Molecular, Cellular and Developmental Biology

Polypurine Tracts (PPTs) Are Designed For High Affinity Binding To Their Cognate Reverse Transcriptases, A Property That Can Be


Authors: Gauri Nair, Yi-tak Lai, Chandravanu Dash, Stuart F. J. Le Grice, and Jeffrey J. DeStefano
Department or Program: CBMG
Presented by: Gauri Nair
Abstract: Polypurine Tracts (PPTs) Are Designed For High Affinity Binding To Their Cognate Reverse Transcriptases, A Property That Can Be Exploited To Produce Novel PPT-Based Antivirals. Gauri Nairª, Yi-tak Laiª, Chandravanu Dash*, Stuart F. J. Le Grice*, and Jeffrey J. DeStefanoª, ªDepartment of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742; *HIV Drug Resistance Program, NCI-Frederick National Institutes of Health, Frederick, MD 21702. Retroviruses carry purine-rich segments in their RNA genomes known as polypurine tracts (PPTs) that are used by reverse transcriptases (RT) for initiation of second strand synthesis. Because RT has to identify and selectively bind this short nucleotide sequence, it is not surprising that the PPT may also be optimized for the tightest possible binding to RT (DeStefano and Cristofaro, Nucleic Acids Res. 34:130). Systematic Evolution of Ligands by Exponential Selection (SELEX) has been used by several groups to identify high affinity RNA and DNA aptamers to RT and other proteins from large random nucleotide pools. Using a unique SELEX approach, we demonstrated that HIV-RT has a very strong sequence preference for specific primer-templates. The selected DNA primers closely resembled the HIV RNA PPT (5’-AAAAGAAAAGGGGGG-3’), raising the possibility that RTs and their cognate PPTs may have co-evolved for tight binding and proper orientation of RT for extension. Conceivably, because of the stringent PPT requirement by RT for priming second strand synthesis, resistance to inhibitors that closely resemble the PPT may be rare. To this end, we are currently testing closely related retroviral RTs from Moloney murine leukemia virus (Mo-MuLV, PPT: 5’-AGAAAAAGGGGGG-3’) and avian myeloblastosis virus (AMV, PPT: 5’-AGGGAGGGGGA-3’), as well as the LTR-retrotransposon Ty3 that has a significantly different PPT (5’-GAGAGAGAGGAA-3’) which does not contain homopolymeric nucleotide runs. These RTs are being used in SELEX experiments to see if they, like HIV-RT, also select for cognate PPT sequences. Binding affinity assays with synthetically designed retroviral DNA-PPT substrates are also being conducted. Data indicates that MuLV- and HIV-RTs have similar affinities for one another’s DNA-PPTs which they bind 25-100 fold more tightly than random sequence primer-template. They have lower affinity for the slightly divergent AMV DNA-PPT, and even lower for the highly dissimilar Ty3 DNA-PPT. In contrast, AMV-RT binds its own DNA-PPT more tightly than those from MuLV or HIV. Finally, single stranded loop-back aptamer inhibitors of HIV-RT based on our SELEX experiments are strong RT inhibitors in vitro, and ongoing cell culture experiments indicate they also inhibit virus replication, thus opening up an exciting new vista of possibilities.

Arc/Arg3.1 is necessary for visual experience-induced homeostatic synaptic plasticity in mouse primary visual cortex


