HSP40: Disease Relevance

Aberrant protein aggregation is the key feature of the pathology of most neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS) ^{336, 337, 338}. Exome-sequencing analyses have identified DnaJC13/Rme-8 to bear disease-causing variants for both, PD and essential tremor ³³⁹. Also mutations in DNAJC13, DNAJC6 and DNAJC5 have been implicated in PD ³⁴⁰. However, to what extend mutations in these genes definitively contribute to PD pathogenesis is controversially discussed in the literature. In contrast to previous findings, sequence analyses of the coding exons of the three genes in early-onset PD cases in a Chinese population did not reveal a possible contribution of these genes to the PD pathogenesis ³⁴¹. In any case, multi-center clinical studies will shed more light on the relevance of DNAJ mutations in the pathogenesis of PD.

The key component of protein aggregates found in ALS, a degenerative disease of spinal and cortical motor neurons and the overlapping clinical syndrome of frontotemporal lobar degeneration (FTLD) and other neurodegenerative disorders, is the RNA-binding protein TDP-43 ³⁴². Recent evidence supports that the CTD of TDP-43 harbors features of a Q/N-rich ‘prion domain’ similar to those observed in yeast prions ^{343, 344, 345}. The key regulators of yeast prion aggregation comprise, amongst others, members of the DNAJ/HSP40 and HSP70/HSPA families ³⁴⁶. Investigations by Udan-Johns and co-workers convincingly demonstrated a reversible nuclear aggregation of TDP-43 after heat shock that is mediated by interactions between HSP40/HSP70 chaperone proteins and the C-terminal prion domain ³⁴³. From these findings the authors hypothesize that TDP-43 aggregation is regulated by HSP40/HSP70 availability, clarifying the recurrent appearance of TDP-43 aggregates in degenerative diseases of both, the brain and muscle characterized by defective protein homeostasis. The same group previously reported that mutations in DnaJB6/Mrj lead to dominantly inherited myopathy with protein aggregates containing TDP-43 and DnaJB6/Mrj ³⁴⁷. Moreover, overexpression of DnaJB6/Mrj can suppress heat-induced TDP-43 aggregation and depletion of DnaJB6/Mrj augments TDP-43 aggregation suggesting TDP-43 as being a client protein of DnaJB6/Mrj which facilitates TDP-43 prion-like aggregation in muscle ³⁴⁸.

DnaJB6/Mrj has recently been found to perturb the formation of fibrils by polyglutamine peptides ³⁴⁹ involved in neurodegenerative disorders such as HD ^{350, 351}. DnaJB6/Mrj could also be identified as an efficient inhibitor of amyloid formation by the aggregation-prone peptide (the amyloid-beta peptide, Aβ42, implicated in AD) as a potent inhibitor of the aggregation of Aβ42 ³⁵². Kinetic analysis and immunochemistry experiments revealed that such high efficiency results from the ability of DnaJB6/Mrj to sequester effectively the Aβ42 aggregates that propagate the amyloid conversion, thereby inhibiting their growth and limiting their proliferation potential ³⁵². These data highlight a tremendous feature of members of the DNAJ/HSP40 family in preventing protein misfolding reactions, implying an crucial role of DNAJ/HSP40 chaperones in both, the sequestration of single monomeric unfolded conformations and interactions with aggregated species.

It should be noted that overexpression of HSP70/HSPA chaperones is almost universally protective against toxic aggregates and filamentous proteins whereas knocking out a chaperone infrequently induces aggregate formation and cell death. In this respect, knocking out DnaJC5/Csp-α is unique in that it causes neurodegeneration in several organisms. For instance, deletion of the csp gene in Drosophila leads to defects in synaptic transmission culminating in paralysis and premature death ³⁵³. Deletion of DnaJC5/Csp-α in mice causes rapid and progressive neurodegeneration acompanied by decreased levels of the plasma membrane SNARE protein (SNARE, soluble NSF [N-ethylmaleimide–sensitive factor] attachment protein receptor) SNAP-25, Hsp70, Hsc70 and α-synuclein suggesting a functional association between these mediators ³⁵⁴. In addition to the decrease in SNAP-25 level in Csp-α KO mice, knocking out DnaJC5/Csp-α exclusively enhances the number of ubiquitinated forms of SNAP-25 and Hsc70, suggesting that SNAP-25 is destabilized and subjected to proteasomal  degradation after Csp-α depletion ³⁵⁵. The same study identified SNAP-25 as a client of the DnaJC5(Csp-α)/Hsc70/SGT chaperone machine which preferentially binds to monomeric SNAP-25 but not to assembled SNARE complexes containing SNAP-25. SGT (small glutamine-rich tetratricopeptide repeat-containing protein) is an ubiquitously expressed co-chaperone of Hsc70. The DnaJC5(Csp-α)/Hsc70/SGT chaperone machine facilitates the folding of SNAP-25 and thus promotes SNARE complex assembly and membrane fusion. The function of α-synuclein may be to maintain SNARE complex assembly in a presynaptic terminal during aging ³⁵⁵. For more detailed information on the structure and function of α-synuclein see review by Witt (2013) ³⁵⁶.

