DegP (also designated as HtrA) and its homologs are found in prokaryotic cells and such eukaryotic organelles as mitochondria and chloroplasts. DegP has been found to be essential for the growth of Gram-negative bacteria under heat shock conditions and arguably considered to possess both protease and chaperone activities. The function of DegP has not been clearly defined. Using genetically incorporated non-natural amino acids as photo-crosslinkers, here we identified the b-barrel outer membrane proteins (OMPs) as the major natural substrates of DegP in Escherichia coli cells. We also demonstrated that DegP primarily functions as a protease, at both low and high temperatures, to eliminate unfolded OMPs, with hardly any appreciable chaperone activity in cells. We also found that the toxic and cell membrane-damaging misfolded OMPs would accumulate in DegP-lacking cells cultured under heat shock conditions. Together, our study defines the primary function of DegP in OMP biogenesis and offers a mechanistic insight into the essentiality of DegP for cell growth under heat shock conditions. Structured digital abstractDegP physically interacts with ompA by pull down (1, 2) DegP physically interacts with ompX, ompA, ompW, ompF, nmpC and ompC by pull down (View interaction) DegP physically interacts with ompC and ompF by pull down (View interaction) DegP physically interacts with ompC and ompA by pull down (View interaction) DegP physically interacts with ompC, malE, fkpA, ompW, ompA, ompF, nmpC and ompX by cross-linking study (View interaction)

Small heat shock proteins (sHSPs) are molecular chaperones ubiquitously present in all forms of life, but their function mechanisms remain controversial. Here we show by cryo-electron microscopy and single particle 3D reconstruction that, at the low temperatures (4–25°C), CeHSP17 (a sHSP from Caenorhabditis elegans) exists as a 24-subunit spherical oligomer with tetrahedral symmetry. Our studies demonstrate that CeHSP17 forms large sheet-like super-molecular assemblies (SMAs) at the high temperatures (45–60°C), and such SMAs are apparently the form that exhibits chaperone-like activity. Our findings suggest a novel molecular mechanism for sHSPs to function as molecular chaperones.

It is essential for organisms to adapt to fluctuating growth temperatures. Escherichia coli, a model bacterium commonly used in research and industry, has been reported to grow at a temperature lower than 46.5°C. Here we report that the heterologous expression of the 17-kDa small heat shock protein from the nematode Caenorhabditis elegans, CeHSP17, enables E. coli cells to grow at 50°C, which is their highest growth temperature ever reported. Strikingly, CeHSP17 also rescues the thermal lethality of an E. coli mutant deficient in degP, which encodes a protein quality control factor localized in the periplasmic space. Mechanistically, we show that CeHSP17 is partially localized in the periplasmic space and associated with the inner membrane of E. coli, and it helps to maintain the cell envelope integrity of the E. coli cells at the lethal temperatures. Together, our data indicate that maintaining the cell envelope integrity is crucial for the E. coli cells to grow at high temperatures and also shed new light on the development of thermophilic bacteria for industrial application.T emperature is considered the most important single environmental factor that profoundly affects the structure and function of biomolecules. Each organism in nature has evolved to live at a certain optimal temperature range. Nonetheless, effective mechanisms have also been evolved for organisms to survive under nonoptimal temperature conditions, typically termed heat shock response (1, 2) and cold shock response (3). It is of great value to understand such mechanisms of living organisms for both unveiling the nature of life and exploring biotechnological application.Escherichia coli, being the most extensively studied bacterium and also a popularly utilized host cell for producing pharmaceutically important recombinant proteins, is known to be unable to grow at a temperature higher than 46.5°C (4-6). It has been widely reported that heterologous overexpression of certain exogenous molecular chaperones (7-11) or an endogenous transcriptional regulator (12) is able to significantly increase the viability of E. coli cells undergoing heat shock treatment at lethal temperatures (around 50°C). It is also well established that preincubating E. coli cells (13,14) and other organisms (reviewed in reference 15) at a sublethal temperature (e.g., 42°C) significantly increases the thermotolerance of the treated organisms at lethal temperatures. However, all these alternations usually would not allow the modified cells to permanently survive and even grow under such lethal temperatures. In two attempts at selecting heat-resistant phenotypes by using extensive experimental evolution, E. coli mutant strains that are able to grow at up to 48°C (16) or 48.5°C (5) were obtained, with growth at the latter temperature being partially related to the high level of expression of the molecular chaperone GroEL/GroES. In contrast, thermophilic bacteria have been found to grow effectively at optimal temperatures much higher than 50°C (17)(18)(19)(20). They are known...

