Maximum-likelihood approach for gene family evolution under functional divergence

According to the observed alignment pattern (i.e., amino acid configuration), we studied two basic types of functional divergence of a protein family. Type I functional divergence after gene duplication results in altered functional constraints (i.e., different evolutionary rate) between duplicate genes, whereas type II results in no altered functional constraints but radical change in amino acid property between them (e.g., charge, hydrophobicity, etc.). Two statistical approaches, i.e., the subtree likelihood and the whole-tree likelihood, were developed for estimating the coefficients of (type I or type II) functional divergence. Numerical algorithms for obtaining maximum-likelihood estimates are also provided. Moreover, a posterior-based site-specific profile is implemented to predict critical amino acid residues that are responsible for type I and/or type II functional divergence after gene duplication. We compared the current likelihood with a fast method developed previously by examples; both show similar results. For handling altered functional constraints (type I functional divergence) in the large gene family with many member genes (clusters), which appears to be a normal case in postgenomics, the subtree likelihood provides a solution that is computationally feasible and robust against the uncertainty of the phylogeny. The cost of this feasibility is the approximation when frequencies of amino acids are very skewed. The potential bias and correction are discussed.     

Statistical methods for testing functional divergence after gene duplication

Functional innovations after gene duplication may result in altered functional constraints between member gene clusters of a gene family. This type (type I) of functional divergence is measured by the coefficient of functional divergence (theta lambda), which can be interpreted as the decrease in rate correlation between gene clusters, or the probability that the evolutionary rate at a site is statistically independent between two gene clusters. A simple stochastic model has been developed for estimating theta lambda and testing its statistical significance. The current model includes the model of rate variation among sites as a special case when theta lambda = 0. Moreover, we have developed a site-specific profile based on the hidden Markov model to identify critical amino acid residues that are responsible for these functional differences between two gene clusters, which may have great potential in functional genomics.     

A simple statistical method for estimating type-II (cluster-specific) functional divergence of protein sequences

Predicting functional amino acid residues in silico is important for comparative genomics. In this paper, we focus on the issue of how to statistically identify cluster-specific amino acid residues that are related to the functional divergence after gene duplication. We approach this problem using a framework based on site-specific shift of amino acid property (type-II functional divergence), as opposed to site-specific shift of evolutionary rate (type-I functional divergence). An efficient statistical procedure is implemented to facilitate the development of phylogenomic database for cluster-specific residues of large-scale protein families. Our method has the following features: 1) statistical testing of the type-II functional divergence and 2) the site-specific Bayesian profile to measure how amino acid residues contribute to type-II (cluster-specific) functional divergence. Consequently, one may obtain the posterior probability for "functional" cluster-specific residues. Case studies are presented and indicate that radical cluster-specific residues are responsible for most of inferred type-II functional divergence, whereas conserved cluster-specific residues appear less than even those imperfect radical cluster-specific residues to this type of functional divergence.     

Evolutionary analysis for functional divergence of Jak protein kinase domains and tissue-specific genes

Jak (Janus kinase) is a nonreceptor tyrosine kinase, which plays important roles in signal transduction pathways. The unique feature of Jak is that, in addition to a fully functional tyrosine kinase domain (JH1), Jak possesses a pseudokinase domain (JH2). Although JH2 lost its catalytic function, experimental evidence has shown that this domain may have acquired some new but unknown functions. This apparent functional divergence after the (internal) domain duplication may result in dramatic changes of selective constraints at some sites. We conducted a data analysis to test this hypothesis. Our result shows that shifted selective constraints (or shifted evolutionary rates) between the JH1 and the JH2 domains are statistically significant. Predicted amino acid sites by posterior analysis can be classified into two groups: very conserved in JH1 but highly variable in JH2, and vice versa. Moreover, we have studied the evolutionary pattern of four tissue-specific genes, Jak1, Jak2, Jak3, and Tyk2, which were generated in the early stages of vertebrates. We found that after the (first) gene duplication, site-specific rate shifts between Jak2/Jak3 and Jak1/Tyk are significant, presumably as a consequence of functional divergence among these genes. The implication of our study for functional genomics is discussed.     

