Convergent signaling pathways—interaction between methionine oxidation and serine/threonine/tyrosine O-phosphorylation

Oxidation of methionine (Met) to Met sulfoxide (MetSO) is a frequently found reversible posttranslational modification. It has been presumed that the major functional role for oxidation-labile Met residues is to protect proteins/cells from oxidative stress. However, Met oxidation has been established as a key mechanism for direct regulation of a wide range of protein functions and cellular processes. Furthermore, recent reports suggest an interaction between Met oxidation and O-phosphorylation. Such interactions are a potentially direct interface between redox sensing and signaling, and cellular protein kinase/phosphatase-based signaling. Herein, we describe the current state of Met oxidation research, provide some mechanistic insight into crosstalk between these two major posttranslational modifications, and consider the evolutionary significance and regulatory potential of this crosstalk.     

The structure of monoamine oxidase from Aspergillus niger provides a molecular context for improvements in activity obtained by directed evolution

Monoamine oxidase from Aspergillus niger (MAO-N) is a flavoenzyme that catalyses the oxidative deamination of primary amines. MAO-N has been used as the starting model for a series of directed evolution experiments, resulting in mutants of improved activity and broader substrate specificity, suitable for application in the preparative deracemisation of primary, secondary and tertiary amines when used as part of a chemoenzymatic oxidation-reduction cycle. The structures of a three-point mutant (Asn336Ser/Met348Lys/Ile246Met or MAO-N-D3) and a five-point mutant (Asn336Ser/Met348Lys/Ile246Met/Thr384Asn/Asp385Ser or MAO-N-D5) have been obtained using a multiple-wavelength anomalous diffraction experiment on a selenomethionine derivative of the truncated MAO-N-D5 enzyme. MAO-N exists as a homotetramer with a large channel at its centre and shares some structural features with human MAO B (MAO-B). A hydrophobic cavity extends from the protein surface to the active site, where a non-covalently bound flavin adenine dinucleotide (FAD) sits at the base of an ‘aromatic cage,’ the sides of which are formed by Trp430 and Phe466. A molecule of l-proline was observed near the FAD, and this ligand superimposed well with isatin, a reversible inhibitor of MAO-B, when the structures of MAO-N proline and MAO-B-isatin were overlaid. Of the mutations that confer the ability to catalyse the oxidation of secondary amines in MAO-N-D3, Asn336Ser reduces steric bulk behind Trp430 of the aromatic cage and Ile246Met confers greater flexibility within the substrate binding site. The two additional mutations, Thr384Asn and Asp385Ser, that occur in the MAO-N-D5 variant, which is able to oxidise tertiary amines, appear to influence the active-site environment remotely through changes in tertiary structure that perturb the side chain of Phe382, again altering the steric and electronic character of the active site near FAD. The possible implications of the change in steric and electronic environment caused by relevant mutations are discussed with respect to the improved catalytic efficiency of the MAO-N variants described in the literature.     

Mass spectrometry analysis of the human endosulfatase Hsulf-2

The human 6-O-endosulfatases HSulf-1 and -2 catalyze the region-selective hydrolysis of the 6-O-sulfate group of the glucosamine residues within sulfated domains of heparan sulfate, thereby ensuring a unique and original post-biosynthetic modification of the cell surface proteoglycans. While numerous studies point out the role of HSulf-2 in crucial physiological processes as well as in pathological conditions particularly in cancer, its structural organization in two chains and its functional properties remain poorly understood. In this study, we report the first characterization by mass spectrometry (MS) of HSulf-2. An average molecular weight of 133,115 Da was determined for the whole enzyme by MALDI-TOF MS, i.e. higher than the naked amino acid backbone (98,170 Da), highlighting a significant contribution of post-translational modifications. The HSulf-2 protein sequence was determined by Nano-LC-MS/MS, leading to 63% coverage and indicating at least four N-glycosylation sites at Asn 108, 147, 174 and 217. These results provide a platform for further structural investigations of the HSulf enzymes, aiming at deciphering the role of each chain in the substrate binding and specificities and in the catalytic activities.     

