Bioassays to Monitor Taspase1 Function for the Identification of Pharmacogenetic Inhibitors

Background: Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available. Methodology/Findings: Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high-throughput microscopy platform (Z'factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamideand 2-benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor. Conclusions: The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance

Human PAPS Synthase Isoforms Are Dynamically Regulated Enzymes with Access to Nucleus and Cytoplasm

In higher eukaryotes, PAPS synthases are the only enzymes producing the essential sulphate-donor 3′-phospho-adenosine-5′-phosphosulphate (PAPS). Recently, PAPS synthases have been associated with several genetic diseases and retroviral infection. To improve our understanding of their pathobiological functions, we analysed the intracellular localisation of the two human PAPS synthases, PAPSS1 and PAPSS2. For both enzymes, we observed pronounced heterogeneity in their subcellular localisation. PAPSS1 was predominantly nuclear, whereas PAPSS2 localised mainly within the cytoplasm. Treatment with the nuclear export inhibitor leptomycin B had little effect on their localisation. However, a mutagenesis screen revealed an Arg-Arg motif at the kinase interface exhibiting export activity. Notably, both isoforms contain a conserved N-terminal basic Lys-Lys-Xaa-Lys motif indispensable for their nuclear localisation. This nuclear localisation signal was more efficient in PAPSS1 than in PAPSS2. The activities of the identified localisation signals were confirmed by microinjection studies. Collectively, we describe unusual localisation signals of both PAPS synthase isoforms, mobile enzymes capable of executing their function in the cytoplasm as well as in the nucleus

Systemic sclerosis presenting as CREST syndrome: A case report and review

Systemic sclerosis (SSc) is a chronic multisystem disorder of unknown etiology, characterized by diffuse fibrosis; degenerative changes; and vascular abnormalities in the skin (scleroderma), articular structures, and internal organs especially the esophagus, GI tract, lung, heart, and kidney. We report the case of a 31 years old female patient who came to the ED with complications of SSc after has been diagnosed with a limited cutaneous scleroderma. This case illustrates the varied multisystem presentation of SSc

Novel Radiolabeled Vanilloid with Enhanced Specificity for Human Transient Receptor Potential Vanilloid 1 (TRPV1)

Transient receptor potential vanilloid 1 (TRPV1) has emerged as a promising therapeutic target. While radiolabeled resiniferatoxin (RTX) has provided a powerful tool for characterization of vanilloid binding to TRPV1, TRPV1 shows 20-fold weaker binding to the human TRPV1 than to the rodent TRPV1. We now describe a tritium radiolabeled synthetic vanilloid antagonist, 1-((2-(4-(methyl-[³H])­piperidin-1-yl-4-[³H])-6-(trifluoromethyl)­pyridin-3-yl)­methyl)-3-(3-oxo-3,4-dihydro-2<i>H</i>-benzo­[<i>b</i>]­[1,4]­oxazin-8-yl)­urea ([³H]­MPOU), that embodies improved absolute affinity for human TRPV1 and improved synthetic accessibility

Age-Based Simulation of Merging Behavior at Freeway Merging Ramps

In this research, VISSIM, a microscopic traffic simulation software, was used to evaluate the influence of drivers\u27 age on the operations of a typical diamond interchange. Data were collected on two merging sections along I-75 in Southwest Florida at free-flow traffic condition to get enough samples for driver\u27s age group. Several measures of effectiveness were collected, including speeds, gaps, and location of entry to the main-line lanes. This information was used as either model input or for verification purposes in the validation process. Two VISSIM models were developed for each site: One model for the existing conditions and verification and another model for a sensitivity analysis, varying the percentage of older drivers and level of service (LOS from A to E) to determine their influence on ramp merging operational characteristics. At 95% confidence level, the results indicate that merging speed is a significant factor influencing the merging location. Older drivers have low merging speeds compared with middle-age and younger drivers. The changes in LOS were found to influence the selection of the merging location for all drivers, whereby at LOS E, most drivers tend to merge near the end of the acceleration lane

Novel Radiolabeled Vanilloid with Enhanced Specificity for Human Transient Receptor Potential Vanilloid 1 (TRPV1)

Transient receptor potential vanilloid 1 (TRPV1) has emerged as a promising therapeutic target. While radiolabeled resiniferatoxin (RTX) has provided a powerful tool for characterization of vanilloid binding to TRPV1, TRPV1 shows 20-fold weaker binding to the human TRPV1 than to the rodent TRPV1. We now describe a tritium radiolabeled synthetic vanilloid antagonist, 1-((2-(4-(methyl-[³H])­piperidin-1-yl-4-[³H])-6-(trifluoromethyl)­pyridin-3-yl)­methyl)-3-(3-oxo-3,4-dihydro-2<i>H</i>-benzo­[<i>b</i>]­[1,4]­oxazin-8-yl)­urea ([³H]­MPOU), that embodies improved absolute affinity for human TRPV1 and improved synthetic accessibility