Interdisciplinary Graduate Programs Symposium
2014 OSU Molecular Life Sciences
Interdisciplinary Graduate Programs Symposium
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Poster abstracts
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Alzheimer’s Disease (AD) is the most common form of dementia, and there is currently no cure or preventative treatment. AD is defined by the appearance of two lesions believed to contribute to the pathogenesis of AD: extracellular beta-amyloid plaques and intracellular neurofibrillary tangles (NFTs) composed of tau protein. However, NFTs better correlate with disease and symtom progression, and are used to diagnose AD. Normal monomeric tau stabilizes microtubules and promotes their assembly. In AD, tau aggregates into long fibrils. Candidate triggers for this change include post translational modifications (PTMs), such as phosphorylation. PTMs of Lys residues can also modulate tau aggregation propensity. But Lys modifications have not been shown at single residue resolution in human brain specimens.
Tau was isolated from normal human brain and analyzed using mass spectrometry, revealing Lys methylation as a novel tau PTM. The modification clustered predominantly in the region of tau that mediates aggregation. Recombinant tau was methylated chemically to create samples with varying methylation stoichiometry. Studies reveal that at low, physiological levels, methylation does not inhibit tau’s ability to promote microtubule assembly, but even low levels of methylation are able to inhibit tau aggregation. Aggregation studies reveal that methylation inhibits aggregation by increasing the amount of protein needed to form aggregates by increasing tau’s dissociation rate from fibrils, and decreasing the fibril extension rate. Methylation also slows aggregation by depressing fibril nucleation, the rate limiting step in fibril formation.
Together these data show that low-occupancy methylation is a normal human tau PTM that does not affect tau’s normal function, but that depresses tau aggregation. Methylation could be a potential regulatory PTM of tau. Maintaining or increasing tau methylation could be a therapeutic approach for slowing NFT formation in AD.
Keywords: Tau protein, aggregation, post-translational modification
Abstract not available online - please check the printed booklet.
Abstract:
Transposable elements (TEs) are mobile, parasitic DNA elements found throughout all life. When active, they cause mutations and destabilize chromosomes. These alterations can damage the germline and cause or accelerate disease, and in many eukaryotes, TEs and remnant sequences make up large portions of the genome. Eukaryotes have developed several homologous small RNA (sRNA) and epigenetic systems to control the expression and transposition of TEs. However, little is known about initial TE recognition by the host.
How does an organism differentiate self from non-self sequence in the nuclear genome? This is relevant for the silencing of TEs, transgenes, and viruses. Our lab has found that newly introduced copies of endogenous TEs are recognized by homology to existing copies, mediated by sRNAs.1 Other labs have transformed active Nicotiana tabacum retrotransposons into Arabidopsis thaliana.2,3 Despite a lack of homology to the host genome, these elements were not only recognized, but heritably silenced after introduction and transposition. However, there is little evidence for how this silencing was triggered or what pathways may be involved.
Using Agrobacterium-mediated transformation, we have introduced one of these TEs, Tto14, into Arabidopsis. Analysis of DNA methylation and sRNA has shown that Tto1 is quickly and deeply silenced in this first generation. This is dependent on the RNA-directed DNA Methylation pathway and is independent of the RNA interference pathway (RNAi). From sRNA northern blot, we infer that this TE is being recognized through its homology to endogenous TEs, in spite of its limited sequence similarity to any region of the Arabidopsis genome. However, when homology based recognition is eliminated, RNAi is active against the element instead. Using truncated forms of this TE, we have also seen that full silencing requires the entire element. In order to minimize homology, further studies will examine the recognition of yeast TEs.
References:
1. Nuthikattu, S., McCue, A. D., Panda, K., Fultz, D., DeFraia, C., Thomas, E. N., & Slotkin, R. K (2013). The Initiation of Epigenetic Silencing of Active Transposable Elements Is Triggered by RDR6 and 21-22 Nucleotide Small Interfering RNAs. PLANT PHYSIOLOGY.
2. Hirochika, H., Okamoto, H., & Kakutani, T (2000). Silencing of Retrotransposons in Arabidopsis and Reactivation by the ddm1 Mutation. The Plant Cell Online.
3.Pérez-Hormaeche, J., Potet, F., Beauclair, L., & Lucas, H (2008). Invasion of the Arabidopsis genome by the tobacco retrotransposon Tnt1 is controlled by reversible transcriptional gene silencing. Plant Physiology.
4.Böhmdorfer, G., Tramontano, A., Luxa, K., & Bachmair, A (2010). A synthetic biology approach allows inducible retrotransposition in whole plants. Systems And Synthetic Biology.
Keywords: Transposon, Epigenetics, Silencing
Abstract:
Paramutation refers to a meiotically heritable epigenetic change in gene regulation involving allelic interactions1. Paramutations are influenced by trans-homolog interactions that are potentially mediated by small RNAs (sRNAs) as part of an RNA-directed DNA methylation pathway (RdDM)2. In Zea mays (maize), paramutation has been detected among alleles of the purple plant1 (pl1) and booster1 (b1) loci, both of which encode transcription factors necessary for anthocyanin pigment production. Paramutation can be affected by mutations in genes encoding presumed RdDM components3. A genetic screen for factors required to maintain repression of a paramutant pl1 allele identified four separate EMS-induced mutant alleles defining the rmr5 locus. Genetic tests show that rmr5 function is required to establish repressed paramutant states at the b1 locus. Fine-scale mapping identified a candidate gene model encoding a DICER-LIKE3 (DCL3) protein having a likely role in RdDM. In plants, specific DCL proteins process distinct sized sRNAs from double-stranded RNA precursors. Sequencing this dcl3 candidate gene, we identified single transition-type lesions predicting protein dysfunction in all four mutant rmr5 alleles. We also found sRNA profiles of rmr5 mutants replete of 24¬ nucleotide (nt) RNAs, strongly indicating that rmr5 encodes DCL3. Unlike in the eudicot Arabidopsis thaliana, DCL3 is required for normal maize growth and development consistent with a potentially broader role for RdDM in the grasses. Additionally, comparisons of 5’-methylcytosine profiles at various positions in the maize genome show that DCL3-derived 24 nt RNAs are not required for DNA methylation typical of RdDM. Our analyses indicate that loss of 24 nt RNAs can be compensated by alternate, yet RdDM effective, small RNA size classes. These findings point to a specific role of the 24nt RNAs in affecting paramutations.
References:
1. Hollick, J.B. and Erhard, K. F. (2011). Paramutation: a Process for Acquiring trans-Generational Regulatory Stress, Curr Opin Plant Biol, 14, 1-7.
2. Bond, D.M. and Baulcombe, D.C. (2014) Small RNAs and Heritable Epigenetic Variation in Plants, Trends Cell Biol, 24, 100-107.
3. Hollick, J.B. (2012). Paramutation: a trans-homolog interaction affecting heritable gene regulation, Curr Opin Plant Biol, 15, 536-543.
Keywords: RdDM, DCL3
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
DNA double strand breaks (DSBs) are a unique problem for cells to address. Unlike most DNA repair pathways, which make use of local complementary DNA as template to correct the incorrect DNA site, DSB pathways must either locate a homologous chromosome to use as template for accurate repair or simply ligate the broken ends to one another. The severity of this DNA damage can cause cell death when unrepaired, and when repaired can form genome rearrangements (translocations, deletions, fusions). These genome rearrangements are often found in many types of cancer, also, mutations in DSB repair genes are commonly found in tumor samples and are often sources of hereditary cancer. Oddly enough in meiotic cells hundreds of DSBs are induced throughout the genome in the onset of Prophase I. These breaks are primarily repaired through homologous recombination and serve two purposes: 1. Promote exchange of genetic information between parental chromosomes and 2. Some crossover points will mature into chiasmata which allow for specific separation of non-sister homologous chromosomes in meiosis I. While the mechanism for mitotic homologous recombination is well understood, the mechanism for meiotic DSB repair is still being uncovered. Evidence from genetic, cellular, and biophysical studies shows that MSH4-MSH5, a MutS homolog, is involved in meiotic homologous recombination. Our group has previously shown MSH4-MSH5 specifically recognizes Holliday Junctions in vitro. In an effort to determine the role MSH4-MSH5 plays upon recognizing Holliday Junction we compare the rate of branch migration with and without purified MSH4-MSH5. We report that MSH4-MSH5 is able to increase the speed of branch migration two-fold. We postulate that this increase is due to MSH4-MSH5 locking the Holliday Junction in a conformation which enhances movement of the branch point.
Keywords: Homologous Recombination, Meiosis, Branch Migration
Abstract:
Collagen fibers, a major component of the extracellular matrix of blood vessels, confer structural and mechanical integrity to the vessel wall. At sites of vascular injury, collagen is exposed to flowing blood, and von Willebrand factor (VWF) and platelet collagen receptor, GPVI, initiate platelet adhesion by binding to the exposed collagen. The native structure of collagen fibers is critical for platelet adhesion. The assembly of collagen fibers is regulated by collagen-binding proteins like discoidin domain receptor 1 (DDR1). Few studies have investigated how alteration in the fiber structure modulates platelet-collagen interactions. This study aimed to determine how DDR1 changes the morphology of collagen fibers in-vivo, and how these changes altered GPVI- and VWF-collagen interactions. To investigate this, mouse aortas from DDR1 KO and WT littermates were isolated and cut into cross-sections. The ultrastructure of collagen fibers was examined by transmission electron microscopy and atomic force microscopy. VWF and GPVI binding to collagen in the vessel wall was determined by incubating aortic sections with recombinant GPVI or platelet-poor plasma and examining protein binding by immunofluorescence. Collagen in the adventitia of DDR1 KO aortas exhibited a reduced width of D-periodicity and an increased depth of D-periodicity. DDR1 KO aortas exhibited greater VWF binding than WT, while no difference was observed for GPVI binding. These studies will elucidate a novel mechanism, namely how changes in the collagen fiber structure modulate platelet-collagen interactions. Based on our results, a down-regulation of DDR1 in the vessel wall could be considered as a biomarker for cardiovascular pathologies prone to thrombogenic events.
Keywords: collagen, platelets, vascular biology
Abstract:
Calmodulin (CaM) is a ubiquitous decoder of the calcium signal within the animal and plant kingdoms. All vertebrates encode a single isoform of CaM, while plants encode up to ten different isoforms, each of which differs in their binding, activation or inhibition of specific target enzymes in response to stimuli. Examples of these stimuli include pathogen attack and wounding, as well as chemical, thermal, and ionic stresses. Our goal is to adapt the design principles that the plant has evolved to selectively modulate the CaM-dependent enzyme hubs within the mammalian cell.
As a first step in understanding the functional differences among these CaMs, we have studied the Ca2+ binding properties of two unique soybean CaMs: sCaM1 and sCaM4, as well as three unique Arabidopsis thaliana CaMs: CaM4, CaM5 and CaM7. We performed steady state fluorescence experiments to follow cation binding and used the stopped-flow technique to measure the kinetics of cation dissociation from these CaMs by means of a variety of fluorescent probes.
We have previously demonstrated that two soybean CaMs bound to several important mammalian enzymes, some of which were inhibited instead of activated by CaM binding. In this study we performed stopped-flow experiments to determine the effect of target binding on the Ca2+ dissociation rates for these various CaMs, as target binding changes CaM’s affinity for Ca2+, drastically slowing the Ca2+ dissociation rate.
