Toward understanding the structure of Amot’s ACCH Domain

poster abstractAmots are a family of adaptor proteins widely involved in cell signaling and lipid binding. Amot80 has been linked to cellular proliferation in breast cancer via the VEGF and MAPK signaling pathways, while Amot130 and AmotL1 have been linked to cellular inhibition via the HIPPO signaling pathway. Amot family members also have a characteristic lipid-binding domain – named the ACCH Domain for its predicted coil-coil structure – that has the ability to selectively target phosphoinositols followed by deformation of the membrane. Understanding the structure-function relationship of this domain may provide options to modulate these signaling pathways, directly affecting cellular differentiation, proliferation, and migration. Extensive crystallization attempts for this domain have failed, leading to a bioinformatics and biophysics-combined approach. Using SAXS, data for the globular structure of Amot80 has been generated and analyzed. Additionally, the threading programs ITASSER and LOMETS were used to develop 20 computational theoretical models. By fitting the computational models to the SAXS data, potential ACCH domain models were generated, and then scored based on accuracy of fit via C-score, TMScore, and RMSD values. This 3D model can then be used to discover how Amot interacts with lipids and further the understanding of Amot’s role in the cancer-signaling cascade

Toward Understanding the Role of Amot80 Lipid Binding in Cellular Proliferation and Migration

poster abstractsAmots are adaptor proteins which coordinate signaling that controls cellular differentiation and proliferation, and their Amot coiled-coil homology (ACCH) domain is able to bind lipids with specificity which leads to membrane deformation and targets transcription factors to the nucleus. Understanding the biophysical mechanisms involved in lipid binding may provide pathways to modulate protein sorting and downstream signaling events inducing cellular differentiation, cancer cell proliferation, and migration. At this time, all work reported on signaling based on Amot expression is unable to distinguish between the role of the Amot80 and the 130 family members as they share a common ACCH domain. The goal of this project is to specifically relate the Amot80 ACCH lipid binding with function related to cancer phenotypes Mutations were carried forward based on lipid sedimentation, FRET, and SAXS assays against the ACCH domain of the protein. Site-directed mutagenesis was then employed to probe the specific contributions of 7 selected lysines and arginines toward lipid head-group binding in the full length protein. The polarity/scaffolding signaling effect of mutations in the Amot80 will be monitored by matrigel, accumulation/cell counting, and titrated thymidine incorporation assays. Cell morphology will be imaged by confocal imaging, and cellular migration will be recorded by video. The effects on YAP1/2 and MAPK activation will be assessed by immunoblot analysis. The changes will then be correlated in extracellular scaffolding and migration with immunoblots and cellular staining. Likewise, effects on proliferation will be monitored by MTT assays. The hypothesis of this aim is that modulation of Amot’s ability to bind selective lipids will interrupt the signaling pathways leading to cellular migration, differentiation, and proliferation. This work was supported by the IUPUI Undergraduate Research Opportunities Program (UROP) and NIH K01CA169078-01

Defining the Roles of Various Lysines and Arginines in Amot Lipid Binding

poster abstractOne of the defining traits of cancerous cells is proliferation. The focus of this study is on the proliferation of mammary cells. As an adaptor protein, the Amot membrane binding event is key to the localization and sorting of proteins responsible for cellular differentiation, proliferation, and migration. The Amot coiled-coil homology domain (ACCH) is a lipid-binding domain responsible for cholesterol affinity and binding to endothelial membranes. Our working hypothesis is that the ability to modulate Amot lipid-binding will lead to means to prevent ductal cell hyperplasia progression into breast cancer tumors. We will determine which residues are responsible for lipid-binding by changing positively charged lysine and arginine into uncharged or negatively charged amino acids. Approximately 40 of these mutations have been screened using a liposome binding assay which mimics how the protein binds with the cell membrane by using an in vitro mixture of lipids similar to that seen in endothelial cells. Forster resonance energy transfer (FRET) was used to confirm significant decreases in lipid binding of ACCH mutants selected from the liposome binding assay, as energy transfer only occurs when the tyrosines in the protein and the Dansylated liposome are in close proximity to each other. In order to saturate the binding affinity of the mutants, the liposomes will be combined with cholesterol in increasing amounts. It has been found that Amot protein is concentrated in areas of PI with higher levels of cholesterol. This will provide a target for the ACCH domain to associate with in the membrane. Mutants deemed important from this study will then be transformed into human cells to study their effects on cell polarity, signal transduction, cell shape, and cellular proliferation

