Dana W. Aswad - US grants

The overall goal of this project is to explore the role of protein carboxyl methylation in regulation of synaptic and neuronal function. Carboxyl methylation is believed to play an important role in modulation of protein function, particularly in neural and endocrine tissues. A widely accepted model views carboxyl methylation as a reversible modification of protein function analogous to phosphorylation-dephosphorylation, yet there are several inconsistencies with this simple model. It has recently been found that carboxyl methylation is not a reversible reaction, but rather that it is the first step in a complex reaction which releases an unmethylated peptide that is different from the original substrate and cannot be remethylated. This finding has important implications for the in vivo function of carboxyl methylation and suggests that a new approach to assaying this enzyme is needed to properly evaluate its role in regulatory events. One immediate aim of the current proposal is to determine the nature of the unusual peptide modification catalyzed by protein carboxyl methyltransferase. Another aim is to use a methanol formation assay to search for endogenous substrates for this enzyme in subfractions of brain tissue and to evaluate the possibility that this reaction is modulated by transmembrane signals such as depolarization and neurotransmitter receptor activation, or by intracellular signals such as cAMP, cGMP and Ca++. This research sould substantially improve our understanding of the biochemical mechanisms which underly regulation of neuronal and synaptic activity in the mammalian brain.

The overall goal of this project is to understand the role of protein carboxyl methylation in neuronal function. There are two current hypotheses of methylation function in eucaryotes: (1) that it mediates some aspect of stimulus-response coupling via the reversible modification of protein function, and (2) that it facilitates the repair or degradation of defective proteins which contain abnormal forms of aspartic acid. In testing the regulation hypothesis, we propose to measure the ability of certain neurotransmitters and depolarizing agents to stimulate transient protein methylation (as indicated by the evolution of radiolabeled methanol), to search in purified subfractions of brain for specific protein which serve as preferential substrates in an in vitro transient methylation assay, and, to determine if the in vitro transient methylation reaction is regulated by the second messengers cAMP, cGMP, Ca++ or phosphatidyl inositol. Our strategy in these experiments is derived from recent indications that carboxyl methylation may serve as an initial activation step in a more complex protein modification reaction than hitherto assumed. An alternative role for carboxyl methylation in the repair or degradatiom of damaged proteins is suggested by the recently discovered selectivity of the methyltransferase enzyme for proteins containing abnormal forms of aspartate, particularly L-isoaspartyl and D-aspartyl residues. We propose to determine if the methyl accepting substrates we have located in synaptic membranes and myelin are enriched in these atypical forms of aspartate, and, to determine if the levels of these abnormal proteins and/or the methyltransferase enzyme changes significantly with age in the human or in association with Alzheimer's disease. Establishing a firm role for carboxyl methylation should provide important new insight on fundamental mechanisms of cellular regulation and/or aging processes in the mammalian nervous system.

Recent findings indicate that formation of isoaspartate (isoAsp) via deamidation of labile Asn-X sequences or direct isomerization at certain Asp-X sequences is a major source of spontaneous protein damage at physiological pH and temperature and that this process may have important consequences for protein function and turnover. This proposal seeks to understand the role of protein sequence and structure on formation of isoaspartate and to explore the effects of isomerization on protein function. Four experimental approaches are proposed. First, we will determine the sites of isoAsp formation in synapsin 1, tissue plasminogen activator, triosephosphate isomerase and the major intrinsic protein of the eye lens. All four of these proteins appear to generate isoAsp at significant rates at physiological pH and temperature. Second, we will synthesize a series homologous Asn- and Asp-containing pentapeptides in which the amino acids neighboring the Asn or Asp are varied in a systematic way. Substitutions will be designed to fill in several critical gaps in our knowledge of how neighboring amino acids influence the propensity of Asn or Asp to isomerize. Knowledge of the sites of isoAsp formation in the above proteins and synthetic peptides will be used to strengthen or refute our current paradigm, which emphasizes the nature of the amino acid linked to the Asn/Asp-carboxyl, together with regional flexibility, to predict sites of isoAsp formation. Our third aim is to determine to what extent isoAsp effects several functional attributes of synapsin 1, including its ability to be phosphorylated by the cAMP- and type II calcium/calmodulin-dependent protein kinases, its ability to bind to small synaptic vesicles, and its ability to bind F-actin. Finally, we will investigate the possibility that protein sequences which are prone to forming isoaspartate may also be prone to crosslinking. These studies should provide important new information on basic mechanisms which govern the stability and integrity of proteins in vitro and in vivo.

