Molecular Chaperones As Regulators of Cell Death

A Hishiya and S Takayama

Cardiovascular Program, The Boston Biomedical Research Institute, Watertown, MA, USA

Molecular chaperones have been reported as multifunc- way of apoptosis, and they will be replaced with newly tional antistress molecules that can regulate diverse proliferated cells. biological processes to maintain cellular homeostasis. Although the protein aggregation and formation of Molecular chaperones have critical roles for maintaining inclusion bodies are commonly observed among these proper protein folding, protein translocation, degradation diseases, recent reports indicated that intracellular of unfolded protein, regulating signal-transduction pro- aggregation does not confer the cellular toxicity teins and so on. Under pathological conditions, inducible (Saudou et al., 1998; Cummings et al., 1999; Chun or constitutively expressed molecular chaperones protect et al., 2002; Watase et al., 2002), rather the protective cells from stress. Non-dividing terminally differentiated effect as they sequester the insoluble aggregation of cells accumulate abnormal proteins due to chronic mutant proteins (Arrasate et al., 2004). Finally, Kayed environmental or physiological stress; thus, proper cha- et al. (2003) showed that the monomeric or oligomeric perone function is critical for maintaining homeostasis of ﬁbrous conformation of mutant proteins termed amy- those cells, such as neuronal and muscular cells. Cancer loid might induceneuronaltoxicity, which is commonly cells also have overexpression of molecular chaperone seen among these diseases. proteins for promoting survival from stress related to It has been reported that apoptotic cell death is growth, cell cycle, hypoxia, metastasis and genetic responsible for the progression of clinical symptom of mutations. Here, we will focus on the function of degenerative disease such as ALS (Ekegren et al., 1999; molecular chaperone proteins for the regulation of cell Martin, 1999), Huntington’s disease (Bova et al., 1999), death in degenerative diseases, ischemic diseases and in Parkinson’s disease (Tatton, 2000; Hartmann et al., cancer. 2001) and Alzheimer’s disease (Rohn et al., 2002; Zhao Oncogene (2008) 27, 6489–6506; doi:10.1038/onc.2008.314 et al., 2003). The intracerebroventricular or intrastriatal administration of caspaseinhibitor (zVAD-fmk) was Keywords: heat-shock proteins; Hsp27; Hsp70; Hsp90; reported as attenuating the symptom of ALS (Li et al., molecular chaperone; co-chaperone 2000) and Huntington’s disease (Ona et al., 1999; Chen et al., 2000; Toulmond et al., 2004).

Molecular chaperones in degenerative diseases As misfolded protein is generally believed to be a critical Degenerative disease pathological factor of aggregation diseases, extensive research has been carried out to examine the potential Molecular chaperones regulate protein folding to roles of molecular chaperones in pathogenesis of maintain proper function of proteins. Misfolded or degenerative diseases. Small heat-shock family molecu- aggregated proteins induce cellular toxicity and cause lar chaperones have 10 members in mammalians. Four degenerative diseases such as polyglutamine disease members of small heat-shock proteins (aA-crystallin (Huntington’s disease and so on), Alzheimer’s disease, (HspB4), aB-crystallin (HspB5), Hsp27 (HspB1) and familial amyotrophic lateral sclerosis (ALS) and Hsp22 (HspB8)) have been found as genes responsible Parkinson’s disease. for human degenerative myopathy, neuropathy and Terminally differentiated and non-dividing cells, such congenital cataract (Table 1). Three different mutations as neurons and muscles, are particularly vulnerable to (R120G, Q151X and 464del) of aB-crystallin havebeen the toxicity of misfolded proteins, as accumulation of discovered in human myofibrillar myopathy. R120G misfolded and aggregated protein increases toxicity in mutation causes desmin-related myopathy, which has such organs. Cell division usually dilutes the toxicity, pathological characteristics of myofibrillar degeneration even though the toxicity exceeds the threshold of cell and is categorized as myofibrillar myopathy. Desmin- death. The cells have the choice of ‘active death’ as a related myopathy is sometimes accompanied with cytosolic aggregated protein, including desmin and aB- crystallin (Vicart et al., 1998; Selcen and Engel, 2003). Correspondence: Dr S Takayama, The Boston Biomedical Research Thebiological characterization of aB-crystallin R120G Institute, 64 Grove Street, Watertown, MA 02472, USA. mutation has indicated that mutant protein lose E-mail: [email protected] chaperone activity, form aggregation alone, perturb Molecular chaperones as regulators of cell death A Hishiya and S Takayama 6490 Table 1 Identified human mutations in molecular chaperones associated with degenerative disease Molecular chaperone Mutation Disease References

aA-Crystallin W9X CC Pras et al. (2000) R49C CC Mackay et al. (2003) R116C CC Litt et al. (1998) R116H CC Gu et al. (2008) G98R CC Santhiya et al. (2006) aB-Crystallin R120G DRM Vicart et al. (1998) 450delAa CC Berry et al. (2001) Q151X MM Selcen and Engel (2003) 464delCTXb MM Selcen and Engel (2003) D140N CC Liu et al. (2006b) P20S CC Liu et al. (2006a) Hsp27 S135F CMT2, DHMN Evgrafov et al. (2004) R136W CMT2 R127W DHMN T151I DHMN P182L DHMN Hsp22 K141N CMT2 Tang et al. (2005) K141N DHMN Irobi et al. (2004) K141E DHMN Hsp60 V72I SPG13 Hansen et al. (2002) Hsf4 A20D MC Bu et al. (2002) I78V L115P R120C SIL1 D170EfsX MSS Anttonen et al. (2005) V186-Q215del A152-Q215del R111X H71QfsX Skipping of exons 6 MSS Senderek et al. (2005) Skipping of exons 9 R111X G116fs L316fs M344fs L456fs G346delQ417X

Abbreviations: CC, congenital cataract; CMT2, Charcot–Marie–Tooth disease type 2; del, deletion; DHMN, distal hereditary motor neuropathy; DRM, desmin-related myopathy; fs, frameshift; MC, Marner cataract; MM, myoﬁbrillar myopathy; MSS, Marinesco–Sjo¨ gren syndrome; SPG13, hereditary spastic paraplegia; X, stop. a450delA, nucleotide deletion at position 450 that resulted in a frameshift in codon 150 and produced an aberrant protein consisting of 184 residues. b464delCTX, 2 bp nucleotide deletion at position 464 (464delCT) that generates eight missense codons (RAHHSHHP) followed by a stop codon.

