Heat Shock Proteins & HSP70 + Factors That Increase Them

HSP70 Resource Guide

Heat Shock Proteins & HSP70 + Factors That Increase Them

Heat Shock Proteins (HSPs) were initially discovered as proteins that were markedly induced by heat shock and other chemical or physical stresses.

Since that time they have viewed as molecular chaperones, guiding and modifying the structures of proteins by their intrinsic ability to re-fold them from a denatured to a native structure. These interactions and the balance toward fully-natured functional proteins necessitates that the HSPs be abundant.

As a result, the HSP27, HSP40, HSP70 and HSP110 genes have evolved to be particularly efficient for mass synthesis after or during stress with powerful transcriptional activation, efficient messenger RNA (mRNA) stabilization, and selective mRNA translation.

HSP27, HSP70, HSP90, and HSP110 increase to become the dominantly expressed proteins after stress. The types of stress are numerous, but include heat shock, metal toxicity, nutrient deprivation, oxidative stress as well as numerous disease states of which cancer has received the most attention.

HSPs are regulated by transcription factors that belong to the heat shock factor family, which allows rapid transcriptional activation as well as ensuring a prompt shut-off post recovery. These heat shock factors include HSF1, HSF2 and HSF4. However, a significant amount of HSPs are constitutively expressed.

Both induced or constitutively expressed HSPs function is several ways, and have the ability to form complexes with client proteins.

These complexes are themselves directly involved in protein folding in the cytosol, endoplasmic reticulum (ER) and mitochondria, the intracellular transport of proteins, protein repair and degradation (of environmentally damaged proteins), regulatory protein control, and the re-folding of misfolded proteins.

Examples of such complexes include HSP10 and HSP60 complexes that mediate protein folding and HSP70- and HSP90-containing complexes that are involved in both generic protein-folding pathways and in specific association with key regulatory proteins within the cell.

HSP90 plays a particularly versatile role in cell regulation – it is the ability to form complexes with a large number of cellular kinases, transcription factors, and other molecules that has led to the protein becoming a drug target of considerable interest. The HSPs have been generally classified into several classes depending on their molecular weights, see Table 1*.

Table 1*.

Protein NameLocalizationFunction
HSP104CytoplasmReleases proteins from aggregates
HSP90α and HSP90ßCytoplasmPrevents protein aggregation, enables protein stabilization and trafficking, facilitates activation of numerous regulated proteins
Grp94Endoplasmic reticulumQuality control of protein processing in the endoplasmic reticulum
TRAP / HSP75MitochondriaUnclear
HSP70 / Hsc70CytoplasmPrevents protein aggregation, aids protein folding
Grp78 / BipEndoplasmic reticulumProtein import and folding in the endoplasmic reticulum
HSP60 / ChaperoninsCytoplasm and mitochondriaPrevents protein aggregation, aids protein folding
HSP47Endoplasmic reticulumFacilitates the folding and assembly of pro-collagen molecules, retaining unfolded molecules within the ER, and assisting the transport of correctly folded molecules from the ER to the Golgi apparatus.
HSP40 / HDJ2CytoplasmHelps protein folding as a co-chaperone of HSP70
HSP32 (HO-1; small HSPs)Endoplasmic reticulum, plasma membrane and mitochondriaCatalyzes first step of heme degradation to bilirubin, which has antioxidant properties.
HSP27 / HSP25 (small HSPs)Endoplasmic reticulumPrevents protein aggregation, may have role in cell growth and differentiation
Alpha B crystallin (small HSPs)CytoplasmMajor eye lens protein. It inhibits TRAIL induced apoptosis in cancer. It confers a cytoprotective effect by suppressing aggregation of denatured proteins. It is constitutively expressed, often at high levels, in human cancers, including gliomas, breast, prostate and renal cell carcinomas.

* Adapted from Annals in Oncology, 14 1169-1176, 2003

HSP70 (also called heat shock protein 70 or HSP72) has a wide range of diverse functions. It is inducibly expressed, whereas Hsc70 (heat shock cognate 70) – sharing a very high degree of homology and function – is not. Hsc70 (also called HSP73) is constitutively expressed.

Generally HSP70 it is seen as a mostly an anti-apoptotic protein. It interacts with the intrinsic and extrinsic pathways of apoptosis at several junctions and inhibits cell death through chaperone dependent as well as independent activities.

HSP70 protects cells from cytotoxicity induced by TNF, monocytes, oxidative stress, chemotherapeutic agents, ceramide and radiation. The apoptotic events generated by nitric oxide and heat stress triggers translocation of Bax from cytoplasm to the mitochondria, which is inhibited by over-expression of HSP70.

Downstream in the pathway, HSP70 also inhibits formation of a functional apoptosome complex by direct interaction with Apaf-1. It prevents late caspase dependent events such as activation of cytosolic phospholipase A2 and changes in nuclear morphology; it can also protect cells from forced expression of caspase-3.

HSP70 can, independent of its chaperoning activity, inhibit JNK mediated cell death, by suppressing JNK phosphorylation either directly and/or through the upstream SEK kinase.

HSP70  and HSP90 have been identified to have a peptide carrier function. They have been found to be involved in the cross-presentation of tumor-derived peptides on MHC class I molecules.

More diverse functions have been reported, however – the binding of mycobacterial HSP70 to CD40 was found to mediate a calcium-dependent cell signaling and the release of CC chemokines, pro-inflammatory cytokines, and nitric oxide, whereas mammalian HSP70s were found to facilitate receptor-mediated endocytosis.

