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Characterization of the autocleavage process of the Escherichia coli HtrA protein: implications for its physiological role.

ABSTRACT: The Escherichia coli HtrA protein is a periplasmic protease/chaperone that is upregulated under stress conditions. The protease and chaperone activities of HtrA eliminate or refold damaged and unfolded proteins in the bacterial periplasm that are generated upon stress conditions. In the absence of substrates, HtrA oligomerizes into a hexameric cage, but binding of misfolded proteins transforms the hexamers into bigger 12-mer and 24-mer cages that encapsulate the substrates for degradation or refolding. HtrA also undergoes partial degradation as a consequence of self-cleavage of the mature protein, producing short-HtrA protein (s-HtrA). The aim of this study was to examine the physiological role of this self-cleavage process. We found that the only requirement for self-cleavage of HtrA into s-HtrA in vitro was the hydrolysis of protein substrates. In fact, peptides resulting from the hydrolysis of the protein substrates were sufficient to induce autocleavage. However, the continuous presence of full-length substrate delayed the process. In addition, we observed that the hexameric cage structure is required for autocleavage and that s-HtrA accumulates only late in the degradation reaction. These results suggest that self-cleavage occurs when HtrA reassembles back into the resting hexameric structure and peptides resulting from substrate hydrolysis are allosterically stimulating the HtrA proteolytic activity. Our data support a model in which the physiological role of the self-cleavage process is to eliminate the excess of HtrA once the stress conditions cease.

SUBMITTER: Jomaa A

PROVIDER: S-EPMC2648369 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Characterization of the autocleavage process of the Escherichia coli HtrA protein: implications for its physiological role.

Jomaa Ahmad A Jomaa Ahmad A Iwanczyk Jack J Tran Julie J Ortega Joaquin J

Journal of bacteriology 20081219 6

The Escherichia coli HtrA protein is a periplasmic protease/chaperone that is upregulated under stress conditions. The protease and chaperone activities of HtrA eliminate or refold damaged and unfolded proteins in the bacterial periplasm that are generated upon stress conditions. In the absence of substrates, HtrA oligomerizes into a hexameric cage, but binding of misfolded proteins transforms the hexamers into bigger 12-mer and 24-mer cages that encapsulate the substrates for degradation or ref ...[more]

PMID: 19103920

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Project description:UnlabelledEscherichia coli segregates protein aggregates to the poles by nucleoid exclusion. Combined with cell divisions, this generates heterogeneous aggregate distributions in subsequent cell generations. We studied the robustness of this process with differing medium richness and antibiotics stress, which affect nucleoid size, using multimodal, time-lapse microscopy of live cells expressing both a fluorescently tagged chaperone (IbpA), which identifies in vivo the location of aggregates, and HupA-mCherry, a fluorescent variant of a nucleoid-associated protein. We find that the relative sizes of the nucleoid's major and minor axes change widely, in a positively correlated fashion, with medium richness and antibiotic stress. The aggregate's distribution along the major cell axis also changes between conditions and in agreement with the nucleoid exclusion phenomenon. Consequently, the fraction of aggregates at the midcell region prior to cell division differs between conditions, which will affect the degree of asymmetries in the partitioning of aggregates between cells of future generations. Finally, from the location of the peak of anisotropy in the aggregate displacement distribution, the nucleoid relative size, and the spatiotemporal aggregate distribution, we find that the exclusion of detectable aggregates from midcell is most pronounced in cells with mid-sized nucleoids, which are most common under optimal conditions. We conclude that the aggregate management mechanisms of E. coli are significantly robust but are not immune to stresses due to the tangible effect that these have on nucleoid size.ImportanceEscherichia coli segregates protein aggregates to the poles by nucleoid exclusion. From live single-cell microscopy studies of the robustness of this process to various stresses known to affect nucleoid size, we find that nucleoid size and aggregate preferential locations change concordantly between conditions. Also, the degree of influence of the nucleoid on aggregate positioning differs between conditions, causing aggregate numbers at midcell to differ in cell division events, which will affect the degree of asymmetries in the partitioning of aggregates between cells of future generations. Finally, we find that aggregate segregation to the cell poles is most pronounced in cells with mid-sized nucleoids. We conclude that the energy-free process of the midcell exclusion of aggregates partially loses effectiveness under stressful conditions.

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Characterization of the autocleavage process of the Escherichia coli HtrA protein: implications for its physiological role.

Publications

Characterization of the autocleavage process of the Escherichia coli HtrA protein: implications for its physiological role.

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