crystal bound to a neighbor dimer and shows thus that binding of a peptide to mouse Clr-g is possible, although not expected or confirmed by other experiments known to us. A model of interaction of Clr-g with NKR-P1F was designed based on electrostatic complementarity of both molecules. This work was supported by the Czech Science Foundation (grant No. P302/11/0855), Ministry of Education, Youth and Sports of the Czech Republic (grant No. LG14009) and by the project BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF.

Mass spectrometric strategies to improve the identification of Pt(II)-modification sites on peptides and proteins.

To further explore the binding chemistry of cisplatin (cis-Pt(NH3)2Cl2) to peptides and also establish mass spectrometry (MS) strategies to quickly assign the platinum-binding sites, a series of peptides with potential cisplatin binding sites (Met(S), His(N), Cys(S), disulfide, carboxyl groups of Asp and Glu, and amine groups of Arg and Lys, were reacted with cisplatin, then analyzed by electron capture dissociation (ECD) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS). Radical-mediated side-chain losses from the charge-reduced Pt-binding species (such as CH3S(•) or CH3SH from Met, SH(•) from Cys, CO2 from Glu or Asp, and NH2(•) from amine groups) were found to be characteristic indicators for rapid and unambiguous localization of the Pt-binding sites to certain amino acid residues. The method was then successfully applied to interpret the top-down ECD spectrum of an inter-chain Pt-crosslinked insulin dimer, insulin + Pt(NH3)2 + insulin (>10 kDa). In addition, ion mobility MS shows that Pt binds to multiple sites in Substance P, generating multiple conformers, which can be partially localized by collisionally activated dissociation (CAD). Platinum(II) (Pt(II)) was found to coordinate to amine groups of Arg and Lys, but not to disulfide bonds under the conditions used. The coordination of Pt to Arg or Lys appears to arise from the migration of Pt(II) from Met(S) as shown by monitoring the reaction products at different pH values by ECD. No direct binding of cisplatin to amine groups was observed at pH 3 ~ 10 unless Met residues were present in the sequence, but noncovalent interactions between cisplatin hydrolysis and amination [Pt(NH3)4](2+) products and these peptides were found regardless of pH.

UV-induced selective oxidation of Met5 to Met-sulfoxide leads to the formation of neurotoxic fibril-incompetent α-synuclein oligomers

Oxidative stress and the formation of cytotoxic aggregates of the presynaptic protein α-synuclein (AS) are two important events associated with the pathogenesis of Parkinson's disease (PD) and several other neurodegenerative diseases. In this context, extensive efforts have been made to elucidate the molecular basis of the cytotoxic synergy between oxidative stress and AS aggregation. In this study, we demonstrate that the exposure of AS to oxidative stress induced by UV radiation (ASUV) blocks the protein fibrillation, leading to the formation of highly toxic fibril-incompetent oligomers. In addition, ASUV exhibited stronger anti-fibrillogenic properties than H2O2-treated AS, inhibiting the fibrillation of unmodified AS at notably low concentrations. Mass spectrometry indicated that Met5 oxidation to Met-sulfoxide was the only modification promoted by UV exposure, which is reinforced by NMR data indicating that Met5 is the only residue whose amide resonance completely disappeared from the 1H-15N HSQC spectrum after UV exposure. This result is supported by previous data that indicate that C-terminal Met residues (Met116 and Met127) and N-terminal Met1 are less susceptible to oxidation than Met5 because of the residual structure of the disordered AS monomer. Overall, our findings suggest that specific oxidation of Met5 might be sufficient to promote the formation of highly neurotoxic oligomers of AS.

Site-specific copper-catalyzed oxidation of α-synuclein: tightening the link between metal binding and protein oxidative damage in Parkinson's disease.