The overall goal of the proposed research is to elucidate the function and physiological substrate specificity of protein L-isoaspartyl methyltransferase (PIMT), an enzyme enriched in brain and neuroendocrine tissues. PIMT transfers methyl groups from Sadenosyl-L-methionine onto the free carboxyl group of atypical beta-linked aspartyl residues. Formation of these isopeptide bonds has been shown to be a major source of spontaneous protein damage under physiological conditions. In vitro, PIMT has been shown to catalyze the conversion of the isopeptide linkage back to a normal linkage, adding support to the idea that it may repair damaged proteins in vivo. In exploring this hypothesis further, our first aim will be to characterize a major substrate for PIMT in rat PC12 cells, determine the site of methylation, and determine if, as predicted by the repair hypothesis, this substrate accumulates isoaspartate when PIMT activity is inhibited. Our second aim is to use a PCR-based approach to determine how many distinct isozymes of PIMT exist, to determine how they differ in sequence, and to determine if they are all generated by alternative splicing of a single gene. Our third aim is to characterize a 30 kD protein from cow brain that binds reversibly to the type II isozyme of PIMT. We wish to know how this protein binds to PIMT II, where it is localized in the cell, and if its sequence is similar to any previously characterized protein of known function. Our final aim is to map the PIMT gene to a defined region of a human chromosome. If it maps near any hereditary diseases of unknown etiology, we will assay tissues or cell lines representative of these diseases to determine if a defect in PIMT expression might be the cause. The proposed studies should provide important new information on an enzyme which appears to play a key role in the control of spontaneous damage to cellular proteins.

The overall goal of the proposed research is to elucidate the function and physiological substrate specificity of protein L-isoaspartyl methyltransferase (PIMT), an enzyme enriched in brain and neuroendocrine tissues. PIMT transfers methyl groups from Sadenosyl-L-methionine onto the free carboxyl group of atypical beta-linked aspartyl residues. Formation of these isopeptide bonds has been shown to be a major source of spontaneous protein damage under physiological conditions. In vitro, PIMT has been shown to catalyze the conversion of the isopeptide linkage back to a normal linkage, adding support to the idea that it may repair damaged proteins in vivo. In exploring this hypothesis further, our first aim will be to characterize a major substrate for PIMT in rat PC12 cells, determine the site of methylation, and determine if, as predicted by the repair hypothesis, this substrate accumulates isoaspartate when PIMT activity is inhibited. Our second aim is to use a PCR-based approach to determine how many distinct isozymes of PIMT exist, to determine how they differ in sequence, and to determine if they are all generated by alternative splicing of a single gene. Our third aim is to characterize a 30 kD protein from cow brain that binds reversibly to the type II isozyme of PIMT. We wish to know how this protein binds to PIMT II, where it is localized in the cell, and if its sequence is similar to any previously characterized protein of known function. Our final aim is to map the PIMT gene to a defined region of a human chromosome. If it maps near any hereditary diseases of unknown etiology, we will assay tissues or cell lines representative of these diseases to determine if a defect in PIMT expression might be the cause. The proposed studies should provide important new information on an enzyme which appears to play a key role in the control of spontaneous damage to cellular proteins.

DESCRIPTION (provided by applicant): The overall goal of this research is to explore the biological function of protein methylation reactions, with particular attention to their possible roles in neuronal function. Our studies will focus on two distinct methyltransferase enzymes. One, protein L-isoaspartyl methyltransferase (PIMT). catalyzes methylation of atypical isoaspartyl sites (isoAsp) that arise in certain proteins as a form of spontaneous damage. IsoAsp can render proteins dysfunctional, and/or highly immunogenic in the same animal from which they are derived. Moreover, PIMT knock-out mice accumulate high levels of intracellular isoAsp sites and develop fatal epileptic seizures at 4-6 weeks. To understand more about the function of PIMT and the consequences of isoAsp accumulation, we will: (1) determine if PIMT functions in vivo to rescue damaged proteins or to facilitate their degradation by comparing the turnover rate of histone H2B (a major in vivo substrate for PIMT) in normal vs. PIMT-deficient cells, and by comparing the racemization of the isoAsp prone Asp-25 residue in H2B; (2) identify how isoAsp accumulation affects the function of synapsin-1 and other synaptosomal proteins; (3) determine if isoAsp sites greatly enhance the immunogenicity of mouse H2B in that same species, the extent to which isoAsp H2B induces an autoimmune pathology, and if the sera of patients afflicted with autoimmune diseases such as systemic lupus erythematosus have antibodies or T cells that selectively recognize the isoAsp form of H2B. The second enzyme to be studied is coactivator-associated arginine methyltransferase 1 (CARM 1), an enzyme that forms complexes with specific transcription factors that mediate glucocorticoid-regulated gene expression. To understand more about the function of CARM 1 we will (1) determine if purified HuD (an mRNA-binding protein implicated in neuronal development) is an in vivo substrate for CARM 1 and search for additional CARM 1 substrates in rat PC12 cells using a previously developed methyltransferase inhibitor-based strategy, and (2) determine if mammalian brain (or other tissues) contain a protein-arginine demethylating enzyme that may reverse the methylation reactions catalyzed by CARM 1 and/or related protein arginine methyltransferases. Our proposed studies on PIMT should provide new insights as to the possible contribution of isoAsp formation and PIMT deficiency in diseases afflicting the brain and possibly the immune system. Similarly, studies on CARM1 should provide important new information on how glucocorticoids regulate gene expression in the brain.