Growth, Yield, and Efficiency of Potassium Fertilizer Use in Burley Tobacco Production

For many crops, band placement of fertilizer offers agronomic, economic, and environmental advantages over commonly used preplant broadcast applications. Drill banding most of the nitrogen (N) fertilizer 12 inches to both sides of the row for tobacco shortly after transplanting greatly aIleviates manganese toxicity and other nutrient imbalances, improves early growth and yield, and offers increased efficiency of N use. Nitrogen fertilizers applied after transplanting are less likely to be leached during excess rainfall events that commonly occur in April and early May in Kentucky

HE AMOT FAMILY OF PROTEINS BINDS AND ACTIVATES NEDD4 FAMILY LIGASES TO PROMOTE THE UBIQUITINATION OF LATS AND YAP

poster abstractAmot adaptor proteins bind and integrate signaling that controls cell po-larity and growth. All three Amot family members (Amot, AmotL1 and AmotL2) directly bind YAP; a transcriptional co-activator that controls the expression of genes involved in organ homeostasis and cell growth. Preven-tion of nuclear accumulation of YAP by either sequestration or degradation in the cytosol abolishes its transcriptional functions and is a major mechanism for growth arrest in response to cellular differentiation. This is mainly thought to be regulated by phosphorylation of YAP by the Hippo kinases LATS1/2. Recently, binding by the Amot proteins was also found to inhibit YAP by sequestering it in the cytosol through both LATS dependent and in-dependent mechanisms. This study identifies a novel mechanism whereby Amot proteins control YAP activation in a Hippo independent mechanism by coupling it to ubiquitination by Nedd4 family ligases. Amot proteins mediate the coupling of Nedd4 ligases with YAP by simultaneously binding both pro-teins via multiple PY motifs that are recognized by WW domains in both YAP and Nedd4. Binding of Nedd4 by Amot is also shown to relieve the auto-inhibition of its ligase activity. This may be a direct consequence of binding Amot or from being re-targeted in cells by Amot proteins to endosomes. Im-portantly, Amot induced ubiquitination of YAP by Nedd4 proteins is shown to enhance the residence of YAP in the nucleus and in YAP activated transcrip-tion. Taken together our data suggest that Amot couples Nedd4 family ubiq-uitin ligases with the transcriptional co-activator YAP to drive the ubiquitination and activation of YAP

Reference values for bone mineral density in healthy Mexican children and adolescents

Introduction: Clinical assessment of bone health by Dual‐Energy X-ray Absorptiometry (DXA) in the paediatric population requires robust reference values. The International Society for Clinical Densitometry (ISCD) recommends that country/regional reference values ideally should be used to improve precision in bone health assessment. / Objective: The aim of this study was to provide reference values for relevant bone health variables for healthy Mexican children and adolescents aged 5 to 18 years. / Methods: This was a cross-sectional, stratified and population-based study, that measured a representative sample of healthy Mexican children and adolescents with DXA. We constructed age- and sex-smoothed reference values for areal bone mineral density (aBMD) of total body less head (TBLH), total body (TB), lumbar spine (LS), and bone mineral apparent density (BMAD) for LS, by means of Generalized Additive Models for Location, Scale and Shape (GAMLSS). / Results: Reference data including the 3th, 5th, 10th, 25th, 50th, 75th, 90th, 95th and 97th centiles, along with lambda (L), mu (M) and sigma (S) values, are given for each variable of interest for each sex at 0.25 years intervals. Reference values relative to height and Tanner-stage for both sexes are also provided. Finally, formulas to enable Z score estimation for clinical use are also presented. / Conclusions: The sex, age, height, Tanner-stage and ethnic-specific reference data provided in this study should enable more precise assessment of bone health in the Mexican paediatric population. The data presented may also allow for future evaluation of potential similarities and differences across different ethnic groups

