In biological fluids, proteins associate with nanoparticles, leading to a protein "corona" defining the biological identity of the particle. However, a comprehensive knowledge of particle-guided protein fingerprints and their dependence on nanomaterial properties is incomplete. We studied the long-lived ("hard") blood plasma derived corona on monodispersed amorphous silica nanoparticles differing in size (20, 30, and 100 nm). Employing label-free liquid chromatography mass spectrometry, one- and two-dimensional gel electrophoresis, and immunoblotting the composition of the protein corona was analyzed not only qualitatively but also quantitatively. Detected proteins were bioinformatically classified according to their physicochemical and biological properties. Binding of the 125 identified proteins did not simply reflect their relative abundance in the plasma but revealed an enrichment of specific lipoproteins as well as proteins involved in coagulation and the complement pathway. In contrast, immunoglobulins and acute phase response proteins displayed a lower affinity for the particles. Protein decoration of the negatively charged particles did not correlate with protein size or charge, demonstrating that electrostatic effects alone are not the major driving force regulating the nanoparticle-protein interaction. Remarkably, even differences in particle size of only 10 nm significantly determined the nanoparticle corona, although no clear correlation with particle surface volume, protein size, or charge was evident. Particle size quantitatively influenced the particle's decoration with 37% of all identified proteins, including (patho)biologically relevant candidates. We demonstrate the complexity of the plasma corona and its still unresolved physicochemical regulation, which need to be considered in nanobioscience in the future.

The chromosomal passenger complex (CPC) of Aurora-B, Borealin, INCENP (inner centromere protein) and Survivin coordinates essential chromosomal and cytoskeletal events during mitosis. Here, we show that the nuclear export receptor Crm1 is crucially involved in tethering the CPC to the centromere by interacting with a leucine-rich nuclear export signal (NES), evolutionarily conserved in all mammalian Survivin proteins. We show that inhibition of the Survivin-Crm1 interaction by treatment with leptomycin B or by RNA-interference-mediated Crm1 depletion prevents centromeric targeting of Survivin. The genetic inactivation of the Survivin-Crm1 interaction by mutation of the NES affects the correct localization and function of Survivin and the CPC during mitosis. By contrast, CPC assembly does not seem to require the Survivin-Crm1 interaction. Our report shows the functional significance of the Survivin-Crm1 interface and provides a novel link between the mitotic effector Crm1 and the CPC.

Survivin functions as an apoptosis inhibitor and a regulator of cell division in many tumours. The intracellular localization of survivin in tumours has been suggested as a prognostic marker. However, current reports are inconsistent and the underlying molecular mechanisms are not understood. The present study has examined the localization and prognostic value of nuclear and cytoplasmic survivin in the pre-therapeutic biopsies from 71 oral and oropharyngeal squamous carcinoma (OSCC) patients. Statistical analysis indicated that preferential nuclear versus cytoplasmic survivin correlated with favourable versus unfavourable disease outcome. Uni- and multi-variate analysis showed that in contrast to total survivin expression, the difference between nuclear and cytoplasmic survivin was a strong predictor for relapse-free survival (p=0.0003). As a potential underlying molecular mechanism, it is shown in OSCC cell lines that predominantly cytoplasmic survivin mediates protection against chemo- and radio-therapy-induced apoptosis. Importantly, the cytoplasmic localization of survivin is regulated by its nuclear export signal (NES), and export-deficient nuclear survivin is not cytoprotective. This study suggests that the difference between cytoplasmic and nuclear survivin is an indicator for survivin activity in tumour cells. Thus, this difference may serve as a predictive marker of outcome in OSCC patients undergoing multi-modality therapy. The pharmacogenetic interference with survivin's cytoplasmic localization is also to be pursued as a potential therapeutic strategy.

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,, 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.By cleaving proteins, proteases are involved in the control of a large number of key physiological processes such as development, metabolism, tissue remodeling, cell proliferation, and apoptosis (1-3). Protease signaling therefore differs from the majority of other signaling pathways by being mostly irreversible (3). Protease signaling is strictly regulated, and the deregulation of protease activity can contribute to various pathologies, including cancer (3).The human Taspase1 gene encodes a protein of 420 amino acids (aa), 4 which is the proenzyme of Taspase1. It belongs to a family of enzymes possessing an asparaginase-2 homology domain. In the MEROPS database, Taspase1 is found as T02.004, classifying this protein as a class PB, subclass PB(T), and T2 family protease. In contrast to the other cis-active type 2 asparaginases, such as amidohydrolases, L-asparaginase, and glycosylasparaginase, only Taspase1 is able to cleave other substrates in trans (4). Therefore, Taspase1 represents a distinct class of proteolytic enzymes. Taspase1-mediated cleavage of proteins follows distinct aspartate residues, suggesting that Taspase1 evolved from hydrolyzing asparagines and glycosylasparagines to recognize a conserved peptide motif with an aspartate at the P1 ...

