Ceramide formation during heat shock: a potential mediator of alpha B-crystallin transcription.

Ceramide has been identified as a potential second messenger that may mediate cell differentiation and apoptosis after exposure to hormonal agonists such as 1 alpha, 25-dihydroxyvitamin D3, tumor necrosis factor alpha, or gamma-interferon. The secondary cellular events that follow ceramide generation remain undefined. We report that in NIH WT-3T3 cells, ceramide induces an enhancement of gene transcription of alpha B-crystallin, a small heat shock protein. The levels of alpha B-crystallin, as measured by Northern blot and immunoblot analyses, were increased by the addition of an exogenous short-chain ceramide, N-acetylsphingosine, or by increasing endogenous intracellular ceramide by inhibition of glucosylceramide synthase. Similar effects were not seen in the expression of the closely related gene, Hsp25. To ascertain whether ceramide-mediated gene transcription was a feature of the heat shock response, cell ceramide was measured in heat shocked cells and observed to be elevated 2-fold immediately upon the return of cells to 37 degrees C. Thus ceramide formed after heat shock treatment of 3T3 cells may mediate the transcription events associated with the cell stress response.

Pharmacological modulation of heat shock factor 1 by antiinflammatory drugs results in protection against stress-induced cellular damage.

The activation of heat shock genes by diverse forms of environmental and physiological stress has been implicated in a number of human diseases, including ischemic damage, reperfusion injury, infection, neurodegeneration, and inflammation. The enhanced levels of heat shock proteins and molecular chaperones have broad cytoprotective effects against acute lethal exposures to stress. Here, we show that the potent antiinflammatory drug indomethacin activates the DNA-binding activity of human heat shock transcription factor 1 (HSF1). Perhaps relevant to its pharmacological use, indomethacin pretreatment lowers the temperature threshold of HSF1 activation, such that a complete heat shock response can be attained at temperatures that are by themselves insufficient. The synergistic effect of indomethacin and elevated temperature is biologically relevant and results in the protection of cells against exposure to cytotoxic conditions.

The DnaJ chaperone catalytically activates the DnaK chaperone to preferentially bind the sigma 32 heat shock transcriptional regulator.

In Escherichia coli the heat shock response is under the positive control of the sigma 32 transcription factor. Three of the heat shock proteins, DnaK, DnaI, and GrpE, play a central role in the negative autoregulation of this response at the transcriptional level. Recently, we have shown that the DnaK and DnaJ proteins can compete with RNA polymerase for binding to the sigma 32 transcription factor in the presence of ATP, by forming a stable DnaJ-sigma 32-DnaK protein complex. Here, we report that DnaJ protein can catalytically activate DnaK's ATPase activity. In addition, DnaJ can activate DnaK to bind to sigma 32 in an ATP-dependent reaction, forming a stable sigma 32-DnaK complex. Results obtained with two DnaJ mutants, a missense and a truncated version, suggest that the N-terminal portion of DnaJ, which is conserved in all family members, is essential for this activation reaction. The activated form of DnaK binds preferentially to sigma 32 versus the bacteriophage lambda P protein substrate.

Degradation of sigma 32, the heat shock regulator in Escherichia coli, is governed by HflB.

The heat shock response in Escherichia coli is governed by the concentration of the highly unstable sigma factor sigma 32. The essential protein HflB (FtsH), known to control proteolysis of the phage lambda cII protein, also governs sigma 32 degradation: an HflB-depleted strain accumulated sigma 32 and induced the heat shock response, and the half-life of sigma 32 increased by a factor up to 12 in mutants with reduced HflB function and decreased by a factor of 1.8 in a strain overexpressing HflB. The hflB gene is in the ftsJ-hflB operon, one promoter of which is positively regulated by heat shock and sigma 32. The lambda cIII protein, which stabilizes sigma 32 and lambda cII, appears to inhibit the HflB-governed protease. The E. coli HflB protein controls the stability of two master regulators, lambda cII and sigma 32, responsible for the lysis-lysogeny decision of phage lambda and the heat shock response of the host.

