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

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. 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 (1Clarke S. Int. J. Pept. Protein Res. 1987; 30: 808-821Crossref PubMed Scopus (294) Google Scholar). 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. 1) (2Clarke S. Stephenson R.C. Lowenson J.D. Ahren T.J. Manning M.C. Stability of Protein Pharmaceuticals: Chemical and Physical Pathways of Protein Degradation. Plenum Press, New York1992: 1-29Google Scholar). Succinimides are unstable intermediates that undergo rapid, nonenzymatic hydrolysis to a mixture of aspartyl and isoaspartyl residues. Isoaspartyls typically account for two-thirds of the emerging products and with the introduction of an extra carbon into the main chain have the greater potential for disruption of protein structure and biological activity (2Clarke S. Stephenson R.C. Lowenson J.D. Ahren T.J. Manning M.C. Stability of Protein Pharmaceuticals: Chemical and Physical Pathways of Protein Degradation. Plenum Press, New York1992: 1-29Google Scholar). The precise ratio of the aspartyl and isoaspartyl products is determined by the accessibility of the succinimide carbonyls to attacking water or hydroxide molecules; attack at the main chain carbonyl produces isoaspartyls, whereas attack at the side chain carbonyl produces aspartyls. The α-carbon of a succinimide is also prone to racemization, resulting in the D-configurations of these amino acids, although at low yields (3Geiger T. Clarke S. J. Biol. Chem. 1987; 262: 785-794Abstract Full Text PDF PubMed Google Scholar). The physiological significance of isoaspartyl formation is emphasized by the presence of an enzyme, l-isoaspartyl O-methyltransferase (PIMT) 1The abbreviations used are: PIMTl-isoaspartyl O-methyltransferaseAdoMetS-adenosyl l-methionineHPrhistidine-containing proteinCAPS3-(cyclohexylamino)propanesulfonic acidHPLChigh pressure liquid chromatography.1The abbreviations used are: PIMTl-isoaspartyl O-methyltransferaseAdoMetS-adenosyl l-methionineHPrhistidine-containing proteinCAPS3-(cyclohexylamino)propanesulfonic acidHPLChigh pressure liquid chromatography. whose function appears to be in limiting the accumulation of isoaspartyl-containing proteins within the cell. PIMT is a highly conserved enzyme found in a wide variety of organisms, including plants (4Urao T. Yamaguchi-Shinozaki K. Dhinozaki K. Trends Plant Sci. 2000; 5: 67-74Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), insects (5Kagan R.M. McFadden H.J. McFadden P.N. O'Conner C. Clarke S. Comp. Biochem. Pysiol. Biochem. Mol. Biol. 1997; 117: 379-385Crossref PubMed Scopus (43) Google Scholar), bacteria (6Gross R. Arico B. Rappuoli R. Mol. Microbiol. 1989; 3: 1661-1667Crossref PubMed Scopus (117) Google Scholar), and mammals (7O'Connor C.M. Clarke S. Biochem. Biophys. Res. Commun. 1985; 132: 1144-1150Crossref PubMed Scopus (32) Google Scholar), that has the ability to specifically recognize and methylate isoaspartyl residues in a variety of peptide and protein contexts (1Clarke S. Int. J. Pept. Protein Res. 1987; 30: 808-821Crossref PubMed Scopus (294) Google Scholar, 2Clarke S. Stephenson R.C. Lowenson J.D. Ahren T.J. Manning M.C. Stability of Protein Pharmaceuticals: Chemical and Physical Pathways of Protein Degradation. Plenum Press, New York1992: 1-29Google Scholar). PIMT catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the α-carboxyl of the l-isoaspartyl group, methylation stimulates reformation of the succinimide, and subsequent hydrolysis converts a fraction of the offending residues to aspartyls. The overall reaction therefore represents true repair when the isoaspartyl results from an isomerized aspartyl and only partial repair of a deamidated asparagine. Because only a fraction of the isoaspartyls are converted to aspartyls with each round of methylation-induced succinimide formation, complete repair requires numerous rounds of cycling utilizing an energetically expensive methyl group donor, making the “repair” reaction quite inefficient. l-isoaspartyl O-methyltransferase S-adenosyl l-methionine histidine-containing protein 3-(cyclohexylamino)propanesulfonic acid high pressure liquid chromatography. l-isoaspartyl O-methyltransferase S-adenosyl l-methionine histidine-containing protein 3-(cyclohexylamino)propanesulfonic acid high pressure liquid chromatography. Genetic studies of the physiological role of PIMT in lower organism have not been clearly supportive of a repair function. In Caenorhabditis elegans, mutants having a disruption of the gene show poor Dauer stage survival, but substrate for the enzyme was found primarily in peptide fragments in both the wild type and PIMT-deficient organisms, suggesting that damaged proteins are cleared by proteolysis rather than repair (8Niewmierzycka A. Clarke S. Arch. Biochem. Biophys. 