Browsing by Subject "Evolution, Molecular"
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Item Distinct Functional Phases in Proteins: A Test by Large-Scale Protein Design(2017-03-24) Subramanian, Subramanian Kanagarajan; Yu, Hongtao; Ranganathan, Rama; Sternweis, Paul C.; Rice, Luke M.The biological properties of proteins - folding, biochemical functions, and evolvability - originate from the global pattern of interactions between amino acids. Coevolution studies suggest a model for this pattern in which the essential constraints are loaded in sparse networks of cooperative residues (termed sectors), embedded within an environment of weakly coupled residues. Here, we test this biphasic model for proteins using a protein design approach in the SHO1-mediated yeast osmo-sensing pathway. We computationally designed libraries of synthetic SHO1 SH3 domains in which the hierarchy of coevolution that defines sectors and their environment is gradually varied. We tested the designed sequences in a quantitative high-throughput assay for SHO1 function in vivo. The data show that sector amino acids contribute in an all-or-nothing fashion while surrounding amino acids have a more graded, near-independent contribution to function. These results support the biphasic model for the information content of protein sequences.Item Doctors need evolution the way engineers need physics, but they don't get it because of politics(2018-09-11) Nesse, Randolph M.[Note: The slides are not available from this event.] The past 25 years have seen many new applications of evolutionary biology in medicine. Some investigate why natural selection has left systems vulnerable, expanding medicine's perspective from that of a mechanic to that of an engineer. Others use phylogenetic methods to trace relationships among organisms, especially pathogens. These advances have inspired a score of books, new journals, a new scientific society, and undergraduate courses in most universities. However, no medical school teaches evolutionary biology the way other basic sciences are taught, so most doctors have misconceptions that are the equivalent of engineers believing in perpetual motion. Historical, practical, political and religious factors conspire to keep evolutionary biology separate from medicine. Clinical mistakes and slowed research progress result. Recognition of the problem and the opportunity are growing but solutions are likely to be piecemeal until a new generation of doctors assumes leadership positions.Item Evolution of Pharyngeal Feeding Behaviors in Free-Living Soil Nematodes(2007-05-22) Chiang, Jing-Tzyh Alan; Avery, LeonTo explore using nematodes as a model for studying behavioral evolution, I examined pharyngeal behaviors in free-living soil nematodes related to Caenorhabditis elegans. The nematode pharynx is divided into three regions: corpus, isthmus, and terminal bulb. Pharyngeal behaviors consist of stereotyped patterns of two motions: pumping and peristalses. I observed pharyngeal behaviors in multiple species, and constructed the following evolutionary model. In the ancestor of free-living soil nematodes, the pharynx had corpus pumping, isthmus peristalses, and terminal bulb pumping, each of which occurred independently. In the Rhabditidae family, the anterior isthmus switched to pumping, and anterior isthmus and terminal bulb pumping became coupled to corpus pumping. In the Diplogasteridae family, the terminal bulb switched to peristalses. In the Cephalobidae family, isthmus peristalses and terminal bulb pumping became coupled. And in the Panagrolaimidae family, the posterior isthmus switched to pumping. The above changes in isthmus and TB behaviors suggested corresponding changes in their neuronal regulation. Using laser ablations, I found that M4’s function evolved significantly: M4 stimulated posterior isthmus peristalsis (Rhabditidae), isthmus/terminal bulb peristalsis (Diplogasteridae), isthmus peristalsis and terminal bulb pumping (Cephalobidae), and posterior isthmus/terminal bulb pumping (Panagrolaimidae). Yet, increased food activated M4 activity in all families. Thus, M4 appeared to be a general “food sensor” neuron, which was co-opted during evolution to perform different downstream functions. Additionally, M2 stimulated anterior isthmus peristalsis in the Panagrolaimidae. Using Caenorhabditis elegans, I investigated possible molecular/genetic causes for the above changes. Why is the terminal bulb unaffected by M4 in Caenorhabditis elegans and the Rhabditidae? I found that mutating slo-1 activated M4-terminal bulb stimulations, suggesting that alterations in synaptic transmission silenced M4-terminal bulb synapses. Also, why does M2 stimulate peristalses in the Panagrolaimidae, and M4 in the other families? I found ceh-28 important for M4 to stimulate peristalsis in Caenorhabditis elegans, but M2 also had the potential for ceh-28 expression. This suggested a genetic/molecular link between the two neurons, and ceh-28 related mechanisms may determine which neuron stimulates peristalsis. Overall, I characterized how pharyngeal behaviors evolved at the behavioral, neuronal, and genetic levels. These