sarcomere architecture and lose antiapoptotic activity changes with mitochondrial dysfunction and disruption (Bova et al., 1999; Perng et al., 1999; Maloyan et al., of the cytoskeletal network (Sanbe et al., 2004, 2005; 2005; Treweek et al., 2005; Simon et al., 2007). Maloyan et al., 2005). Myofibrillar myopathy is also found in homozygous The ocular lens is transparent and avascular tissue. deletion mouse of bag3, which is a member of the BAG Lens epithelial cells proliferate and differentiate into family co-chaperone and a regulator of Hsp70 molecular lens fiber cells, using similar systems such as apoptosis chaperone. Skeletal muscles of bag3À/À miceexhibitthe to eliminate organelles. Apaf1 knockout micehave foci of myofibrillar disruption and represent an early abnormal lens epithelium differentiation and inhibition pathological alteration with altered Z-discs and apop- of apoptosis in their lenses (Cecconi et al., 1998). DNase totic features in muscular tissues (Homma et al., 2006). II-like acid DNase deficiency in mice produced con- Heart-specific overexpression of R120G mutation of genital cataract formation with undigested DNA during aB-crystallin indicated similar phenotype as human lens differentiation (Nishimoto et al., 2003). Missense desmin-related myopathy and cardiac hypertrophy in mutation of aA-crystallin (HspB4) and aB-crystallin mice(Wang et al., 2001). Thesamegroup has usedthis was found in familiar congenital cataracts (Table 1). In transgenic mouse for analysing the biological effects of ocular lenses, crystallin is the major protein component R120G mutation on apoptosis in vivo. Histological in differentiated lens fiber cells. Using missense examination indicated that amyloidogenic oligomer was mutations of aA- or aB-crystallin found in congenital detected in the heart of R120G transgenic mouse, cataracts, many studies have been carried out analysing suggesting that toxic oligomer was generated by thefunctional significanceof thesemutations in vitro or R120G mutation of aB-crystallin and causes apoptotic in vivo (Kumar et al., 1999; Liu et al., 2006b; Singh et al.,

Oncogene Molecular chaperones as regulators of cell death A Hishiya and S Takayama 6491 2006; Gu et al., 2008; Li et al., 2008; Xi et al., 2008), proteins, the active form of small heat-shock protein suggesting that mutants of aA- or aB-crystallin lose may not be increased by simple transgenic methods their chaperone activity and antiapoptotic activity. (Zourlidou et al., 2007). Although in vivo models show Recently, double knockout mouse of aA- and some controversial results, relatively consistent aB-crystallin was generated. In this mouse, caspase 3 results have been reported from cellular experiments, activation and increased apoptotic death were observed indicating that many different molecular chaperones can in lens fibers (Morozov and Wawrousek, 2006). Knock- suppress the apoptosis induced by mutant proteins, in mice were created using aA-crystallin R49C, which which make aggregation in degenerative disease indicated increasing apoptosis and congenital cataract (Table3). (Xi et al., 2008). A similar phenotype of congenital For therapeutic applications, Hsp70 was linked to a cataract was observed in homozygous deletion mice of cell-penetrating peptide derived from the HIV trans- one of the heat-shock transcription factors, heat-shock activator of transcription (Tat) protein. Using in vitro factor 4, probably due to decreased expression of and in vivo system of Parkinsonism model, Tat-Hsp70 g-crystallin (Fujimoto et al., 2004; Min et al., 2004). protected dopaminergic neurons against neurotoxicity Genetic mutation of molecular chaperones has been of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (Nagel found in some other degenerative diseases, but the et al., 2008). Arimoclomol is a co-inducer of heat-shock relation of these mutations and apoptotic death is not protein genes during cell stress, probably through Hsf1 clearly described yet. Missense point mutation of Hsp22 activation. This drug delays the progression of symp- (HspB8) and Hsp27 (HspB1) genes causes peripheral toms in a mouse model of ALS, which has over- neuropathy, distal hereditary motor neuropathy or expression of SOD1 mutation (Kieran et al., 2004), and Charcot–Marie–Tooth disease (Evgrafov et al., 2004; is now under phase IIb clinical trial in human ALS. Tang et al., 2005). Hereditary spastic paraplegia SPG13 Further therapeutic approaches have been tried exploit- is characterized by spasticity and weakness of the lower ing molecular chaperones against ‘degenerative diseases’ limbs with neuron degeneration, and is caused by the (reviewed in Muchowski and Wacker, 2005). mutation in mitochondrial chaperonin hsp60 (Kallio et al., 2002). Mutations in the sil1 gene, a nucleotide exchange factor for Hsp70 homolog in endoplasmic The mechanism of molecular chaperones’ antiapoptotic reticulum (ER; also called GRP78, BiP and HSPA5), function: protein folding and degradation of misfolded cause the autosomal recessive degenerative disease protein Marinesco–Sjo¨ gren syndrome featuring cerebellar The process of protein aggregation has multiple steps ataxia, myopathy and cataract (Anttonen et al., 2005; from unfolded protein, soluble oligomer to insoluble Senderek et al., 2005). aggregation (Caughey and Lansbury, 2003). In this process, soluble oligomer species from the Ab-peptides (amyloid precursor protein b-peptides) and other Can molecular chaperones reverse degenerative disease amyloidogenic peptides have common structures and in vivo? toxicity (Kayed et al., 2003). Molecular chaperones The anti-cell death function of molecular chaperones protect cells from apoptosis induced by toxic oligomers has been studied using an animal model of Huntington’s (Table 3), suggesting that molecular chaperones may disease, spinocerebellar ataxia type I, amyotrophic work prior to thecomposition of solubleamyloid lateral sclerosis (ALS), Parkinson’s disease, spinobulbar oligomer. Wacker et al. (2004) showed that Hsp70 and muscular atrophy (SBMA) and Alzheimer’s disease. Hsp40 act to partition monomeric conformation of Overexpression of molecular chaperones mostly reverses mutant polyQ (polyglutamine) and attenuate two degenerative phenotype in vivo, but somereporthas different oligomer formations (annular and spherical) negative data (Table 2). For example, overexpression of using atomic forcemicroscopy. Schaffar et al. (2004) Hsp70 could not reverse the progression of Hunting- used intramolecular fluorescent resonance energy trans- ton’s disease (Hansson et al., 2003) and ALS (Liu et al., fer and reported that Hsp70 and Hsp40 inhibited the 2005). Although Hsp27 inhibits apoptosis induced by structural rearrangement of mutant Huntingtin protein, mutant Huntingtin protein in cell culture system suggesting that the molecular chaperones could work on (Wyttenbach et al., 2002), overexpression of Hsp27 in the very first step in the process of aggregation. Dedmon the mouse model for Huntington’s disease did not et al. (2005) reported that Hsp70 is bound to prefibrillar improvethephenotype(Zourlidou et al., 2007). species preferentially, but not to monomeric a-Syn The difference among these results may come from (a-Synuclein), and blocked the formation of a-Syn the expression level of chaperones, background of the fibril. However, there is no effect of Hsp70 on the net mouse and differences in experimental approaches. cytotoxicity of the aggregation samples, suggesting that Furthermore, because molecular chaperones such as prefibrillar a-Syn may possess the toxicity (Dedmon Hsp70 require co-chaperones for the folding or degra- et al., 2005). Theaddition of Hsp70 and Hsp40 also dation process of abnormally folded protein, over- inhibited the amyloid oligomer formation of Ab (Evans expression of Hsp70 might not be fully effective et al., 2006) and Huntingtin proteins (Muchowski et al., against the diseases under certain circumstances. As 2000). Thecombination of Hsp70 and Hsp40 also phosphorylation and thefollowing oligomerization are suppressed the assembly of mutant Huntingtin into required for the full activity of small heat-shock detergent-insoluble amyloid-like fibrils, and caused the