Amino acid residue penultimate to the amino-terminal gly residue strongly affects two cotranslational protein modifications, N-myristoylation and N-acetylation

To examine the amino-terminal sequence requirements for cotranslational protein N-myristoylation, a series of site-directed mutagenesis of N-terminal region were performed using tumor necrosis factor as a nonmyristoylated model protein. Subsequently, the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system or in transfected cells. It was found that the amino acid residue at position 3 in an N-myristoylation consensus motif, Met-Gly-X-X-X-Ser-X-X-X, strongly affected the susceptibility of the protein to two different cotranslational protein modifications, N-myristoylation and N-acetylation; 10 amino acids (Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, and His) with a radius of gyration smaller than 1.80 A directed N-myristoylation, two negatively charged residues (Asp and Glu) directed N-acetylation, and two amino acids (Gly and Met) directed heterogeneous modification with both N-myristoylation and N-acetylation. The amino acid requirements at this position for the two modifications were dramatically changed when Ser at position 6 in the consensus motif was replaced with Ala. Thus, the amino acid residue penultimate to the N-terminal Gly residue strongly affected two cotranslational protein modifications, N-myristoylation and N-acetylation, and the amino acid requirements at this position for these two modifications were significantly affected by downstream residues.     

Recombinant chicken egg white cystatin variants of the QLVSG region.

Using recombinant DNA methods, seven cystatin variants were produced by cassette mutagenesis of a chicken egg white cystatin variant which already contains the mutations Ala3, Glu2, Phe1, Ser1-->Met, Met29-->and Met 89-->Leu. When characterized by structural and functional studies, they were all found to harbour mutations in the first hairpin loop, the so-called 'QXVXG' region, which is highly conserved within the cystatin superfamily and thought to be important for its inhibitory activity towards cysteine proteinases. They were purified to more than 90% homogeneity and analysed by SDS/PAGE, HPLC, tryptic peptide mapping, N-terminal amino acid sequencing and ELISA. Structural model building of the variants and their complexes with papain was performed using computer graphics based on the crystallographic coordinates of chicken egg white cystatin and the papain-stefin complex. Only minor conformational changes were required for modelling the mutants or complexes. Equilibrium dissociation constants and rate constants of complex formation of the variants with papain, actinidin as well as cathepsin B and L were determined by kinetic measurements using fluorogenic substrates. The single exchanges Gln53-->Glu, Gln53-->Asn, Val44-->Asp, Gly57-->Ala and the double exchanges Arg52-->Leu, Gln53-->Glu, Gln53-->Asn, Ser56-->Ala, Leu54-->Met, Gly57-->Ala reduced the inhibition of papain, actinidin and cathespin B significantly by 10-1000-fold. With the exception of the Val55-->Asp variant, the differences in the Ki values are mainly due to larger k off values, whereas the kon values seem to be more or less unaffected by the selected mutations. The effect on the inhibition of papain is generally smaller than the effects on actinidin and cathepsin B inhibition. Cathepsin L inhibition is strikingly insensitive to all mutations. These distinct effects of the inhibitor variants indicate differences in proteinase-inhibitor-protein interactions between closely related cysteine proteinases. In addition, the results verify the prediction, made earlier from sequence alignment studies and from a docking model of the chicken cystatin-papain complex, that the first hairpin loop of cystatins is essential for effective inhibition.     

Molecular structure and metal-binding properties of the periplasmic CopK protein expressed in Cupriavidus metallidurans CH34 during copper challenge

The copK gene is localized on the pMOL30 plasmid of Cupriavidus metallidurans CH34 within the complex cop cluster of genes, for which 21 genes have been identified. The expression of the corresponding periplasmic CopK protein is strongly upregulated in the presence of copper, leading to a high periplasmic accumulation. The structure and metal-binding properties of CopK were investigated by NMR and mass spectrometry. The protein is dimeric in the apo state with a dissociation constant in the range of 10^- 5 M estimated from analytical ultracentrifugation. Mass spectrometry revealed that CopK has two high-affinity Cu(I)-binding sites per monomer with different Cu(I) affinities. Binding of Cu(II) was observed but appeared to be non-specific. The solution structure of apo-CopK revealed an all-β fold formed of two β-sheets in perpendicular orientation with an unstructured C-terminal tail. The dimer interface is formed by the surface of the C-terminal β-sheet. Binding of the first Cu(I)-ion induces a major structural modification involving dissociation of the dimeric apo-protein. Backbone chemical shifts determined for the 1Cu(I)-bound form confirm the conservation of the N-terminal β-sheet, while the last strand of the C-terminal sheet appears in slow conformational exchange. We hypothesize that the partial disruption of the C-terminal β-sheet is related to dimer dissociation. NH-exchange data acquired on the apo-protein are consistent with a lower thermodynamic stability of the C-terminal sheet. CopK contains seven methionine residues, five of which appear highly conserved. Chemical shift data suggest implication of two or three methionines (Met54, Met38, Met28) in the first Cu(I) site. Addition of a second Cu(I) ion further increases protein plasticity. Comparison of the structural and metal-binding properties of CopK with other periplasmic copper-binding proteins reveals two conserved features within these functionally related proteins: the all-β fold and the methionine-rich Cu(I)-binding site.     