For each CaM, our analysis revealed that the rate of Ca2+ dissociation was similar to the regional structural change occurring at each domain, which in turn was similar to the rate of closure of each domain’s hydrophobic pocket. All soybean and Arabidopsis CaMs displayed distinct binding behavior for a variety of mammalian target enzymes. These studies can pave the way for further use of these CaMs for potential applications involving activation or inhibition of a subset of CaM-regulated enzymes.
This work was supported by AHA SDG (JPD)
Keywords: Calcium-binding, Calmodulin, Protein engineering
Abstract not available online - please check the printed booklet.
Abstract:
There are at least 11 different variants of linker histone H1 in mammalian cells, the sequences of which vary greatly in their C-terminal domains. The individual functions of H1 variants are not fully understood now. It was believed that H1 served as a global gene regulator by binding to chromatin unspecifically. However, previous researches found that while knocking out a single H1 variant might not lead to any observable phenotype, triple knockouts of H1 variants in mice has shown developmental defects and embryonic lethality. These observations suggest the hypothesis that each H1 variant has its individual function in the cells in addition to its roles as global chromatin modifiers. It is worth investigating whether the dynamics of replication dependent and independent variants of H1 are regulated by distinctive chaperones, similar to how core histones are regulated.
To answer this question, we are identifying the proteins associated with soluble forms of replication-dependent and replication-independent H1 variants. Tetracycline-inducible U2OS cell lines were generated to express 6xHis-tagged human histone H1.1 and H1.2 (replication-dependent variants), and H1.0 and H1.x (replication-independent variants). Soluble whole cell extracts made from those cell lines were purified by chromatography. Proteins associated with these H1 variants were identified by mass spectrometry. We found that some of the H1 variants form two separate complexes while others only exist in one complex. Importantly, we found that some linker histone associated factors associate with both replication-dependent and replication-independent H1 variants while other factors are specific for one type of histone H1. These findings will contribute to the understanding of the non-redundant functions of histone H1 variants, and to the possible new discoveries of proteins that impact chromatin structure by interacting with linker histones.
References:
[1]Yang, SM., et al., H1 linker histone promotes epigenetic silencing by regulating both DNA methylation and histone H3 methylation. PNAS, 2013 Jan 29;110(5):1708-13.
[2]Tagami, H., et al., Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell, 2004. 116(1): p. 51-61.
Keywords: Linker histones, Histone chaperones, DNA-binding protein complexes
Abstract not available online - please check the printed booklet.
Abstract:
TRIC-A and TRIC-B are trimeric intracellular cation channels located at the sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER) of multiple cell types. These channels regulate the permeability of K ions across the SR/ER and consequently the movement of Ca ions during excitation-contraction coupling. Previously we showed that genetic ablation of TRIC led to compromised K-permeability and Ca release across the SR membrane, supporting the hypothesis that TRIC could function as counter-ion channels that allows the flow of K ions into the SR during the acute phase of Ca release. In the absence of TRIC, overload of Ca inside the ER/SR causes instability of Ca storage and release, leading to stress-induced dysfunction of multiple tissues. Spontaneous Ca waves, also called store overload-induced Ca release (SOICR) mediated by the type 2 ryanodine receptor (RyR2), evoke ventricular tachyarrhythmia in individuals with heart failure. Our biochemical studies revealed that the carboxyl-tail domain of TRIC-A could interact with the RyR channel, suggesting the possibility that TRIC-A may directly regulate the Ca release activity. We found that TRIC-A, but not TRIC-B, prevented the appearance of SOICR in HEK293 cells expressing RyR2. Cytosolic Ca measurement by Fura-2 and ER luminal Ca measurement by D1ER revealed that expression of TRIC-A in HEK293 cells could prevent overload of Ca inside the ER by targeting the RyR2 channel function. Such effect was translated into suppression of SOICR. These effects appeared to be specific for TRIC-A, as co-expression of RyR2 with TRIC-B did not affect SOICR. Together, our data suggest that functional interaction between TRIC-A and RyR can modulate the Ca release process from internal stores and regulate Ca homeostasis across the ER/SR.
References:
Yazawa M, Ferrante C, Feng J, et al. TRIC channels are essential for Ca2+ handling in intracellular stores. Nature. 2007;448:78–82.
Yamazaki D, Tabara Y, Kita S, et al. TRIC-A channels in vascular smooth muscle contribute to blood pressure maintenance. Cell Metab. 2011; 14:231–241.
Jiang D, Xiao B, Yang D, Wang R, Choi P, Zhang L, Cheng H, Chen SR. RyR2 mutations linked to ventricular tachycardia and sudden death reduce the threshold for store-overload-induced Ca2+ release (SOICR). Proc Natl Acad Sci. 2004; 101:13062–13067.
Xinyu Zhou, Pei-hui Lin, Daiju Yamazaki, Ki Ho Park, Shinji Komazaki, S.R. Wayne Chen, Hiroshi Takeshima, Jianjie Ma Trimeric Intracellular Cation Channels and Sarcoplasmic/Endoplasmic Reticulum Calcium Homeostasis. Circ Res. 2014; 114: 706-716
Keywords: TRIC, RyR, SOICR
Abstract:
In the course of protein evolution, the interactions between side-chains are optimized for overall organism fitness. An enzyme must both be stable and fulfill its metabolic role within certain kinetic parameters. Because the preferred environment of organisms can vary widely across phylogenies,homologous enzymes can have similar backbones but different side chain packing, thereby fine-tuning interaction strength and dynamic mechanisms for environmental conditions. This evolution-driven varying of side chain packing can create positional correlations in multiple sequence alignments. It has been shown that these correlations may reflect stabilizing interactions, but do not necessarily do so. We present here correlation-guided mutations made in the adenylate kinase enzyme of Bacillus subtilis bacteria (bsADK) which, in the context of a destabilized variant, affect kinetic activity without altering stability. The folded lid domain of bacterial ADK closes over the active site following substrate binding. This domain is stabilized by either a zinc-chelating motif in gram-positive species or by a network of hydrogen bonds in gram-negative species. Mutating the four chelating residues of bsADK to their counterparts in E. coli ADK (ecADK), abolishing metal-binding, results in a severe drop in global stability and the loss of enzymatic activity. Using mutual information analysis, we located two positions in the lid domain strongly correlated to chelating motif. Further mutations were made in bsADK at these positions. These additional mutations were found to have little effect on global stability, but served to partially rescue enzymatic activity. Although it is understood that loss and gain of stability can directly alter the dynamics of adenylate kinase, the fact that these variants do not vary in stability suggests that they could serve as an experimental system for further studying the kinetic pathway of this enzyme. We are currently pursuing biophysical strategies to further characterize these variants and elaborate on their kinetic differences.
Keywords: Evolution, Enzyme, Sequence Alignment
Abstract:
During translation, aminoacyl-tRNA synthetases are responsible for covalently attaching amino acids to cognate tRNAs. Mistakes in this process lead to errors in protein synthesis, and accumulation of such errors can be deleterious to cells. Prolyl-tRNA synthetase (ProRS) mischarges tRNAPro with alanine and cysteine. Proline residues, due to their unique structural properties, are usually highly conserved in protein sequences; thus, errors in translation of Pro codons are not well-tolerated. To ensure high fidelity of this key step in protein synthesis, multiple proofreading mechanisms exist. Most bacterial ProRSs possess a cis-editing domain (INS) to hydrolyze Ala-tRNAPro. Cys-tRNAPro and Ala-tRNAPro in some organisms, however, must be cleared by freestanding, trans-editing domains. These proteins, the INS superfamily, are responsible for hydrolyzing misacylated tRNAPro in all three domains of life. The bacterial protein ProXp-ala is responsible for the deacylation of Ala-tRNAPro, but the structure and mechanism of this enzyme remains unknown. Mutagenesis studies have identified acceptor stem elements of tRNAPro that are critical for ProXp-ala activity. Due to this localized specificity, a microhelix that mimics the acceptor stem of tRNAPro was designed, charged with Ala, and found to be a substrate for ProXp-ala. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to analyze chemical shift perturbations and map the interaction surface for this microhelixPro on ProXp-ala. Additionally, a hydrolysis-resistant, charged Ala-microhelixPro analog is allowing us to determine regions on the protein that specifically involved in alanine recognition. These results provide structural insights into the interaction between ProXp-ala and mischarged tRNAPro and thus allow us to make hypotheses regarding the mechanism of error recognition by other trans-editing enzymes.
Keywords: NMR, tRNA, protein-RNA interaction
Abstract not available online - please check the printed booklet.
Abstract:
Bacteriophage λ encodes a two-component synaptase-exonuclease (Syn-Exo) system used for generating end-to-end concatamers of λ genome before packaging. Redα (λ exo) is a processive 5’-3’ exonuclease that digests linear dsDNA, yielding a 3’ ssDNA overhang. Redβ is a single-strand annealing protein (SSAP) that binds to the resulting 3’ overhang and anneals it to a complementary ssDNA. The current model for Redβ DNA binding and annealing describes β binding weakly to ssDNA as an oligomeric ring of 10-15 subunits, and forming a very tight complex with nascent annealed duplex in the form of a left-handed helical filament. Redβ serves as a model to study the unique DNA repair mechanism of single-strand annealing, which is conserved in higher organisms and includes the eukaryotic homologous pairing protein Rad52. Recently, phage-derived Syn-Exo partners have been used in restriction endonuclease- and ligation-independent genetic engineering, known as recombineering. We have identified a protease-resistant fragment of Redβ, residues 1-177, which is a target for structure determination via x-ray crystallography. We predict that the N-terminal fragment forms the DNA binding domain, while a more flexible C-terminal tail modulates interaction with the partner exonuclease. Full-length Redβ and Redβ(177) are able to assemble into oligomeric structures, but their functional properties differ significantly. Using a fluorescence-based assay, we characterized the DNA-binding and annealing properties of Redβ(177) compared to that of full-length protein and found Redβ(FL) preferentially binds to sequentially-added complementary oligonucleotides, while Redβ177 binds more tightly to ss oligonucleotides. Further, using a Ni-affinity pulldown assay, Redβ(177) fails to interact with λ exonuclease. Utilizing an in vivo assay, we found Redβ(177) is unable to recombine a PCR product or ss oligo with a target plasmid containing regions of homology. Our results provide insight into how SSAP perform their DNA binding and pairing function in vivo.
References:
1. Passy SI, Yu X, Li Z, Chiu SK, Reddy G, and Radding CM (1999). Rings and filaments of beta protein from bacteriophage lambda suggest a superfamily of recombination proteins. Proc Natl Acad Sci USA 96: 4279-4284.
2. Poteete AR (2001). What makes the bacteriophage lambda Red system useful for genetic engineering: molecular mechanism and biological function. FEMS Microbiol Lett. 201, 9-14.
3. Wu Z, Xing X, Wisler JW, Dalton JT, and Bell CE (2006). Domain structure and DNA binding regions of β protein from bacteriophage λ. J Biol Chem 281, 25205-25214.
Keywords: Single-strand annealing, Recombination, DNA Repair
Abstract:
Lysine acetylation is a reversible and highly regulated post-translational modification with a wide diversity of targets. Recently, this modification has emerged as an important potential regulatory mechanism in liver metabolism; different analyses have identified several mitochondrial proteins being acetylated in mouse liver providing a potential link between mitochondrial function and protein acetylation. Importantly, the substrate specificity of Lysine Acetyltransferases (KATs), with the exception of histones, is almost entirely uncharacterized. Our lab has developed a mouse knockout model for the first known protein lysine acetyltransferase HAT1 (also known as KAT1). Subcellular fractionation experiments have allowed us to detect the presence of this enzyme in mitochondrial extracts of mouse embryonic fibroblasts. Additionally knockout cells exhibit increased levels of reactive oxygen species and higher sensitivity to Antimycin A and nutrient deprivation, suggesting that HAT1 promotes mitochondrial function most likely through acetylation of metabolic proteins.