Targeted expression of ced-3 and Ice induces programmed cell death in Drosophila

CED-3 is a cysteine protease required for programmed cell death in the nematode, Caenorhabditis elegans, and shares a sequence similarity with mammalian ICE (interleukin-1beta converting enzyme) family proteases. Both CED-3 and ICE family proteases can induce programmed cell death in mammalian cells. Structural and functional similarities between CED-3 and ICE family proteases indicate that the mechanism of cell death is evolutionarily conserved, suggesting the presence of a similar mechanism involving CED-3/ICE-like proteases in Drosophila. Here we determined whether CED-3 or ICE functions to induce programmed cell death in Drosophila. We have generated transformant lines in which ced-3 or Ice is ectopically expressed using the GAL4-UAS system. Expression of CED-3 and ICE can elicit cell death in Drosophila and the cell death was blocked by coexpressing the p35 gene which encodes a viral inhibitor of CED-3/ICE proteases. Results support the idea that the mechanism of programmed cell death controlled by CED-3/ICE is conserved among widely divergent animal species including Drosophila, and the system described provides a tool to dissect cell death mechanism downstream of CED-3/ICE proteases.     

Mutational analysis of the Caenorhabditis elegans cell-death gene ced-3

Mutations in the gene ced-3, which encodes a protease similar to interleukin-1beta converting enzyme and related proteins termed caspases, prevent programmed cell death in the nematode Caenorhabditis elegans. We used site-directed mutagenesis to demonstrate that both the presumptive active-site cysteine of the CED-3 protease and the aspartate residues at sites of processing of the CED-3 proprotein are required for programmed cell death in vivo. We characterized the phenotypes caused by and the molecular lesions of 52 ced-3 alleles. These alleles can be ordered in a graded phenotypic series. Of the 30 amino acid sites altered by ced-3 missense mutations, 29 are conserved with at least one other caspase, suggesting that these residues define sites important for the functions of all caspases. Animals homozygous for the ced-3(n2452) allele, which is deleted for the region of the ced-3 gene that encodes the protease domain, seemed to be incompletely blocked in programmed cell death, suggesting that some programmed cell death can occur independently of CED-3 protease activity.     

Identification of rice TUBBY-like genes and their evolution

The identification of TUBBY-like genes in organisms ranging from single-celled to multicellular eukaryotes has allowed the phylogenetic history of this gene family to be traced back to the early evolutionary stages of eukaryote development. Rice TUBBY-like genes were located on chromosomes 1, 2, 3, 4, 5, 7, 8, 11 and 12 without any obvious clustering. On a genomic scale, it was revealed that the rice TUBBY-like gene family probably evolved mainly through segmental duplication produced by polyploidy. The altered selective constraints (or site-specific rate changes), related to functional divergence during protein evolution between plant and animal TUBBY-like genes, were statistically significant. Based on posterior probability analysis, five amino acid sites (103, 312, 315, 317 and 319) are thought to be responsible for functional divergence.     

Sequence and structural aspects of the functional diversification of plant alcohol dehydrogenases

The glycolytic proteins in plants are coded by small multigene families, which provide an interesting contrast to the high copy number of gene families studied to date. The alcohol dehydrogenase (Adh) genes encode glycolytic enzymes that have been characterized in some plant families. Although the amino acid sequences of zinc-containing long-chain ADHs are highly conserved, the metabolic function of this enzyme is variable. They also have different patterns of expression and are submitted to differences in nonsynonymous substitution rates between gene copies. It is possible that the Adh copies have been retained as a consequence of adaptative amino acid replacements which have conferred subtle changes in function. Phylogenetic analysis indicates that there have been a number of separate duplication events within angiosperms, and that genes labeled Adh1, Adh2 and Adh3 in different groups may not be homologous. Nonsynonymous/synonymous ratios yielded no signs of positive selection. However, the coefficients of functional divergence (theta) estimated between the Adh1 and Adh2 gene groups indicate statistically significant site-specific shift of evolutionary rates between them, as well as between those of different botanical families, suggesting that altered functional constraints may have taken place at some amino acid residues after their diversification. The theoretical three-dimensional structure of the alcohol dehydrogenase from Arabis blepharophylla was constructed and verified to be stereochemically valid.     

