Funktionelle Analyse der Protease Taspase1 und ihrem Zielprotein Myosin1F

Die Bedeutung von Proteasen für die medizinische und pharmazeutische Forschung ist wegen deren Beteiligung an vielfältigen (patho)biologischen Vorgängen unumstritten. Ein prominentes Beispiel ist die Threonin Protease Taspase1, die aufgrund ihrer Beteiligung an der Entstehung und Progression von MLL-vermittelten Leukämien sowie der erhöhten Expression in soliden Tumoren biomedizinische Relevanz erlangte. Die detaillierten molekularen Mechanismen und Signalwege über die Taspase1 ihre (patho)biologische Wirkung ausübt, sind jedoch noch unverstanden. Erst kürzlich konnte Myosin1F als Substrat der Taspase1 identifiziert werden. Myosine werden als Aktin-abhängige Motorproteine beschrieben, denen vielfältige Funktionen wie z. B. in der Zellbewegung zukommen. Bei Myosin1F handelt es sich jedoch um ein nur unzureichend charakterisiertes Protein. Es gibt jedoch ebenso wie für Taspase1 erste Hinweise auf eine Relevanz bei der Krebsentstehung. Daher wurde im Rahmen dieser Arbeit die (patho)biologische Bedeutung des Taspase1-Myosin1F-Zusammenspiels sowie die Funktion des Myosin1F-Proteins untersucht. Um erste Informationen über die Genexpression zu erlangen, konnte erfolgreich eine qPCR-Methode für Taspase1 und Myosin1F etabliert werden. Diese bestätigte die erhöhte Taspase1-Expression in Tumorzelllinen und konnte ergänzend deutlich geringere Mengen Taspase1-mRNA in Immunzellen sowie die stark erhöhte Menge Taspase1-mRNA in adhärenten Zelllinien im Vergleich zu Suspensionszellen nachweisen. Der Vergleich der Myosin1F und Taspase1 mRNA-Mengen zeigte, dass die Taspase1-Expression weit über dem des Myosin1F liegt, jedoch in adhärenten Zellen mit der mRNA Expression von Myosin1F korreliert. In Immunzellen liegt hingegen ein gegenteiliger Trend vor. Folglich konnte die Genexpression von Myosin1F und Taspase1 als ein wichtiger Unterschied zwischen adhärenten und Suspensions-Tumorzellen identifiziert werden. Dies könnte sich auf Unterschiede im Migrationsverhalten der verschiedenen Zelltypen auswirken, da stark migrierende Zellen wie die des Immunsystems eine starke Migration jedoch kaum Adhäsion aufweisen. Expressionsstudien in Interphasezellen zeigten, dass Myosin1F und die nicht durch Taspase1 spaltbare Variante in Zytosol, an der Membran sowie am Zytoskelett lokalisieren. Analog dazu sind beide Proteine während der Mitose an der Zellmembran und bei Trennung der Tochterzellen zwischen diesen zu finden. Die Mutation in der Taspase1-Schnittstelle im Myosin1F resultiert ebenso wie die Erhöhung der Taspase1-Menge in einer stärker ausgeprägten zytoplasmatischen Lokalisation von Myosin1F. Im Rahmen dieser Arbeit wurde erstmals die Spaltung des Volllänge-Myosin1F-Proteins durch Taspase1 nachgewiesen und Hinweise auf die physiologische Bedeutung der Taspase1-Myosin1F-Wechselwirkung erarbeitet. Für diesen Nachweis wurde u.a. eine als „clevage-IP“ bezeichnete Methode etabliert, die im Folgenden zur Analyse spezifischer Enzym-Substrat-Spaltungen herangezogen werden kann. Interessanterweise konnte mit dieser Methodik nicht nur die dosisabhängige Enzymaktivität gezeigt werden, sondern eine getrennte Expression der Taspase1 - und -Untereinheiten resultierte in einer höheren Hydrolyseaktivität als eine entsprechende Expression des Volllänge-Proteins. Dies weist auf eine limitierende Funktion der intramolekularen cis-Spaltung hin. Weiterführend zeigte sich, dass das durch Spaltung entstehende C-terminale Myosin1F-Fragment stärker als das Volllängekonstrukt im Zytoplasma der Zelle lokalisiert. Das N-terminale Fragment akkumuliert dagegen im Zellkern. Untersuchungen des Spaltprodukts ergaben Hinweise auf ein schwaches, LMB-sensitives NES sowie die Interaktion der Spaltprodukte nach Spaltung des Myosin1F. Dies könnte den Transport des N-terminalen Fragments in den Zellkern regulieren, oder die Bildung eines Multiproteinkomplexes erlauben, wie es für das Taspase1-Substrat MLL bekannt ist. Funktionsanalysen konnten die Funktion des Myosin1F-Proteins in migratorischen Prozessen adhärenter Tumor- und Nicht-Tumorzellen nachweisen. So verstärkte eine ektope Myosin1F-Expression die Substratadhäsion, welche durch Integrin-stimulierende Substanzen beeinflussbar ist. Myosin1F kolokalisiert dabei mit Integrin und sowie Rac1 und Cdc42 und interagiert mit Paxillin, Talin und Aktin. Da es sich bei diesen Proteinen um Marker der frühen fokalen Adhäsionen handelt, scheint Myosin1F dabei eine physiologische Funktion zu spielen. Die Kolokalisation mit Rac1 und Cdc42 deutet zudem auf eine Integrin-vermittelte Filopodienadhäsion zur Stabilisierung von Matrix-Adhäsionspunkten in den Lamellipodien hin. Weiterführend wurde die Beteiligung von Myosin1F an der PI3/Akt-Signalübertragung und die Interaktion mit Akt nachgewiesen sowie die EGF-abhängige Rekrutierung von Myosin1F an die Zellmembran aufgezeigt. Dabei wird die Ausbildung fokaler Adhäsionen scheinbar nicht direkt durch die Myosin1F-Menge in der Zelle beeinflusst, während hingegen die in der Zelle vorliegende Taspase1-Menge von Bedeutung für die Filopodienausbildung ist. Eine gesteigerte Zahl von Filopodien konnte bereits mit erhöhter Invasierung und damit einhergehend verstärkter Aggressivität und deutlich verminderter Überlebensrate in einer Vielzahl unterschiedlicher Krebserkrankungen in Verbindung gebracht werden. Zusammenfassend deuten diese Ergebnisse darauf hin, dass die Prozessierung von Myosin1F durch Taspase1 das Adhäsions- und das Migrationsverhalten von Zellen regulieren kann, was nicht