UC San Diego SearchMenu
2014 Research Showcase
GAMB Abstracts
Abstract Title : Phage Genomics Initiative at UCSD
Abstract : In the Fall of 2013, Mycobacterium smegmatis bacteriophages were collected from various sites in the La Jolla region through soil samples taken from the environment. The soil samples were then enriched, providing conditions favorable for the growth of the phage of interest and unfavorable for competing organisms. After enrichment and filtration, infection of the host bacteria Mycobacterium smegmatis proceeded. This was achieved by mixing the phage filtrates with Mycobacterium smegmatis and top agar, which were then poured onto agar petri dishes and incubated. Through streaking, serial dilutions, and spot titers, 28 phages were isolated and their titers determined. Electron microscopy imaging was performed on the purified samples in order to select the best candidates for sequencing. One phage, Weiss13, was chosen to have its genome completely sequenced.
Abstract Title : Isolation and Characterization of a Bacteriophage 201phi2-1 Protein
Abstract : A number of Pseudomonas bacteriophage with large genomes (>300Kb) have recently been described, but little is known about the genes required for their propagation. Three of these phages -- ΦKZ, ΦPA3, and 201φ2-1 -- are closely related and make up the ΦKZ phage family. ΦKZ and ΦPA3 infect the human opportunistic pathogen Pseudomonas aeruginosa, and bacteriophage 201φ2-1 infects Pseudomonas chlororaphis, an agriculturally-important organism that produces anti-fungal molecules. I have been using a combination of cell biology, genetics, and proteomics to study bacteriophage 201φ2-1 in order to identify novel mechanisms these large phage use to replicate. Proteomics data of 201φ2-1 has shown that during infection, the phage protein gp105 is produced earlier and in greater amounts than any other phage protein. Given its high relative abundance and early expression timeframe, gp105 probably plays an important role during phage infection. However, analysis of the protein gp105 with multiple databases revealed no homologs of this protein other than similar ΦKZ and ΦPA3 proteins. This protein?s function during phage infection is therefore unknown. To gain insight into this, I am examining the localization pattern of gp105 in the host cell and its interactions with phage DNA during infection. I have constructed a fusion protein of gp105 to GFP, which will allow me to track the position of gp105 throughout the infection process using time-lapse microscopy. From these data, I hope to deduce the function of gp105 and its homologs during infection and gain insight into the infection processes of 201φ2-1 and its ΦKZ relatives.
Abstract Title : Cytochrome P450 2B4: A HDX Approach to Plasticity Analysis
Abstract : Cytochrome P450 2B4 displays a marked ability to take on notably different conformations in the presence of ligands. Yet, the enzymatic plasticity of this membrane protein in solution is still not well understood. Here, hydrogen-deuterium exchange mass spectrometry (DXMS) was employed to visualize the conformations of ligand-free 2B4, along with detergent ligands C8E6 and Cymal5. DXMS analysis indicates a reorganization of residues forming the access gates to the inner core of the protein when bound to C8E6 by a decrease in H-D exchange rate, particularly at the access gates to the inner core along residues 45-51 and 200-220. Similar shifts are also observed in interactions with Cymal-5. These shifts are believed to serve as part of the conformational reorganization necessary to preserve structural stability throughout purification and crystallization procedures. It also yields an understanding of how detergents specifically bind to help maintain enzyme activity when samples isolated and stored.