1999; 364: 209-218Crossref PubMed Scopus (16) Google Scholar). An Escherichia coli strain deficient in PIMT did not exhibit greater susceptibility to conditions anticipated to promote deamidation but did demonstrate increased sensitivity to changing conditions in the stationary phase (9Visick J.E. Cai H. Clarke S. J. Bacteriol. 1998; 180: 2623-2628Crossref PubMed Google Scholar). Genetic studies in higher organisms are more supportive of a repair function for PIMT. Mice lacking PIMT show an accumulation of damaged proteins, particularly in the brain; display abnormal brain activity; and suffer fatal seizures at an early age (10Kim E. Lowenson J.D. Maclaren D.C. Clarke S. Young S.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6132-6137Crossref PubMed Scopus (251) Google Scholar). Consistent with the hypothesis that the accumulation of isoaspartyl-containing proteins can be limiting to lifespan, overexpression of PIMT in Drosophila melanogaster extends the lifespan by 32-39% under conditions that accelerate the rates of deamidation, suggesting that PIMT activity is limiting in these situations (11Chavous D.A. Jackson F.R. O'Connor C.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14814-14818Crossref PubMed Scopus (109) Google Scholar). The ability of overexpression of a protein to extend lifespan is rare but not unprecedented. In particular, overexpression of enzymes that function in protein repair have been shown to prolong lifespan, supportive of the hypothesis that senescence results from a decline in cellular repair processes (12Knight J.A. Adv. Clin. Chem. 2000; 35: 1-62Crossref PubMed Google Scholar). Examples include the limiting of the accumulation of potentially damaging agents, as with superoxide dismutase (13Orr W.C. Sohal R.S. Science. 1994; 263: 1128-1130Crossref PubMed Scopus (1257) Google Scholar), as well as general protein repair systems, such as the heat shock proteins (14Tatar M. Khazaeli A.A. Curtsinger J.W. Nature. 1997; 390: 30Crossref PubMed Scopus (268) Google Scholar). The ability for overexpression of these proteins to increase longevity is presumably due to an increased capacity to deal with damaged or misfolded proteins that limit the lifespan of the of PIMT have been determined from C. D.C. Clarke S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), 2000; Full Text Full Text PDF Scopus Google Scholar), and J.E. Clarke S. J. Mol. Biol. 2001; PubMed Scopus Google Scholar). show high in amino acid as well as are of the methyltransferase of PIMT with as well as isoaspartyl-containing structural into the of particularly of co-factor binding in PIMT is the of highly residues in the of the protein within the residues are and in and in and and in T. The side chain of the with the α-carbon amino group of and the side chain of the with the of the protein and co-factor have been for C. 1998; PubMed Scopus Google Scholar, S. S. 1994; Full Text PDF PubMed Scopus Google Scholar, J. J. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google as well as J. T. H. M. 1999; PubMed Scopus Google Scholar). the precise role of these residues has not been and that an role in the reaction The of these residues be the of substrate co-factor binding or perhaps a more role in to the the isoaspartyl substrate has a group, and the co-factor has a of the both of these are Based on sequence the residues in E. coli PIMT are and (Fig. In we the role of and in the catalytic of E. coli as well as the ability of PIMT overexpression in E. coli to in conditions the accumulation of damaged proteins limit and of E. coli were on the E. coli gene sequence C. Clarke S. J. Biol. Chem. Full Text PDF PubMed Google to for of the gene from E. coli The the of the gene and was to include a in The a in a of E. coli as for a of in a of the as by The was and into the the The resulting was with and and the was into the under the of the with the of an The complete sequence of the gene and into the was by was with by of proteins was in phase in of at the were with and for an The were and as J.W. 30: PubMed Scopus Google Scholar). was in and to a with B. The was with of by of The proteins were with and were were determined by and was to be greater than were and a protein for was to a of mixture was and PIMT for thermal were or the of protein. were on a Scholar). reaction mixture of either of wild type PIMT or of the PIMT in a of The was by E. coli at for to promote deamidation of both and S. J.W. