Small Heat Shock Protein IbpB Acts as a Robust Chaperone in Living Cells by Hierarchically Activating Its Multi-type Substrate-binding Residues

Fu, Xinmiao; Shi, Xiaodong; Yin, Linxiang; Liu, Jiafeng; Joo, Keehyoung; Lee, Jooyoung; Chang, Zengyi

doi:10.1074/jbc.m113.450437

Cited by 35 publications

(66 citation statements)

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“…Uniquely, the sHSPs of both prokaryotic and eukaryotic sources have also been found to be associated with cell membranes (34)(35)(36)(37)(38). Over the years, we have tried to delineate the function and mechanism of sHSPs, from largely bacterial sources, via both in vitro and in vivo studies (39)(40)(41)(42)(43)(44)(45). To study the function and mechanism of sHSPs in C. elegans, we first focused on CeHSP17, which remains the least characterized such proteins in terms of function and properties among the small heat shock proteins of C. elegans (46) and is predicted by our bioinformatics analysis to be putatively localized in mitochondria (Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

A Small Heat Shock Protein Enables Escherichia coli To Grow at a Lethal Temperature of 50 C Conceivably by Maintaining Cell Envelope Integrity

Ezemaduka

Shi

et al. 2014

Journal of Bacteriology

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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...

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Section: Resultsmentioning

confidence: 99%

A Small Heat Shock Protein Enables Escherichia coli To Grow at a Lethal Temperature of 50 C Conceivably by Maintaining Cell Envelope Integrity

Ezemaduka

Shi

et al. 2014

Journal of Bacteriology

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“…Protein Purification-The photo-crosslinked SurA-Q35Bpa product was purified via Ni-NTA (GE Healthcare) affinity chromatography in the presence of 8 M urea (a denaturing condition for removing the non-covalently bound proteins) as previously described (14).…”

Section: Methodsmentioning

confidence: 99%

A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria

Wang

Feng

et al. 2016

Journal of Biological Chemistry

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␤-barrel outer membrane proteins (OMPs) are ubiquitously present in Gram-negative bacteria, mitochondria and chloroplasts, and function in a variety of biological processes. The mechanism by which the hydrophobic nascent ␤-barrel OMPs are transported through the hydrophilic periplasmic space in bacterial cells remains elusive. Here, mainly via unnatural amino acid-mediated in vivo photo-crosslinking studies, we revealed that the primary periplasmic chaperone SurA interacts with nascent ␤-barrel OMPs largely via its N-domain but with ␤-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent ␤-barrel OMPs interact with SurA via their N-and C-terminal regions. Additionally, via dual in vivo photo-crosslinking, we demonstrated the formation of a ternary complex involving ␤-barrel OMP, SurA, and BamA in cells. More importantly, we found that a supercomplex spanning the inner and outer membranes and involving the BamA, BamB, SurA, PpiD, SecY, SecE, and SecA proteins appears to exist in living cells, as revealed by a combined analyses of sucrose-gradient ultra-centrifugation, Blue native PAGE and mass spectrometry. We propose that this supercomplex integrates the translocation, transportation, and membrane insertion events for ␤-barrel OMP biogenesis.

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“…The plasmids for expressing Bpa variant proteins of IbpB with a tag of six histidines were constructed as we described previously (34). EF-Tu-His and TnaA-His recombinant proteins were expressed in wild-type BW25113 E. coli cells, purified using nickel-nitrilotriacetic acid affinity chromatography (GE Healthcare), and then subjected to polyclonal antibody production in mice.…”

Section: Methodsmentioning

confidence: 99%

“…Bpa-mediated in Vivo Photocross-linking, Purification of Photocross-linked Products, and LC-MS/MS Analysis-Bpa variant proteins of IbpB were expressed in ⌬ibpB BW25113 E. coli cells, and Bpa-mediated in vivo photocross-linking was performed as we described recently (34). Briefly, the cells (cultured at 30°C or heat shocked at 50°C for 30 min) were transferred to a glass plate of 20 cm in diameter and pre-cooled on ice before being subjected to UV irradiation at 365 nm for 5 min using a Hoefer UVC 500 cross-linker.…”

Section: Methodsmentioning

confidence: 99%

“…We recently characterized the substrate-binding residues of IbpB (34), a representative sHSP from Escherichia coli that confers cell resistance against stresses (11-12, 16, 31-32, 36 -40), by using in vivo site-specific photocross-linking as mediated by the genetically incorporated unnatural amino acid p-benzoyl-L-phenylalanine (Bpa) (35). As a continuous effort, we attempted to identify the substrate proteins that IbpB binds in living cells, as captured by such in vivo photocross-linking, which was successfully utilized in identifying unknown protein-protein interactions (36,37).…”

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confidence: 99%

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In Vivo Substrate Diversity and Preference of Small Heat Shock Protein IbpB as Revealed by Using a Genetically Incorporated Photo-cross-linker

Shi

Yan

et al. 2013

Journal of Biological Chemistry

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Background:The identity of natural substrate proteins of small heat shock proteins is poorly understood. Results: A total of 95 and 54 natural substrate proteins were identified for IbpB in living cells at 50 and 30°C, respectively. Conclusion: IbpB preferentially protects translation-related proteins and metabolic enzymes. Significance: This study offers mechanistic insights into the chaperone functions of small heat shock proteins for cells against stresses.

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Small Heat Shock Protein IbpB Acts as a Robust Chaperone in Living Cells by Hierarchically Activating Its Multi-type Substrate-binding Residues

Cited by 35 publications

References 50 publications

A Small Heat Shock Protein Enables Escherichia coli To Grow at a Lethal Temperature of 50 C Conceivably by Maintaining Cell Envelope Integrity

A Small Heat Shock Protein Enables Escherichia coli To Grow at a Lethal Temperature of 50 C Conceivably by Maintaining Cell Envelope Integrity

A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria

In Vivo Substrate Diversity and Preference of Small Heat Shock Protein IbpB as Revealed by Using a Genetically Incorporated Photo-cross-linker

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