Sequence patterns and signatures: Computational and experimental discovery of amyloid-forming peptides.

Screening amino acid sequence space via experiments to discover peptides that self-assemble into amyloid fibrils is challenging. We have developed a computational peptide assembly design (PepAD) algorithm that enables the discovery of amyloid-forming peptides. Discontinuous molecular dynamics (DMD) simulation with the PRIME20 force field combined with the FoldAmyloid tool is used to examine the fibrilization kinetics of PepAD-generated peptides. PepAD screening of ∼10,000 7-mer peptides resulted in twelve top-scoring peptides with two distinct hydration properties. Our studies revealed that eight of the twelve in silico discovered peptides spontaneously form amyloid fibrils in the DMD simulations and that all eight have at least five residues that the FoldAmyloid tool classifies as being aggregation-prone. Based on these observations, we re-examined the PepAD-generated peptides in the sequence pool returned by PepAD and extracted five sequence patterns as well as associated sequence signatures for the 7-mer amyloid-forming peptides. Experimental results from Fourier transform infrared spectroscopy (FTIR), thioflavin T (ThT) fluorescence, circular dichroism (CD), and transmission electron microscopy (TEM) indicate that all the peptides predicted to assemble in silico assemble into antiparallel ß-sheet nanofibers in a concentration-dependent manner. This is the first attempt to use a computational approach to search for amyloid-forming peptides based on customized settings. Our efforts facilitate the identification of ß-sheet-based self-assembling peptides, and contribute insights towards answering a fundamental scientific question: "What does it take, sequence-wise, for a peptide to self-assemble?".

Adaptive sequence convergence of the tumor suppressor ADAMTS9 between small-bodied mammals displaying exceptional longevity.

Maximum lifespan varies by two orders of magnitude across mammals. How such divergent lifespans have evolved remains an open question, with ramifications that may potentially lead to therapies for age-related diseases in humans. Several species of microbats as well as the naked mole-rat live much longer than expected given their small sizes, show reduced susceptibility to neoplasia, and largely remain healthy and reproductively capable throughout the majority of their extended lifespans. The convergent evolution of extreme longevity in these two groups allows for the opportunity to identify potentially important aging related genes that have undergone adaptive sequence convergence in these long-lived, yet small-bodied species. Here, we have tested 4,628 genes for evidence of convergence between the microbats and naked mole-rat. We find a strong signal of adaptive sequence convergence in the gene A disintegrin-like and metalloprotease with thrombospondin type 1 motifs 9 (ADAMTS9). We also provide evidence that the shared substitutions were driven by selection. Intriguingly, ADAMTS9 is a known inhibitor of the mTor pathway and has been implicated in several aging related processes.

Contrasting patterns of adaptive sequence convergence among echolocating mammals.

Several recent studies have described genes demonstrating adaptive sequence convergence between echolocating bats and dolphin, suggesting that common selective pressures can induce common molecular changes, even in distantly related species. However, in the case of the auditory genes Otoferlin (Otof), Cadherin 23 (Cdh23) and Protocadherin 15 (Pcdh15), the reported sequence convergence was supported only by incongruent gene and species trees and counts of convergent substitutions. Therefore, it remains unclear whether echolocating bats and dolphin really do demonstrate evidence of adaptive sequence convergence, or whether there is simply a high level of random background convergence in these genes. To address this question, we estimated the number of convergent and divergent amino acid substitutions along all independent branches of a sufficiently deep phylogeny containing between 22 and 32 mammals for each gene, and compared convergence between the two proposed suborders of bat, Yangochiroptera and Yinpterochiroptera, and dolphin. We find no support for convergence between bats and dolphin in the gene Pcdh15. For the gene Otof we report minimal evidence for convergent evolution only between the Yinpterochiroptera and dolphin. Cdh23 displayed a high level of convergence between dolphin and the Yinpterochiroptera. In addition, dolphin and certain members of the Yangochiroptera that emit high frequency echolocation calls shared several unique convergent substitutions. These results indicate that the convergent evolution of Cdh23 was likely driven by selection for hearing above a certain frequency threshold. Moreover, the contrasting patterns of convergence between the two bat suborders and dolphin in all auditory genes studied thus far suggest echolocation may have evolved independently in the Yinpterochiroptera and Yangochiroptera.