Abstract Title : The Effects of Caveolin on Aging in C. elegans and Establishment of a Liquid Culture Medium Model
Abstract : Caveolins are scaffolding proteins found in highly ordered membrane domains rich in lipids. These lipid ordered domains are cholesterol and glycosphingolipid enriched signaling microenvironments that allow for adaptation to external stress. The caveolins (Cav-1, -2, -3 in mammals and Cav-1, -2 in C. elegans) interact with and organize various signaling molecules within these domains. Increased caveolin expression in mice protects against cardiac and neuronal stress and increases organismal health and resiliency with age. This project seeks to analyze more global effects of caveolin on aging by using C. elegans as a model species in which Cav-2 is overexpressed (OE). We hypothesize that overexpression of Cav-2 will affect worm aging and function. We performed phenotyping experiments in our N2 wildtype and Cav-2 OE worm strains. These included lifespan, fertility, morphology, and energy utilization assays. We show that Cav-2 OE worms display an increased lifespan, increased fertility, and more efficient mitochondrial respiratory function. Therefore, we conclude that overexpression of Cav-2 in C. elegans facilitates stress adaptation at the whole organism level. This observation underscores the role of caveolin in stress adaptation and has implications to characterize its important role across species. Another goal of this project was to establish a liquid culture medium model to allow completion of function biochemical/biophysical studies in C. elegans that require large quantities of starting material. The liquid culture model has been successfully established to dramatically scale up sample size. Studies are underway to replicate lifespan and functional studies. We conclude that caveolin may be a highly conserved stress adaptive molecule and C. elegans may be an ideal model species to further explore the role of caveolin in biology.
Abstract Title : Effect of diet and adiponectin on fat expansion during gestation
Abstract : High fat maternal diet causes maternal excessive weight gain during gestation. This gain in weight has many effects for the offspring during adulthood such as obesity and metabolic defects. It is not clear whether this increase in maternal gestation weight is attributed by the increase in fat mass or other tissues. It is also unclear during which period of pregnancy most fat expansion occurs and the role of adiponectin on gestational body composition. By using the mouse model, we investigated the effects of adiponectin and maternal over nutrition during gestation on maternal fat mass adaptations. High fat (HF) diet (60% calories from fat) or regular chow (RD) is provided to C57BL/6 dams during their gestational periods. All female mice were adiponectin knockouts and they were either mated with males that were adiponectin knockouts or males that were adiponectin wild types. Echo MRI readings of fat, lean, and water mass were collected at embryonic days 1, 4, 8, 12, 14, 16, 18, and 19. Our results revealed that the average body mass of HF fed group increased by 53.4% of the original mass while that of the control group increased by 35.9% between E1 and E19. It is observed that most fat expansion occurred between E1 and E8 for the HF group in comparison to the control group (74.7% vs 5.2%). The HF group displayed an insignificant higher fat mass at E1 (n=6, 3.09 ± 0.08g vs 2.47 ± 0.20g; p=0.017), and this difference increased significantly until E8 (n=6, 5.40 ± 0.60g vs 2.60 ± 0.31g; p=0.002). In addition, both groups steadily increased in lean mass and the HF group had a larger increase (43.7% vs 30.0%). Differences in total body mass and lean mass are observed in the RD adiponectin WT/KO and KO/KO groups but not in the HF adiponectin WT/KO and KO/KO groups.
Abstract Title : Role and Regulation of FOXA2 in Early Pancreas Induction
Abstract : Forkhead box protein-A2 (FOXA2) is known to play an important role in priming condensed chromatin in preparation of binding by other regulatory transcription factors. Specifically, FOXA2 is known to be a pioneer transcription factor, which differs from normal transcription factors by its ability to bind its target sequences on nucleosomes or compacted chromatin without the need for any cooperative binding. FOXA2 is expressed throughout most stages of pancreas development, and is proposed to be a crucial player in opening specific regions of condensed chromatin for subsequent binding by other regulatory factors during pancreas development. To define the role of FOXA2 during pancreatic differentiation, we must gain a better understanding of when and where FOXA2 binding occurs and whether it is required for initiation of pancreatic gene expression. To address these key questions, we employ ChIP-seq and shRNA mediated knockdown at various stages of pancreatic differentiation of human embryonic stem cells (hESCs). Through these experiments and subsequent analysis, we hope to disentangle the role of FOXA2 in greater detail.