HSP70 and HSP90 are able signal danger to the cell even in the absence of immunogenic peptides. Tumor cells themselves have been identified as a source of extracellular HSP70s, and after treatment with IFN-Hsc70 has been observed.

Furthermore, pro-inflammatory cytokines are released after the interactions of peptide-free HSP70 with CD14 and TLR2/4 on antigen-presenting cells. The process is initiated by the translocation of NF-B into the nucleus.

The cytokine release triggers the stimulation of innate immune system.

HSP70 also competes with the CD40 ligand for binding to antigen-presenting cells. In addition HSP70s appear to be involved in the stimulation of the migration of dendritic cells to the draining lymph nodes and in maturation of those cells They show up-regulation of CD86, CD83, and CD40 after contact with HSP70 MHC class II.

However, the role of HSPs as cytokine- proteins appears to be due, at least in part to contaminating levels of LPS or bacterial lipoprotein contamination in the HSPs preparations. It is also clear that many HSPs can bind LPS. Reduction of LPS levels reduce the stimulatory capacity of HSP70s and HSP90s toward dendritic cells.

Natural Killer (NK) cells are important effector cells of the innate immune system. HSP70 was identified as being a trigger factor for NK cells with a high CD94 surface density. After mapping the interaction if was found that only a 14-mer peptide – T-K-D-N-N-L-L-G-R-F-E-L-S-G (TKD; AA450-463) from the C-terminal domain was required for immuno-stimulus.

When incubated with cytokines and HSP70 or the TKD peptide the cell surface density of NK receptors increases, including CD94. Further blocking studies showed the importance of CD94 in the interaction of NK cells with HSP70 on tumor cells.

Screening of human tumor biopsies have revealed that HSP70 is often present of plasma membranes of the colon, lungs, pancreas, head and neck and metastases thereof. Typical or normal tissue samples are found to be HSP70-negative.

Interestingly, the cell surface densities (from tumor tissues) of HSP70 can be further increased by the administration of reagents (such as membrane-interactive alkyl-lysophospholipids, cytostatic drugs, including taxoides and vincristine sulfate, cyclooxygenase (COX-1/2) inhibitors, acetylsalicylic acid, insulin sensitizers, or subjecting the samples to hyperthermia, radiation, and photodynamic therapy. This heightened HSP70 density correlates with an increased sensitivity to NK cell -mediated cell death, suggesting an NK cell based therapeutic capacity further increased by stimulation with chemical or chemical means.

It has been shown that HSP70 induction is important in neuronal survival after a stroke, which correlates with the classical observations of the importance of HSPs and their role cardio-protection.

HSP70 is also able to improve tissue transplantation efficiency, and eases the serious implications of chronic diseases such as diabetes (resulting form studies using HSP70 inducers such as bimoclomol and BRX-220).

Other neurological diseases including Huntington’s, Parkinson’s and Alzheimer’s or neurological trauma cases show beneficial effects if over-expression of HSP70 (and HSP40) are present.

Inducers of HSP70 are numerous, and include stannous chloride (improving the success rate of tissue transplantations), geranyl-geranyl acetone (protects neurons against cerebral ischemia), the antiulcer drug carbenoxolone and probably the best known one – aspirin, which enhances HSP70 synthesis.

  • Cancer Research
  • Heat Shock
  • Oxidative Stress
  • Chaperones

Source: https://www.stressmarq.com/support/research-tools/hsp70-resource-guide/

The interactive association between heat shock factor 1 and heat shock proteins in primary myocardial cells subjected to heat stress

Heat Shock Proteins & HSP70 + Factors That Increase Them

In animals, different types of stress such as heat,transportation and chemical factors contribute to lethalpathological symptoms related to cardiovascular diseases, such ascardiac arrhythmia, seizure or hypovolemic shock with tachycardiaand, eventually, circulatory collapse (1–3).In the clinical diagnosis and treatment of heat stroke,approximately 25% of patients experience failure of ≥1 organsystems. In mammals, sudden death may occur as a result ofstress-induced damage to cardiac tissue and myocardial cells(4,5).

Heat shock proteins (HSPs) are ubiquitouslyexpressed and highly conserved in prokaryotes and eukaryotes(6). HSP family members aremolecular chaperones that are important for the regulation ofseveral fundamental cellular processes under normal conditions(7).

However, they also play aprotective role during pathological processes (8,9).

HSPs play an important role in intracellular protein transport,cytoskeletal architecture, mutation masking, regulation oftranslation, intracellular redox homeostasis and protection againstspontaneous or induced programmed cell death (10).

HSP70, a member of the HSP70 family, is associatedwith enhanced post-ischemic myocardial recovery in adult rat heartsand with the reduction of infarct size (11).

It interacts with other proteinsand maintains or alters their conformational states (12).

Under normal conditions, heat shockfactor 1 (HSF1) presents as an HSF-HSP70 heterodimer; however, HSF1has been shown to interact with HSP70 under stress conditions(13).

HSP90, which belongs to the HSP90 family, and aspreviously demonstrated, does not act generally in nascent proteinfolding (14). At the molecularlevel, HSP90 binds to substrate proteins, which are in anear-native state and thus at a late stage of folding (15), poised for activation by ligandbinding or interaction with other factors (16).