Short-term hyperthermic treatment of Penaeus monodon increases expression of heat shock protein 70 (HSP70) and reduces replication of gill associated virus (GAV)

Disease is the result of interactions amongst pathogens, the environment and host organisms. To investigate the effect of stress on Penaeus monodon, juvenile shrimp were given short term exposure to hypoxic, hyperthermic and osmotic stress twice over a 1-week period and estimates of total haemocyte count (THC), heat shock protein (HSP) 70 expression and load of gill associated virus (GAV) were determined at different time points. While no significant differences were observed in survival and THC between stressed and control shrimp (P>0.05), HSP 70 expression and GAV load changed significantly (P

Cloning of cytoplasmic heat shock protein 90 (FcHSP90) from Fenneropenaeus chinensis and its expression response to heat shock and hypoxia

Heat shock protein 90 (HSP90) works as a multi-functional chaperone and is involved in the regulation of many essential cellular pathways. In this study, we have identified a full-length complementary DNA (cDNA) of HSP90 (FcHSP90) from Chinese shrimp Fenneropenaeus chinensis. FcHSP90 full-length cDNA comprised 2,552 bp, including a 2,181-bp open reading frame encoding 726 amino acids. Both homology analyses using alignment with previously identified HSP90 and a phylogeny tree indicated that FcHSP90 was a cytoplasmic HSP90. Real-time reverse transcription polymerase chain reaction analysis revealed that FcHSP90 was ubiquitously expressed in all the examined tissues but with highest levels in ovary of F. chinensis. FcHSP90 mRNA levels were sensitively induced by heat shock (from 25A degrees C to 35A degrees C) and reached the maximum at 6 h during heat shock treatment. Under hypoxia conditions, FcHSP90 mRNA levels, in both hemocytes and gill, were induced at 2 h and depressed at 8 h during hypoxia stress. The assessment of FcHSP90 mRNA levels under heat shock and hypoxia stresses indicated that the transcription of FcHSP90 was very sensitive to heat shock and hypoxia, so we deduced that FcHSP90 might play very important roles for shrimp to cope with environmental stress.

Humoral response against heat shock proteins and other mycobacterial antigens after intravesical treatment with bacille Calmette-Guerin (BCG) in patients with superficial bladder cancer

Few studies have analysed the antibody response during intravesical BCG immunotherapy for superficial bladder cancer. We have examined the evolution in serum antibody response against several heat shock proteins (hsp), including the recombinant mycobacterial hsp65 and the native protein P64 from BCG, GroEL from Escherichia coli (hsp60 family), recombinant mycobacterial hsp70 and the E. coli DnaK (hsp70 family), against purified protein derivative of tuberculin (PPD) and the AG85 complex of Mycobacterium bovis BCG, as well as against tetanus toxoid in 42 patients with a superficial bladder tumour, 28 treated with six intravesical BCG instillations and 14 patients used as controls. We also analysed the lymphoproliferative response of peripheral blood mononuclear cells against PPD in this population. Data of antibody responses at 6 weeks post BCG were available in all 28 patients, and at 4 month follow up in 17 patients. All patients who demonstrated a significant increase in IgC antibodies against PPD at 4 months follow up had a significant increase already at 6 weeks of follow up. In contrast, IgG antibodies against hsp increased significantly from 6 weeks to 4 months post- treatment. A significant increase in IgG antibodies against PPD, hsp65, P64, GroEL, and hsp70 at 4 months follow up was observed in 10/17, 8/17, 10/17, 4/17 and 8/17 patients. Native P64 protein elicited a higher antibody response than recombinant mycobacterial hsp65. No increase in antibody response was observed against Dnak from E. coli, against AG85 or tetanus toxoid after BCG therapy. An increase in IgG antibodies against P64 at 4 months follow up compared with pretreatment values was found to be a significant predictor of tumour recurrence (P < 0.01). Further studies with a larger number of patients are needed to confirm the value of the antibody response against P64 as a clinical independent prognostic factor.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine ( DCVC) resulted in a > 1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Transcription of the Hsp30, Hsp70, and Hsp90 heat shock protein genes is modulated by the PalA protein in response to acid pH-sensing in the fungus Aspergillus nidulans

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A specific expression profile of heat-shock proteins and glucose-regulated proteins is associated with response to neoadjuvant chemotherapy in oesophageal adenocarcinomas

BACKGROUND Oesophageal adenocarcinomas often show resistances to chemotherapy (CTX), therefore, it would be of high interest to better understand the mechanisms of resistance. We examined the expression of heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) in pretherapeutic biopsies of oesophageal adenocarcinomas to assess their potential role in CTX response. METHODS Ninety biopsies of locally advanced adenocarcinomas before platin/5-fluorouracil (FU)-based CTX were investigated by reverse phase protein arrays (RPPAs), immunohistochemistry (IHC) and quantitative RT-PCR. RESULTS CTX response strongly correlated with survival (P=0.001). Two groups of tumours with specific protein expression patterns were identified by RPPA: Group A was characterised by low expression of HSP90, HSP27 and p-HSP27((Ser15, Ser78, Ser82)) and high expression of GRP78, GRP94, HSP70 and HSP60; Group B exhibited the inverse pattern. Tumours of Group A were more likely to respond to CTX, resulting in histopathological tumour regression (P=0.041) and post-therapeutic down-categorisation from cT3 to ypT0-T2 (P=0.040). High HSP60 protein (IHC) and mRNA expression were also associated with tumour down-categorisation (P=0.016 and P=0.004). CONCLUSION Our findings may enhance the understanding of CTX response mechanisms, might be helpful to predict CTX response and might have translational relevance as they highlight the role of potentially targetable cellular stress proteins in the context of CTX response.

