RESUMO
Chemical glycosylation of proteins is a powerful tool applied widely in biomedicine and biotechnology. However, it is a challenging undertaking and typically relies on recombinant proteins and site-specific conjugations. The scope and utility of this nature-inspired methodology would be broadened tremendously by the advent of facile, scalable techniques in glycosylation, which are currently missing. In this work, we investigated a one-pot aqueous protocol to achieve indiscriminate, surface-wide glycosylation of the surface accessible amines (lysines and/or N-terminus). We reveal that this approach afforded minimal if any change in the protein activity and recognition events in biochemical and cell culture assays, but at the same time provided a significant benefit of stabilizing proteins against aggregation and fibrillation - as demonstrated on serum proteins (albumins and immunoglobulin G, IgG), an enzyme (uricase), and proteins involved in neurodegenerative disease (α-synuclein) and diabetes (insulin). Most importantly, this highly advantageous result was achieved via a one-pot aqueous protocol performed on native proteins, bypassing the use of complex chemical methodologies and recombinant proteins.
Assuntos
Doenças Neurodegenerativas , Glicosilação , LisinaRESUMO
In this work, synthetic cells equipped with an artificial signaling pathway that connects an extracellular trigger event to the activation of intracellular transcription are engineered. Learning from nature, this is done via an engineering of responsive enzymes, such that activation of enzymatic activity can be triggered by an external biochemical stimulus. Reversibly deactivated creatine kinase to achieve triggered production of adenosine triphosphate, and a reversibly deactivated nucleic acid polymerase for on-demand synthesis of RNA are engineered. An extracellular, enzyme-activated production of a diffusible zymogen activator is also designed. The key achievement of this work is that the importance of cellularity is illustrated whereby the separation of biochemical partners is essential to resolve their incompatibility, to enable transcription within the confines of a synthetic cell. The herein designed biochemical pathway and the engineered synthetic cells are arguably primitive compared to their natural counterpart. Nevertheless, the results present a significant step toward the design of synthetic cells with responsive behavior, en route from abiotic to life-like cell mimics.
Assuntos
Células Artificiais , Precursores Enzimáticos , Precursores Enzimáticos/metabolismoRESUMO
The design of artificial, synthetic cells is a fundamentally important and fast-developing field of science. Of the diverse attributes of cellular life, artificial transmembrane signaling across the biomolecular barriers remains a high challenge with only a few documented successes. Herein, the study achieves signaling across lipid bilayers and connects an exofacial enzymatic receptor activation to an intracellular biochemical catalytic response using an artificial receptor. The mechanism of signal transduction for the artificial receptor relies on the triggered decomposition of a self-immolative linker. Receptor activation ensues its head-to-tail decomposition and the release of a secondary messenger molecule into the internal volume of the synthetic cell. Transmembrane signaling is demonstrated in synthetic cells based on liposomes and mammalian cell-sized giant unilamellar vesicles and illustrates receptor performance in cell mimics with a diverse size and composition of the lipid bilayer. In giant unilamellar vesicles, transmembrane signaling connects exofacial receptor activation with intracellular activation of proteolysis. Taken together, the results of this study take a step toward engineering receptor-mediated, responsive behavior in synthetic cells.
RESUMO
Signal transduction across biological membranes is among the most important evolutionary achievements. Herein, for the design of artificial cells, we engineer fully synthetic receptors with the capacity of transmembrane signaling, using tools of chemistry. Our receptors exhibit similarity with their natural counterparts in having an exofacial ligand for signal capture, being membrane anchored, and featuring a releasable messenger molecule that performs enzyme activation as a downstream signaling event. The main difference from natural receptors is the mechanism of signal transduction, which is achieved using a self-immolative linker. The receptor scaffold is modular and can readily be re-designed to respond to diverse activation signals including biological or chemical stimuli. We demonstrate an artificial signaling cascade that achieves transmembrane enzyme activation, a hallmark of natural signaling receptors. Results of this work are relevant for engineering responsive artificial cells and interfacing them and/or biological counterparts in co-cultures.