HSP40: Function

Members of the DNAJ/HSP40 family have been preserved during evolution and play essential roles in gene expression and translational initiation, folding and unfolding as well as translocation and degradation of proteins. All eukaryotes have several DNAJ/HSP40-encoding genes leading to the expression of tissue- or compartment-specific isoforms fulfilling site-specific functions. DNAJs/HSP40s are chaperones by themselves as they bind to exposed hydrophobic residues of unfolded and nascent polypeptides and share common substrates with HSP70s ^{47, 66, 195,196,197}. In most cases, DNAJs/HSP40s are homodimeric proteins characterized by the presence of the highly conserved J-domain facilitating their interaction with HSPA/HSP70 family members ¹⁹⁸ via binding to the HSP70 N-terminal ATPase domain (Figure 4) ^{55, 199, 200}. This interaction not only regulates and stimulates the intrinsic ATPase activity of HSP70s but also the recruitment of substrate proteins ²⁰¹. DNAJs/HSP40s additionally bind to the C-terminal EEVD-motif present in eukaryotic cytosolic HSP70s via their C-terminal peptide-binding domain ^{64, 202, 203}. ATP hydrolysis requires the presence of nucleotide exchange factors (NEFs) such as GrpE and Bag-1 mediating the dissociation of ADP and the subsequent binding of ATP which governs substrate release from HSP70s and sets back the Hsp70/DnaK chaperone cycle ^204,205,206. Since ATP hydrolysis represents an essential step in the activation of HSP70s, DNAJ/HSP40 family members de facto account for the activity of HSP70s by stabilizing their interplay with substrate proteins. Moreover, DNAJs/HSP40s are implicated in the HSP90 chaperone pathway in conjunction with HSP70 thus cooperating in the folding of numerous substrate proteins in the cytosol of eukaryotes ²⁰⁷ The HSP90 chaperone pathway is also crucially involved in the functional activation of the steroid hormone receptor activation cycle ^{196, 197} (see also Hsp90 Scientific Resource for more details). While numerous DNAJs/HSP40s have been found to bind client proteins directly, others are able to target HSP70 activity to clients at precise subcellular locations thereby delivering specific clients to HSP70 and directly determining their fate (for a review see Kampinga and Craig, 2010 ⁷).

DNAJs/HSP40s are generally grouped in at least three subtypes/subfamilies (also see Family Members). Whereas subtypes 1 and 2 are considered generic co-chaperones, as judged by their ability to bind a broad spectrum of substrates, members of subtype 3 often deliver specific substrates or confer specific cellular localization to DNAJ/HSP40 chaperones ²⁰⁸. In this respect, DnaJC6/auxilin complexed to HspA8/Hsc70 has been characterized to mediate the dissociation of clathrin from the coat of clathrin-coated vesicles as an essential stage in the recycling of endocytic coated vesicles ²⁰⁹. DnaJC7/Tpr-2 containing two chaperone-binding TPR domains has been proposed to facilitate the retrograde transfer of substrate proteins from Hsp90 onto Hsp70 ²¹⁰.

Approximately 30% of de novo-synthesized polypeptides are translocated into the lumen of the ER for folding, oligomerization and subsequent segregation into more distally located compartments (for a review see Qiu et al., 2006 ⁸). Folding and correct assembly of these proteins as well as prevention of their aggregation are mediated by a variety of ER-resident DNAJs/HSP40s ¹³². In humans, seven types of ER-resident DNAJs/HSP40s have been found, while yeast expresses five DNAJs/HSP40s in the ER ¹⁰. Amongst them, DnaJB11/ERdj3 has been shown by Shen and Hendershot as being a soluble ER luminal glycoprotein and a component of unassembled Ig heavy chain/HspA5(BiP) complexes ²¹¹. In addition to its association with unassembled Ig heavy chains, DnaJB11/ERdj3 was shown to bind directly to a number of nascent unfolded and mutant secretory pathway protein substrates, implying its role as a co-factor for HspA5/BiP functions in protein folding and assembly ²¹¹.

A key step for cells in responding to environmental stressors is the phosphorylation of the eukaryotic translation initiation factor eIF2α ²¹² which is crucially involved in the unfolded-protein response (UPR). The UPR which is activated in response to the loss of ER/Ca²⁺ homeostasis and/or the accumulation of misfolded, unassembled, or aggregated proteins in the ER lumen involves both, transcriptional and translational regulation. In this respect, investigations by van Huizen and co-workers identified DnaJC3/p58^IPK as an important component of a negative feedback loop used by cells to block eIF2α signaling thus impairing the UPR ²¹³. DnaJC3/p58^IPK functions as an inhibitor of the eIF2α kinase protein kinase R-like ER kinase (PERK), which is activated during UPR and viral infection in order to attenuate global protein synthesis ²¹⁴. DnaJC3/p58^IPK forms a ternary complex with HspA8/Hsc70 and DnaJ/Hsp40 that is disrupted during virus infection accompanied by the dissociation of DnaJ/Hsp40 from the complex ²¹⁵. The activation of DnaJC3/p58^IPK after viral infections or UPR highlights its role in a broad panel of stress responses ²¹⁵.

