title: Cell-based analysis of structure-function activity of threonine aspartase 1.
abstract: Taspase1 is a threonine protease responsible for cleaving intracellular substrates. As such, (de)regulated Taspase1 function is expected not only to be vital for ordered development but may also be relevant for disease. However, the full repertoires of Taspase1 targets as well as the exact biochemical requirements for its efficient and substrate-specific cleavage are not yet resolved. Also, no cellular assays for this protease are currently available, hampering the exploitation of the (patho)biological relevance of Taspase1. Here, we developed highly efficient cell-based translocation biosensor assays to probe Taspase1 trans-cleavage in vivo. These modular sensors harbor variations of Taspase1 cleavage sites and localize to the cytoplasm. Expression of Taspase1 but not of inactive Taspase1 mutants or of unrelated proteases triggers proteolytic cleavage and nuclear accumulation of the biosensors. Employing our assay combined with scanning mutagenesis, we identified the sequence and spatial requirements for efficient Taspase1 processing in liquid and solid tumor cell lines. Collectively, our results defined an improved Taspase1 consensus recognition sequence, Q(3)(F/I/L/V)(2)D(1)↓G(1)'X(2)'D(3)'D(4)', allowing the first genome-wide bioinformatic identification of the human Taspase1 degradome. Among the 27 most likely Taspase1 targets are cytoplasmic but also nuclear proteins, such as the upstream stimulatory factor 2 (USF2) or the nuclear RNA export factors 2/5 (NXF2/5). Cleavage site recognition and proteolytic processing of selected targets were verified in the context of the biosensor and for the full-length proteins. We provide novel mechanistic insights into the function and bona fide targets of Taspase1 allowing for a focused investigation of the (patho)biological relevance of this type 2 asparaginase.
authors.affiliation: Molecular and Cellular Oncology/Mainzer Screening Center, University Hospital of Mainz, Langenbeckstrasse 1, 55101 Mainz, Germany. bier@uni-mainz.de
authors.forename: Carolin
authors.initials: C
authors.lastname: Bier
authors.forename: Shirley K
authors.initials: SK
authors.lastname: Knauer
authors.forename: Alexander
authors.initials: A
authors.lastname: Klapthor
authors.forename: Andrea
authors.initials: A
authors.lastname: Schweitzer
authors.forename: Alexander
authors.initials: A
authors.lastname: Rekik
authors.forename: Oliver H
authors.initials: OH
authors.lastname: Krämer
authors.forename: Rolf
authors.initials: R
authors.lastname: Marschalek
authors.forename: Roland H
authors.initials: RH
authors.lastname: Stauber
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology*; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D055106:Genome-Wide Association Study; D006801:Humans; D051379:Mice; D009154:Mutation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D010450:Endopeptidases; C480823:taspase1, human