Oncogene Molecular chaperones as regulators of cell death A Hishiya and S Takayama 6492 Table 2 Transgenic mice of molecular chaperones in neurodegenerative disease Neurodegenerative Molecular Engineering Protein Outcome References disease chaperone aggregation

Parkinson’s disease (PD) Hsp70 Transgenic micea Decrease of NA Klucken et al. (2004) insolublefraction

Alzheimer’s disease (AD) Hsp70 Transgenic micea Reduction NA Petrucelli et al. (2004) of Tau level

Amyotrophic lateral Hsp70 Transgenic micea NA Not improved Liu et al. (2005) sclerosis (ALS) Hsp27 Transgenic micea Lack of ubiquiti- Improved the Sharp et al. (2008) nated aggregate phenotype Increased survival of spinal motor neurons (only in early stage)

Huntington’s disease HSF1 Transgenic mice Reduction (in Attenuated weight Fujimoto et al. (2005) (HD) (activeform of skeletal muscle) loss of skeletal HSF1 derived No effect in brain muscleand brain under the b-actin Prolonged lifespan promoter) (not expressed in brain) Hsp104 (yeast) Transgenic mice Reduction Prolonged lifespan Vacher et al. (2005) (CMV promoter) Hsp70 Transgenic micea Transient delay Not improved Hay et al. (2004) Hsp70 Transgenic mice No effect on Modest effect on Hansson et al. (2003) (human b-actin aggregation disease progression promoter) Hsp27 Transgenic micea Not improved Zourlidou et al. (2007)

Spinobulbar muscular Hsp70 Transgenic mice Decrease of AR Ameliorate the Adachi et al. (2003) atrophy (SBMA) (human b-actin nuclear localization motor neuron promoter) and aggregation function

Spinocerebellar ataxia Hsp70 Transgenic micea No effect on NI Improved the Cummings et al. (2001) 1 (SCA1) phenotype

Spinocerebellar ataxia Hsp70 Transgenic mice No effect Not improved Helmlinger et al. (2004) 7 (SCA7) and Hdj2 (human rhodopsin promoter)

Abbreviations: CMV, cytomegalovirus; NA, not applicable. aTransgenic mice, transgene is derived under the human cytomegalovirus enhancer linked to the b-actin promoter.

formation of amorphous, detergent-soluble aggregates receptor (containing polyglutamine repeat) of SBMA, (Muchowski et al., 2000). Thefibril formation of A b was probably by promoting ubiquitination, but not by also inhibited by other chaperones such as Hsp20 (Lee protein folding of the substrate (Howarth et al., 2007). et al., 2005). Interestingly, yeast prion state [PSI þ ] Hsp70 and Hsp40 were also reported as increasing the inheritance depends on the concentration of Hsp104. At solubility of mutant androgen receptor as well as low concentration, Hsp104 promoted the transition promoting thedegradation of theproteinthrough the from oligomeric to fibrillar status of prion proteins proteasome (Bailey et al., 2002). Hsp90 inhibitor (Shorter and Lindquist, 2004). But both amyloidogenic increases proteasome degradation of hyperphosphory- oligomerization and contingent fibrillization were com- lated tau protein, which is a primary component of pletely abolished by high concentration of Hsp104 neurofibrillary tangles in Alzheimer’s disease (Dickey (Shorter and Lindquist, 2004). The proposed pathway et al., 2006). Overexpression of Hsp70 ameliorates and the action of molecular chaperones in fibril phenotypes of SBMA in mice with reduction in the formation are depicted in Figure 1. amounts of mutant androgen receptor (Adachi et al., As well as protein folding of misfolded proteins by 2003). The microtubule-binding protein, tau, is accu- molecular chaperones, molecular chaperones also re- mulated and deposited in the brain of patients with move the abnormal protein aggregates through the Alzheimer’s disease. BAG1 associates with tau protein ubiquitin–proteasome pathway. Hsp40 family protein in an Hsc70-dependent manner, and modulates the Hsj1A contains DnaJ domain and ubiquitin-interacting degradation of tau protein by the 20S proteasome motifs. Overexpression of Hsj1A with Hsp70 reduced (Elliott et al., 2007). Small heat-shock protein Hsp27 polyglutamineinclusion causedby mutant androgen also binds to hyperphosphorylated tau protein, and

Oncogene Molecular chaperones as regulators of cell death A Hishiya and S Takayama 6493 Table 3 Molecular chaperone-related cell death in neurodegenerative disease Neurodegenerative disease Mutant gene Molecular Protein Cell death References chaperone aggregation

Parkinson’s disease (PD) a-Synuclein (a-Syn) Hsp70 Decrease Protect Klucken et al. (2004) (a-Syn insolubility) Hsp70 Decrease Not changed Dedmon et al. (2005) (a-Syn insolubility)

Alzheimer’s disease (AD) Amyloid precursor Hsp70 NA Protect Magrane et al. (2004) protein b-peptide Hsp20 Decrease ﬁbril Protect Lee et al. (2005) formation