Certain DNAJs/HSP40s are key players in regulating protein translation by forming the stable ribosome-associated complex (RAC) in conjunction with HSP70s ¹⁴⁹. Beside their tremendous role in translation, ribosomes also contribute to the folding of nascent polypeptides. Specialized chaperones and co-chaperones associate with nascent polypeptides and thus link translation to protein folding. In yeast, the Hsp70 homologs Ssb1 and Ssb2 associate with ribosomes and are capable of binding nascent polypeptide chains that are released from the organelles indicating an direct effect on protein translation ²¹⁶. Co-chaperones such as the DNAJs/HSP40s Sis1 and Zuo1 that are also associated with ribosomes might contribute to translation initiation ^217,218,219. Accordingly, the human Zuo1 ortholog DnaJC2/Mpp1 has been noted to recruit HspA8/Hsc70 to nascent polypeptide chains as they exit the ribosomes ²²⁰.

Several studies underline the role of DNAJs/HSP40s in protein translocation. For instance, DnaJC1/ERdj1 bears a ribosome-binding site ²²¹ while DnaJC23/ERdj2 can be cross-linked to the Sec61 component of the translocon ²²² composed of Sec61 and the ER membrane-associated subcomplexes Sec62 and Sec63, indicating a possible contribution to protein translocation. As already mentioned, DnaJC6/auxilin and DnaJC26/auxilin-2 play an eminent role in endocytosis by uncoating of clathrin-coated vesicles in conjunction with Hsc70 ^{86, 87}. DnaJC6/auxilin bears a C-terminal J-domain characterized by the presence of an atypical loop ^{88, 89}, a central clathrin-binding domain mediating clathrin polymerization ^{90, 91} as well as an N-terminal PTEN-like domain enabling binding of phosphatidylinositol 4,5-bisphosphate ⁹². DnaJC13/Rem-8 is a further DNAJ/HSP40 family member which is implicated in endocytosis. As demonstrated by Girard et al., DnaJC13/Rem-8 plays a critical role in the trafficking of the cation-independent mannose 6-phosphate receptor between the trans-Golgi network and endosomes and thus in the sorting of lysosomal enzymes ⁹⁵.

DNAJs/HSP40s target proteins for proteasomal degradation in the cytosol by preventing their aggregation. DNAJ/HSP40 co-chaperones such as yeast Scj1, Jem1, and Ydj1 as well as human DnaJB2/Hsj1 have been found to assist the cell to re-translocate misfolded proteins from a certain  compartment (e.g. the ER) to the cytosol ^223,224,225. In this respect, membrane-bound DnaJA1/Hdj2 was detected to promote ubiquitination of cystic fibrosis transmembrane conductance regulator (CFTR) in conjunction with HspA8/Hsc70 and its co-chaperone CHIP (C-terminal Hsp70-interacting protein; possessing an intrinsic E3 ubiquitin ligase activity) ²²⁶.

A wealth of evidence demonstrates that folded proteins are clients of DNAJs/HSP40s. Binding of folded proteins mediates the “remodeling” of large multiprotein complexes that impact the balance of protein/protein interactions ⁷. This destabilization function has originally been discovered in E. coli for DnaK and DnaJ. Both HSPs regulate the destabilization of the λP protein in the initiation complex culminating in the activation of the DNA helicase DnaB ^{37, 227}. In eukaryotes, DnaJC21/Jjj1 and DnaJC6/auxilin are both co-chaperones facilitating the destabilization of protein/protein complexes. While DnaJC21/ Jjj1 is crucially involved in the assembly of the 60 S proteasomal subunit ^{228, 229}, DnaJC6/auxilin plays an eminent role in the uncoating of clathrin-coated vesicles in conjunction with Hsc70 (see above) ^{86, 87}. DnaJC20/Hsc20 is an example of a co-chaperone which facilitates partial unfolding of its client thereby enabling formation of [Fe-S] clusters required for the functionality of a broad panel of proteins ^{230, 231}. For more detailed information see Kampinga and Craig (2010) ⁷.