nur bei Entwicklungsprozessen, sondern vor allem auch im Hinblick auf Metastasierung von Bedeutung ist. Die Daten weisen somit auf einen bis dato unbekannten (Patho)Mechanismus hin, über den Taspase1 durch Spaltung von Myosin1F das Metastasierungspotenzial solider Tumore beeinflussen kann.The importance of proteases for the medical and pharmaceutical research is controversial because of their involvement in diverse (patho)biological processes. A prominent example is the threonine protease Taspase1 that attained biomedical relevance due to their involvement in the development and progression of MLL-mediated leukemias as well as the increased expression in solid tumors. In contrast to other proteases, the understanding of Taspase1’s (patho-) biological relevance and function is limited. Recently Myosin1F could be identified as a substrate of Taspase1. Myosins are described as actin -dependent motor proteins, involved in a variety of functions, such as cell movement. But the Myosin1F is a poorly characterized protein. However there is first evidence of relevance in carcinogenesis like for Taspase1. Therefore the (patho) biological significance of Taspase1-Myosin1F-interaction and the function of the Myosin1F protein were examined in this work. To obtain preliminary information on gene-expression a qPCR method for Taspase1 and Myosin1F could be successfully established. This confirmed the increased Taspase1 expression in tumor cell lines and in addition significantly smaller amounts Taspase1-mRNA in immune cells as well as the greatly increased amount Taspase1-mRNA in adherent cell lines compared to suspension cells. Comparison of Myosin1F and Taspase1-mRNA levels show that Taspase1-expression far exceeds that of the Myosin1F, but is correlated in adherent cell with the mRNA expression of Myosin1F. However, in immune cells is an opposite trend was shown. Consequently, the gene expression of Myosin1F and Taspase1 was identified as an important difference between adherent and suspension tumor cells. This could have an impact on differences in the migration behavior of the different cell types, as the strong migrating cells of the immune system shows strong migration but rarely adhesion. Expressionstudies in interphase cells showed that Myosin1F and the non-cleavable Taspase1-variant localize to the cytosol, the membrane and the cytoskeleton. Analogously, both proteins localize to the cell membrane during mitosis, and are found to concentrate between the daughter cells upon their separation. The mutation in the Taspase1 interface of the Myosin1F results, as well as increasing the Taspase1 amount, in a more pronounced cytoplasmic localization of Myosin1F. In this work, the cleavage of the full-length Myosin1F by Taspase1 protein was first shown and clues to the physiological significance of Taspase1-Myosin1F interaction developed. To analyze the Myosin1F-cleavage, a so called "clevage-IP" method were designated and established that can be used successively for the analysis of specific enzyme-substrate cleavage. Interestingly, with this method not only the dose-dependent enzyme activity was shown, but also the separate expression of Taspase1 - and subunits resulted in a higher hydrolytic activity than a corresponding expression of the full-length protein. This points to a limiting function of the intramolecular cis-cleavage. Related experiments showed that the due to Taspase1-cleavage resulting C-terminal Myosin1F fragment more localized to the cytoplasm than the full-length construct. While the N-terminal fragment accumulate in the nucleus. Investigations of the cleavage product showed evidence of a weak, LMB-sensitive NES and the interaction of the cleavage-products after cleavage of the Myosin1F. This could regulate the transport of the N- terminal fragment into the nucleus or the formation of a multi-protein complex, as it is known for the Taspase1 substrate MLL. Functional analyzes have demonstrated the function of the Myosin1F-protein in migratory processes of adherent tumor and non-tumor cells. Thus, ectopic Myosin1F-expression results in the increased substrate adhesion, which is additional influenced by integrin-stimulating substances. Myosin1F co-localized with integrin and as well as Rac1 and Cdc42 and interacts with paxillin, talin and actin. Because these proteins are a marker of early focal adhesions, Myosin1F seems to play a physiological function thereby. The co-localization with Rac1 and Cdc42 also points to an integrin-mediated adhäsion of the Filopodien for stabilizing matrix adhesions in lamellipodia. Further the participation of Myosin1F in the PI3/Akt-Pathway and the interaction with Akt was detected as well as an EGF-dependent recruitment of Myosin1F to the cell membrane was demonstrated. The formation of focal adhesions is apparently not directly affected by the Myosin1F amount in the cell; while on the other hand, the present Taspase1 amount in the cell seems to be important to the Filopodiaformation. An increased number of filopodia was already linked with increased invasion and aggressiveness in a variety of cancers in combination with a significantly reduced survival. In summary, these results suggest that the processing of Myosin1F by Taspase1 can regulate the adhesion and the migration behavior of cells, which is not only of importance in developmental processes, but also with regard to metastasis. Thus the data indicate a so far unknown (patho)mechanism how cleavage of Myosin1F by the Taspase1 can influence the metastasispotential of solid tumors