The RGS-RhoGEFs control the amplitude of YAP1 activation by serum

Actin-dependent mechanisms drive the nuclear translocation of Yap1 to enable its co-activation of transcription factors that induce pro-growth and survival programs. While Rho GTPases are necessary for the nuclear import of YAP1, the relevant Guanine Exchange Factors (GEFs) and GTPase Activating Proteins (GAPs) that connect this process to upstream signaling are not well defined. To this end, we measured the impact of expressing sixty-seven RhoGEFs and RhoGAPs on the YAP1 dependent activity of a TEAD element transcriptional reporter. Robust effects by all three members of the regulator of G-protein signaling (RGS) domain containing RhoGEFs (ArhGEF1, ArhGEF11 and ArhGEF12) prompted studies relating their known roles in serum signaling onto the regulation of Yap1. Under all conditions examined, ArhGEF12 preferentially mediated the activation of YAP1/TEAD by serum versus ArhGEF1 or ArhGEF11. Conversely, ArhGEF1 in multiple contexts inhibited both basal and serum elevated YAP1 activity through its GAP activity for Gα13. The sensitivity of such inhibition to cellular density and to low states of serum signaling supports that ArhGEF1 is a context dependent regulator of YAP1. Taken together, the relative activities of the RGS-RhoGEFs were found to dictate the degree to which serum signaling promotes YAP1 activity

Integration of systems biology with organs-on-chips to humanize therapeutic development

"Mice are not little people" - a refrain becoming louder as the gaps between animal models and human disease become more apparent. At the same time, three emerging approaches are headed toward integration: powerful systems biology analysis of cell-cell and intracellular signaling networks in patient-derived samples; 3D tissue engineered models of human organ systems, often made from stem cells; and micro-fluidic and meso-fluidic devices that enable living systems to be sustained, perturbed and analyzed for weeks in culture. Integration of these rapidly moving fields has the potential to revolutionize development of therapeutics for complex, chronic diseases, including those that have weak genetic bases and substantial contributions from gene-environment interactions. Technical challenges in modeling complex diseases with "organs on chips" approaches include the need for relatively large tissue masses and organ-organ cross talk to capture systemic effects, such that current microfluidic formats often fail to capture the required scale and complexity for interconnected systems. These constraints drive development of new strategies for designing in vitro models, including perfusing organ models, as well as "mesofluidic" pumping and circulation in platforms connecting several organ systems, to achieve the appropriate physiological relevance. Keywords: organs-on-chips; 3D liver culture; perfusion; drug development; inflammation; organ crosstalk; tissue chip; intestineUnited States. Defense Advanced Research Projects Agency (Award W911NF-12-2- 0039))National Institutes of Health (U.S.) (Grant UH3TR000496

Liver ‘organ on a chip’

© 2017 The liver plays critical roles in both homeostasis and pathology. It is the major site of drug metabolism in the body and, as such, a common target for drug-induced toxicity and is susceptible to a wide range of diseases. In contrast to other solid organs, the liver possesses the unique ability to regenerate. The physiological importance and plasticity of this organ make it a crucial system of study to better understand human physiology, disease, and response to exogenous compounds. These aspects have impelled many to develop liver tissue systems for study in isolation outside the body. Herein, we discuss these biologically engineered organoids and microphysiological systems. Keywords: Microphysiologic systems; Organoids; 3D culture systemsNational Institutes of Health (U.S.) (Grant UH3TR000496)National Institutes of Health (U.S.) (Grant UH3TR000503

Modification of the Creator recombination system for proteomics applications – improved expression by addition of splice sites

BACKGROUND: Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. RESULTS: To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. CONCLUSION: The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics