Bogdan I. Florea

Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4

DNA double-strand break (DSB) repair by nonhomologous end joining (NHEJ) requires the assembly of several proteins on DNA ends. Although biochemical studies have elucidated several aspects of the NHEJ reaction mechanism, much less is known about NHEJ in living cells, mainly because of the inability to visualize NHEJ repair proteins at DNA damage. Here we provide evidence that a pulsed near IR laser can produce DSBs without any visible alterations in the nucleus, and we show that NHEJ proteins accumulate in the irradiated areas. The levels of DSBs and Ku accumulation diminished in time, showing that this approach allows us to study DNA repair kinetics in vivo. Remarkably, the Ku heterodimers on DNA ends were in dynamic equilibrium with Ku70/80 in solution, showing that NHEJ complex assembly is reversible. Accumulation of XRCC4/ligase IV on DSBs depended on the presence of Ku70/80, but not DNA-PKCS. We detected a direct interaction between Ku70 and XRCC4 that could explain these requirements. Our results suggest that this assembly constitutes the core of the NHEJ reaction and that XRCC4 may serve as a flexible tether between Ku70/80 and ligase IV.

Transfection efficiency and toxicity of polyethylenimine in differentiated Calu-3 and nondifferentiated COS-1 cell cultures.

In the present study, we evaluated polyethylenimine (PEI) of different molecular weights (MWs) as a DNA complexing agent for its efficiency in transfecting nondifferentiated COS-1 (green monkey fibroblasts) and well-differentiated human submucosal airway epithelial cells (Calu-3). Studying the effect of particle size, zeta potential, presence of serum proteins or chloroquine, it appeared that transfection efficiency depends on the experimental conditions and not on the MW of the PEI used. Comparing transfection efficiencies in both cell lines, we found that PEI was 3 orders of magnitude more effective in COS-1 than in Calu-3 cells, because Calu-3 cells are differentiated and secrete mucins, which impose an additional barrier to gene delivery. Transfection efficiency was strongly correlated to PEI cytotoxicity. Also, some evidence for PEI-induced apoptosis in both cell lines was found. In conclusion, our results indicate that PEI is a useful vector for nonviral transfection in undifferentiated cell lines. However, results from studies in differentiated bronchial epithelial cells suggest that PEI has yet to be optimized for successful gene therapy of cystic fibrosis (CF).

N-Trimethylated Chitosan Chloride (TMC) Improves the Intestinal Permeation of the Peptide Drug Buserelin In Vitro (Caco-2 Cells) and In Vivo (Rats)

AbstractPurpose. To evaluate N-trimethyl chitosan chloride (TMC) of highdegrees of substitution as intestinal permeation enhancers for thepeptide drug buserelin in vitro using Caco-2 cell monolayers, and toinvestigate TMCs as enhancers of the intestinal absorption of buserelinin vivo, in rats. Methods. TMCs were tested on Caco-2 cells for their efficiency toincrease the paracellular permeability of the peptide buserelin. For thein vivo studies male Wistar rats were used and buserelin wasadministered with or without the polymers intraduodenally. Both types ofexperiments were performed at pH 7.2. Results. Transport studies with Caco-2 cell monolayers confirmed thatthe increase in buserelin permeation is dependent on the degree oftrimethylation of TMC. In agreement with the in vitro results, in vivodata revealed highly increased bioavailability of buserelin followingintraduodenal co-administration with 1.0% (w/v) TMCs.Intraduodenally applied buserelin resulted in 0.8% absolute bioavailability,whereas co-administrations with TMCs resulted in mean bioavailabilityvalues between 6 and 13 %. Chitosan HCl (1.0% pH = 7.2) did notsignificantly increase the intestinal absorption of buserelin. Conclusions. Both the in vitro and in vivo results indicate that TMCsare potent mucosal permeation enhancers of the peptide drug buserelinat neutral pH values.

Ultrasensitive in situ visualization of active glucocerebrosidase molecules

Deficiency of glucocerebrosidase (GBA) underlies Gaucher disease, a common lysosomal storage disorder. Carriership for Gaucher disease has recently been identified as major risk for parkinsonism. Presently, no method exists to visualize active GBA molecules in situ. We here report the design, synthesis and application of two fluorescent activity-based probes allowing highly specific labeling of active GBA molecules in vitro and in cultured cells and mice in vivo. Detection of in vitro labeled recombinant GBA on slab gels after electrophoresis is in the low attomolar range. Using cell or tissue lysates, we obtained exclusive labeling of GBA molecules. We present evidence from fluorescence-activated cell sorting analysis, fluorescence microscopy and pulse-chase experiments of highly efficient labeling of GBA molecules in intact cells as well as tissues of mice. In addition, we illustrate the use of the fluorescent probes to study inhibitors and tentative chaperones in living cells.

