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Approximately 12% of histone H2B molecules in mammalian brain contain a modification wherein Asp25 is present as the d-enantiomer, and is mostly linked to Gly26 via the side-chain carboxyl. Here we (1) demonstrate the high specificity of a polyclonal antibody to this modification, and (2) use this Ab to demonstrate that this modification is enriched in brain relative to liver, thymus, and HeLa cells.

Protein l-isoaspartyl methyltransferase (PIMT/PCMT1), a product of the human pcmt1 gene, catalyzes repair of abnormal l-isoaspartyl linkages in age-damaged proteins. Pcmt1 knock-out mice exhibit a profound neuropathology and die 30-60 days postnatal from an epileptic seizure. Here we express 15 reported variants of human PIMT and characterize them with regard to their enzymatic activity, thermal stability, and propensity to aggregation. One mutation, R36C, renders PIMT completely inactive, whereas two others, A7P and I58V, exhibit activity that is 80-100% higher than wild type. G175R is highly prone to aggregation and has greatly reduced activity. R17S and R17H show markedly enhanced sensitivity to thermal denaturation. Based on previous studies of moderate PIMT variation in humans and mice, we predict that heterozygosity for R36C, G175R, R17S, and R17H will prove detrimental to cognitive function and successful aging, whereas homozygosity (if it ever occurs) will lead to severe neurological problems in the young.

Protein L-isoaspartyl methyltransferase (PIMT/PCMT1), a product of the pcmt1 gene, catalyzes repair of abnormal L-isoaspartyl linkages in age-damaged proteins. Pcmt1 knockout mice exhibit a profound neuropathology and die 30-60 days postnatal from an epileptic seizure. Here we characterize four new SNP variants of human PIMT with respect to enzymatic activity, thermal stability, and propensity to aggregation. Under standard assay conditions, L191S, A150V, P174H and A65V showed activity losses of 72%, 64%, 61%, and 11% respectively. By differential scanning fluorimetry, melting temperature deviations were -5.2, -4.5, +0.5, and -3.4°C. SDS-PAGE of purified protein reveal significant aggregation of L191S, A150V, and P174H, but not A65V. We also report new data on three unusual PIMT variants among the 13 recently characterized by our laboratory. A7P and I58V were previously found to have 1.8-2.0 times the activity of WT PIMT in the standard assay; however, upon kinetic analysis, we find both variants exhibit reduced catalytic efficiency (Vmax/Km) due to weak isoaspartyl substrate binding. The near complete loss of activity (

Autoantibodies against SCLC-associated neuronal antigen ELAVL4 (HuD) have been linked to smaller tumors and improved survival, but the antigenic epitope and mechanism of autoimmunity have never been solved. We report that recombinant human ELAVL4 protein incubated under physiological conditions acquires isoaspartylation, a type of immunogenic protein damage. Specifically, the N-terminal region of ELAVL4, previously implicated in SCLC-associated autoimmunity, undergoes isoaspartylation in vitro, is recognized by sera from anti-ELAVL4 positive SCLC patients and is highly immunogenic in subcutaneously injected mice and in vitro stimulated human lymphocytes. Our data suggest that isoaspartylated ELAVL4 is the trigger for the SCLC-associated anti-ELAVL4 autoimmune response.

Approximately 12 % of histone H2B in mammalian brain contains an unusual D-aspartate residue in its N-terminal tail. Most of this D-aspartate is linked to the C-flanking glycine via an isopeptide bond. To explore the possible significance of these modifications, we generated an antibody to the D-isoaspartyl form of H2B, and used it to assess its levels in H2B associated with "active" vs. "silent" chromatin. We found that the D-isoaspartyl form of H2B appears to be highly enriched in the former. This irreversible modification could serve a novel regulatory function in gene expression.

Formation of atypical isoaspartyl (isoAsp) sites in peptides and proteins via the deamidation-linked isomerization of asparaginyl-Xaa bonds or direct isomerization of aspartyl-Xaa bonds is a major contributor to spontaneous protein damage under mild conditions. This nonenzymatic reaction reroutes the Asx-Xaa peptide bond through the beta-carbonyl of asparaginyl or aspartyl residues, thereby adding an extra carbon to the polypeptide backbone. Formation of isoAsp has been implicated in protein inactivation, aggregation, degradation, and autoimmunity. Knowing the location of isoAsp sites in proteins is important for understanding mechanisms of protein damage and for characterizing protein pharmaceuticals. Here we present a simple nonradioactive method for direct localization of isoAsp residues in peptides or proteins. Using three model peptides, we demonstrate that isoAsp linkages can be cleaved selectively and in high yield by a two-step process in which (i) the isoAsp linkage is converted into a succinimide on incubation with S-adenosyl-l-methionine and the commercially available enzyme, protein l-isoaspartyl-O-methyltransferase, and (ii) the succinimidyl bond is then cleaved by hydroxylamine under conditions that minimize cleavage of the traditional hydroxylamine-sensitive Asn-Gly and related peptide bonds. Location of the isoAsp linkage is then inferred by identifying the cleavage products by mass spectrometry or N-terminal sequencing.

