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1. Correlational networking guides the discovery of unclustered lanthipeptide protease-encoding genes

   https://doi.org/10.1038/s41467-022-29325-1  

   Xue, Dan; Older, Ethan_A; Zhong, Zheng; Shang, Zhuo; Chen, Nanzhu; Dittenhauser, Nolan; Hou, Lukuan; Cai, Peiyan; Walla, Michael_D; Dong, Shi-Hui; et al (March 2022, Nature Communications)

   Abstract Bacterial natural product biosynthetic genes, canonically clustered, have been increasingly found to rely on hidden enzymes encoded elsewhere in the genome for completion of biosynthesis. The study and application of lanthipeptides are frequently hindered by unclustered protease genes required for final maturation. Here, we establish a global correlation network bridging the gap between lanthipeptide precursors and hidden proteases. Applying our analysis to 161,954 bacterial genomes, we establish 5209 correlations between precursors and hidden proteases, with 91 prioritized. We use network predictions and co-expression analysis to reveal a previously missing protease for the maturation of class I lanthipeptide paenilan. We further discover widely distributed bacterial M16B metallopeptidases of previously unclear biological function as a new family of lanthipeptide proteases. We show the involvement of a pair of bifunctional M16B proteases in the production of previously unreported class III lanthipeptides with high substrate specificity. Together, these results demonstrate the strength of our correlational networking approach to the discovery of hidden lanthipeptide proteases and potentially other missing enzymes for natural products biosynthesis. 

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2. Sequence‐Selective Protection of Peptides from Proteolysis

   https://doi.org/10.1002/anie.202102148  

   Li, Xiaowei; Chen, Kaiqian; Zhao, Yan (March 2021, Angewandte Chemie International Edition)

   Abstract Proteolysis of proteins and peptides is involved in the infection of cells by enveloped viruses and also in the invasion and spread of cancer cells. Shutting down broad‐specificity proteases, however, is problematic because normal functions by these proteases will be affected. Herein, nanoparticle receptors were prepared from molecular imprinting for complex biological peptides. Their strong and selective binding enabled them to protect their targeted sequences from proteolysis in aqueous solution at stoichiometric amounts. Generality of the method was demonstrated by the protection of hydrophobic and hydrophilic peptides from different proteases, selective protection of a segment of a long peptide, and selective protection of a targeted peptide in a mixture. Most interestingly, two receptors targeting different parts of a long peptide could work in cooperation to protect the overall sequence, highlighting the versatility of the method. 

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3. Intra-chloroplast proteases: A holistic network view of chloroplast proteolysis

   https://doi.org/10.1093/plcell/koae178  

   van_Wijk, Klaas_J (June 2024, The Plant Cell)

   Abstract Different proteases and peptidases are present within chloroplasts and nonphotosynthetic plastids to process precursor proteins and to degrade cleaved chloroplast transit peptides and damaged, misfolded, or otherwise unwanted proteins. Collectively, these proteases and peptidases form a proteolysis network, with complementary activities and hierarchies, and build-in redundancies. Furthermore, this network is distributed across the different intra-chloroplast compartments (lumen, thylakoid, stroma, envelope). The challenge is to determine the contributions of each peptidase (system) to this network in chloroplasts and nonphotosynthetic plastids. This will require an understanding of substrate recognition mechanisms, degrons, substrate, and product size limitations, as well as the capacity and degradation kinetics of each protease. Multiple extra-plastidial degradation pathways complement these intra-chloroplast proteases. This review summarizes our current understanding of these intra-chloroplast proteases in Arabidopsis and crop plants with an emphasis on considerations for building a qualitative and quantitative network view. 

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4. Conformational ensembles reveal the origins of serine protease catalysis

   https://doi.org/10.1126/science.ado5068  

   Du, Siyuan; Kretsch, Rachael C; Parres-Gold, Jacob; Pieri, Elisa; Cruzeiro, Vinícius_Wilian D; Zhu, Mingning; Pinney, Margaux M; Yabukarski, Filip; Schwans, Jason P; Martínez, Todd J; et al (February 2025, Science)

   Enzymes exist in ensembles of states that encode the energetics underlying their catalysis. Conformational ensembles built from 1231 structures of 17 serine proteases revealed atomic-level changes across their reaction states. By comparing the enzymatic and solution reaction, we identified molecular features that provide catalysis and quantified their energetic contributions to catalysis. Serine proteases precisely position their reactants in destabilized conformers, creating a downhill energetic gradient that selectively favors the motions required for reaction while limiting off-pathway conformational states. The same catalytic features have repeatedly evolved in proteases and additional enzymes across multiple distinct structural folds. Our ensemble-function analyses revealed previously unknown catalytic features, provided quantitative models based on simple physical and chemical principles, and identified motifs recurrent in nature that may inspire enzyme design. 

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5. Secreted Proteases Control the Timing of Aggregative Community Formation in Vibrio cholerae

   https://doi.org/10.1128/mBio.01518-21  

   Jemielita, Matthew; Mashruwala, Ameya A.; Valastyan, Julie S.; Wingreen, Ned S.; Bassler, Bonnie L. (December 2021, mBio)

   Sourjik, Victor; Vogel, Joerg (Ed.)

   ABSTRACT Bacteria orchestrate collective behaviors using the cell-cell communication process called quorum sensing (QS). QS relies on the synthesis, release, and group-wide detection of small molecules called autoinducers. In Vibrio cholerae , a multicellular community aggregation program occurs in liquid, during the stationary phase, and in the high-cell-density QS state. Here, we demonstrate that this aggregation program consists of two subprograms. In one subprogram, which we call void formation, structures form that contain few cells but provide a scaffold within which cells can embed. The other subprogram relies on flagellar machinery and enables cells to enter voids. A genetic screen for factors contributing to void formation, coupled with companion molecular analyses, showed that four extracellular proteases, Vca0812, Vca0813, HapA, and PrtV, control the onset timing of both void formation and aggregation; moreover, proteolytic activity is required. These proteases, or their downstream products, can be shared between void-producing and non-void-forming cells and can elicit aggregation in a normally nonaggregating V. cholerae strain. Employing multiple proteases to control void formation and aggregation timing could provide a redundant and irreversible path to commitment to this community lifestyle. IMPORTANCE Bacteria can work as collectives to form multicellular communities. Vibrio cholerae , the bacterium that causes the disease cholera in humans, forms aggregated communities in liquid. Aggregate formation relies on a chemical communication process called quorum sensing. Here, we show that, beyond overarching control by quorum sensing, there are two aggregation subprograms. One subprogram, which we call void formation, creates a scaffold within which cells can embed. The second subprogram, which allows bacteria to enter the scaffold, requires motility. We discovered that four extracellular proteases control the timing of both void formation and aggregation. We argue that, by using redundant proteases, V. cholerae ensures the reliable execution of this community formation process. These findings may provide insight into how V. cholerae persists in the marine environment or colonizes the human host, as both lifestyles are central to the spread of the disease cholera. 

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