Dana W. Aswad

Determination of d- and l-aspartate in amino acid mixtures by high-performance liquid chromatography after derivatization with a chiral adduct of o-phthaldialdehyde

A sensitive and convenient method for the simultaneous determination of D- and L-aspartic acid in amino acid mixtures is described. The method involves derivatization of the mixture with a chiral fluorogen, followed by high-performance liquid chromatography on a reverse-phase column. The fluorogen used is an adduct of o-phthaldialdehyde with an optically active thiol, N-acetyl-L-cysteine. The sensitivity and accuracy of this method is similar to that using adducts of o-phthaldialdehyde with the achiral thiol, 2-mercaptoethanol. Five picomoles of D-aspartate can be accurately detected in the presence of a 100-fold excess of L-aspartate with a total analysis time (including derivatization) of 10 min.

Hormone-dependent, CARM1-directed, arginine-specific methylation of histone H3 on a steroid-regulated promoter

Activation of gene transcription involves chromatin remodeling by coactivator proteins that are recruited by DNA-bound transcription factors. Local modification of chromatin structure at specific gene promoters by ATP-dependent processes and by posttranslational modifications of histone N-terminal tails provides access to RNA polymerase II and its accompanying transcription initiation complex. While the roles of lysine acetylation, serine phosphorylation, and lysine methylation of histones in chromatin remodeling are beginning to emerge, low levels of arginine methylation of histones have only recently been documented, and its physiological role is unknown. The coactivator CARM1 methylates histone H3 at Arg17 and Arg26 in vitro and cooperates synergistically with p160-type coactivators (e.g., GRIP1, SRC-1, ACTR) and coactivators with histone acetyltransferase activity (e.g., p300, CBP) to enhance gene activation by steroid and nuclear hormone receptors (NR) in transient transfection assays. In the current study, CARM1 cooperated with GRIP1 to enhance steroid hormone-dependent activation of stably integrated mouse mammary tumor virus (MMTV) promoters, and this coactivator function required the methyltransferase activity of CARM1. Chromatin immunoprecipitation assays and immunofluorescence studies indicated that CARM1 and the CARM1-methylated form of histone H3 specifically associated with a large tandem array of MMTV promoters in a hormone-dependent manner. Thus, arginine-specific histone methylation by CARM1 is an important part of the transcriptional activation process.

Isoaspartate in peptides and proteins: formation, significance, and analysis

Formation of isoaspartyl peptide bonds (isoAsp) is one of the most common forms of non-enzymatic degradation of peptides and proteins under mild conditions. IsoAsp arises when certain Asn-Xaa and Asp-Xaa sites undergo a spontaneous intramolecular rearrangement to form a succinimide which subsequently hydrolyzes to generate a mixture of isoAsp-Xaa and Asp-Xaa linkages in a ratio of approximately 2:1. This pathway is responsible for the much greater susceptibility of asparagine, compared with glutamine, to deamidation at neutral and alkaline pH. Rearrangement occurs most readily at Asn-Gly, Asn-Ser, and Asp-Gly sequences where the local polypeptide chain flexibility is high. Formation of isoAsp can decrease the biological activity of a protein pharmaceutical, alter its susceptibility to proteolytic degradation, and elicit autoimmunity. The enzyme protein L-isoaspartyl methyltransferase can be used to measure isoAsp sites in the low pmol range with or without the use of radioisotopes.

Deamidation and isoaspartate formation in proteins: unwanted alterations or surreptitious signals?

Abstract Formation of β-linked Asp-Xaa peptide bonds – isoaspartyl (isoAsp) sites – arise in proteins via succinimide−linked deamidation of asparagine or dehydration of aspartate, reactions which represent a major source of spontaneous protein damage under physiological conditions. Accumulation of atypical isoaspartyl sites is minimized in vivo by the activity of protein L-isoaspartyl O–methyltransferase (PIMT), which regenerates a normal peptide bond. Loss of PIMT has harmful consequences, especially in neurons; thus, formation of isoAsp sites and their subsequent correction by PIMT is widely believed to constitute an important pathway of protein damage and repair. Recent evidence is mounting, however, that deamidation and isoaspartate formation may, in some instances, constitute a novel mechanism for intentional modification of protein structure. Herein we describe the mechanism of Asx rearrangement, summarize the evidence that PIMT serves an important repair function, and then focus on emerging evidence that deamidation and isoAsp formation may sometimes have a useful function.

