The mitogen-activated protein kinome from Anopheles gambiae: identification, phylogeny and functional characterization of the ERK, JNK and p38 MAP kinases

<p>Abstract</p> <p>Background</p> <p><it>Anopheles gambiae </it>is the primary mosquito vector of human malaria parasites in sub-Saharan Africa. To date, three innate immune signaling pathways, including the nuclear factor (NF)-kappaB-dependent Toll and immune deficient (IMD) pathways and the Janus kinase/signal transducers and activators of transcription (Jak-STAT) pathway, have been extensively characterized in <it>An. gambiae</it>. However, in addition to NF-kappaB-dependent signaling, three mitogen-activated protein kinase (MAPK) pathways regulated by JNK, ERK and p38 MAPK are critical mediators of innate immunity in other invertebrates and in mammals. Our understanding of the roles of the MAPK signaling cascades in anopheline innate immunity is limited, so identification of the encoded complement of these proteins, their upstream activators, and phosphorylation profiles in response to relevant immune signals was warranted.</p> <p>Results</p> <p>In this study, we present the orthologs and phylogeny of 17 <it>An. gambiae </it>MAPKs, two of which were previously unknown and two others that were incompletely annotated. We also provide detailed temporal activation profiles for ERK, JNK, and p38 MAPK in <it>An. gambiae </it>cells <it>in vitro </it>to immune signals that are relevant to malaria parasite infection (human insulin, human transforming growth factor-beta1, hydrogen peroxide) and to bacterial lipopolysaccharide. These activation profiles and possible upstream regulatory pathways are interpreted in light of known MAPK signaling cascades.</p> <p>Conclusions</p> <p>The establishment of a MAPK "road map" based on the most advanced mosquito genome annotation can accelerate our understanding of host-pathogen interactions and broader physiology of <it>An. gambiae </it>and other mosquito species. Further, future efforts to develop predictive models of anopheline cell signaling responses, based on iterative construction and refinement of data-based and literature-based knowledge of the MAP kinase cascades and other networked pathways will facilitate identification of the "master signaling regulators" in biomedically important mosquito species.</p

Evolution of protein domain architectures

This chapter reviews current research on how protein domain architectures evolve. We begin by summarizing work on the phylogenetic distribution of proteins, as this will directly impact which domain architectures can be formed in different species. Studies relating domain family size to occurrence have shown that they generally follow power law distributions, both within genomes and larger evolutionary groups. These findings were subsequently extended to multi-domain architectures. Genome evolution models that have been suggested to explain the shape of these distributions are reviewed, as well as evidence for selective pressure to expand certain domain families more than others. Each domain has an intrinsic combinatorial propensity, and the effects of this have been studied using measures of domain versatility or promiscuity. Next, we study the principles of protein domain architecture evolution and how these have been inferred from distributions of extant domain arrangements. Following this, we review inferences of ancestral domain architecture and the conclusions concerning domain architecture evolution mechanisms that can be drawn from these. Finally, we examine whether all known cases of a given domain architecture can be assumed to have a single common origin (monophyly) or have evolved convergently (polyphyly). We end by a discussion of some available tools for computational analysis or exploitation of protein domain architectures and their evolution

EXPANDING THE POTENTIAL PRENYLOME: PRENYLATION OF SHORTENED TARGET SUBSTRATES BY FTASE AND DEVELOPMENT OF FRET-BASED SYSTEM FOR DETECTING POTENTIALLY “SHUNTED” PROTEINS

