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.

FIGURE 1.

In vivo assays for Taspase1 activity. Green/blue fluorescent proteins were visualized by fluorescence microscopy. Scale bars, 10 μm. Dashed lines mark the nuclear/cytoplasmic cell boundaries obtained from the corresponding phase contrast images. a, nuclear/nucleolar localization of Taspase1-GFP (Tasp-GFP) in HeLa cell transfectants visualized by GFP fluorescence (upper panel) or immunofluorescence using α-Taspase1 Ab (lower panel). b, principal and domain organization of the translocation sensor to probe Taspase1 activity. BioTasp is composed of GST, GFP, combinations of a nuclear import (NLS), and a Myc epitope-tagged export (NES) signal, combined with the Taspase1 cleavage site from MLL (CS2; aa ²⁷¹³KISQLDGVDD²⁷²²). BioTasp localizes predominantly to the cytoplasm but is continuously shuttling between the nucleus and the cytoplasm. Coexpression of Taspase1-BFP (Tasp-BFP) results in the removal of the NES by proteolytic cleavage of the biosensor, allowing nuclear accumulation of the green fluorescent fusion.▶, NLS; ◀, NES. c, expression of Taspase1 triggers nuclear accumulation of BioTasp in living HeLa transfectants. The autofluorescent biosensor is predominantly cytoplasmic (upper panel), and coexpression of Taspase1-BFP results in the proteolytic cleavage of the Myc-NES_Rev and nuclear accumulation of BioTasp (lower panel). Myc-NES_Rev was detected using α-Myc tag Ab. d, biosensors containing a nonfunctional Taspase1 cleavage site (BioTasp_mut) or a cleavage site for caspase3 (BioCasp3) remained cytoplasmic upon coexpression of Tasp-BFP. No nuclear accumulation of BioTasp was observed upon coexpression of inactive TaspTV-BFP or the nucleolar Rev-BFP protein. e, trans-cleavage of BioTasp shown by immunoblot analysis of whole cell lysates. 293T cells were transfected with BioTasp together with the indicated Taspase1 expression plasmids or the empty vector (control). Expression of proteins and cleavage products was visualized using α-GST and α-Taspase1 Ab. GAPDH served as loading control. f, efficacy of BioTasp processing in vivo. HeLa cells were transfected with the indicated amounts of BioTasp and Tasp-BFP expression plasmid. 24 h later, the numbers of cells showing cytoplasmic (C), cytoplasmic and nuclear (N/C), or nuclear (N) fluorescence were counted in at least 200 sensor-expressing cells. Results and images from a representative experiment are shown. The numbers of cell showing cytoplasmic fluorescence significantly decreased upon cotransfection of as little as 0.1 μg of Tasp-BFP expression plasmid (***, p < 0.0001).

FIGURE 2.

In vivo mapping of residues critical for trans-cleavage activity of Taspase1. HeLa cells were transfected with the indicated BioTasp cleavage site mutants (BioTasp_CSmut) together with the Tasp- or TaspTV-mCherry expression plasmid. 24 h later, nuclear translocation of the BioTasp_CSmut variants was analyzed in at least 200 fluorescent living cells. Representative examples are shown. a and b, alanine-scanning mutagenesis using active (a) or inactive Taspase (b). c and d, similar exchange mutagenesis using active (c) or inactive Taspase (d). Scale bars, 10 μm. *, residues required for Taspase1 cleavage. ↓ indicates position of Taspase1 cleavage.

FIGURE 3.

Bioinformatical analysis of potential Taspase1 targets. a, predicted cellular localization. Target proteins (gray, see Table 1 for details) are depicted in their respective cellular compartment according to their gene ontology term classification using the GeneSpring GX “Pathway and Network Analysis” tool. Cell membrane (yellow) with pores (blue), cytoplasm (light blue) containing endoplasmic reticulum (brown), mitochondria (violet), and the nucleoplasm (green) surrounded by the nuclear membrane (light green) are represented schematically. b, length distribution of potential Taspase1 targets versus proteins randomly found in a partially scrambled motif search. Results are obtained from database searches employing the sequence Q³(F/I/L/V)²D¹↓G¹′X²′D³′D⁴′/X³′D⁴′/D³′X⁴′ (Taspase1 targets) versus the partially scrambled motif Q³(F/I/L/V)²G¹D¹′X²′D³′D⁴′/X³′D⁴′/D³′X⁴′ (Taspase1 NONtargets).

FIGURE 4.

Verification of in silico predicted Taspase1 targets. a, HeLa cells were transfected with biosensors harboring full-length TF2A (BioTasp_TF) or USF2 (BioTasp_USF2) or the predicted cleavage site from myosin1F (BioTasp_Myo) or NXF5 (BioTasp_NXF5) together with the Tasp-mCherry expression plasmid. 24 h later, trans-cleavage resulting in nuclear translocation of the biosensors was analyzed in at least 200 fluorescent living cells. Representative examples are shown (upper panel). Coexpression of inactive TaspTV-mCherry did not affect the cytoplasmic localization of the biosensors (lower panel). Scale bars, 10 μm. b and c, proteolytic processing of full-length TF2A-GFP (b) or USF2-GFP (c) upon coexpression of Taspase1 but not of the inactive TaspTV mutant demonstrated by immunoblot analysis of transfected 293T cell lysates. Protein expression and cleavage was visualized by α-GFP and α-Taspase1 Ab. GAPDH served as loading control.