Biallelic SUN5 Mutations Cause Autosomal-Recessive Acephalic Spermatozoa Syndrome

Acephalic spermatozoa syndrome is a rare and severe form of teratozoospermia characterized by a predominance of headless spermatozoa in the ejaculate. Family clustering and consanguinity suggest a genetic origin; however, causative mutations have yet to be identified. We performed whole-exome sequencing in two unrelated infertile men and subsequent variant filtering identified one homozygous (c.824C>T [p.Thr275Met]) and one compound heterozygous (c.1006C>T [p.Arg356Cys] and c.485T>A [p.Met162Lys]) SUN5 (also named TSARG4) variants. Sanger sequencing of SUN5 in 15 additional unrelated infertile men revealed four compound heterozygous (c.381delA [p.Val128Serfs^∗7] and c.824C>T [p.Thr275Met]; c.381delA [p.Val128Serfs^∗7] and c.781G>A [p.Val261Met]; c.216G>A [p.Trp72^∗] and c.1043A>T [p.Asn348Ile]; c.425+1G>A/c.1043A>T [p.Asn348Ile]) and two homozygous (c.851C>G [p.Ser284^∗]; c.350G>A [p.Gly114Arg]) variants in six individuals. These 10 SUN5 variants were found in 8 of 17 unrelated men, explaining the genetic defect in 47.06% of the affected individuals in our cohort. These variants were absent in 100 fertile population-matched control individuals. SUN5 variants lead to absent, significantly reduced, or truncated SUN5, and certain variants altered SUN5 distribution in the head-tail junction of the sperm. In summary, these results demonstrate that biallelic SUN5 mutations cause male infertility due to autosomal-recessive acephalic spermatozoa syndrome.     

Extent of N-terminal modifications in cytosolic proteins from eukaryotes

Most proteins in all organisms undergo crucial N-terminal modifications involving N-terminal methionine excision, N-alpha-acetylation or N-myristoylation (N-Myr), or S-palmitoylation. We investigated the occurrence of these poorly annotated but essential modifications in proteomes, focusing on eukaryotes. Experimental data for the N-terminal sequences of animal, fungi, and archaeal proteins, were used to build dedicated predictive modules in a new software. In vitro N-Myr experiments were performed with both plant and animal N-myristoyltransferases, for accurate prediction of the modification. N-terminal modifications from the fully sequenced genome of Arabidopsis thaliana were determined by MS. We identified 105 new modified protein N-termini, which were used to check the accuracy of predictive data. An accuracy of more than 95% was achieved, demonstrating (i) overall conservation of the specificity of the modification machinery in higher eukaryotes and (ii) robustness of the prediction tool. Predictions were made for various proteomes. Proteins that had undergone both N-terminal methionine (Met) cleavage and N-acetylation were found to be strongly overrepresented among the most abundant proteins, in contrast to those retaining their genuine unblocked Met. Here we propose that the nature of the second residue of an ORF is a key marker of the abundance of the mature protein in eukaryotes.     

