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An essential regulatory function of the DnaK chaperone dictates the decision between proliferation and maintenance in Caulobacter crescentus

ABSTRACT: Hsp70 chaperones are well known for their important functions in maintaining protein homeostasis during thermal stress conditions. In many bacteria the Hsp70 homolog DnaK is also required for growth in the absence of stress. The molecular reasons underlying Hsp70 essentiality remain in most cases unclear. Here, we demonstrate that DnaK is essential in the α-proteobacterium Caulobacter crescentus due to its regulatory function in gene expression. Using a suppressor screen we identified mutations in genes coding for different components of the transcriptional machinery as well as an ATP dependent protease that allow growth in the absence of DnaK. These mutations reduced the activity of the heat shock sigma factor σ32 by different mechanisms, suggesting that DnaK's sole essential function in the absence of stress is the inactivation of σ32. We found that σ32 inhibits growth and that its unleashed activity leads to an extensive reprogramming of global gene expression, which re-allocates cellular resources from proliferative to maintenance functions. While this re-allocation provides an advantage during heat stress, it leads to detrimental growth defects under favorable conditions. We conclude that Caulobacter has co-opted the DnaK chaperone system as an essential regulator of gene expression under conditions when its folding activity is dispensable.

ORGANISM(S): Caulobacter vibrioides NA1000

PROVIDER: GSE102372 | GEO | 2017/12/13

SECONDARY ACCESSION(S): PRJNA397583

REPOSITORIES: GEO

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Transcriptomic changes of Escherichia coli expressing BnTR1 at normal and heat stress conditions

Project description:The heat shock response is critical for organisms to survive at a high temperature. Heterologous expression of eukaryotic molecular chaperons protects Escherichia coli against heat stress. Here we report that expression of the plant E3 ligase BnTR1 significantly increase the thermotolerance of Escherichia coli. Different from eukaryotic chaperones, BnTR1 post-transcriptionally regulates the heat shock factor σ32 though zinc fingers of the RING domain, which interacts with DnaK resulting in stabilizing σ32 and subsequently up-regulating heat shock proteins. Our findings indicate the expression of BnTR1 confers thermoprotective effects on E. coli cells, and it may provide useful clues to engineer thermophilic bacterial strains.

2016-08-19 | GSE85807 | GEO

Bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70-Hsp40 substrates preferentially by the HslUV protease

Project description:Whereas in eukaryotic cells Hsp90s are extensively studied in bacteria, the function of Hsp90 (HtpG) and its functional relationship with Hsp70 (DnaK) remains unknown. To uncover physiological processes depending on HtpG and DnaK, we performed comparative quantitative proteomic analyses of insoluble and total protein fractions from unstressed wild type E. coli, and from knockout mutants ΔdnaKdnaJ (ΔKJ), ΔhtpG (ΔG) and ΔdnaKdnaJΔhtpG (ΔKJG) and compared their growth rates under heat-stress also with ΔdnaKdnaJΔhslV.

2021-07-07 | PXD010014 | Pride

The Hsp70 Chaperone System Stabilizes a Thermo-sensitive Subproteome

Project description:Stress-inducible molecular chaperones have essential roles in maintaining protein homeostasis, but the extent to which they affect global proteome stability remains unclear. Here, we analyzed the effects of the DnaK (Hsp70) system on protein stability in Escherichia coli using pulse proteolysis combined with quantitative proteomics. We quantified ~1500 soluble proteins and found ~500 of them to be protease-sensitive under normal growth conditions, indicating a high prevalence of conformationally dynamic states. Acute heat stress resulted in unfolding of an additional ~200 proteins, reflected in exposure of otherwise buried hydrophobic regions. Overexpression of the DnaK chaperone system markedly stabilized most thermo-sensitive proteins, including numerous ribosomal proteins as well as large, hetero-oligomeric proteins that frequently contain the evolutionary ancient c.37 fold (P-loop nucleoside triphosphate hydrolases).

2019-06-24 | PXD012929 | Pride

Assessment of Thermotolerance in preshocked hsp70(-/-) and (+/+) cells

Project description:From preliminary experiments, HSP70 deficient MEF cells display moderate thermotolerance to a severe heatshock of 45.5 degrees after a mild preshock at 43 degrees, even in the absence of hsp70 protein. We would like to determine which genes in these cells are being activated to account for this thermotolerance. Keywords: thermal stress, heat shock response, knockout, cell culture, hsp70

2009-04-09 | GSE11120 | GEO

Transcription profiling of AtHsp70-14 and AtHsp70-15 deficient Arabidopsis thaliana plants

Project description:AtHsp70-14 and -15 (At1g79930 and At1g79920) are two closely related isoforms of the Hsp110/SSE subfamily of the HSP70 chaperones in Arabidopsis thaliana. By analysing T-DNA insertion and amiRNA-AtHsp70-14/15 lines, we could demonstrate an essential function of AtHsp70-15 for plant growth. AtHsp70-15 deficient plants are reduced in size and stomata opening is impaired which results in accelerated wilting of detached leaves. Hsp70-15 deficient plants are also more tolerant to TuMV infection but more sensitive to heat treatment. Therefore comparative transcriptome analysis of amiRNA-AtHsp70-14/15, hsp70-14 knockout, hsp70-15 knockout and wild type plants was performed with leaf samples of 6 week old plants grown under ambient conditions. The analysis of differentially regulated genes compared to the wild type Col-0 plants exhibited a constitutive heat stress response / cytosolic protein response of Hsp70-15 deficient plants.