Authors: Ming Gao, Kenneth Sossa, Lihua Song, Lauren Errington, Laurel Cummings, Hongik Hwang, Dietmar Kuhl, Paul Worley, Hey-Kyoung Lee
Department or Program: BIOL
Presented by: ming gao
Abstract: Visual experience during postnatal development, scales down excitatory synapses in the superficial layers of visual cortex in a process that provides an in vivo paradigm of homeostatic synaptic scaling. The experience-induced increase in neural activity rapidly up-regulates immediate early gene (IEG) products involved in synaptic plasticity, one of which is Arc (activity-regulated cytoskeleton protein or Arg3.1). Cell biological studies indicate that Arc/Arg3.1 functions to recruit endocytic machinery for AMPA receptor (AMPAR) internalization. This action, together with its activity-dependent expression, suggests Arc/Arg3.1 as a regulator of homeostatic synaptic scaling. Since visual experience-induced scaling down of excitatory synapses in layer 2/3 (L2/3) of visual cortex correlates with a reduction in AMPAR at synapses, we investigated whether Arc/Arg3.1 is involved in this process. Using Arc knockout (KO) mice we demonstrate that visual experience-dependent homeostatic synaptic plasticity requires Arc/Arg3.1.

Understanding the functional role of the endo β-1,4 glucanase AtGH9C1 in Arabidopsis thaliana


Authors: Sivacharan Gaddam, Dr. Elena del Campillo
Department or Program: CBMG
Presented by: Sivacharan Gaddam
Abstract: The plant cell wall is a dynamic structure and active rearrangement of cellulose microfibrils and other polysaccharides within the cell wall matrix is essential for plant growth and development. The components and mechanisms involved in these rearrangements remain unclear. Plant glycosyl hydrolases, particularly endo-β-1,4- glucanases (EGases) are hydrolases that catalyze β-1,4- glucan bonds, and potentially mediate cell wall loosening and expansion. A tomato C-type EGase with a unique carbohydrate binding module (CBM49) showed in vitro crystalline cellulose binding (Urbanowicz et al., 2007). BLAST search of the tomato CBM49 revealed 44 orthologs throughout land plants and phylogenetic analysis distinguished two subgroups within the CBM49s in higher plants. Arabidopsis thaliana has three orthologs and one of these, AtGH9C1, showed strong promoter activity specifically in root hair forming cells. The objective of this study was to examine AtGH9C1protein localization with and without the CBM49 using overexpressing GFP fusion protein constructs (AtGH9C1::GFP). This analysis addresses the function of CBM49 in C-Type EGases and the role of AtGH9C1in root hair development. Transient expression of the complete AtGH9C1 (+ CBM49) fusion revealed that CBM49 imposes surface location specificity within the multifaced wall of an epidermal cell. In contrast, the absence of the CBM49 resulted in protein localization throughout the wall surface. A comparison of GFP signal in protein extracts from transiently transformed N. benthamiana, confirmed that AtGH9C1 lacking CBM49 is easily extracted while the complete AtGH9C1 (+CBM49) is not extracted. Thus, removing CBM49 renders the AtGH9C1protein soluble within the wall while the presence of CBM49 seems to anchor AtGH9C1 to the cell wall. Stable lines expressing AtGH9C1 with CBM49 showed localization throughout root hairs including the apical and subapical regions suggesting the AtGH9C1 protein is involved in root hair bulge formation and subsequent elongation.

Dynamin 2 has a possible role in mitosis and cytokinesis other than its endocytic role.


Authors: Tobias Fatscher, Jennifer Chua, Jennifer Lippincott-Schwartz
Department or Program: BIOL
Presented by: Tobias Fatscher
Abstract: Dynamin 2 is involved in different cell processes such as cytokinesis, budding of transport cells, division of organelles, and pathogen resistance. They are also thought to be playing a role in actin assembly at membranes. We transfected MDCKII cells with different mutations of Dynamin and other proteins to research any changes in cytokinesis. The proteins used for our experiment were Rab11a S25N, Ap180C, Esp15, Dyn2 K44a, PRD, and mCeFP as our control. We used immunofluorescent staining to label the parts of interest in the cell. Our proteins were labeled with GFP, the nuclei were stained with Hoechst, and actin was stained with TRITC. We observed cells undergoing cytokinesis with the LSM510 META. The collected data was later compared to check for any differences in cytokinesis within the cells transfected with the different proteins. The cells transfected with K44a seemed mostly to be arrested during the end stage of cytokinesis with a mid body present. Our control cells transfected with mCeFP seemed mostly to be arrested in an early stage of cytokinesis in which the nucleus accumulated in the middle of the cell. The same observation was made for cells transfected with Rab11a S25N treated with aphidicolin. Rab11a S25N transfected cells treated with phalloidin yielded cells mostly in an early stage of cytokinesis, again with a mid body present.

