Jonathan D. Lowenson

Limited accumulation of damaged proteins in l-isoaspartyl (D-aspartyl) O-methyltransferase-deficient mice.

l-Isoaspartyl (d-aspartyl) O-methyltransferase (PCMT1) can initiate the conversion of damaged aspartyl and asparaginyl residues to normal l-aspartyl residues. Mice lacking this enzyme (Pcmt1−/− mice) have elevated levels of damaged residues and die at a mean age of 42 days from massive tonic-clonic seizures. To extend the lives of the knockout mice so that the long term effects of damaged residue accumulation could be investigated, we produced transgenic mice with a mouse Pcmt1 cDNA under the control of a neuron-specific promoter. Pcmt1 transgenic mice that were homozygous for the endogenous Pcmt1 knockout mutation (“transgenic Pcmt1−/− mice”) had brain PCMT1 activity levels that were 6.5–13% those of wild-type mice but had little or no activity in other tissues. The transgenicPcmt1−/− mice lived, on average, 5-fold longer than nontransgenic Pcmt1−/− mice and accumulated only half as many damaged aspartyl residues in their brain proteins. The concentration of damaged residues in heart, testis, and brain proteins in transgenic Pcmt1−/− mice initially increased with age but unexpectedly reached a plateau by 100 days of age. Urine fromPcmt1−/− mice contained increased amounts of peptides with damaged aspartyl residues, apparently enough to account for proteins that were not repaired intracellularly. In the absence of PCMT1, proteolysis may limit the intracellular accumulation of damaged proteins but less efficiently than in wild-type mice having PCMT1-mediated repair.

Phenotypic Analysis of Seizure-prone Mice Lacking l-Isoaspartate (d-Aspartate)O-Methyltransferase

Within proteins and peptides, bothl-asparaginyl and l-aspartyl residues spontaneously degrade, generating isomerized and racemized aspartyl residues. The enzyme protein l-isoaspartate (d-aspartate) O-methyltransferase (E.C. 2.1.1.77) initiates the conversion of l-isoaspartyl andd-aspartyl residues to normal l-aspartyl residues. This “repair” reaction helps to maintain proper protein conformation by preventing the accumulation of damaged proteins containing abnormal amino acid residues. Pcmt1–/– mice manifest two key phenotypes: a fatal seizure disorder and retarded growth. In this study, we characterized both phenotypes and demonstrated that they are linked. Continuous electroencephalogram monitoring of Pcmt1–/– mice revealed that abnormal cortical activity for ∼50% of each 24-h period, even in mice that had no visible evidence of convulsions. The fatal seizure disorder inPcmt1–/– mice can be mitigated but not eliminated by antiepileptic drugs. Interestingly, antiepileptic therapy normalized the growth of Pcmt1–/– mice, suggesting that the growth retardation is due to seizures rather than a global disturbance in growth at the cellular level. Consistent with this concept, the growth rate of Pcmt1–/– fibroblasts was indistinguishable from that of wild-type fibroblasts.

Proteomic Identification of Novel Substrates of a Protein Isoaspartyl Methyltransferase Repair Enzyme

We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein l-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1-/-) accumulate more isoaspartyl residues than their wild-type littermates, with the most “damaged” residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1-/- mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-3H]methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and two-dimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1-/- animals compared with Pcmt1+/+ littermates. Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxyl-terminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, β-synuclein, and α-synuclein, are all substrates for the l-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1-/- brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice.

Protein repair L-isoaspartyl methyltransferase in plants. Phylogenetic distribution and the accumulation of substrate proteins in aged barley seeds.

Protein L-isoaspartate (D-aspartate) O-methyltransferases (MTs; EC 2.1.1.77) can initiate the conversion of detrimental L-isoaspartyl residues in spontaneously damaged proteins to normal L-aspartyl residues. We detected this enzyme in 45 species from 23 families representing most of the divisions of the plant kingdom. MT activity is often localized in seeds, suggesting that it has a role in their maturation, quiescence, and germination. The relationship among MT activity, the accumulation of abnormal protein L-isoaspartyl residues, and seed viability was explored in barley (Hordeum vulgare cultivar Himalaya) seeds, which contain high levels of MT. Natural aging of barley seeds for 17 years resulted in a significant reduction in MT activity and in seed viability, coupled with increased levels of “unrepaired” L-isoaspartyl residues. In seeds heated to accelerate aging, we found no reduction of MT activity, but we did observe decreased seed viability and the accumulation of isoaspartyl residues. Among populations of accelerated aged seed, those possessing the highest levels of L-isoaspartyl-containing proteins had the lowest germination percentages. These results suggest that the MT present in seeds cannot efficiently repair all spontaneously damaged proteins containing altered aspartyl residues, and their accumulation during aging may contribute to the loss of seed viability.