results suggested the utility of nematodes for studying behavioral evolution.Item A Global Experimental Analysis of Protein Function: A Case Study in the PDZ Domain(2011-02-01) McLaughlin, Richard Noel, Jr.; Ranganathan, RamaA complete understanding of the energetic architecture of a protein can be achieved only with a comprehensive description of the interaction of every amino acid with every other amino acid. Many efforts to understand the apparent complexity of protein function have attempted to address this problem with limited mutagenesis studies. A global computational description of amino acid interactions, Statistical Coupling Analysis has shown the existence of a contiguous subset of positions within a protein that displays significant co-evolution, termed protein sectors. Limited mutagenesis studies have shown sectors to be networks of higher-order interaction crucial for protein function; however, a theory of such global scope requires validation with a global experiment. Here, we design an assay system that measures the cellular function of a PDZ domain in a high-throughput and quantitative manner. We perform a comprehensive single amino acid mutagenesis experiment to show that most positions in the protein are robust to most mutations, and the set of positions that shows sensitivity to mutation is enriched for sector positions. Further, we perform a global pairwise epistasis experiment in which we measure the way in which every amino acid mutation in the PDZ domain feels the effect of a second mutation at a key specificity and affinity determining position in the peptide ligand of the PDZ domain. We find that those positions that show strong non-additivity in the context of the peptide mutation are all contained within the PDZ sector. Further, these sector positions that display strong non-additivity all display the property of rapidly changing specificity upon mutation. That is, any mutation at these sector positions has a negative functional effect in the context of the endogenous peptide. However, these positions appear to be spring-loaded for change since these same mutations enhance function in the context of an alternative peptide. We hypothesize that proteins are robust as shown by their insensitivity to general mutation. However, proteins are simultaneously fragile as shown by their sensitivity to specific mutagenesis at sector positions. This fragility, however, is strongly coupled to evolvability as shown by the enhancement of alternative function endowed by these endogenously detrimental mutations.Item Hierarchy of Interactions in Protein Evolution(2016-06-28) Salinas, Victor H.; Yu, Hongtao; Ranganathan, Rama; Takahashi, Joseph; Tu, BenjaminDeciphering the relationship between genotype and phenotype is complicated by the sheer number of possible cooperative interactions amongst the parts that make up biological systems. For even small systems such as individual protein domains, it has been difficult to comprehensively obtain high quality empirical data of amino acid interactions to distinguish different models for the global pattern of cooperativity. The statistical coupling analysis (SCA) - one approach for studying the co-evolution of amino acid positions in homologous sequences - provides a model for this pattern that is distinct from spatial proximity in tertiary structure, positional conservation, or even other forms of co-evolution. Here, we use an extension of deep mutational scanning to analyze nearly 50,000 single and double mutations in several homologs of a model protein - the PDZ family of protein interaction domains. Across the domains queried experimentally, the distributions of couplings between pairs of positions from all possible double mutants are well-approximated by unimodal distributions such that their average provides an estimate of the intrinsic coupling between them. Importantly, the SCA provides the best representation of this experimental pattern of couplings conserved among the homologs. These results highlight the heterogeneous pattern of couplings in protein structures and motivate the re-focus of efforts to understand protein folding and function toward the study of the origin of the co-evolving network of amino acids.Item Low-Density Lipoprotein Receptors in Signaling Modulation and Development(2010-05-14) Dietrich, Martin Frederik; Herz, JoachimThe Low-Density Lipoprotein Receptor gene family is a group of ancient membrane receptors. Originally implied in cargo transport and development of atherosclerosis, the number of members and the diversity of functions have been greatly expanded. LRP1, LRP1b and LRP4 are gene family members that are implicated in the regulation of signaling pathways at the intracellular, extracellular and transcriptional level. These regulations confer viability, control the cellular proliferation at several molecular steps, and allow for proper organ formation by moderating and integrating cellular signaling pathways. / The use of knockin mutant mice has, for the first time, implicated the extracellular domains of LRP1b and LRP4 in signaling modulation in development. While the complete knockout of either