Evolutionary Analysis for Functional Divergence of the Toll-Like Receptor Gene Family and Altered Functional Constraints

The Toll-like receptor (TLR) gene family consists of type 1 transmembrane receptors, which play essential roles in both innate immunity and adaptive immune response by ligand recognition and signal transduction. Using all available vertebrate TLR protein sequences, we inferred the phylogenetic tree and then characterized critical amino acid residues for functional divergence by detecting altered functional constraints after gene duplications. We found that the extracellular domain of TLR genes showed higher functional divergence than that of the cytoplasmic domain, particularly in the region between leucine-rich repeat (LRR) 10 and LRR 15 of TLR 4. Our finding supports the concept that sequence evolution in the extracellular domain may be responsible for the broad diversity of TLR ligand-binding affinity, providing a testable hypothesis for potential targets that could be verified by further experimentation.     

E1B 19,000-Molecular-Weight Protein Interacts with and Inhibits CED-4-Dependent, FLICE-Mediated Apoptosis

Genetic studies of the nematode Caenorhabditis elegans (C. elegans) have identified several important components of the cell death pathway, most notably CED-3, CED-4, and CED-9. CED-4 directly interacts with the Bcl-2 homologue CED-9 (or the mammalian Bcl-2 family member Bcl-x_L) and the caspase CED-3 (or the mammalian caspases ICE and FLICE). This trimolecular complex of CED-4, CED-3, and CED-9 is functional in that CED-9 inhibits CED-4 from activating CED-3 and thereby inhibits apoptosis in heterologous systems. The E1B 19,000-molecular weight protein (E1B 19K) is a potent apoptosis inhibitor and the adenovirus homologue of Bcl-2-related apoptosis inhibitors. Since E1B 19K and Bcl-x_L have functional similarity, we determined if E1B 19K interacts with CED-4 and regulates CED-4-dependent caspase activation. Binding analysis indicated that E1B 19K interacts with CED-4 in a Saccharomyces cerevisiae two-hybrid assay, in vitro, and in mammalian cell lysates. The subcellular localization pattern of CED-4 was dramatically changed by E1B 19K, supporting the theory of a functional interaction between CED-4 and E1B 19K. Whereas expression of CED-4 alone could not induce cell death, coexpression of CED-4 and FLICE augmented cell death induction by FLICE, which was blocked by expression of E1B 19K. Even though E1B 19K did not prevent FLICE-induced apoptosis, it did inhibit CED-4-dependent, FLICE-mediated apoptosis, which suggested that CED-4 was required for E1B 19K to block FLICE activation. Thus, E1B 19K functions through interacting with CED-4, and presumably a mammalian homologue of CED-4, to inhibit caspase activation and apoptosis.     

Mathematical modeling for functional divergence after gene duplication.

In this paper, I present a statistical framework for modeling the functional divergence after gene duplication. A rate-component model to describe the rate covariation among homologous genes of a gene family is implemented when a phylogenetic tree is known. The Markov chain model is rigorous but may require a huge amount of computational time when the number of sequences is large. On the other hand, the Poisson-based model is mathematically analytical so that computation is very fast even for a large dataset. Moreover, under the posterior framework, we have developed a site-specific profile for predicting important amino acid residues responsible for these functional differences between member genes of a gene family. Our study may have great potential for functional genomics because it is cost-effective, and these predictions can be further tested by biological experimentation.     

Phylogenetic analysis and evolution of aromatic amino acid hydroxylase

A study was performed to investigate the phylogenetic relationship among AAAH members and to statistically evaluate sequence conservation and functional divergence. In total, 161 genes were identified from 103 species. Phylogenetic analysis showed that well-conserved subfamilies exist. Exon-intron structure analysis showed that the gene structures of AAAH were highly conserved across some different lineage species, while some species-specific introns were also found. The dynamic distribution of ACT domain suggested one gene fusion event has occurred in eukaryota. Significant functional divergence was found between some subgroups. Analysis of the site-specific profiles revealed critical amino acid residues for functional divergence. This study highlights the molecular evolution of this family and may provide a starting point for further experimental verifications.     

Phylogenetic analysis and evolutionary studies of plant carotenoid cleavage dioxygenase gene

The oxidative breakdown of carotenoid evidences the formation of apocarotenoids through carotenoid cleavage dioxygenases (CCDs). Numerous CCDs and apocarotenoids have been identified and characterized in plants. Using available sequence data, a study was performed to investigate the phylogenetic relationship among CCD genes and to statistically estimate the sequence conservation and functional divergence. In total, 77 genes were identified from 39 species belonging to 21 families. Our result of phylogenetic analysis indicated the existence of well-conserved subfamilies. Moreover, comparative genomic analysis showed that the gene structures of the CCDs were highly conserved across some different lineage species. Through functional divergence analysis, a substantial divergence was found between CCD subfamilies. In addition, examination of the site-specific profile revealed the critical amino acid residues accounting for functional divergence. This study mainly focused on the evolution of CCD genes and their functional divergence which may deliver an initial step for further experimental verifications.     