Keywords: HAT1, acetylation, mitochondria
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
The fragile FHIT gene, positioned at one of the most active common fragile sites, FRA3B, is a frequently altered gene in preneoplasia and cancer. Loss of the Fhit protein causes an imbalance in thymidine triphosphate pools resulting in spontaneous replication stress that leads to chromosomal aberrations such as aneuploidy, copy number variations and point mutations. Thus, Fhit, which is reduced in expression in most human cancers, is a genome ‘caretaker’ whose loss initiates genome instability in preneoplastic lesions. Preliminary studies show that Fhit knockout mice display a moderately increased frequency of spontaneous tumors and greatly increased susceptibility to carcinogen-induced tumors compared to wild type littermates. Early immortalization and increased copy number variations in Fhit-deficient cells suggests the loss of Fhit is linked to the transformation process. Furthermore, exome sequencing analysis of livers from +/+ and -/- mice, post carcinogen injection, also reveal genomic alterations associated with preneoplastic changes observed in vivo. To understand the early events that lead from Fhit loss to tumorigenicity, we are following the sequence of mutational changes in Fhit -/- and WT kidney cells established from mice in culture. We have identified changes in the p53/p21 pathway as well as epithelial to mesenchymal transition states, a characteristic step for tumor invasion. Thus, Fhit loss-induced genome instability facilitates transformation in vitro.
Keywords: genome instability, common fragile sites
Abstract:
Mature vascular smooth muscle cells (VSMCs) have phenotypic plasticity that allows them to transition between contractile, proliferative and synthetic phenotypes in response to environmental cues or blood vessel injury. A primary focus of our lab has been to understand the signaling interactions between endothelial cells and vascular smooth muscle cells. In this current study, we sought to explore how signaling between endothelial cells and VSMCs influenced VSMC phenotypic switching. Previous studies from our lab demonstrated that endothelial cells induce differentiation-specific gene expression when co-cultured with smooth muscle cells or their precursors, mesenchymal stem cells (MSCs). Our results have further shown that endothelial cells not only induce a contractile phenotype in VSMCs and MSCs, but also repress cell proliferation. Moreover, our data indicate that co-cultured endothelial cells can induce collagen synthesis, an indicator of a synthetic phenotype. Our findings demonstrate that endothelial cell-induced Notch signaling plays a critical role in the regulation of SMC differentiation and phenotypic modulation. These studies are poised to elucidate the regulatory role that endothelial cell signaling has on smooth muscle cell phenotypes. A better knowledge of how endothelial cells regulate SMC phenotypic properties will facilitate the development of innovative treatments to improve cardiovascular disease outcomes.
Keywords: Notch, smooth muscle , mesenchymal stem cell
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Pathologic assessment of a resected tumor specimen relies on the judgment of the individual pathologists, which, however can result in inconsistency in diagnosis. Furthermore, the final histopathologic report is generally not available until many days after the performed surgical procedure. Highly sensitive, non-invasive techniques for identifying and distinguishing cancer-bearing tissues from non-cancer-bearing tissues can potentially serve as extremely important adjunct methodologies to that of standard histolopathologic tissue analysis for real-time cancer detection as it relates to the assessment of surgical resection margins and the completeness of surgical resection in both the operating room and the pathology department. In this study, infrared (IR) spectroscopy is used to assess tumor vs. non-tumor regions by k-means clustering analysis. A set of newly developed biomarkers were evaluated. The region between 1476─1776 cm-1 is simultaneously fit with band lineshapes and 2nd derivatives to evaluate the protein contents in tumor and non-tumor regions. Surface plasmonic material is able to improve the diagnostic capacity of the IR spectroscopy and brings this technology into current pathology practices and ultimately into the operating room. Later in the study, such material is introduced. The use of mesh/tissue/window produces an interferometric pattern with regions demonstrating either destructive or constructive interference. New peaks are shown in the IR spectra. Subsequently, they are selected to build up new biomarkers and further analyzed quantitatively.
Keywords: FTIR imaging, surface plasmon resonance, k-means clustering analysis
Abstract not available online - please check the printed booklet.
Abstract:
Binding of transcription factors to their binding sites in promoter regions is the fundamental event in transcriptional gene regulation. When a transcription factor binding site is located within a nucleosome, the DNA has to partially unwrap from the nucleosome to allow transcription factor binding. This reduces the rate of transcription factor binding and is a known mechanism for regulation of gene expression via chromatin structure. Recently a second mechanism has been reported where transcription factor off-rates are dramatically increased when binding to target sites within the nucleosome. There are two possible explanations for such an increase in off-rate short of an active role of the nucleosome in pushing the transcription factor off the DNA: (1) for dimeric transcription factors the nucleosome can change the equilibrium between monomeric and dimeric binding or (2) the nucleosome can change the equilibrium between specific and nonspecific binding to the DNA. We explicitly model both scenarios and find that the second mechanism cannot be reconciled with experimental findings. However, the first mechanism yields increases in off-rate by a factor up to 200. Our results suggest a general mechanism how nucleosomes increase transcription factor dissociation to promote exchange of transcription factors and regulate gene expression.
Keywords: Nucleosome, Transcription factor, kinetics modeling
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Most bacterial prolyl-tRNA synthetase (ProRS) possesses an editing domain (INS), which hydrolyzes the mischarged Ala-tRNAPro. In addition to this in cis editing domain, several freestanding INS domain homologs, such as YbaK, ProXp-ala and ProXp-y are found in various species. Among these proteins, YbaK and ProXp-ala are shown to deacylate Cys-tRNAPro and Ala-tRNAPro separately. We found that ProXp-y has ability to edit Ser- and Thr-tRNA species. In vitro deacylation assays showed that none of the mischarged Ala-, Leu- or Cys- tRNAPro is the substrate of Escherichia coli (E.coli) ProXp-y alone. To explore the role of ProXp-y, we performed halo and growth curve assays for an E.coli proxp-y null strain and compared its behavior with that of the wild-type (wt) strain under high concentrations of 20 natural amino acids individually. Both of these in vivo assays suggested that proxp-y null strain showed growth defect under Ser condition. The slow growth phenotype of proxp-y null strain can be rescued by addition of Ala. Upon overexpression of Ec AlaRS, the null strain displays a more severe growth phenotype in the presence of Ser. In vitro assays confirmed robust deacylation of Ser-tRNAThr and Ser-tRNAAla, which are known to form due to misacylation by ThrRS and AlaRS. In vitro assays also showed that ProXp-y deacylates Thr-tRNAVal and Thr-tRNALys with similar efficiency as Ser-tRNAs. Taken together, these data suggest that ProXp-y may deacylate tRNAs other than tRNAPro and may collaborate with AlaRS to avoid Ala-to Ser mistranslation and with LysRS to avoid Lys-to-Thr mistranslation, as E.coli AlaRS and LysRS have no post-transfer editing domain that is responsible for mischarged Ser- and Thr-tRNAs. In ongoing studies, immunoprecipitation method will be used to identify the RNA and protein partners of ProXp-y. Crystallization trays will be set up to for ProXp-y protein alone and with substrate candidates to obtain the three-dimensional structures.
Keywords: tRNA synthetases
Abstract:
Transcription factors (TF) bind DNA target sites within promoters to activate gene expression. TFs achieve high binding specificity on their recognition DNA sequence by binding with resident times of up to hours in vitro. However, in vivo TFs can exchange on the order of seconds. The factors that regulate TF dynamics in vivo and increase dissociation rates by orders of magnitude are not known. We investigated TF binding and dissociation dynamics at their recognition sequence within duplex DNA, single nucleosomes and short nucleosome arrays with single-molecule Total Internal Reflection Fluorescence (smTIRF) microscopy. We find that the rate of TF dissociation from its site within either nucleosomes or nucleosome arrays is increased by 1000-fold relative to duplex DNA. Our results suggest that TF binding within chromatin could be responsible for the dramatic increase in TF exchange in vivo. Furthermore, these studies demonstrate that nucleosomes regulate DNA-protein interactions not only by blocking DNA-protein binding but by dramatically increasing the dissociation rate of protein complexes from their DNA target sites.
Keywords: Nucleosome Dynamics, Transcription Factor, Single-molecule FRET
Abstract not available online - please check the printed booklet.
Abstract:
Aminoacyl-tRNA synthetases are the enzymes responsible for attaching tRNA species with their appropriate amino acids to form aminoacyl-tRNAs, the substrates for ribosomal translation. Escherichia coli (E. coli) phenylalanyl-tRNA synthetase (PheRS) bears post-transfer editing activity that deacylates mischarged tRNAPhe. Under oxidative stress, the nonprotein amino acid meta-Tyrosine (m-Tyr) accumulates due to direct oxidation of phenylalanine. m-Tyr is a direct threat to translational accuracy and cell survival in a PheRS editing mutant, but not in wild-type E. coli. These data, combined with the observation that PheRS editing activity is dispensable in normal growth conditions, suggest conservation of E. coli PheRS editing is due to the ability to protect the cell from cytotoxic mistranslation of the genetic code under oxidative stress conditions.
Keywords: Protein Mistranslation, Oxidative Stress, Aminoacyl-tRNA Synthetase
Abstract not available online - please check the printed booklet.
Abstract:
The retinal homeobox gene, Rx/RAX, is a conserved transcription factor vital for normal vertebrate eye development. Loss of Rx gene function causes anophthalmia in many vertebrates including humans (Bailey 2004). Rx is also thought to be necessary for maintaining retinal stem cell identity. Unlike humans, frogs maintain a stable population of retinal stem cells (RSCs) and retinal progenitor cells (RPCs) throughout their lifetime for use in retinal growth and maintenance.
The purpose of this study is to understand how an enhancer of Rx, known as ultra conserved element 2 (UCE2), drives Rx mRNA expression in RSCs. It was previously shown that fragment 1 of UCE2 comprising the first 50 bp of UCE2, in combination with an Rx promoter, is sufficient to drive reporter mRNA expression in RSCs (Pan et al., 2010). To further understand which sequences are necessary for driving Rx mRNA expression, mutational analysis of UCE2 fragment 1 was performed. Specifically, deletions were made within UCE2 fragment 1, dividing the fragment into five 10 bp parts termed A-E (UCE2 fragment 1A-E). Our hypothesis is that UCE2 fragment 1D is essential for Rx mRNA expression since it contains a strongly predicted transcription factor binding site.
To study the effect of these mutations, the UCE2 fragment 1 deletion mutants were combined with a Rx promoter driving expression of GFP and then constructed into Xenopus transgenes. Transgenic Xenopus embryos were analyzed by in situ hybridization against GFP mRNA. Rx expression regions were examined for GFP mRNA. Transgenic tadpoles that fail to display GFP mRNA expression comparable to the control, transgenic tadpoles that have intact UCE2, indicate the deleted sequence in the UCE2 fragment 1 is necessary for Rx expression. Thus far, our data indicate that UCE2 fragment 1A is unnecessary for Rx expression while UCE2 fragments 1C and 1D are necessary for RX expression.
Identifying the necessary sequences in UCE2 fragment 1 will lead to a better understanding of how retinal stem cell identity is established and maintained in Xenopus. Importantly, this knowledge could also be applied to the development of retinal stem cells therapies for the treatment of retinal diseases in human.