Defects in cell physiology causedby HSP90 disruption lead to tissue- and organism-level defects.HSP90 is essential for various cellular processes, such as proteinfolding, protein degradation, signal transduction cascades andmorphological evolution.

HSP90 affinity chromatography experimentshave indicated that HSP90 interacts with HSF1 in human cells(17).

HSP60, which belongs to the HSP60 family, isanti-apoptotic and provides protection against cell death bymaintaining mitochondrial oxidative phosphorylation (18–20). HSP60 is typically located in themitochondria of eukaryotic cells (21). It assists in the protectionagainst protein aggregation (22)and in transporting proteins from the cytoplasm to organelles(23).

Crystallin, alpha B (CRYAB, also known as HSPbeta-5) is a member of the small HSP family (24) that has chaperone- properties,including the ability to prevent the accumulation of denaturedproteins and increase cell tolerance to stress. Following itsinduction by cellular stresses, including heat and reactive oxygenspecies, CRYAB promotes cell survival and inhibits apoptosis(25). HSP induction in themyocardium may be a cardioprotective cellular response (26).

Previous studies have indicated that the dramaticincrease in HSP expression is a key part of the heat shockresponse, which is primarily controlled by HSFs (27,28).

HSF1 is a major transcriptionalregulator of HSPs, existing as a trimer with constitutive DNAbinding activity (29). In theabsence of cellular stress, HSF1 is repressed through itsassociation with HSP.

However, in response to stress, HSF1 binds tospecific sequences in HSP promoters and stimulates HSP expression(30). The question as to whetherHSF1 can trigger all HSPs remains unanswered.

In order to addressthis intriguing question, in this study, we detected the levels ofHSPs and HSF1 in heat-stressed rat myocardial cells in vitroand analyzed and compared the data using STRING (version 9.1) todetermine the association between HSF1 and HSPs.

Cell culture and exposure to heat

Primary neonatal rat myocardial cells were providedby Shanghai Fu Meng Gene Biotechnology Co., Ltd. (Shanghai, China).The cells were cultured in Dulbecco's modified Eagle's medium(DMEM, No. 11965-084) 10%, supplemented with 10% fetal calf serum(No.

10270-098) were purchased from Gibco, Thermo Scientific,Shanghai, China, at 37°C in 5% CO2 for 3 days; theviability was >85%. The cells were divided into differentexperimental groups, each consisting of 9 cell culture plates.Heat-stressed cells were exposed to heat at 42°C, whereas thecontrol cells were exposed to a normal temperature of 37°C.

Oneplate from each group was removed from the incubator at the startof the experiment (0 min) and after 10, 20, 40, 60, 120, 240, 360and 480 min.

Semi-quantitative detection of HSP andHSF1 expression levels by western blot analysis

The heat-stressed cells were washed withphosphate-buffered saline (PBS) 3 times, and proteins wereextracted by lysis in sodium dodecyl sulfate (SDS)-polyacrylamidegel Laemmli sample buffer. The protein extracts were boiled for 5min prior to loading equal amounts of protein (10 µg) for10% SDS-polyacrylamide gel electrophoresis.

Proteins weretransferred onto nitrocellulose membranes by electrotransfer andthe membranes were blocked with 5% skimmed milk in Tris-bufferedsaline [20 mM Tris-HCl (pH 7.6), 137 mM NaCl] containing 0.1%Tween-20 (TBST) for 1 h at room temperature. The membranes wereincubated with anti-rat HSF1 monoclonal antibody [1:1,000; ab61382;Abcam Trading (UK) Company Ltd.

], anti-rat HSP90 monoclonalantibody [ab79849; Abcam Trading (UK) Company Ltd.], anti-rat HSP70monoclonal antibody [1:1,000; ab5442; Abcam Trading (UK) CompanyLtd.], anti-rat CRYAB monoclonal antibody [1:1000; ab13496; AbcamTrading (UK) Company Ltd.], or anti-rat β-actin (ACTB) monoclonalantibody [1:1,000, ab8224; Abcam Trading (UK) Company Ltd.] for 16h at 4°C.

After washing with TBST, the membranes were incubatedwith peroxidase-conjugated goat anti-mouse immunoglobulin G at roomtemperature for 1 h, and the antibody-antigen complexes weredetected using Western Blotting Luminol Reagent (Santa CruzBiotechnology, Inc., Santa Cruz, CA, USA). Bands on the developedfilm were quantified using Quantity One software version 4.6.

2(Bio-Rad, Hercules, CA, USA). The intensity of each band wasnormalized to that of β-actin.

Total RNA isolation and reversetranscription-PCR

Total RNA was isolated from the cells in theexperimental and control groups using TRIzol reagent according tothe manufacturer's instructions (Trizol-RNAiso Plus reagent,D9108A; Takara, China). The RNA concentrations were measured at 260nm using a spectrophotometer (M200PRO; Tecan, Austria).

Serialdilutions of RNA were prepared with ribonuclease-free water; 2µg of each sample were reverse transcribed using aTranscript Moloney murine leukemia virus (M-MLV) kit (Invitrogen,Shanghai, China) according to the manufacturer's instructions andstored at −80°C until use.

Random decamers and oligo(dT) wereobtained from a RETROscript kit (AM1710; Ambion, Austin, TX,USA).