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and were by to that complete deamidation of both isoaspartyl substrate has been with to methyl group from the PIMT J.W. Clarke S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). were at for and on for to the PIMT reaction and The were for at in a and to of was and by The were within a of The were at for and on for and the reaction was The were in a liquid on a was in from the high of the protein were used as the of PIMT. of presence and of the in protein were determined by Protein of were with of acid at for and for at and the was a J.E. Clarke S. J. Mol. Biol. 2001; PubMed Scopus Google Scholar). The were a with a The was with a of to from to to and to The was at for The were at a of and was at of the were by a in A. for and S-adenosyl each at a of were S-adenosyl at and at were also under conditions in the protein were not with these the protein was from the by through a with a of the protein by the survival were on a (9Visick J.E. Cai H. Clarke S. J. Bacteriol. 1998; 180: 2623-2628Crossref PubMed Google Scholar). to heat shock was that were to phase in with and to for The from these were by and to in In of were to for in a thermal were on for were by making in and on and at at to the of these results were as the of of the that were not Stability of the E. coli the of the wild type and proteins, thermal were with a of on a The was at a of The proteins were in of the of of was by of the has been Arch. Biochem. Biophys. Scopus Google Scholar). of protein were by the proteins were The were with in and were to were from The by was used to for protein levels of Protein levels were by An system was used to and PIMT catalytic capabilities of wild type and E. coli were deamidated E. coli as an isoaspartyl E. coli wild type PIMT a of and a of with the kinetic determined for the substrate PIMT. The PIMT has a of and a of for the isoaspartyl to a of that of the wild type enzyme with a increase in PIMT to demonstrate methyltransferase activity. on the to also to demonstrate methyltransferase activity of of the wild type and protein wild of is on of under in a a of is on of under and E. coli PIMT the kinetic of these were due to an to the for co-factor was PIMT has a high for the a result of the complete of the from of protein structure for co-factor and by of of to the PIMT proteins was to as an for time and of from the with a time of of of the PIMT a of at with enzymes (Fig. The ability to of the mutants within the cell. for was also on PIMT under disruption of protein and not be in the of either the wild type PIMT or the the is in the although in than with the (Fig. suggesting that has reduced for the co-factor or a for spontaneous as at was not in the of a and PIMT J.E. Clarke S. J. Mol. Biol. 2001; PubMed Scopus Google Scholar), we are the of the of specifically by inactive and not the of PIMT. and has been that in Drosophila increased of PIMT can prolong lifespan under conditions in the formation of damaged proteins limit survival (11Chavous D.A. Jackson F.R. O'Connor C.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14814-14818Crossref PubMed Scopus (109) Google Scholar). the ability of overexpression of wild and inactive to survival capabilities of is a increase in the survival rates of bacteria the wild type enzyme as with or an protein such as the from overexpression of the in in survival capabilities as the wild type Surprisingly, overexpression of the inactive higher survival rates than with the wild type enzyme results that the survival capabilities by PIMT at in are of PIMT repair function and are due to of the in a Stability of the and the that the increased survival capabilities were through the induction of a heat shock as a of overexpression of an unstable protein. The overexpression of unstable or misfolding-prone protein has the potential for induction of a heat shock of the physiological role of the protein. E. coli PIMT is to be a rather unstable enzyme C. Clarke S. