Angelina Hahlbrock

Tuning the Surface of Nanoparticles: Impact of Poly(2-ethyl-2-oxazoline) on Protein Adsorption in Serum and Cellular Uptake.

Due to the adsorption of biomolecules, the control of the biodistribution of nanoparticles is still one of the major challenges of nanomedicine. Poly(2-ethyl-2-oxazoline) (PEtOx) for surface modification of nanoparticles is applied and both protein adsorption and cellular uptake of PEtOxylated nanoparticles versus nanoparticles coated with poly(ethylene glycol) (PEG) and non-coated positively and negatively charged nanoparticles are compared. Therefore, fluorescent poly(organosiloxane) nanoparticles of 15 nm radius are synthesized, which are used as a scaffold for surface modification in a grafting onto approach. With multi-angle dynamic light scattering, asymmetrical flow field-flow fractionation, gel electrophoresis, and liquid chromatography-mass spectrometry, it is demonstrated that protein adsorption on PEtOxylated nanoparticles is extremely low, similar as on PEGylated nanoparticles. Moreover, quantitative microscopy reveals that PEtOxylation significantly reduces the non-specific cellular uptake, particularly by macrophage-like cells. Collectively, studies demonstrate that PEtOx is a very effective alternative to PEG for stealth modification of the surface of nanoparticles.

Cleaving for growth: threonine aspartase 1—a protease relevant for development and disease

From the beginning of life, proteases are key to organismal development comprising morphogenesis, cellular differentiation, and cell growth. Regulated proteolytic activity is essential for the orchestration of multiple developmental pathways, and defects in protease activity can account for multiple disease patterns. The highly conserved protease threonine aspartase 1 is a member of such developmental proteases and critically involved in the regulation of complex processes, including segmental identity, head morphogenesis, spermatogenesis, and proliferation. Additionally, threonine aspartase 1 is overexpressed in numerous liquid as well as in solid malignancies. Although threonine aspartase 1 is able to cleave the master regulator mixed lineage leukemia protein as well as other regulatory proteins in humans, our knowledge of its detailed pathobiological function and the underlying molecular mechanisms contributing to development and disease is still incomplete. Moreover, neither effective genetic nor chemical inhibitors for this enzyme are available so far precluding the detailed dissection of the pathobiological functions of threonine aspartase 1. Here, we review the current knowledge of the structure‐function relationship of threonine aspartase 1 and its mechanistic impact on substrate‐mediated coordination of the cell cycle and development. We discuss threonine aspartase 1‐mediated effects on cellular transformation and conclude by presenting a short overview of recent interference strategies.—Stauber, R. H., Hahlbrock, A., Knauer, S. K., Wünsch, D., Cleaving for growth: threonine aspartase 1‐a protease relevant for development and disease. FASEB J. 30, 1012–1022 (2016). www.fasebj.org

Taspase1: a 'misunderstood' protease with translational cancer relevance

Proteolysis is not only a critical requirement for life, but the executing enzymes also play important roles in numerous pathological conditions, including cancer. Therefore, targeting proteases is clearly relevant for improving cancer patient care. However, to effectively control proteases, a profound knowledge of their mechanistic function as well as their regulation and downstream signalling in health and disease is required. The highly conserved protease Threonine Aspartase1 (Taspase1) is overexpressed in numerous liquid and solid malignancies and was characterized as a ‘non-oncogene addiction’ protease. Although Taspase1 was shown to cleave various regulatory proteins in humans as well as leukaemia provoking mixed lineage leukaemia fusions, our knowledge on its detailed functions and the underlying mechanisms contributing to cancer is still incomplete. Despite superficial similarity to type 2 asparaginases as well as Ntn proteases, such as the proteasome, Taspase1-related research so far gives us the picture of a unique protease exhibiting special features. Moreover, neither effective genetic nor chemical inhibitors for this enzyme are available so far, thus hampering not only to further dissect Taspase1’s pathobiological functions but also precluding the assessment of its clinical impact. Based on recent insights, we here critically review the current knowledge of Taspase1’s structure–function relationship and its mechanistic relevance for tumorigenesis obtained from in vitro and in vivo cancer models. We provide a comprehensive overview of tumour entities for which Taspase1 might be of predictive and therapeutic value, and present the respective experimental evidence. To stimulate progress in the field, a comprehensive overview of Taspase1 targeting approaches is presented, including coverage of Taspase1-related patents. We conclude by discussing future inhibition strategies and relevant challenges, which need to be resolved by the field.