Correlating semiconductor nanoparticle architecture and applicability for the controlled encoding of luminescent polymer microparticles

Luminophore stained micro- and nanobeads made from organic polymers like polystyrene (PS) are broadly used in the life and material sciences as luminescent reporters, for bead-based assays, sensor arrays, printable barcodes, security inks, and the calibration of fluorescence microscopes and flow cytometers. Initially mostly prepared with organic dyes, meanwhile luminescent core/shell nanoparticles (NPs) like spherical semiconductor quantum dots (QDs) are increasingly employed for bead encoding. This is related to their narrower emission spectra, tuneability of emission color, broad wavelength excitability, and better photostability. However, correlations between particle architecture, morphology, and photoluminescence (PL) of the luminescent nanocrystals used for encoding and the optical properties of the NP-stained beads have been rarely explored. This encouraged us to perform a screening study on the incorporation of different types of luminescent core/shell semiconductor nanocrystals into polymer microparticles (PMPs) by a radical-induced polymerization reaction. Nanocrystals explored include CdSe/CdS QDs of varying CdS shell thickness, a CdSe/ZnS core/shell QD, CdSe/CdS quantum rods (QRs), and CdSe/CdS nanoplatelets (NPLs). Thereby, we focused on the applicability of these NPs for the polymerization synthesis approach used and quantified the preservation of the initial NP luminescence. The spectroscopic characterization of the resulting PMPs revealed the successful staining of the PMPs with luminescent CdSe/CdS QDs and CdSe/CdS NPLs. In contrast, usage of CdSe/CdS QRs and CdSe QDs with a ZnS shell did not yield luminescent PMPs. The results of this study provide new insights into structure–property relationships between NP stained PMPs and the initial luminescent NPs applied for staining and underline the importance of such studies for the performance optimization of NP-stained beads