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the of with contribute to thermal wild type E. coli PIMT has a of The is with a of and the reduced with a of these to with the observed survival the of a protein is not of the potential for The of the on are more by the yields of protein from overexpression The levels of and wild type also the hypothesis of of a fraction of the proteins. Over numerous the yields of protein for the were than for the wild the of the was that of the wild and that of the was only The of the on the to is not a of in of the protein. of by for induction of the heat shock are and the presence of or misfolded proteins within the cell. In E. coli, the overexpression of either the or wild type PIMT results in a greater than increase in levels of to induction of the the PIMT the on the heat shock system with an increase in (Fig. levels of induction with the observed survival with in heat shock induction and survival with wild type and overexpression and more with overexpression of the of these levels of the of of protein The temperature-independent induction of the heat shock as a result of overexpression of of misfolding-prone proteins to a of the heat shock The of the heat shock to heat shock be anticipated to a survival to these bacteria the to a high Proteins are susceptible to a variety of spontaneous covalent modifications that result in to both structure and function. are particularly with the potential to undergo deamidation through cyclization of the side chain carbonyl with the nitrogen of the peptide to the formation of a Succinimides are unstable rapidly hydrolysis to a mixture of both aspartyl and isoaspartyl residues. have the potential to undergo a through a succinimide with hydrolysis to the the overall reaction therefore isomerization of an aspartyl to an Because of the of the the rates of succinimide formation from aspartyls are than for of the side chain group can increase have been shown to than the aspartyl residues (2Clarke S. Stephenson R.C. Lowenson J.D. Ahren T.J. Manning M.C. Stability of Protein Pharmaceuticals: Chemical and Physical Pathways of Protein Degradation. Plenum Press, New York1992: 1-29Google Scholar), and a of the system that succinimide formation be from a S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). formation from a has also been in the S. S. J. B. PubMed Scopus Google Scholar). The physiological significance of isoaspartyl formation, of the of these residues, is by the enzyme PIMT. PIMT for isoaspartyl residues in a wide of proteins and as a methyl group donor, PIMT the α-carboxyl of the isoaspartyl reformation of the succinimide with subsequent hydrolysis resulting in a fraction of the isoaspartyl residues converted to structural information on of the reaction are most is the binding of the co-factor within a of the from the of the of the in the reaction is the binding of and of the transfer S-adenosyl from are the protein and most are the residues within the of the protein. residues undergo conserved with the as well as the α-carbon nitrogen of the high of of these residues, with are to a role in the reaction Based on sequence homology the residues in E. coli and were to in an to co-factor binding As anticipated, both the and mutants have methyltransferase The a of of the wild type enzyme and a increase in for the isoaspartyl The is methyltransferase activity be from either the protein from the the the of catalytic function not to be a of an to the ability of the to from the overexpression to a as the wild type enzyme, and as with the wild type enzyme, of from the PIMT was disruption of protein the is to the of the is and is from the enzyme of disruption of the protein The for therefore result from reduced for the The catalytic of PIMT appears to through an with the binding of binding of the isoaspartyl substrate Res. PubMed Scopus Google Scholar). The ability of the to with the in for the isoaspartyl to that the binding of the co-factor the substrate have been as a result of the suggesting that have roles beyond co-factor although is role that is involved in methyl group of the reaction is energetically PIMT is to function a covalent or the of the of the α-carboxyl of isoaspartyl is a for attack on 2000; Full Text Full Text PDF Scopus Google Scholar). the structural the catalytic capabilities of PIMT are the physiological significance of these as well as the in substrate for the enzyme, the of a precise physiological role for the enzyme has not been has been that the enzyme in preventing the accumulation of isoaspartyls that as a of protein but are numerous with function. PIMT is quite in the role of repair of isoaspartyl residues emerging from In the isoaspartyls that from deamidation, conversion to aspartyls not complete the asparagine The complete conversion of a of isoaspartyls to aspartyls requires cycling through the succinimide with each round the of an energetically expensive methyl in of deamidated proteins with PIMT results in the conversion of isoaspartyl to the is to biological activity J. Biol. Chem. 1987; 262: Full Text PDF PubMed Google Scholar). The repair function for PIMT has not been by in particularly with lower An E. coli strain deficient in PIMT did not exhibit greater susceptibility to conditions that promote deamidation (9Visick J.E. Cai H. Clarke S. J. Bacteriol. 