Polyglutaminedisease Polyglutamine tract Hdj-1 Decrease Inhibition of apoptosis Chai et al. (1999) (GFP-conjugated polyglutamine tract) CHIP Decrease Inhibition of cell death Miller et al. (2005) CHIP Decrease Increase of cell viability Jana et al. (2005)

Huntington’s Huntingtin Hsp70 Not changed Increase of cell viability Zhou et al. (2001) disease (HD) Hsp70 Decrease Inhibition of apoptosis Novoselova et al. (2005) Hsp70 Decrease Inhibition of apoptosis Wyttenbach et al. (2002) Hdj-1 Decrease Increase of cell viability Jana et al. (2000) Hsp40 Decrease Increase of cell viability Zhou et al. (2001) Hdj-1 Decrease Inhibition of apoptosis Wyttenbach et al. (2002) Hsp27 Not changed Inhibition of apoptosis Wyttenbach et al. (2002) CCT Decrease Inhibition of apoptosis Kitamura et al. (2006) CCT Multiple foci Increase of cell viability Tam et al. (2006) BAG1 NA Protect Jana and Nukina (2005)

Spinobulbar muscular Androgen receptor Hsp70/Hsp40 Decrease Inhibition of apoptosis Kobayashi et al. (2000) atrophy (SBMA) Hsp110 Decrease Inhibition of apoptosis Ishihara et al. (2003)

Abbreviation: NA, not applicable.

reduces its concentration through the ubiquitin–protea- tion with Hsp70 (Demand et al., 2001; Arndt et al., somesystem(Shimura et al., 2004a). CHIP is an E3 2005; Dai et al., 2005). ubiquitin ligase and interacts with the C-terminal Recently, the relationship between molecular chaper- portion of Hsp70, linking protein folding and ubiqui- ones and autophagy has been extensively studied. tin–proteasome protein degradation systems (reviewed HspB8, one of the small heat-shock proteins, forms a in McDonough and Patterson, 2003). Miller et al. (2005) stable complex with BAG3, and this complex accelerates reported that CHIP suppressed the polyglutamine the degradation of mutant Huntingtin protein through aggregation and toxicity in culture cells. They also autophagy (Carra et al., 2008). showed its protective effect using the zebraﬁsh model (Miller et al., 2005). CHIP is found in theaggregation containing tau protein, and serves E3 ubiquitin ligase Ischemic diseases activity toward tau protein (Hatakeyama et al., 2004; Petrucelli et al., 2004; Shimura et al., 2004b). CHIP also Pathological examination of ischemic lesions indicates promoted the ubiquitination and degradation of mutant two types of cell death, necrosis and apoptosis. It has ataxin-1 (Al-Ramahi et al., 2006) and ataxin-3 (Jana been reported that inhibition of apoptotic pathways et al., 2005). Overexpression of CHIP reduced motor by caspase inhibitors (XIAP and NAIP) or overexpres- symptoms and inhibited the accumulation of nuclear sion of antiapoptotic protein (Bcl-2) in animal models aggregation with degradation of mutant AR in a of ischemic brain injury protects neurons from transgenic mouse model of SBMA (Adachi et al., apoptosis (Martinou et al., 1994; Linnik et al., 1995; 2007). Mutant Ataxia-1 is a polyglutamineproteinthat Xu et al., 1997, 1999; Kitagawa et al., 1998; Hata et al., causes spinocerebellar ataxia type I. Using the Droso- 1999; Trapp et al., 2003). Molecular chaperones have phila model of neurodegeneration, overexpression of also been reported as antiapoptotic in cell culture CHIP reduces the toxicity of mutant ataxin-1 through models or animal models of ischemia-induced apoptosis. direct interaction and ubiquitination of mutant ataxin-1 In this section, we will review molecular chaperones in (Al-Ramahi et al., 2006). CHIP was also reported as an the regulation of ischemia-induced apoptosis and interacting partner of co-chaperone BAG1 and BAG2 summarize how the potential mechanisms of molecular for regulating misfolding-dependent protein degrada- chaperones protect cells from apoptosis in ischemia.

Oncogene Molecular chaperones as regulators of cell death A Hishiya and S Takayama 6494

Native monomer Disordered aggregates?

or Hsp104 (Shorter and Lindquist, 2004) Hsp70/Hsp40 (Schaffar et al., 2004) Misfolded monomer Hsp70/Hsp40 (Schaffar et al., 2004) or (Wacker et al., 2004)

Annular oligomer Hsc70/Hdj-1 (Muchowski et al., 2000) Spherical oligomer Toxic species? (Bucciantini et al., 2002) Amorphous aggregate Hsp70 (Dedmon et al., 2005) Protofibril

Hsp70/Hsp40 or (Wacker et al., 2004)

Amyloid fibrils

Figure 1 Proposed pathway in amyloid assembly and actions of molecular chaperones. Mutated proteins form an abnormal conformation (misfolded monomer), followed by oligomerization. Several types of oligomer conformation were confirmed (for example, annular oligomer, spherical oligomer, amorphous aggregate and protofibril). Soluble oligomers share the common toxic conformation (Kayed et al., 2003). Molecular chaperones prevent the formation of toxic oligomers from misfolded monomer. Molecular chaperones also promote the toxic oligomer to the fibril-type aggregation, which has less toxicity and stable.

The expression of molecular chaperones overexpression of Hsp70 reduces ischemia-induced It has been reported that synthesis of inducible forms of tissueinjury in thebrain (Plumier et al., 1997; Yenari a major molecular chaperone, Hsp70, is increased et al., 1998; Rajdev et al., 2000; Hoehn et al., 2001; Kelly following transient ischemic stress in the brain (Kinou- et al., 2002; Tsuchiya et al., 2003a; Matsumori et al., chi et al., 1993; Nishi et al., 1993; Sharp et al., 1993; 2005), heart (Marber et al., 1995; Plumier et al., 1995; Gaspary et al., 1995), heart (Dillmann et al., 1986; Radford et al., 1996; Trost et al., 1998; Suzuki et al., Mehta et al., 1988; Knowlton et al., 1991), liver (Boeri 2002) and liver (Kuboki et al., 2007). Reduced Hsp70 et al., 2003; Kuboki et al., 2007) and kidney (Zhang expression level induces more infarction volume of focal et al., 2008). To examine the effect of overexpression cerebral ischemia in knockout mice of hsp70.1 (gene of Hsp70 in animal models of ischemic injury, three encoding inducible Hsp70) (Lee et al., 2001, 2004). different methods were used: whole body heat stress Overexpression of Hsp27 and aB-crystallin (HspB5) in (Marber et al., 1994), overexpression of molecular transgenic mice showed a reduced amount of infarction chaperones using viral vector (Yenari et al., 1998; (Ray et al., 2001; Efthymiou et al., 2004) and attenuated Hoehn et al., 2001; Kelly et al., 2002) and transgenic cardiomyocyteapoptosis (Ray et al., 2001). In theheart animals expressing molecular chaperones (brain (Plu- of doubleknockout miceof aB-crystallin and HspB2, mier et al., 1997; Rajdev et al., 2000; Tsuchiya et al., which have shared regulatory elements in the promoter 2003a, b; Matsumori et al., 2005) and heart (Marber region, ischemia-reperfusion exhibits contractile dys- et al., 1995; Plumier et al., 1995; Radford et al., function and increased myocardial infarction (Morrison 1996; Trost et al., 1998)). From these animal studies, et al., 2004; Pinz et al., 2008). Viral vector-mediated