References:
Bailey T.J, El-Hodiri H, Zhang L, Shah R, Mathers P.H, Jamrich M. Regulation of vertebrate eye development by Rx genes. Int. J. Dev. Biol. 2004
Pan Y, Martinez-De Luna RI, Lou CH, Nekkalapudi S, Kelly LE, Sater AK, El-Hodiri HM. Regulation of photoreceptor gene expression by the retinal homeobox (Rx) gene product. Dev Biol. 2010
Keywords: Retinal Stem Cells, Retinal Homeobox , Retinal development
Abstract:
Maternal nanos mRNA is distributed throughout the early Drosophila embryo, but is translated only at the posterior pole in the specialized cytoplasm containing the germline determinants. In the remainder of the embryo, nanos mRNA is repressed by binding of Smaug to sites in the nanos 3’UTR. The piRNA pathway has also been proposed as contributing to the repression of nanos mRNA. Based on preliminary data, we have discovered that the elimination of piRNA binding sites in the 3’ UTR of nanos mRNA has no effect on its repression. Repression of nanos mRNA by Smaug is thought to be partially mediated by recruitment of the eIF-4E inhibitor Cup via direct interaction with the Smaug RNA-binding domain (RBD). At the posterior pole, Oskar has been proposed to interact directly with the Smaug RBD, thereby evicting Smaug from nanos and de-repressing the mRNA. A Smaug chimera we created in which the native RBD is replaced by bacteriophage MS2 Coat Protein regulates an engineered nanos mRNA derivative essentially normally. Additionally, we created a fusion of Cup and MS2 Coat Protein, finding that this fusion protein also regulates the same engineered nanos mRNA. We conclude that Smaug appears to be the major repressor of unlocalized nanos mRNA and that direct interaction of neither Cup nor Oskar with the Smaug RBD is required for nanos mRNA regulation in vivo.
Keywords: nanos, smaug, translational regulation
Abstract not available online - please check the printed booklet.
Abstract:
Breast cancer is a leading cause of cancer-related mortality in women in the United States, despite the development of targeted therapies and earlier diagnoses. One of the major confounding factors in the treatment of this disease is the cellular heterogeneity of the mammary gland. Fibroblasts are cells of mesenchymal origin that are critical for wound healing and are an important component of normal mammary gland microenvironment. In the past decade, fibroblasts have been implicated in accelerating tumor progression. Platelet derived growth factor receptor alpha (PDGFR-α) is a targetable cell surface receptor that is chiefly expressed in mesenchymal cell populations, such as fibroblasts. While mutations in PDGFR-α were shown to be a poor prognostic factor in glioblastomas and Gastro-intestinal stromal tumors, the role of stromal PDGFR-α activation in mammary gland development and mammary carcinomas is yet to be explored. Thus, in this study we are exploring the role of fibroblast-specific PDGFR-α activation in both mammary gland development and cancer using genetic mouse models. We found that constitutive activation of PDGFR-α specifically in fibroblasts alters murine mammary ductal growth. Mutant mice have fewer ducts that fail to grow normally in the mammary fat pad. Mammary glands from these mice have decreased number of ducts, increased extra-cellular matrix (ECM) and increased fibroblast numbers. Stromal PDGFR-α activation also causes increased proliferation of mammary ductal (epithelial) cells suggesting that stromal PDGFR-α activation in combination with oncogenic hits in the epithelium can lead to increased tumor growth. Furthermore fibroblasts derived from the mutant mammary glands have increased levels of AKT, JNK and ERK, which are important pro-tumor pathways in fibroblasts. The mutant mammary fibroblasts also have decreased levels of Transforming Growth Factor- beta (TGF-β 2 and 3) RNA. This observation suggests that TGF-β ligands are potentially a novel downstream target of PDGFR-α signaling in fibroblasts. Since TGF-β biology is very important in both mammary gland development and cancer, our data further supports the hypothesis that stromal PDGFR-α might be a key regulator of mammary gland development and subsequently, play a role in mammary carcinomas.
Keywords: PDGFR, breast cancer, mammary gland development
Abstract:
Normal valve structure consists of stratified layers of extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) surrounded by a monolayer of valve endothelial cells (VEC)(1). VECs serve to sense the hemodynamic environment and communicate with underlying VICs to regulate valve integrity, which is essential for both valve development and maintenance. In contrast, diseased valves are characterized by disorganized ECM, VIC disarray, and disruption of the VEC monolayer; suggesting VEC dysfunction may be an underlying cause of valve disease (2, 3). Although disease pathology has been defined, the underlying molecular mechanisms are largely unknown; however, studies suggest that disease manifested later in life has origins in embryonic development. Despite their importance, the molecular phenotypes of VECs have not been explored. Therefore, the goal of this study is to determine the differential gene expression profile of VECs during hallmark stages of valve development and in models of valve disease. To do this, we have developed a novel method to isolate VECs from embryonic, post natal, and adult mice. Using this method, VECs will be isolated at embryonic day (E)14.5, post natal (PN) and adult (4 month old) stages from Tie2GFP reporter mice and subjected to RNA-seq analysis to generate an expression profile of healthy VECs over time. In addition, profiles of VECs from mouse models of disease will be generated to define molecular differences in VECs between healthy and diseased valves. Generation of these expression profiles will provide important insights into the molecular function(s) of VECs in developing and mature valves, and identify potential mechanisms and novel candidate genes underlying valve disease.
References:
1. Tao G, Kotick JD, Lincoln J. Heart valve development, maintenance, and disease: The role of endothelial cells. Current topics in developmental biology. 2012;100:203-232
2. Weinberg EJ, Mack PJ, Schoen FJ, Garcia-Cardena G, Kaazempur Mofrad MR. Hemodynamic environments from opposing sides of human aortic valve leaflets evoke distinct endothelial phenotypes in vitro. Cardiovasc Eng. 2010;10:5-11
3. Hinton RB, Jr., Lincoln J, Deutsch GH, Osinska H, Manning PB, Benson DW, Yutzey KE. Extracellular matrix remodeling and organization in developing and diseased aortic valves. Circulation research. 2006;98:1431-1438
Keywords: valve endothelial cells, heart valve, RNA-seq
Abstract:
Spermatogenesis is a highly complicated process involving mitosis, meiosis and spermiogenesis. After mitotic division of germ cells (spermatogoinal stem cells), some daughter cells undergo meiosis and others maintain germ cell population. During the first meiotic division, massive DNA double strand breaks (DSB) are induced by SPO11 and repaired by homology-directed repair (HDR). Haploid cells (round spermatids) produced from meiosis form tail and acrosome which covers the anterior portion of sperm head containing digestive enzymes to penetrate the oocyte.
Breast cancer susceptibility gene 1 (Brca1) play essential roles for genome integrity by involving multiple cellular processes including cell cycle check point control, DSB repair and ubiquitination. Mice with missense mutation (Brca1 S1655F or Brca1 M1717R) in C-terminal BRCT domain that disrupts interaction with phospho-ligand showed mitotic HDR defect resulting in tumor development, while mice with Brca1 I26A in N-terminal RING domain that have no E3 ubiquitin ligase activity had functional mitotic HDR. Interestingly, all mutant mice show a common phenotype, infertility. Here, we report multiple functions of BRCA1 during spermatogenesis. First, full-length BRCA1 is absolutely essential for self-renewal of spermatogonial stem cells. BRCA1 truncation mutant mice (Brca1 ex11tr) have completely empty testes showing proliferation defects of germ cells and extensive apoptosis. Second, surprisingly, both E3 ubiquitin ligase activity and BRCT phosphoprotein binding are dispensable for meiotic HDR. All three missense mutant mice can form sex-body and finish meiosis. Lastly, both E3 ubiquitin ligase activity and BRCT phosphoprotein binding are required to inhibit amplification of pericentriolar material (PCM) during spermiogenesis. We observed that PCM is amplified in round spermatids of all three missense mutant mice, and the amplified-extra-PCM display diffuse area having no centriole. Based on this observation, we are currently working on that the extra-PCM which have no centriole could nucleate microtubule disrupting microtubule organization.
References:
Shakya, R. et al, BRCA1 Tumor Suppression Depends on BRCT Phosphoprotein Binding, But Not Its E3 Ligase Activity, Science 334, 525-528
Keywords: Spermatogenesis, BRCA1, Centriole
Abstract:
Opening and closing of heart valve leaflets is largely achieved by the tri-laminar
organization of extracellular matrix (ECM) components including collagens,
proteoglycans and elastins that together, provide all the necessary biomechanical
properties for function throughout life. In contrast, disruption in ECM organization is a
histological landmark of valve disease. In healthy valves, the ECM is maintained by
valve interstitial cells (VICs) that are quiescent and fibroblast-like in the absence of
disease. However in response to pathological stimuli, quiescent VICs (qVICs) are
activated to a myofibroblast-like phenotype indicated by α-smooth muscle actin (α-SMA)
expression and organized stress fibers. In an activated state, VICs breakdown the healthy
ECM structure and replace it with alternative ECM components. These abnormal changes
alter the biomechanics of the valve and lead to dysfunction. Despite these observations,
the mechanisms that regulate VIC activation in post natal valves are unknown. Using
porcine and rat VIC systems, we show that VIC plasticity is dependent on substrate
stiffness and activation can be reversed by culturing on soft substrates. In addition, we
found that the bHLH transcription factor Scleraxis (Scx) is sufficient to promote VIC
activation in vitro and expression is increased with α-SMA in mouse models of valve
disease. Together these studies suggest that the extrinsic and intrinsic factors contribute
to VIC plasticity. Ongoing studies are currently examining the molecular and cellular
processes that drive activation of otherwise qVICs with the goal of providing insights for
the development of new mechanistic-based therapies in the prevention and treatment of
valve disease.
Keywords: Valve Interstitial Cells, Substrate Stiffness, Scleraxis
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Numerous kinetic, structural, and theoretical studies have established that DNA polymerases adjust their domain structures to enclose nucleotides in their active sites and then rearrange critical active site residues and substrates for catalysis. Additionally, structural studies have revealed a large conformational change between the apoprotein and the DNA-protein binary state for Y-family DNA polymerases. To elucidate the details of the conformational transitions of Dpo4 during substrate binding and catalysis, in this study, the stopped-flow FRET technique was used to monitor changes in distance between various pairs of locations in the protein. In addition to providing new insight into the conformational changes involved in nucleotide binding and incorporation, the results here show that the previously described conformational change between the apo and DNA-bound states of Dpo4 occurs in a mechanistic step distinct from initial formation or dissociation of the binary complex of Dpo4 and DNA. Subsequently, simulation and single-molecule fluorescence experiments have added further insight into this proposed model of DNA binding has.