Drug transport and metabolism characteristics of the human airway epithelial cell line Calu-3

Pulmonary drug delivery serves two purposes, namely the application of locally active compounds for treatment of diseases afflicting the lung, and the utilization of the pulmonary epithelia as absorption sites for macromolecular drugs. To elucidate the mechanisms involved in the pulmonary absorption and metabolism of compounds on a cellular level, cell culture models have shown to be, though limited, rather useful in predicting in vivo conditions. The Calu-3 cell line has been employed recently as a model for the airway epithelium in a number of drug transport and metabolism studies. The results of these studies, as well as an evaluation of the predictive potency of the model, are presented here.

Asymmetric Proteasome Segregation as a Mechanism for Unequal Partitioning of the Transcription Factor T-bet during T Lymphocyte Division

Polarized segregation of proteins in T cells is thought to play a role in diverse cellular functions including signal transduction, migration, and directed secretion of cytokines. Persistence of this polarization can result in asymmetric segregation of fate-determining proteins during cell division, which may enable a T cell to generate diverse progeny. Here, we provide evidence that a lineage-determining transcription factor, T-bet, underwent asymmetric organization in activated T cells preparing to divide and that it was unequally partitioned into the two daughter cells. This unequal acquisition of T-bet appeared to result from its asymmetric destruction during mitosis by virtue of concomitant asymmetric segregation of the proteasome. These results suggest a mechanism by which a cell may unequally localize cellular activities during division, thereby imparting disparity in the abundance of cell fate regulators in the daughter cells.

Enhancement of bronchial octreotide absorption by chitosan and N-trimethyl chitosan shows linear in vitro/in vivo correlation

Chitosan is a biocompatible polysaccharide of natural origin that can act as a permeation enhancer. In this study, we used an integral in vitro/in vivo correlation approach to: a) investigate polysaccharide-mediated absorption kinetics of the peptide drug octreotide across mammalian airway epithelium, b) assess formulation toxicity, c) correlate the mechanism of permeation enhancement. The 20% and 60% N-trimethylated chitosan derivatives (TMC20 and TMC60) were synthesized by alkaline methylation using chitosan as starting material. Octreotide was administered in control buffers or in 1.5% (w/v) gel-phase formulations of pH 5.5 for chitosan and pH 7.4 for TMCs. In vitro, reconstituted Calu-3 cell monolayers were used for trans-epithelial electrical resistance (TEER), transport and cytotoxicity assays. Intratracheal instillation in rats was used to determine octreotide kinetics and formulation toxicity in vivo. Chitosan, TMC20 and TMC60 decreased TEER significantly and enhanced octreotide permeation in vitro by 21-, 16- and 30-fold. In vivo, sustained release properties of the formulations were observed and the bio-availability was enhanced by 2.4-, 2.5- and 3.9-fold, respectively. Interestingly, we found a linear in vitro/in vivo correlation between calculated absorption rates (R2=0.93), suggesting that the permeation enhancement by polysaccharides, both in vitro and in vivo, proceeds via an analogous mechanism. Cell viability and histology studies showed that the TMCs are safer than chitosan and that Calu-3 cell monolayers are a valuable model for predicting paracellular transport kinetics in airway epithelia. Additionally, cationic polysaccharides are promising enhancers for peptide drug absorption with prospect for sustained-release formulations.

Specific Cell-Permeable Inhibitor of Proteasome Trypsin-like Sites Selectively Sensitizes Myeloma Cells to Bortezomib and Carfilzomib

Proteasomes degrade the majority of proteins in mammalian cells, are involved in the regulation of multiple physiological functions, and are established targets of anticancer drugs. The proteasome has three types of active sites. Chymotrypsin-like sites are the most important for protein breakdown and have long been considered the only suitable targets for antineoplastic drugs; however, our recent work demonstrated that inhibitors of caspase-like sites sensitize malignant cells to inhibitors of the chymotrypsin-like sites. Here, we describe the development of specific cell-permeable inhibitors and an activity-based probe of the trypsin-like sites. These compounds selectively sensitize multiple myeloma cells to inhibitors of the chymotrypsin-like sites, including antimyeloma agents bortezomib and carfilzomib. Thus, trypsin-like sites are cotargets for anticancers drugs. Together with inhibitors of chymotrypsin- and caspase-like sites developed earlier, we provide the scientific community with a complete set of tools to separately modulate proteasome active sites in living cells.

