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Acquisition of a research and teaching salt water flume at Priest Landing, GA.

2011-12-31 , Weissburg, Marc J. , Webster, Donald R. , Fritz, Hermann M.

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Characterization of the role of acid ceramidase in adrenocortical steroid hormone biosynthesis

2011-11-14 , Lucki, Natasha Chrystman

Sphingolipids modulate multiple cellular functions, including steroid hormone biosynthesis. Sphingosine is an antagonist ligand for the nuclear receptor steroidogenic factor 1 (SF-1), which is the primary transcriptional regulator of most steroidogenic genes. Furthermore, sphingosine-dependent repression of SF-1 function is dependent on the expression of acid ceramidase (ASAH1), an enzyme that forms sphingosine. Based on these data, I hypothesized that ACTH/cAMP signaling regulates ASAH1 function at both transcriptional and post-transcriptional levels. In addition, because SF-1 is predominantly a nuclear protein, I postulated that ASAH1 modulates SF-1 function and, therefore, steroidogenic gene expression by controlling the nuclear concentrations of SPH. To test these hypotheses, I first examined the effect of chronic ACTH/cAMP signaling on the transcription of the ASAH1 gene. Next, the functional significance of ASAH1 expression in adrenocortical cells was probed by generating an ASAH1-knockdown cell line. I subsequently characterized the role of ASAH1 as a transcriptional nuclear receptor coregulator. Finally, I defined the role of sphingosine-1-phosphate, a bi-product of ASAH1 activity, in the acute phase of cortisol biosynthesis. Using a variety of experimental approaches, I identified cAMP response element binding protein as an essential transcriptional activator of the ASAH1 gene. Analysis of adrenocortical cells lacking ASAH1 revealed that ASAH1 is a global regulator of steroidogenic capacity. Furthermore, I identified ASAH1 as a nuclear protein and defined the molecular determinants of the interaction between ASAH1 and SF-1. Collectively, this body of work establishes the integral role of ASAH1 in the regulation of ACTH-dependent adrenocortical cortisol biosynthesis.

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Studies of genetic factors modulating polyglutamine toxicity in the yeast model

2011-09-28 , Gong, He

Polyglutamine-expanded fragments, derived from the human huntingtin protein, are aggregation-prone and toxic in yeast cells, bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline-rich region targets polyglutamine aggregates to the large perinuclear deposit (aggresome). Aggresome targeting ameliorates polyglutamine cytotoxicity in the presence of the prion form of Rnq1 protein, however, aggresome-forming construct remains toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). Disomy by chromosome II partly ameliorates polyglutamine toxicity in the strains containing Sup35 prion. The chromosome II gene, coding for another release factor, and interaction partner of Sup35, named Sup45 (eRF1), is responsible for amelioration of toxicity. Plasmid-mediated overproduction of Sup45, or expression of the Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into prion aggregates, also ameliorate polyglutamine toxicity. Protein analysis indicates that polyglutamines alter aggregation patterns of the Sup35 prion and promote aggregation of Sup45, while excess Sup45 counteracts these effects. In the absence of Sup35 prion, disomy by chromosome II is still able to decrease polyglutamine toxicity. However, SUP45 is no longer the gene responsible for such an effect. Taken together with the finding that the presence of both the Rnq1 prion and the Sup35 prion has an additive effect on polyQ toxicity, one gene or few genes on chromosome II are able to ameliorate polyQ toxicity through a SUP45-independent pathway. The identification of such a gene is currently ongoing. Monosomy by chromosome VIII in diploid heterozygous by AQT (Anti-polyQ Toxicity mutants that are disomic by chromosome II) counteracted the effect of AQT. Similarly, deletion of the arg4 gene in chromosome VIII in AQT haploid was able to eliminate the AQT effect. Moreover, analysis of genes involved in the arginine and polyamine synthesis indicated that loss of genes in later stages of arginine biosynthesis causes increase of polyglutamine toxicity. Deletion of genes arg1, arg4, arg8 (arginine pathway) and spe1 (polyamine pathway) all suppressed the Sup35 prion phenotype expression in the nonsense suppression system. Further analysis regarding the mechanisms behind those effects is needed. Our data uncover the mechanisms by which genetic and epigenetic factors may influence polyglutamine toxicity, and demonstrate that one and the same type of polyglutamine deposits could be cytoprotective or cytotoxic, depending on the prion composition of a eukaryotic cell.

