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Diagnostic testing is essential for management of the COVID-19 pandemic. An agile assay design methodology, optimized for the cobas® 6800/8800 system, was used to develop a dual-target, qualitative SARS-CoV-2 RT-PCR test using commercially available reagents and existing sample processing and thermocycling profiles. The limit of detection was 0.004 to 0.007 TCID50/mL for USA-WA1/2020. Assay sensitivity was confirmed for SARS-CoV-2 variants Alpha, Beta, Gamma, Delta and Kappa. The coefficients of variation of the cycle threshold number (Ct) were between 1.1 and 2.2%. There was no difference in Ct using nasopharyngeal compared to oropharyngeal swabs in universal transport medium (UTM). A small increase in Ct was observed with specimens collected in cobas® PCR medium compared to UTM. In silico analysis indicated that the dual-target test is capable of detecting all >1,800,000 SARS-CoV-2 sequences in the GISAID database. Our agile assay design approach facilitated rapid development and deployment of this SARS-CoV-2 RT-PCR test.

Diagnostic testing is essential for management of the COVID-19 pandemic. An agile assay design methodology, optimized for the cobas® 6800/8800 system, was used to develop a dual-target, qualitative SARS-CoV-2 RT-PCR test using commercially available reagents and existing sample processing and thermocycling profiles. The limit of detection was 30-52 copies/mL for USA-WA1/2020. Assay sensitivity was confirmed for SARS-CoV-2 variants Alpha, Beta, Gamma, Delta and Kappa. The coefficients of variation of the cycle threshold number (Ct) were between 1.1 and 2.2%. There was no difference in Ct using nasopharyngeal compared to oropharyngeal swabs in universal transport medium (UTM). A small increase in Ct was observed with specimens collected in cobas PCR medium compared to UTM. In silico analysis indicated that the dual-target test is capable of detecting all1,800,000 SARS-CoV-2 sequences in the GISAID database. Our agile assay design approach facilitated rapid development and deployment of this SARS-CoV-2 RT-PCR test.

Trichomoniasis, a common curable sexually transmitted infection caused by the protozoan Trichomonas vaginalis (TV), is usually asymptomatic. However, symptomatic women may experience vaginal discharge and/or vulvar irritation. This study evaluated cobas® TV/ Mycoplasma genitalium (MG) (Conformité Européene marking for in vitro diagnostic medical devices [CE-IVD]) against other nucleic acid amplification tests (NAATs) for detecting TV in female urogenital specimens. Matched de-identified specimens from 412 females were collected. cobas® TV/MG results were compared against a composite reference (CR) of 3 different NAATs for TV (Aptima TV, modified S-DiaMGTV™, and a laboratory-developed test). The overall TV prevalence rate was 6.2%, based on cobas® TV/MG results. Relative to the CR, cobas® TV/MG sensitivity/specificity for the specimen types were endocervical swabs (ES) 100%/99.2%, vaginal swabs (VS) 100%/99.7%, urine (U) 100%/99.7%, and cervical specimens in PreservCyt® solution (PC) 100%/99.5%. There was no significant statistical difference between clinician-collected and self-collected VS (p = 0.28). Correlation of cobas® TV/MG vs. Aptima TV demonstrated the following positive, negative, and overall percent agreements, respectively: ES 69.0%, 98.7%, and 96.6%; VS 88.9%, 99.5%, and 98.8%; U 100%, 100%, and 100%; and PC 95.5%, 99.0%, and 98.8%. Detection of TV with cobas® TV/MG for use on the cobas® 6800/8800 systems demonstrated excellent performance in female urogenital specimens (overall sensitivity/specificity of 100%≥99.2%).

BackgroundSARS-CoV-2 causes COVID-19, which can be fatal and is responsible for a global pandemic. Variants with increased transmissibility or the potential to evade immunity have emerged and represent a threat to global pandemic control. Variants of concern (VOC) can be identified by sequencing of viral RNA, or by more rapid methods for detection of subsets of signature mutations.MethodsWe developed a multiplex, real-time RT-PCR assay (cobas®SARS-CoV-2 Variant Set 1) for the qualitative detection and differentiation of three key SARS-CoV-2 mutations in the viral spike protein: del 69-70, E484K and N501Y. Analytical sensitivity and accuracy were evaluated at three testing sites using clinical specimens from patients infected with SARS-CoV-2 variants belonging to several different lineages, including B.1.1.7, B.1.351, and P.1.ResultsThe limit of detection for E484K was between 180 and 620 IU/mL for the three different isolates tested. For N501Y, the LOD was between 270 and 720 IU/mL (five isolates), while for del 69-70, it was 80 - 92 IU/mL (two isolates). Valid test results were obtained with all clinical specimens that were positive using routine diagnostic tests. Compared to sequencing (Sanger and next-generation), test results were 100% concordant at all three loci; no false positive or false negative results were observed.ConclusionsData collected at three independent laboratories indicates excellent performance and concordance of cobas®SARS-CoV-2 Variant Set 1 with sequencing. New sets of primers and probes that target additional loci can be rapidly deployed in response to the identification of other emerging variants.