Primers were designed to anneal specifically to eachtarget mRNA. HSF1, HSP90, HSP70, CRYABand β-actin mRNA sequences were obtained from the NationalCenter for Biotechnology Information (Bethesda, MD, USA) GenBankdatabase (accession nos: NC_005108.2, NP_077369.1 and NC_005111.2).The primers were designed using Primer Premier 5.

0 software forconventional and reverse transcription-PCR amplification.

Thesequences were as follows: HSF1 sense,5′-ACCCCAGCCTCTGCCTGCT-3′ and antisense,5′-TTCCCACTCGGGCTCCAGCA-3′; HSP90 sense, 5′-CCCGGTGCGGTTAGTCACGT-3′ and antisense, 5′-TCCAGAGC GTCTGAGGAGTTGGA-3′;HSP70 sense, 5′-GTCCCTCAAGAGCCCAACCCCAT-3′ and antisense,5′-ACGTGGTCTAGTGGAAGCCACCA-3′; CRYAB sence,5′-CGTCGGCTGGGATCCGGTACT-3′ and antisence,5′-CACGAAGAGCGCCAGGACGA-3′; β-actin sense, 5′-CCCATCTATGAGGGTTCA-3′ and antisense, 5′-TCACGCACGATTTCC-3′. The expected lengthsof the HSF1, HSP90, HSP70, CRYAB andβ-actin PCR products were 153, 214, 124, 153 and 128 bp,respectively. Primers were synthesized by Invitrogen.

Quantitative (real-time) PCR (qPCR)

Each DNA sample (2 µl, 25X dilution) wassuspended in 2X SYBR Premix Ex TaqT™ (DRR041S; Takara, China) with25 pmol of each sense and antisense primer, and double distilledwater was added to a total volume of 25 µl. qPCR wasperformed using an ABI 7300 Real-Time PCR system (AppliedBiosystems Foster City, CA, USA).

The thermal profile wasestablished according to the manufacturer's instructions. Briefly,this protocol consisted of enzyme activation at 95°C for 3 min,followed by 45 cycles of denaturation at 95°C for 5 sec, andannealing and elongation at 52°C for 30 sec.

For each run, anegative control tube without DNA was run along with theexperimental samples. A 2-fold dilution series of the template wasused in the qPCR assays.

The HSPs and HSF1 mRNAexpression levels of all samples were normalized using thefollowing formula: relative quantity of HSF1/HSP mRNA =2−∆∆Ct, where ∆∆Ct = [(Cthsf/hsps mRNA −Ctβ-actin mRNA)test group −(Cthsf/hsps mRNA − Ctβ-actinmRNA)control group].

Analysis of HSF1 and HSP interaction

We used the STRING (version 9.1) database(http://string-db.org/), which aims to provide aglobal perspective for as many organisms as feasible. The databasescores and integrates known and predicted associations, resultingin comprehensive protein networks covering >1,100 organisms(31).

Statistical analysis

Statistical analysis of the differences between theexperimental group and control group values was performed usingone-way analysis of variance followed by the Duncan's multiplecomparison test with SPSS version 20.0 software (IBM, Armonk, NY,USA). A value of P

Source: https://www.spandidos-publications.com/10.3892/ijmm.2015.2414

Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators

Heat Shock Proteins & HSP70 + Factors That Increase Them

  1. Department of Biochemistry, Molecular Biology, and Cell Biology, Rice Institute for Biomedical Research, Northwestern University, Evanston, Illinois 60208 USA

Our cells and tissues are challenged constantly by exposure to extreme conditions that cause acute and chronic stress. Consequently, survival has necessitated the evolution of stress response networks to detect, monitor, and respond to environmental changes (Morimoto et al.

1990, 1994a; Baeuerle 1995; Baeuerle and Baltimore 1996; Feige et al. 1996; Morimoto and Santoro 1998).

Prolonged exposure to stress interferes with efficient operations of the cell, with negative consequences on the biochemical properties of proteins that, under ideal conditions, exist in thermodynamically stable states. In stressed environments, proteins can unfold, misfold, or aggregate.

Therefore, the changing demands on the quality control of protein biogenesis, challenges protein homeostasis, for which the heat shock response, through the elevated synthesis of molecular chaperones and proteases, repairs protein damage and assists in the recovery of the cell.

The inducible transcription of heat shock genes is the response to a plethora of stress signals (Lis and Wu 1993; Morimoto 1993; Wu 1995) (Fig. 1), including (1) environmental stresses, (2) nonstress conditions, and (3) pathophysiology and disease states.

Although changes in heat shock protein (HSP) expression are associated with certain diseases (Morimoto et al.

1990), these observations leave open the question of whether this is an adaptation to the particular pathophysiological state, a reflection of the suboptimal cellular environment associated with the disease, or serves to warn other cells and tissues of imminent danger.

Figure 1.

Conditions that induce the heat shock response. Heat shock gene expression represented here by the activation of HSF and binding to HSE results in the elevated expression of HSPs such as Hsp70. The regulatory conditions are represented by environmental and physiological stress and nonstressful conditions, including cell growth and development and pathophysiological states.

The protective role of HSPs is a measure of their capacity to assist in the repair of protein damage.

Whether in prokaryotes, plants, or animals, overexpression of one or more HSPs is often sufficient to protect cells and tissues against otherwise lethal exposures to diverse environmental stresses including hydrogen peroxide and other oxidants, toxic chemicals, extreme temperatures, and ethanol-induced toxicity (Parsell and Lindquist 1994).