Formation of betalinked 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 RNA-binding protein HuR stabilizes labile mRNAs carrying AU-rich instability elements. This mRNA stabilization can be induced by hypoxia, lipopolysaccharide, and UV light. The mechanism by which these stimuli activate HuR is unclear and might be related to post-translational modification of this protein. Here we show that HuR can be methylated on arginine. However, HuR is not a substrate for PRMT1, the most prominent protein-arginine methyltransferase in mammalian cells, which methylates a number of heterogeneous nuclear ribonucleoproteins. Instead, HuR is specifically methylated by coactivator-associated arginine methyltransferase 1 (CARM1), a protein-arginine methyltransferase previously shown to serve as a transcriptional coactivator. By analyzing methylation of specific HuR arginine-to-lysine mutants and by sequencing radioactively methylated HuR peptides, Arg217 was identified as the major HuR methylation site. Arg217 is located in the hinge region between the second and third of the three HuR RNA recognition motif domains. Antibodies against a methylated HuR peptide were used to demonstrate in vivo methylation of HuR. HuR methylation increased in cells that overexpressed CARM1. Importantly, lipopolysaccharide stimulation of macrophages, which leads to HuR-mediated stabilization of tumor necrosis factor α mRNA in these cells, caused increased methylation of endogenous HuR. Thus, CARM1, which plays a role in transcriptional activation through histone H3 methylation, may also play a role in post-transcriptional gene regulation by methylating HuR.

Isoaspartate formation is a common type of protein damage normally kept in check by the repair enzyme protein-L-isoaspartyl methyltransferase (PIMT). Mice with a knockout of the gene (Pcmt1) for this enzyme (KO, -/-) exhibit a pronounced neuropathology with fatal epileptic seizures at 30-60 days. Heterozygous (HZ, +/-) mice have 50% of the PIMT activity found in wild-type (WT, +/+) mice, but appear normal. To see if HZ mice exhibit accelerated aging at the molecular level, we compared brain extracts from HZ and WT mice at 8 months and 2 years with regard to PIMT activity, isoaspartate levels, and activity of an endogenous PIMT substrate, creatine kinase B. PIMT activity declined modestly with age in both genotypes. Isoaspartate was significantly higher in HZ than WT mice at 8 months and more so at 2 years, rising 5× faster in HZ males and 3× faster in females. Creatine kinase activity decreased with age and was always lower in the HZ mice. These findings suggest the individual variation of human PIMT levels may significantly influence the course of age-related central nervous system dysfunction.

Isoaspartate (isoAsp) formation is a common type of spontaneous protein damage that is normally kept in check by the repair enzyme protein-L-isoaspartyl methyltransferase (PIMT). PIMT-KO (knockout) mice exhibit a pronounced neuropathology highlighted by death from an epileptic seizure at 30 to 60 days after birth. The mechanisms by which isoaspartyl damage disrupts normal brain function are incompletely understood. Proteomic analysis of the PIMT-KO mouse brain has shown that a number of key neuronal proteins accumulate high levels of isoAsp, but the extent to which their cellular functions is altered has yet to be determined. One of the major neuronal targets of PIMT is creatine kinase B (CKB), a well-characterized enzyme whose activity is relatively easy to assay. We show here that (1) the specific activity of CKB is significantly reduced in the brains of PIMT-deficient mice, (2) that in vitro aging of recombinant CKB results in significant accumulation of isoAsp sites with concomitant loss of enzymatic activity, and (3) that incubation of in vitro aged CKB with PIMT and its methyl donor S-adenosyl-L-methionine substantially repairs the aged CKB with regard to both its isoAsp content and its enzymatic activity. These results, combined with similarity in phenotypes of PIMT-KO and CKB-KO mice, suggests that loss of normal CKB structure and function contributes to the mechanisms by which isoAsp accumulation leads to CNS dysfunction in the PIMT-KO mouse. © 2014 Dimitrijevic et al.

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 [1, 2]. While the roles of lysine acetylation, serine phosphorylation, and lysine methylation of histones in chromatin remodeling are beginning to emerge [2–5], low levels of arginine methylation of histones have only recently been documented [4, 6–9], and its physiological role is unknown. The coactivator CARM1 methylates histone H3 at Arg17 and Arg26 in vitro [7] 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 [10, 11]. 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.

Protein L-isoaspartate O-methyltransferase (PIMT) is postulated to repair beta-aspartyl linkages (isoaspartyl (isoAsp)) that accumulate at certain Asp-Xaa and Asn-Xaa sites in association with protein aging and deamidation. To identify major targets of PIMT action we cultured rat PC12 cells with adenosine dialdehyde (AdOx), a methyltransferase inhibitor that promotes accumulation of isoAsp in vivo. Subcellular fractionation of AdOx-treated cells revealed marked accumulation of isoAsp in a 14-kDa nuclear protein. Gel electrophoresis and chromatography of nuclei (3)H-methylated in vitro by PIMT revealed this protein to be histone H2B. The isoAsp content of H2B in AdOx-treated cells was approximately 18 times that in control cells, although no isoAsp was seen in other core histones, regardless of treatment. To confirm the relevance and specificity of this effect, we measured isoAsp levels in histones from brains of PIMT knockout mice. IsoAsp was found at near stoichiometric levels in H2B extracted from knockout brains and was at least 80 times greater than that in H2B from normal mice. Little or no isoAsp was detected in H2A, H3, or H4 from mice of either genotype. Accumulation of isoAsp in histone H2B may disrupt normal gene regulation and contribute to the reduced life span that characterizes PIMT knockouts. In addition to disrupting protein function, isoAsp has been shown to trigger immunity against self-proteins. The propensity of H2B to generate isoAsp in vivo may help explain why this histone in particular is found as a major antigen in autoimmune diseases such as lupus erythematosus.

We found that certain mid-range consumer-level digital single-lens reflex (SLR) cameras using full-frame complementary metal oxide semiconductor (CMOS) sensors outperform X-ray film in acquiring signals from immunoblots that use enhanced chemiluminescence for detection. These cameras exhibit a sensitivity comparable to X-ray film, yet they provide a 3-fold increase in linear dynamic range and substantial cost savings over time, are more convenient to use, and eliminate the chemical waste associated with processing film.