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 TFIIA's nuclear localization but also affects its transcriptional activity. Notably, Taspase 1 processing promotes TFIIA's 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.

Survivin (BIRC5) is an acknowledged cancer therapy-resistance factor and overexpressed in head and neck squamous cell carcinomas (HNSCC). Driven by its nuclear export signal (NES), Survivin shuttles between the nucleus and the cytoplasm, and is detectable in both cellular compartments in tumor biopsies. Although predominantly nuclear Survivin is considered a favorable prognostic disease marker for HNSCC patients, the underlying molecular mechanisms are not resolved. Hence, we performed immunohistochemical and mutational analyses using laser capture microdissection on HNSCC biopsies from patients displaying high levels of nuclear Survivin. We found somatic BIRC5 mutations, c.278T>C (p.Phe93Ser), c.292C>T (p.Leu98Phe), and c.288A>G (silent), in tumor cells, but not in corresponding normal tissues. Comprehensive functional characterization of the Survivin mutants by ectopic expression and microinjection experiments revealed that p.Phe93Ser, but not p.Leu98Phe inactivated Survivin's NES, resulted in a predominantly nuclear protein, and attenuated Survivin's dual cytoprotective activity against chemoradiation-induced apoptosis. Notably, in xenotransplantation studies, HNSCC cells containing the p.Phe93Ser mutation responded significantly better to cisplatin-based chemotherapy. Collectively, our results underline the disease relevance of Survivin's nucleocytoplasmic transport, and provide first evidence that genetic inactivation of Survivin's NES may account for predominantly nuclear Survivin and increased therapy response in cancer patients.

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.

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) KRNKRKLELA ERVDTDFMQLKKRR(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.

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 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.

Roland H. Stauber 1,* , Shirley K. Knauer 2,* , Negusse Habtemichael 1 , Carolin Bier 1 , Britta Unruhe 2 , Simona Weisheit 3 , Stephanie Spange 3 , Frank Nonnenmacher 4 , Verena Fetz 1 , Torsten Ginter 3 , Sigrid Reichardt 3 , Claus Liebmann 3 , Gunter Schneider 5 , Oliver H. Kramer 3,* 1 Molecular and Cellular Oncology/Mainz Screening Center, University Hospital of Mainz, Germany 2 Institute for Molecular Biology, Centre for Medical Biotechnology (ZMB), University Duisburg-Essen, Germany 3 Institute of Biochemistry and Biophysics/Center for Molecular Biomedicine, Friedrich-Schiller-University Jena, Germany 4 Sanofi-Aventis Deutschland GmbH, Industriepark Hoechst, Germany * Denotes equal contribution Received: January 16, 2012; Accepted: January 25, 2012; Published: January 28, 2012; Keywords: BH3-only protein, chemotherapy resistance, hydroxyurea, oral cancer, tumor xenograft, valproic acid Correspondence: Oliver H. Kramer, email: // Roland H. Stauber, email: // Shirley K. Knauer, email: // // Abstract Head and neck squamous cell carcinomas (HNSCCs) are the sixth most common malignant neoplasm and more than 50% of patients succumb to this disease. HNSCCs are characterized by therapy resistance, which relies on the overexpression of anti-apoptotic proteins and on the aberrant regulation of the epidermal growth factor receptor (EGFR). As inherent and acquired resistance to therapy counteracts improvement of long-term survival, novel multi-targeting strategies triggering cancer cell death are urgently required. We investigated how induction of replicational stress by the ribonucleotide reductase inhibitor hydroxyurea (HU) combined with histone deacetylase inhibitors (HDACi) exerts anti-tumor activity. We treated HNSCC cell lines and freshly isolated tumor cells with HDACi, such as the clinically approved anti-epileptic drug valproic acid (VPA), in combination with HU. Our data demonstrate that at clinically achievable levels VPA/HU combinations efficiently block proliferation as well as clonogenic survival, and trigger apoptosis of HNSCC cells. In the presence of VPA/HU, such tumor cells increase expression of the pro-apoptotic BCL-2 family protein BIM, independent of wild-type p53 signaling and in the absence of increased expression of the p53 targets PUMA and BAX. The pro-apoptotic activity of BIM in HNSCCs was found critical for tumor cell death; ectopic overexpression of BIM induced HNSCC apoptosis and RNAi-mediated depletion of BIM protected HNSCC cells from VPA/HU. Also, significantly elevated BIM levels (p

