HSP40: Mechanisms & Interactions

One of the major functions of DNJs/HSP40s comprises the regulation of the ATP-dependent polypeptide-binding by HSP70s in order to promote folding, transport and degradation of the substrate protein. DnaJ/Hsp40 binds to unfolded or non-native polypeptides via its C-terminal substrate binding domain in order to prevent their aggregation (reviewed by Fan et al., 2003 ⁵). DnaJ/Hsp40 is an intermediate protein that complexes with Hsp70 interacting protein (Hip), and transfers the unfolded protein to Hsp70. DnaJ/Hsp40 also binds directly to Hsp70 and stimulates its ATPase activity ^{196, 292}. Afterwards, Hsp70 delivers the unfolded protein to the Hsp90 complex via Hsp70/Hsp90 organizing protein (Hop) ^{132, 197, 293}. DNAJs/HSP40s function as homodimers interacting with the unfolded, non-native polypeptide through hydrophobic interactions. Complex formation obviously primes the unfolded substrate polypeptide into an extended conformation in preparation for the adjacent binding to Hsp70/DnaK ^{65, 114, 294}. DnaJ/Hsp40-bound unfolded substrate is delivered subsequently to Hsp70/DnaK. DnaJ/Hsp40 then interacts, through the conserved HPD motif in the J-domain, with an acidic groove located in the N-terminal ATPase domain (also known as nucleotide binding domain, NBD) of Hsp70/DnaK ⁵⁵. This interaction regulates and stimulates the intrinsic ATPase activity of Hsp70/DnaK ^{32, 36} enabling the formation of stable Hsp70/protein complexes. Recruitment and transfer of substrate proteins to Hsp70/DnaK is not only mediated by interactions with the NBD but also with the substrate binding domain (SBD) of Hsp70/DnaK. DNAJs/HSP40s also interact with the C-terminal octapeptide of human Hsp70, ₆₃₄GPTIEEVD₆₄₁ via a C-terminal peptide-binding domain ²⁰³. It is interesting to note that some DNAJs/HSP40s target HSP70 activity to clients at precise locations in cells and others bind client proteins directly, thereby delivering specific clients to HSP70 and directly determining their fate ⁷.

ATP hydrolysis induces conformational changes in the C-terminal domain (CTD) of Hsp70/DnaK. In the ATP-bound state, the SBD is in an open conformation with low affinity and fast exchange rates for the substrate. After ATP hydrolysis, DnaJ/Hsp40 is released and the SBD switches to a closed conformation with high affinity and low exchange rates for its substrate. ADP release is mediated by the specific interaction of nucleotide exchange factors (NEFs) with the Hsp70/DnaK ATPase domain. Hsp70/protein complexes dissociate upon regeneration of ATP-bound DnaJ/Hsp40. The released substrate can be either folded into the native protein, re-bound to Hsp70/DnaK or aggregate ⁵. A proposed model of the DnaJ/Hsp40-dependent substrate binding to DnaK/Hsp70 is given in Figure 5. For more detailed information on the molecular mechanisms involved in the DnaK/Hsp70 chaperone cycle see also HSP70 Scientific Resource.

The ATPase-stimulating capacity of the DnaJ/Hsp40 J-domain is further boosted by polypeptides bound to the SBD of Hsp70/DnaK ²⁹⁵. As mentioned above, ATP hydrolysis induces a conformational change in DnaK/Hsp70 leading to the presumption of an interdomain communication between the ATPase and SBD of Hsp70/DnaK ^{296, 297}. Previous investigations identified the conserved Glu171 in bacterial DnaK corresponding to mammalian Glu175 as being required for coupling ATPase activity with substrate binding as an essential prerequisite for the chaperone function of DnaK ²⁹⁷. DnaK Glu171, a constituent of the ATP-binding groove, forms parts of the linker region responsible for the conformational changes observed after ATP binding and subsequent binding and release of substrate polypeptides ^{297, 298}. It is interesting to note that Glu171 is located close to the acidic groove containing basic Arg167 in the J-domain binding region of Hsp70/DnaK implying a putative effect of J-domain-mediated Hsp70/Hsp40 (DnaK/DnaJ) interaction on the three-dimensional structure of the linker region regulating the interplay between the ATPase domain and SBD ⁵.

