Engineered reactivity of a bacterial E1-like enzyme enables ATP-driven modification of protein C termini.

In biological systems, ATP provides an energetic driving force for peptide bond formation, but protein chemists lack tools that emulate this strategy. Inspired by the eukaryotic ubiquitination cascade, we developed an ATP-driven platform for C-terminal activation and peptide ligation based on E. coli MccB, a bacterial ancestor of ubiquitin-activating (E1) enzymes that natively catalyzes C-terminal phosphoramidate bond formation. We show that MccB can act on non-native substrates to generate an O-AMPylated electrophile that can react with exogenous nucleophiles to form diverse C-terminal functional groups including thioesters, a versatile class of biological intermediates that have been exploited for protein semisynthesis. To direct this activity towards specific proteins of interest, we developed the Thioesterification C-terminal Handle (TeCH)-tag, a sequence that enables high-yield, ATP-driven protein bioconjugation via a thioester intermediate. By mining the natural diversity of the MccB family, we developed two additional MccB/TeCH-tag pairs that are mutually orthogonal to each other and to the E. coli system, facilitating the synthesis of more complex bioconjugates. Our method mimics the chemical logic of peptide bond synthesis that is widespread in biology for high-yield in vitro manipulation of protein structure with molecular precision.

An N terminomics toolbox combining 2-pyridinecarboxaldehyde probes and click chemistry for profiling protease specificity.

Proteomic profiling of protease-generated N termini provides key insights into protease function and specificity. However, current technologies have sequence limitations or require specialized synthetic reagents for N-terminal peptide isolation. Here, we introduce an N terminomics toolbox that combines selective N-terminal biotinylation using 2-pyridinecarboxaldehyde (2PCA) reagents with chemically cleavable linkers to enable efficient enrichment of protein N termini. By incorporating a commercially available alkyne-modified 2PCA in combination with Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), our strategy eliminates the need for chemical synthesis of N-terminal probes. Using these reagents, we developed PICS2 (Proteomic Identification of Cleavage Sites with 2PCA) to profile the specificity of subtilisin/kexin-type proprotein convertases (PCSKs). We also implemented CHOPPER (chemical enrichment of protease substrates with purchasable, elutable reagents) for global sequencing of apoptotic proteolytic cleavage sites. Based on their broad applicability and ease of implementation, PICS2 and CHOPPER are useful tools that will advance our understanding of protease biology.

Pre-breeding androgen and glucocorticoid profiles in the eastern hellbender salamander (Cryptobranchus alleganiensis alleganiensis).

Seasonally breeding species exhibit cyclical changes in circulating steroid hormone profiles that correspond with changes to their reproductive behavior and ecology. Such information is critical to the conservation of imperiled and data-deficient species, such as the eastern hellbender salamander (Cryptobranchus alleganiensis alleganiensis). We determined changes in plasma testosterone (T), dihydrotestosterone (DHT), 11-ketotestosterone (11-KT), 11-ketoandrostenedione (11-KA), dehydroepiandrosterone (DHEA), cortisol, corticosterone, and progesterone (P4) during a four-month period preceding breeding in adult male and female eastern hellbenders. This pre-breeding period is characterized by increased diel movement and aggression by both sexes, follicular development and yolk production in females, and sperm production, territoriality, and nest site establishment in males. In both males and females, we observed a progressive increase in circulating T and DHT during the pre-reproductive season, both peaking in August (17 days before breeding), but concentrations of both hormones were higher in males. Conversely, 11-KT was higher in females, but did not vary significantly by date. These results suggest that T and DHT are the predominant androgens in eastern hellbenders and are likely important regulators of reproductive processes in both males and females. The detection of significant quantities of DHT and 11-KT in females is particularly interesting, considering that unlike T, neither of these androgens can be converted to estrogens. Therefore, it seems possible that aggression or some aspect of reproduction in the female eastern hellbender may be directly mediated by androgen signaling. Baseline cortisol did not vary throughout the pre-breeding period but was higher in females than males, and also became highly variable in females leading up to breeding. Progesterone, 11-KA, DHEA, and corticosterone were rarely or never detected, and thus, do not appear to be important during the pre-reproductive season. This study provides a physiological framework for future studies of hellbender reproductive biology, which could ultimately be important for their conservation.

Engineered peptide ligases for cell signaling and bioconjugation.

Enzymes that catalyze peptide ligation are powerful tools for site-specific protein bioconjugation and the study of cellular signaling. Peptide ligases can be divided into two classes: proteases that have been engineered to favor peptide ligation, and protease-related enzymes with naturally evolved peptide ligation activity. Here, we provide a review of key natural peptide ligases and proteases engineered to favor peptide ligation activity. We cover the protein engineering approaches used to generate and improve these tools, along with recent biological applications, advantages, and limitations associated with each enzyme. Finally, we address future challenges and opportunities for further development of peptide ligases as tools for biological research.