Chemical modification of insulin in amyloid fibrils

We have investigated the chemical modification of insulin under conditions that promote the conversion of the soluble protein into amyloid fibrils. The modifications that are incorporated into the fibrils include deamidation of Asn A21, Asn B3, and Gln B4. In order to prepare fibrils with minimal deamidation of these residues, the kinetics of aggregation were accelerated by seeding with aliquots of a solution containing preformed fibrils. The resulting fibrils were then reincubated to determine the extent to which chemical modification occurs in the fibril itself. The deamidation of Asn A21 in particular could be followed in detail. Deamidation of this residue in the fibrillar form of insulin was found to occur in only 52 +/- 5% of molecules. This result indicates that there are at least two different packing environments of insulin molecules in the fibrils and suggests that the characterization of chemical modifications may be a useful probe of the environment of polypeptide chains within amyloid fibrils.     

Thr21Met (T21M) but not Ser89Asn (S89N) polymorphisms of the urotensin-II (UTS-II) gene are associated with Behcet's disease (BD)

Behcet's disease (BD) is multisytemic vasculitis or chronic inflammation that may lead to various autoimmune and autoinflammatory syndromes. Exact etiopathogenesis of BD has not been clarified yet. Urotensin II (UTS-II) is predominantly a vasoactive peptide and Thr21Met polymorphism in UTS-II gene was proved to increasing in some autoimmune diseases. Considering these, our objective was to evaluate whether two UTS-II gene polymorphisms (Thr21Met and Ser89Asn) were responsible in genetic susceptibility to BD in a Turkish population. A total of 198 patients with BD and 275 healthy controls were enrolled. We analyzed the genotype and allele frequencies of two UTS-II gene polymorphisms, Thr21Met and Ser89Asn, in BD patients and in controls. We found that Thr21Met but not Ser89Asn polymorphisms of the UTS-II gene were markedly associated with the risk of developing BD (p < 0.0001), The Met21Met genotype was less common among BD patients (6.1% in patients vs. 17.1% in controls; p < 0.0001). There was also an increase in the 21Thr allele (54.8% in BD patients vs. 43.8% in controls) and a decrease in 21Met allele frequencies (45.2% in controls vs. 56.2% in patients) in the BD groups (p < 0.0044). To the best of our knowledge, for the first time in the literature, our study claims that there is an association between Thr21Met, and not between Ser89Asn polymorphisms in the UTS-II gene and BD. These results put a new player to the field of undiscovered pathogenesis of BD and hopefully provide new insights to the treatment options.     

Flash dietary methionine supply over growth requirements in pigs: Multi-facetted effects on skeletal muscle metabolism

Dietary methionine affects protein metabolism, lean gain and growth performance and acts in the control of oxidative stress. When supplied in large excess relative to growth requirements in diets for pigs, positive effects on pork quality traits have been recently reported. This study aimed to decipher the molecular and biochemical mechanisms affected by a dietary methionine supply above growth requirements in the loin muscle of finishing pigs. During the last 14 days before slaughter, crossbred female pigs (n = 15 pigs/diet) were fed a diet supplemented with hydroxy-methionine (Met5; 1.1% of methionine) or not (CONT, 0.22% of methionine). Blood was sampled at slaughter to assess key metabolites. At the same time, free amino acid concentrations and expression or activity levels of genes involved in protein or energy metabolism were measured in the longissimus lumborum muscle (LM). The Met5 pigs exhibited a greater activity of creatine kinase in plasma when compared with CONT pigs. The concentrations of free methionine, alpha-aminobutyric acid, anserine, 3-methyl-histidine, lysine, and proline were greater in the LM of Met5 pigs than in CONT pigs. Expression levels of genes involved in protein synthesis, protein breakdown or autophagy were only scarcely affected by the diet. Among ubiquitin ligases, MURF1, a gene known to target creatine kinase and muscle contractile proteins, and OTUD1 coding for a deubiquitinase protease, were up-regulated in the LM of Met5 pigs. A lower activity of citrate synthase, a reduced expression level of ME1 acting in lipogenesis but a higher expression of PPARD regulating energy metabolism, were also observed in the LM of Met5 pigs compared with CONT pigs. Principal component analysis revealed that expression levels of many studied genes involved in protein and energy metabolism were correlated with meat quality traits across dietary treatments, suggesting that subtle modifications in expression of those genes had cumulative effects on the regulation of processes leading to the muscle transformation into meat. In conclusion, dietary methionine supplementation beyond nutritional requirements in pigs during the last days before slaughter modified the free amino acid profile in muscle and its redox capacities, and slightly affected molecular pathways related to protein breakdown and energy metabolism. These modifications were associated with benefits on pork quality traits.     