Despite their role in neurodegenerative disorders, bacterial and human DNAJs/HSP40s are suspected to contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). The presence of increased levels of antibodies against the E. coli DnaJ has been shown in RA ^{12, 13, 14, 15} with particular high responses to the conserved J-domain of DnaJ ^{13, 15}. Moreover, an upregulation of human DNAJs/HSP40s and significantly increased levels of anti-DnaJA1/Hdj2 and anti-DnaJC14/Hdj3 have been found in the synovial tissue and sera of RA patients ^{15, 357}. 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 RA patients ^{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. Work by the group of Shigeru Kohno convincingly revealed significantly elevated levels of anti-Hsp40 autoantibodies in the periphery of patients 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 it can be hypothesized that autoimmunity against Hsp40 may have a beneficial effect in UC patients by limiting the extent of the disease.

A growing wealth of evidence indicates the pivotal role of DNAJs/HSP40s in tumorigenesis (for a review see Mitra et al., 2009 ¹⁸). In malignantly transformed cells, HSPs enhance cell growth, suppress senescence, and confer resistance to stress-induced apoptosis including protection against cytostatic drugs and radiation therapy ^{18, 26, 358}. With respect to DNAJs/HSP40s, some members of this co-chaperone family have been reported to contribute to numerous aspects of carcinogenesis implying a multi-faceted role of DNAJs/HSP40s in cancer ¹⁸. The study by Oka et al. (2001) was about the first demonstrating elevated levels of HSP40 specifically in human lung cancer specimens ¹⁸⁸. Additionally, significantly elevated levels of anti-Hsp40 autoantibodies could be observed in the periphery of patients with lung cancer suggesting Hsp40 in lung tumor tissue might function as a self-antigen ¹⁸⁸. An upregulated expression of Hsp40 and Hsp70 has been found in gastric and colorectal cancer tissues compared to normal tissues ^{359, 360}. In colorectal cancer, Hsp40 overexpression did not correlate with clinicopathological parameters ³⁵⁹, whereas in gastric cancer low histopathological differentiation was associated with reduced expression of both proteins ³⁶⁰. DnaJC12/Jdp-1 is a type 3 DNAJ/HSP40 which is upregulated in breast tumors in association with the estrogen and progesterone receptor status ²⁵⁹. DNAJC12 is an estrogen target gene whose expression is regulated through estrogen response elements in the corresponding promoter region. The expression of DnaJC12/Jdp-1 might thus function as a marker of the estrogen receptor transactivation activity and might have a predictive value for response to hormonal therapy ²⁵⁹. In renal cell carcinoma (RCC), the cancer-testis antigen DnaJB8/Dj6 contributes to the cancer stem-like cell (CSC) phenotype in RCC ³⁶¹. CSCs are a small population of cancer cells with superior tumor initiating, self-renewal, and differentiation properties. According to Nishizawa et al., overexpression of DnaJB8/Dj6 is more pronounced in CSCs than in the major fraction of RCC cells ³⁶¹. These findings imply a crucial role of DnaJB6/Dj6 in tumor initiation and CSC maintenance rendering it a candidate for CSC-targeting immunotherapy in RCC ³⁶¹. The same group detected a preferential expression of DnaJB8/Dj6 in CSCs/cancer-initiating cells (CICs) derived from colorectal cancer (CRC) cells rather than in non-CSCs/CICs ³⁶².

It should be noted that most cancers have a downregulated expression of DNAJs/HSP40s. In human breast cancer, DNAJB6 is one of the most underexpressed genes in ductal carcinoma compared to normal breast epithelium ³⁶³. Studies by the group of Rajeev S. Samant revealed that the large isoform of DnaJB6/Mrj is lost in invasive ductal carcinoma of the breast ³⁶⁴. Notably, expression of DnaJB6/Mrj reduced growth, migration and motility of cancer cells and was accompanied by a decreased protein secretion profile comprising osteopontin, osteonectin and inducible VGF nerve growth factor. The protein secretion profile of DnaJB6/Mrj-expressing cells also demonstrated enhanced levels of the metastasis suppressor KiSS1 ³⁶⁴. More recently, the same group identified miR-632 as a potentially important epigenetic regulator of DNAJB6 contributing to the downregulation of DNAJB6 in breast cancer and playing a supportive role in malignant progression ²⁶⁸.