Nuclear hormone receptors (NRs) activate transcription by binding to specific enhancer elements associated with target genes. Transcriptional activation is accomplished with the help of complexes of co-activator proteins that bind to NRs. p160 co-activators, a family of three related 160 kDa proteins, serve as primary co-activators by binding directly to NRs and recruiting additional secondary co-activators. Some of these (CBP/p300 and p/CAF) can acetylate histones and other proteins in the transcription complex, thus helping to modify chromatin structure and form an active transcription initiation complex. We recently discovered co-activator-associated arginine methyltransferase 1 (CARM1), which binds to p160 co-activators and thereby enhances transcriptional activation by NRs on transiently transfected reporter genes. CARM1 also methylates specific arginine residues in the N-terminal tail of histone H3 in vitro. A related arginine-specific protein methyltransferase, PRMT1, also binds p160 co-activators and enhances NR function. PRMT1 methylates histone H4 in vitro. The enhancement of NR function by CARM1, PRMT1 and p300 depends on their interactions with p160 co-activators. In the presence of p160 co-activators, some pairs of these three secondary co-activators provide a highly synergistic enhancement of NR function on transiently transfected reporter genes. We have also observed an enhancement of NR function on stably integrated reporter genes by these co-activators. We propose that the synergy of co-activator function between p300, CARM1 and PRMT1 is due to their different but complementary protein modification activities.

A rapid and sensitive HPLC-based method for quantitating isoaspartate levels in peptides and proteins is described. The analyte is incubated for 40 min with S-adenosyl-l-methionine and the commercially available enzyme protein l-isoaspartyl methyltransferase. Methylation of isoaspartyl sites results in stoichiometric production of S-adenosyl-l-homocysteine that is separated from the other components of the reaction by reversed-phase HPLC and quantitated online by absorbance at 260 nm. This method can accurately detect 5 pmol or less of isoaspartate and works with tryptic digests as well as intact proteins. Using a commercially available isoaspartyl peptide, the relationship between isoaspartate levels and S-adenosyl-l-homocysteine production was found to be linear and stoichiometric over a range of 5-250 pmol. Compared to methods that measure [(3)H]methanol production after methylation with S-adenosyl-l-[methyl-(3)H]methionine, the HPLC method is safer, faster, less expensive, and equally sensitive.

Urea is a protein unfolding agent that can accumulate to locally high concentrations in tissues of many organisms. We used Drosophila melanogaster to test the hypothesis that urea loading would promote formation of isoaspartate (β-carboxyl-linked aspartate), a common form of protein damage that occurs most readily in unstructured polypeptides and flexible regions of folded proteins. Ten populations of flies were tested; five control populations of urea-sensitive flies and five previously selected urea-tolerant populations. We measured the effects of urea consumption on levels of both isoaspartate and protein l -isoaspartate methyltransferase (PIMT), an enzyme believed to function in the repair or removal of isoaspartyl proteins. For both sets of populations, urea feeding for 6 days increased isoaspartyl levels by approximately 60%, supporting the idea that disruption of protein secondary and tertiary structures can accelerate the formation of isoaspartate in vivo. Urea feeding tended to increase PIMT activity in both control and urea-tolerant populations. There were no significant differences in PIMT activities or isoaspartyl levels between the control and urea-tolerant flies raised on normal or urea food. The latter findings indicate that urea tolerance evolved in the selected populations without any significant change in PIMT expression or activity.

Isoaspartyl sites, in which an aspartic acid residue is linked to its C-flanking neighbor via its β-carboxyl side chain, are generally assumed to be an abnormal modification arising as proteins age. The enzyme protein l -isoaspartate methyltransferase (PIMT), present in many bacteria, plants, and animals, catalyzes the conversion of isoaspartate to normal α-linked aspartyl bonds and is thought to serve an important repair function in cells. Having introduced a plasmid into Escherichia coli that allows high-level expression of rat PIMT, we explored the possibility that the rat enzyme reduces isoaspartate levels in E. coli proteins, a result predicted by the repair hypothesis. The present study demonstrates that this is indeed the case; E. coli cells expressing rat PIMT had significantly lower isoaspartate levels than control cells, especially in stationary phase. Moreover, the distribution of isoaspartate-containing proteins in E. coli differed dramatically between logarithmic- and stationary-phase cultures. In stationary-phase cells, a number of proteins in the molecular mass range of 66 to 14 kDa contained isoaspartate, whereas in logarithmic-phase cells, nearly all of the detectable isoaspartate resided in a single 14-kDa protein which we identified as ribosomal protein S11. The near stoichiometric levels of isoaspartate in S11, estimated at 0.5 mol of isoaspartate per mol of S11, suggests that this unusual modification may be important for S11 function.

The issue of sex influences on the brain is rapidly moving center stage, driven by abundant results proving that subject sex can and regularly does alter, negate, and even reverse neuroscientific findings and conclusions down to the molecular level and thus can no longer be justifiably marginalized or ignored.

Mammalian brain contains a high mass protein (HMAP) that is unusually rich in atypical L-isoaspartyl (isoAsp) linkages. HMAP has now been purified from bovine brain by anion exchange, hydroxylapatite, and size exclusion chromatography. It is self-aggregating, acidic, and soluble in 5% trichloroacetic acid. Treatment with chondroitinase ABC eliminates the self-aggregation of HMAP and generates several distinct core proteins with estimated masses of 350-450 (doublet), 180, and 100 kDa, indicating that it is composed mainly of chondroitin sulfate proteoglycans (CSPGs). Most of the isoAsp resides in the 350-450-kDa core protein, which was identified by immunoblotting as phosphacan, a CSPG abundant in adult brain. The regional distribution and developmental profile of HMAP in rat brain support this identification. The 180-kDa core protein contains a tenascin-R-related molecule, consistent with recent observations that phosphacan forms a tight complex with tenascin-R. The average phosphacan molecule in adult brain contains at least seven isoAsp sites. Molecular heterogeneity due to isoAsp may explain some of the complex binding properties phosphacan exhibits with its natural ligands. Formation of isoAsp may be important in the roles that phosphacan and other CSPGs play in development of the nervous system.

