Why Stress? Why do we see survival defects for pcm mutants in stationary phase only when an environmental stress is present? Not all stresses produce this effect, and so far, the ones that do (heat, oxidation, salt, methanol) can all affect protein folding. Based on this observation, we hypothesize that isoaspartyl damage may not have grave consequences for a properly folded protein, but can lead to the destabilization of the protein's structure, so that it is either more easily unfolded or more difficult to refold after unfolding. The model below shows how this might work; either: (i) partial unfolding due to environmental stress could allow more (or more rapid) isoAsp formation; (ii) isoAsp formation could locally destabilize protein conformation, magnifying the unfolding effect of the stress; or (iii) isoAsp formation could expose additional sites for oxidation or other stress-induced damage. Alternatively, degradation and recycling of unfolded proteins could be blocked by isoAsp formation. Further experiments will be needed to test this hypothesis and to distinguish among these different models.

When is PCM needed? Two surprising results may shed light on when PCM is needed in aging E. coli. First, we found that isoaspartyl damage accumulates during stationary phase, but that there is no significant difference in accumulation between wild-type cells and pcm mutants¹. Although high pH speeded up isoAsp accumulation, no difference could be seen between wild-type and mutant strains.² Secondly, recent experiments show that aged pcm mutants have an unusually long lag phase when transferred to fresh medium at pH 9.² Taken together, these results suggest that PCM may have limited function in metabolically inactive starving cells but is needed to repair proteins and speed recovery once nutrients are restored. Enhanced stationary-phase stress survival may really be due to improved recovery of those sub-populations of cells that can grow and divide during this period. This is an active area of investigation in the lab currently.

Where is PCM needed? PCM appears, based on its amino-acid sequence, to be a cytoplasmic protein. Yet, if the effect of pH is to increase the rate of isoaspartyl damage, proteins in the inner and outer membranes and periplasm of the E. coli cell would be most readily affected--the cytoplasmic pH is strongly buffered. It may be that a relatively small change in cytoplasmic pH has a significant effect, or it is possible that PCM could also assist in maintaining functional cell-surface proteins in some way. For example, one author has suggested that PCM released from lysed cells could function in the extracellular milleu³. We plan to examine isoaspartyl accumulation and the role of PCM in various cellular compartments.

PCM and SurE. In the genome of E. coli and many other bacteria, pcm is the second gene in an operon with a gene designated surE (see figure at right). Based on its structure, the SurE protein has been classified as an acid phosphatase⁴, but its specific function is unknown. No phenotypes have yet been detected in mutants lacking the surE gene, except that a surE-pcm double mutant shows no loss of stress resistance¹. Interestingly, isoaspartyl damage accumulates to a higher level in a surE-pcm double mutant than in wild-type cells or cells with only one of the two mutations. We suspect that SurE and PCM work together in some way to mitigate the effects of isoaspartyl damage, but the role of SurE is presently unclear.

Want to contribute? The answers to these questions will come from undergraduate research! NCC students who would like to work on protein repair by PCM in E. coli should contact to discuss possible projects. Research can be done during the summer (see the Biology department research page for more info.), during the academic year or during interim.

Next: Current research--Visick lab notebook >>

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¹Visick, J. E., J. K. Ichikawa, and S. Clarke. 1998. Mutations in the Escherichia coli surE gene increase isoaspartyl accumulation in a strain lacking the pcm repair methyltransferase but suppress stress-survival phenotypes. FEMS Microbiol. Lett. 167:19-25.

²Hicks, W. M., M. V. Kotlajich, and J. E. Visick. 2005. Recovery from long-term stationary phase and stress survival in Escherichia coli require the L-isoaspartyl protein carboxyl methyltransferase at alkaline pH. Microbiology 151:2151-2158.

³Clarke, S. 2003. Aging as war between chemical and biochemical processes: protein methylation and the recognition of age-damaged proteins for repair. Ageing Res. Rev. 2:263-285.

⁴Griffith, S. C., M. R. Sawaya, D. R. Boutz, N. Thapar, J. E. Katz, S. Clarke, and T. O. Yeates. 2001. Crystal structure of a protein repair methyltransferase from Pyrococcus furiosus with its L-isoaspartyl peptide substrate. J. Mol. Biol. 313:1103-1016.