Taspase1 mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia-provoking MLL fusions and promotes solid malignancies. Currently, no effective and specific Taspase1 inhibitors are available, precluding its therapeutic exploitation. As the Taspase1 proenzyme is autoproteolytically cleaved and assumed to assemble into an active alpha beta beta alpha heterodimer, we attempted to interfere with its activity by targeting Taspase1's dimerization. Notably, enforced expression of inactive Taspase1 mutants, aiming to inhibit formation of active protease dimers, was not inhibitory. Immunoprecipitation, gel filtration, and in vivo protein interaction assays revealed that active Taspase1 exists predominantly as an alpha beta monomer in living cells, providing an explanation why overexpression of inactive mutants was not trans-dominant. To alternatively test the biological consequences of enforced dimerization, we engineered Taspase1 variants containing the Jun/Fos dimerization motif. In absence of the respective interaction partners, the protease fusions were fully active, while enforcing dimerization by coexpression significantly inhibited processing of several target proteins in living cells. Our study provides the first evidence that Taspase1 is already active as an alpha beta monomer, arguing against heterocomplex formation being required for its pathobiological activity. Thus, it clearly supports strategies aiming to inhibit the cancer-promoting activity of Taspase1 by the identification of chemical decoys enforcing its dimerization.-Bier, C., Knauer, S. K., Wunsch, D., Kunst, L., Scheiding, S., Kaiser, M., Ottmann, C., Kramer, O. H., Stauber, R. H. Allosteric inhibition of Taspase1's pathobiological activity by enforced dimerization in vivo. FASEB J. 26, 3421-3429 (2012). www.fasebj.org

We read with interest the article by Chen and colleagues ([1][1]), identifying a potential Taspase1 inhibitor by using a cellular translocation assay similar to the one described by Bier and colleagues ([2][2]). These results impressively underline the power of cell-based screening, as previous

Signal transducer and activator of transcription 1 (STAT1) is important for innate and adaptive immunity. Histone deacetylase inhibitors (HDACi) antagonize unbalanced immune functions causing chronic inflammation and cancer. Phosphorylation and acetylation regulate STAT1 and different IFNs induce phosphorylated STAT1 homo-/heterodimers, e.g. IFNα activates several STATs whereas IFNγ only induces phosphorylated STAT1 homodimers. In transformed cells HDACi trigger STAT1 acetylation linked to dephosphorylation by the phosphatase TCP45. It is unclear whether acetylation differentially affects STAT1 activated by IFNα or IFNγ, and if cellular responses to both cytokines depend on a phosphatase-dependent inactivation of acetylated STAT1. Here, we report that HDACi counteract IFN-induced phosphorylation of a critical tyrosine residue in the STAT1 C-terminus in primary cells and hematopoietic cells. STAT1 mutants mimicking a functionally inactive DNA binding domain (DBD) reveal that the number of acetylation-mimicking sites in STAT1 determines whether STAT1 is recruited to response elements after stimulation with IFNγ. Furthermore, we show that IFNα-induced STAT1 heterodimers carrying STAT1 molecules mimicking acetylation bind cognate DNA and provide innate anti-viral immunity. IFNγ-induced acetylated STAT1 homodimers are though inactive, suggesting that heterodimerization and complex formation can rescue STAT1 lacking a functional DBD. Apparently, the type of cytokine determines how acetylation affects the nuclear entry and DNA binding of STAT1. Our data contribute to a better understanding of STAT1 regulation by acetylation.

