Monitoring in vivo protein-protein interactions by coupling bimolecular fluorescence complemetation (BIFC) and flow cytometry

Comunicaciones a congreso

PTkChA: editor d’anotacions

El Grup de Processament del Llenguatge Natural, dins del Centre de Tecnologies i Aplicacions del Llenguatge i la Parla (TALP), es dedica al tractament automàtic del llenguatge natural i desenvolupa diferents eines pel tractament d‟aquest. El projecte consisteix en modificar el PTkChA, que és una d‟aquestes eines destinada a l‟etiquetatge de textos. El treball s‟estructura en dues planificacions; en la primera es demana que, a partir d‟uns requisits inicials, s‟analitzi funcionalment l‟aplicació i es determinin totes les modificacions que es poden fer per millorar-la. En la segona, s‟especifiquen i es desenvolupen les modificacions. Així doncs, l‟objectiu d‟aquest projecte és desenvolupar les modificacions

Aggregating sequences that occur in many proteins constitute weak spots of bacterial proteostasis

Aggregation is a sequence-specific process, nucleated by short aggregation-prone regions (APRs) that can be exploited to induce aggregation of proteins containing the same APR. Here, we find that most APRs are unique within a proteome, but that a small minority of APRs occur in many proteins. When aggregation is nucleated in bacteria by such frequently occurring APRs, it leads to massive and lethal inclusion body formation containing a large number of proteins. Buildup of bacterial resistance against these peptides is slow. In addition, the approach is effective against drug-resistant clinical isolates of Escherichiacoli and Acinetobacterbaumannii, reducing bacterial load in a murine bladder infection model. Our results indicate that redundant APRs are weak points of bacterial protein homeostasis and that targeting these may be an attractive antibacterial strategy

Physical characterixation and in vitro biological impact of highly aggregated antibodies separated into size-enriched populations by fluorescence-activated cell sorting

An IgG2 monoclonal antibody (mAb) solution was subjected to stirring, generating high concentrations of nanometer and subvisible particles, which were then successfully size enriched into different size bins by low speed centrifugation or a combination of gravitational sedimentation and Fluorescence-Activated Cell Sorting (FACS). The size-fractionated mAb particles were assessed for their ability to elicit the release of cytokines from a population of donor-derived human peripheral blood mononuclear cells (PBMC) at two phases of the immune response. Fractions enriched in nanometer-sized particles showed a lower response than those enriched in micron-sized particles in this assay. Particles of 5–10 μm in size displayed elevated cytokine release profiles compared to other size ranges. Stir-stressed mAb particles had amorphous morphology, contained protein with partially altered secondary structure, elevated surface hydrophobicity (compared to controls), and trace levels of elemental fluorine. FACS size-enriched the mAb particle samples, yet did not notably alter the overall morphology or composition of particles as measured by Microflow imaging, Transmission Electron Microscopy, and Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy. The utility and limitations of FACS for size separation of mAb particles and potential of in-vitro PBMC studies to rank order the immunogenic potential of various types of mAb particles is discussed

Influence of the Stability of a Fused Protein and Its Distance to the Amyloidogenic Segment on Fibril Formation

Conversion of native proteins into amyloid fibrils is irreversible and therefore it is difficult to study the interdependence of conformational stability and fibrillation by thermodynamic analyses. Here we approached this problem by fusing amyloidogenic poly-alanine segments derived from the N-terminal domain of the nuclear poly (A) binding protein PABPN1 with a well studied, reversibly unfolding protein, CspB from Bacillus subtilis. Earlier studies had indicated that CspB could maintain its folded structure in fibrils, when it was separated from the amyloidogenic segment by a long linker. When CspB is directly fused with the amyloidogenic segment, it unfolds because its N-terminal chain region becomes integrated into the fibrillar core, as shown by protease mapping experiments. Spacers of either 3 or 16 residues between CspB and the amyloidogenic segment were not sufficient to prevent this loss of CspB structure. Since the low thermodynamic stability of CspB (ΔGD = 12.4 kJ/mol) might be responsible for unfolding and integration of CspB into fibrils, fusions with a CspB mutant with enhanced thermodynamic stability (ΔGD = 26.9 kJ/mol) were studied. This strongly stabilized CspB remained folded and prevented fibril formation in all fusions. Our data show that the conformational stability of a linked, independently structured protein domain can control fibril formation

PTkChA: editor d’anotacions

El Grup de Processament del Llenguatge Natural, dins del Centre de Tecnologies i Aplicacions del Llenguatge i la Parla (TALP), es dedica al tractament automàtic del llenguatge natural i desenvolupa diferents eines pel tractament d‟aquest. El projecte consisteix en modificar el PTkChA, que és una d‟aquestes eines destinada a l‟etiquetatge de textos. El treball s‟estructura en dues planificacions; en la primera es demana que, a partir d‟uns requisits inicials, s‟analitzi funcionalment l‟aplicació i es determinin totes les modificacions que es poden fer per millorar-la. En la segona, s‟especifiquen i es desenvolupen les modificacions. Així doncs, l‟objectiu d‟aquest projecte és desenvolupar les modificacions

PTkChA: editor d’anotacions

El Grup de Processament del Llenguatge Natural, dins del Centre de Tecnologies i Aplicacions del Llenguatge i la Parla (TALP), es dedica al tractament automàtic del llenguatge natural i desenvolupa diferents eines pel tractament d‟aquest. El projecte consisteix en modificar el PTkChA, que és una d‟aquestes eines destinada a l‟etiquetatge de textos. El treball s‟estructura en dues planificacions; en la primera es demana que, a partir d‟uns requisits inicials, s‟analitzi funcionalment l‟aplicació i es determinin totes les modificacions que es poden fer per millorar-la. En la segona, s‟especifiquen i es desenvolupen les modificacions. Així doncs, l‟objectiu d‟aquest projecte és desenvolupar les modificacions