STRUCTURAL AND FUNCTIONAL ANALYSIS OF HRG-1 HEME TRANSPORTERS


Authors: Xiaojing Yuan, Olga Protchenko, Caitlin Hall, Caroline Philpott, Iqbal Hamza
Department or Program: ANSC
Presented by: Xiaojing Yuan
Abstract: Heme is an essential cofactor for proteins involved in diverse cellular processes. Heme synthesis utilizes a highly conserved, multistep pathway which culminates in the mitochondria. Free heme is toxic to the cell due to its intrinsic peroxidase activity. How is this hydrophobic macrocycle transported across membranes and incorporated into numerous hemoproteins located in different cellular compartments? Unlike prokaryotes, the mechanism for uptake, trafficking and sequestration of heme in eukaryotes is currently unknown. By using the heme auxotrophy of Caenorhabditis elegans, our laboratory had previously identified HRG-1 as the first eukaryotic heme importer/transporter. To determine how HRG-1 functions in heme transport, we used site-directed mutagenesis and protein chimeras in Saccharomyces cerevisiae heme-deficient strains and mammalian cell lines. Here, we show that histidines are important for HRG-1 function. Furthermore, the carboxyl terminus of HRG-1 is essential for its intracellular localization and membrane trafficking. These results identify the structural and cellular determinants of HRG-1. Future in-depth analysis of HRG-1 mutant proteins will greatly facilitate our understanding of heme in fundamental biological processes.

TSO1: a key regulator of cell cycle control and floral organ differentiation


Authors: Paja Sijacic
Department or Program: CBMG
Presented by: Paja Sijacic
Abstract: In multicellular organisms, cell proliferation has to be coordinated with cell differentiation and organ growth. The Arabidopsis TSO1 gene appears to play a role in coordinating cell proliferation with differentiation. TSO1 encodes a protein containing two cysteine-rich (CXC) DNA-binding domains and is homologous to gene products present in Drosophila, C. elegans, and mammals that are components of a novel chromatin complex dREAM. This novel complex is shown in animal cells to inhibit cell proliferation and promote cell differentiation. Our characterization of tso1 mutants shown here indicates that Arabidopsis TSO1 may also encode a component of a chromatin complex in plants.

Understanding the funtional role of endo β-1,4 glucanase AtGH9C1 in Arabidopsis thaliana


Authors: Sivacharan Gaddam and Dr. Elena del Campillo
Department or Program: CBMG
Presented by: Elena del Campillo
Abstract: The plant cell wall is a dynamic structure and active rearrangement of cellulose microfibrils and other polysaccharides within the cell wall matrix is essential for plant growth and development. The components and mechanisms involved in these rearrangements remain unclear. Plant glycosyl hydrolases, particularly endo-β-1,4- glucanases (EGases) are hydrolases that catalyze β-1,4- glucan bonds, and potentially mediate cell wall loosening and expansion. A tomato C-type EGase with a unique carbohydrate binding module (CBM49) showed in vitro crystalline cellulose binding (Urbanowicz et al., 2007). BLAST search of the tomato CBM49 revealed 44 orthologs throughout land plants and phylogenetic analysis distinguished two subgroups within the CBM49s in higher plants. Arabidopsis thaliana has three orthologs and one of these, AtGH9C1, showed strong promoter activity specifically in root hair forming cells. The objective of this study was to examine AtGH9C1protein localization with and without the CBM49 using overexpressing GFP fusion protein constructs (AtGH9C1::GFP). This analysis addresses the function of CBM49 in C-Type EGases and the role of AtGH9C1in root hair development. Transient expression of the complete AtGH9C1 (+ CBM49) fusion revealed that CBM49 imposes surface location specificity within the multifaced wall of an epidermal cell. In contrast, the absence of the CBM49 resulted in protein localization throughout the wall surface. A comparison of GFP signal in protein extracts from transiently transformed N. benthamiana, confirmed that AtGH9C1 lacking CBM49 is easily extracted while the complete AtGH9C1 (+CBM49) is not extracted. Thus, removing CBM49 renders the AtGH9C1protein soluble within the wall while the presence of CBM49 seems to anchor AtGH9C1 to the cell wall. Stable lines expressing AtGH9C1 with CBM49 showed localization throughout root hairs including the apical and subapical regions suggesting the AtGH9C1 protein is involved in root hair bulge formation and subsequent elongation.

