HSP60: Species Variation

IHC staining of Listeria infected mice spleens 6 days after infection with L-monocytogenes, using Anti-Hsp60 (clone: LK2)

The 60 kDa chaperonin family has homologs in many eukaryotes like Danio rerio, D. melanogaster, yeast, and plants as well as many eubacteria and Archaea. Chaperonins represent a diverse family of molecular chaperones that are present not only in mitochondria as well as cytoplasm of all eukaryotes and eubacteria but also in plastids. Plastidic Cpn60 appears to be distinct from homologous proteins in bacteria and mitochondria in that it is composed of two distinct subunit types, α and β ^{93, 94}. An examination of the Arabidopsis thaliana genome sequence led to the identification of 29 predicted genes with the potential to encode members of the chaperonin family of chaperones (Hsp60, Cpn60) ⁹⁵. Amongst them, eleven Arabidopsis genomic sequences were identified as having potential to encode Cpn60 proteins. Six of these appeared to encode plastidic Cpn60 subunits, including 2 α-subunits and 4 β-subunits (Table 2). Each harbors a putative transit peptide sequence. A seventh genomic sequence appeared to represent a plastidic Cpn60 β-subunit pseudogene. The remaining four genomic sequences comprise three genes encoding mitochondrial Cpn60 polypeptides (Table 2) and an apparent mitochondrial protein pseudogene ⁹⁵. Hsp60-2 is the longest isoform with 585 amino acids, while Hsp60-3B is composed of 572 amino acids and Hsp60 is composed of 577 residues, respectively. The primary structures of plastidic α- and β-subunits and mitochondrial subunits show a high grade of divergence. While α- and β-subunits of A. thaliana Cpn60 have an average 51% identity to each other, they show an approximately 45% identity to the mitochondrial Cpn60 protein. These intersubunit identities are comparable to those found between prokaryotic Cpn60 homologs and any of the eukaryotic subunits ⁹⁵. The Cpn60A paralogs are approximately 57% identical in peptide sequence whereas three Cpn60B paralogs (Cpn60B1 to -3) show an average 88% identity. In contrast, the fourth paralog, Cpn60-B4 is only 60% identical to each of the other three paralogs ⁹⁵. Genes encoding the distinct plastidic subunit types obviously originated from a gene duplication event occurring in the plastid lineages ^{84, 96, 97}. Therefore, α- and β-genes can be considered as being paralogs. It is worth mentioning that genes encoding plastidic and mitochondrial homologs derived from independent endosymbiotic events.

The thorough search of the genome sequence of A. thaliana also revealed nine predicted coding regions distributed among chromosomes 1, 3, and 5 encoding the CCT proteins CCT-α, -β, -γ, -δ, -ε, -η, – θ, -ξ in which CCT-ξ is encoded by two regions ⁹⁵. A similar arrangement exists not only in humans but also in mice where a second CCT-ξ subunit is expressed in testes ^{60, 86}.

The genome of the unicellular green alga Chlamydomonas reinhardtii encodes four Hsp60 chaperones, termed Cpn60A, -B1, -B2, and -C. Cpn60A as well as Cpn60-B1/-2 are targeted to chloroplasts, while Cpn60C is targeted to mitochondria. CPN60Bs from higher plants may form homo- or hetero-oligomeric stacks of two heptameric rings in conjunction with Cpn60A. Similar to higher plants, C. reinhardtii harbors divergent plastidic Cpn60A and Cpn60B subunits ⁹⁸. Cpn60A shares only ≈50% similarity with chloroplast-targeted CPN60Bs wherease Cpn60-B1 shows a 79% identity to Cpn60-B2 and vice versa. Cpn60C is targeted to mitochondria where it forms stacks of two homo-oligomeric heptameric rings. For complete functionality, Cpn60C requires mitochondrial Cpn10 in an ATP-dependent manner. Since the primary structures of the two Chlamydomonas β-subunits only show a ≈25% divergence, it can be hypothesized that the two Cpn60-B subunits have been selectively maintained for a considerable period of time ⁹⁸. It is interesting to note that, unlike plastidic chaperonin transcripts in higher plants, plastidic Cpn60 mRNA levels from C. reinhardtii rapidly increase in response to heat shock ⁹⁸.

Members of the HSP60 family of chaperones are generally not induced by heat shock in Drosophila tissues ¹³. except in Malpighian tubules ⁹⁹. The Berkeley Drosophila Genome Project has reveled four Hsp60 genes, named as Hsp60A, Hsp60B, Hsp60C, and Hsp60D, respectively ¹⁰⁰. Previous studies have shown that the Hsp60A, Hsp60B, and Hsp60C proteins have distinct functions in normal development. The Hsp60A gene is expressed in all cell types of Drosophila and is essential from early embryonic stages ¹⁰¹, while the Hsp60B gene is expressed only in testis, being essential for sperm individualization ¹⁰². Studies by the group of Surajit Sakar and Subash Lakhotia confirmed the crucial role of Hsp60C in proper tracheal development and in early stages of spermatogenesis ¹⁰⁰ as well as in oogenesis, especially in organization and maintenance of cytoskeletal and cell adhesion components ¹⁰³. Hsp60D encodes a protein harboring sequence homology with bacterial GroEL and human HspD1. Transcripts of this gene are ubiquitously found in larval tissues such as imaginal discs and salivary glands ¹⁰⁴. Recent data suggest an essential contribution of Hsp60D to caspase-mediated apoptosis in D. melanogaster through its interaction with Drosophila inhibitor of apoptosis 1 (DIAP-1) ^{104, 105}.