Evidence for widespread subfunctionalization of splice forms in vertebrate genomes.

Gene duplication and alternative splicing are important sources of proteomic diversity. Despite research indicating that gene duplication and alternative splicing are negatively correlated, the evolutionary relationship between the two remains unclear. One manner in which alternative splicing and gene duplication may be related is through the process of subfunctionalization, in which an alternatively spliced gene upon duplication divides distinct splice isoforms among the newly generated daughter genes, in this way reducing the number of alternatively spliced transcripts duplicate genes produce. Previously, it has been shown that splice form subfunctionalization will result in duplicate pairs with divergent exon structure when distinct isoforms become fixed in each paralog. However, the effects of exon structure divergence between paralogs have never before been studied on a genome-wide scale. Here, using genomic data from human, mouse, and zebrafish, we demonstrate that gene duplication followed by exon structure divergence between paralogs results in a significant reduction in levels of alternative splicing. In addition, by comparing the exon structure of zebrafish duplicates to the co-orthologous human gene, we have demonstrated that a considerable fraction of exon divergent duplicates maintain the structural signature of splice form subfunctionalization. Furthermore, we find that paralogs with divergent exon structure demonstrate reduced breadth of expression in a variety of tissues when compared to paralogs with identical exon structures and singletons. Taken together, our results are consistent with subfunctionalization partitioning alternatively spliced isoforms among duplicate genes and as such highlight the relationship between gene duplication and alternative splicing.

Gene duplication followed by exon structure divergence substitutes for alternative splicing in zebrafish.

In this study we report novel findings regarding the evolutionary relationship between gene duplication and alternative splicing, two processes that increase proteomic diversity. By studying teleost fish, we find that gene duplication followed by exon structure divergence between paralogs, but not gene duplication alone, leads to a significant reduction in alternative splicing, as measured by both the proportion of genes that undergo alternative splicing as well as mean number of transcripts per gene. Additionally, we show that this effect is independent of gene family size and gene function. Furthermore, we provide evidence that the reduction in alternative splicing may be due to the partitioning of ancestral splice forms among the duplicate genes - a form of subfunctionalization. Taken together these results indicate that exon structure evolution subsequent to gene duplication may be a common substitute for alternative splicing.

Identification of single nucleotide polymorphisms from the transcriptome of an organism with a whole genome duplication.

BACKGROUND: The common ancestor of salmonid fishes, including rainbow trout (Oncorhynchus mykiss), experienced a whole genome duplication between 20 and 100 million years ago, and many of the duplicated genes have been retained in the trout genome. This retention complicates efforts to detect allelic variation in salmonid fishes. Specifically, single nucleotide polymorphism (SNP) detection is problematic because nucleotide variation can be found between the duplicate copies (paralogs) of a gene as well as between alleles. RESULTS: We present a method of differentiating between allelic and paralogous (gene copy) sequence variants, allowing identification of SNPs in organisms with multiple copies of a gene or set of genes. The basic strategy is to: 1) identify windows of unique cDNA sequences with homology to each other, 2) compare these unique cDNAs if they are not shared between individuals (i.e. the cDNA is homozygous in one individual and homozygous for another cDNA in the other individual), and 3) give a "SNP score" value between zero and one to each candidate sequence variant based on six criteria. Using this strategy we were able to detect about seven thousand potential SNPs from the transcriptomes of several clonal lines of rainbow trout. When directly compared to a pre-validated set of SNPs in polyploid wheat, we were also able to estimate the false-positive rate of this strategy as 0 to 28% depending on parameters used. CONCLUSIONS: This strategy has an advantage over traditional techniques of SNP identification because another dimension of sequencing information is utilized. This method is especially well suited for identifying SNPs in polyploids, both outbred and inbred, but would tend to be conservative for diploid organisms.