PurposeTo determine the propensity of retinal proteins for spontaneous damage via formation of isoaspartyl sites, a common type of protein damage that could contribute to retinal disease.MethodsTissue extracts were obtained from retinas and brains of control mice and from mice in which the gene for protein L-isoaspartate O-methyltransferase (PIMT; an enzyme that repairs isoaspartyl protein damage) was knocked out. PIMT expression in these extracts was measured by Western blot, and its specific activity was assayed by monitoring the rate of [(3)H]methyl transfer from S-adenosyl-[methyl-(3)H]L-methionine to γ-globulin. Isoaspartate levels in extracts were measured by their capacity to accept [(3)H]methyl groups via the PIMT-catalyzed methylation reaction. To compare molecular weight distributions of isoaspartyl-rich proteins in retina versus brain, proteins from PIMT knockout (KO) and control mice were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF). Isoaspartyl proteins were (3)H-labeled on-blot using a PIMT overlay and imaged by autoradiography.ResultsWhen normalized to the β-actin content of each tissue, retina was found to be nearly identical to brain with regard to expression and activity of PIMT and its propensity to accumulate isoaspartyl sites when PIMT is absent. The two tissues show distinct differences in the molecular weight distribution of isoaspartyl proteins.ConclusionsThe retina is rich in PIMT activity and contains a wide range of proteins that are highly susceptible to this type of protein damage. Recoverin may be one such protein. Isoaspartate formation, along with oxidation, should be considered as a potential source of protein dysfunction and autoimmunity in retinal disease.

A previously unidentified endogenous substrate for protein -isoaspartyl methyltransferase in mammalian brain has been characterized and partially purified. This high mass methyl-accepting protein (HMAP) is concentrated in rat brain cytosol and is not detectable in rat liver, heart, lung, kidney, or skeletal muscle. HMAP is acidic and heterogeneous in size, with an average mass, as judged by size-exclusion high performance liquid chromatography, greater than 700 kDa. After partial purification from cow brain by anion-exchange chromatography, ammonium sulfate fractionation, and gel filtration, HMAP could accept 12.1 nmol of methyl groups per mg of protein, suggesting that it contains a level of isoaspartate at least 50 times greater than that of the average protein in brain cytosol. Partially purified HMAP is degraded by trypsin, verifying that it is composed, at least in part, of protein. Additional studies on this unusual macromolecule may shed important new light on mechanisms of isoaspartate formation in cells and the molecular pathology of brain aging.

The formation of isoaspartyl sites during aging of rat tubulin in vitro and in vivo has been studied. When incubated in vitro at pH 7.4, 37 degrees C, purified rat brain tubulin accumulated isoaspartyl sites at a rate > or = 2.4 isoaspartyl sites per 100 tubulin subunits (50 kDa) per day for 30 days. Isoaspartate levels were estimated by the transfer of radiolabeled methyl groups from S-adenosyl-L-[methyl-3H]-methionine in a reaction catalyzed by protein-L-isoaspartyl methyltransferase. isoaspartate formation occurred in parallel with, but was not dependent upon, extensive cross-linking of tubulin via formation of intermolecular disulfide bonds. When rat PC12 cells were incubated for 24 or 72 h in the presence of adenosine dialdehyde, a potent methyltransferase inhibitor, a substantial and consistent increase in the isoaspartate content of tubulin was observed. This suggests that tubulin constantly undergoes isoaspartate formation in vivo, but that the levels are normally kept low by methylation-dependent repair. These findings support the hypothesis that protein-isoaspartyl methyltransferase plays a key role in countering spontaneous damage reactions to proteins associated with cell aging. These results also suggest that tubulin is an important target for protein-isoaspartyl methyltransferase in vivo.

Isoaspartate (isoAsp) formation is a major source of protein damage that is kept in check by the repair function of protein L-isoaspartyl methyltransferase (PIMT). Mice deficient in PIMT accumulate isoAsp-containing proteins, resulting in cognitive deficits, abnormal neuronal physiology and cytoarchitecture, and fatal epileptic seizures 30–60 days after birth. Synapsins I and II, dynamin-1, collapsin response mediator protein 2 (CRMP2), and α/β-tubulin are major targets of PIMT in brain. To investigate links between isoAsp accumulation and the neurological phenotype of the KO mice, we used Western blotting to compare patterns of in vivo phosphorylation or acetylation of the major PIMT targets listed above. Phosphorylations of synapsins I and II at Ser-9 were increased in female KO vs. WT mice, and acetylation of tubulin at Lys-40 was decreased in male KO vs. WT mice. Average levels of dynamin-1 phosphorylation at Ser-778 and Ser-795 were higher in male KO vs. WT mice, but the statistical significance (P>0.1) was low. No changes in phosphorylation were found in synapsins I and II at Ser-603, in CRMP2 at Ser-522 or Thr-514, in DARPP-32 at Thr-34, or in PDK1 at Ser-241. General levels of phosphorylation assessed with Pro-Q Diamond stain, or an anti-phosphotyrosine antibody, appeared similar in the WT and KO mice. We conclude that isoAsp accumulation is associated with altered functional status of several neuronal proteins that are highly susceptible to this type of damage. We also uncovered unexpected differences in how male and female mice respond to isoAsp accumulation in the brain.

