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Cells Overexpressing Hsp27 Show Accelerated Recovery from Heat-Induced Nuclear-Protein Aggregation
Abstract
Protein denaturation/aggregation upon cell exposure to heat shock is a likely cause of cell death. In the nucleus, protein aggregation has often been correlated to inhibition of nuclear located processes and heat-induced cell killing. In Chinese hamster O23 cells made thermotolerant by a prior heating (20'44 degrees C + 10h 37 degrees C) which induces the whole spectrum of heat shock proteins (hsps), the extent of nuclear protein aggregation during heat shock is reduced and the rate of recovery from aggregation after heat shock is enhanced. In contrast, a heat resistant Chinese hamster cell line overexpressing only hsp27 shows an unaltered sensitivity to formation of nuclear protein aggregates by heat, but shows the same enhanced rate of recovery from nuclear protein aggregation as thermotolerant cells. This suggests that accelerated recovery of protein aggregation could be partly responsible for hsp27-mediated thermoprotection.
... Metazoans lack an Hsp104 disaggregase; however, they also exhibit disaggregation activity: e.g., model proteins like firefly luciferase can be reactivated from a heat-aggregated state (Pinto et al., 1991). Also in metazoans, disaggregation is linked to stress resistance in cellular (Kampinga et al., 1987(Kampinga et al., , 1994 and organismal models (Kirstein et al., 2017;Rampelt et al., 2012), documenting the essential need to reverse protein aggregation for cellular survival. Here, we will first describe the nonmetazoan disaggregation machinery before reporting on the metazoan system. ...
... This rapid association preserves a native-like state of bound substrates and protects them from further unfolding (Cheng et al., 2008;Ungelenk et al., 2016) (Figure 3B). sHsps thereby act as a first line of cellular defense against unfolding stress and create a reservoir of proteins competent for disaggregation and refolding upon stress relief (Ehrnsperger et al., 1997;Kampinga et al., 1994;Lee et al., 1997). Of note, however, a holdase activity may not be shared among all members of the sHsp family, and in humans not all members share the same anti-aggregation capacity (Mymrikov et al., 2017;Vos et al., 2008). ...
... Substrate displacement thus requires force application by Hsp70 and Hsp100 disaggregases to disrupt sHsp-substrate interactions, similar to disaggregation of protein aggregates generated in their absence. However, the formation of mixed sHsp-unfolded substrate complexes facilitates poststress disaggregation of substrates in vivo (Kampinga et al., 1994;Mogk et al., 2003a;Ungelenk et al., 2016) and in vitro (Haslbeck et al., 2005;Mogk et al., 2003b;Nillegoda et al., 2015) as compared to substrates aggregated in isolation ( Figure 3B). sHsps seem to prevent the formation of more tight interactions between the unfolded proteins. ...
Both acute proteotoxic stresses that unfold proteins and expression of disease-causing mutant proteins that expose aggregation-prone regions can promote protein aggregation. Protein aggregates can interfere with cellular processes and deplete factors crucial for protein homeostasis. To cope with these challenges, cells are equipped with diverse folding and degradation activities to rescue or eliminate aggregated proteins. Here, we review the different chaperone disaggregation machines and their mechanisms of action. In all these machines, the coating of protein aggregates by Hsp70 chaperones represents the conserved, initializing step. In bacteria, fungi, and plants, Hsp70 recruits and activates Hsp100 disaggregases to extract aggregated proteins. In the cytosol of metazoa, Hsp70 is empowered by a specific cast of J-protein and Hsp110 co-chaperones allowing for standalone disaggregation activity. Both types of disaggregation machines are supported by small Hsps that sequester misfolded proteins.
... HSP27 is ubiquitously expressed in different tissues and cells, and localizes under normal conditions to the cytosol. However, HSP27 is able to translocate into the nucleus in response to heat shock or stress [33,34]. Although we could observe an increase in the levels HSP27 in MCF7 cells compared with MCF10A or MDA231 cells ( Figure 4H), we noted that HSP27 localization paralleled that of NLK ( Figure 4I and Figure 1B). ...
... to heat shock or stress [33,34]. We could also show that NLK is unable to phosphorylate HSP27 at serine 82 in cells transfected with wildtype NLK, but we cannot rule out the possibility that NLK phosphorylate HSP27 at another site beside serine 82. ...
Nemo-like kinase (NLK), a proline-directed serine/threonine kinase regulated by phosphorylation, can be localized in the cytosol or in the nucleus. Whether the localization of NLK can affect cell survival or cell apoptosis is yet to be disclosed. In the present study we found that NLK was mainly localized in the nuclei of breast cancer cells, in contrast to a cytosolic localization in non-cancerous breast epithelial cells. The nuclear localization of NLK was mediated through direct interaction with Heat shock protein 27 (HSP27) which further protected cancer cells from apoptosis. The present study provides evidence of a novel mechanism by which HSP27 recognizes NLK in the breast cancer cells and prevents NLK-mediated cell apoptosis.
... Additionally, HSPB1 forms a complex with the autophagy cargo receptor protein sequestosome-1 (SQSTM1/p62) and regulates the unconventional secretion of HTT [59]. Under stressful conditions, sHSPs bind to the early unfolding intermediates of easily aggregable proteins to form stable sHSP-substrate complexes and maintain the folding capacity of the substrate [60][61][62]. Smaller species, including dimers, commonly prevent aggregation as active forms of sHSPs, possibly because smaller species have more unfolded structures in their subunits, which help enhance the chaperone activity of sHSPs [14,15,63]. ...
The small heat shock proteins (sHSPs), whose molecular weight ranges from 12∼43 kDa, are members of the heat shock protein (HSP) family that are widely found in all organisms. As intracellular stress resistance molecules, sHSPs play an important role in maintaining the homeostasis of the intracellular environment under various stressful conditions. A total of 10 sHSPs have been identified in mammals, sharing conserved α-crystal domains combined with variable N-terminal and C-terminal regions. Unlike large-molecular-weight HSP, sHSPs prevent substrate protein aggregation through an ATP-independent mechanism. In addition to chaperone activity, sHSPs were also shown to suppress apoptosis, ferroptosis, and senescence, promote autophagy, regulate cytoskeletal dynamics, maintain membrane stability, control the direction of cellular differentiation, modulate angiogenesis, and spermatogenesis, as well as attenuate the inflammatory response and reduce oxidative damage. Phosphorylation is the most significant post-translational modification of sHSPs and is usually an indicator of their activation. Furthermore, abnormalities in sHSPs often lead to aggregation of substrate proteins and dysfunction of client proteins, resulting in disease. This paper reviews the various biological functions of sHSPs in mammals, emphasizing the roles of different sHSPs in specific cellular activities. In addition, we discuss the effect of phosphorylation on the function of sHSPs and the association between sHSPs and disease.
... In addition, small HSPs are associated with many essential processes like contraction of smooth muscle, cell proliferation and survival (Champagne et al., 1999;Salinthone, Tyagi, & Gerthoffer, 2008). HSP27 is crucial in maintaining the actin cytoskeleton, averting apoptosis and reducing oxidative stress (Kampinga, Brunsting, Stege, Konings, & Landry, 1994;Lavoie, Lambert, Hickey, Weber, & Landry, 1995). In hereditary motor neuropathy and Charcot-Marie-Tooth disease, the role of HSP27 is well established (Tanabe et al., 2018;Züchner & Vance, 2006). ...
Heat Shock Proteins (HSPs) are evolutionarily conserved proteins from prokaryotes to eukaryotes. They are ubiquitous proteins involved in key physiological and cellular pathways (viz. inflammation, immunity and apoptosis). Indeed, the survivability of the cells under various stressful conditions depends on appropriate levels of HSP expression. There is a growing line of evidence for the role of HSPs in regulating cardiovascular diseases (CVDs) (viz. hypertension, atherosclerosis, atrial fibrillation, cardiomyopathy and heart failure). Furthermore, studies indicate that a higher concentration of circulatory HSP antibodies correlate to CVDs; some are even potential markers for CVDs. The multifaceted roles of HSPs in regulating cellular signaling necessitate unraveling their links to pathophysiology of CVDs. This review aims to consolidate our understanding of transcriptional (via multiple transcription factors including HSF-1, NF-κB, CREB and STAT3) and post-transcriptional (via microRNAs including miR-1, miR-21 and miR-24) regulation of HSPs. The cytoprotective nature of HSPs catapults them to the limelight as modulators of cell survival. Yet another attractive prospect is the development of new therapeutic strategies against cardiovascular diseases (from hypertension to heart failure) by targeting the regulation of HSPs. Moreover, this review provides insights into how genetic variation of HSPs can contribute to the manifestation of CVDs. It would also offer a bird's eye view of the evolving role of different HSPs in the modulation and manifestation of cardiovascular disease.
