Novel phage defense systems featuring diverse enzymatic activities are continually being discovered. Among these, defense systems employing proteolytic enzymes have been identified, revealing a previously unrecognized enzymatic activity in phage defense. These protease-associated defense systems represent an untapped reservoir for new biotechnological tools and may serve as a springboard for the development of proteome editors. This review outlines recent advancements in the discovery and characterization of protease-containing defense systems, proposes methods for further exploration and investigation of protease activity, and considers the prospect of protease defense systems for modulating protein processing and cell fate.

Proteolytic cleavage is an irreversible post-translational modification (PTM), and dysregulation of protease activity is often a hallmark in disease. Aberrant proteolysis can alter protein abundance or function, disturbing cellular state and resulting in disease-specific biomarkers or therapeutic targets. Positional proteomics facilitates global identification and precise quantification of position-specific peptides, such as those located N- or C-terminal in the protein sequence. These techniques enable the study of both natural and neo-protein termini, as well as associated PTMs. Despite its importance, proteolysis remains understudied due to experimental challenges and complex data processing. In this review, we outline key strategies for data analysis and processing in positional proteomics, emphasizing how identification, quantification, and interpretation of proteolytic cleavage sites differ from standard proteomics data analysis pipelines. We discuss differences in common approaches for terminomics-focused workflows, comparing N- versus C-terminomics, as well as different labeling strategies and acquisition methods. Additionally, we highlight considerations for proper normalization approaches, specifically the need to normalize cleavage abundances relative to protein and protease abundance. We explain the importance of integrating structural data, solvent accessibility, and tissue expression profiles during data analysis to better evaluate the biological significance of experimental results.

The evolutionary arms race between bacteria and bacteriophages drives rapid evolution of bacterial defense mechanisms with scattered distribution across genomes. We hypothesized that this variability in bacterial defense systems leads to equally variable counter-defense repertoires in phage genomes. Examining the variable regions in Pseudomonas model phages of the Pbunavirus genus revealed five anti-defense genes, including one inhibiting Druantia type III named DadIII-1, another targeting Thoeris type III named TadIII-1, one inhibiting Zorya type I named ZadI-1, and two related broad defense inhibitors named Bdi1 and Bdi2 targeting four defenses. A typical Pbunavirus encodes up to five known anti-defense genes, some inhibiting four unrelated defense systems with distinct nucleic-acid-targeting mechanisms. Structural homologs of broad-acting Bdi1 and Bdi2 are encoded across diverse phage taxa infecting multiple bacterial hosts. These findings show that phages face a variety of bacterial defenses, driving them to evolve both specific and general strategies to overcome these barriers.