HSP60: Regulation

IHC staining of Listeria infected mice spleens 6 days after infection with L-monocytogenes, using Anti-Hsp60 (clone: LK2)

When cells are subjected to environmental stress, they respond by enhancing expression of HSPs. A total of five HSP families are known to be induced by stress including the HSP60 family of chaperones. The rapid induction of HSP in response to environmental stress is based on a variety of genetic and biochemical processes referred to as the heat shock response (HSR) ¹⁸. HSR is regulated mainly at the transcription level by heat shock factors (HSF). HSFs are transcription factors that bind specific cis-acting sequences upstream of the heat shock gene promoters called heat shock elements (HSE) ²⁵⁵. Among them, HSF-1 is considered as being the key transcription factor of stress-inducible HSPs ^{256, 257}. HSF-1 binds to the 5´-promoter region of any HSP gene thereby inducing immediate transcription. HSF-1 is present in the cytosol in a latent state under normal conditions and is activated by stress-induced trimerization and high-affinity binding to DNA and exposure of domains for transcriptional activity ²⁵⁸. Monomeric HSF-1 is already a phosphoprotein whose activity is repressed by Hsp90 under growth conditions. In response to stress, HSF-1 repression is reversed by Hsp90 dissociation leading to trimerization of HSF-1 ^{259, 260}. HSF-1 homotrimers bind to HSEs of any HSP and trigger immediate and massive transcription of these HSP genes after hyperphosphorylation ^{256, 261, 262}. HSF-1 activation is negatively regulated by Hsp90 and Hsp70 via an autoregulatory loop ²⁶³. Homotrimeric HSF-1 is kept inactive when its regulatory domain is bound by a multi-chaperone complex of Hsp90, co-chaperone p23 (PTGES3) and immunophilin FKBP52 ^{260, 261, 264, 265}. Activated HSF-1 trimers also interact with Hsp70 and the co-chaperone Hsp40 (DnaJ), leading to an inhibition of its transactivation capacity ^266,267,268. HSF-1 transcriptional activity is attenuated not only by the negative feedback loop from HSPs, which represses the transactivation of DNA-bound HSF-1, but also by inhibition of DNA binding through acetylation of Lys80 in the DNA-binding domain of HSF-1. HSF-1 activation is also regulated by sumoylation ²⁶⁹ as well as deacetylation through the catalytic action of the deacetylase sirtuin 1 (Sirt-1) ²⁷⁰.

Apart from its transcriptional regulation, Hsp60 protein levels have also been found as being regulated at the post-transcriptional level. Gene expression studies in cancer revealed an interruption between transcription and translation of HSP60. Tang and collaborators demonstrated that HSP60 mRNA and Hsp60 protein expression varied considerably as HSP60 mRNA levels increased after heat stress while Hsp60 protein levels remained almost unaffected ²⁷¹. In this context, the discovery of micro-RNA (miRNA) identified this RNA subtype as a crucial player in regulating translation of many genes. miRNAs are a class of small non-coding RNAs that negatively regulate gene expression by binding to target mRNAs. Global alterations in miRNAs can be observed in a number of disease states including cancer ^272,273,274. However, little information is available on the role of miRNAs in the regulation of the HSP60 expression. The group of Xi-Yong Yu identified HSP60 (HSPD1) mRNA as being a direct target of miR-1 and miR-206 in cardiomyocytes as upregulation of miR-1 and miR-206 suppressed Hsp60 expression via the MEK-1/-2 pathway thus accelerating cardiomyocyte apoptosis ²⁷⁵. A recent study convincingly demonstrated that miR-1 aggravated cardiac ischemia/reperfusion injury via inhibiting pro-survival proteins, e.g. Hsp60 and PKCε ²⁷⁶. Future approaches analyzing the regulatory potential of miRNAs in HSP60 expression will shed light on the post-transcriptional regulation of HSP60s.