Abstract Title : Characterization of the interaction between the translational repressor complex GIGYF2-4EHP and the ARE-binding protein TTP
Abstract : Gene expression is tightly regulated by different post-transcriptional control pathways. One pathway in particular involves the RNA-binding protein tristetraprolin (TTP), which binds to AU-rich elements (ARE) in the 3' untranslated region of proinflammatory- and cancer-associated mRNAs and promotes their decay by recruiting degradation factors. Recently, the Lykke-Andersen lab (by mass spec in collaboration with the Bennett Lab) revealed a novel interaction between TTP and the translational repressor complex GIGYF2-4EHP. Though TTP is well known to be involved in mRNA decay, its role in translational repression is poorly understood. Whether TTP-mediated mRNA decay and translational repression are inter-dependent is also unknown. To better understand the functional implications of this novel interaction between TTP and GIGYF2-4EHP, first the domains of TTP essential for binding GIGYF2 and 4EHP were mapped out by mutagenesis of TTP. Flag-tagged GIGYF2 and 4EHP and myc-tagged TTP variants were co-transfected into 293T cells and co-immunoprecipitated, and revealed the N-terminal domain of TTP to be sufficient for binding to the GIGYF2-4EHP complex, whereas the C-terminal domain and RNA-binding domain were not sufficient for binding. We also showed this interaction to be independent of other known motifs in TTP, such as the CNOT-interacting motif essential for binding the deadenylase complex, suggesting that TTP binds GIGYF2-4EHP independently of the deadenylase complex. More detailed mutagenesis will be carried out to identify the amino acids necessary for this protein-protein interaction and will allow further elucidation of the relationship between decay and translational repression by TTP.
Abstract Title : Utilizing Tn-seq to create a mutant library of Syneochococus elongatus to elucidate circadian and diurnal response genes
Abstract : Our world is heavily dependent on liquid fossil fuels that both limited and environmentally harmful. The model photosynthetic cyanobacterium, Synechococcus elongatus exhibits tremendous potential as a biofuel source since it has been engineered to produce multiple fuel products while requiring few nutritional inputs beyond CO2 and sunlight. A major setback in using cyanobacteria as an alternative liquid fuel source is the lack of information about the organism in the day-night cycle since it has mostly been examined under constant light. We are using the innovative approach of Tn-seq on S. elongatus to conduct the first genome wide screen on cyanobacteria in the day-night cycle. Tn-seq utilizes transposon mutagenesis combined with high throughput sequencing to conduct a genome wide screen and elucidate genes important to the day-night cycle. As a first step, we have experimented with four different transposons and optimized the conjugation process in order to build a mini-library that has been verified by sequencing. This indicates that our technique and procedure works, and should be scalable to a fully functional mutant library. Once the library is created this technology can be easily reused for genome wide screens under other scientifically and industrially important conditions such as predation and environmental stress.
Abstract Title : Interaction between Enteric Bacteria and Host Engulfment Machinery that Regulate Inflammation
Abstract : Each year 4-6 million people die of enteric infections. The problem is more challenging due to the escalation of antibacterial drug resistance and indicates the need for targeted approaches. After invading intestinal epithelial cells, enteric bacteria infect phagocytes. However, little is known about how phagocytes interact with bacteria to generate inflammatory responses. Previously, we found that BAI1 (Brain Angiogenesis Inhibitor 1) recognizes Gram-negative bacteria through Lipopolysaccharide (LPS). BAI1 facilitates bacterial internalization by the ELMO1 (EnguLfment and cell Motility) /Rac1 pathway and the engulfment of bacteria regulates innate cytokine responses. Using Salmonella as a model of enteric bacteria, we identified Salmonella effector SifA interacts with ELMO1. We hypothesized that the interaction between ELMO1 and the effector from pathogenic bacteria modulates the host inflammatory responses. Infection of ELMO1 KO mice with Salmonella enterica serovar Typhimurium (SL1344) showed attenuated inflammatory responses with lower bacterial load in the ileum and spleen as compared to WT mice. Bacterial internalization in intestinal macrophages was significantly reduced by the ELMO1 knockdown. Using a murine macrophage cell line J774, we found endogenous ELMO1 interacts with the SPI-2 effector SifA. SifA shares a WxxxE signature motif present in several effectors from the enteric pathogens. Interestingly, mutation of the WxxxE motif in SifA, abolished interaction with ELMO1. Control and ELMO1 shRNA (J774) cells were infected either with Salmonella or with SifA mutant and compared to non-pathogenic E. coli for bacterial internalization and cytokine responses. We found TNF-a response from non-pathogenic E. coli is ELMO1-independent whereas the TNF-a from pathogenic bacteria is ELMO1-dependent. These findings suggest a novel role for ELMO1 in the discrimination of pathogenic vs non-pathogenic intestinal bacteria by the interaction with bacterial effectors.