SummaryDauer is a dormancy state that may occur at the end of developmental stage L1 or L2 of Caenorhabditis elegans when the environmental conditions are unfavorable (e.g., lack of food, high temperature, or overcrowding) for further growth. Dauer is a nonaging duration that does not affect the postdauer adult lifespan. Major molecular events would include the sensing of the environmental cues, the transduction of the signals into the cells, and the subsequent integration of the signals that result in the corresponding alteration of the metabolism and morphology of the organism. Genetics approach has been effectively used in identifying many of the so-called daf genes involved in dauer formation using C. elegans as the model. Nevertheless, biochemical studies at the protein and metabolic level has been lacking behind in understanding this important life phenomenon. This review focuses on the biochemical understanding so far achieved on dauer formation and dormancy in general, as well as important issues that need to be addressed in the future.2009 IUBMB IUBMB Life, 61(6): 607-612, 2009

Edited by Miguel De la Rosa Keywords:Small heat shock protein Chaperone Fibril Amyloid Electron microscopy Cryo-electron tomography a b s t r a c t As a class of molecular chaperones, small heat shock proteins (sHsps) usually exist as multi-subunit spherical oligomers. In this study, we report that AgsA, a sHsp of Salmonella enterica serovar Typhimurium, spontaneously forms fibrils in vitro. These fibrils tend to be formed at elevated temperature and do not share the characteristics of amyloid. Interestingly, the fibril-forming AgsA is able to suppress the dithiothreitol-induced aggregation of insulin efficiently within a certain range of temperature. During this process, AgsA fibrils disappear and spherical complexes form between AgsA and insulin molecules. These data suggest that AgsA fibrils may represent a distinctive type of structural and functional form of sHsp from spherical oligomers. Our study provides new insights into sHsp structures and chaperone functions. Structured summary of protein interactions:AgsA and AgsA bind by electron microscopy (View interaction). Insulin and Insulin bind by molecular sieving (View interaction). Insulin and Insulin bind by electron microscopy (View interaction). AgsA and AgsA bind by molecular sieving (View interaction). AgsA and AgsA bind by fluorescence technology (View interaction). AgsA and AgsA bind by circular dichroism (View interaction). Insulin and Insulin bind by fluorescence technology (View interaction). AgsA and Insulin bind by molecular sieving (View interaction). AgsA and AgsA bind by electron tomography (View interaction).

The soil nematode Caenorhabditis elegans was used in 24-h acute exposures to arsenic (As), copper (Cu) and glyphosate (GPS) and to mixtures of As/Cu and As/GPS to investigate the effects of mixture exposures in the worms. A synergistic type of interaction was observed for acute toxicity with the As/Cu and As/GPS mixtures. Sublethal 24-h exposures of 1/1000, 1/100 and 1/10 of the LC50 concentrations for As, Cu and GPS individually and for As/Cu and As/GPS mixtures were conducted to observe responses in locomotory behavior (head thrashing), reproduction, and heat shock protein expression. Head thrash frequency and reproduction exhibited concentration dependent decreases in both individual and combined exposures to the tested chemical stressors, and showed synergistic interactions even at micromolar concentrations. Furthermore, the HSP70 protein level was significantly increased following exposure to individual and combined chemical stressors in wild-type C. elegans. Our findings establish for the first time the effects of exposure to As/GPS and As/Cu mixtures in C. elegans.

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Small heat shock proteins (sHSPs) are known to exhibit in vitro chaperone activity by suppressing the aggregation of misfolded proteins. The 12-kDa sHSPs (Hsp12s) subfamily members from Caenorhabditis elegans, including Hsp12.2, Hsp12.3, and Hsp12.6, however, are devoid of such chaperone activity, and their in vivo functions are poorly understood. Here we verified that Hsp12.1, similar to its homologs Hsp12.2, Hsp12.3, and Hsp12.6, hardly exhibited any chaperone activity. Strikingly, we demonstrated that these Hsp12s seem to play crucial physiological roles in C. elegans, for suppressing dauer formation and promoting both longevity and reproduction. A unique sHSP gene from Filarial nematode worm Brugia malayi was identified such that it encodes two products, one as a full-length Hsp12.6 protein and the other one having an N-terminal arm of normal length but lacks the C-terminal extension. This gene may represent an intermediate form in evolution from a common sHSP to a Hsp12. Together, our study offers insights on what biological functions the chaperone-defective sHSPs may exhibit and also implicates an evolutionary scenario for the unique Hsp12s subfamily.