Hsp60 may also be expressed irrespectively of HSF-1 transcriptional activity. It has been shown previously that Hsp60 expression is upregulated in response to cytokines such as IL-1β, TNF, IL-4, IL-6 and IL-10 ²⁷⁷. In this regard, several inflammatory mediators and signalling molecules such as NF-κB and TNF have been shown as being strictly associated withl HSP gene expression and protein functions. In this context, the NF-κB subunit p65/RelA functions as a transcription factor for numerous HSPs including Hsp60 ^{278, 279} that in turn may have anti-apoptotic functions ^{280, 281}. Hsp60 expression is also regulated by the proto-oncoprotein c-Myc which plays an eminent role in control of cell cycle progression, proliferation, metabolism, and apoptosis ^{282, 283}. cMYC is one of the most frequently altered genes in human cancer, and dysregulated cMYC expression is constantly implicated in tumorigenesis ^{282, 284, 285}. Studies by Tsai et al. revealed that c-Myc directly activates HSP60 transcription through an E-box (CACGTG) site located in the proximal promoter of the HSP60 gene, and that overexpression of Hsp60 induces transformation ²⁸⁶.

The activity of Hsp60 is regulated by several post-translational modifications. Hsp60 is a phosphoprotein ⁷⁰ whose expression and function can be further modulated by ubiquitination ²⁸⁷, sumoylation ⁷¹, acetylation ⁷², malonylation ⁷³, N-glycosylation ⁷⁴ or O-GlcNAcylation ⁷⁵. Hsp60 represents a substrate for DNA-dependent protein kinase which is crucially implicated in regulating Hsp60 levels ²⁸⁸. Mass spectrometric analyses identified Ser70 within Hsp60 (HspD1) as the primary phosphorylation site ^{289, 290}. Phosphorylation of Hsp60 has been identified as being catalyzed by the serine-threonine protein phosphatase 2 A (PP2A) ⁷⁰. Evidence for an acetylation of Hsp60 has emerged after the discovery of the histone deacetylase 1 (HDAC-1) as being an interaction partner of Hsp60 ²⁹¹. Meanwhile, mass spectrometric analysis identified several lysyl residues as acceptor sites for acetyl residues within Hsp60 ⁷². A recent study clearly demonstrated the critical contribution of the small-molecule Mdm-2 antagonist nutlin 2 in lysine acetylation of Hsp60 ²⁹². Malonylation is a further post-translational modification of Hsp60 occurring preferentially at Lys133. Peng and co-workers identified Sirt-5, a member of the class III histone deacetylases (HDAC-3), as the first regulatory enzyme for lysine malonylation because it catalyzes lysine demalonylation and lysine desuccinylation both in vitro and in vivo ⁷³.

Post-translational modifications of Hsp60 also include sumoylation as well as N-glycosylation and O-GlcNAcylation. Hsp60 could be identified as sumoylation target in the fungus C. albicans ⁷¹. Of note, mutation of consensus sumoylation sites in Hsp60 have been shown to affect the resistance of C. albicans to thermal stress thus indicating that sumoylation impacts key cellular processes required for the pathogenicity of this clinically important fungus ⁷¹. Hsp60 has recently been found as being post-translationally modified by N-glycosylation at three potential N-glycosylation sites in human fibrosarcoma cells ⁷⁴. This result together with the observation that Hsp60 is obviously secreted through the conventional ER/Golgi secretory pathway suggests that N-glycosylation affects the immunological properties of Hsp60 in the tumor microenvironment ⁷⁴.

The group of Jin Won Cho detected an O-GlcNAcylation of Hsp60 in rat pancreatic β-cells that increased under hyperglycemic conditions ⁷⁵. It is well known that Hsp60 interacts with pro-apoptotic Bax in the cytosol under normoglycemic conditions. Now it becomes obvious that hyperglycemic conditions trigger detachment of Bax from O-GlcNAcylated Hsp60 and translocation of Hsp60 to mitochondria ⁷⁵. Since hyperglycemia also stimulated cytochrome c release, caspase-3 activation, and cell death, it can be concluded that elevated O-GlcNAcylation of Hsp60 disrupts the Hsp60/Bax interaction, thereby causing apoptosis of pancreatic β-cells. Human Hsp60 is also subjected to ubiquitination. In human monocyte-derived macrophages THP-1, oxidative stress induced overexpression and ubiquitination of Hsp60 and blocked its release from necrotic cells into the extracellular milieu in vitro ²⁸⁷.