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2012 Research Showcase
Molecular Abstracts
Advisor : RU BRYAN
Abstract Title : Activation of AMPK inhibits Monosodium Urate (MSU) crystal-induced Inflammation
Abstract : Purpose: AMP-activated kinase (AMPK), in its phosphorylated and active form, has been shown to inhibit NF-kB activity, causing a decrease in cytokine release which affects inflammation. To investigate the role of monosodium urate (MSU) crystals in inflammation, mouse bone marrow derived macrophages were treated with MSU crystals to test for effects on AMPK. Methods: Macrophages were obtained from bone marrow of mouse femurs and differentiated with granulocyte macrophage colony-stimulating factor (GM-CSF). Some cells were treated with AMPK activator A769662. After MSU treatment, the conditioned media was tested for levels of cytokines, specifically TNF-α, IL-1β, and KC, with ELISA. These cytokines have been shown to mediate inflammatory response. Western blot was performed on macrophage cell lysates to analyze relative levels of phosphorylated AMPK. Results: In both AMPK wild type (WT) and knock-out (KO) mice macrophages, treatment with MSU elevated cytokine release. However, in AMPK KO macrophages, cytokine levels are far greater following treatment than in WT macrophages. In both KO and WT macrophages, phosphorylated AMPK, the active form, decreased with treatment. In relation to the cytokine trend, AMPK KO macrophages have lower phosphorylated AMPK than WT macrophages. Despite a knockout of either the alpha-1 or alpha-2 subunit of AMPK, KO macrophages still express some level of AMPK due to the independent AMPK regulation of each subunit. Various AMPK activators decreased cytokine release and inflammation by increasing AMPK activity. It has been previously shown that macrophages derived from mice with wild-type Toll-like receptors (TLR) 2/4 show increased cytokine levels with MSU treatment, confirming the trend. Conclusion: It was concluded that treatment with MSU crystals causes a decrease in AMPK activation and an increase in cytokine release in bone marrow derived macrophages, implying a greater inflammatory response. In absence of one AMPK subunit, less AMPK is active and more cytokines are released. Treatment with MSU crystals in conjunction with AMPK activators resulted in more AMPK activation and attenuated cytokine release than without treatment with activators. This highlights the role of phosphorylated AMPK in the process of inflammation.
Abstract Title : Exploring the Regulation of yidC2 in Bacillus subtilis
Abstract : The YidC/Oxa1/Alb3 family of proteins found in bacteria, mitochondria, and chloroplasts respectively, perform the essential function of mediating the folding and insertion of transmembrane proteins into their proper locations in the membrane. In Gram-negative bacteria, only one copy of YidC is present; in Gram-positives, there are two, SpoIIIJ/YidC1 and YidC2. Previous research in the Gram-positive species, Streptococcus mutans and Bacillus subtilis has shown that although spoIIIJ/yidC1 and yidC2 share many overlapping functions, they also differ in several ways. Our goal is to determine the conditions that cause B. subtilis to express yidC2 in preference to the constitutively active spoIIIJ. We performed a transposon mutagenesis to isolate mutants that upregulate the yidC2 operon. This screen led to the discovery of several mutants with insertions in the qox operon, that codes for the menaquinol oxidase terminal electron acceptor, cytochrome aa3-600, as well as abrB, a transition state regulatory gene. In addition, Phenotype Microarrays were performed to identify differences in metabolism and chemical sensitivities among ∆spoIIIJ, ∆yidC2 and wild type strains. We observe alterations in sensitivity to a number of compounds, with several affecting membrane integrity and redox balance. These results suggest that changes affecting redox and oxidative metabolism may lead to an upregulation of yidC2.
Abstract Title : Algae-Produced Pfs25 Elicits Antibodies That Inhibit Malaria Transmission
Abstract : Subunit vaccines are significantly more expensive to produce than traditional vaccines because they are based primarily on recombinant proteins that must be purified from the expression system. Despite the increased cost, subunit vaccines are being developed because they are safe, effective, and can elicit antibodies that confer protection against diseases that are not currently vaccine-preventable. Algae are an attractive platform for producing subunit vaccines because they are relatively inexpensive to grow, genetically tractable, easily scaled to large volumes, have a short generation time, and are devoid of inflammatory, viral, or prion contaminants often present in other systems. We tested whether algal chloroplasts can produce malaria transmission blocking vaccine candidates, Plasmodium falciparum surface protein 25 (Pfs25) and 28 (Pfs28). Antibodies that recognize Pfs25 and Pfs28 disrupt the sexual development of parasites within the mosquito midgut, thus preventing transmission of malaria from one human host to the next. These proteins have been difficult to produce in traditional recombinant systems because they contain tandem repeats of structurally complex epidermal growth factor-like domains, which cannot be produced in bacterial systems, and because they are not glycosylated, so they must be modified for production in eukaryotic systems. Production in algal chloroplasts avoids these issues because chloroplasts can fold complex eukaryotic proteins and do not glycosylate proteins. Here we demonstrate that algae are the first recombinant system to successfully produce an unmodified and aglycosylated version of Pfs25 or Pfs28. These antigens are structurally similar to the native proteins and antibodies raised to these recombinant proteins recognize Pfs25 and Pfs28 from P. falciparum. Furthermore, antibodies to algae-produced Pfs25 bind the surface of in-vitro cultured P. falciparum sexual stage parasites and exhibit transmission blocking activity. Thus, algae are promising organisms for producing cysteine-disulfide-containing malaria transmission blocking vaccine candidate proteins.
Abstract Title : Intron retention and alternative AUG site usage characterize the unique regulation of a glycolytic transcription factor in response to glucose availability in S. cerevisiae.