Recent advances on neuronal caspases in development and neurodegeneration.

In view of a large and growing literature, this overview emphasizes recent advances in neuronal caspases and their role in cell death. To provide historical perspective, morphology and methods are surveyed with emphasis on early studies on interleukin converting enzyme (ICE) as a prototype for identifying zymogen subunits. The unexpected homology of ICE (caspase-1) to Caenorhabditis elegans death gene CED-3 provided early clues linking caspases to programmed cell death, and led later to discovery of bcl-2 proteins (CED-9 homologs) and 'apoptosis associated factors' (Apafs). Availability of substrates, inhibitors, and cDNAs led to identification of up to 16 caspases as a new superfamily of unique cysteine proteinases targeting Asp groups. Those acting as putative death effectors dismantle neurons by catabolism of proteins essential for survival. Caspases degrade amyloid precursor protein (APP), presenilins (PS1, PS2), tau, and huntingtin, raising questions on their role in neurodegeneration. Brain contains 'inhibitors of apoptosis proteins' (IAPs) survivin and NAIP associated also with some neuronal disorders. Apoptotic stress in neurons initiates a chain of events leading to activation of distal caspases by pathways that remain to be fully mapped. Neuronal caspases play multiple roles for initiation and execution of cell death, for morphogenesis, and in non-mitotic neurons for homeostasis. Recent studies focus on cytochrome c as pivotal in mediating conversion of procaspase-9 as a major initiator for apoptosis. Identifying signaling pathways and related events paves the way to design useful therapeutic remedies to prevent neuronal loss in disease or aging.     

Functional divergence after gene duplication and sequence-structure relationship: a case study of G-protein alpha subunits

In this article, we use animal G-protein alpha subunit family as an example to illustrate a comprehensive analytical pipeline for detecting different types of functional divergence of protein families, which is phylogeny-dependent, combined with ancestral sequence inference and available protein structure information. In particular, we focus on (i) Type-I functional divergence, or site-specific rate shift, as typically exemplified by amino acid residue highly conserved in a subset of homologous genes but highly variable in a different subset of homologous genes, and (ii) Type-II functional divergence, or the shift of cluster-specific amino acid property, as exemplified by a radical shift of amino acid property between duplicate genes, which is otherwise evolutionally conserved. We utilized the software DIVERGE2 to carry out these analyses. In the case of G-protein alpha subunit gene family, we have predicted amino acid residues that are related to either Type-I or Type-II functional divergence. The inferred ancestral sequences for these sites are helpful to explore the trends of functional divergence. Finally, these predicted residues are mapped to the protein structures to test whether these residues may have 3D structure or solvent accessibility preference.     

Apoptotic suppression by baculovirus P35 involves cleavage by and inhibition of a virus-induced CED-3/ICE-like protease.

Baculovirus p35 prevents programmed cell death in diverse organisms and encodes a protein inhibitor (P35) of the CED-3/interleukin-1 beta-converting enzyme (ICE)-related proteases. By using site-directed mutagenesis, we have identified P35 domains necessary for suppression of virus-induced apoptosis in insect cells, the context in which P35 evolved. During infection, P35 was cleaved within an essential domain at or near the site DQMD-87G required for cleavage by CED-3/ICE family proteases. Cleavage site substitution of alanine for aspartic acid at position 87 (D87A) of the P1 residue abolished P35 cleavage and antiapoptotic activity. Although the P4 residue substitution D84A also caused loss of apoptotic suppression, it did not eliminate cleavage and suggested that P35 cleavage is not sufficient for antiapoptotic activity. Apoptotic insect cells contained a CED-3/ICE-like activity that cleaved in vitro-translated P35 and was inhibited by recombinant wild-type P35 but not P1- or P4-mutated P35. Thus, baculovirus infection directly or indirectly activates a novel CED-3/ICE-like protease that is inhibited by P35, thereby preventing virus-induced apoptosis. Our findings confirmed the inhibitory activity of P35 towards the CED-3/ICE protease, including recombinant mammalian enzymes, and were consistent with a mechanism involving P35 stoichiometric interaction and cleavage. P35's inhibition of phylogenetically diverse proteases accounts for its general effectiveness as an apoptotic suppressor.     