References:
Xu, C., Maxwell, B. A., Brown, J. A., Zhang, L., and Suo, Z. (2009) PLoS Biol. 7, e1000225
Maxwell, B. A., Xu, C., and Suo, Z. (2014) Biochemistry. 53(11):1768-78
Keywords: FRET, conformational dynamics, DNA polymerase
Abstract:
λ Exonuclease is a highly processive 5’-3’ exonuclease that binds double-stranded DNA ends and digests the 5’ end into mononucleotides. The digestion product, a 3’ single-stranded overhang DNA, can serve as the substrate for pairing proteins in homologous recombination. λ Exonuclease forms a homotrimeric ring with a tapered central channel for tracking along the DNA. During catalysis, dsDNA enters the wider side of the channel and the 5’ end is digested at one of the three active centers. Then the resulting 3’ single-stranded overhang exits through the narrower side of the channel to emerge out the back of the trimer. Two mechanisms could be envisioned for enzymes that form oligomeric rings to use their multiple active sites: the sequential mechanism, where all active sites in the oligomer are engaged in catalysis sequentially; and the non-sequential mechanism, where the substrate DNA locks onto one active site for multiple rounds of catalysis. To understand how the λ Exonuclease trimer uses its three active sites, we used a “mutant poisoning” approach, where inactive subunits are mixed with active subunits to form hybrid trimers. In the sequential mechanism, one inactive subunit will cause the trimer to lose all activity; whereas in the non-sequential mechanism, the trimer remains active as long as there is at least one active subunit. A K131A mutant of λ Exonuclease, which is completely inactive for nucleotide hydrolysis, but maintains its DNA binding ability, was introduced. Nickel spin pull down assays, where K131A mutant with a Histidine tag was mixed with untagged wild-type λ Exonuclease and run on a nickel column, showed observable subunit exchange after 1h incubation at 37℃. The λ Exonuclease activity was measured by determining the rate of digestion of a linear pUC19 duplex DNA. The results showed that the λ Exonuclease hybrid trimers still remain active, suggesting that a non-sequential mechanism of active sites is used during λ Exonuclease catalysis.
References:
Smith, G.R. (1988) Homologous Recmombination in Procaryotes. Microbiol. Rev. 52: 1-28.
Zhang, J., Xing, X., Herr, A.B., and Bell, C.E. (2009). Crystal structure of E. coli RecE protein reveals a toroidal tetramer for processing double-stranded DNA breaks. Structure 5: 690-702.
Zhang J., McCabe K.A., and Bell C.E. (2011) Crystal structures of lambda exonuclease in complex with DNA suggest an electrostatic ratchet mechanism for processivity. Proc. Natl. Acad. Sci. 29: 11872-7.
Crampton DJ, Mukherjee S, Richardson CC (2006) DNA-induced switch from independent to sequential dTTP hydrolysis in the bacteriophage T7 DNA helicase. Mol Cell 21:165–174.
Zhou, M.; Wysocki, V.H., (2014) Surface induced dissociation: Dissecting noncovalent protein complexes in the gas phase. Acc. Chem. Res.
Keywords: Homologous Recombination, Lambda Exonuclease, mutant poisoning
Abstract not available online - please check the printed booklet.
Abstract:
Pyrrolysine (pyl), sometimes called the 22nd amino acid, seemingly exists solely to assist in the microbial transfer of a methyl group from methylated amines to a corrinoid protein (as in the first step of methanogenesis from methylamines). MttB, the trimethylamine methyltransferase, is a TIM barrel containing pyl in the putative active site cleft. The proposed mechanism for methyl transfer involves pyl acting as a “handle,” forming a covalent bond with trimethylamine, orienting it for attack by the highly nucleophilic Co(I) of the corresponding corrinoid protein, MttC.
Pyl may have come to this function by entering an existing family of methyltransferases. Indeed, we identified many MttB homologues that lack pyl. One such homolog, Desulfitobacterium hafniense MtgB, was subsequently shown to act as a glycine betaine-corrinoid methyltransferase. In silico analysis of MtgB revealed four conserved amino acid residues likely to participate in binding and catalysis. An arginine (R309) located within the barrel is likely to coordinate the carboxylate group of TMG. Two aromatic residues within the barrel but nearer to the surface (Y96 and F353) may stabilize the methylated nitrogen of TMG via cation-π interactions. A nearby histidine (H345) would then be well positioned to participate in catalysis. Such binding of TMG could orient the methyl group for attack by the Co(I) of a corrinoid protein, consistent with the conserved architecture of corrinoid-dependent methyltransferases.
To investigate this model, mutations were generated at each of the four positions. Preliminary in vitro methylation assays indicate that each mutation reduces the activity of the enzyme below the level of detection. Recently, MtgB was crystallized in the presence of TMG and the corrinoid cofactor. The forthcoming solution of the substrate-bound structure will further refine this model, ultimately providing insight into the larger family of methyltransferases that include pyl.
Keywords: Pyrrolysine, Methyltransferase, Corrinoid
Abstract:
Eukaryotes require highly accurate and processive DNA polymerases to ensure faithful and efficient replication of their genomes. DNA polymerase ε (Polε) has been shown to catalyze leading-strand DNA synthesis during replication in vivo, but little is known about the kinetic mechanism of polymerization catalyzed by this replicative enzyme. To elucidate this mechanism, we have generated a truncated, exonuclease-deficient mutant of the catalytic subunit of human Polε (Polε exo-) and carried out pre-steady-state kinetic analysis of this enzyme. Our results show that Polε exo-, as other DNA polymerases, follows an induced-fit mechanism when catalyzing correct nucleotide incorporation. Polε exo- binds DNA with a KdDNA of 79 nM and dissociates from the E·DNA binary complex with a rate constant of 0.021 s−1. Although Polε exo- binds a correct incoming nucleotide weakly with a KddTTP of 31 μM, it catalyzes correct nucleotide incorporation at a fast rate constant of 248 s−1 at 20 °C. Both a large reaction amplitude difference (42%) between pulse-chase and pulse-quench assays and a small elemental effect (0.9) for correct dTTP incorporation suggest that a slow conformational change preceding the chemistry step limits the rate of correct nucleotide incorporation. In addition, our kinetic analysis shows that Polε exo- exhibits low processivity during polymerization. To catalyze leading-strand synthesis in vivo, Polε likely interacts with its three smaller subunits and additional replication factors in order to assemble a replication complex and significantly enhance its polymerization processivity.
References:
Zahurancik, W. J., Klein, S. J., and Suo, Z. (2013) “Kinetic Mechanism of DNA Polymerization Catalyzed by Truncated Human DNA Polymerase ε”, Biochemistry. 52, 7041-7049.
Keywords: DNA polymerase , Mechanism, Pre-steady-state
Abstract:
The YidC family members function to insert proteins into membranes in bacteria, chloroplasts, and mitochondria and they can also act as a platform to fold and assemble proteins into higher order complexes. Here, we provide information about the proximity relationships and dynamics of the five conserved C-terminal transmembrane (TM) regions within E. coli YidC. By using a YidC construct, with tandem thrombin protease sites introduced into the cytoplasmic loop C1, crosslinking between paired-Cys residues located within TM segments or in the membrane border regions was studied using thiospecific homobifunctional crosslinking agents with different spanner lengths or by iodine-catalyzed disulfide formation. These crosslinking studies show that TM3, TM4, TM5, and TM6 each have a face oriented toward TM2 of the in vivo expressed YidC. The studies also reveal that YidC is a dynamic protein, as crosslinking was observed between cytoplasmic Cys residues with a variety of crosslinkers. A large number of conserved proline residues on the cytoplasmic side of the 5 conserved core TM segments could explain the observed flexibility and the structural fluctuations of the TM segments could provide an explanation of how YidC is able to recognize a variety of different substrates.
Keywords: YidC, Cysteine Crosslinking, Helical Packing
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
In cancer cachexia, a muscle wasting syndrome seen in end-stage cancer, patients suffer from fatigue and weakness coupled with anorexia and weight loss. A previous study by our lab showed that the hearts of c26 adenocarcinoma tumor-bearing mice, a common model of cancer cachexia, exhibit early signs of diastolic dysfunction at the cellular level and systolic dysfunction on echocardiography. Echocardiography of tumor burdened mice showed decreased fractional shortening and posterior wall thickness and increased left ventricular systolic diameter without changes in absolute and normalized heart weight, suggesting early systolic dysfunction. At the mycocyte level, tumor burdened animals showed significant increases in time-to-90% shortening and time-to-90% relengthening, indicating myocyte dysfunction. Here we attempted to ameliorate these effects with losartan, an angiotensin receptor blocker, which prevents the development of cardiac hypertrophy via inhibition of the angiotensin II pathway. Daily treatment of tumor burdened mice with losartan appears to prevent much of the observed cardiovascular remodeling due to the effects of the c26 adenocarcinoma tumor.
Keywords: Cachexia, Myocardial Dysfunction, Losartan
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Purpose: Deficient BRCA confers a 54-90% lifetime risk of breast cancer but only accounts for 20% of familial breast cancer. We identify other genetic factors in breast cancer by studying proteins that could perform similar functions to BRCA and might function in the same pathways as known breast cancer susceptibility genes.
Methods: We screened 70 breast cancer gene expression datasets for candidate genes that were coexpressed with genes in the BRCA DNA repair pathways. MYCBP, c-Myc Binding Protein, coexpressed with four DNA repair factors; differentially expressed in breast carcinomas versus normal tissue; and demonstrated an effect on 3 DNA repair pathways.
We hypothesize that MYCBP plays a role in DNA repair and propose testing function in DNA repair pathways, investigating MYCBP regulation of repair gene transcription, and evaluating the potential of MYCBP to be a biomarker in breast cancer.
Results & Implications:
DNA repair: Similar to known DNA repair genes, MYCBP depletion results in a 5-fold decrease in DNA repair.
Transcriptional regulation: MYCBP depletion results in a loss of DNA repair factors BRCA1, RAD51, and 53BP1 (protein, RNA). The decrease in repair factor expression shows the basis for MYCBP having such a profound effect on multiple DNA repair pathways. The loss of these genes essential to three different DNA repair pathways increases the likelihood of downstream carcinogenesis.
Tumor sample expression: We stained human breast tissue samples with MYCBP antibody found differential staining between normal and cancer tissue. Data on MYCBP expression patterns in tumor samples can provide insight in patient diagnosis.
Cancer therapy response: We have tested cisplatin on MYCBP-deficient cells, which show sensitivity to the drug. Cisplatin is a common cancer therapeutic; however, some patients have shown resistance to its application. Determining root cause and likely response through MYCBP will help personalize patient care.
Keywords: MYCBP, doublestrand break repair
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
To identify novel and perhaps targetable mechanisms of oncogenic expression contributing to androgen-dependent and castration-resistant prostate cancer (ADPC and CRPC, respectively), we have extended previous findings from Zhang, et al., Cancer Research 2011 to comprehensively identify gene expression profiles determined by the collaborative activities of the transcription factors (TFs) FoxA1 and CREB1 in the LNCaP (ADPC model) and LNCaP-abl (CRPC model, and LNCaP derivative) cell lines. By applying an integrated genomics approach, we have identified relevant pathways downstream of this co-regulatory pathway, as well as CREB1/FoxA1 target genes whose expression patterns correlate with clinical outcomes. That this pathway appears to be sensitive to compounds inhibiting CREB1 transactivation suggests a therapeutic potential in targeting cooperative TF activity in cases of advanced prostate cancer.
Keywords: Prostate Cancer, FoxA1 , CREB1
Abstract not available online - please check the printed booklet.