... Posttranslational modifications can strongly influence the functions of HSP27. It can act as a chaperone, stabilizing denatured or aggregated proteins and folding them back to their original form [12,19]. Phosphorylation, which is triggered by oxidative stress [28], abolishes oligomerization, resulting in increased chaperone activity of monomers [2,11]. The localization of HSP27 is not limited to the cytosol, but has also been detected in the nucleus [1]. ...
Purpose:
Orthodontic tooth movement is a complex process involving the remodeling of extracellular matrix and bone as well as inflammatory processes. During orthodontic treatment, sterile inflammation and mechanical loading favor the production of receptor activator of NF-κB ligand (RANKL). Simultaneously, expression of osteoprotegerin (OPG) is inhibited. This stimulates bone resorption on the pressure side. Recently, heat shock protein 27 (HSP27) was shown to be expressed in the periodontal ligament after force application and to interfere with inflammatory processes.
Methods:
We investigated the effects of phosphorylated HSP27 on collagen synthesis (COL1A2 mRNA), inflammation (IL1B mRNA, IL6 mRNA, PTGS2 protein) and bone remodeling (RANKL protein, OPG protein) in human periodontal ligament fibroblasts (PDLF) without and with transfection of a plasmid mimicking permanent phosphorylation of HSP27 using real-time quantitative polymerase chain reaction (RT-qPCR), western blot and enzyme-linked immunosorbent assays (ELISAs). Furthermore, we investigated PDLF-induced osteoclastogenesis after compressive strain in a co-culture model with human macrophages.
Results:
In particular, phosphorylated HSP27 increased gene expression of COL1A2 and protein expression of PTGS2, while IL6 mRNA levels were reduced. Furthermore, we observed an increasing effect on the RANKL/OPG ratio and osteoclastogenesis mediated by PDLF.
Conclusion:
Phosphorylation of HSP27 may therefore be involved in the regulation of orthodontic tooth movement by impairment of the sterile inflammation response and osteoclastogenesis.
... Hsp70 further interacts with Hsp100 for efficient disaggregation and refolding of the substrate [16,21]. The interaction between Hsp70 and Hsp100 is well-characterized in several organisms and crucial for efficiently refolding the denatured substrate [22][23][24][25][26][27][28][29][30]. Similar interactions of Hsp70 with Hsp90 and Hsp60 are also well-documented [31][32][33][34][35][36][37]. ...
Heat shock proteins maintain protein homeostasis and facilitate the survival of an organism under stress. Archaeal heat shock machinery usually consists of only sHsps, Hsp70, and Hsp60. Moreover, Hsp70 is absent in thermophilic and hyperthermophilic archaea. In the absence of Hsp70, how aggregating protein substrates are transferred to Hsp60 for refolding remains elusive. Here, we investigated the crosstalk in the heat shock response pathway of thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. In the present study, we biophysically and biochemically characterized one of the small heat shock proteins, Hsp14, of S. acidocaldarius. Moreover, we investigated its ability to interact with Hsp20 and Hsp60 to facilitate the substrate proteins' folding under stress conditions. Like Hsp20, we demonstrated that the dimer is the active form of Hsp14, and it forms an oligomeric storage form at a higher temperature. More importantly, the dynamics of the Hsp14 oligomer are maintained by rapid subunit exchange between the dimeric states, and the rate of subunit exchange increases with increasing temperature. We also tested the ability of Hsp14 to form hetero‐oligomers via subunit exchange with Hsp20. We observed hetero‐oligomer formation only at higher temperatures (50 °C–70 °C). Furthermore, experiments were performed to investigate the interaction between small heat shock proteins and Hsp60. We demonstrated an enthalpy‐driven direct physical interaction between Hsp14 and Hsp60. Our results revealed that Hsp14 could transfer sHsp‐captured substrate proteins to Hsp60, which then refolds them back to their active form.
... N-and C-termini confer to sHSPs solubility facilitating their oligomerization (Figure 1a) [15,22]. sHSP oligomers can be engaged within protein aggregates in order to facilitate protein folding [23,24]. HSP27 is the most referenced member of the sHSP family in cases of autoimmunity (see below). ...
Within the last two decades, there has been increasing evidence that heat-shock proteins can have a differential influence on the immune system. They can either provoke or ameliorate immune responses. This review focuses on outlining the stimulatory as well as the inhibitory effects of heat-shock proteins 27, 40, 70, 65, 60, and 90 in experimental and clinical autoimmune settings.
... HSP27 has previously been considered as an adaptive cellular response protein that helps the organism to survive due to its protective effects against heat-induced cellular damage and its primary function as a molecular chaperone involved in protein folding and a variety of cellular processes (Singh et al., 2017). Additionally, the phosphorylated form of HSP27 can interact with denatured and aggregated proteins to protect cells against severe damage from stress (Kampinga et al., 1994;Trott et al., 2009). In previous studies, we have demonstrated that HSP27 is significantly elevated in photoaged rat skin and HaCaT cells and exerts protective effects against photoaging by modulating antiapoptotic pathways (Liu et al., 2019a,b). ...
Skin aging caused by UV radiation is called photoaging is characterized by skin roughness and dryness accompanied by a significant reduction of dermal collagen. Rapamycin is a macrolide immunosuppressant which has been shown to exhibit “anti-aging” effects in cells and organisms, however, its roles in the skin photoaging remains unclear. Here, we investigate the role of rapamycin and HSP27, which we have previously identified as an inhibitor of UV-induced apoptosis and senescence in HaCat cells, in a UVA-induced photoaging model of primary human dermal fibroblasts (HDFs). Results from senescence-associated beta-galactosidase (SA-β-gal) staining revealed that rapamycin significantly reduced senescence in UVA-treated HDFs. In addition, treatment with rapamycin significantly increased cell autophagy levels, decreased the expression of p53 and phosphorylated HSP27, and reduced genotoxic and oxidative cellular stress levels in UVA-induced HDFs. Knockdown of HSP27 resulted in a significant increase of MMP-1 and MMP-3 as well as a decrease in type I collagen expression. Rapamycin mitigated these effects by activation of the classical TGF-β/Smad signaling pathway and increasing the transcriptional activity of MAPK/AP-1. Taken together, these results suggest that rapamycin may potentially serve as a preventive and therapeutic agent for UVA-induced photoaging of the skin.
... Refolding of proteins from sHsp-substrate complexes as well as from sHsp-incorporating aggregates can be conducted by ATP-dependent chaperones. In mammalian cells, the recovery of substrate proteins is mediated by the Hsp70/Hsp40 chaperone system [11,163,164]. For some sHsps like Hsp22 (HspB8) the interaction between sHsps and Hsp40/Hsp70 is assumed to be facilitated by the Hsp70 co-chaperone Bag3 [165][166][167][168][169]. Additionally, some sHsps interact as well with proteins in their native conformation like pHsp20 (phosphorylated HspB6) with 14-3-3 (see above) or Hsp27 (HspB1) and αB with cytoskeleton components like actin [63,111,[170][171][172][173][174]. ...
The proteostasis network allows organisms to support and regulate the life cycle of proteins. Especially regarding stress, molecular chaperones represent the main players within this network. Small heat shock proteins (sHsps) are a diverse family of ATP-independent molecular chaperones acting as the first line of defense in many stress situations. Thereby, the promiscuous interaction of sHsps with substrate proteins results in complexes from which the substrates can be refolded by ATP-dependent chaperones. Particularly in vertebrates, sHsps are linked to a broad variety of diseases and are needed to maintain the refractive index of the eye lens. A striking key characteristic of sHsps is their existence in ensembles of oligomers with varying numbers of subunits. The respective dynamics of these molecules allow the exchange of subunits and the formation of hetero-oligomers. Additionally, these dynamics are closely linked to the chaperone activity of sHsps. In current models a shift in the equilibrium of the sHsp ensemble allows regulation of the chaperone activity, whereby smaller oligomers are commonly the more active species. Different triggers reversibly change the oligomer equilibrium and regulate the activity of sHsps. However, a finite availability of high-resolution structures of sHsps still limits a detailed mechanistic understanding of their dynamics and the correlating recognition of substrate proteins. Here we summarize recent advances in understanding the structural and functional relationships of human sHsps with a focus on the eye-lens αA- and αB-crystallins.
... However, rather than keeping clients in a soluble state, sHSPs complexed with their clients are often found to coaggregate with them. This prevents cytotoxicity of the aggregates and facilitates later recovery and refolding or degradation of the aggregated proteins [145][146][147][148]. Moreover, as best characterized with yeast Hsp42, sHSPs may even function as "sequestrases" that actively drive the controlled aggregation of non-native proteins, thereby preventing the overburdening of the refolding machinery during cellular stress [148][149][150][151][152][153]. ...