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Development of the new yeast-based assays for prion properties

2011-08-29 , Sun, Meng

Prion is an infectious isoform of a normal cellular protein which is capable of converting the non-prion form of the same protein into the alternative prion form. Mammalian prion protein PrP is responsible for prion formation in mammals, causing a series of fatal and incurable prion diseases. (1) We constructed, for the first time, a two-component system to phenotypically monitor the conformational status of PrP in the yeast cells. In this system, the prion domain of Sup35 (Sup35N) was fused to PrP90-230, and the initial formation of the PrPSc-like conformation stimulated prion formation of Sup35N, which in turn converted soluble Sup35 into the prion isoform, leading to a detectable phenotype. Prion-like properties of PrP were studied in this novel yeast model system. Additionally, we employed this system to study amyloidogenic protein Aβ42 aggregation in the yeast model. It has been suggested that the ability to form transmissible amyloids (prions) is widespread among yeast proteins and is likely intrinsic to proteins from other organisms. However, the distribution of yeast prions in natural conditions is not yet clear, which prevents us from understanding the relationship between prions and their adaptive roles in various environmental conditions. (2) We modified and developed sequence and phenotype-independent approaches for prion detection and monitoring. We employed these approaches for prion-profiling among yeast strains of various origins. (3) Lastly, we found a prion-like state [MCS+] causing nonsense suppression in the absence of the Sup35 prion domain. Our results suggested that [MCS+] is determined by both a prion factor and a nuclear factor. The prion-related properties of [MCS+] were studied by genetic and biochemical approaches.

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Yeast model for studying heritable mammalian prion disease

2011-12-01 , Chernoff, Yury O.

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Regulation of the cardiac isoform of the ryanodine receptor by S-adenosyl-l-methionine

2011-11-08 , Gaboardi, Angela Kampfer

Activity of the Ryanodine Receptor (RyR2) (aka cardiac Ca2+ release channel) plays a pivotal role in contraction of the heart. S-adenosyl-l-methionine (SAM) is a biological methyl group donor that has close structural similarity to ATP, an important physiological regulator of RyR2. This work provides evidence that SAM can act as a RyR2 regulatory ligand in a manner independent from its recognized role as a biological methyl group donor. RyR2 activation appears to arise from the direct interaction of SAM, via its adenosyl moiety, with the RyR2 adenine nucleotide binding sites. Because uncertainty remains regarding the structural motifs involved in RyR2 modulation by ATP and its metabolites, this finding has important implications for clarifying the structural basis of ATP regulation of RyR2. During the course of this project, direct measurements of single RyR2 activity revealed that SAM has distinct effects on RyR2 conductance. From the cytosolic side of the channel, SAM produced a single clearly resolved subconductance state. The effects of SAM on channel conductance were dependent on SAM concentration and membrane holding potential. A second goal of this work was to distinguish between the two possible mechanisms by which SAM could reduce RyR2 conductance: i) SAM interfering directly with ion permeation via binding within the conduction pathway (pore block), or ii) SAM binding a regulatory (or allosteric) site thereby stabilizing or inducing a reduced conductance conformation of the channel. It was determined that SAM does not directly interact with the RyR2 conduction pathway. To account for these observations an allosteric model for the effect of SAM on RyR2 conductance is proposed. According to this model, SAM binding stabilizes an inherent RyR2 subconductance conformation. The voltage dependence of the SAM related subconductance state is accounted for by direct effects of voltage on channel conformation which indirectly alter the affinity of RyR2 for SAM. Patterns in the transitions between RyR2 conductance states in the presence of SAM may provide insight into the structure-activity relationship of RyR2 which can aid in the development of therapeutic strategies targeting this channel.

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Evolutionary synthetic biology: structure/function relationships within the protein translation system

2011-09-06 , Cacan, Ercan

Production of mutant biological molecules for understanding biological principles or as therapeutic agents has gained considerable interest recently. Synthetic genes are today being widely used for production of such molecules due to the substantial decrease in the costs associated with gene synthesis technology. Along one such line, we have engineered tRNA genes in order to dissect the effects of G:U base-pairs on the accuracy of the protein translation machinery. Our results provide greater detail into the thermodynamic interactions between tRNA molecules and an Elongation Factor protein (termed EF-Tu in bacteria and eEF1A in eukaryotes) and how these interactions influence the delivery of aminoacylated tRNAs to the ribosome. We anticipate that our studies not only shed light on the basic mechanisms of molecular machines but may also help us to develop therapeutic or novel proteins that contain unnatural amino acids. Further, the manipulation of the translation machinery holds promise for the development of new methods to understand the origins of life. Along another line, we have used the power of synthetic biology to experimentally validate an evolutionary model. We exploited the functional diversity contained within the EF-Tu/eEF1A gene family to experimentally validate the model of evolution termed ‘heterotachy’. Heterotachy refers to a switch in a site’s mutational rate class. For instance, a site in a protein sequence may be invariant across all bacterial homologs while that same site may be highly variable across eukaryotic homologs. Such patterns imply that the selective constraints acting on this site differs between bacteria and eukaryotes. Despite intense efforts and large interest in understanding these patterns, no studies have experimentally validated these concepts until now. In the present study, we analyzed EF-Tu/eEF1A gene family members between bacteria and eukaryotes to identify heterotachous patterns (also called Type-I functional divergence). We applied statistical tests to identify sites possibly responsible for biomolecular functional divergence between EF-Tu and eEF1A. We then synthesized protein variants in the laboratory to validate our computational predictions. The results demonstrate for the first time that the identification of heterotachous sites can be specifically implicated in functional divergence among homologous proteins. In total, this work supports an evolutionary synthetic biology paradigm that in one direction uses synthetic molecules to better understand the mechanisms and constraints governing biomolecular behavior while in another direction uses principles of molecular sequence evolution to generate novel biomolecules that have utility for industry and/or biomedicine.