The GroEL and GroES proteins and regulation of their expression have been studied in most detail in E. coli. The groES and groEL genes form the groESL (GroE) operon essential for E. coli viability at any temperature ¹⁰⁶. The groESL operon of E. coli and other bacteria analyzed are arranged in the order promoter–groES–groEL ¹⁰⁷. Several bacteria harbor a further, monocistronic groEL operon including Synechocystis sp. ¹⁰⁸, Synechococcus vulcanus ¹⁰⁹, Rhizobium meliloti ¹¹⁰, and the cyanobacterium Anabaena sp. ¹¹¹.

Many bacteria have multiple copies of the groEL gene which are active under different environmental conditions consequently leading to the expression of various Hsp60 homologs. This includes a number of Gram-positive bacteria such as M. leprae, S. albus, M. tuberculosis and cyanobacteria ^{108, 112, 113, 114} (see Table 2) as well as several Gram-negative bacteria also expressing multiple Hsp60 homologs ^{110, 115, 116}. Unlike all other prokaryotes, the nitrogen fixing soybean root nodule bacterium, Bradyrhizobium diazoefficiens (old name B. japonicum), possesses a multigene family consisting of seven very similar groEL-like genes ⁶⁴. These homologs are expressed to different degrees by differentially regulated groESL operons. Amongst them, one family member (GroEL-3) is induced by a mechanism that does not involve the well-known heat shock response ¹¹⁶. Interestingly, its synthesis is co-regulated at the transcriptional level with the process of symbiotic nitrogen fixation via the oxygen-responsive transcriptional activator NifA and the σ-factor RpoN (afM4).

Unlike the homo-oligomer GroEL, archaeal chaperonins are often composed of several distinct, but homologous subunits. The archaeal chaperonins belong to the group II chaperonins (called the thermosome, TF55 or CCT complex – analogous to the eukaryotic molecules), and are strongly induced by heat shock ⁶¹. Archaea posses one to three homologous chaperonin-encoding genes (see also Table 2) except for Methanosarcina acetivorans which expresses five genes ⁶⁵. Phylogenetic analyses imply that multiple independent duplications in archaeal chaperonin genes occurred in archaeal lineages facilitating the presence of multiple chaperonin genes ^{97, 117}. In archaeal genomes, duplicate chaperonin genes (paralogs) are often more similar to each other than to those in other archaea, suggesting recent (lineage-specific) duplications ⁸⁴. As stated by Archibald and collaborators, the persistence of chaperonin paralogs in multiple archaeal lineages may involve a process of co-evolution, where chaperonin heterogeneity is altered independently of selection on function ¹¹⁷. Although archaeal chaperonins are strongly induced by heat in vitro ¹¹⁸, their in vivo function remains unclear. In a recent study, the group of Igor K. Lendnev observed that Hsp60 from hyperthermophile Pyrococcus furiosus binds to and degrades insulin fibrils. Prolonged incubation with the chaperonin was found to produce large amorphous aggregates with polydisperse topologies ¹¹⁹. Based on these findings the authors postulate a novel approach to the disassembly of refractory protein aggregates under physiological conditions. However, a contribution of archaeal chaperonins to ribosomal RNA processing is also quite likely ^{120, 121}.

Compared to archaeal chaperonins, the eukaryotic CCT chaperonin complex TRiC is even more hetero-oligomeric ^{52, 122}. Previous comparative sequence analyses identified this complex in the common ancestor of animals and fungi ⁸³. The results by Archibald and colleagues ascribe the origin of the CCT gene duplications to the common ancestor of animals, fungi, plants, parabasalids, and diplomonads, and likely to the common ancestor of all extant eukaryotes ⁸⁴. Sequence comparisons of CCT genes in mouse, encoding the subunits of TRiC, confirmed the existence of eight distinct subunit species (α, β, γ, δ, ϵ, η, θ, and ζ), each thought to occupy a unique position in the octameric CCT rings ^{52, 83, 85, 123, 124}. As suggested by the group of W. Ford Doolittle ⁸⁴, the divergent nature of these genes, together with the discovery of clear yeast orthologs (see Table 2) to each of the mouse subunits ^{82, 83, 125}, might imply an ancient paralogy within eukaryotes. Apart from the classical TRiC-interacting cytoskeletal proteins actin and tubulin, a genome-wide analysis of chaperonin function in yeast identified a third connection between TRiC and the cytoskeleton, the septin ring whose assembly and function has been shown to clearly depend on the interaction with TRiC ¹²⁶. S. cerevisiae also expresses the mitochondrial Hsp60 which exhibits striking amino acid sequence similarity to its counterparts in humans, plants, and bacteria indicating a high degree of conservation ¹²⁷. The yeast Hsp60 protein is encoded by the HSP60 gene (also known as MIF4, CPN60 or MNA2) in which mutations give rise to phenotypes characterized by mitochondrial dysfunction ¹²⁸.

Higher eukaryotes such as C. elegans and zebrafish (D. rerio) also express multiple HSP60 genes (Table 2). A genetic approach revealed an upregulated expression of hspd1 (nbl) in blastema cells during zebrafish fin regeneration ³². The study also highlighted the pivotal role of HspD1 in blastema formation and viability in D. rerio. Mutations in CCT-3 (Cct-γ) lead to the no tectal neuron phenotype in zebrafish, interfering with retinotectal development ¹²⁹. A growing body of evidence now indicates that HspD1 is critically involved in gametogenesis of C. elegans ¹³⁰, and in spermatogenesis of rats ^{131, 132} and men ¹³³. In the latter, a significant reduction in HspD1-immunopositive spermatogonia could be observed in testes with maturation arrest of spermatogenesis at the level of primary spermatocytes compared with testes exhibiting normal spermatogenesis ¹³³.

Table 2: HSP60s of various pro- and eukaryotic organisms