FFAME.org :: Eric Gaucher's Publications

Paleotemperature trend for Precambrian life inferred from resurrected proteins
Gaucher, EA Ganesh, O Govindarajan, S
Nature (2007) In press

Computational reconstruction of ancestral genomic regions from evolutionarily conserved gene clusters
Danchin, EGJ Gaucher, EA Pontarotti, P
Ancestral Sequence Reconstruction, ed. David A. Liberles, Oxford University Press 139-150 (2007)

Experimental resurrection of ancient biomolecules: gene synthesis, heterologous protein expression, and functional assays
Gaucher, EA
Ancestral Sequence Reconstruction, ed. David A. Liberles, Oxford University Press 153-163 (2007)

A thermophilic last universal ancestor inferred from its estimated amino acid composition
Brooks, DJ Gaucher, EA
Ancestral Sequence Reconstruction, ed. David A. Liberles, Oxford University Press 200-207 (2007)

Ancestral sequence reconstruction as a tool to understand natural history and guide synthetic biology: realizing and extending the vision of Zuckerkandl and Pauling
Gaucher, EA
Ancestral Sequence Reconstruction, ed. David A. Liberles, Oxford University Press 20-33 (2007)

Molecular evolutionary models to guide experiments in protein engineering and directed evolution
Gaucher, EA
(2007) In review

Molecular Paleoscience: Systems Biology from the Past
Benner, SA Sassi, SO Gaucher, EA
Adv. Enzymol. Relat. Areas Mol. Biol. 75 (2006)

Analysis of transitions at two-fold redundant sites in mammalian genomes. Transition redundant approach-to-equilibrium (TREx) distance metrics
Li, T Chamberlin, SG Caraco, MD Liberles, DA Gaucher, EA Benner, SA
BMC Evol. Biol. 6 25 (2006)
<Abstract>

Background: The exchange of nucleotides at synonymous sites in a gene encoding a protein is believed to have little impact on the fitness of a host organism. This should be especially true for synonymous transitions, where a pyrimidine nucleotide is replaced by another pyrimidine, or a purine is replaced by another purine. This suggests that transition redundant exchange ( TREx) processes at the third position of conserved two-fold codon systems might offer the best approximation for a neutral molecular clock, serving to examine, within coding regions, theories that require neutrality, determine whether transition rate constants differ within genes in a single lineage, and correlate dates of events recorded in genomes with dates in the geological and paleontological records. To date, TREx analysis of the yeast genome has recognized correlated duplications that established a new metabolic strategies in fungi, and supported analyses of functional change in aromatases in pigs. TREx dating has limitations, however. Multiple transitions at synonymous sites may cause equilibration and loss of information. Further, to be useful to correlate events in the genomic record, different genes within a genome must suffer transitions at similar rates. Results: A formalism to analyze divergence at two fold redundant codon systems is presented. This formalism exploits two-state approach-to-equilibrium kinetics from chemistry. This formalism captures, in a single equation, the possibility of multiple substitutions at individual sites, avoiding any need to "correct" for these. The formalism also connects specific rate constants for transitions to specific approximations in an underlying evolutionary model, including assumptions that transition rate constants are invariant at different sites, in different genes, in different lineages, and at different times. Therefore, the formalism supports analyses that evaluate these approximations. Transitions at synonymous sites within two-fold redundant coding systems were examined in the mouse, rat, and human genomes. The key metric (f(2)), the fraction of those sites that holds the same nucleotide, was measured for putative ortholog pairs. A transition redundant exchange ( TREx) distance was calculated from f(2) for these pairs. Pyrimidine-pyrimidine transitions at these sites occur approximately 14% faster than purine-purine transitions in various lineages. Transition rate constants were similar in different genes within the same lineages; within a set of orthologs, the f(2) distribution is only modest overdispersed. No correlation between disparity and overdispersion is observed. In rodents, evidence was found for greater conservation of TREx sites in genes on the X chromosome, accounting for a small part of the overdispersion, however. Conclusion: The TREx metric is useful to analyze the history of transition rate constants within these mammals over the past 100 million years. The TREx metric estimates the extent to which silent nucleotide substitutions accumulate in different genes, on different chromosomes, with different compositions, in different lineages, and at different times.