Chromosomal rearrangements of the mixed lineage leukemia (MLL) gene with numerous partner genes are frequently found in acute myeloid (AML) and acute lymphoblastic leukemia (ALL). Although the pathomechanism of t(4;11)-mediated leukemia is still discussed, expression of the AF4•MLL fusion enhanced the repopulating potential of CD34+-cells, and led to the development of predominantly proB-ALL in a mouse model. The AF4•MLL protein contains cleavage-sites for Threonine Aspartase-1 (Taspase1). Upon processing by Taspase1, the AF4•MLL cleavage products form a protein complex resistant to SIAH-mediated degradation and activate oncogenic programs. Furthermore, Taspase1 is overexpressed in liquid and solid human cancers suggesting that Taspase1 is coopted to promote and sustain tumorigenesis. As genetic deletion of Taspase1 in the mouse produced no overt deficiencies, inhibition of Taspase1 may offer novel anticancer strategies, including the treatment of leukemias. Human Taspase1 encodes a protease of 420 amino acids (aa) cleaving substrates in trans by recognizing a conserved peptide motif (Q3[F,I,L,V]2D1↓G1’x2’D3’D4’). Unfortunately, Taspase1’s activity is not affected by common protease inhibitors, currently precluding the assessment of its clinical and therapeutic relevance. Here, we present our endeavors to target Taspase1’s oncogenic potential by (i) overexpression of inactive Taspase1 variants, and (ii) testing a putative Taspase1 inhibitor.

Background The chromosomal translocation t(4;11)(q21;q23) is associated with high-risk acute lymphoblastic leukemia of infants. The resulting AF4•MLL oncoprotein becomes activated by Taspase1 hydrolysis and is considered to promote oncogenic transcriptional activation. Hence, Taspase1’s proteolytic activity is a critical step in AF4•MLL pathophysiology. The Taspase1 proenzyme is autoproteolytically processed in its subunits and is assumed to assemble into an αββα-heterodimer, the active protease. Therefore, we investigated here whether overexpression of catalytically inactive Taspase1 variants are able to interfere with the proteolytic activity of the wild type enzyme in AF4•MLL model systems. Methodology/Findings The consequences of overexpressing the catalytically dead Taspase1 mutant, Taspase1T234V, or the highly attenuated variant, Taspase1D233A, on Taspase1’s processing of AF4•MLL and of other Taspase1 targets was analyzed in living cancer cells employing an optimized cell-based assay. Notably, even a nine-fold overexpression of the respective Taspase1 mutants neither inhibited Taspase1’s cis- nor trans-cleavage activity in vivo. Likewise, enforced expression of the α- or β-subunits showed no trans-dominant effect against the ectopically or endogenously expressed enzyme. Notably, co-expression of the individual α- and β-subunits did not result in their assembly into an enzymatically active protease complex. Probing Taspase1 multimerization in living cells by a translocation-based protein interaction assay as well as by biochemical methods indicated that the inactive Taspase1 failed to assemble into stable heterocomplexes with the wild type enzyme. Conclusions Collectively, our results demonstrate that inefficient heterodimerization appears to be the mechanism by which inactive Taspase1 variants fail to inhibit wild type Taspase1’s activity in trans. Our work favours strategies targeting Taspase1’s catalytic activity rather than attempts to block the formation of active Taspase1 dimers to interfere with the pathobiological function of AF4•MLL.

Background Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available. Methodology/Findings Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high-throughput microscopy platform (Z'factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamide and 2-benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor. Conclusions The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance., PLoS ONE, 6 (5), ISSN:1932-6203

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.

Hearing impairment is a worldwide health problem. Employing semi-quantitative immunological detection methods, we found that the apoptosis inhibitor protein Birc5 is expressed in cell types critical for hearing perception. In the guinea pig model, moderate noise exposure causing only a temporary mean hearing impairment of 33dB significantly enhanced Birc5 expression in the spiral ligament, nerve fibers and the organ of Corti. In contrast, intratympanic gentamicin injection inducing permanent cell damage and mean hearing loss of 24dB correlated with a significant Birc5 downregulation in the ligament, nerve fibers and the organ of Corti. The cytoprotective activity of the guinea pig and human Birc5 protein was confirmed by cloning of the gene and by subsequent ectopic expression and challenging studies against the ototoxin gentamicin in epithelial and auditory cell models. As the mammalian cochlea is unable to regenerate upon damage, these data suggest that modulation of Birc5 expression may represent a novel physiological mechanism to protect the inner ear against stress-induced cell damage. Hence, the targeted modulation of Birc5 levels may lead to novel otoprotective therapeutic strategies.