In vertebrate tissue culture cells and animal models, elevating HSPs level, either by modulation of the heat shock response or by constitutive overexpression of specific heat shock proteins, restricts or substantially reduces the level of pathology and cell death (Mizzen and Welch 1988; Huot et al. 1991; Jaattela et al.

1992; Parsell and Lindquist 1994; Mestril et al. 1994; Plumier et al. 1995; Marber et al. 1995; Mehlen et al. 1995; Mosser et al. 1997). This has led to the recognition that HSPs, via their chaperoning effects on proteins, protect cells from many forms of stress-induced cell damage and could influence the course of disease.

The stress signal that activates the heat shock response is widely held to be the flux of non-native, such as, nipfridid proteins (Morimoto et al. 1994b).

Adaptation to this stress, in turn, leads to the elevated expression of heat shock genes such that molecular chaperones are rapidly synthesized and deployed to prevent protein misfolding and to assist in their refolding to the native state.

Stress-induced transcription requires activation of heat shock factor (HSF) (Lis and Wu 1993; Morimoto 1993; Voellmy 1994, 1996; Wu 1995) that binds to the heat shock promoter element (HSE), characterized as multiple adjacent and inverse iterations of the pentanucleotide motif 5′-nGAAn-3′ (Fernandes et al. 1994).

An unexpected complexity of the regulation of the heat shock response was the finding that plants and larger animals, un yeast and Drosophila, have multiple HSFs (Sorger and Pelham 1988; Wiederrecht et al. 1988; Clos et al. 1990; Scharf et al. 1990, 1993; Sarge et al. 1991; Schuetz et al.

1991; Nakai and Morimoto 1993; Treuter et al. 1993; Czarnecka-Verner et al. 1995; Nover et al. 1996; Nakai et al. 1997). Among vertebrates, HSFs 1, 2, and 4 are ubiquitous, whereas HSF3 has been characterized only in avian species (Table 1). Thus far, our understanding of the role and function of each of these HSFs is incomplete.

However, it appears that the diversity of HSFs provides redundancy and specialization of stress signals, a means to differentially control the rate of transcription of heat shock genes, and provides novel interactions with other regulatory factors thus expanding the link between cell stress and other genetic networks.

Among the various cloned HSF genes, there is an overall sequence identity of 40% with structural conservation (Fig.2) in the winged helix–turn–helix DNA binding domain (Harrison et al. 1994; Vuister et al. 1994; Schultheiss et al.

1996), an adjacent 80 residue hydrophobic repeat (HR-A/B) essential for trimer formation (Sorger and Nelson 1989; Clos et al. 1990; Peteranderl and Nelson 1992), and the carboxy-terminal transactivation domain (Chen et al. 1993; Green et al. 1995; Shi et al. 1995; Zuo et al. 1995; Wisniewski et al. 1996).

With the exception of the HSF in budding yeast and human HSF4, another hydrophobic repeat (HR-C) is located adjacent to the transactivation domain; this repeat has been suggested to suppress trimer formation by interacting with HR-A/B (Nakai and Morimoto 1993; Rabindran et al. 1993).

Also, positioned between HR-A/B and HR-C are sequences that negatively regulate DNA binding and transcriptional activation (Nieto-Sotelo et al. 1990; Hoj and Jakobsen 1994; Green et al. 1995;Shi et al. 1995; Zuo et al. 1995).

Other features unique to certain HSFs are the presence of an amino-terminal transactivation domain in the Saccharomyces cerevisiae HSF (Sorger 1990) and spliced variants of mouse and human HSF2 and HSF4, which have variable transactivation properties (Fiorenza et al. 1995; Goodson et al. 1995; A. Nakai, pers. comm.). Apart from S.

cerevisiae, the HSF form present in unstressed cells is a latent monomer that lacks both DNA binding and transcriptional activity (Larson et al. 1988; Clos et al. 1990; Rabindran et al. 1991; Sarge et al. 1991, 1993; Baler et al. 1993; Westwood and Wu 1993; Zuo et al. 1994).

Figure 2.

General structural and regulatory features of HSFs.

Schematic representation of HSF1 structural motifs corresponding to the DNA-binding domain, hydrophobic heptad repeats (HR-A/B and HR-C), the carboxy-terminal transcriptional activation domain, and the negative regulatory domains that influence HSF1 activity.

The relative positions of these domains in HSF1 are indicated by the amino acid residues. Shown below is a schematic of the intramolecular negatively regulated monomer that, upon stress exposure, is activated to form homotrimers with DNA-binding activity.

Of the HSFs coexpressed in vertebrates, HSF1 is functionally analogous to yeast and Drosophila HSF as the principal stress-induced transcription factor (Nakai et al. 1993; Rabindran et al. 1991; Sarge et al. 1991).

Yeast HSF is essential and in Drosophila, loss of HSF exhibits an early developmental phenotype and is essential for induction of the heat shock response and stress tolerance (Wiederrecht et al. 1988; Jedlicka et al. 1997; Sorger et al. 1988). Mice lacking HSF1 can develop normally and attain adulthood.

However, fibroblasts derived from HSF1-deficient mice are incapable of stress-induced transcription of heat shock genes, consistent with the expectations of the central role of HSF1 in the heat shock response (McMillan et al. 1998).