The unusual substrate specificity of eukaryotic protein carboxyl methyltransferases

Abstract Recent studies reveal that the eukaryotic protein carboxyl methyltransferase exhibit an unexpected specificity for l-isoaspartyl sequences (aspartyl sequences in which the peptide linkage occurs via the side-chain β-carboxyl group of the aspartate). Deamidation of labile asparagine sites is probably the major source of isoaspartate in vivo . The specificity of this enzyme implies that it may play a key role in the degradation or repair of deamidation-damaged proteins.

Protein Repair in the Brain, Proteomic Analysis of Endogenous Substrates for Protein L-Isoaspartyl Methyltransferase in Mouse Brain

Protein l-isoaspartyl methyltransferase (PIMT) catalyzes repair of l-isoaspartyl peptide bonds, a major source of protein damage under physiological conditions. PIMT knock-out (KO) mice exhibit brain enlargement and fatal epileptic seizures. All organs accumulate isoaspartyl proteins, but only the brain manifests an overt pathology. To further explore the role of PIMT in brain function, we undertook a global analysis of endogenous substrates for PIMT in mouse brain. Extracts from PIMT-KO mice were subjected to two-dimensional gel electrophoresis and blotted onto membranes. Isoaspartyl proteins were radiolabeled on-blot using [methyl-3H]S-adenosyl-l-methionine and recombinant PIMT. Fluorography of the blot revealed 30-35 3H-labeled proteins, 22 of which were identified by peptide mass fingerprinting. These isoaspartate-prone proteins represent a wide range of cellular functions, including neuronal development, synaptic transmission, cytoskeletal structure and dynamics, energy metabolism, nitrogen metabolism, pH homeostasis, and protein folding. The following five proteins, all of which are rich in neurons, accumulated exceptional levels of isoaspartate: collapsin response mediator protein 2 (CRMP2/ULIP2/DRP-2), dynamin 1, synapsin I, synapsin II, and tubulin. Several of the proteins identified here are prone to age-dependent oxidation in vivo, and many have been identified as autoimmune antigens, of particular interest because isoaspartate can greatly enhance the antigenicity of self-peptides. We propose that the PIMT-KO phenotype results from the cumulative effect of isoaspartate-related damage to a number of the neuron-rich proteins detected in this study. Further study of the isoaspartate-prone proteins identified here may help elucidate the molecular basis of one or more developmental and/or age-related neurological diseases.

Purification and characterization of two distinct isozymes of protein carboxymethylase from bovine brain.

Protein carboxymethylase (EC 2.1.1.24) from cytosol of bovine brain was found to exist as two apparent isozymes that could be separated by chromatography on DEAE‐cellulose at pH 8.O. Rechromatography of the two forms, designated PCM I and PCM II, indicated that they are not interconvertible. Both enzymes have a molecular weight of 24,300 by sodium dodecyl sulfate‐polyacryl‐amide gel electrophoresis. PCM I consists mainly of one isoelectric form, pI 6.5, whereas PCM II resolves into two forms of pI 5.6 and 5.7. The relative amounts of PCM I and PCM II show a marked tissue dependence. Brain has approximately twice as much PCM I as II, whereas liver contains only the type II enzyme. The two enzymes were found to have similar substrate specificities when tested with five different methyl‐accepting proteins. Synapsin I, a basic protein associated with synaptic vesicles, was found to be an excellent methyl‐accepting protein with regard to its Km (1.2 μM), but it exhibited a low stoichiome‐try of methyl incorporation.