Abstract : In higher eukaryotes, regulated alternative splicing is a critical means by which cells achieve genomic reprogramming in response to environmental changes. In the yeast Saccharomyces cerevisiae, genome-wide microarray analyses indicate that splicing outcomes are affected under different stresses, but our understanding of how splicing is regulated in response to a dynamic environment is still limited to only a handful of genes. Notably, each of these genes contains an intron with a non-consensus splice site. We identified GCR1 as a novel candidate for regulated splicing in yeast since the GCR1 intron contains a non-consensus 5? splice site and expression of Gcr1 is sensitive to glucose availability. Here we examine how expression of Gcr1 is regulated in response to glucose at the level of splicing, RNA decay, and translation. Surprisingly, we observe that the GCR1 intron is preferentially retained in the presence of glucose and that an alternative start codon in the GCR1 intron permits the generation of a 94kDa protein. The levels of this protein decrease rapidly upon glucose depletion, as do the levels of the GCR1 pre-mRNA. This work provides the first example of alternative AUG site usage that depends on regulated intron retention in yeast.
Abstract Title : Vax1 and cFos regulation of GnRH transcription changes during GnRH neuron maturation
Abstract : Sexual maturation and fertility are dependent on the proper function of the hypothalamic-pituitary-gonadal axis, initiated by a critical population of gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus. Dysregulation of GnRH neuron maturation and GnRH secretion in humans leads to reproductive disorders and reduced fertility. As GnRH neurons mature during fetal development, they migrate from the olfactory placode to the hypothalamus and increase the expression of GnRH. The level of GnRH expression marks the status of GnRH neuron maturation. In the process of indentifying the genes potentially involved in GnRH neuron maturation, the group of researchers from the Mellon lab found that ventral anterior homeobox1 (vax1) is highly transcribed in mature immortalized GnRH neurons but absent in immature immortalized GnRH neurons, making vax1 of interest for the study of GnRH neuron maturation. In this project which I conduct with and under the guidance of post-doctoral fellow Dr. Hanne Hoffmann, we investigate the regulation of GnRH expression by Vax1 in both immature and mature murine GnRH neurons. We have found that over-expressing vax1 induces GnRH promoter driving luciferase expression in immature GnRH neurons, whereas it has an inhibitory effect in mature GnRH neurons. Comparison of the 5? upstream region of the GnRH promoter between human, mouse and rat permitted the identification of a number of conserved Vax1 binding sites in the GnRH enhancers and promoter. Using 5? truncations of the GnRH promoter driving luciferase, we limited Vax1 binding in the upstream regulatory region of GnRH gene to both the promoter and proximal enhancer E1. It is known that GnRH transcription is also regulated by an immediate early gene cfos. We were interested in finding whether there is any cooperation between Vax1 and cFos in regulating GnRH transcription. We found that over-expressing vax1 induces cfos promoter driving luciferase expression in both immature and mature GnRH neurons. Moreover, over-expressing cfos by itself has a similar effect to over-expressing vax1 on the GnRH E1/P driving luciferase?inductive in immature GnRH neurons and inhibitory in mature GnRH neurons. This suggests that Vax1 indirectly regulates GnRH expression through cFos. This is confirmed by a time course trace of endogenous cfos and GnRH transcription after Vax1 over-expression in mature GnRH neurons, in which endogenous cFos is induced previous to GnRH repression. On the other hand, these findings do not eliminate the possibility that Vax1 also binds directly to the GnRH promoter and regulates GnRH expression. This possibility is yet to be studied by EMSA showing Vax1 binding to GnRH promoter. In addition, we plan to extend the project from in-vitro cell line studies to in-vivo experiments on mice. In order to do so, we will determine whether vax1 transcription co-localize with that of GnRH during GnRH neuron development and observe changes in GnRH neuron maturation and migratiom in vax1 knock-out mice.
Abstract Title : The Unique Shuttling Pathway of the Peroxisomal PTS2 Receptor, Pex7, in Pichia pastoris
Abstract : Peroxisomal matrix protein import relies on two pathways that use one of two peroxisomal targeting signals (PTSs), known as PTS1 and PTS2, present on cargo proteins. The majority of import occurs through the PTS1 pathway, dependent on the receptor Pex5. The PTS2 pathway is dependent on the receptor Pex7 and its co-receptor Pex20. Pex5 and Pex20 bind their respective cargo in the cytosol and shuttle them into the peroxisome matrix to be released. After cargo release, Pex5 and Pex20 are subject to mono-ubiquitination which allows for receptor recycling - a process dependent on the ubiquitin-conjugating enzyme, Pex4, and the AAA ATPases, Pex1 and Pex6. When receptor recycling is blocked, Pex5 and Pex20 are poly-ubiquitinated and degraded by the proteasome, in a process called the RADAR (Receptor Accumulation and Degradation in the Absence of Recycling) pathway. However, we found that the shuttling pathway of Pex7 differs from that of Pex5 and Pex20 as Pex7 is present at low levels in wild-type cells but stabilized in pex mutants affecting normal peroxisomal matrix protein import, suggesting that Pex7 is constitutively degraded during its normal import cycle. Specifically, Pex7 is stabilized in the receptor recycling mutants, Δpex4 and Δpex6, due to its inability to enter into peroxisomes, as seen by fractionation and protease protection assays. However, in the mutants affecting peroxisomal membrane proteins containing a RING domain, such as Δpex2, Pex7 can enter peroxisomes but presumably cannot be exported, thus causing accumulation of Pex7. Additionally, the import and export of Pex7 may have a previously unknown dependence on Pex5 and Pex20, which may be mediated by the special cargo, Pex8, which contains both a PTS1 and PTS2. The shuttling pathway of the PTS2 receptor, Pex7, demonstrates a sharp divergence from the previously studied shuttling pathways of Pex5 and Pex20 and could represent a mechanism to regulate Pex7 levels when the PTS2 pathway is not needed.