Fly versus man: Evolutionary impairment of nucleolar targeting affects the degradome of Drosophila’s Taspase1

Human Taspase1 is essential for development and cancer by processing critical regulators, such as the mixed‐lineage leukemia protein. Likewise, its ortholog, trithorax, is cleaved by Drosophila Taspase1 (dTaspase1), implementing a functional coevolution. To uncover novel mechanism regulating protease function, we performed a functional analysis of dTaspase1 and its comparison to the human ortholog. dTaspase1 contains an essential nucleophile threonine195, catalyzing cis cleavage into its α‐ and β‐subunits. A cell‐based assay combined with alanine scanning mutagenesis demonstrated that the target cleavage motif for dTaspase1 (Q3[F/I/L/M]2D1↓ G1′X2′X3′) differs significantly from the human ortholog (Q3[F,I,L,V]2D1↓G1′X2′D3′D4′), predicting an enlarged degradome containing 70 substrates for Drosophila. In contrast to human Taspase1, dTaspase1 shows no discrete localization to the nucleus/nucleolus due to the lack of the importin‐α/nucleophosmin1 interaction domain (NoLS) conserved in all vertebrates. Consequently, dTaspase1 interacts with neither the Drosophila nucleoplasmin‐like protein nor human nucleophosmin1. The impact of localization on the proteases degradome was confirmed by demonstrating that dTaspase1 did not efficiently process nuclear substrates, such as upstream stimulatory factor 2. However, genetic introduction of the NoLS into dTaspase1 restored its nucleolar localization, nucleophosmin1 interaction, and efficient cleavage of nuclear substrates. We report that evolutionary functional divergence separating vertebrates from invertebrates can be achieved for proteases by a transport/localization‐regulated mechanism.—Wünsch, D., Hahlbrock, A., Heiselmayer, C., Bäcker, S., Heun, P., Goesswein, D., Stöcker, W., Schirmeister, T., Schneider, G., Krämer, O. H., Knauer, S. K., Stauber, R. H. Fly versus man: evolutionary impairment of nucleolar targeting affects the degradome of Drosophilas Taspase1. FASEB J. 29, 1973‐1985 (2015). www.fasebj.org

Effects of paclitaxel on permanent head and neck squamous cell carcinoma cell lines and identification of anti-apoptotic caspase 9b

PurposePaclitaxel is an effective chemotherapeutic agent against various human tumors inducing apoptosis via binding to β-tubulin of microtubules and arresting cells mainly in the G2/M phase of the cell cycle. However, the underlying specific molecular mechanisms of paclitaxel on head and neck squamous cell carcinoma (HNSCC) have not been identified yet.MethodsThe apoptotic effects and mechanisms of paclitaxel on different permanent HPV-negative HNSCC cell lines (UT-SCC-24A, UT-SCC-24B, UT-SCC-60A and UT-SCC-60B) were determined by flow cytometry assays, polymerase chain reaction analysis, immunofluorescence-based assays and sequencing studies.ResultsPaclitaxel induced a G2/M arrest in HNSCC cell lines followed by an increased amount of apoptotic cells. Moreover, the activation of caspase 8, caspase 10 and caspase 3, and the loss of the mitochondrial outer membrane potential could be observed, whereas an activation of caspase 9 could barely be detected. The efficient activation of caspase 9 was not affected by altered methylation patterns. Our results can show that the promoter region of apoptotic protease activating factor 1 (Apaf-1) was not methylated in the HNSCC cell lines. By sequencing analysis two isoforms of caspase 9, the pro-apoptotic caspase 9 and the anti-apoptotic caspase 9b were identified. The anti-apoptotic caspase 9b is missing the catalytic site and acts as an endogenous inhibitor of apoptosis by blocking the binding of caspase 9 to Apaf-1 to form the apoptosome.ConclusionOur data indicate the presence of anti-apoptotic caspase 9b in HNSCC, which may serve as a promising target to increase chemotherapeutic apoptosis induction.

Disease-relevant signalling-pathways in head and neck cancer: Taspase1’s proteolytic activity fine-tunes TFIIA function

Head and neck cancer (HNC) is the seventh most common malignancy in the world and its prevailing form, the head and neck squamous cell carcinoma (HNSCC), is characterized as aggressive and invasive cancer type. The transcription factor II A (TFIIA), initially described as general regulator of RNA polymerase II-dependent transcription, is part of complex transcriptional networks also controlling mammalian head morphogenesis. Posttranslational cleavage of the TFIIA precursor by the oncologically relevant protease Taspase1 is crucial in this process. In contrast, the relevance of Taspase1-mediated TFIIA cleavage during oncogenesis of HNSCC is not characterized yet. Here, we performed genome-wide expression profiling of HNSCC which revealed significant downregulation of the TFIIA downstream target CDKN2A. To identify potential regulatory mechanisms of TFIIA on cellular level, we characterized nuclear-cytoplasmic transport and Taspase1-mediated cleavage of TFIIA variants. Unexpectedly, we identified an evolutionary conserved nuclear export signal (NES) counteracting nuclear localization and thus, transcriptional activity of TFIIA. Notably, proteolytic processing of TFIIA by Taspase1 was found to mask the NES, thereby promoting nuclear localization and transcriptional activation of TFIIA target genes, such as CDKN2A. Collectively, we here describe a hitherto unknown mechanism how cellular localization and Taspase1 cleavage fine-tunes transcriptional activity of TFIIA in HNSCC.