Histone H2B is a common target of autoantibodies in both spontaneous and drug-induced systemic lupus erythematosus (SLE). Recent studies demonstrate that Asp(25) of histone H2B (H2B) spontaneously converts to an isoaspartic acid (isoAsp) in vivo. Our laboratory has demonstrated that the posttranslational modification of an aspartic acid to an isoaspartic acid within self-peptides renders otherwise ignored peptides immunogenic. Analysis of serum from lupus-prone mice and histone antibody positive SLE patients revealed antibodies specific to the Asp and isoAsp H2B(21-35) peptide, and that the expression of these antibodies is dependent on TLR9. IsoAsp H2B(21-35) is immunogenic in non-autoimmune prone mice and mice lacking the ability to repair isoAsp have significantly reduced levels of antibodies to H2B. Asp H2B(21-35) incubated at physiological temperatures and pH acquires the isoAsp modification, demonstrating that H2B(21-35) is prone to spontaneous isoAsp formation in vivo. Autoimmunity to isoAsp H2B suggests that this form of the autoantigen may be critical in the induction of anti-histone autoantibodies in human SLE and in murine models of disease.

Protein L-isoaspartyl methyltransferase (PIMT) repairs abnormal isoaspartyl peptide bonds in age-damaged proteins. It has been reported that synuclein, a protein implicated in neurodegenerative diseases, is a major target of PIMT in mouse brain. To extend this finding and explore its possible relevance to neurodegenerative diseases, we attempted to determine the stoichiometry of isoaspartate accumulation in synuclein in vivo and in vitro. Brain proteins from PIMT knockout mice were separated by 2D electrophoresis followed by on-blot [(3)H]-methylation to label isoaspartyl proteins, and by immunoblotting to confirm the coincident presence of synuclein. On-blot (3)H-methylation revealed numerous isoaspartyl proteins, but no signal in the position of synuclein. This finding was corroborated by immunoprecipitation of synuclein followed by on-blot (3)H-methylation. To assess the propensity of synuclein to form isoaspartyl sites in vitro, samples of recombinant mouse and human α-synucleins were aged for two weeks by incubation at pH 7.5 and 37 °C. The stoichiometries of isoaspartate accumulation were extremely low at 0.02 and 0.07 mol of isoaspartate per mol of protein respectively. Using a simple mathematical model based on the first order kinetics of isoaspartyl protein methyl ester hydrolysis, we ascribe the discrepancy between our results and the previous report to methodological limitations of the latter stemming from an inherent, and somewhat counterintuitive, relationship between the propensity of proteins to form isoaspartyl sites and the instability of the (3)H-methyl esters used to tag them. The results presented here indicate that synuclein is not a major target of PIMT in vivo, and emphasize the need to minimize methyl ester hydrolysis when using methylation to assess the abundance of isoaspartyl sites in proteins.

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.

When incubated at pH 7.3, 37 degrees C, human recombinant tissue plasminogen activator accumulated 0.77 mol of isoaspartate per mol of plasminogen activator over a 14-day period. Isoaspartate was detected by enzymatic transfer of 3H-labeled methyl groups from S-adenosyl-L-methionine in a reaction catalyzed by protein L-isoaspartyl methyltransferase. Analysis of tryptic peptides derived from aged plasminogen activator revealed that the two major sites of isoaspartate accumulation resulted from deamidation of Asn58 in the sequence -FNGG- and Asn177 in the sequence -GNSD-. Significant levels of isoaspartate also accumulated via deamidation of Asn37 in the sequence -CNSG-. All three sites occur in sequences predicted from studies with synthetic peptide to be unstable. All three sites appear to be on the surface of the protein, and all three occur in regions of the protein predicted to have higher than average chain mobility. These findings add support to the idea that sequence and flexibility play major roles in determining susceptibility to deamidation and peptide bond isomerization at Asn and Asp sites under mild conditions. These studies also illustrate the utility of enzymatic methylation for characterizing sites of deamidation in a large protein that contains numerous disulfide bonds and several sites of glycosylation.

Several proteins that interact with RNA, e.g. the heterogenous ribonucleoprotein particle A and B proteins, fibrillarin and nucleolin, contain the modified amino acid NG,NG-dimethylarginine. Here, we report that two synthetic peptides, Ac-GGRGGFGGRGGFGGRGGFG-NH2 (R3) and GGFGGRGGFG-NH2 (R1), which are based on methylated sequences in fibrillarin and nucleolin, inhibit the methylation of a large majority of the methyl-accepting proteins observed in extracts of adenosine dialdehyde-treated PC12 cells. Concomitantly, the peptides themselves become methylated, suggesting that they compete for the same enzyme that carries out the bulk of N-methylation in PC12 cells. R3 potently inhibits formation of NG,NG-dimethylarginine in PC12 substrates, with a lesser effect on NG-monomethylarginine and NG,N'G-dimethylarginine. Bovine brain contains an activity that methylates PC12 methyl acceptors. After partial purification, the bovine methyltransferase efficiently modifies R3 and R1, yielding half-maximal rates of methylation at approximately 0.2 and approximately 2 microM peptide, respectively. A search of the GenPept database for the FGGRGGF motif revealed 13 candidate methyl acceptors containing arginine and at most two similar substitutions or one mismatch. Of these, 10 are known or presumed to interact with RNA. These findings are consistent with the hypothesis that a majority of proteins containing NG,NG-dimethylarginine interact with RNA.