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Studies on the mechanisms of RNA-driven DNA repair and modification

2011-11-14 , Shen, Ying

Our previous studies have demonstrated that RNA can serve as a template for double-strand break (DSB) repair in the yeast Saccharomyces cerevisiae using synthetic RNA-containing oligonucleotides (oligos). Following this initial work, we show that the RNA tract of RNA-containing oligos can be copied into DNA to transfer a genetic change at the chromosomal level also in the bacterium Escherichia coli and in human cells. Exploiting the use of oligos containing ribonucleoside monophosphates (rNMPs), we developed a molecular approach to generate RNA/DNA hybrids of chosen sequence and structure at the chromosomal level in both yeast and E. coli cells. Such technique allows us to study how rNMPs present in the DNA genome of cells are tolerated by cells, what factors recognize and target rNMPs in DNA and to what extent the embedded rNMPs may alter genome integrity. Here we proved that mispaired rNMPs embedded into genomic DNA, if not removed, serve as templates for DNA synthesis during chromosomal replication and produce a genetic change. We discovered that mispaired rNMPs that are embedded in genomic DNA are not only targeted by ribonucleases H (RNases H) but also by the mismatch repair (MMR) system both in yeast and in E. coli. Our data reveal novel substrates for the MMR system, and also uncover an unpredicted competition between RNase H and MMR for the RNA/DNA mispairs.

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GOAP: A Generalized Orientation-Dependent, All-Atom Statistical Potential for Protein Structure Prediction

2011-10 , Zhou, Hongyi , Skolnick, Jeffrey

An accurate scoring function is a key component for successful protein structure prediction. To address this important unsolved problem, we develop a generalized orientation and distance-dependent all-atom statistical potential. The new statistical potential, generalized orientation-dependent all-atom potential (GOAP), depends on the relative orientation of the planes associated with each heavy atom in interacting pairs. GOAP is a generalization of previous orientation-dependent potentials that consider only representative atoms or blocks of side-chain or polar atoms. GOAP is decomposed into distance- and angle-dependent contributions. The DFIRE distance-scaled finite ideal gas reference state is employed for the distance-dependent component of GOAP. GOAP was tested on 11 commonly used decoy sets containing 278 targets, and recognized 226 native structures as best from the decoys, whereas DFIRE recognized 127 targets. The major improvement comes from decoy sets that have homology-modeled structures that are close to native (all within ∼4.0 Å) or from the ROSETTA ab initio decoy set. For these two kinds of decoys, orientation-independent DFIRE or only side-chain orientation-dependent RWplus performed poorly. Although the OPUS-PSP block-based orientation-dependent, side-chain atom contact potential performs much better (recognizing 196 targets) than DFIRE, RWplus, and dDFIRE, it is still ∼15% worse than GOAP. Thus, GOAP is a promising advance in knowledge-based, all-atom statistical potentials. GOAP is available for download at http://cssb.biology.gatech.edu/GOAP.

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A test of optimal defense theory vs. the growth-differentiation balance hypothesis as predictors of seaweed palatability and defenses

2011-08-31 , Heckman, Melanie L.

Because organisms have limited resources to allocate to multiple life history traits, the Optimal Defense Theory (ODT) and the Growth-Differentiation Balance Hypothesis (GDBH) were developed by terrestrial plant ecologists to predict intraindividual defense allocation based on the cost of defense and these life history trade-offs. However, these theories have garnered equivocal experimental support over the years and are rarely experimentally extended from predictions of plant physiology to the palatability of the tissues an herbivore experiences. We therefore examined tissue palatability, nutritional value, and defense mechanisms in multiple Dictyotalean seaweeds in two Caribbean locations, using two herbivores. Relative palatability of tissues varied greatly with algal species, grazer species, and location. Because older bases were not consistently defended, GDBH did not predict relative palatability. We could not reject ODT without intensive measures of tissue fitness value and herbivore risk, and this theory was therefore not useful in making broad predictions of tissue palatability. In testing the physiological predictions of these theories, we found the young, growing apices of these seaweeds to be generally more nutritionally valuable than the old, anchoring bases and found organic-rich apices to be more chemically deterrent, thus supporting ODT. However, the combined chemical, nutritional, and structural traits of these algae all influenced herbivore choice. As a result, these patterns of apical value and chemical defense reflected palatability of live tissues for only one of five algal species, which rendered ODT and GDBH poor predictors of relative palatability for most algae.

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