DESCRIPTION (provided by applicant): This proposal requests partial funding for a conference on "Biological Methylation" to be held from July 10- 15, 2004, at the Vermont Academy in Saxton's River, Vermont. This will be the 6th biennial conference on this topic, which is held under the auspices of the Federation of American Societies for Experimental Biology (FASEB). "Biological Methylation" is unique in its integrative treatment of S-adenosyI-L-methionine (AdoMet)-dependent methyltransferases, biological processes affected by methylation, and AdoMet metabolism. New methyltransferase enzymes and methyl-accepting substrates are being identified at accelerating rates through the application of modern genomic, proteomic and bioinformatics approaches. Substrates for the methyltransferases now include DNA, RNAs, a wide range of proteins, lipids and a large number of small organic molecules. Because of the increasingly recognized importance of DNA and chromatin methylation in the control of gene expression and chromosomal stability, approximately 70% of the meeting's sessions deal with these, and closely related medical topics, especially carcinogenesis. Other sessions deal with small molecule methylation, structural studies of methyltransferases, and fundamental biochemical and cellular processes related to AdoMet metabolism. This conference is attended by a diverse group of junior and senior scientists from a range of academic and research institutions. A major goal of the meeting is to promote interaction between scientists studying methylation reactions at the molecular, cellular and physiological levels. The conference format includes nine plenary sessions (34 invited speakers), two mid-afternoon "Recent Advances" minisymposia (for which 12 speakers will be selected from submitted abstracts), and two late-afternoon poster sessions. Ample time will be provided for the 120-150 expected participants to socialize and interact on an informal basis.

Project Summary Our long-range goal is to elucidate the potential role of isoaspartyl (isoAsp) protein formation in brain aging and age-related neurological disease. Accumulation of isoAsp sites is a major form of protein damage that is normally kept in check by protein L-isoaspartyl methyltransferase (PIMT), a repair enzyme that is highly enriched in brain. PIMT knockout mice accumulate high levels of isoAsp-damaged proteins, especially in the brain and testes, and their phenotype is mainly neurological; increased brain size, abnormal neuronal physiology and metabolic signaling pathways, decreased cognitive function, and fatal epilepsy at 4-10 weeks after birth. IsoAsp formation can disrupt protein function, can elicit auto-immunity, and is often accompanied by formation of protein aggregates in vitro. We hypothesize that inefficiencies in the repair of isoAsp sites in neurons contributes significantly to the neurodegeneration that occurs in advanced age and in certain brain diseases. Our recent findings also suggest that isoAsp formation may underlie a novel form of protein aggregation that involves covalent cross-linking. We propose to explore these ideas via the following four specific aims. Aim 1 will compare brain extracts of PIMT -/- mice with wild type littermates to see if complete loss of this key repair enzyme leads to (a) isoAsp accumulation in synuclein and tau, two well studied proteins involved in several forms of neurodegeneration that have been reported by others to be highly susceptible to isoAsp formation, (b) aggregation of synuclein, tau, and collapsin-response mediator protein 2 (CRMP2), and (c) hyperphosphorylation of proteins in general, and site-specific hyperphosphorylation of tau and CRMP2. Aim 2 will compare PIMT +/- mice (which express 50-55% of normal PIMT activity) vs wild type littermates to see how a moderate reduction of PIMT activity in vivo alters isoAsp accumulation, protein aggregation, and protein hyperphosphorylation (as in Aim 1) as a function of age. Aims 3 stems from our recent findings that recombinant mouse CRMP2 undergoes isoAsp formation concomitant with formation of SDS-insoluble aggregates when it is aged in vitro at pH 7.4 and 37¿C. We will carry out a series of studies to determine if, as we suspect from recent data, this aggregation involves covalent cross-linking and is mechanistically related to isoAsp formation. Aim 4 will compare PIMT -/- mice with wild type littermates to look for functional changes in neuronal enzymes that accumulate isoAsp in vivo. We will initially focus on creatine kinase B (brain specific isoform) and the 70 kDa heat shock cognate protein (HSC70) which are among the 22 proteins we have found that accumulate high levels of isoAsp in the PIMT -/- mouse brain. If the hypotheses that guide these 4 aims are proven to be correct, this would call for pharmacological or genetic interventions that could boost the PIMT repair system to help stave off the decline in human cognitive function and neurological status that occurs in advanced age.