Regulation of the Carpel Development Gene SPATULA by SEUSS and LEUNIG co-repressors


Authors: Courtney Hollender, Vidyadhar Karmarkar, and Zhongchi Liu
Department or Program: CBMG
Presented by: Courtney Hollender
Abstract: SPATULA (SPT) encodes a bHLH transcription factor expressed primarily in the stigma and transmitting tract of the Arabidopsis gynoecium, the female reproductive organ. This gene specifies stigma and proper style development, as spt-2 mutants have reduced stigmatic tissue and unfused carpels in the style. Previously, APETALA2 (AP2) was show to repress SPT expression in sepals and petals as ap2-2 mutants showed ectopic SPT expression and ectopic stigmatic tissues. How AP2 represses SPT is unknown. We present evidence that SEUSS (SEU) and LEUNIG (LUG) co-repressors associate with the SPT promoter to repress SPT expression in sepals and petals. Furthermore, we demonstrate a direct interaction between SEU and AP2 via yeast-two-hybrid and bimolecular fluorescence complementation, suggesting that AP2 represses SPT by recruiting SEU and LUG co-repressors. It was previously hypothesized that a high auxin level at the apex of gynoecium is crucial for the activation of SPT, which is required to specify stigma. ETTIN (ETT), an auxin response factor, was previously shown to repress SPT in carpel valves, the part of gynoecium that does not develop stigma. This repression of SPT by ETT may also be mediated by SEU and LUG co-repressors. We present preliminary evidence supporting a model of SPT regulation during gynoecium development.

Aptamers with Polypurine Tract-Like Sequences as Effective HIV-1 Inhibitors in Cell Culture


Authors: Yi-Tak Lai, Gauri R. Nair and Jeffrey J. DeStefano
Department or Program: CBMG
Presented by: Yi-Tak Lai
Abstract: Aptamers are synthetic, single stranded nucleic acids with unique three-dimensional structures, allowing the folded sequence to bind specifically to the target protein. Since the initial discovery of aptamers targeting HIV reverse transcriptase (HIV-RT) by Larry Gold’s group at University of Colorado, several RNA and DNA aptamers specific to HIV-RT and many other proteins have been developed. It has been proposed that the broader and more specific structurally-based interactions between the reverse transcriptase and aptamers may help to evade drug resistance; however, this remains to be demonstrated. Our lab has recently used a unique SELEX (Systematic Evolution of Ligands by EXponential enrichment) technique to show that HIV-RT can bind with high affinity to specific primer-template sequences (DeStefano and Nair, Oligonucleotides 2008, 18(2):133-44). Based on these sequences, which resembled the HIV polypurine tract (PPT), DNA aptamers that demonstrate tight binding and inhibition of HIV-RT in vitro were developed. We are currently investigating if aptamers designed in our lab that are potent HIV-1 RT inhibitors in vitro can also inhibit viral infection in cell culture. Low micromolar concentrations of aptamers in the absence of a transfection agent inhibited replication in Jurkat cells without significant cellular toxicity. Using fluorescently tagged aptamers, we observed entry of the aptamers into Jurkat cells; this process was enhanced in the presence of HIV-1. It is still unclear if the effect of aptamer inhibition on the replication cycle is due to RT inhibition or to other factors such as alteration of cellular pathways. We are currently investigating this by studying effects of the aptamer on individual steps in replication and screening for revertants. We are also trying to improve aptamer delivery to cells by attaching cargo proteins that facilitate cell entry. This could reduce the amount of aptamer required for inhibition. The study of these aptamers could help us to understand the interaction between RT and the different nucleic acid substrates, ultimately allowing for the potential development of small molecule inhibitors that would be useful in the treatment of HIV-1.