Skeletal muscle and the nervous system depend on efficient protein quality control, and they express chaperones and cochaperones at high levels to maintain protein homeostasis. Mutations in many of these proteins cause neuromuscular diseases, myopathies, and hereditary motor and sensorimotor neuropathies. In this review, we cover mutations in DNAJB6, DNAJB2, αB-crystallin (CRYAB, HSPB5), HSPB1, HSPB3, HSPB8, and BAG3, and discuss the molecular mechanisms by which they cause neuromuscular disease. In addition, previously unpublished results are presented, showing downstream effects of BAG3 p.P209L on DNAJB6 turnover and localization.
... HSP27 can interact with Bid or cytochrome c to play an antiapoptotic role or as a chaperone to prevent misfolded proteins. [8][9][10][11][12] So far, close relationship of HSP27 with cancers has been found in breast cancer, 13,14 esophageal cancer, 15,16 CRC, 17 head and neck cancer, 18 and other solid tumors. A study in the Chinese population showed that the C allele of the rs2868371 functional promoter polymorphism of HSPB1 was associated with an increased risk of lung cancer, but better survival in patients with advanced NSCLC compared with the G allele. ...
Background:
Colorectal cancer (CRC) is the third most common cancer in the world. Genome-wide association studies are a powerful method to analyze the status of single-nucleotide polymorphisms (SNPs) in specific genes. Heat shock proteins (HSPs) were found to be involved in the cancer progression and chemoresistance. However, there is still no further study about polymorphisms of HSP beta-1 (HSPB1) in colorectal cancer. We proposed the SNP of HSPB1 may be correlated with the progression and metastasis in colon cancer.
Methods:
We recruited 379 colorectal cancer patients and categorized as four stages following the UICC TNM system. Then, we selected tagging SNPs of HSPB1 by 10% minimum allelic frequency in Han Chinese population from the HapMap database and analyze with the Chi-square test.
Results:
We demonstrated the association of HSPB1 genetic polymorphisms rs2070804 with tumor depth with colorectal cancer. But, there is a lack of association between HSPB1 genetic polymorphisms and colorectal cancer invasion, recurrence or metastasis.
Conclusions:
The polymorphisms of HSPB1 seemed to change the tumor behavior of colorectal cancer. HSPB1 rs2070804 polymorphism is associated with the depth of the primary tumor. But, there is no further correlation with other to the clinical parameters such as cancer invasiveness, local recurrence, or distant metastasis.
... Our findings provide an answer to the long-standing question of why ubiquitinated proteins are deposited in the cytosol by proteasome impairment; they provide definite identification of underlying molecular machinery and show its essential involvement in the proteostasis in animal cells. and the cytosol (17)(18)(19)(20). In addition to the relocation of molecular chaperones, misfolded proteins in the cytosoland the ER are also imported into the nucleus for degradation by the proteasome as shown recently in yeast (21,22). ...
Significance
It is commonly observed that proteasome impairment results in accumulation of ubiquitinated proteins in the cytosol. Even proteins originally located in the nucleus show similar cytosolic accumulation, suggesting that unidentified machinery proactively transports them to the cytosol. Here, we report that a protein complex, UBIN–polyubiquitinated substrate transporter, harboring ubiquitin binding domain and nuclear export signal specifically mediates this process. In addition, their worm homologues showing similar transportation activity are important to maintain the lifespan of worms under natural condition. Our findings provide an answer to the long-standing question of why ubiquitinated proteins are deposited in the cytosol by proteasome impairment; they provide definite identification of underlying molecular machinery and show its essential involvement in the proteostasis in animal cells.
... et al., 2011). Small heat shock proteins (sHsps) have been reported to enhance recovery from heat-induced nuclear protein aggregation (Kampinga et al., 1994;Stege et al., 1995) likely by maintaining denatured proteins in a folding competent state (Ehrnsperger et al., 1997;Lee et al., 1997;Deunnwald et al., 2012;Rampelt et al., 2012). The present results indicate that HSPB1 (Hsp27) co-localized at nuclear speckles after heat shock with disaggregation/refolding machine components including HSPA1A, DNAJB1, and HSPH1. ...
Heat shock proteins (Hsps) co-operate in multi-protein machines that counter protein misfolding and aggregation and involve DNAJ (Hsp40), HSPA (Hsp70), and HSPH (Hsp105α). The HSPA family is a multigene family composed of inducible and constitutively expressed members. Inducible HSPA6 (Hsp70B') is found in the human genome but not in the genomes of mouse and rat. To advance knowledge of this little studied HSPA member, the targeting of HSPA6 to stress-sensitive neuronal sites with components of a disaggregation/refolding machine was investigated following thermal stress. HSPA6 targeted the periphery of nuclear speckles (perispeckles) that have been characterized as sites of transcription. However, HSPA6 did not co-localize at perispeckles with DNAJB1 (Hsp40-1) or HSPH1 (Hsp105α). At 3 h after heat shock, HSPA6 co-localized with these members of the disaggregation/refolding machine at the granular component (GC) of the nucleolus. Inducible HSPA1A (Hsp70-1) and constitutively expressed HSPA8 (Hsc70) co-localized at nuclear speckles with components of the machine immediately after heat shock, and at the GC layer of the nucleolus at 1 h with DNAJA1 and BAG-1. These results suggest that HSPA6 exhibits targeting features that are not apparent for HSPA1A and HSPA8.
... The ability of sHsps to associate to early unfolding intermediates of substrates and to preserve their structure thus seems to represent a conserved feature (Fig. 1). Notably, sHsp binding to substrates during stress conditions has been initially suggested to create a reservoir of proteins that are competent for refolding upon return to physiological growth conditions (Ehrnsperger et al. 1997;Haslbeck et al. 2005a;Kampinga et al. 1994;Lee et al. 1997). The recent findings that sHsps trap substrates in minimally misfolded, near-native conformations are in accordance with and further specify the original model. ...
Small heat shock proteins (sHsps) exhibit an ATP-independent chaperone activity to prevent the aggregation of misfolded proteins in vitro. The seemingly conflicting presence of sHsps in insoluble protein aggregates in cells obstructs a precise definition of sHsp function in proteostasis networks. Recent findings specify sHsp activities in protein quality control systems. The sHsps of yeast, Hsp42 and Hsp26, interact with early unfolding intermediates of substrates, keeping them in a ready-to-refold conformation close to the native state. This activity facilitates substrate refolding by ATP-dependent Hsp70-Hsp100 disaggregating chaperones. Hsp42 can actively sequester misfolded proteins and promote their deposition at specific cellular sites. This aggregase activity represents a cytoprotective protein quality control strategy. The aggregase function of Hsp42 controls the formation of cytosolic aggregates (CytoQs) under diverse stress regimes and can be reconstituted in vitro, demonstrating that Hsp42 is necessary and sufficient to promote protein aggregation. Substrates sequestered at CytoQs can be dissociated by Hsp70-Hsp100 disaggregases for subsequent triage between refolding and degradation pathways or are targeted for destruction by selective autophagy termed proteophagy.
... Furthermore, HSP32 has been distinguished from other HSPs based on its cytoprotective effects against ischemic reperfusion injuries, acute renal failures, and gastrointestinal diseases [39][40][41][42]. HSP27 is a small HSP that promotes the recovery process in cells subjected to stressful conditions by regulating several crucial cellular processes, which include proteasomal degradation of unwanted proteins and minimizing the aggregation of misfolded proteins [43][44][45]. HSP70 and HSP32 are also noteworthy for their antiapoptotic functions and cytoprotective effects [30,31,[36][37][38][39][40][41][42]. Therefore, it could be possible that the mechanism by which these two HSPs improve postthaw viability is by reducing the damage caused by oxidative stress and apoptosis; however, the number of apoptotic cells did not reach statistical significance between the experimental groups. ...
Extensive research has been performed to determine the effect of freezing protocol and cryopreservation agents on the viability of ASCs as well other cells. Unfortunately, the conclusion one may draw after decades of research utilizing fundamentally similar cryopreservation techniques is that a barrier exists, which precludes full recovery. We hypothesize that agents capable of inducing a subset of heat shock proteins (HSPs) and chaperones will reduce the intrinsic barriers to the post-thaw recovery of ASCs. ASCs were exposed to 43 °C for 1 hour to upregulate heat shock proteins (HSPs) and the temporal HSP expression profile post heat shock was determined by performing qPCR and western blotting assays. The expression of HSP70 and HSP32 were found to be maximum at 3 hours after the heat shock whereas HSP90 and HSP27 remain unchanged. The heat shocked ASCs cryopreserved during maximal HSPs expression exhibited increased post thaw viability than the non-heat shocked samples. Histochemical staining and qRT-PCR indicated that the ASC differentiation potential was retained. Thus, suggesting that the up-regulation of HSPs prior to a freezing insult is beneficial to ASCs and a potential alternative to the use of harmful cryoprotective agents.