Protein isoaspartyl methyltransferase is implicated in the repair of age-damaged proteins containing atypical, isoaspartyl linkages. To test the prediction that isoaspartyl proteins would accumulate during methyltransferase inhibition, rat PC12 cells were treated with the indirect methylation inhibitor, adenosine dialdehyde. We observed a marked, dose- and time-dependent, reversible accumulation of substrates for the enzyme that closely paralleled the elevation of its competitive inhibitor, S-adenosylhomocysteine. The accumulation of substrates also paralleled a cytostatic action of adenosine dialdehyde; however, 30 microM 3-deazaadenosine, another indirect methylation inhibitor, also caused an accumulation of substrates without affecting cell division, and other cytostatic agents did not affect substrate levels. Acidic gel electrophoresis revealed increased methyl-accepting capacity in a broad spectrum of proteins, with predominant increases in discrete bands of M(r) 46,000 and 110,000. A major substrate (M(r) 17,400) in untreated cells did not increase in methyl-accepting capacity during treatment. Methyl groups at the accumulated sites did not survive conventional electrophoresis, indicating the lability characteristic of isoaspartyl methyl esters in damaged proteins. These results are consistent with an involvement of the methyltransferase in the metabolism of damaged proteins, and they provide a basis for the characterization of physiological substrates for the enzyme.

Protein L-isoaspartyl methyltransferase, an enzyme enriched in brain, is implicated in the repair of age-damaged proteins containing atypical, isoaspartyl peptide bonds. We have investigated the kinetics of methylation using a synthetic peptide substrate having the structure Trp-Ala-Gly-Gly-isoAsp-Ala-Ser-Gly-Glu. Double-reciprocal plots of initial velocity versus concentration of S-adenosylmethionine (AdoMet) at different fixed concentrations of peptide gave straight lines converging at a positive 1/v value and a negative 1/AdoMet value. The product S-adenosylhomocysteine (AdoHcy) was a competitive inhibitor towards AdoMet and a linear mixed-type inhibitor towards peptide. These results are consistent with the rapid-equilibrium random sequential bi-bi mechanism previously proposed for the enzyme, but they also reveal the formation of the dead-end, enzyme-peptide-AdoHcy, complex. The rate constants were: Vmax = 32-34 nmol/min/mg, Kpeptide = 7.6-9.4 microM, KAdoMet = 1.9-2.2 microM, alpha = 0.43-0.53, KAdoHcy = 0.08 microM, gamma = 2.9. The interaction factors alpha and gamma indicate that binding of enzyme to peptide increases its affinity for AdoMet and decreases its affinity for AdoHcy. Methylation was linear with time throughout the transfer of 2 mol of methyl groups/mol of enzyme. This absence of burst kinetics suggests that slow release of products cannot explain the low turnover number.

Bovine brain is known to contain two major isoforms of protein L-isoaspartyl methyltransferase (PIMT), an enzyme that facilitates repair of atypical L-isoaspartyl peptide bonds in proteins. Although the two isoforms can be separated by anion-exchange chromatography, they appear to have similar, if not identical, substrate specificities in vitro. The more basic type I isoform has been extensively characterized, and its complete sequence has been reported. The present study was undertaken in an attempt to understand the structural and functional uniqueness of the more acidic type II isoform. Electrospray mass spectrometry of the intact enzymes revealed that the type II isoform is approximately 43 amu heavier than the type I isoform. Cyanogen bromide cleavage followed by HPLC with on-line mass analysis revealed that the type II isoform contains a unique C-terminal fragment which is 43 amu heavier than the corresponding fragment from the type I isoform. Amino acid composition analysis and direct sequencing of this fragment indicate that the type II isoform ends in the sequence ...RDEL, while the type I is known to end in ...RWK. Since ...RDEL, like ...KDEL, serves as an effective endoplasmic reticulum retention signal, we propose that the type II isoform serves to repair damaged proteins within the endoplasmic reticulum or, perhaps, within some other specialized compartment of the cell. Comparison of the protein sequences of the two bovine brain isoforms to DNA sequences for rodent PIMT reported by others suggests that the type II isoform may be produced by splicing within the codon for Arg224.

It was demonstrated recently that substrates for protein N -methyltransferases ( J. Najbauer and D. W. Aswad, 1990, J. Biol. Chem. 265, 12,717–12,721 ) and protein carboxyl methyltransferases ( J. Najbauer, B. A. Johnson, and D. W. Aswad, 1991, Anal. Biochem. 197, 412–420 ) accumulate when rat PC12 cells are cultured in the presence of the methylation inhibitor, adenosine dialdehyde. In the present report, we have further characterized this phenomenon in PC12 cells and in two other, widely used cell types. Adenosine dialdehyde was found to increase the methyl-accepting capacity of proteins in human skin fibroblasts and mouse Sp2/0 myeloma cells. However, both the level of methyl incorporation in untreated cells and the amount of stimulation afforded by inhibitor treatment were substantially lower in these cells than in PC12 cells. All three cell lines accumulated methyl acceptor(s) at 17–21 kDa. The PC12 cells and the fibroblasts also exhibited stimulation of three apparently similar proteins in the 33- to 38-kDa region, where several arginine-methylated proteins involved in RNA processing would be expected. The optimal conditions for methylation of PC12 cell extracts with regard to pH, time of methylation, and S -[ methyl - 3 H]adenosyl- l -methionine concentration were characterized. Increased methyl incorporation was detected after adenosine dialdehyde treatments as short as 2 h, and methylation of most substrates continued to increase as the time of treatment was extended to 72 h. The kinetics of accumulation varied from substrate to substrate. Fluorograms of two-dimensional gels of extracts from untreated PC12 cells incubated in the presence of S -[ methyl - 3 H] adenosyl- l -methionine revealed patterns of methyl incorporation similar to those of treated cells, but longer exposure times were necessary (e.g., 35 days vs 7 days). These findings suggest that the inhibitor treatment works mainly by inhibiting the post- or cotranslational methylation of a “normal” array of cellular proteins.