Abstract:
Metastasis suppressor genes are those that inhibit metastasis and secondary progression whose loss can lead to metastatic progression. Defining their mechanisms of actions may yield critical findings that can lead to translational opportunities to improve treatment of progressive metastatic cancers. We recently identified RCAN1-4 (Regulator of Calcineurin 1-4) as a potential metastasis suppressor. Microarray analysis found that RCAN1-4 expression level was consistently upregulated in human thyroid cancer cells treated with metastin. In thyroid cancer tissues, metastatic lesions were shown to have much lower RCAN1-4 levels compared with paired primary tumors and in vitro studies showed RCAN1-4 suppressed cancer cell growth and motility. Other groups have shown that RCAN1-4 inhibits endothelial cell proliferation, migration, and angiogenesis in B16-F10 melanoma subcutaneous implants. Moreover, RCAN1, the gene from which RCAN1-4 and other splice variants are encoded, is partially responsible for the reduced solid tumor rate in Down syndrome, potentially through its effects on endothelial cells. Taken together, these data demonstrate that RCAN1-4 may function uniquely as a bona-fide tumor metastasis suppressor through effects on both tumor cells and the host. To determine its role in tumor cells in vivo, we have generated stable RCAN1-4 knockdown and inducible overexpression thyroid cancer cell lines, which will be used for xenograft studies to identify the role of RCAN1-4 in tumor cells during tumor progression. To determine the role of RCAN1-4 specifically in the host as a regulator of cancer progression, we have created a novel RCAN1-4 specific knockout mouse. These mice are viable and will be used to investigate the effects of RCAN1-4 knockout in the host on metastatic progression and tumor angiogenesis using syngenic model systems. Utilizing these systems, we anticipate being able to determine the mechanisms by which RCAN1-4 metastasis suppression occurs.
Keywords: RCAN1-4, Metastasis suppressor, Angiogenesis
Abstract:
Winter is a challenging time to survive and breed outside of the tropics. Animals monitor day length (photoperiod) to regulate seasonally appropriate adaptations in anticipation of harsh winter conditions. The net result of these photoperiod-mediated adjustments is enhanced immune function and increased survival in the absence of breeding. Thus, the ability to discriminate day length information is critical for survival and reproduction in small animals. However, during the past century, urban and suburban development has rapidly expanded and filled the night sky with light from various sources, obscuring crucial light-dark signals, which alters interpretation of day lengths. Furthermore, reduced space, increased proximity to people, and the presence of light at night may act as stressors for small animals. Whereas acute stressors typically enhance immune responses, chronic exposure to stressors typically impairs immune responses. Therefore, we hypothesized the combination of dim light at night and chronic stress interferes with enhanced cell-mediated immunity observed during short days. Siberian hamsters (Phodopus sungorus) were assigned to short or long days with dark nights or dim (5 lux) light at night for 10 weeks. Following restraint (6 h/day, 2 wks), delayed type hypersensitivity (DTH) responses were assessed. Both dim light at night and restraint reduced DTH responses. Dim light at night during long nights produced an intermediate short day phenotype including impaired DTH response and partially regressed reproductive organs. These results suggest the constant presence of light at night could negatively affect small species survival by disrupting the timing of breeding and immune responses.
Keywords: dim light at night, cell-mediated immunity, chronic stress
Abstract:
Developing pre-clinical spinal cord injury (SCI) models that better reflect the human condition will aid in the translation of potential therapeutics from bench to bedside. Immunocompromised mice, such as NOD-SCID-IL2rg/null (NSG), support engraftment of human blood stem cells and the development of a fully functional human immune system. Thus, humanized NSG (hNSG) mice could be utilized to more accurately reflect the inflammatory response during spinal cord injury and repair.
Newborn NSG mice were engrafted with human CD34+ hematopoietic stem cells, underwent a moderate SCI 12-14 weeks later, and allowed to recovery for 28 days. SCI resulted in an immediate loss of coordinated hind limb function with gradual recovery that plateaus at a BMS score of 4 (occasional plantar stepping). Mean lesion volume was 0.67 mm3 and lesion length 2076 µm. Spinal cord lesions were devoid of neurons and myelinated axons, with a rim of spared tissue containing a GFAP labeled glial scar. The lesion core consisted of Iba-1 immunolabeled phagocytic cells characteristic of macrophages that infiltrate the lesion after injury.
Flow cytometry of blood samples confirmed that 50% of total circulating cells were of human origin (hCD45+), and three distinct monocyte subtypes were identified by levels of cell surface markers CD14 and CD16. Labeling of spinal cord tissue with hCD45 identified the presence of human immune cells in the lesion core, many with a phagocytic phenotype typical of macrophages.
We have demonstrated that hNSG mice engraft human blood stem cells, resulting in the development of circulating human immune cells with specific populations of monocytes resembling those observe in humans. hNSG mice have recovery profiles and spinal cord lesions similar to what has been observed in other mouse strains. Finally, human immune cells were present in the lesion core and displayed a phenotype similar to phagocytic macrophages. In all, humanized mice represent a novel model for the study of inflammation after spinal cord injury.
Keywords: Spinal Cord Injury, Inflammation, Immunology
Abstract:
As one of the most potent signals to the circadian system, light exposure during the night significantly disrupts circadian rhythms. The circadian system regulates metabolism, driving physiologically relevant oscillations in gene transcription and availability of metabolic hormones and enzymes. Exposure to dim light at night (DLAN) has been associated with increased body mass and altered feeding rhythms in adult mice. High fat diet (HFD) also disrupts behavioral and molecular rhythms in adult mice. When combined with DLAN, HFD exacerbates body mass gain and impaired glucose processing. But the effect of these two factors during development remains unspecified. Early life is a critical time for the development of endogenous circadian rhythms, as well as metabolic priming. Disruption of early postnatal clock gene expression precedes an adult obesity phenotype. We predict that disruption of circadian rhythmicity through DLAN and/or HFD primes for increased adiposity in adulthood. Mice were bred in our lab and litters were normalized to 10 pups (CL) or reduced to 3 pups (SL). CL mice were weaned onto chow diet whereas SL mice were given HFD to maintain a lifelong HFD condition. Both groups were also split by light condition; half were maintained in a standard light dark (LD) cycle or exposed to nightly dim (5 lux) light (DLAN). After four weeks in respective lighting conditions, food intake and locomotor activity were assessed. At nine weeks of age mice either underwent glucose tolerance testing or hippocampal, fat, and liver tissues were collected for qPCR. Here we report that male mice exposed to lifelong HFD exhibit hyperphagia and impaired glucose tolerance. Males exposed to DLAN and HFD increased daytime food intake despite no total increase in calories consumed. Unlike reported in adults, DLAN males decreased HFD induced weight gain. Further studies are underway to elucidate the divergent weight phenotypes observed at different ages of exposure to DLAN.
Keywords: light at night, development, obesity
Abstract:
Spasticity is clinically defined as a velocity dependent increase in tonic stretch reflexes with exaggerated tendon jerks and is a common sequela of spinal cord injury (SCI). Spasticity occurs in up to 75% of individuals with SCI with most reporting that it negatively impacts quality of life. While the pathophysiology of spasticity is largely unknown, one possible mechanism involves dysfunction of the spinal interneurons (Neilson 2004). Previously we showed an inability of the remote lumbar spinal cord to learn an instrumental task at chronic time points following mid-thoracic transection (TX). Here, we examined interneurons of the lumbar cord for morphological changes as key players in this known deficit. We used Golgi’s staining technique combined with neuron tracing software to quantify dendritic spines of interneurons located in laminas VI and VII after different injury severities. We looked at acute (7d) and chronic (42d) time points in both complete and incomplete injuries as a model of reactive plasticity following SCI as well as to examine the effects of sparing on this response. This experiment is compelling as it is the first to attempt quantification of plasticity in the lumbar locomotor networks that likely contribute to spasticity in humans. Preliminary data showed an increase in the number of dendritic spines following transection suggesting a potentially maladaptive response. Additionally, these interneurons had smaller cell body size compared to those of naïve animals. Consistent with the noted morphological changes, data acquired from sister groups showed a decrease in stimulation amplitude required to produce a predetermined ankle flexion force of 0.4-0.5N in the tibialis anterior muscle after chronic transection injury (n=5; Mean Stim = 0.64mA +/- 0.19) compared to naïve (n=8; Mean Stim = 2.30 +/- 0.10; p<.001). Together, these findings show that morphological changes of lumbar interneurons appear to produce greater neuronal excitability, which would be consistent with hyperreflexia and spasticity. They also provide a novel target for interventions designed to reduce spasticity and promote functional recovery following spinal cord injury.
References:
1) Neilson et al (2007) Acta Physiol 189(2):171-80.
2) Roy and Edgerton (2012) Exp Neurol 235(1):116-22.
3) Hansen et al (2014) Submitted.
Keywords: Spinal Cord Injury, Plasticity, Spasticity
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Hereditary neuropathies, or Charcot-Marie-Tooth disease, are some of the most common inherited human neurological disorders, affecting approximately 1 in 2,500 people in the United States. There at least 50 genes identified that, when mutated, result in neuropathy [1, 2]. Though these genes are diverse, many appear to involve the disruption of native protein-protein interactions. Intriguingly, three of the ten small heat shock proteins (sHSPs) expressed in humans have been associated with hereditary motor neuropathies. Designated HSPB1, HSPB3 and HSPB8, these small proteins readily interact with other sHSPs to form homo- and hetero-oligomeric complexes [3]. Of these 3 proteins, HSPB3 remains the least understood. The development of an axonal motor neuropathy characterized by adult-onset, slowly progressive distal arm and leg weakness is associated with a missense mutation, R7S, in the N-terminal region of HSPB3 [4]. In this study, we investigate the interaction between mutant HSPB3 and other sHSPs using co-immunoprecipitation and immunofluorescence techniques. We find alterations in the protein interactome of wild type HSPB3 compared to mutant HSPB3. We propose that this disruption in sHSP interactions plays a mechanistic role in the associated motor neuropathies.
References:
1. Gess, B., A. Schirmacher, and P. Young, [Genetics of neuropathies]. Nervenarzt, 2013. 84(2): p. 157-65.
2. Rossor, A.M., et al., The distal hereditary motor neuropathies. J Neurol Neurosurg Psychiatry, 2012. 83(1): p. 6-14.
3. Delbecq, S.P. and R.E. Klevit, One size does not fit all: The oligomeric states of alpha B crystallin. Febs Letters, 2013. 587(8): p. 1073-1080.
4. Kolb, S.J., et al., Mutant small heat shock protein B3 causes motor neuropathy: utility of a candidate gene approach. Neurology, 2010. 74(6): p. 502-6.
Keywords: Small Heat Shock Proteins, HSPB3, Motor Neuropathies
Abstract:
Chronic neuroinflammation and calcium dysregulation are shared components of Alzheimer’s disease and other neurodegenerative processes. Prolonged neuroinflammation produces elevation of pro-inflammatory cytokines and reactive oxygen species which are capable of altering neuronal calcium homeostasis via L-type voltage dependent calcium channels (L-VDCCs) and ryanodine receptors (RyRs). Chronic neuroinflammation also leads to deficits in spatial memory, which may be related to calcium dysregulation. The studies herein use an in vivo model of chronic neuroinflammation: rats were treated with intraventricular infusion of lipopolysaccharide (LPS) for 28 days. Synaptosomes from LPS-infused rats have increased calcium uptake, which is decreased by pharmacological blockade of the L-VDCC either in vivo or ex vivo. LPS-infused rats had significant memory deficits in the Morris water maze; this deficit was ameliorated by treatment with an L-VDCC antagonist. Taken together, these data indicate that calcium dysregulation during chronic neuroinflammation is at least partially dependent on increases in L-VDCC function. However, blockade of the RyRs also slightly improves spatial memory of LPS-infused rats, demonstrating that other calcium channels are dysregulated during chronic neuroinflammation. Calcium-dependent immediate early gene expression was reduced to control levels in LPS-infused rats treated with L-VDCC or RyR antagonists, indicating normalized synaptic function that may underlie improvements in spatial memory. Pro-inflammatory markers are also reduced in LPS-infused rats treated with either drug. Overall, these data suggest that calcium dysregulation via L-VDCCs and RyRs plays a crucial role in neuroinflammation-induced memory deficits.