receptor is embryonically lethal, the expression of a truncated receptor, spanning only the extracellular domain, confers viability and only a mitigated phenotype. / For LRP4, the difference is most visible in the kidney. The present LRP4 extracellular preserves thresholds critical for organogenesis, yet, the complete absence displays a subpenetrant phenotype of kidney agenesis. In this thesis work, results demonstrate the ability of the LRP4 extracellular domain to not only bind a broad variety of soluble ligands in the extracellular space, but further to influence the Wnt, and possibly others, signaling pathways that are required for kidney development. / In an osteoblast-specific model of LRP1 knock-out, the relationship between theLRP1and the PDGF receptor has been further investigated. LRP1 is known to negatively regulate the PDGF receptor. However, the exact mechanism(s) are not fully understood. In the wild-type, PDGF receptor beta binds directly to LRP1 upon ligand stimulation. LRP1 knockout leads to significant upregulation of the PDGF receptor beta at the protein level. The stimulation of the receptor with PDGF-BB, its corresponding ligand, leads to overactivation of the signaling pathway with both increased turnover and phosphorylation/activation of the receptor, demonstrated by cellular proliferation and p21 downregulation. In vivo, the LRP1 knockout leads to a bone-derived hyperproliferation with formation of tumors at the epiphysis. The in vitro experiments are supporting evidence, combined with previously published literature, to imply the LRP1/PDGF receptor pathway.Item Molecular Underpinnings of Human Brain Evolution and Cognition at Cellular Resolution(December 2023) Caglayan, Emre; Chahrour, Maria; Hon, Gary C.; Madabhushi, Ram; Sun, Lu O.; Konopka, GenevieveMolecular and functional characterization of the human brain is challenging due to its experimental inaccessibility. Most of our understanding about human brain function relies on the assumption that biological processes uncovered in model organisms are conserved in humans. Comparisons of the humanii brain with non-human primate brains offer to both uncover the novelties in human brain evolution and better evaluate the insights obtained from model organisms about human brain function. To achieve this, highthroughput sequencing methods on post-mortem brain tissues provide a rewarding readout to understand human brain evolution at the molecular level. In addition to their use in comparative studies, these technologies were also utilized with a hope to understand molecular underpinnings of measurable human brain activity metrics. During my dissertation, I read relevant literature extensively (Chapter 1) and sought to understand human-specific epigenomic and transcriptomic changes at cellular resolution in the cortical brain (Chapter 2). Additionally, after in-depth analysis of many human brain single-nuclei RNA-seq datasets, I found a pervasive ambient RNA contamination problem, and devised in silico solutions to tackle this problem. My efforts improved the analytical approach in the field as well as in my research (Chapter 3). I have also been involved in efforts to identify transcriptomic correlates of brain activity in human subjects (Chapters 4-5). After detailing these efforts, I discuss the implications of these findings, weigh their impact on our understanding of human brain function and offer ideas for further research (Chapter 6).Item Multi-Scale Structure and Dynamics of Visual Signaling in Drosophila Photoreceptor Cells(2012-07-16) Helms, Stephen Jess; Ranganathan, RamaA general problem in science today is how to understand complex systems. An emerging and promising approach makes the bold assumption that complex systems adhere to particular design principles. The power of this is that design principles by definition impose an intuitive nature on a system by presupposing purpose. Existing studies have fruitfully shown the application of engineering principles in biology, but biological systems have many distinct features, particularly due to evolution. In this work, I used Drosophila phototransduction, a well-studied sensory system renowned for its high performance, to search for evolutionary design principles. I focused on three levels of structure in the system: compartmentalization of molecules into microvilli, modularity of dynamic scaffolding by InaD, and functional integration within a single domain of InaD. Using rigorous quantitative measurement and theory with an evolutionary mindset, I uncovered intuitive, simplifying design principles at each level: Microvilli are used to build fast, homogeneous signaling compartments whose dimensions are constrained by these requirements. Dynamic scaffolding is a modular feature of InaD PDZs 4-5 which have been co-inherited in many scaffolds. Within PDZ5, ligand binding and oxidation of the domain are linked through pairwise coupling with a conformational equilibrium—a generic property found in all proteins—and not each other. These results show that this approach can be successful in revealing novel design principles in complex evolved systems.Item A Tool-Box for