Protein prenylation is a posttranslational modification involving the attachment of a C15 or C20 isoprenoid group to a cysteine residue near the C-terminus of the target substrate by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I), respectively. Both of these protein prenyltransferases recognize a C-terminal CaaX sequence in their protein substrates, but recent studies in yeast- and mammalian-based systems have demonstrated FTase can also accept sequences that diverge in length from the canonical four-amino acid motif, such as the recently reported five-amino acid C(x)3X motif. In this work, we further expand the substrate scope of FTase by demonstrating sequence-dependent farnesylation of shorter three-amino acid Cxx C-terminal sequences using both genetic and biochemical assays. Surprisingly, biochemical assays utilizing purified mammalian FTase and Cxx substrates reveal prenyl donor promiscuity leading to both farnesylation and geranylgeranylation of these sequences. The work herein expands the substrate pool of sequences that can be potentially prenylated, further refines our understanding of substrate recognition by FTase and GGTase-I and suggests the possibility of a new class of prenylated proteins within proteomes. To identify potential new Cxx substrates in human proteomes, we explored a FRET-based system using phosphodiesterase delta subunit (PDE) as the acceptor protein for potentially prenylated Cxx sequences. While not conclusive, this work lays the foundation for an assay not dependent on membrane localization as a signal for prenylation inside cells and suggests future studies to improve upon the utility of this assay. Lastly, this work demonstrates FTase’s flexibility in accepting a prenyl donor analogue with an azobenzene moiety that can be modulated with light. This establishes a potential new avenue for mediating membrane localization behavior of prenylated proteins

Replisome-mediated homeostasis of DNA/RNA hybrids in eukaryotic genomes is critical for cell fates and chromatin stability

During DNA replication, forks often stall upon encountering obstacles blocking their progression. Cells will act to speedily remove or overcome such barriers, thus allowing complete synthesis of chromosomes. This is the case for R-loops, DNA/RNA hybrids that arise during transcription. One mechanism to remove such R-loops involve DNA/RNA helicases. Here, I have shown that one such helicase, Sen1, associates with replisome components during S phase in the model organism S. cerevisiae. I demonstrate that the N-terminal domain of Sen1 is both sufficient and necessary for the interaction of the protein with the replisome. I also identified Ctf4 as one of at least two replisome interactors of Sen1. By mutational analysis, a mutant of Sen1 (Sen1-3) that cannot interact with the replisome was created. This mutant is healthy on its own but is lethal in the absence of both RNase H1 and H2. Overexpression of the sen1-3 allele from the constitutive ACT1 promoter is able to suppress this synthetic lethality, suggesting that Sen1 travels with replisomes in order to be quickly recruited at sites of R-loops that impair fork progression so as to remove those R-loops. In some cases, cells exploit fork stalling for biologically important processes. This is the case in Sz. pombe, where an imprint prevents complete DNA replication, triggering cell-type switching. This imprint is dependent on Pol1, a component of the replisome. Importantly, a single imprinting-defective allele of pol1 has been identified to date. Using in vitro assays, I have shown that this Pol1 mutant has reduced affinity for its substrates and is a correspondingly poor polymerase. By generating novel alleles of pol1, I have also demonstrated that switching-deficiency correlates with the affinity of Pol1 for its substrates in vivo. Finally, two interactors of Pol1 (Mcl1Ctf4 and Spp1Pri1 ) have been shown to have switching defects. S. cerevisiae and Sz. pombe have similar yet distinct genetic nomenclature conventions. Given that both model organisms were used in this study, it is important to highlight the conventions for both organisms to prevent confusion. In S. cerevisiae, wildtype gene names are expressed as a three letter, uppercase and italic name followed by a number (e.g. SEN1). The three letter name often corresponds to the screen through which the gene in question was originally identified. Mutants are generally designated with the same three letter but in lower case (unless the mutant is dominant) and with an allele designation (e.g. sen1∆, sen1-1 and sen1-2). Because of historical context, the allele designations vary in format (e.g. leu2-3,112 is a mutant of LEU2). Protein names are given as a three letter name with the first letter in uppercase (e.g. Sen1). This is also true for mutant proteins, with the added allele designation (e.g Sen1-1 and Sen1-2). In this study, I have generated constructs of the SEN1 gene and these constructs are referred to as SEN1 (X-Y), where X and Y refer to the first and last residues being encoded for. The corresponding proteins are referred to as Sen1 (X-Y). Different promoters have been used and, where appropriate, the promoters are expressed similarly to their wildtype gene names (e.g. GAL1, SEN1 and ACT1). In Sz. pombe, wildtype gene names are expressed as a three letter, lowercase and italic name followed by a number (e.g. pol1). Mutants are generally designated in the same format but with an allele designation. Like in S. cerevisiae, the allele designation varies widely (e.g. pol1-1, pol1-H4 and pol1-ts13). Additionally, because of the historical context, some (but not all) alleles of pol1 are referred to as swi7 to reflect the fact that they are defective for cell-type switching. Similar to the situation in S. cerevisiae, proteins names are given as a three letter name with the first letter in uppercase for both wildtype and mutants (e.g. Pol1 and Swi7-1). Sometimes, for the sake of comparison, genes or proteins are referred to their S. cerevisiae orthologues (e.g. swi1TOF1 and Swi1Tof1 , respectively). Several protein tags have been used in this study. When written in gene form, they were written in capital letters and italicized, irrespective of the host (e.g. 5FLAG) and when in protein form, they were written in capital, irrespective of the host (e.g. 5FLAG)