Application of DETECTER, an Evolutionary Genomic Tool to Analyze Genetic Variation, to the Cystic Fibrosis Gene Family
Gaucher, EA DeKee, DW Benner, SA
BMC Genomics 7 44 (2006)
<Abstract>

Background: The medical community requires computational tools that distinguish genetic differences having phenotypic impact within the vast number of mutations that do not. Tools that do this will become increasingly important for those seeking to use human genome sequence data to predict disease, make prognoses, and customize therapy to individual patients.
Results: An approach, termed DETECTER, is proposed to identify sites in a protein sequence where amino acid replacements are likely to have a significant effect on phenotype, including causing genetic disease. This approach uses a model-dependent tool to estimate the normalized replacement rate at individual sites in a protein sequence, based on a history of those sites extracted from an evolutionary analysis of the corresponding protein family. This tool identifies sites that have higher-than-average, average, or lower- than-average rates of change in the lineage leading to the sequence in the population of interest. The rates are then combined with sequence data to determine the likelihoods that particular amino acids were present at individual sites in the evolutionary history of the gene family. These likelihoods are used to predict whether any specific amino acid replacements, if introduced at the site in a modern human population, would have a significant impact on fitness. The DETECTER tool is used to analyze the cystic fibrosis transmembrane conductance regulator (CFTR) gene family.
Conclusions: In this system, DETECTER retrodicts amino acid replacements associated with the cystic fibrosis disease with greater accuracy than alternative approaches. While this result validates this approach for this particular family of proteins only, the approach may be applicable to the analysis of polymorphisms generally, including SNPs in a human population.

The diverse biological functions of phosphatidylinositol transfer proteins in eukaryotes
Phillips, SE Vincent, P Rizzieri, KE Schaaf, G Bankaitis, VA Gaucher, EA
Crit. Rev. Biochem. Mol. Biol. 41 (1) 21-49 (2006)
<Abstract>

Phosphatidylinositol/phosphatidylcholine transfer proteins (PITPs) remain largely functionally uncharacterized, despite the fact that they are highly conserved and are found in all eukaryotic cells thus far examined by biochemical or sequence analysis approaches. The available data indicate a role for PITPs in regulating specific interfaces between lipid-signaling and cellular function. In this regard, a role for PITPs in controlling specific membrane trafficking events is emerging as a common functional theme. However, the mechanisms by which PITPs regulate lipid-signaling and membrane-trafficking functions remain unresolved. Specific PITP dysfunctions are now linked to neurodegenerative and intestinal malabsorbtion diseases in mammals, to stress response and developmental regulation in higher plants, and to previously uncharacterized pathways for regulating membrane trafficking in yeast and higher eukaryotes, making it clear that PITPs are integral parts of a highly conserved signal transduction strategy in eukaryotes. Herein, we review recent progress in deciphering the biological functions of PITPs, and discuss some of the open questions that remain.

A call for likelihood phylogenetics even when the process of sequence evolution is heterogeneous
Gaucher, EA Miyamoto, MM
Mol. Phylogenet. Evol. 37 (3) 928-931 (2005)
<Abstract>

All methods of phylogenetic inference make assumptions about the underlying evolutionary process of their characters and it is these assumptions that determine their relative successes and failures in the estimation of the true phylogeny for a group. This dependency of phylogenetic accuracy and robustness on evolutionary assumptions has been most extensively studied for the classic case of Felsenstein (1978) and its four-taxon phylogeny with two long, unrelated, terminal branches interspersed with two short ones. Given this model phylogeny, "long branch attraction" can occur and thereby lead to the convergence of a phylogenetic method onto an incorrect tree with the two long and two short terminal branches directly connected rather than interspersed. The extent to which a particular phylogenetic method is susceptible to this problem depends on what assumptions it makes about the evolution of the characters and data themselves.

Resurrecting ancestral alcohol dehydrogenases from yeast
Thomson, JM Gaucher, EA Burgan, MF De Kee, DW Li, T Aris, JP Benner, SA
Nature Genet. 37 (6) 630-635 (2005)
<Abstract>

Modern yeast living in fleshy fruits rapidly convert sugars into bult ethanol through pyruvate. Pyruvate loses carbon dioxide to become acetaldehyde, which is reduced by alcohol dehydrogenase 1 (Adh1) to ethanol, which accumulates. Yeast later consumes the accumulated ethanol, exploiting Adh2, an Adh1 homolog differing by 24 (of 348) amino acids. Because many microorganisms cannot grow in ethanol, accumulated ethanol may help yeast defend resources in the fruit. We report here the reconstruction of the last common ancestor of Adh1 and Adh2, called AdhA. The kinetic behavior of AdhA suggests that it was optimized to make (not consume) ethanol. This is consistent with the hypothesis that before the Adh1-Adh2 duplication, yeast did not accumulate ethanol for later consumption but rather used AdhA to recycle NADH generated in the glycolytic pathway. Silent nucleotide dating suggests that the Adh1-Adh2 duplication occurred near the time of duplication of several other proteins involved in the accumulation of ethanol, possibly in the Cretaceous age when fleshy fruits arose. These results help to connect the chemical behavior of these enzymes through systems analysis to a time of global ecosystem change, a small but useful step towards a planetary systems biology.

