HSP40: Regulation

When cells are subjected to environmental stress, they respond by enhancing expression of HSPs. HSP families known to be induced by stress comprise not only HSPA/HSP70, HSPB (small HSPs), HSPC/HSP90, and HSPD/HSP60 but also HSPH/HSP110 and the DNAJ/HSP40 family of chaperones. The corresponding genes have evolved a highly efficient mechanism for mass synthesis during stress, with powerful transcriptional activation, efficient messenger ribonucleic acid (mRNA) stabilization, and selective mRNA translation ²⁴². 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) ³¹. 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) ²⁴³. Among them, HSF-1 is considered as being the key transcription factor of stress-inducible HSPs ^{244, 245}. 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 ²⁴⁶. 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 ^{247, 248}. HSF-1 homotrimers bind to HSEs of any HSP and trigger immediate and massive transcription of these HSP genes after hyperphosphorylation ^{244, 249, 250}. HSF-1 activation is negatively regulated by Hsp90 and Hsp70 via an autoregulatory loop ²⁵¹. 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 ^{248, 249, 252, 253}. Activated HSF-1 trimers also interact with Hsp70 and the co-chaperone Hsp40/DnaJ, leading to an inhibition of its transactivation capacity ^{254, 255, 256}. 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 ²⁵⁷ as well as deacetylation through the catalytic action of the deacetylase sirtuin 1 (Sirt-1) ²⁵⁸. In this context, DNAJs/HSP40s have been identified to regulate their own transcription. Work by Zhong and collaborators clearly demonstrated that a specific cis-element containing the SIS1 HSE plus an additional promoter sequence function as the autoregulatory element in SIS1 ²¹⁹. In addition, SIS1 transcription is also regulated by the HSPA/HSP70 member Ssa, and this induction requires only the HSE of SIS1. These findings underline the specificity of the induction of heat shock genes and the multiple mechanisms involved in their transcriptional regulation ²¹⁹. Steroid hormones such as estrogen have also been reported to regulate transcription of DNAJ/HSP40. Studies by De Bessa et al. revealed an association between the estrogen receptor-positive status and high levels of DNAJC12/JDP1 mRNA expression ²⁵⁹. Furthermore, DNAJC12/JDP1 mRNA levels increased markedly in breast cancer cells MCF-7 after exposure to 17-beta-estradiol. Expression of DnaJC12/Jdp-1 depends on the presence of potential estrogen response elements (EREs) in the promoter region of the DNAJC12/JDP1 gene suggesting an estrogen-mediated regulatory mechanism ²⁵⁹. Enhancer elements also contribute to the transcriptionally upregulation of DNAJs/HSP40s genes. For instance, curcumin (diferuloylmethane) an active component of the spice turmeric has been identified to transcriptionally increase DnaJB4/Hlj1 expression through an activator protein (AP-1) site within the DNAJB4/HLJ1 enhancer ²⁶⁰.

As already mentioned, the E. coli chromosome contains genes for six DnaJ/Hsp40 homologs including cbpA and dnaJ whose expression increases tenfold after heat shock ²⁶¹. The increase in dnaJ expression is rapid and regulated primarily at the transcriptional level. CbpA (curved DNA binding protein A) functions not only as a multicopy suppressor for dnaJ mutations in vivo, but also as a co-chaperone for the DnaK system in vitro ^{115, 118, 262}. CbpA is encoded in an operon together with the CbpA regulator CbpM, interacting with CbpA and thereby inhibiting both the co-chaperone and DNA-binding activities of CbpA ^{118, 263}. Transcription of the cbpAM operon has been found to be under control of the σ^S and Lrp global regulators, and both leucine availability and growth temperature affect transcription ^{118, 264}. The same study revealed that CbpA and CbpM accumulates to similar levels in stationary phase, and that unbound CbpM is unstable and degraded by the Lon and ClpAP proteases indicating a multilevel regulation of the CbpA activity ^{118, 264}.

Apart from its transcriptional regulation, DNAJ/HSP40 protein levels have also been found as being regulated at the post-transcriptional level. 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 ^{265, 266, 267}. However, little information is available on the role of miRNAs in the regulation of the DNAJ/HSP40 expression. The group of Rajeev S. Samant identified DnaJB6/Mrj as being a direct target of miR-632 in metastatic breast cancer cells responsible for the downregulation of DnaJB6/Mrj observed in human breast cancer ²⁶⁸. However, future approaches analyzing the regulatory potential of miRNAs in DNAJ/HSP40 expression will shed light on the post-transcriptional regulation of these co-chaperones.

DNAJs/HSP40s may also be expressed irrespectively of HSF-1 transcriptional activity. It has been shown previously that DnaJA1/Hdj2 as well as DnaJB1/Hdj1 are upregulated in response to bacterial lipopolysaccharide (LPS), a well-known inducer of inflammatory immune responses ²⁶⁹. LPS is known to bind to Toll-like receptor 4 (TLR-4) in association with its glycosyl-phosphatidylinositol-anchored co-receptor CD14 ²⁷⁰, culminating in the activation and translocation of NF-κB into the nucleus ²⁷¹. In this regard, several inflammatory mediators and signaling molecules such as NF-κB and TNF have been shown as being strictly associated with HSP gene expression and protein functions. In this context, the NF-κB subunit p65/RelA functions as a transcription factor for numerous HSPs including DnaJ/Hsp40 ^{272, 273} that in turn may have anti-apoptotic functions ^{25, 274}.

