Homopolymeric Amino Acid Repeats Research Articles

A poly-histidine motif of HOXA1 is involved in regulatory interactions with cysteine-rich proteins

Homopolymeric amino acid repeats are found in about 24 % of human proteins and are over-represented in transcriptions factors and kinases. Although relatively rare, homopolymeric histidine repeats (polyH) are more significantly found in proteins involved in the regulation of embryonic development. To gain a better understanding of the role of polyH in these proteins, we used a bioinformatic approach to search for shared features in the interactomes of polyH-containing proteins in human. Our analysis revealed that polyH protein interactomes are enriched in cysteine-rich proteins and in proteins containing (a) cysteine repeat(s). Focusing on HOXA1, a HOX transcription factor displaying one long polyH motif, we identified that the polyH motif is required for the HOXA1 interaction with such cysteine-rich proteins. We observed a correlation between the length of the polyH repeat and the strength of the HOXA1 interaction with one Cys-rich protein, MDFI. We also found that metal ion chelators disrupt the HOXA1-MDFI interaction supporting that such metal ions are required for the interaction. Furthermore, we identified three polyH interactors which down-regulate the transcriptional activity of HOXA1. Taken together, our data point towards the involvement of polyH and cysteines in regulatory interactions between proteins, notably transcription factors like HOXA1.

Proteome-scale relationships between local amino acid composition and protein fates and functions.

Proteins with low-complexity domains continue to emerge as key players in both normal and pathological cellular processes. Although low-complexity domains are often grouped into a single class, individual low-complexity domains can differ substantially with respect to amino acid composition. These differences may strongly influence the physical properties, cellular regulation, and molecular functions of low-complexity domains. Therefore, we developed a bioinformatic approach to explore relationships between amino acid composition, protein metabolism, and protein function. We find that local compositional enrichment within protein sequences is associated with differences in translation efficiency, abundance, half-life, protein-protein interaction promiscuity, subcellular localization, and molecular functions of proteins on a proteome-wide scale. However, local enrichment of related amino acids is sometimes associated with opposite effects on protein regulation and function, highlighting the importance of distinguishing between different types of low-complexity domains. Furthermore, many of these effects are discernible at amino acid compositions below those required for classification as low-complexity or statistically-biased by traditional methods and in the absence of homopolymeric amino acid repeats, indicating that thresholds employed by classical methods may not reflect biologically relevant criteria. Application of our analyses to composition-driven processes, such as the formation of membraneless organelles, reveals distinct composition profiles even for closely related organelles. Collectively, these results provide a unique perspective and detailed insights into relationships between amino acid composition, protein metabolism, and protein functions.

POU3F2 participates in cognitive function and adult hippocampal neurogenesis via mammalian-characteristic amino acid repeats.

POU3F2/BRN-2 is a transcription factor that is mainly expressed in the central nervous system and plays an important role in brain development. The transactivation domain of POU3F2 includes multiple mammalian-characteristic tandem amino acid repeats (homopolymeric amino acid repeats). We previously generated knock-in mice (Pou3f2Δ/Δ mice) in which all three homopolymeric amino acid repeats were deleted from the Pou3f2 transactivation domain and identified phenotypic impairments in maternal behavior and pup recognition. Yet, the exact biological implications of homopolymeric repeats are not completely understood. In this study, we investigated cognitive function and hippocampal neurogenesis in Pou3f2Δ/Δ mice. Pou3f2Δ/Δ mice exhibited cognitive impairment in object recognition and object location tests. Immunohistochemistry for doublecortin, a marker of immature neurons, showed a lower number of newborn neurons in the dentate gyrus of adult Pou3f2Δ/Δ mice compared with wild-type mice. Consistent with this observation, adult Pou3f2Δ/Δ mice had lower numbers of 5-bromo-2'-deoxyuridine (BrdU) and NeuN double-positive cells at 4 weeks after BrdU injection compared with control mice, indicating the decreased generation of mature granule cells in Pou3f2Δ/Δ mice. Taken together, these results suggest that POU3F2 is involved in cognitive function as well as adult hippocampal neurogenesis, and that homopolymeric amino acid repeats in this gene play a functional role.

Editorial: Protein Interaction Networks in Health and Disease.

Editorial: Protein Interaction Networks in Health and Disease.

Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats.

