Due to its dual role as an apoptosis inhibitor and mitotic regulator, Survivin is accepted as a nodal protein hub, with high relevance in basic cell and translational cancer biology. Particularly, our work on the impact of Survivin's nuclear export signal not only resulted in several publications but also provided the basis for innovative chemico-genetic translational interference strategies. Successfully embedded in collaborative DFG research projects, such as the RTG/GRK1739 Radiation Biology and the CRC/SFB1093 Supramolecular Chemistry on Proteins, we are currently pursuing the concept of supramolecular inhibition and innovative nanocarrier based delivery tools. In addition, recent publications revealed a so far unknown role in the DNA damage response and as such now set the stage for in-depth investigations of Survivin's role in cancer biology and therapy response. Here, we will additionally focus on Survivin’s apoptotic/mitotic switch, whether it really exists as proposed and how it can be induced. This has so far eluded experimental investigation, although it would be of utmost importance to finally understand how Survivin decides between its two (or more) biological roles.
Recently, we succeeded in the development of supramolecular inhibitors targeting the Survivin/Histone H3 binding site and the Survivin's dimer interface, partially overlapping with its nuclear export signal (NES), thus blocking the pivotal interaction with the export receptor Crm1 ( see below).
Although less is known about the molecular (patho)mechanisms of the threonine protease Taspase1, the ZMB’s outstanding expertise in protease research represents an ideal platform to study its unresolved impact on ‘Cell fate decisions in development and disease’. Employing cutting-edge live cell microscopy (in cooperation with our high-end microscopes implemented at the Imaging Center Campus Essen) and biosensor systems, we not only uncovered novel mechanisms regulating Taspase1’s structure activity relationships (SAR), but also identified previously unknown targets with cellular functions in e.g., transcriptional regulation and cell differentiation and migration. Hence, Taspase1 also represents an important molecular switch determining cell fate and in addition serves as a valuable model in protease research to unravel novel fine-tuning mechanisms with translational relevance.
In the collaborative framework of the DFG CRC/SFB1093 Supramolecular Chemistry on Proteins, we are currently pursuing the concept of supramolecular inhibition of pivotal Taspase1 protein interactions. Here, we particularly focus on its Importin-mediated nuclear transport, but also on the interplay of prerequisites for autocatalytic activation of the protease and proteolytic activity in general. Is it possible to interfere with Importin-related nucleolar association and at which steps of the protease activation process? And finally, how is substrate specificity for only one or a certain subset of Taspase1 target(s) achieved and at which developmental stages?
Recently, we succeeded in the development of supramolecular binders blocking the pivotal interaction of Taspase1's nuclear import signal (NLS) with the import receptor Importin α ( see below).
Cell delivery systems & Nanoparticles
Together with the Faculty of Chemistry and as a member of CENIDE, we develop small ligands as transporters for nucleic acids and proteins and optimize their cellular uptake mechanism. A long-term goal of this project is to achieve not only efficient cell transfection, but also cell-specific delivery by e.g., the addition of distinct targeting sequences. In addition, there has been recent success in the use of nanoparticles as carriers for insoluble or poorly soluble drugs.
As nanoparticles bind proteins in biological fluids, such as the blood system, knowledge of the biomolecule corona covering the surface of the nanoparticles is critical for the success of nano-biomedical applications, including tumor targeting and in vivo imaging. For one, we could provide the first time-resolved, standardized analysis of the protein corona formed on different types of nanomaterials. Moreover, particularly humans are increasingly exposed to nanomaterials via the inhalative and oro-gastrointestinal route, representing also major infection paths for fungal spores and enteric bacterial pathogens. Just recently we substantiated the (patho)biological consequences of microbe-nanomaterial interactions and thus propose potential nanomaterial applications as anti-biotics or anti-mycotics.
Complementing the strategies pursued together with the Faculty of Chemistry we venture on the field of cell-penetrable VHH single domain antibodies (so-called nanobodies) as nanoscale biological binders. Combining our expertise in cell delivery systems, protein modification and cellular localization signals we explore possibilities to kidnap proteins from the site of action in order to reduce their functional availability.