1998; 180: 2623-2628Crossref PubMed Google Scholar). In C. elegans, substrate for the enzyme was found primarily in peptide fragments in both the wild type and PIMT-deficient organisms, suggesting that damaged proteins are cleared by proteolysis rather than repair (8Niewmierzycka A. Clarke S. Arch. Biochem. Biophys. 1999; 364: 209-218Crossref PubMed Scopus (16) Google Scholar). have to alternate as to the physiological function of the enzyme, including an in E. Lowenson J.D. Clarke S. Young S.G. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), in the of the system J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), or as an Proc. Natl. Acad. Sci. U. S. A. 2001; 98: PubMed Scopus Google Scholar). a that overexpression of PIMT extends the lifespan of Drosophila under conditions anticipated to promote deamidation is in the role of the enzyme in the repair of damaged proteins (11Chavous D.A. Jackson F.R. O'Connor C.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14814-14818Crossref PubMed Scopus (109) Google Scholar). that accumulation of isoaspartyl residues limiting to the survival of an organism under conditions and that PIMT overexpression increased survival capabilities through increased to deal with these residues, we the ability of overexpression of both and inactive of PIMT to survival to stress. overexpression of wild type we observed a increase in the heat shock survival rates as with an a increase in survival capabilities is observed with overexpression of the and bacteria the inactive the survival capabilities, that the increased survival is of PIMT repair function. The increased survival was therefore to as a of induction of the heat shock system as a of overexpression of of unstable or misfolding-prone protein. the induction of the E. coli protein in a that with the observed increase in survival The for induction of the heat shock are and the presence of protein within the cell. In E. coli, overexpression of proteins result in the induction of the the of the The system has both in limiting protein and as a thermal and in as the and system of the E. coli heat shock Adv. 2001; PubMed Scopus Google Scholar). The of the system through binding to misfolded proteins is to be a of the of heat shock gene in levels of of wild have on the levels and activity of heat shock at T. A. H. B. Mol. Microbiol. 2001; PubMed Scopus Google Scholar). In Drosophila, overexpression of PIMT only in of lifespan at with lifespan observed at (11Chavous D.A. Jackson F.R. O'Connor C.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14814-14818Crossref PubMed Scopus (109) Google Scholar). that either isoaspartyl formation only limiting to survival at higher rates of deamidation be anticipated or that an induction of the heat shock system is at for the The that is the for induction of the Drosophila heat shock system but the that PIMT in with the heat shock system the observed survival results the of the heat shock system in the Drosophila that the induction is a of the of the PIMT of methyltransferase activity. The physiological role of the isoaspartyl methyltransferase enzyme as well as in An alternate hypothesis for PIMT function from the hypothesis that succinimide formation a physiological for the of most that are by As in as well as the the presence of an aspartyl can formation of a succinimide, resulting in the of the have that have the potential to formation of a succinimide ring as a of the group and that possibly a physiological of activity as observed in both the proteins of as well as the of the succinimide to an aspartyl results in of the formation be by protein J. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), either a for the of a of proteins or perhaps occurring as an of the In l-isoaspartyl methyltransferase as a enzyme rather than a repair enzyme, possibly a of of enzyme activity.

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