DnaJA3/Tid-1 is implicated in the pathogenesis of gliomas ³⁶⁵ as well as in skin cancer ³⁶⁶ and colon cancer ³⁶⁷. In basal cell carcinoma (BCC), the loss of DnaJA3/Tid-1 expression correlates with the loss of differentiation capacity of the neoplastic cells ³⁶⁶. A tumor-associated mutation at the human DNAJA3/TID1 locus gives rise to aberrantly high levels of the Tid-1(L) mutant variant in SF767 glioma cells ³⁶⁵. Adenoviral-mediated delivery of the two major isoforms of DnaJA3/Tid-1,Tid-1(L) and Tid-1(S), into SF767 cells showed differential effects. While Tid-1(S) induced caspase-dependent apoptosis as well as mitochondrial cytochrome c release in DnaJA3/Tid-1 mutant SF767 cells, and caused growth arrest in wild-type DnaJA3/Tid-1-expressing control cells, Tid-1(L) infection had no apparent effect on glioma cell growth ³⁶⁵. In contrast, progression of colorectal cancer has been reported to correlate with an upregulation of DnaJA3/Tid-1 and a loss of polarization of the DnaJA3/Tid-1 expression ³⁶⁷. The work by Kurzik-Dumke also provide evidence that DnaJA3/Tid-1 acts as a ligand of the tumor suppressor adenomatous polyposis coli (APC) underlining its role in tumorigenesis and the evolutionarily conserved Wnt/Wingless (Wg) signaling pathway ³⁶⁷ involved in diverse biological processes, including determination, proliferation, migration and differentiation during embryonic development and adult homeostasis (summarized by Clevers, 2006 ³⁶⁸).

DnaJC15/Mcj is a small mitochondrial-resident DNAJ/HSP40 of 16 kDa which is highly abundant in the heart ⁹⁶ and testis ⁹⁷. A loss of DnaJC15/Mcj expression can be found in the majority of primary human ovarian tumors ⁹⁷. DnaJC15/Mcj expression has been associated with enhanced sensitivity to paclitaxel, topotecan, and cisplatin, implying a critical role in de novo chemoresistance in ovarian carcinoma ⁹⁷. Epigenetic inactivation of DnaJC15/Mcj has been reported as being a crucial event in the development of various malignant paediatric brain tumors ³⁶⁹. Thus, downregulation of DnaJC15/Mcj obviously represents a key feature in malignant progression of certain cancers.

DnaJB4/Hlj1 has been characterized as a novel tumor suppressor whose expression was found to correlate negatively with lung cancer cell invasion ³⁷⁰. In the same study, overexpression of DnaJB4/Hlj1 suppressed cancer cell invasive potential through upregulation of E-cadherin expression in vitro. DnaJB4/Hlj1 expression is downregulated in the majority of NSCLC patients correlating with prognosis ³³¹. DnaJB4/Hlj1 inhibits invasion, motility, growth, tumorigenesis, and cell-cycle progression via a novel  STAT-1/p21^Waf-1 pathway independent of p53 and interferon ³³¹. Interestingly, NSCLC patients with high DnaJB4/Hlj1-expressing tumors had reduced cancer and prolonged overall survival than those with low-expressing tumors. 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.

The type 1 DNAJ/HSP40 family member DnaJA1/Hdj2 has been characterized to be implicated in the resistance of glioblastomas to radiotherapy ³⁷¹. DnaJA1/Hdj2 is subjected to farnesylation at specific Ser residues for membrane anchorage ²⁸⁹ and can be used as surrogate marker for farnesylated proteins ³⁷². DnaJA1/Hdj2 has also been targeted as a biomarker for pancreatic cancer to evaluate the effects of farnesyl protein transferase inhibitors ^{372, 373}. Of note, DnaJA1/Hdj2 has been shown to be downregulated 5-fold in a genomics analysis of pancreatic cancer cells relative to normal healthy cells and cells undergoing pancreatitis ³⁷⁴. The potential importance of DnaJA1/Hdj2 to pancreatic cancer was demonstrated by stress response cell-based assays using cell lines overexpressing DnaJA1/Hdj2 ³⁷⁵. In this study, DnaJA1/Hlj2 was shown to be involved in a stress response signaling pathway that suppresses the anti-apoptosis state found in pancreatic cancer cells.

Using RNA samples isolated from tumor specimens used for representative oligonucleotide microarray analysis (ROMA), Ivanov and collaborators showed a reduced expression of DNAJA1 in the short-term recurrence cohort of patients with pleural malignant mesothelioma ³⁷⁶. Based on these observations one can speculate that the downregulation of the DNAJA1 gene may be associated with early stages of the disease and its adverse prognosis.

DnaJB9/ERdj4/Mdg1 belongs to the class of ER-resident DNAJs/HSP40s and is upregulated after exposure to stress and during angiogenesis ³⁷⁷. As demonstrated by Pröls et al., DnaJB9/ERdj4 is involved in the control of cell cycle arrest occurring during terminal cell differentiation and under stress conditions ³⁷⁷. Injection of DnaJB9/ERdj4-overexpressing Rous sarcoma virus-transformed embryo hamster fibroblasts (HET-SR) into animals revealed that overexpression of the DNAJB9/MDG1 gene significantly reduced the metastatic activity of HET-SR cells without modifying cell proliferation and motility ³⁷⁸.

Apart from their role in carcinogenesis, DNAJs/HSP40s are implicated in the pathogenesis of microbial infections. Recent investigations clearly demonstrate the pivotal role of DnaJ in the pathogenesis of pneumococcal infections ¹¹.