Keywords: calcium, neuroinflammation, memory
Abstract:
Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder characterized by loss of lower motor neurons.It occurs due to deletion or mutation in the SMN1 gene and retention of the SMN2 gene.SMA is caused by a decrease in the levels of the SMN protein; complete absence of SMN is lethal.SMN is ubiquitous but a decrease in SMN causes degeneration of the motor neurons (MNs) and muscle atrophy.Hence understanding the spatial requirement of SMN is critical to localize the functional importance of SMN and for the development of therapies.Using tissue specific Cre-drivers and the Cre-loxP recombination system, we deleted mouse Smn in just the neurons or just the muscle; with SMN2 transgene in the background providing low SMN everywhere.As a reciprocal experiment, we restored normal levels of SMN in neurons or muscle with low SMN levels in other tissues.We observed that decreasing SMN in muscle has no phenotypic effect.This was corroborated by muscle function studies (twitch, tetanic and eccentric force).Replacement of Smn in muscle did not rescue SMA mice.Thus the muscle does not require high levels of SMN.SMN from two copies of SMN2 in the background is sufficient for normal muscle function.Deletion of Smn with Human Synapsin-iCre (all neurons plus vasculature) gave a phenotype similar to SMA while replacement gave rescue of the phenotype.Deletion of Smn with Nestin-Cre (all neurons, but weak in MNs) or with ChAT-Cre (all MNs) did not give an SMA phenotype and replacement did not rescue.Nestin-Cre together with ChAT-Cre (thus covering all neurons) gave an SMA phenotype upon deletion and rescue upon replacement.Electrophysiological measurements of the motor unit (MUNE: motor unit number estimate and CMAP: compound motor action potential) were performed on the deletion and rescue lines.These, along with weight and survival curves of deletion and replacement experiments show that SMN is a must in MNs.Hence increasing SMN in neurons is sufficient to rescue SMA mice.
References:
1. Burghes A.H.,and Beattie C.E. (2009) Spinal muscular atrophy : Why do low levels of survival motor neuron protein make motor neurons sick?, Nature Reviews. Neuroscience 10, 597-609.
2. Gavrilina, T. O., McGovern, V. L., Workman, E., Crawford, T. O., Gogliotti, R. G., DiDonato, C. J., Monani, U. R., Morris, G. E., and Burghes, A. H. (2008) Neuronal SMN expression corrects spinal muscular atrophy in severe SMA mice while muscle-specific SMN expression has no phenotypic effect, Human molecular genetics 17, 1063-1075.
Keywords: SMA, spinal muscular atrophy, MNs, motor neurons
Abstract not available online - please check the printed booklet.
Abstract:
In the elderly, peripheral infection is associated with a higher incidence of neuropsychiatric complications, but the mechanisms underlying these complications are unknown. Using a mouse model of aging, we have reported that acute activation of the innate immune system caused an exaggerated neuroinflammatory response and the development of depressive-like behavior in aged mice. These age-related inflammatory deficits may be caused by impaired regulation of central nervous system (CNS) immune cells, called microglia. For example, following immune challenge, aged microglia produce amplified levels of both pro- and anti-inflammatory cytokines. Despite higher levels of Interleukin (IL)-10, an anti-inflammatory cytokine, microglial activation was unresolved in the aged brain. In adult mice, astrocytes express the IL-10 receptor (IL-10R) and increase production of the regulatory molecule Transforming Growth Factor (TGF) b in response to IL-10 treatment. Therefore, the purpose of this study was to investigate the degree to which IL-10 signaling and subsequent TGFb upregulation is impaired in the brain of aged mice. First, we examined astrocytes in adult and aged mice and found that astrocytes in aged mice had increased expression of the inflammatory markers GFAP and vimentin. Astrocytes from aged mice also had decreased IL-10R expression compared to adults. Following immune challenge in vivo, astrocytes from adult mice upregulated IL-10R and TGFb mRNA. Astrocytes from aged mice, however, failed to upregulate both IL-10R and TGFb mRNA. This lack of regulation by TGFb was associated with exaggerated pro-inflammatory gene expression and neuroinflammation. These data indicate that astrocytes from aged mice are more inflammatory and less sensitive to IL-10, and in turn, TGFb is not upregulated following immune challenge. Future studies will use AAV9 gene delivery to enhance IL-10R expression preferentially on astrocytes to restore IL-10 responsiveness and TGFb production.
Keywords: Microglia, Astrocytes, neuroinflammation
Abstract not available online - please check the printed booklet.
Abstract:
Cysteine-rich 61 (CCN1) is a 4-domain matricellular protein that promotes angiogenesis, cell adhesion, migration, invasion, and inflammation. CCN1 is upregulated in virally infected glioma cells and is pro-inflammatory (Haseley et al. Cancer Res, 2012) both by directly and indirectly activating macrophages. CCN1 induces indirect activation by binding the α6β1 integrin receptor on glioma cells to initiate a type-1 interferon response, measured by significant upregulation of interferon response genes in CCN1 overexpressing glioma cells. Macrophage migration was directly induced by purified CCN1 protein alone, and this induction was significantly reduced using neutralizing anti-αM or β2 antibodies (P<0.05). We hypothesized that secreted CCN1, through its pro-inflammatory mechanisms, inhibits viral replication and propagation in tumors treated with oncolytic herpes simplex virus type 1 (HSV1), thereby reducing antitumor efficacy of the virus. We investigated the use of a function-blocking anti-CCN1 antibody in vivo in combination with oncolytic HSV1 to treat subcutaneous gliomas in NUDE mice. IP administration of anti-CCN1 antibody improved oncolytic HSV1 replication, measured by luciferase imaging and HSV1 staining, and reduced macrophage and NK infiltration in SQ tumors over an IgG control group. Combination anti-CCN1 antibody and oncolytic HSV1 therapy significantly improved time to progression (p=0.047) without significant enhancement in survival (p=0.10). Our results implicate CCN1 as a regulator of the antiviral innate immune response which can be partially overcome in vivo by administering a function-blocking anti-CCN1 antibody. This treatment was able to slow regrowth of tumors and is a promising indication that CCN1 blocking therapy in vivo may further enhance oncolytic viral efficacy in the treatment of glioblastoma.
Keywords: Glioblastoma, Oncolytic, Cancer
Abstract:
Amyotrophic lateral sclerosis (ALS) is a late onset, fast progressing and always fatal motor neuron (MN) disease. Recently, dramatic reduction of major histocompatibility complex class I (MHCI) in MNs was reported in rapidly progressive ALS mice, suggesting beneficial role of MHCI in preserving MNs during disease. In consistent with previous findings from ALS mice, we observed the substantial reduction of MHCI signals in human MNs in ALS postmortem tissue. Interestingly, in vitro co-culture model of ALS demonstrated that this is mediated by non-cell autonomous effects, specifically by ALS astrocytes.
In ALS, astrocytes have been identified as mediators of MN death. However, the mechanism how ALS astrocytes specifically recognize and kill MNs has remained unknown. We hypothesized that ALS astrocytes discriminate target MNs based on the reduced level of MHCI. Mouse and human ALS astrocytes expressed MHCI inhibitory receptors, which are known to recognize low level of MHCI in target cells and activate killing pathway. Overexpression of MHCI in MNs known to bind to these receptors protected MNs from ALS astrocytes toxicity in vitro and extended survival and delayed the disease progression by 38% in ALS mice.
We extended our studies to determine how astrocytes kill MNs in ALS. It is well known that natural killer (NK) cells utilize two cytolytic proteins, pore forming protein, perforin (PRF1) and cell death-inducing proteases granzyme B (GZMB), which are secreted by reduced MHCI in target cells. Like NK cells, low MHCI in MNs triggered both mouse and human ALS astrocytes to actively release PRF1 and GZMB. Suppression of PRF1 or GZMB expression in these ALS astrocytes resulted in MN protection during in vitro co-culture.
Taken together, our results show that ALS astrocytes specifically display toxicity towards MNs by detecting decreased levels of MHCI and engage a killing mechanism, which is a mechanism reminiscent of the innate immune system. These findings have great implication for ALS research on the aspect of potential therapeutic opportunities.
References:
1.Haidet-Phillips, A.M., et al. Astrocytes from familial and sporadic ALS patients are toxic to motor neurons. Nat Biotechnol 29, 824-828 (2011).
2.Garden, G.A. & La Spada, A.R. Intercellular (mis)communication in neurodegenerative disease. Neuron 73, 886-901 (2012).
3.Glass, C.K., Saijo, K., Winner, B., Marchetto, M.C. & Gage, F.H. Mechanisms underlying inflammation in neurodegeneration. Cell 140, 918-934 (2010).
4.Andersen, M.H., Schrama, D., Thor Straten, P. & Becker, J.C. Cytotoxic T cells. J Invest Dermatol 126, 32-41 (2006).
5.Philips, T. & Robberecht, W. Neuroinflammation in amyotrophic lateral sclerosis: role of glial activation in motor neuron disease. Lancet Neurol 10, 253-263 (2011).
Keywords: amyotrophic lateral sclerosis, astrocyte, major histocompatibility complex class 1
Abstract not available online - please check the printed booklet.
Abstract:
Human T-cell Leukemia Virus Type-1 (HTLV-1) is a complex retrovirus infecting 15-25 million people worldwide. HTLV-1 is the etiological agent of an aggressive malignancy of CD4+ T cells termed Adult T-Cell Leukemia (ATL). ATL patients, on average, survive one year from disease onset. The HTLV-1 regulatory protein Tax is required for HTLV-1-mediated cellular transformation both in vitro and in vivo. Tax primarily functions to promote transcription of viral genes, but has also been shown to deregulate cellular genes leading to cell growth and genetic instability. Previous studies showed that Tax induces NF-κB by two distinct signaling/activation pathways: the classical pathway, which is rapid, and the alternative pathway, which is delayed. The exact role of the alternative pathway in HTLV-1-mediated transformation is unknown. We propose that Tax interaction with the alternative NF-κB pathway is important for HTLV-induced pathogenesis. To test this hypothesis we will identify and utilize Tax mutants that are deficient in their ability to activate the NF-κB pathways. Viruses containing these mutant forms of tax will be used to infect primary peripheral blood mononuclear cells (PBMCs) and transformation will be monitored via a cellular proliferation assay. We also plan to identify Tax-1 binding partners that are important for Tax-mediated activation of the NF-κB pathway. To identify these binding partners we generated S-protein epitope tagged Tax expression vectors for both wild type and NF-κB activation deficient tax genes. We expressed tagged Tax proteins in 293T cells and performed immunoprecipitation followed by mass spectrometry to identify binding partners. By comparing the differences between the wild type Tax and mutant Tax binding partners we will be able to identify candidate proteins required for Tax-mediated activation of the alternative NF-κB pathway. We hypothesis that these candidates could then be used in future studies as clinical targets to treat patients with ATL.