Homopolymeric amino acids repeats (AARs), which are widespread in proteomes, have often been viewed simply as spacers between protein domains, or even as “junk” sequences with no obvious function but with a potential to cause harm upon expansion as in genetic diseases associated with polyglutamine or polyalanine expansions, including Huntington disease and cleidocranial dysplasia. A growing body of evidence indicates however that at least some AARs can form organized, functional protein structures, and can regulate protein function. In particular, certain AARs can mediate protein-protein interactions, either through homotypic AAR-AAR contacts or through heterotypic contacts with other protein domains. It is still unclear however, whether AARs may have a generalized, proteome-wide role in shaping protein-protein interaction networks. Therefore, we have undertaken here a bioinformatics screening of the human proteome and interactome in search of quantitative evidence of such a role. We first identified the sets of proteins that contain repeats of any one of the 20 amino acids, as well as control sets of proteins chosen at random in the proteome. We then analyzed the connectivity between the proteins of the AAR-containing protein sets and we compared it with that observed in the corresponding control networks. We find evidence for different degrees of connectivity in the different AAR-containing protein networks. Indeed, networks of proteins containing polyglutamine, polyglutamate, polyproline, and other AARs show significantly increased levels of connectivity, whereas networks containing polyleucine and other hydrophobic repeats show lower degrees of connectivity. Furthermore, we observed that numerous protein-protein, -nucleic acid, and -lipid interaction domains are significantly enriched in specific AAR protein groups. These findings support the notion of a generalized, combinatorial role of AARs, together with conventional protein interaction domains, in shaping the interaction networks of the human proteome, and define proteome-wide knowledge that may guide the informed biological exploration of the role of AARs in protein interactions.

Highlight: nonmammalian transcription factors make mice mothers less loving.

For the first time, a complex behavior, mothers caring for their offspring, has been linked to a semimysterious repeating amino acid sequence common to mammals. These repeats, found more often in mammals than other vertebrates, have been seen in the past to affect protein–protein interactions, transcriptional regulation, and phenotypic variation. Japanese biologists Shintaroh Ueda, Den'etsu Sutoo, and colleagues from the University of Tokyo and the University of Tsukuba wanted to know whether these repeats had evolutionary significance. To investigate, the researchers replaced a gene in mice with its ortholog from western clawed frogs. The amphibian version of the transcription factor Pou3f2 is repeat-free, whereas the mammalian version is rich in glycine, glutamine, and proline repeats. They report their findings in a recent issue of Genome Biology Evolution (Makoto et al. 2014). Before starting the experiments, says Ueda, it was hard to guess what changes they would see, but he felt confident something would appear. The effects were dramatic: Carrying nonmammalian Pou3f2 essentially turned female mice into bad mothers. Though giving birth to healthy, normal-sized pups, a dam was less interested in her pups, and less likely to fetch them and bring them to her nest. Most pups born to “knock-in” mothers did not survive to weaning, whereas most pups born to normal mothers did. “This is significant, I think, for reminding us that we shouldn't just be looking at the regions of proteins traditionally considered functional,” says Noel Faux, a research fellow at the Florey Institute for Neuroscience and Mental Health in Melbourne, Australia, who was not involved in the research. “Regions we wouldn't normally consider obviously do have a function here.” Without the repeating homopolymeric amino acids, the mice did not have as much dopamine and serotonin in their brains—past studies have shown that these two neurotransmitters heavily affect how mammalian mothers care for their offspring. Dopamine and serotonin regulate many other complex thoughts and behaviors: Anxiety, fear, mood control, motivation, cognition, reward, movement, regulation of body temperature, and drug abuse. “We hypothesize that the regulation of these functions is not sufficiently strict in nonmammals including amphibians,” write Ueda et al. Although animals such as fish and amphibians behave more or less instinctively, mammals behave in ways that are not necessarily instinctive, they show emotion, bond socially, and nurturing their young. “This study opens a host of interesting questions,” says Faux. “If you give these [knock-in] mice tests for memory, anxiety or depression, how do they perform?” In a more technical vein, he is curious to know how the structure of transcription factor Pou3f2 differs between knock in and normal mice. Although orthologous proteins fold in a similar manner, the repeats presumably, he says, affect structure in some way. Does the length of the repeats affect neurotransmitter levels and behavior, he wonders. Also, in this study, all three repeat-rich regions of the mammalian Pou3f2 gene were swapped for no-repeat amphibian versions. He would like to see experiments removing each of those regions individually, to see which has the strongest effect on behavior. Faux says many of his colleagues will be intrigued by the study. It may, he hopes, bolster support for their research: “The more evidence that these [repeats] do have an impact the more the granting bodies are likely to take it on board. It's sort of a new area and people are still a bit skeptical that these things can have an effect on fairly large behaviors.” Ueda also believes that this field of research is just beginning. There are hundreds of mammalian genes with homopolymeric amino acid repeats, he says, and the majority are evolutionarily conserved in length across mammals and have important roles in transcription, translation, and signaling processes. These knock-in mice, says Ueda, are available as models for researchers to deeply dive into the characteristics that make mammals unique.