Oncogene Molecular chaperones as regulators of cell death A Hishiya and S Takayama 6495 overexpression of Hsp60 also showed neuroprotection inhibitor of Hsp90, geladanamycin, induces Hsp70 of CA1 pyramidal neurons from ischemic damage expression and reduced ischemia-induced apoptosis (Hwang et al., 2007). in vivo and in vitro (Lu et al., 2002; Xu et al., 2003). The roles of molecular chaperones on antiapoptotic effect against ischemic stress were summarized in The mechanism of antiapoptosis Table4. Lee et al. (2004) directly showed the anti- After ischemic damage, reactive oxygen species increase apoptotic effect of Hsp70 in focal cerebral ischemia mitochondrial pathways of apoptotic signals, releasing using gene knockout mice. In contrast, apoptosis was cytochrome c, caspase9 and thesecondmitochondria- attenuated in transgenic mice of Hsp70 during cerebral derived activator of caspases (Smac). SOD (copper/zinc ischemia and reperfusion (Tsuchiya et al., 2003a, b). superoxide dismutase) has an important function in attenuating ischemic stress in mitochondria (Suzuki Antiapoptotic function et al., 1999). Using transgenic rats overexpressing SOD, As an in vitro experiment, overexpression of Hsp70 ischemia-induced hippocampal CA1 neurons were inhibits apoptosis induced by ischemic stress in cardio- protected from apoptosis by the suppression of super- myocytes (Suzuki et al., 2002), astrocytes (Papadopou- oxideproduction, cytochrome c and Smac release los et al., 1996) and kidney cells (Meldrum et al., 2001). (Sugawara et al., 2002). Mitochondrial Hsp70 (Hsp75/ The mitochondrial chaperones Hsp60 and Hsp10 also Grp75) inhibited apoptosis induced by oxygen–glucose protected cells from ischemic stress-induced apoptosis in deprivation or glucose deprivation in primary astrocytes cardiomyocytes (Lau et al., 1997; Lin et al., 2001). The (Voloboueva et al., 2008) or ischemia reperfusion in inhibition of ischemia-induced apoptosis by Hsp70 was cardiomyocytes (Williamson et al., 2008) with decreased demonstrated using transgenic mice overexpressing rat reactive oxygen species. Overexpression of Hsp70 in Hsp70 (Tsuchiya et al., 2003a, b) and hsp70.1 gene isolated rat hearts inhibited apoptosis after ischemia deletion mice (Lee et al., 2004). Herpes simplex virus- reperfusion while improving the activity and amount of mediated overexpression of Hsp70 improved neuronal SOD (Suzuki et al., 2002). PEP-1-Hsp27 (amphipathic survival in a rat model of ischemic brain injury peptide carrier conjugated Hsp27 for the delivery into (Yenari et al., 1998; Kelly et al., 2002). Pharmacological cells) also protected astrocytes from apoptosis induced

Table 4 Molecular chaperone-related apoptosis in ischemia Ischemia Animals or cells Molecular Method Apoptosis References chaperone

Animal studies Focal cerebral I/R Rat Hsp70 Herpes simplex Improvement of Yenari et al. (1998) virus neuronal survival Permanent middle MouseHsp70 Transgenic Inhibition of apoptosis Tsuchiya et al. (2003b) cerebral artery mousea occlusion Focal cerebral I/R MouseHsp70 Transgenic Inhibition of apoptosis Tsuchiya et al. (2003a) global cerebral I/R mousea Focal cerebral I/R Mouse Hsp70.1 Knockout mouse Increase of apoptosis Lee et al. (2004) Myocardial I/R Mouse aB-crystallin Transgenic mousea Inhibition of apoptosis Ray et al. (2001)

Cell culture model Ischemic condition Renal tubular Hsp70 Heat shock Inhibition of apoptosis Meldrum et al. (2001) (immersed in mineral oil) cells (LLC-PK1) Ischemic condition Renal tubular Hsp70 Transfection Inhibition of apoptosis Meldrum et al. (2003) (immersed in mineral oil) cells (LLC-PK1) I/R Isolated rat Hsp70 Transfection Inhibition of apoptosis Suzuki et al. (2002) heart Oxygen glucose Astrocytes Hsp70 Retrovirus Inhibition of apoptosis? Papadopoulos et al. deprivation (1996) Oxygen glucose Astrocytes Grp75 Retrovirus Inhibition of apoptosis Voloboueva et al. (2008) deprivation (mitochondrial Hsp70) Hypoxia and Cardiomyocytes Grp75 Adenovirus Inhibition of apoptosis Williamson et al. (2008) reoxygenation (mitochondrial Hsp70) Simulated ischemia Cardiomyocytes Hsp60 and Adenovirus Inhibition of apoptosis Lin et al. (2001) and reoxygenation Hsp10 Ischemic condition Cardiomyocytes Hsp60 and Adenovirus Inhibition of apoptosis Lau et al. (1997) (hypotonic and Hsp10 hypoxic condition) ATP depletion and Kidney cells Heat shock Inhibition of apoptosis Wang et al. (1999) recovery

Abbreviation: I/R, ischemia and reperfusion. aTransgenic mice, transgene is derived under the human cytomegalovirus enhancer linked to the b-actin promoter.

