HSP40: Isoforms

The DNAJ/HSP40 family represents the largest HSP family in humans comprising 50 family members, even though the exact number is still unclear ⁸. All eukaryotes have several DNAJ/HSP40 genes leading to the expression of compartment-specific isoforms fulfilling organelle-specific functions. The single members of this family differ from each other by expression level, molecular mass, amino acid constitution, subcellular location, and tissue distribution. Historically, DNAJs/HSP40s have been classified into three subtypes (1, 2 and 3; also referred as to subfamily A, B, and C ^33,34,35). According to Kampinga and colleagues, the human genome encodes four type 1, 14 type 2 as well as 23 type 3 DNAJ/HSP40 proteins and nine J-like proteins (Table 2) ⁶³. Amongst them is DnaJC5G whose J-domain harbors a 16 amino acid residue insertion, as well as DnaJC6, 8, 13, 15, 19, and 20 with poorly conserved secondary structure elements within their J-domains. Unlike DnaJC5G and DnaJC8, all of the remaining J-domain-containing proteins have been proposed to act as true DNAJ/HSP40 chaperones (for a review see Qiu et al., 2006 ⁸). The human genome possesses many DNAJ pseudogenes lacking major parts of the coding sequence and showing only partial homology to DNAJ. In addition, three J-like proteins were identified in yeast that contain regions closely resembling a J-domain, but in which the HPD motif is non-conservatively replaced ¹⁰. Genome-wide analysis revealed at least seven HSP40/DNAJ proteins bearing partially conserved J-like domains in humans ⁸.

The ER-resident DNAJ/HSP40 (also termed ERdj) proteins comprise seven members (DnaJC1/ERdj1/Mtj1, DnaJC23/ERdj2/Sec63, DnaJB11/ERdj3/HEDJ, DnaJB9/ERdj4/Mdg1, DnaJC10/ERdj5/JPDI, DnaJC3/ERdj6/p58, and DnaJB14/EGNR9427), four (DnaJC1/ERdj1, DnaJC23/ERdj2, DnaJB9/ERdj4, and DnaJB14/EGNR9427) of which have been reported as being integral membrane proteins ⁷². DnaJB11, DnaJC1, and DnaJB11 (ERdj1–3) as well as DnaJC3/ERdj6 have been linked to folding of nascent proteins, often at the translocation channel ^{68, 72, 73}. In contrast, DnaBJ9/ERdj4 and DnaJC10/ERdj5 play important roles in the ER-associated degradation of unfolded secretory proteins ^74,75,76. Interestingly, DnaJB9/ERdj4 has been found as being membrane-associated ⁷⁷, whereas DnaJC10/ERdj5 represents a soluble protein ⁷⁴. DnaJB9/ERdj4 is a 26 kDa type 2 DNAJ/HSP40 which translocates to the nucleolus upon heat shock ¹⁸. DnaJB11/ERdj3 as well as DnaJC3/ERdj6 and DnaJC10/ERdj10 are somewhat unusual among the DNAJ/HSP40 family in that they form intramolecular disulfide bonds. DnaJB11/ERdj3 and DnaJC10/ERdj10 are glycosylated and, in the case of DnaJB11/ERdj3, contain high-mannose Endo H-sensitive carbohydrates ⁷⁸.

Subfamily B bears the most widely expressed and most heat-inducible DnaJ/Hsp40, cytosolic DnaJB1/Hdj1 (Figures 2+3). As demonstrated by different groups, DnaJB1/Hdj1 is also present in extracellular vesicular exosomes ^{71, 79}. Two isoforms as the result of alternative splicing have been described for this protein differing by 100 amino acid residues. The DNAJB1 gene is conserved in chimpanzee, dog, cow, mouse, rat, chicken, zebrafish, S. cerevisiae, Kluyveromyces lactis, Ashbya gossypii, Schizosaccharomyces pombe, Magnaporthe oryzae, Neurospora crassa, rice, and frog. 99 organisms have orthologs with the human DNAJB1 gene. A further member of this subfamily, DnaJB13/Tsarg- 6 is highly homologous to DnaJB5 with a 45% amino acid identity, and bears an HPL tripeptide in place of the HPD motif that plays a crucial role in J-domain functions ⁸. DnaJB10 is actually the mouse ortholog of human DnaJB2 and therefore missing amongst the human HSP40/DNAJ family members.

DNAJs/HSP40s can be expressed tissue-specifically or universally in all tissues in multicellular organisms. For instance, DnaJB2/Hsj1 has been reported to be predominantly expressed in human brain ⁸⁰. The tissue-specific expression can be achieved by alternative splicing of the corresponding DNAJ mRNA in certain cases. In this regard, two transcript variants encoding DnaJA1/Hdj2 have been identified for the DNAJA1 gene as the result of alternative splicing leading to the expression of two DnaJA1/Hdj2 isoforms differing from each other by 95 amino acids ⁶⁹. While one of the alternatively spliced isoforms was found to reside in the nucleus, indicating that it acts as a nuclear co-chaperone, the other isoform was observed throughout the cell most likely due to the elimination of a putative nuclear localization signal sequence ⁶⁹. Moreover, isoform 1 of DnaJA1/Hdj2 was found as being highly expressed in brain and other tissues while the other alternatively spliced isoform was highly abundant in testis and sperm ⁷⁰. The DNAJA1 gene is conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, zebrafish, and frog. 144 organisms have orthologs with the human DNAJA1 gene. Nuclear DnaJA1/Hdj2 translocates to the cytoplasm and cell membrane after heat shock through a non-classical and lipid-dependent pathway ⁸¹. DnaJA1/Hdj2 contains an N-terminal J-domain and is upregulated at both, mRNA and protein levels upon various stress and mitogenic stimulations ⁸¹.

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. Isoform 1 and 2 only differ at their C-termini ⁸² while isoform 3 lacks residues 454-480 and harbors multiple amino acid substitutions ⁸³. Although the vast majority of DnaJA3/Tid-1 is located to mitochondria ^{82, 84}, its reported functions and protein interactions are predominantly non-mitochondrial ⁸⁵.

DnaJC6/auxilin and DnaJC26/auxilin-2 are further prominent representatives of the huge DNAJ/HSP40 family that play an eminant 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. While DnaJC6/auxilin is significantly more abundant in brain as compared to non-neural tissues ⁹³, DnaJC26/auxilin-2 is ubiquitously expressed with the highest expression levels being found in testis ⁹⁴. Notably, two alternatively spliced isoforms have been found in humans differing from each other by 79 amino acid residues.

DnaJC13/Rem-8 is the largest member of subfamily C with a molecular mass of 254 kDa. It is tightly associated with microsomal membranes and co-localizes with markers of the endosomal system ⁹⁵. DnaJC13/Rem-8 is crucially involved 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. DnaJC13/Rem-8 has also been identified in human urinary exosomes ⁷¹.

DnaJC15/Mcj is a small mitochondrial-resident DNAJ/HSP40 of 16 kDa which is highly abundant in the heart ⁹⁶ and testis ⁹⁷. It is a type 3 DNAJ/HSP40 family member and bears a C-terminal J-domain. Table 2 enumerates the most common members of the DNAJ/HSP40 family.