Overexpression of l-Isoaspartate O-Methyltransferase in Escherichia coli Increases Heat Shock Survival by a Mechanism Independent of Methyltransferase Activity

Abstract

Over time and under stressing conditions proteins are susceptible to a variety of spontaneous covalent modifications. One of the more commonly occurring types of protein damage is deamidation; the conversion of asparagines into aspartyls and isoaspartyls. The physiological significance of isoaspartyl formation is emphasized by the presence of the conserved enzyme L-isoaspartyl O-methyltransferase (PIMT), whose physiological function appears to be in preventing the accumulation of deamidated proteins. Seemingly consistent with a repair function, overexpression of PIMT in Drosophila melanogaster extends lifespan under conditions expected to contribute to protein damage. Based on structural information and sequence homology we have created mutants of residues proposed to be involved in co-factor binding in Escherichia coli PIMT. Both mutants retain S-adenosyl L-methionine binding capabilities but demonstrate dramatically reduced kinetic capabilities, perhaps suggestive of catalytic roles beyond co-factor binding. As anticipated, overexpression of the wild type enzyme in E. coli results in bacteria with increased tolerance to thermal stress. Surprisingly, even greater levels of heat tolerance were observed with overexpression of the inactive PIMT mutants. The increased survival capabilities observed with overexpression of PIMT in E. coli, and possibly in Drosophila, are not due to increased isoaspartyl repair capabilities but rather a temperature-independent induction of the heat shock system as a result of overexpression of a misfolding-prone protein.An alternate hypothesis as to the physiological substrate and function of L-isoaspartyl methyltransferase is proposed.Proteins are susceptible to a variety of spontaneous, covalent modifications that have the potential for disruption of both structure and biological activity. The formation of isoaspartyl residues, through either the deamidation of asparagines or isomerization of aspartates, are among the most rapidly occurring types of damage that afflict proteins under physiological conditions (1).The metamorphosis of asparagine and aspartate residues is initiated by the nucleophilic attack of the neighboring peptide nitrogen on the side chain carbonyl, resulting in the cyclization of the side chain with the main chain to the formation of a succinimide ring (Fig.

Biological Significance of Isoaspartate and Its Repair System

“…48) As a similar phenotype, it is reported that overexpression of PIMT in E. coli increases heat shock survival. 49) These results suggest that overexpression of PIMT provides protection from environmental stress in aging tissues. It is still unclear, however, whether PIMT activity is responsible for the increase in survival because even greater levels of heat tolerance were observed with the overexpression of inactive PIMT mutants in E. coli.…”

“…It is still unclear, however, whether PIMT activity is responsible for the increase in survival because even greater levels of heat tolerance were observed with the overexpression of inactive PIMT mutants in E. coli. 49) …”

Protein Repairl-Isoaspartyl Methyltransferase1 Is Involved in Both Seed Longevity and Germination Vigor inArabidopsis

“…Furthermore, in Escherichia coli, Caenorhabditis elegans, Drosophila melanogaster, and mice (Reissner and Aswad, 2003;Shimizu et al, 2005), there is genetic evidence that PIMT plays a protective role in vivo to overcome environmental stress in aging tissues. Overaccumulation of PIMT in E. coli (Kindrachuk et al, 2003) and in Drosophila (Chavous et al, 2001) is associated with phenotypes of high heat-shock survival and extended lifespan under high temperature conditions, respectively. The role of PIMT as an aging-related repair enzyme is also supported by the characterization of PIMT-deficient mutants, which exhibit a reduced dauer phase survival in C. elegans (Kagan et al, 1997a), a higher sensitivity to oxidative stress in stationary E. coli cells (Visick et al, 1998a), and epileptic seizures in mice that severely limit their survival to only 12 weeks (Kim et al, 1997(Kim et al, , 1999.…”

PROTEIN l-ISOASPARTYL METHYLTRANSFERASE2 Is Differentially Expressed in Chickpea and Enhances Seed Vigor and Longevity by Reducing Abnormal Isoaspartyl Accumulation Predominantly in Seed Nuclear Proteins

“…For example, overaccumulation of PIMT in Escherichia coli is associated with high heat shock survival under mild heat stress conditions, while a PIMT-deficient E. coli mutant showed higher sensitivity toward oxidative stress. PIMT-deficient mice were shown to suffer with epileptic seizures and a reduced life span (Kim et al, 1997;Visick et al, 1998;Kindrachuk et al, 2003). In such PIMT-deficient mutants, isoAsp residues become overrepresented in proteins, suggesting that PIMT maintains a low level of isoAsp in proteins, thus combating the effect of aging or stress particularly in cells with low metabolic activity.…”