- Tuning Nanobodies’ bioactivity against Survivin
- Selective disruption of Survivin's protein-protein interactions
- Inhibition of the Survivin-CRM1 interaction by peptide-modified molecular tweezers
Tuning Nanobodies’ bioactivity against Survivin
Nanobodies are highly affine binders, often used to track disease-relevant proteins inside cells. However, they often fail to interfere with pathobiological functions, required for their clinical exploitation. Here, a nanobody targeting the disease-relevant apoptosis inhibitor and mitosis regulator Survivin (SuN) is utilized. Survivin’s multifaceted functions are regulated by an interplay of dynamic cellular localization, dimerization, and protein–protein interactions. However, as Survivin harbors no classical “druggable” binding pocket, one must aim at blocking extended protein surface areas. Comprehensive experimental evidence demonstrates that intracellular expression of SuN allows to track Survivin at low nanomolar concentrations but failed to inhibit its biological functions. Small angle X-ray scattering of the Survivin-SuN complex locates the proposed interaction interface between the C-terminus and the globular domain, as such not blocking any pivotal interaction. By clicking multiple SuN to ultrasmall (2 nm) gold nanoparticles (SuN-N), not only intracellular uptake is enabled, but additionally, Survivin crosslinking and interference with mitotic progression in living cells are also enabled. In sum, it is demonstrated that coupling of nanobodies to nanosized scaffolds can be universally applicable to improve their function and therapeutic applicability.
Stahl P, Kollenda S, Sager J, Schmidt L, Schroer M, Stauber RH, Epple M, Knauer SK. (2023). Tuning nanobodies' bioactivity: Coupling to ultrasmall gold nanoparticles allows the intracellular interference with Survivin.Small, 2300871, doi: 10.1002/smll.202300871.
Selective disruption of Survivin's protein-protein interactions
Targeting specific protein binding sites to interfere with protein-protein interactions (PPIs) is crucial for the rational modulation of biologically relevant processes. Survivin, which is highly overexpressed in most cancer cells and considered to be a key player of carcinogenesis, features two functionally relevant binding sites. Here, based on structural analysis and computational modelling, we identified two ligands (LNES and LHIS) that selectively inhibit different biologically relevant PPIs of Survivin to partner proteins (Histone H3 and the nuclear export receptor Crm1). Accurate binding free energy calculations with CL-FEP confirm a preferential binding of LNES to Survivin's NES compared to LHIS, which is in perfect agreement with experimental results. Accordingly, the binding free energy calculations also hind towards a stronger binding of LHIS to Survivin's Histone H3 binding site compared to LNES. The ability of the ligands to distinguish between the Histone H3 and Crm1 binding site leading to a selective inhibition of only one PPI was shown via proximity ligation assay (PLA). Using a biosensor assay we proved that LNES is the first known supramolecular ligand able to reduce Survivin’s nuclear export activity in a cellular context. We show that LHIS and LNES are both able to inhibit two cancer-relevant PPIs of Survivin, with a consequently reduced cell proliferation in different cancer cell lines.
Aschmann D, Vallet C, Tripathi SK, Ruiz-Blanco YB, Brabender M, Schmuck C, Sánchez-Garcia E,* Knauer SK,* Giese M* (2022). A supramolecular approach based on guanidiniocarbonylpyrrole ligands allows selective disruption of Survivin’s protein-protein interactions. ChemBioChem, doi.org/10.1002/cbic.202100618. *corresponding authors
Inhibition of the Survivin-CRM1 interaction by peptide-modified molecular tweezers
Survivin’s dual function as apoptosis inhibitor and regulator of cell proliferation is mediated via its interaction with the export receptor CRM1. This protein-protein interaction represents an attractive target in cancer research and therapy. Here, we report a sophisticated strategy addressing Survivin’s nuclear export signal (NES), the binding site of CRM1, with advanced supramolecular tweezers for lysine and arginine. These were covalently connected to small peptides resembling the natural, self-complementary dimer interface which largely overlaps with the NES. Several biochemical methods demonstrated sequence-selective NES recognition and interference with the critical receptor interaction. These data were strongly supported by molecular dynamics simulations and multiscale computational studies. Rational design of lysine tweezers equipped with a peptidic recognition element thus allowed us to address a previously unapproachable protein surface area. As an experimental proof-of-principle for specific transport signal interference, this concept should be transferable to any protein epitope with a flanking well-accessible lysine.