Several investigations within the past years showed that DnaJ/Hsp40 can interact directly with molecules in various cell compartments. However, the list of client proteins for DnaJ/Hsp40 is still incomplete. In this respect, DnaJB6/Mrj has recently been identified to interact with urokinase-type plasminogen activator receptor (uPAR) via its CTD resulting in enhanced uPAR-dependent adhesion to the extracellular matrix protein vitronectin ²⁹⁹. uPA-mediated cell adhesion to vitronectin represents an important event in wound healing, tissue remodeling, immune response, and cancer. It is unclear up to date how DnaJB6/Mrj triggers uPAR to augment adhesion to vitronectin. In human embryonic kidney 293 (HEK293) cells, the expression of uPAR results in the generation of tight complexes with β1-integrins thereby promoting the adhesion to vitronectin ^{300, 301}. Human DnaJB6/Mrj was also found to interact with the intermediate filament keratin 18 (K18), and to regulate K8/18 filament organization ³⁰². Moreover, DnaJB6/Mrj has been reported to mediate proteasomal degradation of K18 intermediate filaments ³⁰³. Thus, these observations provide insight into the putative role of the DnaJB6/uPAR interaction in modulating tumor growth by either altering keratin filament organization and/or promoting vitronectin binding via β1-integrins ²⁹⁹. A yeast two-hybrid screening identified the rel homology domain of the transcription factor NFATc3 (nuclear factor of activated T cells c3) as an interacting factor of DnaJB6/Mrj ²⁸⁰. DnaJB6/Mrj and NFATc3 were shown to directly associate with each other not only in vitro but also in vivo. Also, DnaJB6/Mrj served as a potent inhibitor of NFAT transcriptional activity within the nucleus through a mechanism involving class II HDAC recruitment (HDAC-4/HDAC-5/HDAC-7/HDAC-9) in conjunction with heat shock stimulation highlighting its function as an NFATc3 co-repressor and HDAC interacting factor ²⁸⁰. Breast cancer metastasis suppressor 1 (Brms-1) is an additional interaction partner of DnaJB6/Mrj recently identified in a yeast two-hybrid screen ³⁰⁴. Brms-1, a member of the mSin3/HDAC complex ³⁰⁵, interacts with class I and class II HDACs ³⁰⁴. Hurst et al. found out that the Brms-1 turnover is proteasome-dependent and that Hsp90 contributes to the stability of Brms-1 suggesting a crucial role in maintaining the biological functions of Brms-1 in metastasis suppression ³⁰⁴.

A most recent investigation revealed an upregulation of the molecular chaperones Hsp40, Hsp70, and Hsp90 in HeLa cells overexpressing anti-apoptotic Bag-1 ³⁰⁶. Herein, Hsp90 and Hsp40 have been found as being involved in the regulation of the Bag-1 interactome including various anti-apoptotic BCL-2 family members and c-Raf. Bag-1 is the eukaryotic ortholog of bacterial GrpE and was discovered originally as a Bcl-2-associated protein ³⁰⁷ regulating HSP70 nucleotide exchange and ATPase activity ^{308, 309}. Bag-1 interacts with a wide range of cellular targets and regulates cell survival, signaling, metastasis, proliferation, and transcription mechanisms. It has been shown previously that Bag-1 stimulates the anti-apoptotic effect of BCL-2 family members and augments the activity of the protein kinase Raf-1 involved in signal transduction as well as modulating cell growth and differentiation ³¹⁰. Stress-induced upregulation of Hsp70 results in the formation of Bag-1/Hsp70 complexes that can compete against Bag-1/Raf-1 complex formation, thereby suppressing Raf-1 kinase activity ³¹¹.