Synapsin I Is a Major Endogenous Substrate for Protein L-Isoaspartyl Methyltransferase in Mammalian Brain

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.

Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning

The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when PIMT protein binding was poisoned with S-adenosyl homocysteine. After four rounds, phage titer plateaued in AdoMet-containing pans, whereas titer declined in both controls. This strategy identified 17 in-frame PIMT target proteins, including a cupin-family protein similar to those identified previously using on-blot methylation. All recovered phage had at least one susceptible Asp or Asn residue. Five targets were recovered independently. Two in-frame targets were produced in Escherichia coli as recombinant proteins and shown by on-blot methylation to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution upon thermal insult. Mutant analysis of plants deficient in any of three in-frame PIMT targets resulted in demonstrable phenotypes. An over-representation of clones encoding proteins involved in protein production suggests that the translational apparatus comprises a subgroup for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would hinder protein function in these targets, possibly resulting in poor seed performance.

Intracellular protein modification associated with altered T cell functions in autoimmunity

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.

Chemical modifications of deposited amyloid-beta peptides.

Publisher Summary This chapter describes the procedures that demonstrate that few of the A β peptides present in dense neuritic plaques in a brain with Alzheimers disease are untouched by one or more of several spontaneous chemical modifications. Only 10% of the peptides possess the normal L-aspartyl residue expected at the N terminus, whereas 20% start with an L-isoaspartyl residue, 51% start with a pyroglutamyl residue, and the rest suffer various amounts of proteolysis. Only 20% of the residues in the seventh position are in the normal L-aspartyl configuration, with the rest being isomerized and/or racemized. Racemization also affects 4–6% of the serine residues. If these modifications arise randomly through the A β population in a stable amyloid deposit, fewer than 2% of the peptides should remain unaltered. Even if chemical modification is not random (i.e., younger peptides are undamaged, whereas older peptides are multiply modified), no more than 10% of the A β will be unaltered at the N terminus. A β from vascular amyloid deposits is less damaged, and thus perhaps younger than that in neuritic deposits, but modification of 37% of these peptides at the N-terminus is still significant. It is therefore important to take these modifications into account when studying the role of A β in the development and progression of Alzheimers disease.

Spontaneous degradation and enzymatic repair of aspartyl and asparaginyl residues in aging red cell proteins analyzed by computer simulation

The inherent instability of proteins may be a limiting factor in the longevity of an organism. Spontaneously altered forms may themselves be toxic, or their accumulation may simply crowd out normal proteins. Two of the major sites of nonenzymatic degradation are aspartyl and asparaginyl residues, which are susceptible to an intramolecular reaction that results in the deamidation of asparaginyl residues and the isomerization and racemization of both aspartyl and asparaginyl residues. In all eucaryotic cells examined so far, an enzyme is present that can recognize at least some of these damaged sites and initiate their conversion to normal forms. This enzyme, the type II protein carboxyl methyltransferase, catalyzes the methyl esterification of L-isoaspartyl and D-aspartyl residues, enabling them to spontaneously revert to their normal L-aspartyl configurations. In this study, we utilize data on the rates of spontaneous degradation and enzymatic methylation in a computer program that simulates these reactions in the intact human erythrocyte. The results show that the methyltransferase may have an important role in limiting the accumulation of proteins containing altered aspartyl and asparaginyl residues.

Protein aging: Extracellular amyloid formation and intracellular repair

Soluble proteins can undergo spontaneous structural and conformational alterations that lead to their stable aggregation into amyloid fibrils. Amyloidogenic proteins have been implicated in several types of age-related pathologic changes. For example, transthyretin amyloid accumulation in the heart can lead to cardiac failure, while β-amyloid deposition within the microvasculature and gray matter of the brain is linked to cerebral hemorrhage and neuronal death. Over the course of evolution, protein structures have developed that largely resist such aggregation. Spontaneous chemical modifications correlated with the normal aging process, however, including the deamidation, isomerization, and racemization of asparaginyl and aspartyl residues, as well as the oxidation and glycation of various amino acid residues, may contribute to amyloid formation by altering protein structure. In fact, a recent chemical analysis of neuritic plaque and vascular β-amyloid deposits from the brains of Alzheimers disease victims has revealed that the majority of the aspartyl residues in β-amyloid are in the isomerized and/or racemized configuration. Although enzymes exist that can reverse at least part of this damage for intracellular proteins, the accumulation of extracellular proteins containing altered residues might contribute to the deterioration of heart, brain, and other tissues that occurs with aging and disease.