Oncogene Molecular chaperones as regulators of cell death A Hishiya and S Takayama 6496 by oxidativestress in vitro as well as in vivo with Quercetin also decreased the size of intraperitoneal decreasing lipid peroxidation (An et al., 2008). There are xenograft tumor of MiaPaCa-2 tumor cells (pancreatic several reports indicating the accumulation of protein adenocarcinoma cancer cells) in vivo (Aghdassi et al., aggregation and ubiquitination, a hallmark of cellular 2007). The diminished expression of Hsp70 by triptolide, response against misfolded proteins, after ischemic a small molecule discovered from the Chinese plant stress (Hu et al., 2000; Ouyang and Hu, 2001; Giffard Tripterygiumwilfordii , also resulted in the passive et al., 2004), suggesting that molecular chaperones have apoptosis of pancreatic cancer cells in vivo as well as in functions in regulating protein folding and aggregation vitro (Phillips et al., 2007). Thesuppression of Hsp110 during ischemic stress. expression by RNAi also enhanced apoptosis in cancer Hsp70 transgenic mice (cytomegalovirus enhancer cells, but not in normal cells (Hosaka et al., 2006). and chicken b-actin promoter) have been used for ischemia-reperfusion injury experiments, and significant The mechanism of antiapoptosis neuroprotection was observed with the inhibition of Protein folding apoptosis (Tsuchiya et al., 2003a, b). In this model, after . Primary structure (peptide sequences) ischemia reperfusion, Hsp70 is bound to AIF (apopto- determines the final conformation of protein (Anfinsen’s sis-inducing factor) in cytosol and prevented the nuclear dogma), concluded from the result of Anfinsen’s translocation of AIF (Matsumori et al., 2005). BAX experiment ‘enzymatic activity of ribonuclease was translocation from cytosol to mitochondria is regulated preserved after denature and renature cycle in vitro’. by Hsp60, which binds to BAX in cytosol and inhibits Are all mutated proteins unfolded? The genomic translocation of BAX to mitochondria. BAX dissociates instability is commonly observed in cancer cells, from Hsp60, and translocates to the mitochondria of resulting in amino-acid substitution and deletion. Thus, adult cardiomyocytes under hypoxic conditions (Gupta it is expected that cancer cells have abnormally folded and Knowlton, 2002). proteins due to genetic mutations, but mutated protein can get proper folding by molecular chaperones. Blagosklonny et al. (1996) reported that p53 mutant (p53V143A) failed to obtain or stabilize correct con- Cancer formation without functional Hsp90, suggesting that molecular chaperones can make mutant proteins survive Since molecular chaperones were discovered, elevated in cancer cells. In the bacterium Salmonella typhimur- expression of molecular chaperones has been widely ium, a decreased level of fitness was observed when reported in cancer tissues and cells, suggesting the mutations were accumulated (Maisnier-Patin et al., contribution of aberrant expression of heat-shock 2005). Intriguingly, levels of the heat-shock proteins proteins to tumorigenesis (reviewed in Jaattela, 1999). DnaK and GroEL were increased in lineages harboring The high expression of molecular chaperones is many mutations, and the overexpression of GroEL induced by physiological stress, which is caused by a further increased the fitness of lineages (Maisnier-Patin shortage of oxygen, nutrition or other essential mole- et al., 2005). The overexpression of GroEL in Escher- cules from their proliferative activity. Additionally, ichia coli attenuated the growth retardation caused by tumor cells have to maintain the high turnover of harmful mutations as well (Fares et al., 2002). Induction DNA replication, RNA transcription and protein of molecular chaperones is beneficial for the survival of translation. cancer cells as well as bacterial cells. Tumor transformation has a selective advantage in terms of proliferation, antiapoptosis and against other ER stress. Because of highly proliferative growth, physiological stress. In this context, molecular chaper- tumor cells are under hypoxia and hyponutrition until ones have oncogenic activity. Forced expression of they can further acquire the additional ability of either Hsp70 in Rat-1 cells transformed the cells in terms of angiogenesis or infiltration to another tissue. Further- loss of contact inhibition and formation of foci (Volloch more, tumor cells have to meet the demand of high and Sherman, 1999). Transgenic mice expressing Hsp70 expression of proteins, translation of RNA and replica- under insulin promoter (where transgene was also tion of DNA because of their highly proliferating expressed in T cells) developed T-cell lymphoma (Seo growth. These stressful conditions easily cause ER et al., 1996). In this section, we will focus on the stress, which is originated from ER. Membrane or correlation of molecular chaperones and apoptosis in secretary proteins, which have signal sequence or signal tumor cells. patch, are translated, folded and modified in ER. Cells tightly regulate the process of protein synthesis and Antiapoptotic function of molecular chaperones in tumor maturation, and only properly folded proteins can be cells transported to the next organelle, the Golgi apparatus. The antiapoptotic function of molecular chaperones in Thedisruption of ER functions, such as malnutrition, tumor cells has been reported (Table 5). Hsp70 is hypoxia, hypoglycosylation, virus infection and unba- strongly induced in pancreatic adenocarcinoma cell lanced Ca2 þ homeostasis, can induce the accumulation lines, and inhibition of the expression by quercetin, of misfolded proteins in ER lumen and ER stress. one of the most active flavonoids, or RNAi, resulted in Unfolded protein in ER is sensed by three major ER the prominent apoptosis of cells (Aghdassi et al., 2007). membrane proteins, PERK, ATF6 and IRE, followed

Oncogene Molecular chaperones as regulators of cell death A Hishiya and S Takayama 6497 Table 5 Molecular chaperone-related apoptosis in cancer cells Molecular chaperone Cancer (cell line) Method Apoptosis References

Hsp70 Acute lymphoblastic leukemia (Molt-4) Antisense Increase Wei et al. (1995) Breast carcinoma (MCF-7) Antisense Increase Nylandsted et al. (2000) Breast carcinoma (MDA-MB-468) Antisense Increase Nylandsted et al. (2000) Colon adenocarcinoma (HT-29) Triptolide Increase Phillips et al. (2007) Colon carcinoma (rat; PROb) ADD70 Increase Schmitt et al. (2006) Glioblastoma (U373MG) Antisense adenovirus Increase Nylandsted et al. (2002) (in vivo) Melanoma (mouse; B16F10) ADD70 Increase Schmitt et al. (2006) Pancreatic adenocarcinoma (MiaPaCa-2) Quercetin RNAi Increase Aghdassi et al. (2007) Pancreatic adenocarcinoma (Panc-1) Quercetin RNAi Increase Aghdassi et al. (2007) Pancreatic adenocarcinoma (MiaPaCa-2) Triptolide Increase Phillips et al. (2007) Pancreatic adenocarcinoma (Panc-1) Triptolide Increase Phillips et al. (2007) Prostate adenocarcinoma (PC-3) Antisense Increase Jones et al. (2004) Quercetin Oral squamous cell carcinoma (HSC-2) Antisense Increase Kaur et al. (2000) Oral squamous cell carcinoma Antisense Increase Kaur et al. (2000) (primary human cells)

