Abstract
BackgroundThe huntingtin-associated protein 40 (HAP40) abundantly interacts with huntingtin (HTT), the protein that is altered in Huntington’s disease (HD). Therefore, we analysed the evolution of HAP40 and its interaction with HTT.ResultsWe found that in amniotes HAP40 is encoded by a single-exon gene, whereas in all other organisms it is expressed from multi-exon genes. HAP40 co-occurs with HTT in unikonts, including filastereans such as Capsaspora owczarzaki and the amoebozoan Dictyostelium discoideum, but both proteins are absent from fungi. Outside unikonts, a few species, such as the free-living amoeboflagellate Naegleria gruberi, contain putative HTT and HAP40 orthologs.Biochemically we show that the interaction between HTT and HAP40 extends to fish, and bioinformatic analyses provide evidence for evolutionary conservation of this interaction. The closest homologue of HAP40 in current protein databases is the family of soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs).ConclusionOur results indicate that the transition from a multi-exon to a single-exon gene appears to have taken place by retroposition during the divergence of amphibians and amniotes, followed by the loss of the parental multi-exon gene. Furthermore, it appears that the two proteins probably originated at the root of eukaryotes. Conservation of the interaction between HAP40 and HTT and their likely coevolution strongly indicate functional importance of this interaction.
Highlights
The huntingtin-associated protein 40 (HAP40) abundantly interacts with huntingtin (HTT), the protein that is altered in Huntington’s disease (HD)
Using cryo-electron microscopy, we recently determined the structure of human HTT [11], which showed a largely alpha-helical protein with three major domains composed of a protein tandem repeat structural motif, the Huntingtin, elongation factor 3 (EF3), protein phosphatase 2A (PP2A), and the yeast kinase TOR1 (HEAT) repeat: a large N-terminal domain with 21 HEAT repeats (N-HEAT), a smaller C-terminal domain with 12 HEAT repeats (C-HEAT), and a connecting bridge domain
Conversion of Factor VIII intronic transcript A (F8A) from a multi‐exon to a single‐exon gene during the divergence of amphibians and amniotes While in humans and mice HAP40 has previously been shown to be encoded by single exon genes (SEGs), with three copies present in humans and only one in mice, we noted early on in our study that in zebrafish (Danio rerio) the F8A ortholog comprises 11 exons and spans about 9200 nucleotides, versus only about 1700 nucleotides in humans and mice
Summary
The huntingtin-associated protein 40 (HAP40) abundantly interacts with huntingtin (HTT), the protein that is altered in Huntington’s disease (HD). Huntingtin (HTT) is a large intracellular protein with a molecular weight (MW) of 348 kDa, which is functionally involved in diverse cellular processes. These include endocytosis, vesicle transport, autophagy, and HTT orthologs are present throughout protists and animals, but absent in plants and fungi [3, 4]. Due to the inherent flexibility of HTT [11], structure determination only became possible following the purification of HTT as a tight complex with the cognate huntingtin-associated protein 40 (HAP40). HAP40, formed of 4 canonical and 2 decayed tetratricopeptide repeats (TPR), binds in a cleft between the three domains, forming mainly hydrophobic contacts to N-HEAT and C-HEAT and electrostatic interactions with the bridge domain, thereby stabilizing the conformation of HTT. A large number of HTT interactors [1, 12] strongly suggest that HTT serves as a multivalent interaction hub for the coordination of many different functions
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