Abstract Title : Application of proteomic mass spectrometry to the characterization of Mycobacterium smegmatis bacteriophage
Abstract : As one of the most abundant and genetically diverse life forms, bacteriophages hold great potential for advancing our understanding of bacterial-phage interactions and for treating bacterial infections in these times of rising antibiotic resistance. Mycobacteriophages--viruses that infect such bacterial hosts as Mycobacterium tuberculosis and Mycobacterium smegmatis--are of particular interest, and efforts are underway to characterize their diverse structures, functions, and replicative processes. Here, we use protein mass spectrometry to examine the proteomes of both sequenced and newly isolated M. smegmatis bacteriophages, with the goal of characterizing and classifying them on the basis of conserved proteins identified from genomic studies. Tandem mass spectrometry was conducted on tryptic digests of prepared phage lysates, and peptides were identified with ABSCIEX Protein Pilot 4.0 software. The spectra were analyzed using a protein database compiled from the published genome sequences of M. smegmatis phages in the NCBI repository. Proteomic analysis of multiple sequenced phages allowed us to annotate putative gene products with likely roles in the replication and structure of the phage particle. This approach is effective as a rapid, high-throughput for characterization of phage proteomes and permits the enhanced annotation of the putative gene products of sequenced phages.
Abstract Title : Towards the Three Dimensional Structure and Function of Hop2/Mnd1
Abstract : Meiosis is a crucial two-step cell division program in eukaryotes that results in the production of haploid gametes , specialized reproductive cells with only half the usual chromosome number. Gametes have only one copy of each chromosome, instead of two homologs as in a normal diploid cell. Proper reduction of chromosome number in meiosis depends on each pair of homologous chromosomes associating and forming crossovers. These crossovers are recombination products involving reciprocal exchange of the homologs?s DNA, thereby physically linking the two chromosomes. Recombination is initiated by DNA cleavage, then the DMC1 (disrupted meiotic cDNA) protein, in coordination with the Hop2 and Mnd1 proteins, enables a broken DNA end to seek out and pair with a homologous duplex DNA. We are seeking to discover the 3D structure of the Hop2-Mnd1 complex, which will provide insights on the mechanism through which it binds DNA and how it specifically interacts with DMC1. To aid crystallization, we inserted a highly soluble fusion partner, b(562)RIL, into both Hop2 and Mnd1 in two separate locations in each protein. b(562)RIL has been shown to increase the stability of proteins in solution, and has been instrumental in the crystallization of a number of proteins. Using combinations of the four insertion constructs and wild-type Hop2 and Mnd1, 8 co-expression constructs were successfully cloned and expressed with high yield in E. coli. For 4 of these, we have performed initial crystallization screening using both high-throughput robotic and manual crystal tray setup. Though we have not yet obtained promising crystallization hits, the fusion proteins are significantly more soluble and better-behaved in crystallization trays than the wild-type Hop2:Mnd1 complex. Solving the structure of Hop2:Mnd1 will provide insight into how Dmc1 specifically mediates inter-homolog recombination, as well as the exact biochemical and structural roles of Hop2 and Mnd1.
Abstract Title : Drugs and You: A DXMS study of protein-drug interaction
Abstract : Cytochrome P450 2B4 possesses significant plasticity that allows for extensive ligand interactions. However, efforts to resolve its various structures are still incomplete. To address this, 2B4 structure was assessed through the use of hydrogen-deuterium exchange mass spectrometry (DXMS). DXMS allowed for visualization of the conformations of Cymal-5-bound 2B4, along with interactions with the drug amlodipine. It was noted that amlodipine initially nestled into a region lining the gateway to the protein core and induced a structural reorganization resulting in stabilization of the region. This resulted in a reduced deuteration along residues 45-51. Higher concentrations of amlodipine yielded an additional stabilization at 200-220, indicating that these two binding sites, which lie in close proximity to each other, have differing Kd values. It also causes a similar phenomena in several other regions. This may prove crucial in assessing how a drug like amlodipine can potentially influence enzyme substrate accessibility, and how treatment regimens can potentially influence normal metabolic functions.