Hsp27 Colon adenocarcinoma REG (no expression) Increase in REG Garrido et al. (1998) PRO (expression conﬁrmed) (in vivo)

Hsp110 Colon cancer (HCT116) RNAi Increase Hosaka et al. (2006) Colon cancer (SW620) RNAi Increase Hosaka et al. (2006) Gastric cancer (KATO-III) RNAi Increase Hosaka et al. (2006) Gastric cancer (MKN28) RNAi Increase Hosaka et al. (2006) Hepatoma (SK-Hep1) RNAi Increase Hosaka et al. (2006) Pancreatic cancer (PK8) RNAi Increase Hosaka et al. (2006)

ADD70, a peptide containing the AIF sequence involved in its interaction with Hsp70. Quercetin, a widely distributed bioflavonoid that inhibits heat-shock protein induction (not related to HSF1 activity). Triptolide, a diterpene triepoxide from the plant Triptergiumwilfordii , which is highly effective in inhibiting Hsp70 expression. REG and PRO, a sub-population of a chemically induced rat colon carcinoma selected and isolated according to their susceptibility to trypsin-mediated detachment from plastic surfaces (Martin et al., 1983). PRO gives rise to progressive tumors in most of the animals, whereas REG gives rise to no tumors or to tumors that regress in less than 30 days (Caignard et al., 1985). by the activation of several pathways called unfolded apoptosis, especially in the presence of anticancer protein response, which induce transient attenuation of therapeutic drugs (doxorubicin, taxol, cisplatin and so protein translation, increase folding activity by the on) (reviewed in Li and Lee, 2006). Luo et al. (2006) induction of molecular chaperones and degradation of showed that grp78-deficient mice were embryonically malfolded protein by ubiquitin–proteasome machinery lethal with massive apoptosis when they are 3.5 days ER-associated degradation (ERAD). But when abnor- old, suggesting the antiapoptotic function of Grp78. In mal proteins are still persisting or exceed the capacity in fact, cancer progression was significantly attenuated ER, apoptotic cascade is activated. Because of limitation with reduction of tumor cell proliferation, inhibition of of space, please refer other review articles for ER stress- tumor angiogenesis and massive apoptosis in tumor cells induced apoptosis (Xu et al., 2005), in cancer (Moenner in grp78 heterozygous mice, in which the expression et al., 2007), in neurodegenerative diseases (Lindholm level of Grp78 is reduced by about half (Dong et al., et al., 2006), in diabetes mellitus (Eizirik et al., 2008) and 2008). Thequality control of ER proteinis tightly ischemia (DeGracia and Montie, 2004). regulated by ER chaperones, and abnormal function From the Hsp70 family of molecular chaperone of ER chaperones induces apoptosis. As described in proteins, glucose-regulated protein 78 (Grp78) is one Table 6, we summarized apoptosis-related phenotypes in of the best characterized ER chaperone proteins, which genetic disease of molecular chaperones including ER is involved in ER function and has an antiapoptotic chaperones. function in tumor cells (Jamora et al., 1996; Suzuki et al., 2007). Theantiapoptotic mechanism of Grp78 may fall into two main pathways. Because of its Direct inhibition of an apoptotic pathway chaperone function, Grp78 directly works on protein In the mitochondrial pathway, cell death signals induce folding, and attenuates ER stress. On the other hand, the release of cytochrome c, Smac/DIABLO or AIF. as Grp78 also serves as a sensor for ER stress, the Small heat-shock proteins, Hsp27, inhibit apoptosis by accumulation of misfolded proteins in ER lumen keeps directly binding to cytosolic cytochrome c and seques- Grp78 away from IRE, PERK and ATF6, which results tering Apaf1 (Garrido et al., 1999; Bruey et al., 2000). in the activation of these membrane proteins, and is A recent report indicated that Hsp27 also inhibited followed by unfolded protein response or apoptosis. The Smac release from mitochondria (Chauhan et al., 2003). overexpression of Grp78 has been observed in many Molecular chaperone Hsp70 inhibits apoptosome tumor cell types, and protects tumor cells against formation by direct binding to Apaf1 (Beere et al.,

Oncogene Molecular chaperones as regulators of cell death A Hishiya and S Takayama 6498 Table 6 Phenotypes of molecular chaperone knockout mice Molecular chaperone Engineering Phenotype Apoptosis References

110-kDa heat-shock proteins Hspa4l (Apg1, Osp94) Deﬁcient Male infertility (about 42% Held et al. (2006) of men) Hydronephrosis (about 12% of mice)

90-kDa heat-shock proteins Hsp90b Deﬁcient Embryonic lethal with pla- Voss et al. (2000) cental development defect

70-kDa heat-shock proteins Hsp70.1 Deficient No morphological Huang et al. (2001) abnormalities Hsp70.2 Deficient Male infertility (lack of Spermatocytes Dix et al. (1996) spermatogenesis) Hsp70.3 Deficient No morphological Huang et al. (2001) abnormalities Hsc70 Knockout cells Non-viable Florin et al. (2004)

40-kDa heat-shock proteins DjA1 (DnaJA1, Hdj2) Deficient Male infertility (defects Terada et al. (2005) in spermatogenesis) DjA3 (DnaJA3, Tid1) Deficient Embryonic lethal Lo et al. (2004) Deficient Death before 10 weeks Hayashi et al. (2006) (heart specifically) of agewith dilatedcardio- myopathy DjB1 (DnaJB1, Hdj1) Deficient No abnormalities Uchiyama et al. (2006) DjB6 (DnaJB6, Mrj) Deficient Embryonic lethal with Hunter et al. (1999) defects in placental development DjC3 (DnaJC3, p58IPK) Deficient Diabetes Pancreatic islet cells Ladiges et al. (2005) DjC5 (DnaJC5, CSPa) Deficient Death within 2 months of Fernandez-Chacon et al. (2004) age neurodegeneration