... The cellular function of these molecular chaperones is to bind proteins, maintain their structural integrity, and keep them functionally active (Jakob et al., 1993;Ehrnsperger et al., 1997;Lee et al., 1997). Thus, they act as molecular traps that protect the cell from the possibilities of irreversible protein aggregation and ultimately sustaining protein homeostasis (Jakob and Buchner, 1994;Kampinga et al., 1994;Ehrnsperger et al., 1997;Ehrnsperger et al., 1998). The combination of highly stable, long-lived proteins and a high concentration of molecular chaperones implies that the enclosed lens is well equipped to deal with any misfolding of the lens proteins Cataract is defined as opacity of the eye lens and is the leading cause of vision impairment worldwide. ...
α-Crystallin, a member of small heat shock proteins, is the major structural protein within the eye lens and is believed to play an exceptional role in the stability of lens proteins and its transparency. In the current manuscript, we have investigated the effect of an organic solvent, trifluoroethanol (TFE), on the structure and function of α-crystallin isolated from camel eye lens. Incubation of this protein with TFE changed the secondary and tertiary structures, which resulted in the aggregation of α-crystallin as evidenced by intrinsic fluorescence, Rayleigh's scattering, Thioflavin T assay, and circular dichroism spectroscopic studies. The treatment with different concentrations of TFE led to increased exposure of hydrophobic domains of α-crystallin, which was observed by 8-anilino 1-napthalene sulfonic acid extrinsic fluorescence assay. These results clearly indicate that TFE induced significant changes in the secondary and tertiary structures of α-crystallin, leading to aggregation and amyloid formation. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay established the cytotoxicity of the aggregated α-crystallin towards HepG2 cell lines through reactive oxygen species production. In conclusion, α-crystallin protein was found to be susceptible to conformational changes by TFE, suggesting that α-crystallin, although basically acting like a heat shock protein and functionally displaying chaperone-like activity, might capitulate to change in lens environment induced by diseased conditions or age-related changes, resulting in cataract formation. Copyright © 2015 John Wiley & Sons, Ltd.
... In general, unfolded proteins complexed by sHsps remain foldingcompetent and in mammalian cells, the ATP-dependent Hsp70/Hsp40 system is required for their re-folding [241][242][243]. The release of substrates from α-crystallins is, however, not yet sufficiently understood. ...
The two α-crystallins (αA- and αB-crystallin) are major components of our eye lenses. Their key function there is to preserve lens transparency which is a challenging task as the protein turnover in the lens is low necessitating the stability and longevity of the constituent proteins. α-crystallins are members of the small heat shock protein family. αB-crystallin is also expressed in other cell types.
The review summarizes the current concepts on the polydisperse structure of the α-crystallin oligomer and its chaperone function with a focus on the inherent complexity and highlighting gaps between in vitro and in vivo studies.
Both α-crystallins protect proteins from irreversible aggregation in a promiscuous manner. In maintaining eye lens transparency, they reduce the formation of light scattering particles and balance the interactions between lens crystallins. Important for these functions is their structural dynamics and heterogeneity as well as the regulation of these processes which we are beginning to understand. However, currently, it still remains elusive to which extent the in vitro observed properties of α-crystallins reflect the highly crowded situation in the lens.
Since α-crystallins play an important role in preventing cataract in the eye lens and in the development of diverse diseases, understanding their mechanism and substrate spectra is of importance. To bridge the gap between the concepts established in vitro and the in vivo function of α-crystallins, the joining of forces between different scientific disciplines and the combination of diverse techniques in hybrid approaches are necessary.
Copyright © 2015. Published by Elsevier B.V.
... Stored misfolded proteins are then either refolded or degraded (Jakob et al., 1993;Kampinga et al., 1994;Ehrnsperger et al., 1997;Rogalla et al., 1999;Sun and MacRae, 2005;Paul et al., 2010). ...
The human small heat shock protein 27 (HSP27 or HSPB1) is a multifunctional protein that participates in a variety of cellular processes such as controlling protein folding, F-actin-dependent processes, cytoprotection/anti-apoptosis, differentiation, cell proliferation, and gene expression. The structural and physiological properties of HSP27 are partially controlled by phosphorylation and several protein kinases that mediate phosphorylation have been identified. While phosphorylation of serine residues 15, 78 and 82 has been most extensively studied, other in vivo phosphorylation modifications have been identified. Here we review the different phosphorylations of HSP27 and consider the consequences of phosphorylation on HSP27’s conformation, subcellular localization, and cellular roles. We also address the phosphorylation pattern under pathogenic conditions and discuss the possible implications of HSP27 phosphorylation in human disease.
... As molecular chaperones, small heat shock proteins (sHsps) protect stressed cells from unspecific aggregation of unfolding proteins (Basha et al., 2012;Haslbeck et al., 2005a;McHaourab et al., 2009) by stably binding them in an ATP-independent manner and keeping them in a refolding-competent state (Ehrnsperger et al., 1997;Haslbeck et al., 2005a;Kampinga et al., 1994;Lee et al., 1997;McHaourab et al., 2009). This holdase activity sets sHsps apart from ATP-dependent chaperones with folding capacity (Hartl et al., 2011;Richter et al., 2010). ...
Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the aggregation of unfolding proteins during proteotoxic stress. In Caenorhabditis elegans, Sip1 is the only sHsp exclusively expressed in oocytes and embryos. Here, we demonstrate that Sip1 is essential for heat shock survival of reproducing adults and embryos. X-ray crystallography and electron microscopy revealed that Sip1 exists in a range of well-defined globular assemblies consisting of two half-spheres, each made of dimeric "spokes." Strikingly, the oligomeric distribution of Sip1 as well as its chaperone activity depend on pH, with a trend toward smaller species and higher activity at acidic conditions such as present in nematode eggs. The analysis of the interactome shows that Sip1 has a specific substrate spectrum including proteins that are essential for embryo development.
Copyright © 2015 Elsevier Inc. All rights reserved.
... In addition, the strongest positive aB-crystallin signals were observed in the cytoplasm, around and above the nucleus of H 9 C 2 cells at 100 min of heat stress. Previous data have suggested that sHSPs can function as a protein chaperone in the nuclear compartment [55]. It is also possible for cytoplasmic chaperones to have related nuclear functions, such as importins which are both cytoplasmic chaperones for exposed basic domains as well as nuclear import receptors [56]. ...
Relationships between αB-crystallin expression patterns and pathological changes of myocardial cells after heat stress were examined in vitro and in vivo in this study using the H9C2 cell line and Sprague-Dawley rats, respectively. Histopathological lesions, characterized by acute degeneration, karyopyknosis and loss of a defined nucleus, became more severe in rat hearts over the course of heat stress treatment from 20 min to 100 min. The expression of αB-crystallin in rat hearts showed a significant decrease (P<0.05) throughout the heat stress treatment period, except at the 40 min time point. Likewise, decreased αB-crystallin expression was also observed in the H9C2 cell line exposed to a high temperature in vitro, although its expression recovered to normal levels at later time points (80 and 100 min) and the cellular damage was less severe. The results suggest that αB-crystallin is mobilized early after exposure to a high temperature to interact with damaged proteins but that the myocardial cells cannot produce sufficient αB-crystallin for protection against heat stress. Lower αB-crystallin expression levels were accompanied by obvious cell/tissue damage, suggesting that the abundance of this protein is associated with protective effects in myocardial cells in vitro and in vivo. Thus, αB-crystallin is a potential biomarker of heat stress.
... We and others have reported that HSPB1 expression enhances cellular resistance to heat shock [1,2], oxidative stress [3], apoptosis inducers456 and a pleiotropic number of conditions or compounds that are deleterious to cells, such as anti-cancer drugs [7] . Recent studies have shown that HSPB1 has multiple strategies to attenuate the damage induced by stress challenges. ...
... In addition to Hsp70/40, substrate refolding can be assisted by the Hsp100/ClpB and Hsp60/GroE chaperones in cells and compartments where these proteins are found. Several in vitro and in vivo studies have demonstrated that renaturation of denatured model substrates by high-molecular-weight chaperones was more efficient in the presence of sHsps (Kampinga et al. 1994Stege et al. 1995;Lee et al. 1997; Lee and Vierling 2000;Mogk et al. 2003;Haslbeck et al. 2005). ...
The small heat shock proteins (sHsps), which are ubiquitous stress proteins proposed to act as chaperones, are encoded by an unusually complex gene family in plants. Plant sHsps are classified into different subfamilies according to amino acid sequence similarity and localization to distinct subcellular compartments. In the whole Arabidopsis thaliana genome, 19 genes were annotated to encode sHsps, of which 14 belong to previously defined plant sHsp families. In this paper, we report studies of the five additional sHsp genes in A. thaliana, which can now be shown to represent evolutionarily distinct sHsp subfamilies also found in other plant species. While two of these five sHsps show expression patterns typical of the other 14 genes, three have unusual tissue specific and developmental profiles and do not respond to heat induction. Analysis of intracellular targeting indicates that one sHsp represents a new class of mitochon-drion-targeted sHsps, while the others are cytosolic/nuclear, some of which may cooperate with other sHsps in formation of heat stress granules. Three of the five new proteins were purified and tested for chaperone activity in vitro. Altogether, these studies complete our basic understanding of the sHsp chaperone family in plants.