Taspase1 is a threonine protease suspected to process (patho)biologically relevant nuclear and cytoplasmic substrates, such as the mixed lineage leukemia protein. However, neither the mechanisms regulating Taspase1's intracellular localization nor their functional consequences are known. Analysis of endogenous and ectopically expressed Taspase1 detected the protease predominantly in the nucleus accumulating at the nucleolus. Microinjection and ectopic expression studies identified an evolutionarily conserved bipartite nuclear import signal (NLS) (amino acids 197 K RNKRK LELA ERVDTDFMQLK KRR 220 ) interacting with importin-α. Notably, an NLS-mutated, import-deficient Taspase1 was biologically inactive. Although the NLS conferred nuclear transport already of the proenzyme, Taspase1's nucleolar localization required its autoproteolytic processing, triggering its interaction with the nucleolar shuttle protein nucleophosmin. In contrast, (auto)catalytically inactive Taspase1 mutants neither accumulated at the nucleolus nor bound nucleophosmin. Active nuclear import and interaction with nucleophosmin was found to be required for the formation of proteolytically active Taspase1 ensuring to efficiently process its nuclear targets. Intriguingly, coexpression of pathological nucleophosmin variants increased the amount of cytoplasmic Taspase1. Hence, Taspase1 appears to exploit the nuclear export activity of nucleophosmin to gain transient access to the cytoplasm required to also cleave its cytoplasmic substrates. Collectively, we here describe a hitherto unknown mechanism regulating the biological activity of this protease.

CRC). 18 For treatment specification see the respective references. The respective study protocols were approved by the local ethics committee after obtaining the patients informed consent and samples were processed anonymously. All cases were diagnosed histopathologically, and staged according to the TNM or disease specific (ALL) classification. Tissue specimens or cells from low-density bone marrow samples 16 were stored in liquid nitrogen until extraction of mRNA.RNA extraction and RT-qPCR analysis. Total RNA was purified, and changes in mRNA levels were compared by reverse transcription (RT) and quantitative real-time PCR (qPCR) analysis as described. 19 Primers (Invitrogen) were: 5'-GACGACCCCATAG AGGAACA-3' (sense) and 5'-CCATGGCAGCCAGCTGCTCG -3' (antisense) for wt survivin; 5'-GACCACCGCATCTCTACAT TC-3' (sense) and 5'-AAGTGCTGGTATTACAGGCGT-3' (antisense) for survivin -2B ; 5'-GAGGCTGGCTTCATCCACTG-3' (sense) and 5'-GCTCTCTCAATTTTGTTCTTG-3' (antisense) for survivin -3B ; 5'-GACCACCGCATCTCTACATTC-3' (sense) and 5'-TGTTGGTTTCCTTTGCATGG-3' (antisense) for survivin -DEx3 ; 5'-GATGACATCAAGAAGGTGGTG-3' (sense) and 5'-GCTGTA GCCAAATTCGTTGTC-3'(antisense) for GAPDH. SYBR green was used as fluorescent dye.

The dual role of the inducible NO synthase (iNOS) and NO signaling in head and neck squamous cell carcinoma (HNSCC) is a complex and can both promote or inhibit tumor progression. However, the underlying molecular mechanisms are not yet resolved in detail. We show for the first time that conditions, favoring low NO levels conferred resistance against cisplatin/taxol-induced apoptosis in HNSCC cell lines. Cytoprotection was mediated by survivin, because we observed its upregulation subsequent to low doses of the NO donors S-nitroso-N-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) or ectopic expression of physiologic amounts of iNOS. Also, RNAi-mediated depletion of survivin blocked NOs anti-apoptotic effects. Induction of survivin involves activation of the phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway, which was antagonized by the PI3K-inhibitor LY294002. Importantly, application of the iNOS-specific inhibitor 1400W combined with RNAi-mediated downregulation of survivin cooperatively enhanced drug-induced cell death. The iNOS/survivin-axis appears to be also of clinical relevance since immunohistochemistry revealed that iNOS expression correlated with enhanced survivin levels in HNSCC specimens. In contrast, high NO concentrations suppressed survivin levels in HNSCC but also in non-malignant cells resulting in apoptosis. Cell death induced by high amounts of SNAP/SNP or by strong overexpression of iNOS involved activation of p38MAP-kinase, which was counteracted by the p38MAP-kinase inhibitor SB202190. Here, we provide evidence for a novel molecular mechanism how NO signaling may contribute to therapy resistance in HNSCC by modulating survivin expression. Our data further suggest pursuing pharmacogenetic iNOS/survivin-targeting approaches as potential therapeutic strategies in head and neck cancer.

Survivin appears to function as a regulator of cell division and as an apoptosis inhibitor in many species. Here, we characterized the nucleocytoplasmic transport of mouse survivin 140 , and its splice variants survivin 121 and survivin 40 . We show that the dynamic intracellular localization of survivin 140 is mediated by a Crm1-dependent nuclear export signal (NES) present also in survivin 121 , but absent in survivin 40 . In contrast, neither survivin nor survivin splice variants contain an active nuclear import signal and seem to enter the nucleus by passive diffusion. The activity of the NES is required for survivin-mediated protection against cell death inducing stimuli and influences protein degradation. During mitosis, NES-deficient survivin variants fail to correctly localize to the mitotic machinery and promote proper cell division. In vivo and in vitro protein interaction assays show that survivin 140 and survivin 121 as well as their export-deficient mutants are able to form homo-as well as heterodimers. The transdominant negative phenotype observed upon expression of export-deficient survivin appears, therefore, to be mediated by the formation of inactive survivin heterodimers. The survivin-Crm1 axis is essential for the biological activities of murine survivin, and mouse models will allow investigating its functional implications during development and tumorigenesis.