Activation of HSF1 (see below) is in response to a multitude of stress conditions, such as heat shock, oxidative stress, and amino acid analogs that lead to the synthesis of non-native proteins (Morimoto et al. 1990, 1994b, 1996; Wu 1995).

Because heat shock also leads to the inhibition of protein synthesis, and in doing so prevents the appearance of nascent polypeptides that could misfold, HSF1 has an important role in the molecular response to non-native proteins.

In the stressed cell, the fate of non-native proteins therefore depends exquisitely upon molecular chaperones to capture and maintain intermediate folded states, and upon recovery to facilitate their refolding or degradation.

In avian cells, HSF1 and HSF3 are coexpressed and coactivated by chemical and physiological stress, which led to the suggestion of HSF regulatory and function redundancy (Nakai et al. 1995; Tanabe et al. 1997).

However, it is now clear that cells lacking HSF3 are severely compromised for induction of the heat shock response even though HSF1 is expressed (Tanabe et al. 1998). HSF3 also interacts with other transcription factors and can be activated by the Myb oncogene, independent of stress, via direct protein–protein interaction between the HSF3 and Myb DNA binding domains (Kanei-Ishii et al. 1997). The interactions between Myb and HSF3 reveal novel genetic regulatory pathways that connect events of cell growth and cell stress.

Relative to the more complete characterization of HSF1 during the heat shock response, the regulation and role of HSF2 has been an enigma.

Induction of the DNA binding properties of HSF2 is accompanied by a transition from an inert dimeric state to an activated trimer and occurs during early mouse embryonic development, spermatogenesis, and in human erythroleukemia K562 cells exposed to hemin (Theodorakis et al. 1989; Sistonen et al. 1992; 1994; Mezger et al. 1994; Sarge et al.

1994; Rallu et al. 1997). Although these results suggested that HSF2 activity was associated with development and differentiation, the events responsible for activation of HSF2 were uncharacterized.

The stress signal responsible for HSF2 activation was recently shown to result from downregulation of the ubiquitin-dependent protein degradation machinery (Mathew and Morimoto 1998).

Incubation of mammalian cells expressing HSF2 with specific inhibitors of the ubiquitin-proteasome pathway, such as MG132 or lactacystin, resulted in the activation of HSF2 DNA binding activity in a cell-type-independent mechanism. Un HSF1, HSF2 is a labile protein whose concentration increases upon inhibition of proteasome activity (Mathew et al. 1998).

These results establish a role for HSF2 in the heat shock response as a molecular response to the flux of non-native proteins targeted for protein degradation, as a complement to HSF1 that is principally activated by the flux of newly synthesized non-native proteins.

Consequently, the kinetics of HSF1 activation typically is very rapidly relative to the delayed activation profile of HSF2 (Sistonen et al. 1994). Selex experiments performed to identify the optimal nucleotide binding sites for HSF1 and HSF2 have suggested potential distinctions in preferences for the consensus HSE and differences in the numbers of HSE pentamer binding sites required for optimal binding of either HSF (Kroeger and Morimoto 1994). These studies have raised the possibility that HSF2 may have distinct target genes from those of HSF1, as well as differing specificities for common target genes (Leppa et al. 1997; Liu et al. 1997).

General features of heat shock gene transcription following binding of HSF to its target have been elucidated elegantly and will only be summarized here briefly as this has been the topic of recent reviews (Lis and Wu 1993; Wu 1995).

The inducible binding of HSF leads to changes in the organization of chromatin structure localized to the 5∼-flanking regions of heat shock genes (Wu 1980, 1984; Giardina et al. 1992). Although HSF associates with components of the chromatin remodeling machinery (Becker and Wu 1992; Tsukiyama et al.

1994; Brown and Kingston 1997) and the basal transcriptional machinery, association specifically with TBP also has been detected (Mason and Lis 1997).

Such interactions may be critical for the release of the paused RNA polymerase II that represents a pre-initiated nascent transcript that elongates in a stress-dependent manner to yield inducible heat shock mRNAs (Gilmour and Lis 1986; Rougvie and Lis 1988).

Does HSF directly sense its biochemical environment? Although heat shock has been the typical stress condition employed, HSF trimer formation cannot solely be a temperature-regulated event because the majority of stressors have their effect in cells grown at 37°C.

Yet, there exists a substantial amount of data indicating that HSF translated in vitro in reticulocyte lysates can be activated by heat shock and that purified Drosophila HSF or recombinant mouse and human HSF1 can acquire DNA binding upon in vitro heat shock (Mosser et al. 1990; Goodson and Sarge 1995; Larson et al. 1995; Zhong et al. 1998).

wise, in vitro translated HSF1 or purified HSF can be activated by low pH (pH 6.5) or salicylate (Mosser et al. 1990; Zhong et al. 1998). As salicylate is an organic acid, its effect on HSF activity could be analogous to the activating effects of low pH, perhaps acting on HSF1 conformation.

Although these results suggest that the intrinsic properties of HSF can be modulated by its biochemical environment, they do not exclude a role for other negative regulators that may function to keep HSF in the repressed state.

