Hsp70 and Hsp90—a relay team for protein folding

The heat shock protein 70 (Hsp70/DnaK) gene of Bacillus licheniformis is 1,839 bp in length encoding a polypeptide of 612 amino acid residues. The deduced amino acid sequence of the gene shares high sequence identity with other Hsp70/DnaK proteins. The characteristic domains typical for Hsps/DnaKs are also well conserved in B. licheniformis DnaK (BlDnaK). BlDnaK was overexpressed in Escherichia coli using pQE expression system and the recombinant protein was purifi ed to homogeneity by nickel-chelate chromatography. The optimal temperature for ATPase activity of the purifi ed BlDnaK was 40°C in the presence of 100 mM KCl. The purifi ed BlDnaK had a V max of 32.5 nmol Pi/min and a K M of 439 μM. In vivo, the dnaK gene allowed an E. coli dnaK756-ts mutant to grow at 44°C, suggesting that BlDnaK should be functional for survival of host cells under environmental changes especially higher temperature. We also described the use of circular dichroism to characterize the conformation change induced by ATP binding. Binding of ATP was not accompanied by a net change in secondary structure, but ATP together with Mg 2+ and K + ions had a greater enhancement in the stability of BlDnaK at stress temperatures. Simultaneous addition of DnaJ, GrpE, and NR-peptide (NRLLLTG) synergistically stimulates the ATPase activity of BlDnaK by 11.7-fold.

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“…6). This result corroborates that the cation plays a role in mediating contacts between Hsp70 and nucleotide [56,57].…”

Section: Nucleotide-induced Stability Changessupporting

confidence: 83%

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

et al. 2009

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“…Analysis by semi-quantitative methods at the mRNA level by Northern blot or at the protein level by Western blot clearly demonstrated complex regulation. Furthermore, more recent evidence showed that HSPs are involved in the cell response to most environmental and physiological stressors (reviewed by Wegele et al 2004). This includes studies in molluscs (CruzRodriguez et al 2000;Snyder et al 2001;Boutet et al 2003;Piano et al 2004).…”

Section: Discussionmentioning

confidence: 99%

Transcriptional expression levels of cell stress marker genes in the Pacific oyster Crassostrea gigas exposed to acute thermal stress

Farcy

Voiseux

Lebel

et al. 2009

Cell Stress and Chaperones

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During the annual cycle, oysters are exposed to seasonal slow changes in temperature, but during emersion at low tide on sunny summer days, their internal temperature may rise rapidly, resulting in acute heat stress. We experimentally exposed oysters to a 1-h acute thermal stress and investigated the transcriptional expression level of some genes involved in cell stress defence mechanisms, including chaperone proteins (heat shock proteins Hsp70, Hsp72 and Hsp90 (HSP)), regulation of oxidative stress (Cu-Zn superoxide dismutase, metallothionein (MT)), cell detoxification (glutathione S-transferase sigma, cytochrome P450 and multidrug resistance (MDR1)) and regulation of the cell cycle (p53). Gene mRNA levels were quantified by reverse transcription-quantitative polymerase chain reaction and expressed as their ratio to actin mRNA, used as a reference. Of the nine genes studied, HSP, MT and MDR1 mRNA levels increased in response to thermal stress. We compared the responses of oysters exposed to acute heat shock in summer and winter and observed differences in terms of magnitude and kinetics. A larger increase was observed in September, with recovery within 48 h, whereas in March, the increase was smaller and lasted more than 2 days. The results were also compared with data obtained from the natural environment. Though the functional molecule is the protein and information at the mRNA level only has limitations, the potential use of mRNAs coding for cell stress defence proteins as early sensitive biomarkers is discussed.

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“…Hop optimises the functional cooperation between Hsp70 and Hsp90 which requires highly co-ordinated interactions for the folding and conformational regulation of a variety of proteins [5]. The molecular chaperone complex of Hop, Hsp70 and Hsp90 is involved in the folding and maturation of key regulatory proteins involved in cell viability [6], such as steroid hormone receptors, transcription factors and kinases (some of which are involved in cancer progression). The role of Hop in tumour progression is not fully elucidated; however, Hop has been shown to be over expressed in colonic carcinoma cells [7] and in hepatocellular carcinoma (HCC) [8].…”

Section: Introductionmentioning

confidence: 99%

RNAi knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via MMP-2 down regulation

et al. 2011

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Keywords:Hop/STIP1/STI1 (Hsp70/Hsp90-organising protein) Pancreatic cancer Immunohistochemistry In vitro invasion MMP-2 a b s t r a c tWe previously identified Hop as over expressed in invasive pancreatic cancer cell lines and malignant tissues of pancreatic cancer patients, suggesting an important role for Hop in the biology of invasive pancreatic cancer. Hop is a co-chaperone protein that binds to both Hsp70/Hsp90. We hypothesised that by targeting Hop, signalling pathways modulating invasion and client protein stabilisation involving Hsp90-dependent complexes may be altered.In this study, we show that Hop knockdown by small interfering (si)RNA reduces the invasion of pancreatic cancer cells, resulting in decreased expression of the downstream target gene, matrix metalloproteinases-2 (MMP-2). Hop in conditioned media co-immunoprecipitates with MMP-2, implicating a possible extracellular function for Hop. Knockdown of Hop expression also reduced expression levels of Hsp90 client proteins, HER2, Bcr-Abl, c-MET and v-Src. Furthermore, Hop is strongly expressed in high grade PanINs compared to lower PanIN grades, displaying differential localisation in invasive ductal pancreatic cancer, indicating that the localisation of Hop is an important factor in pancreatic tumours.Our data suggests that the attenuation of Hop expression inactivates key signal transduction proteins which may decrease the invasiveness of pancreatic cancer cells possibly through the modulation of Hsp90 activity. Therefore, targeting Hop in pancreatic cancer may constitute a viable strategy for targeted cancer therapy.

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Hsp70 and Hsp90—a relay team for protein folding

Cited by 566 publications

References 409 publications

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

Transcriptional expression levels of cell stress marker genes in the Pacific oyster Crassostrea gigas exposed to acute thermal stress

RNAi knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via MMP-2 down regulation

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