Background The guinea pig is widely used as a model to study (patho)physiological processes, such as neurodegenerative disorders. Survivin's dual function as an apoptosis inhibitor and a mitotic regulator is crucial not only for ordered development but its modulation seems crucial also under disease conditions. However, data on the expression and function of the guinea pig Survivin protein (SurvivinGp) are currently lacking. Results Here, we here report the cloning and functional characterization of SurvivinGp. The respective cDNA was cloned from spleen mRNA, containing a 426 bp open reading frame encoding for a protein of 142aa. SurvivinGp displays a high homology to the human and murine orthologue, especially in domains critical for function, such as binding sites for chromosomal passenger complex (CPC) proteins and the nuclear export signal (NES). Notably, phylogenetic analyses revealed that SurvivinGp is more related to humans than to rodents. Ectopic expression studies of a SurvivinGp-GFP fusion confirmed its dynamic intracellular localization, analogous to the human and murine counterparts. In interphase cells, SurvivinGp-GFP was predominantly cytoplasmic and accumulated in the nucleus following export inhibition with leptomycin B (LMB). A typical CPC protein localization during mitosis was observed for SurvivinGp-GFP. Microinjection experiments together with genetic knockout demonstrated that the NES is essential for the anti-apoptotic and regulatory role of SurvivinGp during cell division. In vivo protein interaction assays further demonstrated its dimerization with human Survivin and its interaction with human CPC proteins. Importantly, RNAi-depletion studies show that SurvivinGp can fully substitute for human Survivin as an apoptosis inhibitor and a mitotic effector. Immunohistochemistry, immunofluorescence, and western blotting were employed to detect Survivin expression in guinea pig tissues. Besides its expression in proliferating tissues, such as spleen and liver, we also found Survivin in terminally differentiated cell types. Importantly, Survivin was detectable also in the cochlea, suggesting a potential role for Survivin in the auditory system. Conclusions We provide the first experimental evidence for the expression of Survivin in the guinea pig. As SurvivinGp can substitute for known functions of human Survivin, the guinea pig model will now also allow investigating Survivin's (patho)physiological role and to test Survivin-directed potential therapeutic strategies.

Survivin is described as a bifunctional protein inhibiting apoptosis and regulating mitosis. However, the biological functions and contributions to cancer progression of survivin splice variants are controversially discussed. We here show that the intracellular localization of these splice variants depends on a Crm1-dependent nuclear export signal (NES) present in survivin, surviving(-2B) and survivin(-3B), but absent in survivin(-deltaEx3) and survivin(-2alpha). Survivin isoforms lack an active nuclear import signal and are able to enter the nucleus by passive diffusion. Only survivin(-3B) but none of the other splice variants is cytoprotective and able to efficiently interact with chromosomal passenger complex (CPC) proteins. The NES together with efficient CPC formation is required for the cytoprotective activity of survivin isoforms, as well as for their correct localization and function during cell division. In the tumours from breast, colorectal, head and neck cancer, lymphoma and leukemia patients, survivin and survivin(-2B) were found overexpressed. However, survivin was the predominant form detected, and the other survivin isoforms were only expressed at low levels in tumours. Our data provide a molecular rationale for the localization and activity of survivin variants, and conclude that survivin isoforms are unlikely to modulate survivin in trans in cancer patients.

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 trans-dominant 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.

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.

Hearing impairment caused by ototoxic insults, such as noise or gentamicin is a worldwide health problem. As the molecular circuitries involved are not yet resolved, current otoprotective therapies are rather empirical than rational. Here, immunohistochemistry and western blotting showed that the cytoprotective protein survivin is expressed in the human and guinea pig cochlea. In the guinea pig model, moderate noise exposure causing only a temporary hearing impairment transiently evoked survivin expression in the spiral ligament, nerve fibers and the organ of Corti. Mechanistically, survivin upregulation may involve nitric oxide (NO)-induced Akt signaling, as enhanced expression of the endothelial NO synthase and phosphorylated Akt were detectable in some surviving-positive cell types. In contrast, intratympanic gentamicin injection inducing cell damage and permanent hearing loss correlated with attenuated survivin levels in the cochlea. Subsequently, the protective activity of the human and the guinea pig survivin orthologs against the ototoxin gentamicin was demonstrated by ectopic overexpression and RNAi-mediated depletion studies in auditory cells in vitro. These data suggest that survivin represents an innate cytoprotective resistor against stress conditions in the auditory system. The pharmacogenetic modulation of survivin may thus provide the conceptual basis for the rational design of novel therapeutic otoprotective strategies. OA gold