Is activation of HSF1 a titrated phenomenon such that simultaneous or subsequent exposure to multiple stressors at subthreshold levels leads to a complete heat shock response? This issue addresses whether stress has multiple distinct targets or stress receptors and whether these stress signals converge upon common pathways. The temperature for activation of HSF is not an absolute; to illustrate this point, growing HeLa cells at temperatures

Source: http://genesdev.cshlp.org/content/12/24/3788.full

The Role of Inducible Hsp70, and Other Heat Shock Proteins, in Adaptive Complex of Cold Tolerance of the Fruit Fly (Drosophila melanogaster)

Heat Shock Proteins & HSP70 + Factors That Increase Them

The ubiquitous occurrence of inducible Heat Shock Proteins (Hsps) up-regulation in response to cold-acclimation and/or to cold shock, including massive increase of Hsp70 mRNA levels, often led to hasty interpretations of its role in the repair of cold injury expressed as protein denaturation or misfolding.

So far, direct functional analyses in Drosophila melanogaster and other insects brought either limited or no support for such interpretations. In this paper, we analyze the cold tolerance and the expression levels of 24 different mRNA transcripts of the Hsps complex and related genes in response to cold in two strains of D.

melanogaster: the wild-type and the Hsp70- null mutant lacking all six copies of Hsp70 gene.

We found that larvae of both strains show similar patterns of Hsps complex gene expression in response to long-term cold-acclimation and during recovery from chronic cold exposures or acute cold shocks. No transcriptional compensation for missing Hsp70 gene was seen in Hsp70- strain.

The cold-induced Hsps gene expression is most probably regulated by alternative splice variants C and D of the Heat Shock Factor. The cold tolerance in Hsp70- null mutants was clearly impaired only when the larvae were exposed to severe acute cold shock.

No differences in mortality were found between two strains when the larvae were exposed to relatively mild doses of cold, either chronic exposures to 0°C or acute cold shocks at temperatures down to -4°C.

The up-regulated expression of a complex of inducible Hsps genes, and Hsp70 mRNA in particular, is tightly associated with cold-acclimation and cold exposure in D. melanogaster.

Genetic elimination of Hsp70 up-regulation response has no effect on survival of chronic exposures to 0°C or mild acute cold shocks, while it negatively affects survival after severe acute cold shocks at temperaures below -8°C.

Citation: Štětina T, Koštál V, Korbelová J (2015) The Role of Inducible Hsp70, and Other Heat Shock Proteins, in Adaptive Complex of Cold Tolerance of the Fruit Fly (Drosophila melanogaster). PLoS ONE 10(6): e0128976. https://doi.org/10.1371/journal.pone.0128976

Academic Editor: Giancarlo López-Martínez, New Mexico State University, UNITED STATES

Received: January 22, 2015; Accepted: May 1, 2015; Published: June 2, 2015

Copyright: © 2015 Štětina et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by the Czech Science Foundation (http://www.gacr.cz/en/) grant 13-01057S to V.K.

and by Ministry of Education, Youth and Sports of the Czech Republic (http://www.msmt.cz/research-and-development-1) grant KONTAKT II LH 12103 to V.K. The work of T.Š.

was supported by University of South Bohemia (https://www.jcu.cz/?set_language=en) grant GAJU 038/2014/P.

Competing interests: The authors have declared that no competing interests exist.

Insects, as very small ectotherms with little possibility to maintain body heat, have evolved different and complex physiological mechanisms to cope with low body temperatures [1].

These mechanisms help to prevent occurrence of various types of cold injury: (a) indirect chilling injury: accumulates over long, chronic exposures (days to months) to mild cold (hypothermic temperatures around or above zero) and is mechanistically linked most ly with disturbed coordination of various metabolic pathways, accumulation of toxic intermediates including reactive molecules (oxidative stress), depletion of free chemical energy, and consequent disturbance of ion homeostasis [2–6]; (b) direct chilling injury: results from brief, acute exposures (minutes to hours) to severe cold (cryothermic temperatures close to supercooling point but without freezing), which causes dissociation of multimeric macromolecular complexes, loss of enzymatic activity, protein denaturation [7–9], phase transitions in membrane lipids, massive ion leakage, and rapid cell death [10,11]; and (c) freezing injury: is linked with growing ice crystals causing mechanical damage to subcellular structures, freeze-concentration of solutes that may exceed toxic limits, and severe freeze-dehydration that may cause fusions of incompatible membraneous compartments [12].

This study will focus on survival after chronic and acute exposures to cold (without freezing) in the larvae of fruit fly, D. melanogaster, an insect species with tropical origin. Tropical insects typically exhibit only limited tolerance to cold.

Nevertheless, they posses clear capacity for enhancing cold tolerance in response to long-term acclimation (LTA) on a time scale of days to weeks [13–15].

In addition, practically all insects studied so far, including fruit fly, exhibited capacity to undergo very rapid adjustments of cold tolerance on a time scale of minutes to hours (so called rapid cold hardening, RCH) [16,17], which helps to prevent cold injury during diurnal changes of environmental temperature [18].

The physiological mechanisms underpinning LTA and RCH are most probably distinct [17,19].

While the RCH is primarily driven by rapid cellular processes signaling cascades and changes in protein phosphorylation [19], the LTA or seasonal cold acclimation is more systemic, highly complex and often triggered in advance, prior to the advent of cold season, together with transition from active life to developmental arrest called diapause [20,21]. Diapause and seasonal acclimatization represent deep phenotypic transfigurations global changes in gene transcription, protein expression, and metabolom composition [22–24]. The adaptive complex of LTA includes several physiological mechanisms such as regulation of activity of ice nucleators affecting the supercooling capacity [25,26], synthesis of low-molecular mass cryoprotectants [27,28], synthesis of proteins which regulate the process of ice formation [29,30], compositional remodeling of cell membranes [31], and, last but not least, up-regulation of cellular protective systems to prevent apoptosis [32], oxidative damage [33], and protein denaturation [34,35]. Due to complexity, redundancy and interplay between various processes, it is inherently difficult to study the role of individual mechanisms of LTA in separation [36].