Four unreported types of glycans containing mannose-6-phosphate are heterogeneously attached at three sites (including newly found Asn 233) to recombinant human acid alpha-glucosidase that is the only approved treatment for Pompe disease

Myozyme is a recombinant human acid alpha-glucosidase (rhGAA) that is currently the only drug approved for treating Pompe disease, and its low efficacy means that a high dose is required. Mannose-6-phosphate (M6P) glycosylation on rhGAA is a key factor influencing lysosomal enzyme targeting and the efficacy of enzyme replacement therapy (ERT); however, its complex structure and relatively small quantity still remain to be characterized. This study investigated M6P glycosylation on rhGAA using liquid chromatography (LC)–electrospray ionization (ESI)–high-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS). The glycans released from rhGAA were labeled with procainamide to improve mass ionization efficiency and the sensitivity of MS/MS. The relative quantities (%) of 78 glycans were obtained, and 1.0% of them were glycans containing M6P (M6P glycans). These were categorized according to their structure into 4 types: 3 newly found ones, comprising high-mannose-type M6P glycans capped with N-acetylglucosamine (GlcNAc) (2 variants, 17.5%), hybrid-type M6P glycans (2 variants, 11.2%), and hybrid-type M6P glycans capped with GlcNAc (3 variants, 6.9%), as well as high-mannose-type M6P glycans (3 variants, 64.4%). HCD-MS/MS spectra identified six distinctive M6P-derived oxonium ions. The glycopeptides obtained from protease-digested rhGAA were analyzed using nano-LC-ESI-HCD-MS/MS, and the extracted-ion chromatograms of M6P-derived oxonium ions confirmed three M6P glycosylation sites comprising Asn 140, Asn 233 (newly found), and Asn 470 attached heterogeneously to nine M6P glycans (two types), eight M6P glycans (four types), and seven M6P glycans (two types), respectively. This is the first study of rhGAA to differentiate M6P glycans and identify their attachment sites, despite rhGAA already being an approved drug for Pompe disease.     

Two rat surfactant protein A isoforms arise by a novel mechanism that includes alternative translation initiation

A single gene for rat surfactant protein A (SP-A) encodes two isoforms that are distinguished by an isoleucine-lysine-cysteine (IKC) N-terminal extension (SP-A and IKC-SP-A). Available evidence suggests that the variants are generated by alternative signal peptidase cleavage of the nascent polypeptide at a primary site (Cys(-)(1)-Asn(1)) and a secondary site (Gly(-)(4)-Ile(-)(3)). In this study, we used site-directed mutagenesis and heterologous expression in vitro and in insect cells to the examine mechanisms that may lead to alternative signal peptidase cleavage including alternative translation initiation at two in-frame AUGs (Met(-)(30) and Met(-)(20)), a suboptimal context for hydrolysis at the primary cleavage site, or cotranslational protein modifications that expose an otherwise cryptic secondary cleavage site. In vitro translation of a rat cDNA for SP-A resulted in both 28 and 29 kDa primary translation products on SDS-PAGE analysis, while translation of cDNAs encoding Met-30Ala and Met-20Ala mutations resulted in only the single 28 and 29 kDa molecular mass species, respectively. These data are consistent with translation initiation at both Met(-)(30) and Met(-)(20) during in vitro synthesis of SP-A. The Met-30Ala mutation reduced expression of the longer isoform in insect cells, indicating that the Met(-)(30) site also contributes to eucaryotic protein expression. Forcing translation initiation at Met(-)(30) by optimizing the Kozak consensus sequence surrounding that codon or by mutating the Met(-)(20) codon resulted in preferential expression of the longer SP-A isoform but reduced overall expression of the protein almost 10-fold. Both isoforms were generated to some degree whether translation was initiated at the codon for Met(-)(30) or Met(-)(20), indicating that the site of translation initiation is not the sole determinant of isoform generation and suggesting that either the context of the primary cleavage site is suboptimal or that cotranslational modifications affect cleavage. Preventing N-terminal glycosylation at Asn(1) did not affect the site of signal peptidase cleavage. Disruption of interchain disulfide formation at Cys(-)(1) by substitution with serine markedly enhanced cleavage at the Gly(-)(4)-Ile(-)(3) bond, but substitution with alanine enhanced cleavage at the Cys(-)(1)-Asn(1) bond. We conclude that rat SP-A isoforms arise by a novel mechanism that includes both alternative translation initiation at two in-frame AUGs and a suboptimal context for signal peptidase hydrolysis at the primary cleavage site.     