Cellular inter-relationship between butyrophilin 1A1 (BTN) and xanthine oxidoreductase (XOR) in lactating mammary epithelial cells


Authors: Jeong, J., *Kadegowda, A.K.G., Xu, J., and Mather, I.H. * co-primary author
Department or Program: ANSC
Presented by: Jaekwang Jeong
Abstract: Butyrophilin 1a1 (BTN) and xanthine oxidoreductasae (XOR) are expressed in the lactating mammary gland and are secreted into milk in association with lipid droplets. BTN-/- and XOR+/- mutant mice show similar lactation phenotypes with severe defects in the secretion of milk lipid. This suggests the possible involvement of BTN and/or XOR in milk fat secretion during lactation. We have recently shown that XOR binds to the B30.2 domain of BTN (Jeong et al., J. Biol. Chem., 284, 22444-22456, 2009). We now show that the domain in XOR that binds to BTN is the iron-sulfur cluster domain in the N-terminus. In addition, we demonstrate that BTN is exclusively localized in the apical plasma membrane while XOR is localized in the cytoplasm and that XOR interacts with BTN at the apical plasma membrane. XOR protein is increased by 2 and 4 fold, respectively, in BTN+/- and BTN-/- mouse lines and enzyme activity is corresponding increased by 2 fold and 3 fold. Thus, loss of BTN leads to accumulation of intra-cellular XOR, presumably because XOR cannot be exported from the cell associated with milk-lipid droplets in the absence of its binding partner. Further studies are required to test if the increased levels of XOR in BTN mutant mice lead to elevated levels of reactive oxygen species (ROS), which may cause tissue damage and contribute to the mutant phenotype. We postulate that the interaction between BTN and XOR is either required to form a structural scaffold, or signaling complex which functions not only in milk-lipid droplet secretion but also may regulate the production of ROS in wild-type mammary epithelial cells.

A Comparative Study of the Binding Affinity of HIV-1 Reverse Transcriptase to DNA vs. RNA Templates


Authors: Jeffrey T. Olimpo and Jeffrey J. DeStefano
Department or Program: CBMG
Presented by: Jeffrey Olimpo
Abstract: During HIV-1 replication, the ability of its reverse transcriptase to bind DNA and RNA substrates is integral for the generation of viral double stranded DNA that can ultimately be integrated into the host genome. While this is the case, studies on the binding stability of HIV-1 reverse transcriptase (HIV-RT) to RNA versus DNA substrates demonstrate an increased affinity of HIV-RT to RNA-DNA versus DNA-DNA hybrids. Previous research in our lab reported that this binding may require only a short, 5 base pair RNA-DNA hybrid region, where positioning of the polymerase active site of the enzyme directly over the RNA-DNA (3’ terminus of the DNA primer directly over the first nucleotide of the 5 base RNA segment) region yields optimal binding (~20-fold better binding than DNA-DNA). In an effort to further explore the requirements for tighter binding of HIV-RT to RNA versus DNA templates, our current research is focused on analyzing issues of nucleotide composition and positioning within the aforementioned 5 base pair region. Preliminary findings indicate that increased binding affinity of HIV-RT to RNA-DNA versus DNA-DNA may possibly be attributed to a single nucleotide at position 4 (bound to the 4th base back from the 3’ terminus of the DNA primer) of the RNA-DNA hybrid region, where tighter binding remains contingent upon positioning of the polymerase domain over this region. Additionally, preliminary evidence suggests that such binding may be due to increased template flexibility rather than increased interactions with 2’-OH groups, as a substrate with a single RNA nucleotide at position 4 with a 2’ methyl group replacing the hydroxyl bound as tightly as the normal 2’ hydroxyl. Finally, there was a clear binding advantage for uracil versus other RNA nucleotides at position 4. Taken together, these findings suggest a role for uracil and position 4 in promoting template flexibility and, subsequently, increased binding affinity of HIV-RT to RNA vs. DNA templates. It is anticipated that such findings may have potential implications in the development of nucleic acid-based inhibitors of HIV-1 reverse transcriptase.

