László Patthy

Genome evolution and the evolution of exon-shuffling--a review.

Recent studies on the genomes of protists, plants, fungi and animals confirm that the increase in genome size and gene number in different eukaryotic lineages is paralleled by a general decrease in genome compactness and an increase in the number and size of introns. It may thus be predicted that exon-shuffling has become increasingly significant with the evolution of larger, less compact genomes. To test the validity of this prediction, we have analyzed the evolutionary distribution of modular proteins that have clearly evolved by intronic recombination. The results of this analysis indicate that modular multidomain proteins produced by exon-shuffling are restricted in their evolutionary distribution. Although such proteins are present in all major groups of metazoa from sponges to chordates, there is practically no evidence for the presence of related modular proteins in other groups of eukaryotes. The biological significance of this difference in the composition of the proteomes of animals, fungi, plants and protists is best appreciated when these modular proteins are classified with respect to their biological function. The majority of these proteins can be assigned to functional categories that are inextricably linked to multicellularity of animals, and are of absolute importance in permitting animals to function in an integrated fashion: constituents of the extracellular matrix, proteases involved in tissue remodelling processes, various proteins of body fluids, membrane-associated proteins mediating cell-cell and cell-matrix interactions, membrane associated receptor proteins regulating cell cell communications, etc. Although some basic types of modular proteins seem to be shared by all major groups of metazoa, there are also groups of modular proteins that appear to be restricted to certain evolutionary lineages. In summary, the results suggest that exon-shuffling acquired major significance at the time of metazoan radiation. It is interesting to note that the rise of exon-shuffling coincides with a spectacular burst of evolutionary creativity: the Big Bang of metazoan radiation. It seems probable that modular protein evolution by exon-shuffling has contributed significantly to this accelerated evolution of metazoa, since it facilitated the rapid construction of multidomain extracellular and cell surface proteins that are indispensable for multicellularity.

Intron‐dependent evolution: Preferred types of exons and introns

Exon insertions and exon duplications, two major mechanisms of exon shuffling, are shown to involve modules that have introns of the same phase class at both their 5′‐ and 3′‐ends. At the sites of intronic recombinations exon insertions and duplications create new introns which belong to the same phase class as the recipient introns. As a consequence of repeated exon insertions and exon duplications introns of a single phase class predominate in the resulting genes, i.e. gene assembly by exon shuffling is reflected both by this nonrandom intron phase usage and by the correlation between the domain organization of the proteins and exon‐intron organization of their genes. Genes that appeared before the eukaryote‐prokaryote split do not show these diagnostic signs of exon shuffling. Since ancestral introns (e.g. self‐splicing introns) did not favour intronic recombination, exon shuffling may not have been significant in the early part of protein evolution.

The implications of alternative splicing in the ENCODE protein complement.

Alternative premessenger RNA splicing enables genes to generate more than one gene product. Splicing events that occur within protein coding regions have the potential to alter the biological function of the expressed protein and even to create new protein functions. Alternative splicing has been suggested as one explanation for the discrepancy between the number of human genes and functional complexity. Here, we carry out a detailed study of the alternatively spliced gene products annotated in the ENCODE pilot project. We find that alternative splicing in human genes is more frequent than has commonly been suggested, and we demonstrate that many of the potential alternative gene products will have markedly different structure and function from their constitutively spliced counterparts. For the vast majority of these alternative isoforms, little evidence exists to suggest they have a role as functional proteins, and it seems unlikely that the spectrum of conventional enzymatic or structural functions can be substantially extended through alternative splicing.

COMMON EVOLUTIONARY ORIGIN OF THE FIBRIN-BINDING STRUCTURES OF FIBRONECTIN AND TISSUE-TYPE PLASMINOGEN ACTIVATOR

Comparison of the primary structures of high‐M r urokinase and tissue‐type plasminogen activator reveals a high degree of structural homology between the two proteins, except that tissue activator contains a 43 residue long amino‐terminal region, which has no counterpart in urokinase. We show that this segment is homologous with the finger‐domains responsible for the fibrin‐affinity of fibronectin. Limited proteolysis of the amino‐terminal region of plasminogen activator was found to lead to a loss of the fibrin‐affinity of the enzyme. It is suggested that the finger‐domains of fibronectin and tissue‐types plasminogen activator have similar functions and that the finger‐domains of the two proteins evolved from a common ancestral fibrin‐binding domain.

Kringles: modules specialized for protein binding: Homology of the gelatin-binding region of fibronectin with the kringle structures of proteases

Prothrombin, plasminogen, urokinase‐ and tissue‐type plasminogen activators contain homologous structures known as kringles. The kringles correspond to autonomous structural and folding domains which mediate the binding of these multidomain proteins to other proteins. During evolution the different kringles retained the same gross architecture, the kringle‐fold, yet diverged to bind different proteins. We show that the amino acid sequences of the type II structures of the gelatin‐binding region of fibronectin are homologous with those of the protease‐kringles. Prediction of secondary structures revealed a remarkable agreement in the positions of predicted β‐sheets, suggesting that the folding of kringles and type II structures may also be similar. As a corollary of this finding, the disulphide‐bridge pattern of type II structures is shown to be homologous to that in kringles. It is noteworthy that protease‐kringles and fibronectin type II structures have similar functions inasmuch as they mediate the binding of multidomain proteins to other proteins. It is proposed that the kringles of proteases and type II structures of fibronectin evolved from a common ancestral protein binding module.

