The emergence of bioorthogonal reactions has greatly advanced research in the fields of biology and medicine. They are not only valuable for labeling, tracking, and understanding biomolecules within ...living organisms, but also important for constructing advanced bioengineering and drug delivery systems. As the systems studied are increasingly complex, the simultaneous use of multiple bioorthogonal reactions is equally desirable. In this review, we take a look at the different bioorthogonal reactions that have recently been developed, the methods of cellular incorporation and the strategies to create orthogonality within the bioorthogonal landscape.
Bioorthogonal reactions are widely used for the chemical modification of biomolecules. The application of vinylboronic acids (VBAs) as non‐strained, synthetically accessible and water‐soluble ...reaction partners in a bioorthogonal inverse electron‐demand Diels–Alder (iEDDA) reaction with 3,6‐dipyridyl‐s‐tetrazines is described. Depending on the substituents, VBA derivatives give second‐order rate constants up to 27 m−1 s−1 in aqueous environments at room temperature, which is suitable for biological labeling applications. The VBAs are shown to be biocompatible, non‐toxic, and highly stable in aqueous media and cell lysate. Furthermore, VBAs can be used orthogonally to the strain‐promoted alkyne–azide cycloaddition for protein modification, making them attractive complements to the bioorthogonal molecular toolbox.
Bioorthogonal iEDDA reagents: Vinylboronic acids (VBAs) were studied as non‐strained, synthetically accessible, and water‐soluble bioorthogonal reagents in the Carboni–Lindsey reaction with dipyridyl‐s‐tetrazines. The VBAs were shown to be biocompatible, non‐toxic, and highly stable in aqueous media and cell lysate. Furthermore, VBAs were used orthogonally to the strain‐promoted alkyne–azide cycloaddition for protein modification.
Profiling the nascent cellular proteome and capturing early proteomic changes in response to external stimuli provides valuable insights into cellular physiology. Existing metabolic protein labeling ...approaches based on bioorthogonal methionine- or puromycin analogs allow for the selective visualization and enrichment of newly synthesized proteins. However, their applications are limited as they often require methionine-free conditions, auxotrophic cells and/or are toxic to cells. Here, we introduce THRONCAT, a threonine-derived non-canonical amino acid tagging method based on the bioorthogonal threonine analog β-ethynylserine (βES) that enables efficient labeling of the nascent proteome in complete growth media within minutes. We use THRONCAT for the visualization and enrichment of nascent proteins in bacteria, mammalian cells and Drosophila melanogaster. We profile immediate proteome dynamics of B-cells in response to B-cell receptor activation simply by adding βES to the culture medium, demonstrating the ease-of-use of the method and its potential to address diverse biological questions. In addition, using a Drosophila model of Charcot-Marie-Tooth peripheral neuropathy, we show that THRONCAT enables visualization and quantification of relative protein synthesis rates in specific cell types in vivo.
Activity-based protein profiling (ABPP) is a powerful technique to label and detect active enzyme species within cell lysates, cells, or whole animals. In the last two decades, a wide variety of ...applications and experimental read-out techniques have been pursued in order to increase our understanding of physiological and pathological processes, to identify novel drug targets, to evaluate selectivity of drugs, and to image probe targets in cells. Bioorthogonal chemistry has substantially contributed to the field of ABPP, as it allows the introduction of tags, which may be bulky or have unfavorable physicochemical properties, at a late stage in the experiment. In this review, we give an overview of the bioorthogonal reactions that have been implemented in ABPP, provide examples of applications of bioorthogonal chemistry in ABPP, and share some thoughts on future directions.
Cell-penetrating peptides are able to transport a wide variety of cargo across cell membranes. Although promising, they are not often considered for therapeutic purposes as they lack controllable ...activity and cell selectivity. We have developed an activation strategy based on a split octa-arginine cell-penetrating peptide (CPP) that can be activated by means of bioorthogonal ligation. To this end we prepared two non-penetrating tetra-arginine halves, functionalized either with a tetrazine or with a complementary bicyclo6.1.0nonyne (BCN) group. We demonstrate that an active octa-arginine can be reconstituted
upon mixing the complementary split peptides. The resulting activated peptide is taken up as efficiently as the well-established cell-penetrating peptide octa-arginine. The activation of the oligo-arginines can also be achieved using
-cyclooctene (TCO) as a ligation partner, while norbornene appears too kinetically slow for use
. We further show that this strategy can be applied successfully to transport a large protein into living cells. Our results validate a promising first step in achieving control over cell penetration and to use CPPs for therapeutic approaches.
The ability to rapidly regulate the functions of specific proteins in living cells is a valuable tool for biological research. Here we describe a new technique by which the degradation of a specific ...protein is induced by a small molecule. A protein of interest is fused to a ligand-induced degradation (LID) domain, resulting in the expression of a stable and functional fusion protein. The LID domain is comprised of the FK506- and rapamycin-binding protein (FKBP) and a 19-amino-acid degron fused to the C terminus of FKBP. In the absence of the small molecule Shield-1, the degron is bound to the FKBP fusion protein and the protein is stable. When present, Shield-1 binds tightly to FKBP, displacing the degron and inducing rapid and processive degradation of the LID domain and any fused partner protein. Structure-function studies of the 19-residue peptide showed that a 4-amino-acid sequence within the peptide is responsible for degradation.
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•Small heat shock protein hetero-oligomerization affects their chaperone function.•The HspB2:HspB3 expression ratio determines phase separation and aggregation.•HspB2 condensates and ...HspB2:HspB3 aggregates are fully reversible.•Proximity labelling unveils autophagy factor recruitment to HspB2:HspB3 aggregates.•Stoichiometry-dependant regulation of phase behaviour may be widespread in biology.
Small heat shock proteins (sHSPs) are essential ATP-independent chaperones that protect the cellular proteome. These proteins assemble into polydisperse oligomeric structures, the composition of which dramatically affects their chaperone activity. The biomolecular consequences of variations in sHSP ratios, especially inside living cells, remain elusive. Here, we study the consequences of altering the relative expression levels of HspB2 and HspB3 in HEK293T cells. These chaperones are partners in a hetero-oligomeric complex, and genetic mutations that abolish their mutual interaction are associated with myopathic disorders.
HspB2 displays three distinct phenotypes when co-expressed with HspB3 at varying ratios. Expression of HspB2 alone leads to formation of liquid nuclear condensates, while shifting the stoichiometry towards HspB3 resulted in the formation of large solid-like aggregates. Only cells co-expressing HspB2 with a limited amount of HspB3 formed fully soluble complexes that were distributed homogeneously throughout the nucleus. Strikingly, both condensates and aggregates were reversible, as shifting the HspB2:HspB3 balance in situ resulted in dissolution of these structures.
To uncover the molecular composition of HspB2 condensates and aggregates, we used APEX-mediated proximity labelling. Most proteins interact transiently with the condensates and were neither enriched nor depleted in these cells. In contrast, we found that HspB2:HspB3 aggregates sequestered several disordered proteins and autophagy factors, suggesting that the cell is actively attempting to clear these aggregates. This study presents a striking example of how changes in the relative expression levels of interacting proteins affects their phase behavior. Our approach could be applied to study the role of protein stoichiometry and the influence of client binding on phase behavior in other biomolecular condensates and aggregates.
Post-translational regulation of protein abundance in cells is a powerful tool for studying protein function. Here, we describe a novel genetically encoded protein domain that is degraded upon ...exposure to nontoxic blue light. We demonstrate that fusion proteins containing this domain are rapidly degraded in cultured cells and in zebrafish upon illumination.
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