hsp70 genes in the human genome: Conservation and differentiation patterns predict a wide array of overlapping and specialized functions

Abstract

Background: Hsp70 chaperones are required for key cellular processes and response to environmental changes and survival but they have not been fully characterized yet. The human hsp70-gene family has an unknown number of members (eleven counted over ten years ago); some have been described but the information is incomplete and inconsistent. A coherent body of knowledge encompassing all family components that would facilitate their study individually and as a group is lacking. Nowadays, the study of chaperone genes benefits from the availability of genome sequences and a new protocol, chaperonomics, which we applied to elucidate the human hsp70 family.

Results: We identified 47 hsp70 sequences, 17 genes and 30 pseudogenes. The genes distributed into seven evolutionarily distinct groups with distinguishable subgroups according to phylogenetic and other data, such as exon-intron and protein features. The N-terminal ATP-binding domain (ABD) was conserved at least partially in the majority of the proteins but the C-terminal substrate-binding domain (SBD) was not. Nine proteins were typical Hsp70s (65-80 kDa) with ABD and SBD, two were lighter lacking partly or totally the SBD, and six were heavier (>80 kDa) with divergent C-terminal domains. We also analyzed exon-intron features, transcriptional variants and protein structure and isoforms, and modality and patterns of expression in various tissues and developmental stages. Evolutionary analyses, including human hsp70 genes and pseudogenes, and other eukaryotic hsp70 genes, showed that six human genes encoding cytosolic Hsp70s and 27 pseudogenes originated from retro-transposition of HSPA8, a gene highly expressed in most tissues and developmental stages.

Conclusion: The human hsp70-gene family is characterized by a remarkable evolutionary diversity that mainly resulted from multiple duplications and retrotranspositions of a highly expressed gene, HSPA8. Human Hsp70 proteins are clustered into seven evolutionary Groups, with divergent C-terminal domains likely defining their distinctive functions. These functions may also be further defined by the observed differences in the N-terminal domain.

Figure 1

Phylogenetic tree of the 17 human Hsp70 proteins encoded in the genes studied in this work. The seven distinct evolutionary groups supported by bootstrap values (1000 samples) equal or greater than 85% are indicated by brackets to the right. Alignments were obtained with Clustal W [42] and the evolutionary tree was obtained using the neighbor-joining algorithm [38] with the distance transformation method of Kimura [39], as implemented in Clustal W. A similar tree (not shown) was obtained using the maximum likelihood approach implemented in the program PHYML [40].

Figure 2

Phylogenetic tree of the 17 human Hsp70 proteins and the proteins that would be encoded in the related pseudogenes identified in this work. Same as tree shown in Figure 1 but including the most conserved pseudogenes (see text). Shaded areas highlight each group of pseudogenes with the gene from which they originated, indicated by an asterisk. A similar tree (not shown) was obtained using the maximum-likelihood approach implemented in the program PHYML [40]. See legend for Figure 1 and text for details on Methods.

Figure 3

Phylogenetic tree of atypical eukaryotic hsp70 genes. Included are the proteins encoded in the atypical human hsp70 genes (Groups III, IV, and V in Figure 1) and in the hsp70 genes of eighteen other completely sequenced eukaryotic genomes. Sequences from the latter eighteen non-human genomes were identified using as queries human proteins from Groups II, VI, and VII and the SSPA procedure [36]. The tree is rooted by the DnaK sequence (AAC73125.1) from E. coli (DnaK E. coli) and by representatives of typical human Hsp70s (from Groups II, VI, and VII). See legend to Figure 1 and text for details on methods and calculation of bootstrap values. Human proteins are in red. Acronyms in black indicate the eukaryotic genomes in which other hsp70 genes were found, as follows: ANOGA: Anopheles gambiae (Insects); ARATH: Arabidopsis thaliana (Plants); CANGL: Candida glabrata (Fungi); CAEEL: Caenorhabditis elegans (Nematoda); CHICK: Gallus gallus (Birds); CYAME: Cyanidioschyzon merolae (Red alga); DANRE: Danio rerio (Fish); DROME: Drosophila melanogaster (Insects); ENTHI: Entamoeba histolytica (Protists); LEIMA: Leishmania major (Protists); MOUSE: Mus musculus (Mammals); NEUCR: Neurospora crassa (Fungi); SCHPO: Schizosaccharomyces pombe (Fungi); TRYCR: Trypanosoma cruzi (Protists); XENTR: Xenopus tropicalis (Amphibians); YEAST: Saccharomyces caerevisiae (Fungi).

Figure 4

Phylogenetic tree of human Hsp70 proteins and prokaryotic DnaK. Included are all human Hsp70 proteins except the highly diverged HSPA12A and HSPA12B. One sequence was chosen for each prokaryotic (bacterial in black, and archaeal in blue) group. Human proteins are in red.

Figure 5

Phylogenetic tree of eukaryotic Hsp70 protein 12. The tree includes the human HSPA12A and HSPA12B proteins and the homologs found in eighteen sequenced eukaryotic genomes. See legend for Figure 3 and text for methods and other details. Homologs of human HSPA12A and HSPA12B were found only in vertebrates. The evolutionary tree suggests that the human genes originated from a unique progenitor also appearing in fish possibly due to a duplication event that occurred in Tetrapoda before the divergence of Amphibia. Only one HSPA12 copy was found in reptiles and birds, suggesting gene loss (or extreme divergence) of one HSPA12 copy from this lineage.

Figure 6

Structural features of human Hsp70 proteins. Typical Hsp70 structures comprise two major domains: ATP-binding domain (ABD) and substrate-binding domain (SBD). The regions comprising the crystal structure of the ABD (1s3x [21], human HSPA1A, positions 1–382) and the NMR structure of the SBD (1ckr [22], rat HSPA8, corresponding to human HSPA8 and HSPA1A homologous positions 385–543) are shown as lines at the top of the figure. Domains conserved in human Hsp70 sequences within these regions are shown as aligned white boxes. Other domains conserved among groups of sequences are shown as aligned boxes of matching colors. Roman numerals to the left indicate the Hsp70 evolutionary groups. UBP, ubiquitin-binding peptide; TPR1, tetratrico peptide repeat 1; ER, endoplasmic reticulum. See text for other details.