Nanoparticle decoration impacts airborne fungal pathobiology

Significance In this work, we demonstrate that nanoparticles rapidly assemble on spores under physiologically and ecologically relevant conditions. We provide in vitro and in vivo evidence that nanoparticle coating of the clinically most relevant airborne fungal pathogen, Aspergillus fumigatus, can affect the pathobiological identity and fate of both fungal spores and nanoparticles. Our findings suggest that nanoparticle coating of bioaerosols may be relevant for ecology and human health. Airborne fungal pathogens, predominantly Aspergillus fumigatus, can cause severe respiratory tract diseases. Here we show that in environments, fungal spores can already be decorated with nanoparticles. Using representative controlled nanoparticle models, we demonstrate that various nanoparticles, but not microparticles, rapidly and stably associate with spores, without specific functionalization. Nanoparticle-spore complex formation was enhanced by small nanoparticle size rather than by material, charge, or “stealth” modifications and was concentration-dependently reduced by the formation of environmental or physiological biomolecule coronas. Assembly of nanoparticle-spore surface hybrid structures affected their pathobiology, including reduced sensitivity against defensins, uptake into phagocytes, lung cell toxicity, and TLR/cytokine-mediated inflammatory responses. Following infection of mice, nanoparticle-spore complexes were detectable in the lung and less efficiently eliminated by the pulmonary immune defense, thereby enhancing A. fumigatus infections in immunocompromised animals. Collectively, self-assembly of nanoparticle-fungal complexes affects their (patho)biological identity, which may impact human health and ecology.

TFIIA transcriptional activity is controlled by a ‘cleave-and-run’ Exportin-1/Taspase 1-switch

Transcription factor TFIIA is controlled by complex regulatory networks including proteolysis by the protease Taspase 1, though the full impact of cleavage remains elusive. Here, we demonstrate that in contrast to the general assumption, de novo produced TFIIA is rapidly confined to the cytoplasm via an evolutionary conserved nuclear export signal (NES, amino acids 21VINDVRDIFL30), interacting with the nuclear export receptor Exportin-1/chromosomal region maintenance 1 (Crm1). Chemical export inhibition or genetic inactivation of the NES not only promotes TFIIAs nuclear localization but also affects its transcriptional activity. Notably, Taspase 1 processing promotes TFIIAs nuclear accumulation by NES masking, and modulates its transcriptional activity. Moreover, TFIIA complex formation with the TATA box binding protein (TBP) is cooperatively enhanced by inhibition of proteolysis and nuclear export, leading to an increase of the cell cycle inhibitor p16INK, which is counteracted by prevention of TBP binding. We here identified a novel mechanism how proteolysis and nuclear transport cooperatively fine-tune transcriptional programs.

Protein Translocation Assays to Probe Protease Function and Screen for Inhibitors

In this chapter, you will learn how to use translocation biosensors to investigate protease functions in living cells. We here present modular protein translocation biosensors tailored to investigate protease activity and protein-protein interactions. Besides the mapping of protease function, the biosensors are also applicable to identify chemicals and/or (nano)materials modulating the respective protein activities and can also be exploited for RNAi-mediated genetic screens.

Threonine Aspartase1: An unexplored protease with relevance for oral oncology?

Proteases are involved in numerous pathobiological processes in oral cancer. Due to disappointing results with metalloproteinase inhibitors new protease targets need to be explored. The protease Threonine Aspartase1 (Taspase1) is critically involved in tumour promotion and, interestingly, has first been described to be overexpressed in head and neck squamous cell carcinoma (HNSCC). However, our knowledge on how the protease contributes to oral malignancies is still incomplete. In addition, no effective inhibitors for this enzyme are available so far, hampering the assessment of its clinical impact. However, the fact that in contrast to other proteases no additional homologs are predicted in the human genome, Taspase1 represents a promising and yet unexploited target for oral cancer. Here, we introduce Taspase1’s current knowledge to stimulate the field, discussing not only the enzyme’s cancer relevance but also potential future inhibition strategies. Oral cancer (OC), including cancers of the head and neck, are among the most common malignant neoplasm in humans [1]. OCs are epithelial cancer arising in the oral cavity, oropharynx, larynx, or hypopharynx (Fig. 1) [2]. The majorities of OC cases are induced byexposure to an excess of carcinogensand/or seemassociatedwith oncogenic human papillomaviruses [3,4]. Albeit the use of kinase inhibitors or antibodies has gained increasing clinical relevance [1], the additional targeting of proteases is actively discussed [5–7]. More than 500 different proteases are encoded in the human genome with multiple physiological roles (Suppl. Fig. 1) [8,9]. In cancer, proteases fulfill diverse functions including angiogenesis, modulation of signalling pathways, and metastasis [10]. Notably, it is estimated that 5–10% of all pharmaceutical targets being pursued for drug development are proteases [11]. Initially, the main focus was on metalloproteinases from the matrix metalloproteinases (MMP) family. Also in OC, an increased expression of multiple MMPs prompted the development of MMP inhibitors (MMPIs) as cancer therapeutics [12,13]. However, the results of MMPIs in clinical trials were disappointing [12,14].