Keywords: HTLV, Tax, NF-B
Abstract:
The primer for reverse transcription in HIV-1, human tRNALys3, is selectively packaged into virions along with tRNALys1,2. Human lysyl-tRNA synthetase (LysRS), the only cellular factor known to interact specifically with all three tRNALys isoacceptors, is also packaged into HIV-1. Selective packaging of tRNALys depends on the ability of the tRNA to bind to LysRS and the presence of both host cell factors within virions is required for optimal viral infectivity. LysRS is normally part of a dynamic mammalian multisynthetase complex (MSC). Recent studies show that extra-cellular stimuli (e.g., interferon-γ, TNF-α) can trigger phosphorylation of LysRS, releasing it from the complex to act in non-translational pathways. Elucidating the detailed molecular mechanism for the alternative function of LysRS in HIV-1 infectivity is needed in order to develop effective therapeutics aimed at targeting this essential host cell factor. The mechanism by which the LysRS/tRNA complex is diverted from its normal function in translation and recruited into particles is unclear. Here, we show that the expression of LysRS is unaltered upon HIV-1 infection of HEK293T and CD4+ HuT/CCR5+ T cells, suggesting that the LysRS species packaged is recruited from an existing pool of LysRS. Interestingly, using immunofluorescence and confocal microscopy, we find that LysRS trafficking is altered upon HIV-1 infection. Confocal analysis and cellular fractionation studies indicate that LysRS localizes to the nucleus during HIV-1 infection. Studies with a LysRS mutant lacking a nuclear localization signal are planned, and LysRS-Gag and LysRS-genomic RNA co-localization studies are currently underway. Our studies also indicate that phosphorylation of LysRS on Ser and/or Thr residues occurs after HIV-1 infection. We hypothesize that phosphorylation results in release from the MSC, which may re-direct LysRS to assembling virions. Studies to understand the significance of these findings for HIV infectivity are in progress.
Keywords: LysRS, HIV-1, Assembly
Abstract not available online - please check the printed booklet.
Abstract:
Epigenetic modifications are influential in organizing and controlling gene expression. An example of epigenetic control is repressive cytosine and histone methylation that leads to transcriptional gene silencing (TGS) of potentially harmful DNA, including transposable elements and DNA viruses. Geminiviruses have circular ssDNA genomes that replicate via dsDNA intermediates that associate with histones to form minichromosomes, which can be repressed by epigenetic modifications leading to TGS. Arabidopsis encodes two plant-specific RNA polymerases, Pol IV and Pol V, which carry out non-coding transcription that results in cytosine methylation. We are using geminiviruses as de novo methylation models to more precisely define the roles of these polymerases in chromatin methylation. Results indicate that Pol IV and V are not required to initiate viral DNA methylation, but are necessary for the hypermethylation of viral promoters that accompanies host recovery from infection. Additionally, virus-specific siRNAs are produced in pol IV and pol V mutants, albeit at reduced levels. Thus we hypothesize that another polymerase, likely Pol II, can perform non-coding transcription leading to methylation initiation, while Pol IV and V are needed for its amplification and spread. However, Pol IV and V are individually required for the establishment of a repressive mark, dimethyl histone 3 lysine 9 (H3K9me2), on viral chromatin. Interestingly, Pol IV and V are multi-subunit enzymes that are related to, and share subunits with, Pol II. In addition to transcribing mRNA, Pol II orchestrates the formation of heterochromatin and transcriptional gene silencing in fission yeast. Experiments with hypomorphic Arabidopsis Pol II mutants are in progress to examine its role in methylation-mediated defense. Pol II mutants are hypersusceptible to geminivirus infection and also are unable to deposit H3K9me2 on viral chromatin, suggesting that this enzyme may recruit Pol IV and V to amplify repressive cytosine methylation and establish H3K9me2 marks.
Keywords: geminivivirus, rddm, pol ii
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
The tumor suppressor p53 is a critical 54 kDa protein responsible for the regulation of cellular differentiation during times of cellular stress. This protein is so critical as to be found to be mutated in approximately 50% of all human cancer cases. The majority of these mutations are found in the central, ordered DNA-binding domain of the protein. One way in which these mutations can disrupt DNA binding is to lower the overall stability of the protein, making it unable to properly interact with DNA. Consequently, any knowledge that can be gained in stabilizing the p53 DNA-binding domain could potentially be very valuable. It has been observed that the p53 homolog in C. elegans is considerably more stable than its human counterpart. By comparing the sequences of human p53 and worm p53, it was proposed that shortening the S7S8 turn is an important factor in this stability.1 This initial work was done in silico, and the Magliery lab is constructing mutants based on this hypothesis to be expressed and screened in vitro. Compared to human p53, worm p53 has four fewer residues in the S7S8 loop. By expressing and screening various four- and five- residue deletion mutants, insight was gained into which residues are important in the loop. These results will be used to design a loop mutant library that will probe sequence space for mutants that are more stable than the control construct. There is also evidence that the final few residues of the disordered N-terminal domain of p53 has a profound effect on p53 solubility and a moderate effect on stability.2 Potential effects of this change to the DNA binding domain of p53 have been explored. In addition to the worm p53 comparison strategy, algorithmic improvement of p53 has been attempted using methodology developing in the Magliery lab.3 The resulting construct is algorithm derived p53, or Algo p53, has been expressed and characterized.
References:
1. Pan, Y.; Ma, B.; Levine, A. J.; Nussinov, R., Comparison of the human and worm p53 structures suggests a way for enhancing stability. Biochemistry 2006, 45 (12), 3925-33.
2. Wells, M.; Tidow, H.; Rutherford, T. J.; Markwick, P.; Jensen, M. R.; Mylonas, E.; Svergun, D. I.; Blackledge, M.; Fersht, A. R., Structure of tumor suppressor p53 and its intrinsically disordered N-terminal transactivation domain. Proc Natl Acad Sci U S A 2008, 105 (15), 5762-7.
3. Durani, V.; Magliery, T. J., Protein engineering and stabilization from sequence statistics: variation and covariation analysis. Methods Enzymol 2013, 523, 237-56.
Keywords: tumor suppressor p53, protein stability , protein engineering
Abstract:
Isopropanol is one of the most widely used solvents in the world. It is also one of the secondary alcohols that can be used as a direct or partial replacement for gasoline. Since the metabolic pathways and regulation of natural isopropanol producing strains are not well known, the final isopropanol titer can not satisfy rapidly increasing industrial demand. In this work, we studied the feasibility of enabling Clostridium tyrobutyricum to produce isopropanol. C. tyrobutyricum is an anaerobic acidogen which can convert glucose and xylose to acetate and butyrate. Plasmids were constructed with C. acetobutylicum ctf (acetoacetyl-CoA transferase), adc (acetoacetate decarboxylase) and C. beijerinckii adh (secondary alcohol dehydrogenase) genes. The plasmids were transformed into C. tyrobutyricum wild type, ack (acetate kinase) knockout, pta (phosphotransacetylase) knockout and ptb (phosphotransbutyrylase) knockout. The abilities of isopropanol production of these mutants were compared. And process engineering was perfomed to further boost final isopropanol titer, yield and productivity.
Keywords: isopropanol, biofuel, C tyrobutyricum
Abstract:
Multiple Sclerosis (MS) is an immune-mediated, demyelinating disease of the central nervous system that causes severe neurological deficits and is thought to result, in part, from chronic inflammation due to an antigen-specific T cell immune response. Current treatments for MS suppress the immune system without antigen specificity, increasing the risks of cancer, chronic infection and other long-term side-effects. In this study, we developed an antigen specific treatment of experimental autoimmune encephalomyelitis (EAE), a murine model of MS, by co-encapsulating the immunodominant peptide of myelin oligodendrocyte glycoprotein (MOG) with dexamethasone (DXM) into acetalated dextran (Ac-DEX) microparticles (MPs) (MOG/DXM/MPs). DXM-encapsulated MPs (DXM/MPs) reduced nitric oxide and IL-6 production by activated antigen-presenting cells, verifying that these microparticles MPs can effective deliver DXM to the cells that drive T cell activation. Clinical scores of the mice were reduced from 3.4 to 1.6 after 3 injections 3 days apart with DXM/MOG/MPs (MOG 17.6 µg and DXM 8 µg). This change in clinical score was significantly greater than observed with PBS, empty MPs, free DXM and MOG, DXM/MPs and MOG/MPs. Additionally, treatment with MOG/DEX/MPs significantly inhibited disease-associated T cell cytokine expression in an antigen-specific manner. Here we show a promising approach for the therapeutic treatment of MS using a polymer-based microparticle delivery platform to reduce myelin antigen-specific T cell responses.
Keywords: Immunomodulation, Polymeric Microparticles, Multiple Sclerosis
Abstract not available online - please check the printed booklet.
Abstract:
Clostridium tyrobutyricum ATCC 25755 is an acidogen with butyrate and acetate as the two main fermentation products from glucose and xylose. In addition to a high butyrate yield, C. tyrobutyricum also has a high butyrate and butanol tolerance, making it an ideal candidate for the industrial production of biofuels such as n-butanol. In a previous study, an adhE2 gene encoding a bifunctional aldehyde/alcohol dehydrogenase from C. acetobutylicum was overexpressed in an ack knock-out strain of C. tyrobutyricum, and the mutant produced 10 g/L butanol and more than 15 g/L acids from glucose. To further improve butanol production, it is desirable to re-utilize the butyrate and acetate and convert them to their corresponding alcohols. In this study, three different plasmids containing ctfAB genes which encode for a CoA-transferase from Clostridium acetobutylicum were constructed. The CoA-transferase catalyzing the formation of acetoacetate by transferring a CoA moiety from acetoacetyl-CoA to either butyrate or acetate was co-expressed with the adhE2 gene. As expected, ctfAB overexpression raised butanol production to 12-14 g/L, with an enhanced butanol yield (from 0.10 g/g to 0.26 g/g glucose) and productivity (from 0.13 g/L•h to 0.35 g/L•h), and reduced butyrate and acetate accumulation by 80% and 57%, respectively. Interestingly, ctfAB overexpression also induced acetone production to a level of 4-7.5 g/L. Batch fermentation kinetics in pH-controlled fermentors with these mutants was further investigated at different pH values, and the results are presented in this study.
Keywords: Butanol, Clostridium tyrobutyricum, CoA-transferase
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Exogenous application of an electric field may direct cell migration and improve wound healing. However, this principle remains to be optimally utilized to improve patient care. Optimal device design that would fit the needs of broader wound care remains a challenge. To that end it is important to understand the cellular and molecular basis of how therapeutic electric field may influence wound tissue and cells. Here we sought to characterize a novel FDA-approved, redox-active bioelectric dressing (BED) and is currently used for patient care at our wound center. Scanning electron microscope images, and energy dispersive spectroscopic X-ray analysis identified silver and zinc as the principle components of BED. These metals are placed in proximity of about 1 mm to each other thus forming a redox couple and generating an ideal potential in the order of 1 Volt in the presence of a conductive liquid medium (e.g. wound fluid). With emphasis on wound re-epithelialization, results on keratinocyte cell migration are reported. In a standard scratch assay, BED accelerated human keratinocyte migration (p<0.05, n=9). Such effect was enabled by three inter-related mechanisms: (i) elevated endogenous H2O2 (catalase and N-acetyl cysteine sensitive), known to be a potent driver of redox signaling, (ii) increased phosphorylation of IGF1R as recognized based on a human phospho-RTK array screen and (iii) reduction of protein thiols and increase in integrinαv expression (p<0.05, n=4), both of which are known to be drivers of cell migration. Furthermore, BED elevated keratinocyte mitochondrial membrane potential (n=6, p<0.05), and glucose uptake (n=4, p<0.05). Findings of this work raises an interesting possibility that electron flow in BED may cross-talk with biological redox processes thus influencing cell and tissue function.
Keywords: wound healing, bioelectric dressing, cell migration