Abstract Title : Comparative Genomics of Mycobacteriophage Weiss13 Identifies Mosaic Structures
Abstract : Mycobacteriophages are small viri that infect bacteria by injecting their host with double-stranded DNA. In the fall of 2013, first-year students in the Phage Genomics Research Initiative course isolated the mycobacteriophage Weiss13, purified its DNA and had it sequenced. In the Spring quarter of 2014, we used DNA Master to produce an initial list of gene coding regions in Weiss13; genes were called based on the longest open reading frame and a strong ribosomal binding site. Gene calls were then manually adjusted based on coding potential and local sequence alignment. Using the BLASTp search function of the phage genome database, we matched Weiss13 genes to identified genes in other phages, which enabled prediction of gene functions. A phage cluster is identified by overall DNA sequence similarity, and Weiss13 has been identified as a member of Cluster R. In comparison with Cluster A with 249 members, Cluster R only contains three other members: Send513, Nilo, and Papyrus. Using a program called Phamerator, we compared Weiss13 to other mycobacteriophages and looked in similarities and differences among their genomes. Our results indicate that Weiss13 has a mosaic structure; its genes have different evolutionary relationships. Some genes in Weiss13 have sequences similar to a limited number of other phages, while other Weiss13 genes have sequences similar to a broad number of other phages. Because phages represent a large untapped genomic reservoir, understanding the phylogenetic relationships among phages will enable development of phages as antibacterial agents.
Abstract Title : Analysis of Pyruvate Carboxylase and Pyruvate Dehydrogenase Kinase Functions in Diet Induced Obesity and Cardiac Lipotoxicity
Abstract : Obesity has become an increasingly prevalent problem on both global and national level due to higher caloric consumption and decreased activity level. Obesity has dangerous side effects including heart dysfunction, inflammation, and diabetes. Humans share anatomical and genetic characteristics with Drosophila melanogaster, which allows this fly species to be a good model for studying the genetics behind high-fat diet (HFD) induced obesity and the associated heart dysfunction. Previous studies in the laboratory have shown that HFD feeding leads to obesity and cardiac liptoxicity via deregulation of the TOR (Target of Rapamycin) - PGC-1 (PPARγ Co-activator 1) axis. A microarray analysis was performed to identify downstream targets of PGC-1. My project consisted of analyzing the role of two of these genes: PDK (Pyruvate Dehydrogenase Kinase) and PC (Pyruvate Carboxylase), both regulators of glucose metabolism. I tested each gene through loss-of-function mutations and analyzed the effect on fat content. I found that both PDK and PC mutants exhibited decreased fat content under both diet conditions (NF and HFD). Additionally, I used the UAS Gal4 system to do a heart-specific knockdown of each of these genes individually. My preliminary results show that the increased heart rate and concomitant decreased heart period caused by HFD is abolished under PC knockdown. This data suggests that PC and PDK play key roles in the mechanism of HFD induced obesity and heart dysfunction, downstream of PGC-1. To confirm this data, future research will include genetic epistasis experiments with PGC-1 and other members of the insulin-TOR pathway.
Abstract Title : The LysE Superfamily of Transport Proteins Involved in Cell Physiology and Pathogenesis
Abstract : We have characterized the LysE superfamily of transmembrane transport proteins which catalyze export of amino acids, lipids and heavy metal ions. Statistical means were used to show that it includes newly identified families specific for (1) tellurium, (2) iron/lead, (3) manganese, (4) calcium, (5) nickel/cobalt, (6) neutral amino acids, and (7) peptidoglycolipids. Internal repeats were identified, and multiple alignments, phylogenetic trees, average hydropathy, amphipathicity and similarity plots, and conserved motifs provided evidence that all members of the superfamily derived from a single common 3- TMS peptide via intragenic duplication. Their common origin implies that they share common structural, mechanistic and functional attributes. The transporters of this superfamily play important roles in ionic homeostasis, protection from excessive cytoplasmic heavy metal/ metabolite concentrations and cell envelope assembly. They thus influence the physiology and pathogenesis of numerous microbes, being potential targets of drug action.