Inferred thermophily of the Last Universal Ancestor based on estimated amino acid composition
Brooks, DJ Gaucher, EA
(2005) Submitted
<Abstract>

The environmental temperature of the last universal ancestor (LUA) of all extant organisms is the subject of heated debate. Because the amino acid composition of proteins differs between mesophiles and thermophiles, the inferred amino acid composition of proteins in the LUA could be used to classify it as one or the other. We applied expectation maximization (EM) to estimate the amino acid composition of a set of thirty-one proteins in the LUA based on alignments of their modern day descendants, a phylogenetic tree relating those descendants and a model of evolution. Separate estimates of amino acid composition in LUA proteins were derived using modern day sequences of eight mesophilic species, eight thermophilic species or the sixteen species combined. We show that the relative mean Euclidean distance between the amino acid composition in one species and that of a set of mesophiles or thermophiles can be employed as a classifier with 100% accuracy. Applying this classifier to the estimated amino acid composition of the ancestral protein set in the LUA, we find it to be classified as a thermophile even when only the proteins of mesophilic species are used to derive the estimate. Based on the estimated amino acid composition of proteins in the LUA, we infer that it was a thermophile. We discuss our findings in the context of previous data pertaining to the OGT of the LUA, particularly the inferred G + C content of its rRNA. We conclude that the gathering evidence strongly supports a thermophilic LUA.

Cytoplasmic glycosylation of protein-hydroxyproline and its relationship to other glycosylation pathways
West, CM van der Wel, H Sassi, S Gaucher, EA
Biochim. Biophys. Acta 1673 (1-2) 29-44 (2004)
<Abstract>

The Skp1 protein, best known as a subunit of E3(SCF)-ubiquitin ligases, is subject to complex glycosylation in the cytoplasm of the cellular slime mold Dictyostelium. Pro143 of this protein is sequentially modified by a prolyl hydroxylase and five soluble glycosyltransferases (GT), to yield the structure Galalpha1,Galalpha1,3Fucalpha1,2Galbeta1,3GlcNAcalpha1-HyPro143. These enzymes are unusual in that they are expressed in the cytoplasmic compartment of the cell, rather than the secretory pathway where complex glycosylation of proteins usually occurs. The first enzyme in the pathway appears to be related to the soluble animal prolyl 4-hydroxylases (P4H), which modify the transcriptional factor subunit HIF-1alpha in the cytoplasm, and more distantly to the P4Hs that modify collagen and other proteins in the rER, based on biochemical and informatics analyses. The soluble alphaGlcNAc-transferase acting on Skp1 has been cloned and is distantly related to the mucin-type polypeptide N-acetyl-alpha-galactosaminyltransferase in the Golgi of animals. Its characterization has led to the discovery of a family of related polypeptide N-acetyl-alpha-glucosaminyltransferases in the Golgi of selected lower eukaryotes. The Skp1 GlcNAc is extended by a bifunctional diglycosyltransferase that sequentially and apparently processively adds beta1,3Gal and alpha1,2Fuc. Though this structure is also formed in the animal secretory pathway, the GTs involved are dissimilar. Conceptual translation of available genomes suggests the existence of this kind of complex cytoplasmic glycosylation in other eukaryotic microorganisms, including diatoms, oomycetes, and possibly Chlamydomonas and Toxoplasma, and an evolutionary precursor of this pathway may also occur in prokaryotes. (C) 2004 Elsevier B.V. All rights reserved.

The planetary biology of cytochrome P450 aromatases
Gaucher, EA Graddy, LG Li, T Simmen, RC Simmen, FA Schreiber, DR Liberles, DA Janis, CM Benner, SA
BMC Biology 2 (1) 19 (2004)
<Abstract>

