ICC staining of Hsp47 in heat shocked HeLa cells using Anti-Hsp47 (clone: 1C4-1A6) and a FITC secondary (green: cytoplasm; blue: DAPI nuclear stain)
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 σS 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.