In this paper, we assess the role of Heat Shock Proteins (Hsps), especially of the inducible form of Hsp70, in the adaptive complex of LTA in larvae of D. melanogaster.

There are several good reasons for choosing the fruit fly as a model: (1) Fruit fly represents well established, genetically tractable, model organism offering good knowledge on genetic structure and physiological functions of Hsps [37–41].

(2) Fruit fly larvae possess considerable capacity to improve their cold tolerance in response to cold acclimation, both RCH and LTA [14,42,43], but the physiological basis of this phenotypic plasticity is largely unknown.

(3) The species richness in the family Drosophilidae is often exploited in comparative studies on geographic clines in stress tolerance, including cold tolerance, with the aim to understand evolutionary patterns of speciation, to explain principles of ecological niche occupation, and/or to predict future responses to climate changes [44,45].

(4) The main reason, however, was that the earlier studies on the role of Hsps in the fruit fly cold tolerance brought variable results. Burton et al. [46] showed that 70 kDa Hsps were synthesized during recovery from chronic exposure to 0°C in the absence of heat shock and that a mild heat pre-treatment helped to prevent mortality caused by subsequent cold exposure.

Since this pioneering study, the cold-stimulated up-regulation of Hsps complex was repeatedly confirmed in drosophilids at the levels of mRNA and proteins [47–50]. Moreover, Colinet et al. [51] reported that knocking down the expression of small Hsp22 and Hsp23 genes by RNAi increases chill coma recovery time.

These results, together with functional (RNAi) studies performed on Hsps in other insect species [34,35], supported the view that Hsps play important role in insect cold tolerance. This view, however, was challenged in a study by Nielsen et al. [52] conducted with heat-sensitive mutant strain of D. melanogaster that harbors a mutation in the hsf gene that renders the gene product, heat shock transcription factor HSF, non-functional above 30°C [52,53]. Nielsen's et al. study [52] convincingly showed that HSF activation and subsequent Hsp70 expression did not occur during RCH and, although the HSF activation and Hsp70 up-regulation did occur during the LTA, no beneficial effect on fly cold tolerance was detected.

The main objective of this study was to clarify whether or not the up-regulation of inducible Hsp70 associated with LTA and recovery after cold exposure plays a role in the adaptive complex of cold tolerance of D. melanogaster.

The responses to cold were compared in two fly strains: Hsp70- null mutant lacking all six copies of Hsp70 gene [54] and the wild-type strain Oregon R [55]. We focused on cold tolerance in fully grown 3rd instar larvae that were acclimated (LTA) at low temperatures (15°C followed by 6°C) in order to express their maximum cold tolerance [14].

The effects of chronic exposures to mild cold were distinguished from the effects of acute exposures to severe subzero temperatures. In addition, we used two different cold pre-treatments at sub-lethal doses of cold, again chronic or acute, in order to stimulate the expression of inducible Hsps prior to cold exposure.

The expression levels of Hsp70 mRNA transcripts and another 19 genes belonging to Hsps complex plus 4 other genes (Frost, Menin, Cold shock protein, and Starvin) potentially linked to cold acclimation/cold injury repair, were quantified by qRT-PCR but we found no compensation response for missing Hsp70 gene in Hsp70- strain.

We suggest that the up-regulation of Hsp70 mRNA, which is ubiquitously observed in response to cold-acclimation and recovery after cold exposure, need not always be directly linked to, or necessary for, repair of cold injury. We observed that cold tolerance in Hsp70- null mutants of D.

melanogaster is compromised only when the larvae are exposed to severe cold shocks of or below -8°C. The exposures to milder doses of cold, either chronic exposures to 0°C or acute cold shocks at temperatures down to -4°C, caused similar rates of survival/mortality in the wild type larvae and the Hsp70- null mutants.

The main experiments were conducted with two strains of Drosophila (Sophophora) melanogaster (Meigen, 1830): the wild-type (Oregon R strain) [55] is routinely maintained in our laboratory for decades, and Hsp70-null mutant strain (Hsp70- strain) [54] was obtained from Bloomington Drosophila Stock Center as a stock no. 8841 with a genotype: w[1118]; Df(3R)Hsp70A, Df(3R)Hsp70B. The wild-type fruit fly has six nearly identical gene copies that encode Hsp70 protein and Hsp70 accounts for the bulk of Hsps that are expressed upon heat shock [56]. All six copies of Hsp70 gene were deleted by homologous recombination in the Hsp70-null mutants and the Hsp70- homozygous strain was established [54]. The larvae of White strain (mutation in locus white; [57]), which served as genetic background to create the Hsp70- strain, were used in our study to verify the constitutive levels of target genes' expression in unstressed larvae. In consequence of lacking Hsp70 gene, the Hsp70- larvae and adult flies showed reduced thermotolerance, which was specifically expressed as low or almost no survival after a severe heat shock (39–39.5°C) that was preceded by a sub-lethal heat pre-treatment at 35–36°C. Interestingly, the survival after milder heat shocks (

Source: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0128976