Following incubation of rat brain membranes with the catalytic subunit of cAMP-dependent protein kinase and [32P]ATP, a previously unreported phosphoprotein, pp59, was found to be enriched in cerebellar synaptic plasma membrane preparations, but not in those prepared from cerebral cortex, hippocampus, olfactory bulb, or striatum. This protein, which has an Mr of 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is not phosphorylated by the cGMP-dependent protein kinase. While pp59 was consistently detected in cerebellar membranes from adult Sprague-Dawley rats, it was not detected in bovine or rabbit cerebellar membranes. Moreover, pp59 did not comigrate with any of the autophosphorylated subunits of the Ca2+/calmodulindependent protein kinase in rat cerebellar membranes. Extraction of pp59 from these membranes could be accomplished with 6 M urea, but not with 0.4 M NaCl or 0.5% (v/v) Triton X-100. The urea solubility suggests that pp59 is not an integral membrane protein. Acid hydrolysis of the protein phosphorylated in vitro yielded phosphoserine but no significant amount of phosphothreonine or phosphotyrosine. Further analysis of pp59 may provide new insights into the role of cAMP in modulation of synaptic function in the cerebellum.

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)

The specific activity of protein L-isoaspartyl methyltransferase, an enzyme implicated in the metabolism of damaged, isoaspartate-containing proteins, has been measured in postmortem samples of parietal cortex from 30 individuals (19 with Alzheimer's disease and 11 controls). Methyltransferase specific activity was positively correlated with age at death, increasing by 2.9 pmol/min/mg of protein for every ten years of age (r = .51, p less than 0.005). This correlation was significant in the control and Alzheimer's disease groups alike. Specific activity also appeared to be about 15% higher in females than in age- and diagnosis-matched males (p less than 0.05). No significant differences were observed between age- and sex-matched Alzheimer patients and controls, suggesting that a deficiency in this enzyme is not responsible for the accumulation of abnormal proteins in Alzheimer's disease.

Formation of l-isoaspartyl (isoAsp) peptide bonds is a major source of protein damage in vivo and in vitro. Accumulation of isoAsp in cells is limited by a ubiquitous repair enzyme, protein l-isoaspartyl methyltransferase (PIMT). Reduction of PIMT activity in mouse brain or rat PC12 cells leads to a dramatic and selective accumulation of isoAsp sites in histone H2B. To learn more about the mechanism and specificity of isoAsp formation in histones, we purified mononucleosomes from rat liver and chicken erythrocytes and subjected them to in vitro aging for 0-16 days. In rat nucleosomes, the pattern of isoAsp accumulation duplicated that observed in vivo; only H2B accumulated significant isoAsp that we have now localized to the Asp25-Gly26 bond in the N-terminal tail. In chicken nucleosomes, isoAsp accumulated mainly in histone H2A and, to a lesser extent, in histone H2B. Minor sequence differences are consistent with the species-specific patterns of isoAsp accumulation and suggest that, in chicken, isoAsp occurs at the Asp121-Ser122 bond in the flexible C-terminal tail of H2A and at the Asp26-Lys27 bond in the N-terminal tail of H2B. The aging-induced accumulation of isoAsp in rat and chicken nucleosomes is repaired upon incubation of the damaged nucleosomes with PIMT and AdoMet. Our findings suggest that in vivo generation of isoAsp sites in histones occurs as a self-catalyzed process at the level of the nucleosome and is driven by the same structural features that have been shown to promote isoAsp formation in purified proteins and synthetic peptides.

Protein N-methylation is a widespread modification whose functions are poorly understood. To overcome the inherent technical difficulties in identification of N-methylated proteins, we cultured PC12 cells with a methylation inhibitor, in expectation that proteins would accumulate in a hypomethylated state. Cell extracts were then incubated with [methyl-3H]S-adenosyl-L-methionine to label methyl-accepting sites via endogenous methyltransferases. Using two-dimensional gel electrophoresis we detected over 50 methyl acceptors, ranging from 18 to 120 kDa. Most had isoelectric points greater than 7.0. NG,NG-Dimethylarginine and NG-monomethylarginine accounted for about 90% of the methyl-3H-amino acids recovered after acid hydrolysis and thin-layer chromatography. The production of hypomethylated proteins should prove useful, not only in the identification of new methyl acceptors, but also in the isolation and characterization of new methyltransferases.

Posttranslational protein modifications influence a number of immunologic responses ranging from intracellular signaling to protein processing and presentation. One such modification, termed isoaspartyl (isoAsp), is the spontaneous nonenzymatic modification of aspartic acid residues occurring at physiologic pH and temperature. In this study, we have examined the intracellular levels of isoAsp residues in self-proteins from MRL+/+, MRL/lpr, and NZB/W F1 mouse strains compared with nonautoimmune B10.BR mice. In contrast to control B10.BR or NZB/W mice, the isoAsp content in MRL autoimmune mice increased and accumulated with age in erythrocytes, brain, kidney, and T lymphocytes. Moreover, T cells that hyperproliferate to antigenic stimulation in MRL mice also have elevated intracellular isoAsp protein content. Protein l-isoaspartate O-methyltransferase activity, a repair enzyme for isoAsp residues in vivo, remains stable with age in all strains of mice. These studies demonstrate a role for the accumulation of intracellular isoAsp proteins associated with T cell proliferative defects of MRL autoimmune mice.

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.