The cytosolic domain of Pex22p stimulates the Pex4p-dependent ubiquitination of the PTS1-receptor

Peroxisomal biogenesis is an ubiquitin-dependent process because the receptors required for the import of peroxisomal matrix proteins are controlled via their ubiquitination status. A key step is the monoubiquitination of the import receptor Pex5p by the ubiquitin-conjugating enzyme (E2) Pex4p. This monoubiquitination is supposed to take place after Pex5p has released the cargo into the peroxisomal matrix and primes Pex5p for the extraction from the membrane by the mechano-enzymes Pex1p/Pex6p. These two AAA-type ATPases export Pex5p back to the cytosol for further rounds of matrix protein import. Recently, it has been reported that the soluble Pex4p requires the interaction to its peroxisomal membrane-anchor Pex22p to display full activity. Here we demonstrate that the soluble C-terminal domain of Pex22p harbours its biological activity and that this activity is independent from its function as membrane-anchor of Pex4p. We show that Pex4p can be functionally fused to the trans-membrane segment of the membrane protein Pex3p, which is not directly involved in Pex5p-ubiquitination and matrix protein import. However, this Pex3(N)-Pex4p chimera can only complement the double-deletion strain pex4Δ/pex22Δ and ensure optimal Pex5p-ubiquitination when the C-terminal part of Pex22p is additionally expressed in the cell. Thus, while the membrane-bound portion Pex22(N)p is not required when Pex4p is fused to Pex3(N)p, the soluble Pex22(C)p is essential for peroxisomal biogenesis and efficient monoubiquitination of the import receptor Pex5p by the E3-ligase Pex12p in vivo and in vitro. The results merge into a picture of an ubiquitin-conjugating complex at the peroxisomal membrane consisting of three domains: the ubiquitin-conjugating domain (Pex4p), a membrane-anchor domain (Pex22(N)p) and an enhancing domain (Pex22(C)p), with the membrane-anchor domain being mutually exchangeable, while the Ubc- and enhancer-domains are essential

The 10,000-year biocultural history of fallow deer and its implications for conservation policy

Over the last 10,000 y, humans have manipulated fallow deer populations with varying outcomes. Persian fallow deer (Dama mesopotamica) are now endangered. European fallow deer (Dama dama) are globally widespread and are simultaneously considered wild, domestic, endangered, invasive and are even the national animal of Barbuda and Antigua. Despite their close association with people, there is no consensus regarding their natural ranges or the timing and circumstances of their human-mediated translocations and extirpations. Our mitochondrial analyses of modern and archaeological specimens revealed two distinct clades of European fallow deer present in Anatolia and the Balkans. Zooarchaeological evidence suggests these regions were their sole glacial refugia. By combining biomolecular analyses with archaeological and textual evidence, we chart the declining distribution of Persian fallow deer and demonstrate that humans repeatedly translocated European fallow deer, sourced from the most geographically distant populations. Deer taken to Neolithic Chios and Rhodes derived not from nearby Anatolia, but from the Balkans. Though fallow deer were translocated throughout the Mediterranean as part of their association with the Greco-Roman goddesses Artemis and Diana, deer taken to Roman Mallorca were not locally available Dama dama, but Dama mesopotamica. Romans also initially introduced fallow deer to Northern Europe but the species became extinct and was reintroduced in the medieval period, this time from Anatolia. European colonial powers then transported deer populations across the globe. The biocultural histories of fallow deer challenge preconceptions about the divisions between wild and domestic species and provide information that should underpin modern management strategies

Bioassays to Monitor Taspase1 Function for the Identification of Pharmacogenetic Inhibitors

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]benzenesulfonamideand 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

The 10,000-year biocultural history of fallow deer and its implications for conservation policy