BACKGROUND: Joining a model for the molecular evolution of a protein family to the paleontological and geological records (geobiology), and then to the chemical structures of substrates, products, and protein folds, is emerging as a broad strategy for generating hypotheses concerning function in a post-genomic world. This strategy expands systems biology to a planetary context, necessary for a notion of fitness to underlie (as it must) any discussion of function within a biomolecular system.
RESULTS: Here, we report an example of such an expansion, where tools from planetary biology were used to analyze three genes from the pig Sus scrofa that encode cytochrome P450 aromatases-enzymes that convert androgens into estrogens. The evolutionary history of the vertebrate aromatase gene family was reconstructed. Transition redundant exchange silent substitution metrics were used to interpolate dates for the divergence of family members, the paleontological record was consulted to identify changes in physiology that correlated in time with the change in molecular behavior, and new aromatase sequences from peccary were obtained. Metrics that detect changing function in proteins were then applied, including KA/KS values and those that exploit structural biology. These identified specific amino acid replacements that were associated with changing substrate and product specificity during the time of presumed adaptive change. The combined analysis suggests that aromatase paralogs arose in pigs as a result of selection for Suoidea with larger litters than their ancestors, and permitted the Suoidea to survive the global climatic trauma that began in the Eocene.
CONCLUSIONS: This combination of bioinformatics analysis, molecular evolution, paleontology, cladistics, global climatology, structural biology, and organic chemistry serves as a paradigm in planetary biology. As the geological, paleontological, and genomic records improve, this approach should become widely useful to make systems biology statements about high-level function for biomolecular systems.

Significance of cytoplasmic prolyl hydroxylation and complex glycosylation in the cellular slime mold Dictyostelium
West, CM van der Wel, H Sassi, S Gaucher, E Ercan, A
Glycobiology 14 (11) 1063-1063 (2004)

Evolutionary, structural and biochemical evidence for a new interaction site of the leptin obesity protein
Gaucher, EA Miyamoto, MM Benner, SA
Genetics 163 (4) 1549-1553 (2003)
<Abstract>

The Leptin protein is central to the regulation of energy metabolism in mammals. By integrating evolutionary, structural, and biochemical information, a surface segment, outside of its known receptor contacts, is predicted as a second interaction site that may help to further define its roles in energy balance and its functional differences between humans and other mammals.

Initiation of mucin-type O-glycosylation in lower eukaryotes (O-alpha-GlcNAc-type) and higher eukaryotes (O-alpha-GalNAc-type) is homologous
West, CM Wang, F van der Wel, H Gaucher, E Sassi, S Metcalf, T Heise, N Mendonca-Previato, L Previato, JO
Glycobiology 13 (11) 875-876 (2003)

Inferring the palaeoenvironment of ancient bacteria on the basis of resurrected proteins
Gaucher, EA Thomson, JM Burgan, MF Benner, SA
Nature 425 (6955) 285-288 (2003)
<Abstract>

Features of the physical environment surrounding an ancestral organism can be inferred by reconstructing sequences(1-9) of ancient proteins made by those organisms, resurrecting these proteins in the laboratory, and measuring their properties. Here, we resurrect candidate sequences for elongation factors of the Tu family (EF-Tu) found at ancient nodes in the bacterial evolutionary tree, and measure their activities as a function of temperature. The ancient EF-Tu proteins have temperature optima of 55-65degreesC. This value seems to be robust with respect to uncertainties in the ancestral reconstruction. This suggests that the ancient bacteria that hosted these particular genes were thermophiles, and neither hyperthermophiles nor mesophiles. This conclusion can be compared and contrasted with inferences drawn from an analysis of the lengths of branches in trees joining proteins from contemporary bacteria(10), the distribution of thermophily in derived bacterial lineages(11), the inferred G+C content of ancient ribosomal RNA(12), and the geological record combined with assumptions concerning molecular clocks(13). The study illustrates the use of experimental palaeobiochemistry and assumptions about deep phylogenetic relationships between bacteria to explore the character of ancient life.

Complex glycosylation of Skp1 in Dictyostelium: implications for the modification of other eukaryotic cytoplasmic and nuclear proteins
West, CM van der Wel, H Gaucher, EA
Glycobiology 12 (2) 17R-27R (2002)
<Abstract>

Recently, complex O-glycosylation of the cytoplasmic/nuclear protein Skp1 has been characterized in the eukaryotic microorganism Dirtyostelium. Skp1's glycosylation is mediated by the sequential action of a prolyl hydroxylase and five conventional sugar nucleotide-dependent glycosyltransferase activities that reside in the cytoplasm rather than the secretory compartment. The Skp1-HyPro GlcNAc-Transferase, which adds the first sugar, appears to be related to a lineage of enzymes that originated in the prokaryotic cytoplasm and initiates mucin-type O-linked glycosylation in the lumen of the eukaryotic Golgi apparatus. GlcNAc is extended by a bifunctional glycosyltransferase that mediates the ordered addition of beta1,3-linked Gal and alpha1,2-linked Fuc. The architecture of this enzyme resembles that of certain two-domain prokaryotic glycosyl-transferases. The catalytic domains are related to those of a large family of prokaryotic and eukaryotic, cytoplasmic, membrane-bound, inverting glycosyltransferases that modify glycolipids and polysaccharides prior to their translocation across membranes toward the secretory pathway or the cell exterior. The existence of these enzymes in the eukaryotic cytoplasm away from membranes and their ability to modify protein acceptors expose a new set of cytoplasmic and nuclear proteins to potential prolyl bydroxylation and complex O-linked glycosylation.