The activity of DNAJs/HSP40s is regulated by several post-translational modifications. In many cases, DNAJs/HSP40s are phosphoproteins (e.g. DnaJA1, DnaJB4, DnaJC1, DnaJC29) whose expressions and functions can be further modulated co- and post-translationally by acetylation (e.g. DnaJA1, DnaJB2, DnaJB12, DnaJC5, DnaJC8, DnaJC13), glycosylation (DnaJB11, DnaJC10, DnaJC16), palmitoylation (DnaJC5, DnaJC5B, DnaJC5G), methylation (DnaJA1-4), prenylation (DnaJA1, DnaJA2, DnaJA4), and formation of intramolecular disulfide bonds (DnaJB11, DnaJC3, DnaJC10), respectively. It has recently been shown that the redox state of the cell obviously regulates the co-chaperone activity of DnaJA1/Hdj2 through thioredoxin-dependent oxidation of cysteine thiols and concomitant release of coordinated zinc, suggesting a contribution of cysteine residues in the ZF domain of DnaJA1/Hdj2 ²⁷⁵.

As already mentioned, some of the DNAJs/HSP40s exist as phosphoproteins. In the case of DnaJA1/Hdj2, mass spectrometric analyses identified multiple phosphorylation sites in DnaJA1/Hdj2 including Ser381, Ser430, Ser479, Ser480, and Ser484 ^{276, 277, 278}. However, only little information is available on the protein kinases mediating this process and its functional relevance. Studies by Kostenko and co-workers most recently identified Ser149 or/and Ser151 and Ser171 in the CTD as the primary phosphorylation sites in DnaJB1/Hdj1 catalyzed by the mitogen-activated protein kinase-activated protein kinase 5 (MK5) ²⁷⁹. MK5 has been shown in this study to stimulate the ATPase activity of the DnaJB1/Hsp70 complex and enhance the repression of HSF-1-driven transcription by DnaJB1/Hdj1. Evidence for an acetylation of Hsp60 has emerged after the discovery of the histone deacetylase 4 (HDAC-4) as being an interaction partner of DnaJB6/Mrj ²⁸⁰. Meanwhile, mass spectrometric analyses identified Lys56 within DnaJC5/Csp-alpha ²⁸¹ and Ala2 in DnaJC7/Tpr-2 as acceptor sites for acetyl residues ²⁸². Acetylation of the latter was reported to be catalyzed by the N-terminal acetyltransferase NatB ²⁸².

Glycosylation is a further post-translational modification of DNAJs/HSP40s including ER-resident DnaJB11/ERdj3 and DnaJC10/ERdj5 as well as DnaJC16. DnaJB11/ERdj3 has recently been found as being N-glycosylated at Asp261 by the addition of high-mannose Endo H-sensitive carbohydrate side chains ⁷⁸. ER-resident DNAJs/HSP40s such as DnaJC3/ERdj6, DnaJB11/ERdj3 and DnaJC10/ERdj5 form intramolecular disulfide bonds ^{61, 283, 284}. In DnaJC10/ERdj5, a member of the protein disulfide isomerase family of proteins localized to the ER of mammalian cells and catalyzing the removal of non-native disulfides, four disulfide bonds can be found that contribute to the reductase activity of the chaperone attributed to the presence of four thioredoxin domains ^{283, 284}.

Post-translational modifications of DNAJs/HSP40s also comprise palmitoylation which occurs particularly in membrane-associated DnaJC5, DnaJC5B, and DnaJC5G functioning as potential lipid anchors ²⁸⁵. However, palmitoylation does not seem to be necessarily required for membrane anchorage as demonstrated in this study. Certain members of the DNAJ/HSP40 subfamily A such as DnaJA1/Hdj2, DnaJA2/Dnj3, DnaJA4/Hsj4 are subjected to farnesylation at specific Ser residues thereby facilitating membrane anchorage ^{286, 287, 288, 289}. In Ydj1, farnesylation obviously promotes regulation and activation of diverse Hsp90 clients as alteration of the C-terminal farnesylation signal disrupts the functional and physical interaction of Ydj1 and Hsp90 with certain client proteins such as S protein kinase Ste11 and the glucocorticoid receptor ²⁹⁰. Mass spectrometric analyses demonstrated that DnaJA3/Tid-1 is methylated at Arg58, Arg238 and Arg293 by co-activator-associated arginine methyltransferase 1 (CARM1) ²⁹¹. In addition, S-methylation of cysteine residues has also been reported amongst members of subfamily A (DnaJA1/Hdj2, DnaJA2/Dnj3, DnaJA4/Hsj4) as can be seen in the UniProt database. Notwithstanding this issue, the functional significance of post-translational methylation remains unclear to date.