Over the last 10,000 y, humans have manipulated fallow deer populations with varying outcomes. Persian fallow deer ( Dama mesopotamica ) are now endangered. European fallow deer ( Dama dama ) are globally widespread and are simultaneously considered wild, domestic, endangered, invasive and are even the national animal of Barbuda and Antigua. Despite their close association with people, there is no consensus regarding their natural ranges or the timing and circumstances of their human-mediated translocations and extirpations. Our mitochondrial analyses of modern and archaeological specimens revealed two distinct clades of European fallow deer present in Anatolia and the Balkans. Zooarchaeological evidence suggests these regions were their sole glacial refugia. By combining biomolecular analyses with archaeological and textual evidence, we chart the declining distribution of Persian fallow deer and demonstrate that humans repeatedly translocated European fallow deer, sourced from the most geographically distant populations. Deer taken to Neolithic Chios and Rhodes derived not from nearby Anatolia, but from the Balkans. Though fallow deer were translocated throughout the Mediterranean as part of their association with the Greco-Roman goddesses Artemis and Diana, deer taken to Roman Mallorca were not locally available Dama dama , but Dama mesopotamica . Romans also initially introduced fallow deer to Northern Europe but the species became extinct and was reintroduced in the medieval period, this time from Anatolia. European colonial powers then transported deer populations across the globe. The biocultural histories of fallow deer challenge preconceptions about the divisions between wild and domestic species and provide information that should underpin modern management strategies

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

The 10,000-year biocultural history of fallow deer and its implications for conservation policy

Over the last 10,000 years, humans have manipulated fallow deer populations with varying outcomes. Persian fallow deer (Dama mesopotamica) are now endangered. European fallow deer (Dama dama) are globally widespread and are simultaneously considered wild, domestic, endangered, invasive, and are even the national animal of Barbuda and Antigua. Despite their close association with people, there is no consensus regarding their natural ranges or the timing and circumstances of their human-mediated translocations and extirpations. Our mitochondrial analyses of modern and archaeological specimens revealed two distinct clades of European fallow deer present in Anatolia and the Balkans. Zooarchaeological evidence suggests these regions were their sole glacial refugia. By combining biomolecular analyses with archaeological and textual evidence, we chart the declining distribution of Persian fallow deer and demonstrate that humans repeatedly translocated European fallow deer, sourced from the most geographically distant populations. Deer taken to Chios and Rhodes in the Neolithic derived not from nearby Anatolia, but from the Balkans. Though fallow deer were translocated throughout the Mediterranean as part of their association with the Greco-Roman goddesses Artemis and Diana, deer taken to Roman Mallorca were not locally available Dama dama, but Dama mesopotamica. Romans also initially introduced fallow deer to Northern Europe but the species became extinct and was reintroduced in the medieval period, this time from Anatolia. European colonial powers then transported deer populations across the globe. We argue that these biocultural histories of fallow deer should underpin modern management strategie

Overexpression of the catalytically impaired Taspase1 T234V or Taspase1 D233A variants does not have a dominant negative effect in T(4;11) leukemia cells.

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, Taspase1(T234V), or the highly attenuated variant, Taspase1(D233A), 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

Probing Taspase1 multimerization in living cells.

<p><b>A.</b> Heterocomplex formation of Taspase1 and Taspase1 variants shown by co-immunoprecipitation (IP). IPs of 293T cell extracts co-transfected with the indicated expression constructs were carried out using α-GFP Ab-coated magnetic beads and μ-MACS columns. Precipitated proteins were identified by immunoblot using the indicated antibodies. Input: Total amount of cell lysate. IP: immunoprecipitated proteins. *: GFP-degradation products <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034142#pone.0034142-Landgraf1" target="_blank">[33]</a>. <b>B.</b> Principle of the translocation based protein-protein interaction assay. The Tasp_Cyt fusion is composed of GFP, Taspase1 and a NES (?) and thus, continuously shuttling between the nucleus and the cytoplasm. The red-fluorescent Taspase1 variants (Tasp-mCherry prey) accumulate at the nucleus/nucleolus. Upon efficient protein-protein interaction, the GFP-tagged cytoplasmic Tasp_Cyt co-localizes with the Tasp-mCherry prey to the nucleus/nucleolus in living cells. <b>C.</b> Localization of indicated proteins in the absence of potential interaction partners. <b>D.</b> Neither co-expression of WT nor inactive Taspase1 variants resulted in strong nuclear/nucleolar translocation of Tasp_Cyt. Co-expression of NPM1-RFP, known to strongly interact with Taspase1, triggered nuclear/nucleolar translocation of Tasp_Cyt (positive control). In contrast, co-expression of the non-interacting nucleolar RevM10BL-RFP protein showed no effect (negative control) as visualized by fluorescence microscopy in living HeLa transfectants. Scale bars, 10 µm.</p