Heat Shock Protein 90 Inhibitors as Broad Spectrum Anti-Infectives

Combating stress is one of the prime requirements for any organism. For parasitic microbes, stress levels are highest during the growth inside the host. Their survival depends on their ability to acclimatize and adapt to new environmental conditions. Robust cellular machinery for stress response is, therefore, both critical and essential especially for pathogenic microorganisms. Microbes have cleverly exploited stress proteins as virulence factors for pathogenesis in their hosts. Owing to its ability to sense and respond to the stress conditions, Heat shock protein 90 (Hsp90) is one of the key stress proteins utilized by parasitic microbes. There are growing evidences for the critical role played by Hsp90 in the growth of pathogenic organisms like Candida, Giardia, Plasmodium, Trypanosoma, and others. This review, therefore, explores potential of exploiting Hsp90 as a target for the treatment of infectious diseases. This molecular chaperone has already gained attention as an effective anti-cancer drug target. As a result, a lot of research has been done at laboratory, preclinical and clinical levels for several Hsp90 inhibitors as potential anti-cancer drugs. In addition, lot of data pertaining to toxicity studies, pharmacokinetics and pharmacodynamics studies, dosage regime, drug related toxicities, dose limiting toxicities as well as adverse drug reactions are available for Hsp90 inhibitors. Therefore, repurposing/repositioning strategies are also being explored for these compounds which have gone through advanced stage clinical trials. This review presents a comprehensive summary of current status of development of Hsp90 as a drug target and its inhibitors as candidate anti-infectives. A particular emphasis is laid on the possibility of repositioning strategies coupled with pharmaceutical solutions required for fulfilling needs for ever growing pharmaceutical infectious disease market.

Trans-spliced Heat Shock Protein 90 Modulates Encystation in Giardia lamblia

Background: Hsp90 from Giardia lamblia is expressed by splicing of two independently transcribed RNA molecules, coded by genes named HspN and HspC located 777 kb apart. The reasons underlying such unique trans-splicing based generation of GlHsp90 remain unclear. Principle Finding: In this study using mass-spectrometry we identify the sequence of the unique, junctional peptide contributed by the 5' UTR of HspC ORF. This peptide is critical for the catalytic function of Hsp90 as it harbours an essential ``Arg'' in its sequence. We also show that full length GlHsp90 possesses all the functional hall marks of a canonical Hsp90 including its ability to bind and hydrolyze ATP. Using qRT-PCR as well as western blotting approach we find the reconstructed Hsp90 to be induced in response to heat shock. On the contrary we find GlHsp90 to be down regulated during transition from proliferative trophozoites to environmentally resistant cysts. This down regulation of GlHsp90 appears to be mechanistically linked to the encystation process as we find pharmacological inhibition of GlHsp90 function to specifically induce encystation. Significance: Our results implicate the trans-spliced GlHsp90 from Giardia lamblia to regulate an essential stage transition in the life cycle of this important human parasite.

Functional characterization of heat-shock protein 90 from Oryza sativa and crystal structure of its N-terminal domain

Heat-shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that is essential for the normal functioning of eukaryotic cells. It plays crucial roles in cell signalling, cell-cycle control and in maintaining proteome integrity and protein homeostasis. In plants, Hsp90s are required for normal plant growth and development. Hsp90s are observed to be upregulated in response to various abiotic and biotic stresses and are also involved in immune responses in plants. Although there are several studies elucidating the physiological role of Hsp90s in plants, their molecular mechanism of action is still unclear. In this study, biochemical characterization of an Hsp90 protein from rice (Oryza sativa; OsHsp90) has been performed and the crystal structure of its N-terminal domain (OsHsp90-NTD) was determined. The binding of OsHsp90 to its substrate ATP and the inhibitor 17-AAG was studied by fluorescence spectroscopy. The protein also exhibited a weak ATPase activity. The crystal structure of OsHsp90-NTD was solved in complex with the nonhydrolyzable ATP analogue AMPPCP at 3.1 angstrom resolution. The domain was crystallized by cross-seeding with crystals of the N-terminal domain of Hsp90 from Dictyostelium discoideum, which shares 70% sequence identity with OsHsp90-NTD. This is the second reported structure of a domain of Hsp90 from a plant source.