Modular exchange principles in proteins

Abstract The past year has brought a wealth of publications that bear on modular exchange of proteins and on the role of introns in this process. Analysis of the new data helps clarify the mechanism, evolutionary significance and history of exon-shuffling.

Modular assembly of genes and the evolution of new functions

Modular assembly of novel genes from existing genes has long been thought to be an important source of evolutionary novelty. Thanks to major advances in genomic studies it has now become clear that this mechanism contributed significantly to the evolution of novel biological functions in different evolutionary lineages. Analyses of completely sequenced bacterial, archaeal and eukaryotic genomes has revealed that modular assembly of novel constituents of various eukaryotic intracellular signalling pathways played a major role in the evolution of eukaryotes. Comparison of the genomes of single-celled eukaryotes, multicellular plants and animals has also shown that the evolution of multicellularity was accompanied by the assembly of numerous novel extracellular matrix proteins and extracellular signalling proteins that are absolutely essential for multicellularity. There is now strong evidence that exon-shuffling played a general role in the assembly of the modular proteins involved in extracellular communications of metazoa. Although some of these proteins seem to be shared by all major groups of metazoa, others are restricted to certain evolutionary lineages. The genomic features of the chordates appear to have favoured intronic recombination as evidenced by the fact that exon-shuffling continued to be a major source of evolutionary novelty during vertebrate evolution.

The PAN module: the N‐terminal domains of plasminogen and hepatocyte growth factor are homologous with the apple domains of the prekallikrein family and with a novel domain found in numerous nematode proteins

Based on homology search and structure prediction methods we show that (1) the N‐terminal N domains of members of the plasminogen/hepatocyte growth factor family, (2) the apple domains of the plasma prekallikrein/coagulation factor XI family, and (3) domains of various nematode proteins belong to the same module superfamily, hereafter referred to as the PAN module. The patterns of conserved residues correspond to secondary structural elements of the known three‐dimensional structure of hepatocyte growth factor N domain, therefore we predict a similar fold for all members of this superfamily. Based on available functional informations on apple domains and N domains, it is clear that PAN modules have significant functional versatility, they fulfill diverse biological functions by mediating protein‐protein or protein‐carbohydrate interactions.

Identification of Cathepsin B as a Mediator of Neuronal Death Induced by Aβ-activated Microglial Cells Using a Functional Genomics Approach

Alzheimers disease is a progressive neurodegenerative disease characterized by senile plaques, neurofibrillary tangles, dystrophic neurites, and reactive glial cells. Activated microglia are found to be intimately associated with senile plaques and may play a central role in mediating chronic inflammatory conditions in Alzheimers disease. Activation of cultured murine microglial BV2 cells by freshly sonicated Aβ42 results in the secretion of neurotoxic factors that kill primary cultured neurons. To understand molecular pathways underlying Aβ-induced microglial activation, we analyzed the expression levels of transcripts isolated from Aβ42-activated BV2 cells using high density filter arrays. The analysis of these arrays identified 554 genes that are transcriptionally up-regulated by Aβ42 in a statistically significant manner. Quantitative reverse transcription-PCR was used to confirm the regulation of a subset of genes, including cysteine proteases cathepsin B and cathepsin L, tissue inhibitor of matrix metalloproteinase 2, cytochrome c oxidase, and allograft inflammatory factor 1. Small interfering RNA-mediated silencing of the cathepsin B gene in Aβ-activated BV2 cells diminished the microglial activation-mediated neurotoxicity. Moreover, CA-074, a specific cathepsin B inhibitor, also abolished the neurotoxic effects caused by Aβ42-activated BV2 cells. Our results suggest an essential role for secreted cathepsin B in neuronal death mediated by Aβ-activated inflammatory response.

Functions of agrin and agrin-related proteins

Agrin, a molecule produced by motoneurons that induces the aggregation of nicotinic acetylcholine receptors (nAChRs), has recently been structurally characterized. Agrin-related proteins (ARPs) that arise from differential splicing are synthesized by neurons and muscle. The C-terminal region of agrin that instructs muscle to aggregate nAChRs contains three laminin A modules separated by epidermal growth factor-like modules. Alternative splicing in the laminin A modules leads to the formation of at least three ARPs that are devoid of nAChR-aggregating activity. In their N-terminal regions, both agrin and ARPs contain nine follistatin-related modules that, like those in follistatin and in another related protein, osteonectin, may have the capability to bind members of the transforming growth factor beta (TGF-beta) or platelet-derived growth factor (PDGF) families. This review proposes that these follistatin-like regions of agrin and ARPs might bind and localize growth factors, and thus provide a matrix-bound concentration of them. Beyond agrins role in inducing AChR aggregation, the function of agrin and ARPs to provide a localized reservoir of growth factors could contribute to the formation and maintenance of the long-lasting synaptic architecture by specifying and limiting the area of influence of these molecules.