The studies by Qi et al. identified a novel client of DnaJB1/Hdj1, the negative p53 regulator Mdm-2 ³¹². p53 is a transcription factor which helps to eliminate potential tumor progenitor cells by acting as an apoptosis inductor ^{313, 314}. DnaJB1/Hdj1 has been demonstrated to stabilize Mdm-2 at the post-translational level and to inhibit the Mdm-2-mediated ubiquitination and degradation of p53 thus contributing to p53 activation in cancer cells ³¹⁵. Notably, depletion of DnaJB1/Hdj1 in cancer cells suppressed p53 activity, enhanced the activity of the Rb/E2F pathway, and promoted cancer cell growth in vitro and in vivo in a p53-dependent manner ³¹⁵. These findings provide evidence for the p53-dependent tumor suppressor function of DnaJB1/Hdj1. In this context, the mitochondrial DnaJ/Hsp40 homolog, DnaJA3/Tid-1 has been identified as a novel regulator of p53-mediated apoptosis. Studies by Ahn and collaborators clearly demonstrated that DnaJA3/Tid-1 forms a complex with p53 under hypoxic conditions that directs p53 translocation to the mitochondria followed by initiation of the mitochondrial apoptosis pathway ³¹⁶. It is worth mentioning that depletion of DnaJA3/Tid-1 abolished p53 translocation to the mitochondria and blocked apoptosis, while its overexpression promoted p53 mitochondrial localization and apoptosis. Overexpression  of  DnaJA3/Tid-1 in cancer cell lines expressing mutant p53 defective in transcriptional activity led to restoration of mitochondrial localization and pro-apoptotic activities of the mutant p53 proteins ³¹⁶. The same group found out that DnaJA3/Tid-1 directly interacts with p53 through its J-domain and its depletion induces resistance to stresses by inhibiting the p53 localization to the mitochondria ³¹⁷. These findings render DnaJA3/Tid-1 a promising target in anti-tumor therapies of p53-related cancers ^{316, 317}. The group of Sidney Pestka was about the first who reported of a human DNAJ/HSP40 interacting with the Jak-2 and IFN-γ receptor complex. This group presents evidence that DnaJA3/Tid-1 functions as a negative modulator of the JAK/STAT pathway ³¹⁸. DnaJA3/Tid-1 was found to interact with Jak-2 and with the IFN-γ receptor chain IFN-γR2 with which Jak-2 remains associated. Moreover, DnaJA3/Tid-1 interacts with the HSP70/HSPA family of chaperones in IFN-γ-responsive HEp2 cells, and this interaction is reduced in the presence of IFN-γ ³¹⁸. From these data the authors proposed a model for the modulation of the Jak-2 activity by DnaJA3/Tid-1. It is well established that the IFN-γ receptor complex is composed of two chains, IFN-γR1 and IFN-γR2 ^{319, 320}. Jak-2 and IFN-γR2 associate with DnaJA3/Tid-1 and HSP70 to form a complex in conjunction with IFN-γR1 and Jak-1. Following IFN-γ treatment, HSP70 and DnaJA3/Tid-1 dissociate successively from the complex thereby activating the kinase function of Jak-2. Once DnaJA3/Tid-1 is released from the complex, signal transduction is initiated. In cells overexpressing DnaJA3/Tid-1, excess DnaJA3/Tid-1 interferes with the formation of the active complex, consequently suppressing downstream events ³¹⁸. As already discussed, three alternatively spliced isoforms have been described for the mitochondrial DnaJ/Hsp40 homolog, DnaJA3/Tid-1 that are 480, 453 and 300 amino acids in length, respectively. Amongst them, the two longer isoforms exert opposing biological effects. Isoform 1 (also known as Tid-1(L)) has been found to promote apoptosis, while isoform 2 (also known as Tid-1(S)) suppresses it ^{82, 321}. Moreover, Tid-1(L) is more stable than Tid-1(S) and has been demonstrated to interact with the transcription factors STAT-1 and STAT-3 through its CTD thereby facilitating their association with cytosolic Hsc70/HspA8 ^{85, 322}. Studies by the group of Jiing-Dwan Lee clearly revealed that DnaJA3/Tid-1 negatively regulates the motility and metastasis of breast cancer cells by downregulating the expression of pro-angiogenic IL-8, most likely through diminishing the activity of NF-κB on the promoter of the IL8 gene ³²³. IL-8 functions as a key player in carcinogenesis as it is required for initiation of tumor-associated inflammation and neovascularization ³²⁴. The same group also provide evidence for the suppressive action of upregulated DnaJA3/Tid-1 on the expression of ErbB-2/Her-2 in ErbB-2/Her-2 overexpressing human mammary carcinomas by attenuating ErbB-2-dependent oncogenic extracellular signal-regulated kinase 1/2 (ERK1/2) and mitogen-activated protein kinase 1 (MAPK1) signaling pathways culminating in apoptosis ^{323, 325}. Investigations in recent years indicate an overexpression of the receptor tyrosine kinase ErbB-2/Her-2 in a wide range of solid human tumors and its association with an unfavorable prognosis particularly in breast cancer ^{326, 327, 328}.