Meiners A, Bäcker S, Hadrovic I, Heid C, Beuck C, Ruiz-Blanco Y.B, Mieres-Perez J, Pörschke M, Grad J.-N, Vallet C, Hoffmann D, Bayer P, Sanchez-Garcia E,* Schrader T,* Knauer SK* (2020). Specific inhibition of the Survivin-CRM1 interaction by peptide-modified molecular tweezers. *corresponding authors, Nat Commun 12:1505, doi: 10.1038/s41467-021-21753-9.
Taspase1 facilitates Topoisomerase IIß-mediated DNA 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 IIβ-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 multi- functional co-activator of estrogen-stimulated transcription.
Oelschläger L, Stahl P, Kaschani F, Stauber RH, Knauer SK, Hensel A. (2023). Taspase1 facilitates Topoisomerase IIß-mediated DNA breaks driving estrogen-induced transcription.Cells 2023, 12(3), 363, doi: 10.3390/cells12030363.
Recognition of a flexible protein loop in Taspase1 by multivalent supramolecular tweezers
Many natural proteins contain flexible loops utilizing well-defined complementary surface regions of their interacting partners and usually undergo major structural rearrangements to allow perfect binding. The molecular recognition of such flexible structures is still highly challenging due to the inherent conformational dynamics. Notably, protein-protein interactions are on the other hand characterized by a multivalent display of complementary binding partners to enhance molecular affinity and specificity. Imitating this natural concept, we here report the rational design of advanced multivalent supramolecular tweezers that allow to address two lysine and arginine clusters on a flexible protein surface loop. The protease Taspase 1, which is involved in cancer development, carries a basic bipartite nuclear localization signal (NLS) and thus interacts with Importin α, a prerequisite for proteolytic activation. Newly established synthesis routes enabled us to covalently fuse several tweezer molecules into multivalent NLS ligands. The resulting bi- up to pentavalent constructs were then systematically compared in comprehensive biochemical assays. In this series, the stepwise increase in valency was robustly reflected by the ligands’ gradually enhanced potency to disrupt the interaction of Taspase 1 with Importin α, correlated with both higher binding affinity and inhibition of proteolytic activity.
Finally, we can thus state: “The more the better”: Multivalent, kraken-like supramolecular tweezers are able to recognize the flexible protein loop in the oncologically relevant protease Taspase 1 and inhibit its interaction with Importin α as well as the proteolytic activity of the adjacent active site. Created with BioRender.com.
Höing A, Kirupakaran A, Beuck C, Pörschke M, Niemeyer FC, Seiler T, Hartmann L, Bayer P, Schrader T, Knauer SK (2022). Recognition of a flexible protein loop in Taspase 1 by multivalent supramolecular tweezers. Biomacromolecules 23(11): 4504-18, doi: 10.1021/acs.biomac.2c00652.
The Taspase1/Myosin1f-axis regulates filopodia dynamics
The unique threonine protease Tasp1 impacts not only ordered development and cell proliferation but also pathologies. However, its substrates and the underlying molecular mechanisms remain poorly understood. We demonstrate that the unconventional Myo1f is a Tasp1 substrate and unravel the physiological relevance of this proteolysis. We classify Myo1f as a nucleo-cytoplasmic shuttle protein, allowing its unhindered processing by nuclear Tasp1 and an association with chromatin. Moreover, we show that Myo1f induces filopodia resulting in increased cellular adhesion and migration. Importantly, filopodia formation was antagonized by Tasp1-mediated proteolysis, supported by an inverse correlation between Myo1f concentration and Tasp1 expression level. The Tasp1/Myo1f-axis might be relevant in human hematopoiesis as reduced Tasp1 expression coincided with increased Myo1f concentrations and filopodia in macrophages compared to monocytes and vice versa. In sum, we discovered Tasp1-mediated proteolysis of Myo1f as a fine-tuning mechanism to regulate cellular Myo1f concentration and activity post-transcriptionally, thereby dynamically modulating filopodia density and length, e.g., essential during differentiation of innate immune cells.
Hensel A,* Stahl P, Moews L, König L, Höing A, Schulze N, Nalbant P, Stauber RH, Knauer SK* (2022). Taspase1 cleavage regulates Myosin1f-induced filopodia. iScience 25(6), doi:10.1016/ j.isci.2022.104355. *corresponding authors