Protein tango: the toolbox to capture interacting partners

The evaluation of protein function in the context of the whole cell is crucial for understanding of living systems. In this context, the identification and modulation of protein-protein interactions in and outside cells is of ample importance. Several methods have been developed in the past years to detect and/or actively induce protein-protein interactions in living cells. As a result, tools are now available to manipulate intracellular events by reversible or irreversible cross-linking of proteins in a specific manner. These techniques open many new doors and enable the dissection of complicated protein networks. Herein we describe which cross-linkers and inducers of dimerization are out there and how to make use of this great toolbox.     

Dendrimer-based macromolecular conjugate for the kidney-directed delivery of a multitargeted sunitinib analogue

The development of a macromolecular conjugate of a multitargeted tyrosine kinase inhibitor is described that can be used for renal‐specific delivery into proximal tubular cells. A novel sunitinib analogue, that is, 17864, is conjugated to a NH₂‐PAMAM‐G3 dendrimer via the platinum (II)‐based Universal Linkage System (ULS?). The activity of 17864 is retained after coordination to the ULS linker alone or when coupled to NH₂‐PAMAM‐G3. 17864‐UlS‐NH₂‐PAMAM‐G3 is non‐toxic to proximal tubular cells in vitro. After intravenous administration to mice, 17864‐UlS‐NH₂‐PAMAM‐G3 rapidly and efficiently accumulates in the kidneys. These results are encouraging for future studies focusing on the development of novel therapeutics for the treatment of renal diseases.

     

Pyrenyl Carbon Nanotubes for Covalent Bilirubin Oxidase Biocathode Design: Should the Nanotubes be Carboxylated?

Understanding the characteristics of nanomaterials in the context of electrode designs for bio‐electrocatalysis is an emerging research direction. Applications for fuel cells, batteries, and biosensors are directly benefited. The objective of this study is to understand the influence of unfunctionalized multiwalled carbon nanotubes (MWNT) in comparison to carboxylated nanotubes (MWNT?COOH) for pi‐pi stacking with 1‐pyrenebutyric acid (Py) and covalent immobilization of bilirubin oxidase (BOD) enzyme toward the resulting oxygen reduction currents. We designed pyrolytic graphite‐edge electrodes modified with MWNT/Py, MWNT?COOH/Py, or only MWNT?COOH for carbodiimide activation and BOD immobilization. The relative increase in surface ?COOH groups as we move from MWNT to MWNT/Py to MWNT?COOH/Py modification is voltammetrically estimated. Although the MWNT?COOH/Py displayed the highest relative amount of surface ?COOH groups, the oxygen reduction current was the largest for the BOD‐immobilized MWNT/Py electrode than others. Results indicate that unfunctionalized MWNT is the optimal choice for pi‐pi stacking with pyrene linkers and covalent BOD immobilization as biocathode for energy devices. Favorable hydrophobic MWNT surface to interact more closely with the electron‐receiving T1 Cu site of BOD, as opposed to the relatively polar and more defective MWNT?COOH material due to functionalization, is suggested to be one of the underlying factors for the observed electrocatalytic trend.     

Branched Linkers for Site-Specific Fluorescent Labeling of Antibodies

Fluorescent antibodies have proved to be an invaluable tool for molecular biology and diagnostics. They are routinely produced by modification of lysine residues, which leads to high heterogeneity. As such, their affinity may be compromised if the antigen-binding site is affected, the probability of which increases along with the degree of labeling. In this work, we propose a methodology for the synthesis of site-specific antibody-dye conjugates with a high degree of labeling. To this end, we synthesized two oxyamine-based branched triazide linkers and coupled them with a periodate-oxidized anti-PRAME antibody 6H8; two oxyamine-based linear monoazide linkers of similar structure were used as controls. The azide-labeled antibodies were subsequently conjugated with fluorescent dyes via SPAAC, a copper-free click reaction. Compared to their counterparts made with linear linkers, the branched conjugates possessed a higher degree of labeling. The utility of the methodology was demonstrated in the detection of the PRAME protein on the surface of the cell by flow cytometry.