... The small heat shock/ α-crystallin proteins function as molecular chaperones, wherein they bind denaturing proteins and prevent their aggregation within cells under stress (Lee et al. 1997; Muchowski et al. 1997; Plater et al. 1996; Rajaraman et al. 1996). When stress is relieved, the partially denatured proteins may either be passed to ATP-dependent chaperones for refolding or spontaneous refolding occurs (Kampinga et al. 1994; Ehrnsperger et al. 1997; Lee et al. 1997; Liang et al. 1997a ). It is possible that the small heat shock/α-crystallin proteins, with their ability to curb irreversible denaturation of other proteins, are one of the first lines of defence against stress. ...
Immunological evidence is provided for the first time of a small heat shock/α-crystallin protein in the scleractinian coral Madracis mirabilis. The protein, termed cp26, had a molecular weight of 26 000; it reacted with an antibody raised to a small heat shock/α-crystallin protein fromArtemia franciscana and its production in corals was temperature sensitive. Corals collected from seawater at 25.5oC or lower lacked cp26, but the protein was produced in some of these animals when they were heat shocked experimentally. When exposed naturally to high environmental temperatures for relatively short times, corals contained cp26 and responded to heat shock in the laboratory. Corals growing at elevated temperatures tended to die when subjected to additional heat stress. Specifically, M. mirabilis died at about 31-33oC, as indicated by visual inspection of the animals, low recovery of protein in cell-free extracts, and loss of protein bands in SDS-polyacrylamide gels. Death was accompanied by the appearance of a diffuse, unidentified protein band on western blots that reacted with an antibody to cp26. Madracis mirabilis clearly reacts to heat shock by production of cp26; further study is required to determine if this small heat shock/α-crystallin protein will be a useful biomarker of stress in corals.
... We and others have reported that HSPB1 expression enhances cellular resistance to heat shock [1,2], oxidative stress [3], apoptosis inducers456 and a pleiotropic number of conditions or compounds that are deleterious to cells, such as anti-cancer drugs [7] . Recent studies have shown that HSPB1 has multiple strategies to attenuate the damage induced by stress challenges. ...
Human HSP27 (HSPB1) is a molecular chaperone sensor which, through dynamic changes in its phosphorylation and oligomerization, allows cells to adapt to changes in their physiology and/or mount a protective response to injuries. In pathological conditions, the high level of HSPB1 expression can either be beneficial, such as in diseases characterized by cellular degenerations, or be malignant in cancer cells where it promotes tumourigenesis, metastasis and anti-cancer drug resistance. Structural changes allow HSPB1 to interact with specific client protein partners in order to modulate their folding/activity and/or half-life. Therefore, the search is open for therapeutic compounds aimed at either down- or upregulating HSPB1 activity. In this respect, we have previously described two peptide aptamers (PA11 and PA50) that specifically interact with HSPB1 small oligomers and decrease its anti-apoptotic and tumourigenic activities. A novel analysis of the different HSPB1-interacting aptamers that were isolated earlier revealed that one aptamer (PA23) has the intriguing ability to stimulate the protective activity of HSPB1. We show here that this aptamer abolishes the dominant negative effect induced by the R120G mutant of αB-crystallin (HSPB5) by disrupting its interaction with HSPB1. Hence, developing structure-based interfering strategies could lead to the discovery of HSPB1-based therapeutic drugs.
... We and others have reported that HspB1 expression enhances cellular resistance to heat shock [1,2], oxidative stress [3], apoptosis inducers [4][5][6] and a pleiotropic number of conditions or compounds that are deleterious to cells, such as anti-cancer drugs [7]. Recent studies have shown that HspB1 has multiple strategies to attenuate the damages induced by stress challenges. ...
... It seems unlikely, however, that this change is a major contributor to the MF-induced increase in post-I–R cell survival. In clear contrast to the results obtained by others for cells placed under stress [43], cellular levels of Hsp25 did not change significantly in response to I–R, with or without MF preconditioning. ...
The potential for 60 Hz magnetic field (MF) preconditioning to protect heart-derived, H9c2 cultures from damage by simulated ischemia and reperfusion (I-R) was examined. The most effective MF exposure conditions (120 μT, 4-8 h) increased cell survival by 40-50 % over that seen with I-R alone. Potential targets of MF preconditioning were assessed by investigating the apoptosis-related drop in Bcl-2 levels and elevation of the specific activities of caspases 3, 8 and 9 produced by I-R. In response to MF exposure Bcl-2 levels rose 2 to 2.6-fold, and caspase specific activities fell 51-72 % from the values seen after I-R alone. Levels of Hsp's 25, 32 and 72 were examined in response to the MF, but showed little-to-no elevation beyond that produced by I-R. However, MF preconditioning produced a 77 % decrease in the I-R-induced translocation of phosphorylated Hsp25 (Hsp25-P) from the cytosolic to the nuclear-cytoskeletal cell fraction. This might protect by maintaining active Hsp25-P in the cytosol to function as a chaperone or to bind cytochrome c. Blocking Hsp25 phosphorylation with SB203580, an inhibitor of p38 MAPK, resulted in increases of 64 and 80 % in the respective specific activities of caspases 3 and 9 in cells subjected to I-R, and eliminated the MF-induced reduction in caspase 3 activity.
... Several studies demonstrate an interaction between
alpha-Hsps and nucleic acids. It is known that an overexpression of Hsp27 in cells
can promote the accelerated recovery of heat shock-produced intranuclear protein
aggregates
27
. An involvement of Hsp25 granules in binding and targeting denatured
substrates for accelerated degradation has also been shown. ...
In order to evaluate the effects of hyperthermia on adriamycin cardiomyopathy and its relationship with heat shock protein induction and myosin accumulation, female Sprague-Dawley rats (21-24 days) were randomized into four groups: the control, adriamycin, temperature and temperature-adriamycin groups. Adriamycin was injected i.v. at a dose of 27 mg/Kg (0.1 ml). The rats were exposed to a temperature of 45ºC for 35 min, followed by a recovery (1 h) at room temperature prior to adriamycin treatment. Body weight was recorded weekly. The thickness of the ventricular wall and percentage of cellular damage were biometrically and ultrastructurally evaluated, respectively. Heat shock protein 25 and myosin accumulation were determined through Western blot analysis. The determinations were carried out monthly until the third month after treatment. At eight and twelve weeks after treatment, the thickness of the ventricular wall seemed to decrease in the adriamycin-treated rats in relation to the other groups. An electron microscopic analysis of the adriamycin group's left ventricular wall samples, showed more sarcomeric changes and loss of myofibrils than the control, temperature and temperature-adriamycin groups. At 24 hours after treatment with adriamycin, higher levels of heat shock protein 25 and myosin were observed (week 0) in the temperature-adriamycin group than in the control and adriamycin groups (4, 8 and 12 weeks). Hyperthermia was confirmed by a multivariate approach to induce heat shock protein 25 and myosin, which would strengthen cardiac-sarcomeric myosin arrangement.
Biomolecular condensation allows for the dynamic organization of molecules in time and space. Condensate formation is regulated through many mechanisms including the action of molecular chaperones. While molecular chaperones have long been viewed through the lens of their roles in protein folding, misfolding, and quality control, their ability to manipulate protein-protein interactions is increasingly recognized to play a major role in the precise control of condensate biology. In this review we highlight recent studies investigating the roles of canonical and non-canonical chaperones in regulating condensate formation, material state, and dispersal. We discuss the broadening of longstanding conceptions of chaperone functions to include condensate regulation, and the discovery of previously unappreciated chaperone activities in well-known proteins. We close by considering the biological activities being uncovered during the ongoing upheaval at the boundary between chaperone biology and biomolecular condensation.
In human cells under stress conditions, misfolded polypeptides can form potentially cytotoxic insoluble aggregates. To eliminate aggregates, the HSP70 chaperone machinery extracts and resolubilizes polypeptides for triage to refolding or degradation. Yeast and bacterial chaperones of the small heat-shock protein (sHSP) family can bind substrates at early stages of misfolding, during the aggregation process. The co-aggregated sHSPs then facilitate downstream disaggregation by HSP70. Because it is unknown whether a human sHSP has this activity, we investigated the disaggregation role of human HSPB1. HSPB1 co-aggregated with unfolded protein substrates, firefly luciferase and mammalian lactate dehydrogenase. The co-aggregates formed with HSPB1 were smaller and more regularly shaped than those formed in its absence. Importantly, co-aggregation promoted the efficient disaggregation and refolding of the substrates, led by HSP70. HSPB1 itself was also extracted during disaggregation, and its homo-oligomerization ability was not required. Therefore, we propose that a human sHSP is an integral part of the chaperone network for protein disaggregation.