Abstract Title : What do mRNA quality control and neurodegeneration have in common? A tale of a ribosome-associated E3 ubiquitin ligase
Abstract : Aleksandar Jamborcic1,2, Mario H. Bengtson1 and Claudio Joazeiro1 1. Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 2. University of California San Diego, La Jolla, CA Messenger RNA lacking stop codons ('non-stop mRNA') can arise from errors in gene expression, and encode aberrant proteins whose accumulation could be deleterious to cellular function. How eukaryotic cells eliminate non-stop proteins has remained unknown. Our lab has recently reported that the S. cerevisiae Ltn1 RING-domain-type E3 ubiquitin ligase acts in the quality control of non-stop proteins (Bengtson & Joazeiro 2010. Nature 467:470-3). Ltn1 is predominantly associated with ribosomes, and marks nascent non-stop proteins with ubiquitin to signal their proteasomal degradation. The biological relevance of this process is underscored by the finding that loss of Ltn1 function confers sensitivity to stress caused by increased non-stop protein production. We speculate that defective protein quality control may underlie the neurodegenerative phenotype that results from mutation of the mouse Ltn1 homologue, Listerin. We are interested in investigating the mechanism of Ltn1 binding to ribosomes. Among other things, we would like to know what ribosomal subunits are implicated, or are in Ltn1?s vicinity. Ltn1-mediated ubiquitylation of non-stop proteins seems to be triggered by their stalling in ribosomes on translation through the poly(A) tail. Because Ltn1 targets nascent polypeptides that are translationally-arrested, we hypothesize that ribosomal subunits around the nascent polypeptide exit tunnel are involved. Strains previously described in the literature as viable and containing either deleted, or GFP/Flag-tagged ribosomal subunits were collected. Interestingly, we found that in a strain expressing Rpl35 as a GFP fusion there was marked accumulation and defective ubiquitylation of Ltn1?s substrates. We will next directly examine whether or not the Rpl35 mutation directly interferes with Ltn1 binding to ribosomes.
Abstract Title : The Cap Binding Complex (CBC) regulates translation of the unspliced GCR1 transcript in Saccharomyces cerevisiae to regulate metabolic gene expression during glucose starvation.
Abstract : Genome-wide arrays have shown that environmental stresses induce changes in yeast gene expression. Our lab demonstrated that the same change in gene expression observed during glucose starvation is also observed when the Cap Binding Complex (CBC), involved in splicing of intron-containing genes, is deleted in Saccharomyces cerevisiae. We have also demonstrated that CBC protein levels decrease when glucose is depleted from the media as cells grow to saturation. Since the critical glycolytic transcription factor, Gcr1, is encoded by an intron-containing gene, we hypothesized that the CBC influences metabolic gene expression during glucose depletion via regulated splicing of GCR1. Although we have confirmed that the CBC is required for expression of Gcr1 protein, we were surprised to find that GCR1 preferentially retains its intron even in a WT strain under glucose-rich conditions. Furthermore, an alternative start codon within the GCR1 intron generates a unique protein product, and translation from this intronic AUG requires the CBC. These results raise questions about the biological function of the unspliced variant and whether the sequences that contribute to the regulation of Gcr1 expression are evolutionarily conserved. Preliminary data from recent analyses of an intronless GCR1 suggest that the unspliced transcript plays a critical role in metabolic regulation in response to glucose availability.
Advisor : DR. PAUL PRICE
Abstract Title : Role of Tissue-nonspecific Alkaline Phosphatase and Fetuin in Biomineralization
Abstract : Our goal is to understand the mechanisms by which proteins control the the normal mineralization of bones and teeth and prevent the abnormal mineralization of soft tissues. We studied two proteins which have been implicated in the regulation of biomineralization: tissue-nonspecific alkaline phosphatase (TNAP) and fetuin. Fetuin is a 48kDa glycoprotein that is synthesized in the mammalian liver and found in high concentrations in serum and bone and is an important inhibitor of apatite growth in-vitro. TNAP has been identified in previous studies as a component of serum calcification activity and its effect in increasing the rate of mineral formation is observed only exclusively in the presence of fetuin. Apatite crystal formation was investigated using an in-vitro assay that closely resembles the calcium, phosphate and pH of human serum. We have demonstrated that fetuin is a substrate for alkaline phosphatase activity and that de-phosphorylation of fetuin greatly reduces its mineral inhibition activity. The high concentration of alkaline phosphatase and fetuin in bones suggest that bone fetuins may have a more dephosphorylated state. The reduced mineral inhibition activity of fetuin within bone may allow for normal mineralization to occur. Our findings also suggest that the phosphorylation state of serum fetuin may play a role in abnormal soft tissue mineralization such as the formation atherosclerotic plaques.