Some DNAJs/HSP40s have evolved J-domain-independent functions that impact the stability of protein complexes and the prevention of their aggregation, respectively ⁷. While type 1 DNAJs/HSP40s work independently of HSP70s for polypeptide binding and preventing aggregation, type 2 family members are able to bind to polypeptides irrespective of HSP70s but must be associated with HSP70s to prevent aggregation ¹⁴¹. However, type 3 DNAJs/HSP40s are strictly dependent on the presence of an HSP70 chaperone for complete functionality ^{5, 141}. In this context, DnaJB6/Mrj and DnaJB8/Dj6 are capable of preventing the aggregation of polyglutamine proteins irrespective of the presence of their J-domain ²³². The work by Hageman and colleagues further revealed that a C-terminal serine-rich (SSF-SST) region and the C-terminal tail are essential for this function. In addition, the SSF-SST region was shown to be involved in substrate binding, formation of polydisperse oligomeric complexes, and interaction with several histone deacetylases including HDAC-4, HDAC-6, and Sirt-2. As blockage of HDAC-4 reduced DnaJB8/Dj6 function these data provide a functional link between HDACs and DNAJs in suppressing cytotoxic protein aggregation ²³².

In addition to their intracellular location, 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. Such remodeling processes downregulate the release of misfolded, aggregation-prone proteins during ER stress. Genereux and collaborators most recently demonstrated an UPR-induced upregulation and secretion of DnaJB11/ERdj3 into the extracellular environment which augments the extracellular proteostasis capacity ¹⁸². Remarkably, DnaJB11/ERdj3 was found as being co-secreted in a stable complex with misfolding-prone protein clients thus connecting intracellular and extracellular proteostasis capacity during ER stress ¹⁸². In this context, the non-classical molecular chaperone Hsp47, also known as serpin H1, which is essential for biosynthesis and secretion of collagen molecules, has been identified in the sera of rheumatoid arthritis (RA) patients where it might function as an indicator of cartilage destruction in RA ^{183, 184}. Elevated serum levels of Hsp47 were also present in patients with mixed connective tissue disease ¹⁸⁵, acute exacerbation (AE) of idiopathic pulmonary fibrosis ¹⁸⁶ or acute idiopathic interstitial pneumonias ¹⁸⁷ suggesting that high levels of Hsp47 may be useful blood markers of these disorders.

Following environmental stress, expression of DNAJs/HSP40s is dramatically upregulated in order to promote cell survival in the face of endogenous or exogenous injury. In this regard, exposure of cells to the chemicals ibuprofen, indomethacin, ZnSO₄, and 8-hydroxy-quinoline induced expression of Hsp40/DnaJB1 with concomitant translocation to the nucleus ¹⁹¹. Moreover, the ER stress gene DNAJC10/ERDJ5 was found to be induced in neuroectodermal tumor cells by the retinoid analog fenretinide ¹⁹², which has an increasingly important profile as a cancer preventive and chemotherapeutic drug ¹⁹³.

Chemical stress also induces release of DNAJs/HSP40s into the extracellular milieu. As demonstrated by Saito and colleagues, treatment of cells with acrylamide caused necrotic and thus irreversible cell death accompanied by the release of several HSPs including DNAJs/HSP40s ¹⁹⁴. Since extracellular residing HSPs act as danger signals for the immune system ^{194, 233}, these stress proteins have been added to the list of ‘alarmins’. Endogenous alarmins and exogenous pathogen-associated molecular patterns (PAMPs) both comprise the group of danger-associated molecular patterns (DAMPs) ²³⁴. HSPs are considered to be a “danger signal” for the immune system because of their ability to induce inflammatory phenotypes in innate immune cells. In this regard, T cell responses to E. coli DnaJ-derived peptides were found as being eminently pro-inflammatory whereas peptides derived from human DnaJ induced anti-inflammatory interleukin-10 (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. In general, extracellular HSPs are considered as molecules with immunomodulatory functions ^{236, 237}, either as cross-presenters of immunogenic peptides via MHC antigens ^{238, 239} or in a peptide-free version as chaperokines ²⁴⁰ or stimulators of innate immune responses ²⁴¹. DNAJs/HSP40s also show potent cytokine activity by upregulating the expression of inflammatory cytokines in human monocytes and can thus be added to the list of chaperokines and immune modulators.

As stated in the section Localization, significantly elevated levels of anti-Hsp40 autoantibodies could be observed in the periphery of patients with lung cancer ¹⁸⁸ and those with ulcerative colitis (UC) campared to normal individuals ¹⁸⁹. In UC patients with inactive disease, those with proctitis or left-sided colitis harbored higher titres of anti-Hsp40 autoantibodies than those with total colitis. From these data the authors suggest that autoimmunity against Hsp40 may have a beneficial effect in UC patients by limiting the extent of the disease. Elevated levels of anti-DnaJ, anti-DnaJA1/Hdj2, and anti-DnaJC14/Hdj3 serum antibodies could also be detected in RA patients providing evidence for an immunomodulatory function of DNAJs/HSP40s in RA ^{15, 190}.