Psoriasis is a chronic disfiguring skin condition which may be induced or exacerbated by stress. Heat shock proteins (HSPs), as molecular chaperones, play a central role in protein folding and cellular protein homeostasis. The many different functions of HSPs in the cell depend on the specific HSP involved. HSPs play crucial roles in inflammation and immune reactions, and have emerged as promising therapeutic targets. In this review, we compile current lines of evidence concerning the roles and molecular mechanisms of HSPs that lead to the occurrence and development of psoriasis.
Small heat shock proteins (sHsps) are a ubiquitous and ancient family of ATP-independent molecular chaperones. A key characteristic of sHsps is that they exist in ensembles of iso-energetic oligomeric species differing in size. This property arises from a unique mode of assembly involving several parts of the subunits in a flexible manner. Current evidence suggests that smaller oligomers are more active chaperones. Thus, a shift in the equilibrium of the sHsp ensemble allows regulating the chaperone activity. Different mechanisms have been identified that reversibly change the oligomer equilibrium. The promiscuous interaction with non-native proteins generates complexes that can form aggregate-like structures from which native proteins are restored by ATP-dependent chaperones such as Hsp70 family members. In recent years, this basic paradigm has been expanded and new roles, new cofactors as well as variations in structure and regulation of sHsps have emerged.
Heat shock proteins are important for maintaining protein homeostasis and cell survival. Among different classes of highly conserved Hsps, small low molecular weight Hsps have significant place, particularly of Hsp27, whose role has been demonstrated in wide range of biological processes, including development, immunity, diseases and therapy. In this review, the structure and functions of Hsp27 and related genes, their role in different cellular processes as well as in stress tolerance is highlighted.
All organisms rely on a conserved cellularmachinery supporting and controlling the life cycle of proteins: the proteostasis network. Within this network, the main players that determine the fate of proteins are molecular chaperones, the ubiquitin-proteasome and the lysosome- autophagy systems. sHsps (small heat-shock proteins) represent one family of molecular chaperones found in all domains of life. They prevent irreversible aggregation of unfolded proteins and maintain proteostasis by stabilizing promiscuously a variety of non-native proteins in an ATP-independent manner. In the cellular chaperone network, sHsps act as the first line of defence and keep their substrates in a folding-competent state until they are refolded by downstream ATP-dependent chaperone systems. Besides this interaction with unfolding substrates upon stress, sHsps show a different mode of binding for specific clients which are also recognized under physiological conditions. In vertebrates, sHsps are especially needed to maintain the refractive index of the eye lens. Additionally, sHsps are linked to a broad variety of diseases such as myopathies and neuropathies. The most striking feature of sHsps is their ability to form dynamic ensembles of higher oligomers. The activity of sHsps is regulated by changes in the composition of the ensembles.
Acute myocardial infarction remains one of the major causes of death among men in the Western World. The most common cause of acute myocardial infarction is thrombosis or occlusion of the coronary arteries that feed the left ventricle of the heart. The lack of blood flow to the cardiac muscle can result in severe cellular damage that eventually may compromise the muscle’s ability to contract. Coronary thrombosis is a direct consequence of coronary artery disease or more specifically atherosclerosis. Recent research aimed at identifying the cause of atherosclerosis and efforts to prevent it have contributed to a decreased incidence of myocardial infarction in the past decade. However, in spite of this recent improvement, the incidence of myocardial infarction leading to subsequent severe cardiac failure still remains a significant medical problem. Therefore, the salvage of additional myocardium following an infarction is a highly desirable aim. Recent evidence indicates that endogenous protective mechanisms are readily activated in cardiomyocytes during an ischemic event, thus a better understanding of these endogenous protective mechanisms will most likely lead to additional myocardial salvage in the reperfused myocardium.
As was reviewed in chapters 4-6: first, transient ATP depletion induces heat shock gene expression and HSP synthesis; and, second, the elevated level of HSP(s) is associated with the protection of various mammalian cells from injury and death under metabolic (or ischemic) stress. Both conclusions were well-grounded and in fact we address here a special adaptive reaction which confers tolerance to energy starvation, thus allowing a cell to withstand sustained deprivation of dATP. Never theless, it is still poorly understood how excess HSP(s) compensates for ATP deficiency and maintains the viability of ATP-deprived cells. In this chapter, we present some speculations and hypotheses which might, at least in part, clarify this intriguing problem.
Our first chapter is devoted to the general description of stress proteins and peculiarities of their expression in eukaryotic cells. We also introduce readers to the modern views of the problem of “negative regulation” of heat shock gene transcription to facilitate an understanding of the subsequent sections of this book.
Since the evolution of the cellular phenotype, prokaryotic and eukaryotic cells have had to cope with adverse changes in their environment. Although cells have evolved many distinct stress responses, this chapter will focus on three major, highly conserved, response systems, i.e., the genotoxic response system, which is activated by DNA damage; the oxidative stress response system, which is activated by excess reactive oxygen species (ROS) and imbalances in the oxidant/antioxidant status within cells; and the heat shock response, which is activated by exposure to heat and other agents that adversely affect protein folding (Fig. 1). The sections dealing with each of the stress response systems begin with a description of the prokaryotic stress response because, in most instances, the prokaryotic systems are the best understood. This is followed by a discussion of the eukaryotic stress response systems, focusing on yeast and mammals. Finally, each section concludes with a discussion about what is known concerning the induction of these stress response systems in mammalian embryos, particularly postimplantation mammalian embryos. Normal embryonic development requires a precisely orchestrated chain of temporal and spatial events, and any alterations in this chain could lead to altered development and subsequent pathogenesis. Although the mammalian embryo develops within the protective environment of the uterus, this protection is not absolute and we now know that mammalian development can be perturbed by a wide variety of chemical and physical agents, many of which are known to induce one or more of these stress systems in nonembryonic systems. Thus understanding the embryo’s stress response capabilities is essential to the understanding of how developmental toxicants exert their toxicity.
Small heat shock proteins (sHSPs) have been studied for 40 years now, initially as proteins up-regulated upon heat stress in which they can help cells to better withstand or recover from heat damage. In humans, we now know that there is a ‘colorful’ family of ten different sHSP members (referred to as HSPB1-HSPB10) that together seem to fulfill many more, only partially overlapping, functions than just protecting cells from heat damage. Here, we summarize how the ten human HSPB members are thought to contribute to the protein quality control in cells and how they may do so via different mechanisms. Also, we will summarize what is known to date about the role of individual HSPB members in neurodegenerative diseases and in cardiomyopathies.
Colorectal cancer (CRC) causes over half a million deaths worldwide and has a particularly poor prognosis when diagnosed at an advanced stage. Heat shock proteins (HSP) have been found to be elevated in CRC patients and HSPB1, HSPA1A and HSPC1 has been shown to have some prognostic value. CRC, in common with all cancers, has important associated oncogene and tumor suppressor gene associations and we show how many of these interact directly with one or more of the HSP. We discuss the current chemotherapeutic options available to the clinician when presented with CRC and how these may be improved with a consideration of the role of HSP in the development of the tumor as well as the response to therapy. Direct manipulation of HSP has the potential to decrease the therapeutic dose of anti-tumor drugs and we propose novel strategies that have the potential to be adapted to the clinic.
Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that are implicated in a variety of diseases. Upon stress, they stabilize unfolding proteins and prevent them from aggregating. However, under physiological conditions without severe stress, some sHsps interact with other proteins. In a perspective view, their ability to bind specific client proteins might allow them to fine-tune the availability of the client for other, client-dependent cellular processes. Additionally, some sHsps seem to interact with specific co-chaperones. These co-chaperones are usually part of large protein machineries that are functionally modulated upon sHsps interaction. Finally, secreted human sHsps seem to interact with receptor proteins, potentially as signal molecules transmitting the stress status from one cell to another. This review focuses on the mechanistic description of these different binding modes for human sHsps and how this might help to understand and modulate the function of sHsps in the context of disease.