Triple Bioorthogonal Ligation Strategy for Simultaneous Labeling of Multiple Enzymatic Activities

Bioorthogonal chemistry plays an important role in chemical biology research by creating the means to carry out selective chemical transformations in complex biological samples. A ligation reaction classifies as being bioorthogonal when it can be performed in a biological sample in a chemoselective manner without any interference with the biological system. Bioorthogonal reactions have been used in cell-surface labeling of glycoproteins and studies of biological processes that involve post-translational modifications. Another area of research that has benefited from bioorthogonal chemistry is two-step activity-based protein profiling (ABPP), where it enables the temporal separation of a reporter group and a chemical probe that is directed to the active site of an enzyme (such a chemical probe is also called activity-based probe, ABP). Two-step ABPP strategies are of particular interest when the presence of a tag interferes with selectivity, affinity, cell-permeability, or bioavailability of the probe. A further advantage of tandem labeling strategies is the option to use different reporter groups depending on the type of experiment and the desired method of analysis while using a single ABP. Several bioorthogonal ligation strategies have been described, and continuing efforts are being made to develop ligations that are more selective and efficient than existing methods. At the same time the high complexity of biological processes often requires the study of multiple targets simultaneously, thereby creating a need for ligation reactions that are orthogonal with respect to each other and can thus be used concurrently in a single experiment. Over the past decade, a number of tandem ligation strategies has been described for use in bioconjugation. The first report of a tandem bioorthogonal ligation in complex biological samples involved a Staudinger–Bertozzi ligation and Diels– Alder cycloaddition procedure, which utilizes mutually orthogonal reagents but suffers from the need to mask free thiol groups prior to the ligation step to avoid nonspecific labeling. More recently, it was reported that a copper-free azide–cyclooctyne cycloaddition can be used concurrently with an inverse-electron-demand Diels–Alder reaction between tetrazine and trans-cyclooctene for the simultaneous labeling of two different receptors on cell surfaces, provided that the proper reagents are carefully selected so that crossreactivity is minimized. Herein, we describe a triple ligation strategy employing the tetrazine ligation, Staudinger–Bertozzi ligation, and copper(I)-catalyzed Huisgen [2+3] cycloaddition (“click” reaction) for the selective and simultaneous labeling of three different enzymatic activities in a single experiment (Scheme 1a). Several examples of two-step ABPP strategies using click chemistry and Staudinger–Bertozzi ligation have been described previously. The tetrazine ligation, however, has thus far not been used for this purpose. Therefore we set out to develop a two-step ABPP strategy in which an ABP is functionalized with norbornene as a ligation handle that can react with a tetrazine reagent conjugated to a reporter group to enable detection and analysis of labeled proteins. As a model system for our studies we selected the 20S proteasome, containing three catalytically active subunits (b1, b2, and b5) that can be targeted by either broad-spectrum or subunit-specific ABPs. We designed two proteasome ABPs that are functionalized with norbornene as a ligation handle: ABP 1 is derived from the pan-reactive proteasome inhibitor epoxomicin, and ABP 2 has a different scaffold based on a b5-subunit-selective proteasome inhibitor (Scheme 1 b). Furthermore, we chose to create a panel of three tetrazine reagents functionalized with different tags, being BodipyTMR (3 a), BodipyFL (3 b), and biotin (3c). Other reagents used herein for two-step labeling of the proteasome by click chemistry and Staudinger ligation are shown in Scheme 1c. The synthesis of all reagents and competition experiments confirming the ability of the ABPs to target all proteolytically active proteasome b subunits (1, 4, 5) or only the b5 subunit (2) in cell extracts and/or in living cells can be found in the Supporting Information. The applicability of the tetrazine ligation for two-step labeling of endogenous proteasome activity was tested by exposing human embryonic kidney (HEK) cell lysates to norbornene-functionalized ABP 1 in a concentration that results in complete proteasome binding followed by ligation with one of the tetrazine reagents 3a–c for one hour at 37 8C. Analysis of labeled proteins by SDS-PAGE using either fluorescent readout or detection by streptavidin Western blotting (Figure 1a and Figure S2 in the Supporting Information) showed that ligation with all three tetrazine reagents results in labeling of the three catalytically active proteasome b subunits in a concentration-dependent manner. In this [*] L. I. Willems, N. Li, Dr. B. I. Florea, M. Ruben, Prof. G. A. van der Marel, Prof. H. S. Overkleeft Leiden Institute of Chemistry and Netherlands Proteomics Centre Gorlaeus Laboratories Einsteinweg 55, 2333 CC Leiden (The Netherlands) E-mail: h.s.overkleeft@chem.leidenuniv.nl

Novel Activity‐Based Probes for Broad‐Spectrum Profiling of Retaining β‐Exoglucosidases In Situ and In Vivo

A high-end label: Cyclophellitol aziridine-type activity-based probes allow for ultra-sensitive visualization of mammalian β-glucosidases (GBA1, GBA2, GBA3, and LPH) as well as several non-mammalian β-glucosidases (see picture). These probes offer new ways to study β-exoglucosidases, and configurational isomers of the cyclophellitol aziridine core may give activity-based probes targeting other retaining glycosidase families.