Abstract Title : Characterization of Hepatocyte-Specific SRSF1 (ASF/SF2) Knockout Mice
Abstract : Alternative splicing is widely believed to have a major impact on almost all biological processes since it increases proteome complexity and thereby controls protein function. SRSF1 (SF2/ASF) is a member of the SR protein family, which are essential for constitutive splicing and fulfilling regulatory functions in alternative splicing. Previously, we have shown that SRSF1 binds to the alternatively spliced exon 11 of the insulin receptor (INSR) and promotes its incorporation in hepatoma cells. To demonstrate the physiological importance of alternative splicing to the regulation of metabolism, we created a hepatocyte specific knockout of SRSF1 (SRSF1LKO) by crossing SRSF1flox/flox mice with albumin-cre mice, which specifically express the cre recombinase in adult hepatocytes. Mice are born in the expected mendelian ratio and do not show any growth retardation unlike the hepatocytes specific SRSF3 knockout that we have previously characterized. We will present data showing efficient tissue specific deletion of the SRSF1 allele, the reduction in SRSF1 expression, and alterations in INSR splicing. We will also present data on the metabolic and physiological parameters and liver morphology between SFSF1LKO and littermate control mice, as well as alterations in global gene expression and splicing by exon array analysis. SFSF1LKO is an important model to study alternative splicing in vivo.
Abstract Title : Modulation of hypoxia-inducible factor-1 expression by a leading Gram-positive bacterium
Abstract : Group A Streptococcus (GAS) is a leading human bacterial pathogen responsible for a wide spectrum of local and systemic infections. The host innate immune system, which includes epithelial barriers and phagocytic cells, provides a critical first line of defense against invading microorganisms. Our laboratory has shown that transcription factor hypoxia-inducible factor-1 (HIF-1) serves a master regulator of many bactericidal and inflammatory activities in phagocytes and skin epithelial cells (keratinocytes). However, the specific bacterial factors recognized by host cells for HIF-1 activation and the potential for certain pathogens to interfere with the HIF-1 response are unknown. To identify such bacterial factors, we developed a luciferase-based reporter assay in keratinocytes to screen a panel of isogenic GAS mutants against the wild-type parent strain for differences in HIF-1 activation. We have identified several putative virulence factors that influence HIF-1 levels, and are currently testing the effects of these differences on bacterial killing by keratinocytes. Soon we will expand our studies by using in vitro and in vivo HIF-1 knockout systems to probe the functional consequences of the modulations in HIF-1 expression on the host innate immune response and infection outcome. Since GAS can sometimes produce severe, life-threatening infections, even in previously healthy individuals, these findings will contribute to our understanding of the mechanisms through which the coordinated actions of multiple bacterial virulence determinants are able to resist the multifaceted constituents of the host's innate immunity.
Advisor : DR. WILLIS LI
Abstract Title : Screening for Heterochromatin Promoting Drug in Drosophila
Abstract : It was previously found that the JAK/STAT pathway directly plays a role in heterochromatin formation, a form of chromatin that is tightly compact and is essential for gene silencing, chromosome organization and preservation of genome integrity. Antagonizing heterochromatin formation induces tumor genesis in Drosophila melanogaster hematopoietic tumor model. Observing this, we intend to screen a small set of molecule compounds for their ability to promote heterochromatin formation in hopes of discovering a potential anti cancer drug. Using Drosophila, we are initially screening for drugs by observing the eye color phenotype of the DX1 gene and position-effect variegation (PEV) mutant flies. Eye color change in these flies upon treatment of the drug should correlate to heterochromatin formation change. Knowing that heterochromatin is much more resistant to cleavage of nucleases and restriction enzymes compared to euchromatin, DNAse digestion assays and restriction enzyme assays will be done to more directly observe heterochromatin formation. Confirming the drug?s ability to promote heterochromatin formation, tumor proliferation assays will be performed to determine its ability to prevent cancer. Finally, after discovering this drug?s capability of suppressing cancer, structure and mechanism of heterochromatin formation will later be explored.
Advisor : ANDREW M. LOWY
Abstract Title : A Novel Anti-RON Antibody, RON8, Inhibits Tumor Growth in an Orthotopic Patient-Derived Xenograft Model of Pancreatic Cancer
Abstract : A Novel Anti-RON Antibody, RON8, Inhibits Tumor Growth in an Orthotopic Patient-Derived Xenograft Model of Pancreatic Cancer. Patrick Holman*, Jaclyn Miyamoto*, Michele Babicky, Megan Harper, Dawn Jaquish, Evangeline Mose, Randall French, and Andrew Lowy. (*Coauthors) Introduction Pancreatic cancer is the fourth leading cause of cancer related deaths in the United States. This aggressive cancer has a five-year survival rate less than five percent and limited therapeutic options. Current standard of care includes Gemcitabine, a cytotoxic nucleoside analog that incorporates into DNA during cell division and inhibits replication. We previously demonstrated that the RON receptor tyrosine kinase is overexpressed in approximately 80 percent of human pancreatic cancers, implicating this receptor as a potential therapeutic target. Additionally, we have shown that silencing RON in pancreatic cancer cell lines is capable of sensitizing them to Gemcitabine therapy in vivo. In this study, we tested the efficacy of RON8, a novel anti-RON therapy, RON8 is a human-derived monoclonal antibody antagonist of the RON receptor. We hypothesized that the RON8 antibody would inhibit tumor growth in an orthotopic xenograft model, and would demonstrate synergistic inhibition when combined with Gemcitabine. Methods Orthotopic tumor xenografts were established by either injection of immortalized human pancreatic cancer cell lines (BxPC3, FG) or surgical implantation of patient-derived tumor tissue into the pancreata of NOD/SCID mice. Patient-derived tumors were propagated in NOD/SCID mice by serial passaging to obtain a sufficient amount of tumor tissue to implant for treatment groups. Tumor growth and progression were monitored by weekly ultrasounds. After a tumor threshold size (75-200 mm3) was reached, mice were randomly assigned to one of four treatment groups: human IgG antibody, RON8, Gemcitabine, or Gemcitabine+RON8. Tumor volume was calculated for each animal (n= 4-5 per treatment group) and plotted over time. After 4-8 weeks of treatment, the mice were sacrificed and the final primary tumor volume and weight was measured. Repeated measures and one way ANOVA were performed to determine statistical significance. RON expression was verified via western blot analysis of tumor tissues and cell lysates. Results All tumor tissues and cell lines expressed the RON receptor. In the orthotopic cell injection model, treatment with the RON8 antibody or RON8+Gemcitabine did not significantly inhibit tumor growth. In the patient-derived xenograft model, two of four tumors treated with RON8 exhibited attenuated tumor growth compared with IgG control. Gemcitabine was highly effective in inhibiting tumor growth in the patient-derived xenograft model, an effect that was not observed in the cell line injection model. Conclusion We have developed a unique orthotopic model for preclinical testing of potential therapeutic agents for pancreatic cancer. All tumors tested expressed RON and displayed rapid tumor growth when implanted into immunodeficient mice. The patient-derived xenograft model showed high sensitivity to Gemcitabine therapy, an effect that was not replicated in cell line injection models. In two of four patient-derived xenografts, the novel RON8 antibody significantly inhibited tumor growth. This orthotopic xenograft model can be used to screen human tumors for sensitivity to targeted therapy and identify potential biomarkers that may be predictive of therapeutic response.