Mammalian-specific sequences in pou3f2 contribute to maternal behavior.

Various mutations have occurred during evolution among orthologs, genes in different species that diverged from a common ancestral gene by speciation. Here, we report the remarkable deterioration of a characteristic mammalian maternal behavior, pup retrieval, in nonmammalized mice, in which the transcription factor Pou3f2 was replaced with the Xenopus ortholog lacking all of the homopolymeric amino acid repeats of mammalian POU3F2. Most of the pups born to the nonmammalized mice died within days after birth, depending on the dam genotype alone. Quantitative immunohistochemical analysis revealed decreases in the rate-limiting enzymes of dopamine and serotonin synthesis in various brain structures. Similar results were obtained in knock-in mice in which all of the homopolymeric amino acid repeats of mammalian POU3F2 were removed. Pup retrieval behavior in mammals is thus strongly related to monoamine neurotransmitter levels via the acquisition of homopolymeric amino acid repeats during mammalian evolution.

Elevated Evolutionary Rate in Genes with Homopolymeric Amino Acid Repeats Constituting Nondisordered Structure

Homopolymeric amino acid repeats are tandem repeats of single amino acids. About 650 genes are known to have repeats of this kind comprising seven residues or more in the human genome. According to the evolutionary conservativeness, we classified the repeats into three categories: those whose length is conserved among mammals (CM), those whose length differs among nonprimate mammals but is conserved among primates (CP), and those whose length differs among primates (VP). The frequency of each repeat, especially Ala, Leu, Pro, and Glu repeats, varies greatly in each category. The 3D structure of homopolymeric amino acid repeats is considered to be intrinsically disordered. As expected, a large proportion of the repeats had a disordered structure, and nearly half of the repeats were predicted as completely disordered. However, a number of the repeats predicted to have nondisordered structure: 13% and 25% of the repeats for categories CM and VP, respectively. Comparison of the substitution rates showed a higher Ka/Ks ratio for the genes with not disordered repeats than the genes with disordered repeats. These results indicate that amino acid substitution rates have been elevated in the genes with nondisordered repeats.

Morphological Change Caused by Loss of the Taxon-Specific Polyalanine Tract in Hoxd-13

Sequence comparison of Hoxd-13 among vertebrates revealed the presence of taxon-specific polyalanine tracts in amniotes. To investigate their function at the organismal level, we replaced the wild-type Hoxd-13 gene with one lacking the 15-residue polyalanine tract by using homologous recombination. Sesamoid bone formation in knock-in mice was different from that in the wild type; this was observed not only in the homozygotes but also in the heterozygotes. The present study provides the first direct evidence that taxon-specific homopolymeric amino acid repeats are involved in phenotypic diversification at the organismal level.

Class III POU genes: Generation of homopolymeric amino acid repeats under GC pressure in mammals

The class III POU transcription factor genes play an important role in the nervous system. Comparison of their entire amino acid sequences disclosed a remarkable feature of particular mammalian class III POU genes. Alanine, glycine, and proline repeats were present in the mammalian Brain-1 gene, whereas most of these repeats were absent in the nonmammalian homologue. The mammalian Brain-2 gene had alanine, glycine, proline, and glutamine repeats, which were missing in the nonmammalian homologue. The mammalian Scip gene had alanine, glycine, proline, and histidine repeats, but the nonmammalian homologue completely lacked these repeats. In contrast, the mammalian Brain-4 gene had no amino acid repeats like its nonmammalian homologue. The mammalian genes containing the characteristic amino acid repeats had another feature, higher GC content. We found a positive correlation between the GC content and the amino acid repeat ratio. Those amino acids were encoded by triplet codons with relatively high GC content. These results suggest that the GC pressure has facilitated generation of the homopolymeric amino acid repeats.

The E75 gene of Manduca sexta and comparison with its Drosophila Homolog

The ecdysone-inducible E75 gene responsible for the 75B puff of Drosophila melanogaster encodes a family of proteins which are members of the steroid receptor superfamily. These proteins are believed to be involved in the regulation of ecdysone response. In order to investigate the evolutionary conservation of E75, we have identified the E75 gene of Manduca sexta. We show here the structure of a cDNA believed to encode the Manduca homolog of the E75 B protein, and demonstrate that the putative DNA binding, hormone binding and amino and carboxy terminal flanking domains are conserved. However, due to a relative reduction in intron size and number and the absence of homopolymeric amino acid repeats, the E75 B transcription unit and protein are considerably smaller in M. sexta than in D. melanogaster. These findings have implications for the identification of critical structural features of E75 and also suggest that E75 has a conserved function and a shared ligand in Lepidoptera.