Large scale prediction and reverse genetics analysis of programmed cell death genes in Chlamydomonas reinhardtii

Programmed cell death (PCD) refers to any form of cell death that is coordinated by the genome. PCD consists of complex molecular pathways which directly cause the death of a cell. The most well-known form of PCD is an animal-specific process known as apoptosis, of which the underlying molecular pathways are well-characterized. However, despite the observation that PCD occurs ubiquitously throughout the tree of life, little is known regarding the molecular mechanisms of PCD in non-animal systems. In response to a number of different environmental stressors, Chlamydomonas reinhardtii undergoes a form of PCD which exhibits characteristics of apoptosis, including DNA laddering, accumulation of reactive oxygen species, and externalization of phosphatidylserine. The presence of these shared features between C. reinhardtii PCD and apoptosis suggests that similar molecular pathways may underlie the two processes. Despite this, many of the genes required for apoptosis in animals appear to be absent in C. reinhardtii. In the present study, we first employed a large-scale, homology-based, bioinformatics approach to predict the gene products that contribute to C. reinhardtii PCD. From the list of sequences that were obtained by these methods, we selected several entries to study in further detail using a reverse genetic approach. We obtained C. reinhardtii mutant strains, each with an insertional mutation in one of the selected genes, from the Chlamydomonas Library Project (CLiP) and validated that the insert had been mapped accurately in each of the mutant strains. To determine the effects of losing any of these selected genes, we subjected the mutant and parental background strains to a PCD-inducing heat stress. First, we sought to determine if the loss of a single selected gene would affect the ability of cells to undergo PCD in response to stress. Second, we wanted to determine if the loss of one of the selected genes would alter either the timing or intensity of phenotypes characteristic of C. reinhardtii PCD. Our results suggest a role for several of the selected genes in PCD, and future studies will be aimed at further characterizing these roles in more detail

Ethical issues of synthetic biology: a personalist perspective

The main objective of this thesis is to assess the bioethical issues raised by Synthetic Biology from a specific bioethical approach, personalism, specifically ontological personalism, a philosophy that shows the objective value of the person on the basis of its ontological structure. The person, as a being endowed with reason, freedom and awareness, has a special value which is above that of other beings.El objetivo principal de este trabajo es evaluar las cuestiones bioéticas planteadas por la Biología Sintética desde un enfoque bioético específico, el personalismo, específicamente el personalismo ontológico, una filosofía que muestra el valor objetivo de la persona sobre la base de su estructura ontológica.Ciencias ExperimentalesPrograma Oficial de Doctorado en Bioétic