Abstract : Stomata, present in the aerial epidermis of land plants, provide gateways for regulating carbon dioxide (CO2) and water exchange between plants and the atmosphere. The stress induced phytohormone abscisic acid (ABA) reduces transpirational water loss crucial for the fitness of plants by inducing stomatal closure . Cytosolic Ca2+ has been reported to play a major role in ABA-induced stomatal closure. The objective is to elucidate the roles of members of two different protein families that represent calcium sensors in stress responses of the model plant Arabidopsis thaliana - Calcium Dependent Protein Kinases (CPKs) and Calmodulin-like (Cml) proteins. cpk3/6 and cpk 4/11 double mutant plants show impaired ABA- and Ca2+-induced stomatal closing and Cml9 knock-out plants (cml9-1 and cml9-2) are reported to be drought sensitive and show an ABA-dependent seedling growth phenotype. Underlying molecular mechanisms leading to these aletered responses are still poorly understood. To improve the understanding of these proteins and their mechanisms, two different quadruple mutant plant lines, cpk3/cpk4/cpk6/cpk11 and cpk5/cpk6/cpk11/cpk23, will be established by crossing parent plant lines, and subsequent screening using PCR based genotyping. Gene expression knock-out will be confirmed by RT-PCR. For both, the quadruple CPK and the Cml9 knock-out lines bioassays determining stomatal movements in response to several stimuli will be carried out improving the understanding of the role of these proteins within plants' abiotic stress tolerance to environmental challenges, such as droughts. Consequently, strategies can be developed and engineered to promote strong resistance against environmental stresses in plants, resulting in increased crop yields.
Abstract Title : Insulin Effector, FOXO1, Decreases Activin Induction of Follicle-stimulating Hormone Beta Gene Expression
Abstract : Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are produced exclusively in pituitary gonadotrope cells and are essential for reproduction in mammals. FSH and LH are heterodimeric glycoproteins composed of a common alpha and a unique beta subunit (FSHB and LHB). Regulation of Fshb and Lhb gene expression is the rate limiting step in the production of the mature hormones. Metabolic disorders including obesity are associated with disturbed gonadotropin hormone production and infertility. In previous studies, insulin signaling has been reported to modulate LH levels, potentially through the FOXO1 transcription factor. The Thackray lab has shown that insulin signaling via PI3K/AKT regulates FOXO1 phosphorylation and cellular localization in gonadotropes and that FOXO1 suppresses basal transcription and gonadotropin-releasing hormone (GnRH) induction of Fshb and Lhb gene expression. For my research project, I am investigating whether FOXO1 can modulate activin induction of Fshb. My results demonstrated that activin induction of Fshb gene expression was repressed with overexpression of a constitutively active form of FOXO1 (FOXO1-CA) in LbT2 cells, an immortalized gonadotrope cell line. This suppression required the DNA-binding domain (DBD) of FOXO1, suggesting that FOXO1 either binds directly to the Fshb promoter or FOXO1 is tethered to the promoter via a protein-protein interaction. The FOXO1 suppression mapped to a proximal -304/-95 bp region of the Fshb promoter, which corresponds to a region containing multiple activin responsive sites including a consensus SMAD Binding Element (SBE) at -267 as well as other SMAD half sites and FOXL2 binding elements. Since FOXO1 has been shown to interact with SMAD3/4, I tested whether the FOXO1 repression is occurring through SMAD proteins. Co-expression of exogenous SMADS3/4 in LbT2 cells resulted in a 28 fold induction of the Fshb promoter. This induction was completely repressed by FOXO1-CA. In addition, I demonstrated that activin induction of a 4x SBE multimer was also suppressed by FOXO1-CA. These studies suggest that FOXO1 is most likely interacting with SMADS to elicit a repressive effect on the Fshb promoter. Future experiments will utilize various molecular and biochemical assays to further investigate mechanisms of FOXO1 suppression of the Fshb promoter.
Abstract Title : Comparison of the activities of the human TUTases
Abstract : Terminal uridylyl transferases (TUTases) are template-independent polymerases capable of catalyzing the transfer of single or multiple UMP residues to RNAs in eukaryotes. Previous studies have shown that these enzymes play a role in messenger RNA and small RNA turnover. The in vitro activities of a few of the seven human TUTases (TUTase 1 - TUTase 7) have already been characterized, but done so individually. Here we compared the activities of five of the seven human TUTases. Our data indicate that, in vitro, TUTase 4 and 7 prefer to add UMP residues to an RNA template, while TUTase 2, 3 and 5 prefer to add AMP residues. Furthermore, we show that TUTase 7 is unique in its ability to not only add short tails to an RNA template, but also very long tails over 100 nucleotides long.