High levels of physical activity accelerate telomere shortening in Cast/ei J mice


Authors: A.T. Ludlow, M. Marshall, S. Witkowski, E.E. Spangenburg, S.M. Roth.
Department or Program: KNES
Presented by: Andrew Ludlow
Abstract: Three reports, including one from our lab (Ludlow et al 2008), have shown physical activity to be associated with telomere length in humans. We observed that moderate levels of physical activity were associated with longer telomeres compared to both very low and very high physical activity levels (P < 0.05). Our primary purpose was to determine the feasibility of mechanistic studies on telomeres in mice known to voluntarily run at high levels. To achieve this aim, we investigated telomere length and telomerase enzyme activity in two commonly used inbred mouse strains (Balb/c J and C57/Bl6 J) and one strain known to have short telomeres (Cast/ei J). The Balb/c J and C57/Bl6 J mouse strains had significantly longer telomere lengths when compared to both humans (P < 0.001) and the Cast/ei J animals (P = 0.01). This result was consistent across multiple tissue types in the mice, with the Cast/ei J having significantly shorter telomere lengths. No differences were observed in telomerase enzyme activity among strains, though tissue-specific differences were noted within the Cast/ei J strain (e.g., highest values in brain and liver vs. skeletal muscle). The second purpose of our research was to determine if long-term voluntary physical activity influenced telomeres in one of the above selected strains. Cast/ei J mice were subjected to a 1 yr voluntary wheel running intervention in comparison to an aged-matched sedentary group. Telomere length was measured in the exercise (wheel access, n = 10) and sedentary (n = 11) groups in red gastrocnemius skeletal muscle homogenates. The exercise group averaged 6.2 ± 2.3 km running per day over the 1 yr period. Body weight measured bi-weekly did not differ between the groups (P = 0.42). At the end of the 1 yr intervention, telomere length (Telomere PCR Ct to Single copy gene PCR Ct Ratio; Cawthon 2002; Callicott and Womack 2006) was significantly longer in the sedentary vs. exercise animals (sedentary = 6.55 ± 0.61 vs. exercise = 4.24 ± 0.50 AU; P=0.007). The exercise mice exhibited significantly higher telomerase activity in skeletal muscle tissue compared to sedentary mice (0.083 ± 0.025 vs. 0.018 ± 0.005 amoles/0.43 ug protein; P=0.03). We also examined mRNA levels of Trf2, Trf1, Ku70, Ku80 and Gapdh in skeletal muscle of these same mice and observed an up-regulation of all telomere-related targets in the exercise vs. sedentary mice. These results provide the first evidence of change in telomere length in response to voluntary physical activity in mice. Our preliminary findings provide support for a role of physical activity in modifying telomere length, telomerase activity, and expression of telomere-related proteins. The Cast/ei J strain, a highly spontaneously active strain of mouse, likely performs a ‘moderate’ level of PA in a non-wheel cage and when given wheel access, exercises at an extremely high level which increases telomere attrition.