Optimal conditions for the use of protein l-isoaspartyl methyltransferase in assessing the isoaspartate content of peptides and proteins

Protein L-isoaspartyl methyltransferase provides a basis for enzymatic measurement of atypical, isoaspartyl linkages which make a major contribution to protein microheterogeneity. The low Vmax of the methyltransferase reaction and the instability of the methyl ester can hinder accurate determinations, and different laboratories using different conditions have achieved discrepant values for the isoaspartate content of the same proteins. To investigate the effects of these conditions, and to optimize the assay, isoaspartyl delta sleep-inducing peptide was methylated under a variety of conditions. We found that 1 microM methyltransferase was required to obtain stoichiometric modification of 2 microM peptide in 40-min reactions at pH 6.2 and 30 degrees C. A computer model utilizing kinetic constants obtained from studies on initial rates of methylation predicted the same requirement for enzyme concentration. Carrier protein was necessary for optimal methyltransferase activity at enzyme concentrations below 0.4 microM. Stoichiometric methylation required concentrations of S-adenosylmethionine to be in substantial excess over those of peptide; 50 microM S-adenosylmethionine is the minimum needed for complete modification of 10 microM peptide. Spontaneous demethylation was significant under all conditions tested, so that the methyl ester itself never reached a ratio of 1 mol/mol of total peptide. These results demonstrate that the most accurate measurements of isoaspartate are obtained when reactions are carried out at low peptide concentrations, high S-adenosylmethionine concentrations, and high enzyme concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Deamidation of calmodulin at neutral and alkaline pH: Quantitative relationships between ammonia loss and the susceptibility of calmodulin to modification by protein carboxyl methyltransferase

Measurements of ammonia release provide the first direct evidence that calmodulin becomes extensively deamidated during incubations at 37 degrees C, pH 7.4 or pH 11. A stoichiometry of 0.5 mol of NH3 released/mol of calmodulin is observed after 2 h at pH 11 or after 8-9 days at pH 7.4. These treatments also increase the ability of calmodulin to serve as a substrate for the isoaspartate-specific protein carboxyl methyltransferase from bovine brain. The stoichiometries of methylation are highly correlated with the stoichiometries of ammonia release. Deamidation and increased methyl-accepting capacity also occur in parallel for seven other proteins (aldolase, bovine serum albumin, cytochrome c, lysozyme, ovalbumin, ribonuclease A, and triosephosphate isomerase) upon incubation at pH 11. However, in comparison to calmodulin, these other proteins show very little deamidation and increased methylation capacity following incubation at pH 7.4. Deamidation of calmodulin at pH 7.4 is unaffected by the addition of 10(-7) M Ca2+; however, at 4 X 10(-6) M Ca2+, the rate of deamidation is inhibited by approximately 70%. The Ca2+-protection effect is consistent with the suggestion (B. A. Johnson, N. E. Freitag, and D. W. Aswad, (1985) J. Biol. Chem. 260, 10913-10916) that deamidation occurs preferentially at Asn-60 and/or Asn-97, each of which resides in a distinct Ca2+-binding domain.

Major Degradation Products of Basic Fibroblast Growth Factor: Detection of Succinimide and Iso-aspartate in Place of Aspartate15

The degradation products of basic fibroblast growth factor (bFGF) were isolated by ion exchange HPLC (HP-IEC) and characterized. The predominant product at pH 5 was a succinimide in place of aspartate15 as determined by LC/MS, N-terminal sequencing, and susceptibility to degradation at pH >6.5. The rate of appearance of the succinimidyl-bFGF at 22 °C was comparable to that reported for small peptides, consistent with a high flexibility predicted for asp15-gly. Tryptic mapping together with [3H]-methylation indicated that iso-aspartate was formed at the position of asp15. Size exclusion HPLC indicated the presence of intact and truncated dimers and trimers which associated through disulfide linkages. Two truncated monomer forms were found that co-eluted by HP-IEC; the cleavages were determined to be at asp28-pro and asp15-gly using LC/MS and N-terminal sequencing. These degradation products which occurred at sites that are away from receptor or heparin binding domains of bFGF remained bioactive in a cell proliferation assay.