Abstract Title : Protein Kinase C-Theta phosphorylates and inhibits GIVīs Guanidine Exchange Factor (GEF) Activity
Abstract : GIV/Girdin is a unique multidomain signal transducer that activates trimeric G protein, Gi, via its GEF motif (Guanine nucleotide Exchange Factor) downstream of growth factor receptors (RTKs, e.g., InsulinR). The key function of this pathway is enhancement of PI3K-Akt signals during a diverse set of biological processes, e.g., epithelial wound healing, macrophage chemotaxis, autophagy, neuronal development, tumor cell migration and tumor angiogenesis. The primary goal of my research has been to understand how GIV?s GEF activity is modulated in physiology and pathology, and how that translates to PI3K activity in cells. We previously identified key residues in GIV that are necessary for its GEF function, and for the enhancement of the PI3K-Akt pathway. Recently, by using extensive mutagenesis as an approach, Serine (S)1689 within the GEF motif was identified as a key determinant of GIVīs GEF activity. As part of my research in the lab, we have demonstrated that the novel PKC isoform, PKCθ (Theta), is the kinase responsible for phosphorylating S1689 and downregulating GIVīs GEF function in cells. Using a combination of cell and molecular biology approaches (immunoprecipitation, immunofluorescence, pulldown and immunoblotting) we have investigated this key phosphoevent both in vitro and in vivo, and identified that phosphorylation of GIV by PKCθ inhibits the GIVGEF-dependent PI3K-Akt signal enhancement. The pivotal role that GIV?s GEF motif plays in physiology and disease makes it important to further evaluate how phosphoregulation of GIV-GEF by PKCθ affects other biological roles of GIV. For example, it has been demonstrated that PKCθ contributes to insulin resistance via downregulation of the PI3K-Akt signalling pathway. As a continuum of this research, we will now study the interplay between PKCθ and GIV in mediating insulin resistance and its clinical implications in Type 2 Diabetes.
Advisor : JEFF LONG
Abstract Title : Stem Cell Regulation by Class III HD-ZIPs in Arabidopsis thaliana
Abstract : One of the earliest steps in plant development is the formation of the upward Shoot Apical Meristem (SAM) and downward Root Apical Meristem (RAM), which act as the sites of all postembryonic development. The SAM contains a population of stem cells at its tip, which are controlled by a variety of regulatory mechanisms. One component of this regulatory network is a family of transcription factors known as CLASS III HOMEODOMAIN-LEUCINE ZIPPERS (HD-ZIP IIIs), which also function in patterning differentiating leaf tissues. Loss-of function, gain-of-function, and misexpression studies indicate that HD-ZIP IIIs control SAM stem cell fate in parallel and partially overlapping pathways. This suggests the existence of both shared and unique target genes. To better understand stem cell regulation by HD-ZIP IIIs, we seek to identify HD-ZIP III binding sites in various tissues at different times in development. Here, we have established tissue specific expression of fluorescent-tagged HD-ZIP IIIs using promoter elements from other regulatory genes, which permits ChIP-Seq identification of each individual HD-ZIP IIIs binding sites in these tissues. Further, we seek to better understand the role of LITTLE ZIPPER (ZPR) transcription factors in regulating HD-ZIP III function. Through these efforts, we hope to provide insight into the dynamics of this important regulatory network essential to stem cell regulation.
Abstract Title : PTHrP promotes proliferation and confers radioresistance in vestibular schwannoma in vitro
Abstract : Vestibular schwannomas (VS) are slow-growing tumors of the vestibulocochlear nerve that can cause significant morbidity due to their critical location. VS that arise due to NF2, an autosomal dominant familial disease caused by mutation of the neurofibromin 2 (NF2) gene with loss of its functional gene product, merlin, are notoriously more difficult to manage than sporadic VS. Currently, the only available treatment options for VS are microsurgery and irradiation, both of which carry additional risks, including recurrence. PTHrP, widely expressed in neoplastic tissues, has been implicated in various aspects of tumor progression and response to treatment. Although PTHrP has been detected and studied in various solid tumors and cancer cell lines, it has yet to be investigated in vestibular schwannoma. We aim to determine whether PTHrP expression in vestibular schwannomas may contribute to cell proliferation and radiation resistance, two fundamental processes underlying VS tumor recurrence. Based on our results, PTHrP is expressed in VS tissue as well as the human VS cell line HEI-193. Immunofluorescence of NF2-transfected HEI-193 cells reveals an inhibitory effect of the tumor suppressor merlin on PTHrP, suggesting PTHrP may facilitate VS tumorigenesis. Treatment with PTHrP inhibitor and peptide resulted in a consistently smaller and larger surviving fraction, respectively, of HEI-193 cells exposed to ionization radiation, compared to control. PTHrP was revealed to be modulated by epidermal growth factor receptor (EGFR) signaling as EGF-treated HEI-193 cells expressed higher levels of PTHrP transcript. In addition, EGF-induced cell proliferation was shown to be mediated in part by PTHrP. Our results implicate PTHrP as a potential target for effective VS treatment. By inhibiting PTHrP activity, it may be possible to increase the effectiveness of radiation therapy, thereby reducing the need for surgical intervention, and lessen the likelihood of VS tumor recurrence. Further studies are needed to investigate effects of PTHrP on VS growth and radiation-induced cell death in primary VS cultures and in vivo.