The accumulation of potentially deleterious L-isoaspartyl linkages in proteins is prevented by the action of protein L-isoaspartyl O-methyltransferase, a widely distributed enzyme that is particularly active in mammalian brain. Methyltransferase-deficient (knock-out) mice exhibit greatly increased levels of isoaspartate and typically succumb to fatal epileptic seizures at 4-10 weeks of age. The link between isoaspartate accumulation and the neurological abnormalities of these mice is poorly understood. Here, we demonstrate that synapsin I from knock-out mice contains 0.9 +/- 0.3 mol of isoaspartate/mol of synapsin, whereas the levels in wild-type and heterozygous mice are undetectable. Transgenic mice that selectively express methyltransferase only in neurons show reduced levels of synapsin damage, and the degree of reduction correlates with the phenotype of these mice. Isoaspartate levels in synapsin from the knock-out mice are five to seven times greater than those in the average protein from brain cytosol or from a synaptic vesicle-enriched fraction. The isoaspartyl sites in synapsin from knock-out mice are efficiently repaired in vitro by incubation with purified methyltransferase and S-adenosyl-L-methionine. These findings demonstrate that synapsin I is a major substrate for the isoaspartyl methyltransferase in neurons and suggest that isoaspartate-related alterations in the function of presynaptic proteins may contribute to the neurological abnormalities of mice deficient in this enzyme.

Protein l-isoaspartyl methyltransferase (PIMT) has been implicated in the repair or metabolism of proteins containing atypical l-isoaspartyl peptide bonds. The repair hypothesis is supported by previous studies demonstrating in vitro repair of isoaspartyl peptides via formation of a succinimide intermediate. Utilization of this mechanism in vivo predicts that PIMT modification sites should exhibit significant racemization as a side reaction to the main repair pathway. We therefore studied the d/l ratio of aspartic acid at specific sites in histone H2B, a known target of PIMT in vivo. Using H2B from canine brain, we found that Asp25 (the major PIMT target site in H2B) was significantly racemized, exhibiting d/l ratios as high as 0.12, whereas Asp51, a comparison site, exhibited negligible racemization (d/l ≤ 0.01). Racemization of Asp25 was independent of animal age over the range of 2–15 years. Using H2B from 2–3-week mouse brain, we found a similar d/l ratio (0.14) at Asp25 in wild type mice, but substantially less racemization (d/l = 0.035) at Asp25 in PIMT-deficient mice. These findings suggest that PIMT functions in the repair, rather than the metabolic turnover, of isoaspartyl proteins in vivo. Because PIMT has numerous substrates in cells, these findings also suggest that d-aspartate may be more common in cellular proteins than hitherto imagined and that its occurrence, in some proteins at least, is independent of animal age.

The RNA-binding protein HuR stabilizes labile mRNAs carrying AU-rich instability elements. This mRNA stabilization can be induced by hypoxia, lipopolysaccharide, and UV light. The mechanism by which these stimuli activate HuR is unclear and might be related to post-translational modification of this protein. Here we show that HuR can be methylated on arginine. However, HuR is not a substrate for PRMT1, the most prominent protein-arginine methyltransferase in mammalian cells, which methylates a number of heterogeneous nuclear ribonucleoproteins. Instead, HuR is specifically methylated by coactivator-associated arginine methyltransferase 1 (CARM1), a protein-arginine methyltransferase previously shown to serve as a transcriptional coactivator. By analyzing methylation of specific HuR arginine-to-lysine mutants and by sequencing radioactively methylated HuR peptides, Arg(217) was identified as the major HuR methylation site. Arg(217) is located in the hinge region between the second and third of the three HuR RNA recognition motif domains. Antibodies against a methylated HuR peptide were used to demonstrate in vivo methylation of HuR. HuR methylation increased in cells that overexpressed CARM1. Importantly, lipopolysaccharide stimulation of macrophages, which leads to HuR-mediated stabilization of tumor necrosis factor alpha mRNA in these cells, caused increased methylation of endogenous HuR. Thus, CARM1, which plays a role in transcriptional activation through histone H3 methylation, may also play a role in post-transcriptional gene regulation by methylating HuR.

The preferential in vitro methylation of histone H3 by coactivator-associated arginine methyltransferase 1 (CARM1) has been proposed as a basis for its ability to enhance gene transcription [Chen, D., et al. (1999) Science 284, 2174-2177]. To further evaluate the significance of H3 methylation, we studied the kinetics and site specificity of its modification by CARM1. Affinity-purified CARM1 methylated recombinant chick H3, which is free of posttranslational modifications, and calf thymus H3, which is heterogeneous with regard to preexisting modifications, equally well, exhibiting a V(max) of 4500 pmol min(-1) (mg of enzyme)(-1) and an apparent K(m) for H3 ofor = 0.2 microM. The catalytic efficiency (k(cat)/K(m)) of CARM1 toward H3 was at least 1000 times that toward R1 (GGFGGRGGFGG-amide), a highly effective substrate for protein arginine methyltransferase 1. Peptide mapping of 3H-methyl-labeled H3 indicated methylation at Arg-2, Arg-17, and Arg-26 in the N-terminal region and at one or more of four arginines (128/129/131/134) at the C-terminus. Two of the N-terminal sites, Arg-17 and Arg-26, occur in the sequence KAXRK and appear to be more efficiently methylated than Arg-2. CARM1 catalyzed formation of N(G),N(G)-dimethylarginine (asymmetric) but little or no N(G),N'(G)-dimethylarginine (symmetric) and no form of methyllysine. Amino acid analysis of untreated calf thymus H3 revealed that 3.7% of the molecules naturally contain asymmetric dimethylarginine and/or monomethylarginine. Our findings support the hypothesis that methylation of H3 may be involved in the mechanism of transcriptional coactivation by CARM1 of genes whose expression is under the control of nuclear receptors.

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.