Sequence and structural aspects of functional diversification in class I alpha-mannosidase evolution.

MOTIVATION: Class I alpha-mannosidases comprise a homologous and functionally diverse family of glycoside hydrolases. Phylogenetic analysis based on an amino acid sequence alignment of the catalytic domain of class I alpha-mannosidases reveals four well-supported phylogenetic groups within this family. These groups include a number of paralogous members generated by gene duplications that occurred as far back as the initial divergence of the crown-group of eukaryotes. Three of the four phylogenetic groups consist of enzymes that have group-specific biochemical specificity and/or sites of activity. An attempt has been made to uncover the role that natural selection played in the sequence and structural divergence between the phylogenetically and functionally distinct Endoplasmic Reticulum (ER) and Golgi apparatus groups. RESULTS: Comparison of site-specific amino acid variability profiles for the ER and Golgi groups revealed statistically significant evidence for functional diversification at the sequence level and indicated a number of residues that are most likely to have played a role in the functional divergence between the two groups. The majority of these sites appear to contain residues that have been fixed within one organelle-specific group by positive selection. Somewhat surprisingly these selected residues map to the periphery of the alpha-mannosidase catalytic domain tertiary structure. Changes in these peripherally located residues would not seem to have a gross effect on protein function. Thus diversifying selection between the two groups may have acted in a gradual manner consistent with the Darwinian model of natural selection. CONTACT: bishogr@millsaps.edu.     

Functional Evolution of Phosphatidylethanolamine Binding Proteins in Soybean and Arabidopsis

Gene duplication provides resources for novel gene functions. Identification of the amino acids responsible for functional conservation and divergence of duplicated genes will strengthen our understanding of their evolutionary course. Here, we conducted a systemic functional investigation of phosphatidylethanolamine binding proteins (PEBPs) in soybean (Glycine max) and Arabidopsis thaliana. Our results demonstrated that after the ancestral duplication, the lineage of the common ancestor of the FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) subfamilies functionally diverged from the MOTHER OF FT AND TFL1 (MFT) subfamily to activate flowering and repress flowering, respectively. They also underwent further specialization after subsequent duplications. Although the functional divergence increased with duplication age, we observed rapid functional divergence for a few pairs of young duplicates in soybean. Association analysis between amino acids and functional variations identified critical amino acid residues that led to functional differences in PEBP members. Using transgenic analysis, we validated a subset of these differences. We report clear experimental evidence for the functional evolution of the PEBPs in the MFT, FT, and TFL1 subfamilies, which predate the origin of angiosperms. Our results highlight the role of amino acid divergence in driving evolutionary novelty after duplication.     

ICE/CED-3 Family Executes Oligodendrocyte Apoptosis by Tumor Necrosis Factor

Abstract: Tumor necrosis factor (TNF) is thought to be one of the mediators responsible for the damage of oligodendrocytes (OLGs) in multiple sclerosis (MS). We report here the involvement of the interleukin 1β-converting enzyme (ICE)/ Caenorhabditis elegans gene ced-3 (CED-3) family in TNF-mediated cell death of OLGs. The addition of TNF-α to primary cultures of OLGs that express ice and cpp32 significantly decreased the number of live OLGs in 72 h. DNA fragmentation was detected in TNF-treated OLGs at 36 h with the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. Benzyloxycarbonyl-Asp-CH₂OC(O)-2,6-dichlorobenzene, an inhibitor of the ICE/CED-3 family that shows p35 -like inhibitory specificity, protected against the TNF-induced cell death of OLGs. Furthermore, acetyl-YVAD-CHO (a specific inhibitor of ICE-like proteases) as well as acetyl-DEVD-CHO (a specific inhibitor of CPP32-like proteases) enhanced the survival of OLGs treated with TNF-α, indicating that ICE- and the CPP32-mediated cell death pathways are activated in TNF-induced OLG cell death. Our results suggest that the inhibition of ICE/CED-3 proteases may be a novel approach to treat neurodegenerative diseases such as MS.