Quantifying the Relationship Between Gene Expression, Nutrient Conditions, and Cellular Growth Rate in Bacteria(2020-08-01T05:00:00.000Z) Mathis, Andrew David; Mishra, Prashant; Lin, Milo; Reynolds, Kimberly A.; Ross, Elliott M.A central aspect of the genotype to phenotype problem is relating changes in gene expression to cellular division (or growth rate). The relationship between gene expression and growth rate can be complex, nonlinear, and dependent on environmental conditions, however, most high-throughput studies condense this complexity into a single discrete measurement per gene. This greatly limits the utility of high-throughput screening data in applications like rational engineering of cellular systems, modeling of cellular behavior, and genetic screening for specific phenotypes. I developed a series of techniques, based on highthroughput CRISPR interference gene knockdowns, to more continuously quantify the effects of gene expression and nutrient condition on bacterial growth rate. These techniques allow high-throughput titration of gene expression, precise modulation of environment conditions, and corresponding quantification of growth rate, epistasis, and gene-by-environment interactions all in the same experiment. Using these techniques, I have demonstrated that epistasis can explain co-evolution between a pair of enzymes, that gene by environment interactions are often specific to certain gene expression regimes, and that the sign and magnitude of epistasis can be dependent gene expression levels. In sum, these techniques are an essential step towards developing predictive models that relate gene expression, nutrient condition, and growth rate.Item Toward Structural and Functional Predictions from Biological Sequences(2018-05-25) Li, Wenlin; Otwinowski, Zbyszek; Grishin, Nick V.; Thomas, Philip J.; Rosenbaum, Daniel M.Biological sequences, including DNA and protein sequences, are believed to encode sufficient information to determine the structure and function of biological molecules, which in turn decide the phenotypic traits of animals. Deciphering the biological sequences is an important and multiscale problem that connecting the information flow from genotypes to phenotypes. Current advances in next-generation sequence technology provided tons of sequencing data, demanding innovations in computational algorithm for better interpretation. I developed computational methodologies to understand the biological sequences in various levels. In the primary sequence level, I analyzed the evolutionary information encoded in protein families and predicted the function (and active sites) of the proteins. To aid my sequence analysis, I developed a set of computational methodologies and deployed them as public web-servers. In the protein structure level, I studied the plasticity of the 3D structures, as well as demonstrated its effect on the uncertainty of computational scoring algorithms. In the organism level, I innovated the computational methodology to assemble and analyze complete genomes of butterflies and discovered convergence evolution in butterfly wing patterns. In conclusion, I advanced the knowledge of biological sequences in multi-layers by computational approaches.Item Using Evolutionary Statistics to Understand Cellular Systems(2019-11-18) Schober, Andrew Frank, Jr.; Lin, Milo; Reynolds, Kimberly A.; Reese, Michael L.; Tu, BenjaminMetabolic enzyme function is dependent on the larger context of a biochemical pathway. Despite detailed characterization of the requisite molecular "parts," it remains difficult to predict the adaptive response to a simple perturbation. That is: if the activity or expression of a single enzyme is changed, what other proteins (if any) require compensatory mutation? Comparative genomics and experimental evolution provide two powerful approaches to begin addressing these questions. In my thesis work, I examined adaptive interactions with the essential enzyme dihydrofolate reductase (DHFR). Analyses of gene synteny and co-occurrence across 1445 bacterial genomes indicated that DHFR coevolves with thymidylate synthase (TYMS), but is relatively decoupled from the rest of the folate metabolic pathway (and genome). Through directed evolution of E. coli, I demonstrated that these two enzymes adapt cooperatively in response to antibiotic stress. An allele replacement experiment confirmed that a pair of mutations to DHFR and TYMS were sufficient to reconstitute the entire trimethoprim resistance phenotype, establishing that the two enzymes are capable of independently driving adaptation. In the final component of my thesis, I drew on the 'mirror-tree' method to define a new measure of residue-residue coevolution which corrects for the phylogenetic relationship among species. In summary, my results verify that small groups of genes within larger metabolic pathways can form adaptive modules that evolve as a unit in response to environmental or mutational stress. Moreover, my mirror-tree inspired analysis provides a path forward for understanding how coupled adaptation between genes manifests at the resolution of site specific constraints on the protein sequence.