Taspase1 Facilitates Topoisomerase IIß-Mediated DNA Double-Strand Breaks Driving Estrogen-Induced Transcription.

The human protease Taspase1 plays a pivotal role in developmental processes and cancerous diseases by processing critical regulators, such as the leukemia proto-oncoprotein MLL. Despite almost two decades of intense research, Taspase1's biology is, however, still poorly understood, and so far its cellular function was not assigned to a superordinate biological pathway or a specific signaling cascade. Our data, gained by methods such as co-immunoprecipitation, LC-MS/MS and Topoisomerase II DNA cleavage assays, now functionally link Taspase1 and hormone-induced, Topoisomerase IIß-mediated transient DNA double-strand breaks, leading to active transcription. The specific interaction with Topoisomerase IIα enhances the formation of DNA double-strand breaks that are a key prerequisite for stimulus-driven gene transcription. Moreover, Taspase1 alters the H3K4 epigenetic signature upon estrogen-stimulation by cleaving the chromatin-modifying enzyme MLL. As estrogen-driven transcription and MLL-derived epigenetic labelling are reduced upon Taspase1 siRNA-mediated knockdown, we finally characterize Taspase1 as a multifunctional co-activator of estrogen-stimulated transcription.

A Bivalent Supramolecular GCP Ligand Enables Blocking of the Taspase1/Importin α Interaction.

Taspase1 is a unique protease not only pivotal for embryonic development but also implicated in leukemia as well as solid tumors. As such, it is a promising target in cancer therapy, although only a limited number of Taspase1 inhibitors lacking general applicability are currently available. Here we present a bivalent guanidiniocarbonyl-pyrrole (GCP)-containing supramolecular ligand that is capable of disrupting the essential interaction between Taspase1 and its cognate import receptor Importin α in a concentration-dependent manner in vitro with an IC50 of 35 µM. Here, size of the bivalent vs the monovalent construct as well as its derivation with an aromatic cbz-group arose as critical determinants for efficient interference of 2GC. This was also evident when we investigated the effects in different tumor cell lines, resulting in comparable EC50 values (∼40-70 µM). Of note, in higher concentrations, 2GC also interfered with Taspase1's proteolytic activity. We thus believe to set the stage for a novel class of Taspase1 inhibitors targeting a pivotal protein-protein interaction prerequisite for its cancer-associated proteolytic function.

The other side of the corona: nanoparticles inhibit the protease taspase1 in a size-dependent manner.

When nanoparticles enter a physiological environment, they rapidly adsorb biomolecules, in particular cellular proteins. This biological coating, the so-called nanoparticle protein corona, undoubtedly affects the biological identity and potential cytotoxicity of the nanomaterial. To elucidate a possible impact on the adsorbed biomolecules, we focused on an important group of players in cellular homeostasis, namely proteolytic enzymes. We could demonstrate that amorphous silica nanoparticles are not only able to bind to the oncologically relevant threonine protease Taspase1 as revealed by microscale thermophoresis and fluorescence anisotropy measurements, but moreover inhibit its proteolytic activity in a non-competitive manner. As revealed by temperature-dependent unfolding and CD spectroscopy, binding did not alter the stability of Taspase1 or its secondary structure. Noteworthy, inhibition of protein function seems not a general feature of nanoparticles, as several control enzymes were not affected in their proteolytic activity. Our data suggests that nanoparticles bind Taspase1 as an αß-dimer in a single layer without conformational change, resulting in noncompetitive inhibition that is either allostery-like or occludes the active site. Nanoparticle-based inhibition of Taspase1 could be also achieved in cell lysates and in live cells as shown by the use of a protease-specific cellular cleavage biosensor. Collectively, we could demonstrate that nanoparticles could not only bind but also selectively inhibit cellular enzymes, which might explain observed cytotoxicity but might serve as a starting point for the development of nanoparticle-based inhibitors as therapeutics.

Cancer-Cell-Specific Drug Delivery by a Tumor-Homing CPP-Gossypol Conjugate Employing a Tracelessly Cleavable Linker.

Tumor-targeted drug delivery is highly important for improving chemotherapy, as it reduces the dose of cytotoxic agents and minimizes the death of healthy tissues. Towards this goal, a conjugate was synthesized of gossypol and a MCF-7 cancer cell specific CPP (cell penetrating peptide), thus providing a selective drug delivery system. Utilizing the aldehyde moiety of gossypol, the tumor homing CPP RLYMRYYSPTTRRYG was attached through a semi-labile imine linker, which was cleaved in a traceless fashion under aqueous conditions and had a half-life of approximately 10 hours. The conjugate killed MCF-7 cells to a significantly greater extent than HeLa cells or healthy fibroblasts.

Cysteine-dependent ubiquitination of Pex18p is linked to cargo translocation across the peroxisomal membrane.

The peroxisomal matrix protein import is facilitated by cycling receptor molecules that shuttle between the cytosol and the peroxisomal membrane. In the yeast Saccharomyces cerevisiae, the import of proteins harboring a peroxisomal targeting signal of type II (PTS2) is mediated by the receptor Pex7p and its co-receptor Pex18p. Here we demonstrate that Pex18p undergoes two kinds of ubiquitin modifications. One of these ubiquitination events depends on lysines 13 and 20 and forces rapid Pex18p turnover by proteasomal degradation. A cysteine residue near the extreme Pex18p amino-terminus is required for the second type of ubiquitination. It turned out that this cysteine residue at position 6 is essential for the function of Pex18p in peroxisomal protein import but does not contribute to receptor-cargo association and binding to the peroxisomal import apparatus. However, in contrast to the wild-type protein, cysteine 6-mutated Pex18p is arrested in a membrane-protected state, whereas Pex7p is accessible in a protease protection assay. This finding indicates that Pex18p export is linked to cargo translocation, which supports the idea of an export-driven import of proteins into peroxisomes.

Ubp15p, a ubiquitin hydrolase associated with the peroxisomal export machinery.

Peroxisomal matrix protein import is facilitated by cycling receptors shuttling between the cytosol and the peroxisomal membrane. One crucial step in this cycle is the ATP-dependent release of the receptors from the peroxisomal membrane. This step is facilitated by the peroxisomal AAA (ATPases associated with various cellular activities) proteins Pex1p and Pex6p with ubiquitination of the receptor being the main signal for its export. Here we report that the AAA complex contains dislocase as well as deubiquitinating activity. Ubp15p, a ubiquitin hydrolase, was identified as a novel constituent of the complex. Ubp15p partially localizes to peroxisomes and is capable of cleaving off ubiquitin moieties from the type I peroxisomal targeting sequence (PTS1) receptor Pex5p. Furthermore, Ubp15p-deficient cells are characterized by a stress-related PTS1 import defect. The results merge into a picture in which removal of ubiquitin from the PTS1 receptor Pex5p is a specific event and might represent a vital step in receptor recycling.