2011-03-28 | E-MEXP-3082 | biostudies-arrayexpress

Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays

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2007-03-07 | E-BUGS-2 | biostudies-arrayexpress

DnaK interaction and folding of firefly luciferase studied by hydrogen/deuterium exchange – mass spectrometry

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2020-01-15 | PXD016509 | Pride

Hsp70 ATP-dependent dimerization and Chaperone/Co-chaperone Interactions

Project description:Eukaryotic protein homeostasis (proteostasis) is largely dependent on the action of highly conserved Hsp70 molecular chaperones. Recent evidence indicates that apart from conserved molecular allostery, Hsp70 proteins retained and adapted throughout the evolution the ability to assemble as functionally relevant ATP-bound dimers. Here we have compared the ATP-dependent dimerization of DnaK, human stress-inducible Hsp70, Hsc70 and BiP Hsp70 proteins showing that their dimerization propensities differ with stress-inducible Hsp70 being predominantly dimeric in the presence of ATP. The structural analyses using hydrogen/deuterium exchange mass spectrometry, native electrospray ionization mass spectrometry, chemical cross-linking and small-angle X-ray scattering revealed that stress-inducible Hsp70 assembles in solution as an antiparallel dimer with the intermolecular interface closely resembling the ATP-bound dimer interfaces captured in DnaK and BiP crystal structures. ATP-dependent dimerization of stress-inducible Hsp70 is necessary for its efficient interaction with Hsp40 as shown by experiments with dimerization-deficient mutants. Moreover, dimerization of ATP-bound Hsp70 is required for its participation in high molecular weight protein complexes detected ex vivo supporting its functional role in vivo. As human cytosolic Hsp70 has the ability to interact with tetratricopeptide repeat (TPR) domain containing co-chaperones, we tested the interaction of Hsp70 ATP-dependent dimer with Chip and Tomm34 co-chaperones. While Chip associates with intact Hsp70 dimer to form a larger complex, binding of Tomm34 disrupts Hsp70 dimer and this event plays an important role in Hsp70 activity regulation. In summary, this study provides structural evidence of robust ATP-dependent antiparallel dimerization of human inducible Hsp70 protein and suggests novel role of TPR domain co-chaperones in multichaperone complexes involving Hsp70 ATP-bound dimers.

2018-12-11 | PXD010069 | Pride

Transcription profiling of Hsp70-14 and -15 silenced Arabidopsis plants

Project description:AtHsp70-14 and -15 are two closely related isoforms of the Hsp110/SSE subfamily of the HSP70 chaperones in Arabidopsis thaliana. By analysing T-DNA insertion and amiRNA-AtHsp70-14/15 lines, we could demonstrate an essential function of AtHsp70-15 for plant growth. AtHsp70-15 silenced plants are reduced in size and stomata opening is impaired which results in accelerated wilting of detached leaves. AtHsp70-15 silenced plants are also more tolerant to TuMV infection but more sensitive to heat treatment. Therefore comparative transcriptome analysis of amiRNA-AtHsp70-14/15 and control plants, PME1, was performed with leaf samples of 8 week old plants grown under ambient conditions. The analysis of differentially regulated genes compared to control plants exhibited a constitutive heat stress response / cytosolic protein response.

2011-04-29 | E-MEXP-2866 | biostudies-arrayexpress

Rapid acetylation of yeast Hsp70 mediates the cellular response to heat stress

Project description:Hsp70 is a highly conserved molecular chaperone critical for the folding of new and denatured proteins. While traditional models state that cells respond to stress by upregulating inducible HSPs, this response is relatively slow and is limited by transcriptional and translational machinery. Recent studies have identified a number of post-translational modifications (PTMs) on Hsp70 that act to fine-tune its function. We utilized mass spectrometry to determine whether yeast Hsp70 (Ssa1) is differentially modified upon heat shock. We uncovered four lysine residues on Ssa1, K86, K185, K354 and K562 that are deacetylated in response to heat shock. Mutation of these sites cause a substantial remodeling of the Hsp70 interaction network of co-chaperone partners and client proteins while preserving essential chaperone function. Acetylation/deacetylation at these residues alter expression of other heat-shock induced chaperones as well as directly influencing Hsf1 activity. Taken together our data suggest that cells may have the ability to respond to heat stress quickly though Hsp70 deacetylation, followed by a slower, more traditional transcriptional response.

2020-02-24 | PXD015185 | Pride

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