Renewable Fuels and Chemicals from the Organic Fraction of Municipal Solid Waste

Municipal solid waste (MSW) is any non-industrial waste produced in households and public or commercial institutions. 3.4 billion tonnes of MSW will be produced annually by 2050, but unsustainable practices like landfilling and incineration currently dominate MSW management. The organic fraction of MSW (OMSW) typically comprises ~50% lignocellulose-rich material but is underexplored as a biomanufacturing feedstock. This thesis investigated OMSW as a feedstock for producing renewable biofuels and chemicals. Uniquely, the OMSW-derived fibre used in this project was produced via a commercial autoclave pre-treatment from a realistic and reproducible MSW mixture. The OMSW fibre was subjected to comprehensive compositional analysis and hydrolysis, and OMSW hydrolysate was analysed for sugars, metals and marker inhibitors to evaluate fermentability. Waste residues were investigated as a feedstock for biogas production. Next, the growth and productivity of eight diverse and biotechnologically useful microbial species was characterised on OMSW fibre hydrolysate supplemented with 1% yeast extract and the best candidate was further characterised and improved for industrial applications. The OMSW fibre contained a large polysaccharide fraction, comprising 38% cellulose and 4% hemicellulose. Hydrolysate of OMSW fibre was high in D-glucose and D-xylose, low in inhibitors, deficient in nitrogen and phosphate and abundant in potentially toxic metals. Hydrolysis residues contained a six-fold greater metal concentration but generated 33.4% more biomethane in anaerobic digestion compared to unhydrolysed fibre. Microbial screening identified three species that robustly and efficiently fermented OMSW fibre hydrolysate: Saccharomyces cerevisiae, Zymomonas mobilis and Rhodococcus opacus. These species could theoretically produce 139 Kg and 136 Kg of ethanol and 91 Kg of triacylglycerol (TAG) per tonne of OMSW, respectively. R. opacus had the highest fermentation productivity, concurrently using D-glucose and D-xylose and producing TAG to 72% of maximum theoretical yield. Expression of a heterologous thioesterase in R. opacus to augment lauric acid production proved unsuccessful and requires further work. Overall, this study showed that OMSW is a promising renewable feedstock for biomanufacturing. The microorganisms identified through this work grew robustly and efficiently on OMSW fibre hydrolysate and are promising candidates for developing an OMSW biorefining platform

Quantification of Conformational Heterogeneity and its Role in Protein Aggregation and Unfolding

Proteins can exhibit significant conformational heterogeneity either under denaturing conditions or in aqueous solutions. The latter is true for a class of proteins whose sequences predispose them to form heterogeneous ensembles of conformations. Characterization of conformational heterogeneity in a protein ensemble requires the quantification of the amplitudes of spontaneous fluctuations in conjunction with information regarding coarse grain measures that report on the average sizes, shapes, and densities. This often demands multiplexed experimental approaches whose readouts are interpreted or annotated using ensembles drawn from atomistic or coarse grain computational simulations. Efforts to characterize conformational heterogeneity contribute directly to our understanding of disorder-to-order transitions in protein folding and self-assembly. These efforts are also crucial to our understanding of the heterotypic interactions involving intrinsically disordered proteins and non-native states of well-folded proteins. These heterotypic interactions are important in signal transduction and the regulation of protein homeostasis. The onset and progression of several systemic and neurodegenerative conformational diseases are linked to the nature and degree of conformational heterogeneity in specific proteins or proteolytic products of proteins. This thesis work focuses on the quantitative characterization of conformational heterogeneity in simulated ensembles of inducibly unfolded and intrinsically disordered proteins. Advances in nuclear magnetic resonance spectroscopy afford the possibility of detailed measurements of inter-residue distances and modulations to the relaxation dynamics of paramagnetic spins that are inserted as probes into a protein. These state-of-the-art measurements show interesting features within denatured state ensembles that cannot be explained using canonical random coil models. Here, we use computer simulations to generate plausible facsimiles of denatured state ensembles that reproduce experimental data and demonstrate that the ensembles that are consistent with the data are characterized by the presence of low-likelihood, long-range intra-chain contacts between hydrophobic groups. When placed in the context of sequence conservation information, it appears that these contacts act as gatekeepers that protect proteins from the deleterious consequences of protein aggregation by sequestering hydrophobic groups in an assortment of intra-chain long-range contacts. We also characterize the nature and degree of conformational heterogeneity in glutamine- and asparagine-rich containing systems. These efforts lead to insights regarding the role of conformational heterogeneity in mediating intermolecular associations that are implicated in aggregation and self-assembly of these systems. Analysis of results from atomistic simulations leads to a phenomenological model for the modulation of conformational heterogeneity and degeneracies of intermolecular interactions by naturally occurring sequences that flank polyglutamine domains. Finally, we develop a formal order parameter to quantify the conformational heterogeneity in simulated ensembles of proteins. When combined with measures of density and fluctuations thereof, it can be used to provide a complete description of the degree and nature of conformational heterogeneity in different ensembles, thus affording the ability to compare different ensembles to each other while also providing a way to categorize conformational transitions