ER chaperones Grp78 (Bip) Deficient Embryonic lethal Inner cell mass Luo et al. (2006) Calreticulin Deficient Embryonic lethal Mesaeli et al. (1999) Misdevelopment of the heart Calnexin Deficient Early postnatal death Denzel et al. (2002) Motor disorder (loss of large myelinated nerve fibers) Calmegin Deficient Male infertility Ikawa et al. (1997) Grp94 Deficient Embryonic lethal Wanderling et al. (2007) Hsp47 Deficient Embryonic lethal with Nagai et al. (2000) defects in collagen biosynthesis

Small heat-shock proteins aA-Crystallin Deficient Cataract Brady et al. (1997) (Cryaa, HspB4) aB-Crystallin R120G transgenic Death at 5–7 months of age Wang et al. (2001) (Cryab, HspB5) Desmin-related cardiomyopathy R120G transgenic in Death of congestive heart Wang et al. (2003b) DRM background failure in 7 weeks after birth Cardiac hypertrophy aA-Crystallin/ Deficient Significantly smaller than Boyle et al. (2003) aB-crystallin littermate Cataract aB-Crystallin/HspB2 Deficient Muscle degeneration Brady et al. (2001) (MKBP) (in tongueand soleus) HspB1 (Hsp25) Deficient No morphological Huang et al. (2007) abnormalities

Heat-shock factors HSF1 Deﬁcient Placental development Christians et al. (2000) defect

Oncogene Molecular chaperones as regulators of cell death A Hishiya and S Takayama 6499 Table 6 Continued Molecular chaperone Engineering Phenotype Apoptosis References

Deficient Astrogliosis Santos and Saraiva (2004) Neurodegeneration Deficient Defects of the Xiao et al. (1999) chorioallantopic placenta Prenatal lethality Growth retardation Female infertility Deficient Abnormal olfactory TurbinateTakaki et al. (2006) neurogenesis Deficient Demyelination CNS Homma et al. (2007) Astrogliosis Constitutive Abnormal Spermatocytes Nakai et al. (2000) expression of an spermatogenesis activeform HSF2 Deficient Morphological Testes Kallio et al. (2002) Abnormalities in brain apoptosis in spermatocytes (but no maleinfertility) Female subfertility Deficient No abnormalities McMillan et al. (2002) Deficient Increased embryonic Wang et al. (2003a) lethality Morphological abnormalities in brain Reduced female fertility HSF1/HSF2 Deficient Male infertility Wang et al. (2004) HSF4 Deficient Cataract Fujimoto et al. (2004) Deficient Cataract Min et al. (2004)

Co-chaperones CHIP Deficient Peripartum period death Thymus Dai et al. (2003) (about 20% of total deficient mice) Thymic atrophy in dead mice Deficient Premature aging (reduced Min et al. (2008) lifespan) p23 (Hsp90 Deficient Perinatal lethality with Grad et al. (2006) co-chaperone) defect of the lung maturation BAG1 Deficient Embryonic lethal with Fetal liver developing Gotz et al. (2005) defects in forebrain and nervous system liver BAG3 (CAIR1, Bis) Deficient Myofibrillar myopathy Heart Homma et al. (2006) BAG4 (SODD) Deficient No abnormalities Takada et al. (2003) (abnormal cytokine production in response to TNFa)

Abbreviations: CNS, central nervous system; DRM, desmin-related myopathy; TNFa, tumor necrosis factor-a.

2000; Saleh et al., 2000). Another report has negative et al., 2001). A peptide containing the AIF sequence observation about the involvement of Hsp70 in the involved in its interaction with Hsp70 induced apoptosis regulation of apoptosomes. High salt concentration in both rat colon cancer cells and mouse melanoma cells (from the preparation of recombinant Hsp70) itself is under cisplatin stimuli (Schmitt et al., 2006). Nylandsted enough for the inhibition of apoptosome (Steel et al., et al. (2000) reported that Hsp70 inhibited apoptosis in 2004). A recent report indicated that Hsp70 and tumor human breast tumor cells, but it was neither dependent suppressor protein PHAPI and CAS (cellular apoptosis on caspases nor on p53, suggesting versatile functions susceptibility protein) stimulate the apoptosome by of Hsp70 or thefunction in thedownstreamof promoting nucleotide exchange of Apaf1 (Kim et al., unknown caspases. Endonuclease G (EndoG) is a 2008). Thefunction of Hsp70 in apoptosomeis to mitochondrial enzyme that has nuclease activity and is promoteapoptosomeactivity. induced by apoptotic signals. Hsp70 binds to EndoG Hsp70 directly binds to AIF and inhibits AIF- and AIF, preventing DNase activity (Kalinowska et al., dependent caspase-independent apoptosis (Ravagnan 2005).

Oncogene Molecular chaperones as regulators of cell death A Hishiya and S Takayama 6500 Bax translocates to the mitochondria and participates Conclusions in apoptosis induction. Heat-shock proteins have been reported as inhibitors of Bax translocation. Hsp27 Molecular chaperones have critical function in protein inhibits bax translocation from cytosol to mitochondria folding, protein degradation and cell death. CHIP was through the PI3 kinase-dependent Akt pathways found as a co-chaperone of Hsp70 and a ubiquitin ligase (Havasi et al., 2008). Hsp70 is also reported as an targeting abnormal proteins, which failed to be refolded inhibitor of Bax translocation (Stankiewicz et al., 2005). by Hsp70. Recent reports indicated that ubiquitin– Regulation of ischemia-induced apoptosis by Hsp60 in bax proteasome degradation of apoptosis-related signal is translocation was described above. Recently, cytokine- also directly regulated by CHIP. Thus, CHIP or other induced destabilization of mRNA of BH3-only Bcl-2 chaperone-associating ubiquitin–proteasome-related family protein, Bim, is reported as directly regulated by proteins might have critical functions in regulating Hsc70 and its co-chaperones, BAG4, CHIP, HIP and apoptosis when unfolded or aggregated protein is Hsp40 (Matsui et al., 2007). Co-chaperone, CHIP E3 ligase accumulated in cells. Therefore, research in the mechan- is also reported as a ubiquitinating protein in apoptosis ism of regulating ‘fold or degradation’ decision by signaling and inhibits apoptosis, such as Ask1 (apoptosis molecular chaperones and their co-chaperones may give signal-regulating kinase) and DAPK (death-associated us more precise mechanism of molecular chaperone protein kinase) (Hwang et al., 2005; Zhang et al., 2007). function in regulating apoptotic signals.

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