Hsp27, a highly conserved small molecular weight heat shock protein, is widely known to be developmentally regulated and heat inducible. Its role in thermo-tolerance is also implicated. The present study is a sequel of our earlier studies to understand the molecular organization of heat shock genes/proteins and their role in development and thermal adaptation in a sheep pest, Lucilia cuprina (blowfly), which exhibits unusually high adaptability to a variety of environmental stresses, including heat and chemicals. In this report our aim was to understand the evolutionary relationship of Lucilia hsp27 gene/protein with those of other species and its role in thermal adaptation. We sequence characterized the Lchsp27 gene (coding region) and analyzed its expression in various larval and adult tissues under normal as well as heat shock conditions. The nucleotide sequence analysis of 678 bps long coding region of Lchsp27 exhibited closest evolutionary proximity with Drosophila (90.09%), which belongs to the same order, Diptera. Heat shock caused significant enhancement in the expression of Lchsp27 gene in all the larval and adult tissues examined, however, in a tissue specific manner. Significantly, in Malpighian tubules, while the heat induced level of hsp27 transcript (mRNA) appeared increased as compared to control, the protein level remained unaltered and nuclear localized. We infer that Lchsp27 may have significant role in the maintenance of cellular homeostasis, particularly, during summer months, when the fly remains exposed to high heat in its natural habitat. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
The number of reports on the effects of temperature is still increasing because of the temperature is one of the most encountered stressful factors in the environment, thus it deemed important to survey the literatures for effects of temperature on the biological consequences. The objective of this review was to establish the thermoregulatory response and adaptation of some non-mammalian species during temperature. Although, there was relative scarcity of information on the relation between oxidative stress and antioxidant enzymes during temperature, this review great interest to elicit this relation in non-mammalian species. Here, this review suggests that, the increase in the oxidative stress due to temperature maybe a reason for such decrease and exhaustion of antioxidant enzymes and a sequence of cellular injury or death, because of increased endogenous production of the free radicals. However, there was exception in this hypothesis because this argument is still ambiguous because of the difficulties of the direct observation of the active oxygen species in the biological systems due to their short lifetime. Taken together, because of one of the most important functions of heat shock protein is to protect the organisms from the deleterious effects of temperature, thus, it can be hypothesized that the formation of heat shock protein and antioxidant enzymes may be related to the changes in the levels of free radicals in non-mammalian species during temperature.
Immunological evidence is provided for the first time of a small heat shock/α-crystallin protein in the scleractinian coral Madracis mirabilis. The protein, termed cp26, had a molecular weight of 26 000; it reacted with an antibody raised to a small heat shock/α-crystallin protein fromArtemia franciscana and its production in corals was temperature sensitive. Corals collected from seawater at 25.5oC or lower lacked cp26, but the protein was produced in some of these animals when they were heat shocked experimentally. When exposed naturally to high environmental temperatures for relatively short times, corals contained cp26 and responded to heat shock in the laboratory. Corals growing at elevated temperatures tended to die when subjected to additional heat stress. Specifically, M. mirabilis died at about 31-33oC, as indicated by visual inspection of the animals, low recovery of protein in cell-free extracts, and loss of protein bands in SDS-polyacrylamide gels. Death was accompanied by the appearance of a diffuse, unidentified protein band on western blots that reacted with an antibody to cp26. Madracis mirabilis clearly reacts to heat shock by production of cp26; further study is required to determine if this small heat shock/α-crystallin protein will be a useful biomarker of stress in corals.
Investigations of the cellular response to thermal and other types of stresses have allowed the identification of families of proteins (the heat shock or stress proteins, Hsp) whose expression is enhanced when environmental conditions become deleterious (reviewed in Georgopoulos and Welch 1993; Morimoto et al. 1994). Hsp are subdivided in two groups, based on their apparent molecular mass, i.e., the large and small heat shock proteins. Small heat shock proteins, now denoted small stress proteins (sHsp), are characterized by a domain of homology to aA,B-crystallin proteins from vertebrate eye (reviewed in Arrigo and Landry 1994). Despite this particular homology, sHsp are less conserved than the large Hsp (i.e., Hsp70) since, among species, they show greater variations in sequence, in number and in molecular mass. All sHsp analyzed so far share the extreme tendency to form oligomers. Like a-crystallin, sHsp are in the form of aggregates with heterodispersed native molecular masses (which can reach up to 800 kDa or more). This structural organization of sHsp depends on the physiology of the cell and probably also on the phosphorylation of these proteins (Siezen et al. 1978a; Arrigo 1987; Arrigo and Welch 1987; Arrigo et al. 1988; Kato et al. 1994; Lavoie et al. 1995; Mehlen and Arrigo 1994; Mehlen et al. 1995b,c).
Small heat shock proteins (sHsps) are molecular chaperones that prevent the aggregation of nonnative proteins. The sHsps investigated to date mostly form large, oligomeric complexes. The typical bacterial scenario seemed to be a two-component sHsps system of two homologous sHsps, such as the Escherichia coli sHsps IbpA and IbpB. With a view to expand our knowledge on bacterial sHsps, we analyzed the sHsp system of the bacterium Deinococcus radiodurans, which is resistant against various stress conditions. D. radiodurans encodes two sHsps, termed Hsp17.7 and Hsp20.2. Surprisingly, Hsp17.7 forms only chaperone active dimers, although its crystal structure reveals the typical α-crystallin fold. In contrast, Hsp20.2 is predominantly a 36mer that dissociates into smaller oligomeric assemblies that bind substrate proteins stably. Whereas Hsp20.2 cooperates with the ATP-dependent bacterial chaperones in their refolding, Hsp17.7 keeps substrates in a refolding-competent state by transient interactions. In summary, we show that these two sHsps are strikingly different in their quaternary structures and chaperone properties, defining a second type of bacterial two-component sHsp system.
Proteine sind lineare Polymere, die von den Ribosomen aus aktivierten Aminosäuren synthetisiert werden. Das Produkt dieses Biosyntheseprozesses ist eine Polypeptidkette, die anschließend in ihre individuelle dreidimensionale Struktur falten muss, die sie zur Ausübung ihrer zellulären Funktion befähigt. Christian Anfinsen konnte zeigen, dass dieser Faltungsprozess keine zusätzlichen Faktoren und keine Energiezufuhr benötigt und somit autonom ist. Im Jahre 1972 wurde ihm für diese Leistung der Nobelpreis für Chemie zuerkannt. Ausgehend von In-vitro-Experimenten mit gereinigten Proteinen glaubte man lange, dass sich auch in der lebenden Zelle die richtige Raumstruktur spontan bildet, sobald die neu synthetisierte Proteinkette das Ribosom verlässt. Darüber hinaus dachte man, dass Proteine ihre gefaltete (native) Konformation beibehalten, bis sie schließlich von speziellen Enzymen abgebaut werden. In den letzten zehn Jahren hat sich diese Sichtweise der zellulären Proteinfaltung stark geändert. Heute weiß man, dass eine Zelle über komplexe und hoch entwickelte Proteinmaschinen verfügt, die die Proteinfaltung unterstützen und die strukturelle Integrität von Proteinen unter Bedingungen aufrechterhalten, unter denen diese zu entfalten und zu aggregieren drohen. Diese Proteinmaschinen bezeichnet man als molekulare Chaperone, da sie, wie ihre menschlichen Pendants, unerwünschte Kontakte zwischen ihren „Schützlingen“ verhindern. In diesem Aufsatz sollen die wichtigsten Eigenschaften dieser Klasse von Proteinen vorgestellt und an ausgewählten Beispielen die Struktur-Funktions-Beziehungen sowie die zugrunde liegenden molekularen Mechanismen erläutert werden.
The heat shock response (HSR) and the acute inflammatory response (APR) are both key homeostatic mechanisms for resisting
extracellular insult. There is evolving understanding regarding the relationship between these two responses. Activation of
the HSR exerts some pro-inflammatory effects when HSP are released during cell insult and such extracellular HSP induce cytokine
release in inflammatory and immune modulating cells. However, the intracellular mediators of the HSR including the transcription
factor heat shock factor 1 (HSF1) and the HSP have profoundly anti-inflammatory effects. HSF1 can be induced by the elevated
temperatures encountered in inflamed tissues and in fever as well as by anti-inflammatory prostaglandins. Such activated HSF1
represses cytokine release both directly by inhibiting nuclear factor of interleukin 6 (NF-IL6) and indirectly when elevated
HSP inhibit the potent inflammatory factor NF-kB. Reciprocal effects are observed on activation of the APR which leads to
inhibition of HSF1 through stimulation of inactivating phosphorylation events involving the mitogen activated kinase (MAPK)
pathways. Activation of the HSR thus constitutes a feedback regulatory mechanism for the APR and limits the lethal over stimulation
of cytokine release which may occur during infection. However, in order for rapid activation of the APR, mechanisms also exist
for HSF1 repression, permitting controlled activation of the response during infection
The heat shock proteins are a family of highly conserved proteins with critical roles in maintaining cellular homeostasis
and in protecting the cell from stressful conditions. While the critical intracellular roles of heat shock proteins are undisputed,
evidence suggests that the cell possess the necessary machinery to actively secrete specific heat shock proteins both basally
and in response to cellular stress. This chapter will discuss the secretory mechanisms identified to date that allows cells
to release specific heat shock proteins. Importantly, several studies have established that this release is the result of
an active secretory process, as opposed to non-specific processes such as cell lysis. Importantly, while the classical protein
secretory pathway via the endoplasmic reticulum and Golgi apparatus does not seem to be involved in the stress-induced release
of heat shock proteins, we discuss the evidence that lipid-rafts and exosomes are important mediators of both basal and stress-induced
heat shock protein release
The heat shock protein (Hsp) family is an evolutionarily conserved system that is charged with preventing unfolded or misfolded proteins in the cell from aggregating. In Alzheimer's disease, extracellular accumulation of the amyloid β peptide (Aβ) and intracellular aggregation of the microtubule associated protein tau may result from mechanisms involving chaperone proteins like the Hsps. Due to the ability of Hsps to regulate aberrantly accumulating proteins like Aβ and tau, therapeutic strategies are emerging that target this family of chaperones to modulate their pathobiology. This article focuses on the use of Hsp-based therapeutics for treating primary and secondary tauopathies like Alzheimer's disease. It will particularly focus on the pharmacological targeting of the Hsp70/90 system and the value of manipulating Hsp27 for treating Alzheimer's disease.