The transcription factor NF-κB functions as a key orchestrator in innate immunity and inflammation and has emerged as a crucial tumor promoter ³²⁹. In this respect, DnaJA3/Tid-1 has been noted to associated with the cytoplasmic NF-κB/IκB complex through direct interaction with IκBα/β and the α/β subunits of the IκB kinase (IKK) complex ³³⁰. In addition, upregulated expression of DnaJA3/Tid-1 by using recombinant baculovirus or adenovirus led to inhibition of cell proliferation and induction of apoptosis of human osteosarcoma cells regardless of the p53 expression status.

The anti-apoptotic functions of DNAJs/HSP40s became apparent by the work of Gotoh and colleagues who could show that DnaJB1/Hdj1 and DnaJA1/Hdj2 each complexed to Hsp70 prevents LPS/IFN-γ- and NO-induced apoptosis upstream of cytochrome c release from mitochondria but downstream of CHOP (CCAAT-enhancer binding protein homologous protein) induction through interactions with Bax and inhibition of translocation to mitochondria ²⁸⁸. In a previous study, DnaJB4/ Hlj1 has been characterized as a novel tumor suppressor that inhibits cancer cell-cycle progression, proliferation, invasion and tumorigenesis, and that is significantly correlated with prognosis in non-small cell lung carcinoma (NSCLC) patients ³³¹. More recently, DnaJB4/Hlj1 was found to promote the sensitivity of cancer cells to UV stress-induced apoptosis through enhancing JNK activation and caspase activity. It is cleaved by caspase-3 at a non-typical caspase-3 cleavage site (MEID) at amino acids 125–128, followed by protein degradation during the apoptotic process ³³². Based on these findings one can speculate that a therapeutic strategy aiming to induce expression of DnaJB4/Hlj1 might represent a useful approach in order to improve the outcome of radiotherapy and patient survival.

As already mentioned, DNAJs/HSP40s have been found in the extracellular milieu, either in exosomes (DnaJA1, DnaJA2, DnaJB1, DnaJC7, and DnaJC13) ⁷¹ or in a soluble form upon UPR activation which is known to impact extracellular proteostasis through transcriptional remodeling of the ER proteostasis pathways ¹⁸². In this respect, E. coli DnaJ-derived peptides have been found to induce pro-inflammatory T cell responses whereas peptides derived from human DnaJ induced anti-inflammatory IL-10 production from synovial fluid mononuclear cells ²³⁵. In addition, bacterial DnaJ as well as human DnaJB1/Hdj1, DnaJA1/Hdj2, and DnaJC14/Hdj3 inhibited proliferation of stimulated CD4+ or CD8+ T cells in cultures of peripheral blood mononuclear cells (PBMCs) of RA patients compared to healthy controls ¹⁵. Both DnaJ and DnaJA1/Hdj2 significantly stimulated secretion of anti-inflammatory IL-10 by PBMCs of RA patients, and of pro-inflammatory IL-6 by PBMCs of RA and control groups. DnaJ reduced secretion of pro-inflammatory TNF by both groups of PBMCs implying an increased humoral response to human DNAJ/HSP40 family members DnaJA1/Hdj2 and DnaJC14/ Hdj3 in RA patients ¹⁵. How extracellular residing DNAJs/HSP40s mediated cytokine release from immune cells is far from being answered. Extracellular HSPs are known to bind to a broad spectrum of cell surface receptors including CD14, CD40, the C-type lectin receptor Lrp-1 (CD91/a₂-macroglobulin receptor) as well as TLR-2 and TLR-4 ^{237, 333, 334, 335}. However, a specific receptor for DNAJs/HSP40s has not been identified up to date.