Identification of a Golgi-associated UDP-GlcNAc : polypeptide mucin-type alpha-N-acetylglucosaminyltransferase that modifies cell surface proteins in Dictyostelium
West, CM van der Wel, H Metcalf, T Kaplan, L Gaucher, EA
Glycobiology 12 (10) 697-697 (2002)

The crystal structure of eEF1A refines the functional predictions of an evolutionary analysis of rate changes among elongation factors
Gaucher, EA Das, UK Miyamoto, MM Benner, SA
Mol. Biol. Evol. 19 (4) 569-573 (2002)

Evolution - Planetary biology - Paleontological, geological, and molecular histories of life
Benner, SA Caraco, MD Thomson, JM Gaucher, EA
Science 296 (5569) 864-868 (2002)
<Abstract>

The history of life on Earth is chronicled in the geological strata, the fossil record, and the genomes of contemporary organisms. When examined together, these records help identify metabolic and regulatory pathways, annotate protein sequences, and identify animal models to develop new drugs, among other features of scientific and biomedical interest. Together, planetary analysis of genome and proteome databases is providing an enhanced understanding of how life interacts with the biosphere and adapts to global change.

Predicting functional divergence in protein evolution by site-specific rate shifts
Gaucher, EA Gu, X Miyamoto, MM Benner, SA
Trends Biochem. Sci. 27 (6) 315-321 (2002)
<Abstract>

Most modern tools that analyze protein evolution allow individual sites to mutate at constant rates over the history of the protein family. However, Walter Fitch observed in the 1970s that, if a protein changes its function, the mutability of individual sites might also change. This observation is captured in the 'non-homogeneous gamma model', which extracts functional information from gene families by examining the different rates at which individual sites evolve. This model has recently been coupled with structural and molecular biology to identify sites that are likely to be involved in changing function within the gene family. Applying this to multiple gene families highlights the widespread divergence of functional behavior among proteins to generate paralogs and orthologs.

A bifunctional diglycosyltransferase forms the Fuca1,2Galb,3-disaccharide on Skp1 in the cytoplasm of Dictyostelium
van der Wel, H Fisher, SZ Gaucher, EA West, CM
Glycobiology 11 (10) 884-884 (2001)

Function-structure analysis of proteins using covarion-based evolutionary approaches: Elongation factors
Gaucher, EA Miyamoto, MM Benner, SA
Proc. Natl. Acad. Sci. USA 98 (2) 548-552 (2001)
<Abstract>

The divergent evolution of protein sequences from genomic databases can be analyzed by the use of different mathematical models. The most common treat all sites in a protein sequence as equally variable. More sophisticated models acknowledge the fact that purifying selection generally tolerates variable amounts of amino acid replacement at different positions in a protein sequence. In their "stationary" versions, such models assume that the replacement rate at individual positions remains constant throughout evolutionary history. "Nonstationary" covarion versions, however, allow the replacement rate at a position to vary in different branches of the evolutionary tree. Recently, statistical methods have been developed that highlight this type of variation in replacement rates. Here, we show how positions that have variable rates of divergence in different regions of a tree ("covarion behavior"), coupled with analyses of experimental three-dimensional structures, can provide experimentally testable hypotheses that relate individual amino acid residues to specific functional differences in those branches. We illustrate this in the elongation factor family of proteins as a paradigm for applications of this type of analysis in functional genomics generally.

Evolution, language and analogy in functional genomics
Benner, SA Gaucher, EA
Trends in Genetics 17 (7) 414-418 (2001)
<Abstract>

Almost a century ago, Wittgenstein pointed out that theory in science is intricately connected to language. This connection is not a frequent topic in the genomics literature. But a case can be made that functional genomics is today hindered by the paradoxes that Wittgenstein identified. If this is true, until these paradoxes are recognized and addressed, functional genomics will continue to be limited in its ability to extrapolate information from genomic sequences.