Abstract
BackgroundThreonine 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/FindingsEfficient 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]benzenesulfonamide and 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.ConclusionsThe 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.
Highlights
Besides their critical role in intra- and intercellular ‘‘waste management’’, proteases are currently accepted as important signaling molecules involved in numerous biological and pathological functions [1,2]
Incubation of the substrate, GST-2Cl, containing the mixed lineage leukemia (MLL) cleavage sites 1 and 2 (MLL aa 2650–2808), with increasing amounts of rTasp1 resulted in the proteolytic cleavage of the substrate as well as in the autocatalytic processing of the proenzyme
These results indicated the possibility that bacterially expressed Taspase1 displays only an attenuated catalytic activity
Summary
Besides their critical role in intra- and intercellular ‘‘waste management’’, proteases are currently accepted as important signaling molecules involved in numerous biological and pathological functions [1,2] These include metabolism, tissue remodeling, apoptosis, cell proliferation and migration [1,3]. The discovery of Taspase founded a new class of endopeptidases that utilize the N-terminal threonine of its mature b-subunit as the active site [5]. Mutation of this catalytic nucleophile, Thr234, abolishes Taspase1’s proteolytic activity [5,6]. 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 Taspase are currently available
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