Amyloid aggregation of α-synuclein (AS) has been linked to the pathological effects associated with Parkinson's disease (PD). Cu(II) binds specifically at the N-terminus of AS and triggers its aggregation. Site-specific Cu(I)-catalyzed oxidation of AS has been proposed as a plausible mechanism for metal-enhanced AS amyloid formation. In this study, Cu(I) binding to AS was probed by NMR spectroscopy, in combination with synthetic peptide models, site-directed mutagenesis, and C-terminal-truncated protein variants. Our results demonstrate that both Met residues in the motif (1)MDVFM(5) constitute key structural determinants for the high-affinity binding of Cu(I) to the N-terminal region of AS. The replacement of one Met residue by Ile causes a dramatic decrease in the binding affinity for Cu(I), whereas the removal of both Met residues results in a complete lack of binding. Moreover, these Met residues can be oxidized rapidly after air exposure of the AS-Cu(I) complex, whereas Met-116 and Met-127 in the C-terminal region remain unaffected. Met-1 displays higher susceptibility to oxidative damage compared to Met-5 because it is directly involved in both Cu(II) and Cu(I) coordination, resulting in closer exposure to the reactive oxygen species that may be generated by the redox cycling of copper. Our findings support a mechanism where the interaction of AS with copper ions leads to site-specific metal-catalyzed oxidation in the protein under physiologically relevant conditions. In light of recent biological findings, these results support a role for AS-copper interactions in neurodegeneration in PD.

Endogenous and synthetic MMP inhibitors in CNS physiopathology.

Matrix metalloproteinases (MMPs, including the membrane-type MMPs (MT-MMPs)), a disintegrin and metalloproteinase (ADAM), and ADAM with thrombospondin motifs belong to the metzincins, a subclass of metalloproteinases that contain a Met residue and a Zn(2+) ion at the catalytic site necessary for enzymatic reaction. MMP proteolytic activity is mainly controlled by their natural tissue inhibitors of metalloproteinase (TIMP). A number of synthetic inhibitors have been developed to control deleterious MMP activity. The roles of MMPs and some of their ECM substrates in CNS physiology and pathology are covered by other chapters of the present volume and will thus not be addressed in depth. This chapter will focus (i) on the endogenous MMP inhibitors in the CNS, (ii) on MMP and TIMP regulations in three large classes of neuropathologic processes (inflammatory, neurodegenerative, and infectious), and (iii) on synthetic inhibitors of MMPs and the perspective of their use in different brain diseases.

Promiscuous methionyl-tRNA synthetase mediates adaptive mistranslation to protect cells against oxidative stress.

ABSTRACTAminoacyl-tRNA synthetases (ARSs) acylate transfer (t)RNAs with amino acids. Charging tRNAs with the right amino acids is the first step in translation; therefore, the accurate and error-free functioning of ARSs is an essential prerequisite for translational fidelity. A recent study found that methionine (Met) can be incorporated into non-Met residues of proteins through methionylation of non-cognate tRNAs under conditions of oxidative stress. However, it was not understood how this mis-methionylation is achieved. Here, we report that methionyl-tRNA synthetase (MRS) is phosphorylated at Ser209 and Ser825 by extracellular signal-related kinase (ERK1/2) under conditions of stress caused by reactive oxygen species (ROS), and that this phosphorylated MRS shows increased affinity for non-cognate tRNAs with lower affinity for tRNAMet, leading to an increase in Met residues in cellular proteins. The expression of a mutant MRS containing the substitutions S209D and S825D, mimicking dual phosphorylation, reduced ROS levels and cell death. This controlled inaccuracy of MRS seems to serve as a defense mechanism against ROS-mediated damage at the cost of translational fidelity.

Circles within circles: crosstalk between protein Ser/Thr/Tyr-phosphorylation and Met oxidation

BackgroundReversible posttranslational protein modifications such as phosphorylation of Ser/Thr/Tyr and Met oxidation are critical for both metabolic regulation and cellular signalling. Although these modifications are typically studied individually, herein we describe the potential for cross-talk and hierarchical regulation.ResultsThe proximity of Met to Ser/Thr/Tyr within the proteome has not previously been addressed. In order to consider the possibility of a generalized interaction, we performed a trans-kingdom sequence analysis of known phosphorylation sites in proteins from bacteria, fungi, plants, and animals. The proportion of phosphorylation sites that include a Met within a 13-residue window centered upon Ser/Thr/Tyr is significantly less than the occurrence of Met in proximity to all Ser/Thr/Tyr residues. Met residues are present at all positions (-6 to +6, inclusive) within the 13-residue window that we have considered. Detailed analysis of sequences from eight disparate plant taxa revealed that many conserved phosphorylation sites have a Met residue in the proximity. Results from GO enrichment analysis indicated that the potential for phosphorylation and Met oxidation crosstalk is most prevalent in kinases and proteins involved in signalling.ConclusionThe large proportion of known phosphorylation sites with Met in the proximity fulfils the necessary condition for cross-talk. Kinases/signalling proteins are enriched for Met around phosphorylation sites. These proteins/sites are likely candidates for cross-talk between oxidative signalling and reversible phosphorylation.