Site‐specific rapid deamidation and isomerization in human lens αA‐crystallin in vitro

Recent studies have suggested that the isomerization/racemization of aspartate residues in proteins increases in aged tissues. One such residue is Asp151 in lens‐specific αA‐crystallin. Although many isomerization/racemization sites have been reported in various proteins, the factors that lead to those modifications in proteins in vivo remain obscure. Therefore, an in vitro system is needed to assess the mechanisms of modifications of Asp under various conditions. Deamidation of Asn to Asp in proteins occurs more rapidly than isomerization/racemization of Asp, although the reaction passes through the same intermediate in both pathways. Here, therefore, we replaced Asp151 in human lens αA‐crystallin with Asn by using site‐directed mutagenesis. The recombinant protein was expressed in Escherichia coli and used to investigate the deamidation/isomerization/racemization of Asn151 after incubation at 50°C for various durations and under different pH. After incubation, the mutant αA‐crystallin was subjected to enzymatic digestion followed by liquid chromatography–MS/MS to evaluate the ratio of modifications in Asn151‐containing peptides. The Asp151Asn αA‐crystallin mutant showed rapid deamidation to Asp with the formation of specific Asp isomers. In particular, deamidation increased greatly under basic conditions. By contrast, subunit–subunit interactions between αA‐crystallin and αB‐crystallin had little effect on the modification of Asn151. Our findings suggest that the Asp151Asn αA‐crystallin mutant represents a good in vitro model protein to assess deamidation, isomerization, and the racemization intermediates. Furthermore, our in vitro results show a different trend from in vivo data, implying the presence of specific factors that induce racemization from L‐Asp to D‐Asp residues in vivo.     

The effect of hypusine modification on the intracellular localization of eIF5A

Eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein essential for eukaryotic cell proliferation and is the only protein containing hypusine, [N^ε-(4-amino-2-hydroxybutyl)lysine]. eIF5A is activated by the post-translational synthesis of hypusine. eIF5A also undergoes an acetylation at specific Lys residue(s). In this study, we have investigated the effect of hypusine modification and acetylation on the subcellular localization of eIF5A. Immunocytochemical analyses showed differences in the distribution of non-hypusinated eIF5A precursor and the hypusine-containing mature eIF5A. While the precursor is found in both cytoplasm and nucleus, the hypusinated eIF5A is primarily localized in cytoplasm. eIF5A mutant proteins, defective in hypusine modification (K50A, K50R) were localized in a similar manner to the eIF5A precursor, whereas hypusine-modified mutant proteins (K47A, K47R, K68A) were localized mainly in the cytoplasm. These findings provide strong evidence that the hypusine modification of eIF5A dictates its localization in the cytoplasmic compartment where it is required for protein synthesis.     

Function of Modified Nucleosides 7-Methylguanosine, Ribothymidine, and 2-Thiomethyl-N6-(Isopentenyl)adenosine in Procaryotic Transfer Ribonucleic Acid