The role of hsp27, one of Drosophila melanogaster's small heat shock proteins, in the process of thermotolerance was investigated. The coding sequence of hsp27 was subcloned downstream of the human hsp27 promoter which has been shown to be constitutively expressed in Chinese hamster O23 cells. Cellular resistance to a thermal stress was measured two days after transfection by a survival assay following a 3.5 h heat treament at 44°C. Expression of Drosophila hsp27 was shown to confer thermal resistance to O23 cells in a manner which was dependent on the level of expression of this hsp. Immunoblot analysis confirmed that the thermal resistance was related to the expression of Drosophila hsp27 as none of the endogeneous hsps showed an increased level under these conditions.
Previous studies have shown a relative increase in the protein content of chromatin isolated from heated cells compared with unheated cells. Using flow cytometric and biochemical methods, we have observed an increased protein content of HeLa nuclei isolated from heated cells. HeLa cells were heated for 7.5, 15, or 30 min at 45/sup 0/C, nuclei isolated fixed, stained with fluorescence channel of FITC-stained nuclei from cells heated for 7.5, 15, or 30 min at 45/sup 0/C relative to control was 1.46 +- 0.05, 1.88 +- 0.14, and 2.02 +- 0.14, respectively. Labeling studies that FITC may not be specific for lysine. The mean light scatter channel (for angles of 1 to 15/sup 0/) relative to control nuclei was 1.18 +-0.04, 1.34 +- 0.08, and 1.46 +- 0.10 (for 7.5, 15, or 30 min at 45/sup 0/C, respectively). Electronic volume determinations in conjunction with microscopically measured volume indicate that the electrical resistance is reduced in nuclei from heated cells. The relative increase in the protein content of nuclei from heated cells determined biochemically was 1.18 +- 0.04, 1.30 +- 0.06, and 1.39 +- 0.05 (for 7.5, 15, or 30 min at 45/sup 0/C, respectively). The heat-induced increase in protein content of nuclei can be accounted for by the increased protein content observed in chromatin assuming the contribution from nonchromatin nuclear protein is constant.
The alpha-crystallins (alpha A and alpha B) are major lens structural proteins of the vertebrate eye that are related to the small heat shock protein family. In addition, crystallins (especially alpha B) are found in many cells and organs outside the lens, and alpha B is overexpressed in several neurological disorders and in cell lines under stress conditions. Here I show that alpha-crystallin can function as a molecular chaperone. Stoichiometric amounts of alpha A and alpha B suppress thermally induced aggregation of various enzymes. In particular, alpha-crystallin is very efficient in suppressing the thermally induced aggregation of beta- and gamma-crystallins, the two other major mammalian structural lens proteins. alpha-Crystallin was also effective in preventing aggregation and in refolding guanidine hydrochloride-denatured gamma-crystallin, as judged by circular dichroism spectroscopy. My results thus indicate that alpha-crystallin refracts light and protects proteins from aggregation in the transparent eye lens and that in nonlens cells alpha-crystallin may have other functions in addition to its capacity to suppress aggregation of proteins.
The heat-shock protein Hsp90 is the most abundant constitutively expressed stress protein in the cytosol of eukaryotic cells, where it participates in the maturation of other proteins, modulation of protein activity in the case of hormone-free steroid receptors, and intracellular transport of some newly synthesized kinases. A feature of all these processes could be their dependence on the formation of protein structure. If Hsp90 is a molecular chaperone involved in maintaining a certain subset of cellular proteins in an inactive form, it should also be able to recognize and bind non-native proteins, thereby influencing their folding to the native state. Here we investigate whether Hsp90 can influence protein folding in vitro and show that Hsp90 suppresses the formation of protein aggregates by binding to the target proteins at a stoichiometry of one Hsp90 dimer to one or two substrate molecule(s). Furthermore, the yield of correctly folded and functional protein is increased significantly. The action of Hsp90 does not depend on the presence of nucleoside triphosphates, so it may be that Hsp90 uses a novel molecular mechanism to assist protein folding in vivo.
Hyperthermia increases levels of nuclear-associated proteins in a manner that correlates with cell killing. If the increase in nuclear-associated proteins represents a lethal lesion then treatments that protect against killing by heat should reduce and/or facilitate the recovery of levels of the proteins in heated cells. This hypothesis was tested using three heat protection treatments: cycloheximide, D2O, and thermotolerance. All three treatments reduced levels of the proteins measured immediately following hyperthermia at 43.0 or 45.5 degrees C, with the greatest reduction occurring at 43.0 degrees C. In addition to reducing the proteins, thermotolerance facilitated the recovery of the proteins to control levels following hyperthermia. Thus thermotolerance may protect cells by both reducing the initial heat damage and facilitating recovery from that damage. Cycloheximide and D2O did not facilitate recovery of nuclear-associated proteins, suggesting that their protection against cytotoxicity related to the proteins resulted solely from their reduction of increases in levels of the proteins. All three treatments have been shown to stabilize cellular proteins against thermal denaturation. The results of this study suggest that the increase in nuclear-associated proteins may result from thermally denatured proteins adhering to the nucleus and that it is the ability of cycloheximide, D2O, and thermotolerance to thermostabilize proteins that reduces the increase in levels of the proteins within heated cells.
Pelham previously proposed that the hsp70 family of heat shock proteins could prevent the formation and/or allow the dissolution of protein aggregates created during stress conditions. We confirmed this hypothesis by showing that the E. coli hsp70 homolog, the dnaK gene product, protects the host RNA polymerase enzyme from heat inactivation in an ATP-independent reaction. In addition, we show that heat-inactivated and aggregated RNA polymerase is both disaggregated and reactivated following simultaneous incubation with DnaK protein and hydrolyzable ATP. The DnaK756 mutant protein has lost the ability to disaggregate the inactivated RNA polymerase enzyme. Our results demonstrate that the DnaK protein contributes to E. coli's growth not only by protecting some enzymes from denaturation but also by reactivating some once they are misfolded or aggregated.
The role of a heat-induced increase in nuclear protein mass in killing of cells by hyperthermia was investigated jointly by two groups that had previously reported apparently conflicting results. A correlation between the fraction of HeLa S3 cells killed and the protein content of nuclei isolated immediately after heat exposure was found. This correlation held when thermal sensitivity was modified by the sensitizers 0.41 M ethanol and 5 mM procaine or by the protector 0.6 M glycerol. However, when the HeLa cells were made thermotolerant by a priming heat exposure of 15 min at 45 degrees C followed by 5 h at 37 degrees C, the correlation no longer held. At the 10% survival level a 1.27-fold greater nuclear protein content was observed in tolerant cells relative to nontolerant cells. Thus no general correlation between initial heat-induced nuclear protein mass changes and hyperthermic cell killing exists. When heated cells were returned to 37 degrees C, a time-dependent reduction in the protein content was observed in nuclei isolated after incubation for various times at 37 degrees C. This rate of reduction in nuclear protein content was found to be accelerated in the tolerant cells. Heat-induced changes in cell-cycle progression had no significant effects on the data obtained. It is concluded from the total data base that not only the absolute increment in nuclear protein mass must be taken into account but also the duration of the binding expressed in the rate of recovery.(ABSTRACT TRUNCATED AT 250 WORDS)
When nuclei were isolated from exponentially growing HeLa S3 cells immediately after a treatment with hyperthermia and/or procaine-HCl, an increase in nuclear protein binding was observed. The extent of this increase, however, did not correlate with cell survival under all conditions of the various treatments. For example, an increase up to 40 per cent in nuclear protein binding as a result of procaine treatment did not lead to a decrease of survival, while a 40 percent increase of nuclear protein binding as a result of hyperthermia corresponded with over 90 per cent cell killing. In addition the extent of heat-induced enhancement of nuclear protein content was approximately equal for thermotolerant and heated control cells, or for cells heated in the presence of procaine. The rate of decay in nuclear protein binding upon post-heat incubations at 37 degrees C of the cells, however, was enhanced in tolerant cells and retarded in cells heated in the presence of procaine as compared to heated control cells. These results show that, in spite of suggestions in other reports, neither the initial rate of enhanced protein binding nor the extent of the protein bound to the nucleus seems a reliable measure for heat toxicity. The capacity of the cell to reverse this heat-induced protein binding must be considered.