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

Glut4 trafficking: Roles of LRP1, endosomal pH and Rab GTPases

Insulin regulates Glut4 trafficking between the intracellular storage compartments and the PM of fat and muscle cells, which is a key process in glucose homeostasis. Insulin is released in response to post-prandial excess blood sugar and facilitates glucose clearance by stimulating Glut4 translocation. The detailed Glut4 trafficking itinerary and how insulin regulates this process is not known. Our research was focused in understanding the effect of insulin in Glut4 trafficking and the pathways Glut4 take during its trafficking between the Glut4 storage compartments (GSVs) and the PM. Using a flow cytometric based assays, we show that insulin increased Glut4 exocytic constant (kex) and the amount of Glut4 cycling consistent with previous studies, but also showed that insulin does not affect the Glut4 endocytic constant (ken). Previous studies have estimated ken values but in this study we measured it directly using multiple experimental approaches and showed it is not inhibited by insulin. We also showed all the AS160 substrate Rab GTPases found on Glut4 vesicles are involved in Glut4 trafficking. Even though Rab10 was already previously shown to be involved in Glut4 trafficking, we show here that Rabs 8A, 8B and 14 are also involved in Glut4 trafficking in adipocytes. These knockdowns also affected LRP1 trafficking. Using the Rab knockdown's effect on Glut4 and LRP1 trafficking, we modeled their effect in membrane trafficking in the constitutive and regulated pathways. We also show evidence that one of the major cargo proteins of GSVs, LRP1, might be involved in Glut4 trafficking using its luminal domain. Endosomal pH is required for receptor mediated endocytosis but in this study we showed for the first time that it is also required for Glut4 trafficking. We show evidence that there are two Glut4 trafficking pathways: pH-dependent and pH-independent trafficking