To elucidate subtle functions of transfer ribonucleic acid (tRNA) modifications in protein synthesis, pairs of tRNA's that differ in modifications at specific positions were prepared from Bacillus subtilis. The tRNA's differ in modifications in the anticodon loop, the extra arm, and the TUC loop. The functional properties of these species were compared in aminoacylation, as well as in initiation and peptide bond formation, at programmed ribosomes. These experiments demonstrated the following. (i) In tRNA(f) (Met) the methylation of guanosine 46 in the extra arm to 7-methylguanosine by the 7-methylguanosine-forming enzyme from Escherichia coli changes the aminoacylation kinetics for the B. subtilis methionyl-tRNA synthetase. In repeated experiments the V(max) value is decreased by one-half. (ii) tRNA(f) (Met) species with ribothymidine at position 54 (rT54) or uridine at position 54 (U54) were obtained from untreated or trimethoprim-treated B. subtilis. The formylated fMet-tRNA(f) (Met) species with U54 and rT54, respectively, function equally well in an in vitro initiation system containing AUG, initiation factors, and 70s ribosomes. The unformylated Met-tRNA(t) (Met) species, however, differ from each other: "Met-tRNA(f) (Met) rT" is inactive, whereas the U54 counter-upart effectively forms the initiation complex. (iii) Two isoacceptors, tRNA(1) (Phe) and tRNA(2) (Phe), were obtained from B. subtilis. tRNA(1) (Phe) accumulates only under special growth conditions and is an incompletely modified precursor oftRNA(2) (Phe): in the first position of the anticodon, guanosine replaces Gm, and next to the 3' end of the anticodon (isopentenyl)adenosine replaces 2-thiomethyl-N(6)-(isopentenyl)adenosine. Both tRNA's behave identically in aminoacylation kinetics. In the factor-dependent AUGU(3)-directed formation of fMet-Phe, the undermodified tRNA(1) (Phe) is always less efficient at Mg(2+) concentrations between 5 and 15 mM than its mature counterpart.     

Proteomics of beta2-microglobulin amyloid fibrils

Knowledge on the chemical structure of beta2-microglobulin in natural amyloid fibrils is quite limited because of the difficulty in obtaining tissue samples suitable for biochemical studies. We have reviewed the available information on the chemical modifications and we present new data of beta2-microglobulin extracted from non-osteotendinous tissues. beta2-microglobulin can accumulate in these compartments after long-term haemodialysis but rarely forms amyloid deposits. We confirm that truncation at the N-terminus is an event specific to beta2-microglobulin derived from fibrils but is not observed in the beta2-microglobulin from plasma or from the insoluble non-fibrillar material deposited in the heart and spleen. We also confirm the partial deamidation of Asn 17 and Asn 42, as well as the oxidation of Met 99 in fibrillar beta2-microglobulin. Other previously reported chemical modifications cannot be excluded, but should involve less than 1-2% of the intact molecule.     

Proteomics of β2-microglobulin amyloid fibrils

Knowledge on the chemical structure of β2-microglobulin in natural amyloid fibrils is quite limited because of the difficulty in obtaining tissue samples suitable for biochemical studies. We have reviewed the available information on the chemical modifications and we present new data of β2-microglobulin extracted from non-osteotendinous tissues. β2-microglobulin can accumulate in these compartments after long-term haemodialysis but rarely forms amyloid deposits. We confirm that truncation at the N-terminus is an event specific to β2-microglobulin derived from fibrils but is not observed in the β2-microglobulin from plasma or from the insoluble non-fibrillar material deposited in the heart and spleen. We also confirm the partial deamidation of Asn 17 and Asn 42, as well as the oxidation of Met 99 in fibrillar β2-microglobulin. Other previously reported chemical modifications cannot be excluded, but should involve less than 1–2% of the intact molecule.     

Chemical modifications in therapeutic protein aggregates generated under different stress conditions

In this study, we characterized the chemical modifications in the monoclonal antibody (IgG(2)) aggregates generated under various conditions, including mechanical, chemical, and thermal stress treatment, to provide insight into the mechanism of protein aggregation and the types of aggregate produced by the different stresses. In a separate study, additional biophysical characterization was performed to arrange these aggregates into a classification system (Joubert, M. K., Luo, Q., Nashed-Samuel, Y., Wypych, J., and Narhi, L. O. (2011) J. Biol. Chem. 286, 25118-25133). Here, we report that different aggregates possessed different types and levels of chemical modification. For chemically treated samples, metal-catalyzed oxidation using copper showed site-specific oxidation of Met(246), His(304), and His(427) in the Fc portion of the antibody, which might be attributed to a putative copper-binding site. For the hydrogen peroxide-treated sample, in contrast, four solvent-exposed Met residues in the Fc portion were completely oxidized. Met and/or Trp oxidation was observed in the mechanically stressed samples, which is in agreement with the proposed model of protein interaction at the air-liquid interface. Heat treatment resulted in significant deamidation but almost no oxidation, which is consistent with thermally induced aggregates being generated by a different pathway, primarily by perturbing conformational stability. These results demonstrate that chemical modifications are present in protein aggregates; furthermore, the type, locations, and severity of the modifications depend on the specific conditions that generated the aggregates.