Existence of Abnormal Protein Aggregates in Healthy Escherichia coli Cells

Abstract

Protein aggregation is a phenomenon observed in all organisms and has often been linked with cell disorders. In addition, several groups have reported a virtual absence of protein aggregates in healthy cells. In contrast to previous studies and the expected outcome, we observed aggregated proteins in aerobic exponentially growing and "healthy" Escherichia coli cells. We observed overrepresentation of "aberrant proteins," as well as substrates of the major conserved chaperone DnaK (Hsp70) and the protease ClpXP (a serine protease), in the aggregates. In addition, the protein aggregates appeared to interact with chaperones known to be involved in the aggregate repair pathway, including ClpB, GroEL, GroES, and DnaK. Finally, we showed that the levels of reactive oxygen species and unfolded or misfolded proteins determine the levels of protein aggregates. Our results led us to speculate that protein aggregates may function as a temporary "trash organelle" for cellular detoxification.Formation or accumulation of aggregated proteins, which are present in all organisms, has become an important area of intensive research, mainly due to observations that there is a link with many disorders, including aging or neurodegenerative diseases (16).Major substrates for aggregation are unfolded or misfolded proteins. Indeed, misfolded proteins inappropriately expose hydrophobic surfaces normally buried in the protein's interior, leading to nonnative conformations able to interact and form aggregates. In cells, protein folding may fail because of amino acid misincorporation. Moreover, DnaK/Hsp70 and GroEL/ Hsp60 chaperone protein substrates, as well as partially degraded proteins, are more prone to unfolding and thus to aggregation (15).A common theory is that protein aggregation may be the consequence of a failed quality control mechanism normally charged with repairing or removing the misfolded or unfolded proteins. Indeed, to maintain functional proteins, the cells contain chaperones or proteases that assist proper protein folding and disaggregation of aggregates (17). For instance, Hsp104/ClpB has the capacity to rescue unfolded or misfolded proteins from an aggregated state in cooperation with the Hsp70/DnaK chaperone system (9). However, as recently shown in Huntington's disease, protein aggregates could serve as "temporary storage zones" within cells in order to maintain their function and integrity for prolonged periods of time (1, 13).A common opinion is that in healthy cells, the levels of formation and accumulation of protein aggregates are extremely low, although protein unfolding occurs constantly. Indeed, several groups have observed a virtual absence of protein aggregates in healthy cells (2, 5, 15), although it is possible that insufficient material was used for detection.Here we isolated and characterized protein aggregates in exponentially growing "healthy" Escherichia coli cells using classical procedures to extract E. coli insoluble cell fractions and protein aggregates. Our results led us to speculate that the...

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