Abstract Title : Functional characterization of Pex19p N and C-terminal deletions in Pichia pastoris
Abstract : The study of peroxisome biogenesis seeks to understand the underlying processes involved in the initial formation, maturation, and proliferation of peroxisomes. Peroxisome biogenesis requires a set of proteins known as peroxins, many of which are involved in the import of peroxisomal matrix proteins. Recent studies have highlighted the importance of the endoplasmic reticulum (ER) in the de novo synthesis of peroxisomes. According to this model, pre-peroxisomal vesicles are budded from the ER in a Pex19p dependent manner and mature by importing matrix proteins sequentially from the cytosol, requiring Pex3p. Pex19p is a cytosolic protein with an N-terminal Pex3p binding domain and a C-terminal domain known to interact with the membrane targeting signal (mPTS) of other peroxisomal membrane proteins (PMPs). In this study, we have characterized several Pex19p N and C-terminal deletions to determine the minimal structural requirement of Pex19p that supports its function. Deletions in the N-terminal domain of Pex19p disrupted the interaction between Pex19p and Pex3p while deletions in the C-terminal domain of Pex19p left this interaction intact. We found that deletions in either of the two domains caused a delay in peroxisome biogenesis although import competent peroxisomes were still formed. In conclusion, physical segregation of the Pex3p and PMP binding domains of Pex19p has provided novel insights into the role of Pex19p in peroxisome biogenesis.
Abstract Title : The role of secondary metabolites in the interspecies interactions of Serratia marcescens and Bacillus subtilis
Abstract : We investigated the interspecies interaction between two soil bacteria, Bacillus subtilis, which is used as a probiotic, and Serratia marcescens, an opportunistic pathogen. We found that B. subtilis and S. marcescens form a co-colony when grown alongside one another. However when either one of the species is placed at a higher concentration, relative to the other, it is able to inhibit growth of the other species. Therefore the outcome of this interspecies interaction varies under different conditions. We tested transcription factor mutants of B. subtilis and found a biofilm development gene, sinR, and two environmental stress response genes, ylaC and sigB, to be necessary to inhibit growth of S. marcescens. We also tested secondary metabolite mutants of B. subtilis and discovered two genes to be necessary for B. subtilis to inhibit growth of S. marcescens; sfp, which is necessary for synthesis of several secondary metabolites, and pksX, responsible for a polyketide synthase which synthesizes bacillaene. In order to isolate secondary metabolites from each species we performed solid-phase extraction of bacterial cultures grown both independently and in interaction. Matrix assisted laser desorption ionization ? time of flight (MALDI-TOF) mass spectrometry was performed to identify the masses in all the fractions obtained from solid phase extraction, and correlate specific masses to the fractions that cause growth inhibition. Further purification by High Purification Liquid Chromatography (HPLC) of those fractions indicate that S. marcescens produces at least two metabolites capable of inhibiting B. subtilis growth. Specifically, we discovered purified prodigiosin, a secondary metabolite from S. marcescens, to strongly inhibit growth of B. subtilis. Secondary metabolites are known to contribute to bacterial survival and defenses in interspecies interactions, and the work presented here further elucidate the variety of impacts that secondary metabolites can have in nature.
Abstract Title : Androgen-mediated transcriptional regulation of gonadotropin-releasing hormone receptor
Abstract : For the past year I have been looking at an unprecedented ?super-induction? of the 600-base pair construct of the GnRH receptor gene promoter in the presence of synthetic androgen R1881. This super-induction is interesting due to its absence in the full-length 1.1 kbp promoter when treated with R1881 (the 1.1 kbp expression does still get induced above baseline with R1881, but not by half as much). This observation led to the hypothesis that an upstream regulatory mechanism suppresses a normally super-induced region downstream of 600 bp. With my supervisor Emily Witham?s guidance I mapped this region to a Hormone Response Element at 499 base pairs. Currently we are in the process of confirming its binding to androgen receptor, which we use in the form of bee whole cell extract, using an Electric Mobility Shift Assay; The androgen receptor used in this assay is in the form of bee whole cell extracts, which expectedly binds to radioactively labeled 499 HRE sequence and also gets recognized by the flag antibody, showing a second band on the gel known as a gel super-shift. My masters research in the following year will be a further exploration of the effects of this upstream self-regulatory mechanism when paired with androgen, and its consequences in mouse reproductive behavior, as delineated in my research plan.
Advisor : STEVE A. KAY
Abstract Title : Investigating the regulation of the Circadian clock output
Abstract : Many organisms, from single cellular cyanobacteria to multicellular animals, are able to predict environmental changes caused by Earth?s rotation. Plants utilize the circadian clock to coordinate their biology with the temporal environment making them more fit to survive. They have a clock- regulated ability to anticipate dawn and dusk; and this results in increased chlorophyll content, enhanced photosynthetic activity, and optimization of nightly starch use. Certain clock components also influence responses to biotic stimuli that providing disease and pathogen resistance, and regulate adaptive responses to abiotic stresses such as drought and low temperature. While many of the core components of the circadian clock are known, how the internal timekeeping mechanism is able to convey its ?time information? to its stress response pathways is not well understood. We have identified a family of transcriptional co-activators that may play a role in regulating output from the circadian clock. To test the functional role of this family, we have generated plants with both constitutive and disrupted expression of these co-activators, and are examining these plants for altered circadian regulation of stress responses. By identifying new components of the circadian clock mechanism, we can utilize this knowledge to enhance agriculture and plant growth.
