Film-like organelles equip cells with multiple genetic codes.

The simultaneous incorporation of distinct noncanonical amino acids into different proteins within eukaryotic cells remains challenging. This new study by Reinkemeierand Lemke demonstrates how 2D phase separation can be used to engineer spatially separated organelles. These film-like organelles translate proteins independently from each other and the canonical genetic code.

Investigating eosin Y as a photocatalyst for the radical-dependent activity-based probing of deubiquitinating enzymes.

Eosin Y was assessed for its ability to induce a thiol-ene dependent protein-protein reaction in a metal-free, oxygen-tolerant, visible light mediated system. Protein-protein coupling efficiency under these mild conditions was comparable to previously reported UV-dependent conditions. The desired thiol-ene reaction was however limited within more complex biological systems.

Probing enzymatic activity - a radical approach.

Deubiquitinating enzymes (DUBs) are known to have numerous important interactions with the ubiquitin cascade and their dysregulation is associated with several diseases, including cancer and neurodegeneration. They are an important class of enzyme, and activity-based probes have been developed as an effective strategy to study them. Existing activity-based probes that target the active site of these enzymes work via nucleophilic mechanisms. We present the development of latent ubiquitin-based probes that target DUBs via a site selective, photoinitiated radical mechanism. This approach differs from existing photocrosslinking probes as it requires a free active site cysteine. In contrast to existing cysteine reactive probes, control over the timing of the enzyme-probe reaction is possible as the alkene warhead is completely inert under ambient conditions, even upon probe binding. The probe's reactivity has been demonstrated against recombinant DUBs and to capture endogenous DUB activity in cell lysate. This allows more finely resolved investigations of DUBs.

Strategies to Target Specific Components of the Ubiquitin Conjugation/Deconjugation Machinery.

The regulation of ubiquitination status in the cell is controlled by ubiquitin ligases acting in tandem with deubiquitinating enzymes. Ubiquitination controls many key processes in the cell from division to death making its tight regulation key to optimal cell function. Activity based protein profiling has emerged as a powerful technique to study these important enzymes. With around 100 deubiquitinating enzymes and 600 ubiquitin ligases in the human genome targeting a subclass of these enzymes or even a single enzyme is a compelling strategy to unpick this complex system. In this review we will discuss different approaches adopted, including activity-based probes centered around ubiquitin-protein, ubiquitin-peptide and mutated ubiquitin scaffolds. We examine challenges faced and opportunities presented to increase specificity in activity-based protein profiling of the ubiquitin conjugation/deconjugation machinery.