Abstract Title : In-vivo expression analysis of potential virulence genes of Leptospira interrogans in a hamster model of infection
Abstract : Leptospirosis is a widespread zoonotic disease caused by virulent spirochaetes of the genus Leptospira. Despite over 1 million severe cases of Leptospirosis occurring annually and case mortality rates approaching 20%, much remains unknown with regards to its molecular mechanisms of pathogenesis. Previous experiments aimed at elucidating virulence factors using comparative genomics between saprophytic and virulent Leptospira, transposon mediated mutagenesis, and microarray studies under various environmental conditions have been largely unsuccessful. To date, only 5 virulence factors have been identified for pathogenic leptospires: a lipopolysaccharide, heme oxygenase, flagella, InvA, and the OmpA-like Loa22. In an effort to characterize additional virulence factors, an attenuation experiment was conducted using a highly pathogenic (LD50 < 100 bacteria) strain of L. interrogans serovar Lai that was passed bi-weekly until it became avirulent (LD50 >108 bacteria). Both genomes were then sequenced using Solexa nex-gen sequencing. This revealed that the attenuated organism had accumulated 35 single nucleotide polymorphisms (SNPs) resulting in non-synonymous, protein coding amino acid substitutions. Next, an experiment was conducted on the original virulent strain to determine if the RNA products of these genes were upregulated in a host organism using relative changes in fold expression with reverse transcriptase-PCR, resulting in robust upregulation of many of the candidate genes.
Abstract Title : Activation of the Akt Pathway by Small Molecule Compounds
Abstract : Rationale- Activation of the Akt pathway is known to lead to cell survival. Two small molecule compounds were recently discovered that were found to inhibit PHLPP (PH Domain Leucine-Rich Repeat Protein Phosphatase) in immortal cells. PHLPP negatively regulates the Akt pathway and removal or inhibition leads to increased phosphorylation and activation of Akt. These compounds have not previously been tested in primary cells. Objective- To test the small molecule compounds in primary cells in vitro and in vivo and ultimately assess protection from ischemic injury. Methods and Results- The Diversity Set of the National Cancer Institute was recently scanned for small molecule compounds that would inhibit PHLPP. Two compounds were identified, NCS 117079 (referred to as #1) and NCS 45536 (#13), both resulting in increased phosphorylation of Akt Ser473, as well as protection from apoptosis in immortalized cells. #1 was found to be a selective inhibitor, increasing p-Akt Ser473, while Thr308 levels remained stable. With this existing knowledge, we treated neonatal rat ventricular myocytes (NRVM) with #1 and #13 in varying concentrations. Both showed phosphorylation of Ser473 and Thr308 at similar levels. We then chose doses which showed significant increases in Akt phosphorylation, pretreated cells and then stimulated with IGF-1 and LIF at 15 and 30 minute time points. There was a significant increase in phosphorylation of Thr308 and Ser473 at all time points and controls. Conclusion- This preliminary data shows that the compounds, while not inhibiting PHLPP selectively, activate the Akt pathway. It is our hope that we can use these compounds ex vivo and then in vivo in order to protect from ischemic damage.
Abstract Title : Algae-Produced Pfs25 Elicits Anitbodies That Inhibit Malaria Transmission
Abstract : Subunit vaccines are significantly more expensive to produce than traditional vaccines because they are based primarily on recombinant proteins that must be purified from the expression system. Despite the increased cost, subunit vaccines are being developed because they are safe, effective, and can elicit antibodies that confer protection against diseases that are not currently vaccine-preventable. Algae are an attractive platform for producing subunit vaccines because they are relatively inexpensive to grow, genetically tractable, easily scaled to large volumes, have a short generation time, and are devoid of inflammatory, viral, or prion contaminants often present in other systems. We tested whether algal chloroplasts can produce malaria transmission blocking vaccine candidates, Plasmodium falciparum surface protein 25 (Pfs25) and 28 (Pfs28). Antibodies that recognize Pfs25 and Pfs28 disrupt the sexual development of parasites within the mosquito midgut, thus preventing transmission of malaria from one human host to the next. These proteins have been difficult to produce in traditional recombinant systems because they contain tandem repeats of structurally complex epidermal growth factor-like domains, which cannot be produced in bacterial systems, and because they are not glycosylated, so they must be modified for production in eukaryotic systems. Production in algal chloroplasts avoids these issues because chloroplasts can fold complex eukaryotic proteins and do not glycosylate proteins. Here we demonstrate that algae are the first recombinant system to successfully produce an unmodified and aglycosylated version of Pfs25 or Pfs28. These antigens are structurally similar to the native proteins and antibodies raised to these recombinant proteins recognize Pfs25 and Pfs28 from P. falciparum. Furthermore, antibodies to algae-produced Pfs25 bind the surface of in-vitro cultured P. falciparum sexual stage parasites and exhibit transmission blocking activity. Thus, algae are promising organisms for producing cysteine-disulfide-containing malaria transmission blocking vaccine candidate proteins.
Abstract Title : Development of a p27-GFP Reporter Fusion Protein to Detect Transition Across the G1 Phase Restriction Point
Abstract : The widely accepted model of G1 cell cycle progression proposes cyclin D:Cdk4/6 inactivates the retinoblastoma tumor suppressor protein (Rb) during early G1 phase by progressive multi-phosphorylation or hypo-phosphorylation to release E2F transcription factors, resulting in the activation of cyclin E:Cdk2 and transition into late G1 phase. However, due to the use of supra-physiologic overexpression studies, this model remains largely unproven biologically and there has been no method to microscopically view cells as they transit across the restriction point in real time. To help address this problem, I designed a GFP fusion protein with p27, a protein that is stable in early G1 phase, but rapidly degraded at the restriction point due to phosphorylation by active cyclin E:Cdk2 complexes. Transfection of the p27-GFP fusion reporter into cells shows cell cycle specific expression. The p27-GFP fusion protein allows for the first time, the potential to visually dissect events that occur before and immediately after the restriction point.