Regulated proteolysis is a vital process that affects all living things. Bacteria use energy-dependent AAA+ proteases to power degradation of misfolded and native regulatory proteins. Given that proteolysis is an irreversible event, specificity and selectivity in degrading substrates are key. Specificity is often augmented through the use of adaptors that modify the inherent specificity of the proteolytic machinery. Regulated protein degradation is intricately linked to quality control, cell-cycle progression, and physiological transitions. In this review, we highlight recent work that has shed light on our understanding of regulated proteolysis in bacteria. We discuss the role AAA+ proteases play during balanced growth as well as how these proteases are deployed during changes in growth. We present examples of how protease selectivity can be controlled in increasingly complex ways. Finally, we describe how coupling a core recognition determinant to one or more modifying agents is a general theme for regulated protein degradation.

The LonA (or Lon) protease is a central post-translational regulator in diverse bacterial species. In Vibrio cholerae, LonA regulates a broad range of behaviors including cell division, biofilm formation, flagellar motility, c-di-GMP levels, the type VI secretion system (T6SS), virulence gene expression, and host colonization. Despite LonA's role in cellular processes critical for V. cholerae's aquatic and infectious life cycles, relatively few LonA substrates have been identified. LonA protease substrates were therefore identified through comparison of the proteomes of wild-type and ΔlonA strains following translational inhibition. The most significantly enriched LonA-dependent protein was TfoY, a known regulator of motility and the T6SS in V. cholerae. Experiments showed that TfoY was required for LonA-mediated repression of motility and T6SS-dependent killing. In addition, TfoY was stabilized under high c-di-GMP conditions and biochemical analysis determined direct binding of c-di-GMP to LonA results in inhibition of its protease activity. The work presented here adds to the list of LonA substrates, identifies LonA as a c-di-GMP receptor, demonstrates that c-di-GMP regulates LonA activity and TfoY protein stability, and helps elucidate the mechanisms by which LonA controls important V. cholerae behaviors.

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Protein degradation is an essential process in all organisms. This process is irreversible and energetically costly; therefore, protein destruction must be tightly controlled. While environmental stresses often lead to upregulation of proteases at the transcriptional level, little is known about post-translational control of these critical machines. In this study we show that in Caulobacter crescentus levels of the Lon protease are controlled through proteolysis. Lon turnover requires active Lon and ClpAP proteases. We show that specific determinants dictate Lon stability with a key carboxy-terminal histidine residue driving recognition. Expression of stabilized Lon variants results in toxic levels of protease that deplete normal Lon substrates such as the replication initiator DnaA to lethally low levels. Taken together, this work demonstrates a feedback mechanism in which ClpAP and Lon collaborate to tune Lon proteolytic capacity for the cell. Importance Proteases are essential, but unrestrained activity can also kill cells by degrading essential proteins. The quality control protease Lon must degrade many misfolded and native substrates. We show that Lon is itself controlled through proteolysis and that bypassing this control results in toxic consequences for the cell.

The lung extracellular matrix (ECM) maintains the structural integrity of the tissue and regulates the phenotype and functions of resident fibroblasts. Lung‐metastatic breast cancer alters these cell‐ECM interactions, promoting fibroblast activation. There is a need for bio‐instructive ECM models that match the ECM composition and biomechanics of the lung to study these cell‐matrix interactions in vitro. Here, a synthetic, bioactive hydrogel is synthesized that mimics the native lung modulus and includes a representative distribution of the most abundant ECM peptide motifs responsible for integrin‐binding and matrix metalloproteinase (MMP)‐mediated degradation in the lung, which enables quiescent culture of human lung fibroblasts (HLFs). Stimulation with transforming growth factor β1 (TGF‐β1), metastatic breast cancer conditioned media (CM), or tenascin‐C‐derived integrin‐binding peptide activated hydrogel‐encapsulated HLFs demonstrates multiple environmental methods to activate HLFs in a lung ECM‐mimicking hydrogel. This lung hydrogel platform is a tunable, synthetic approach to studying the independent and combinatorial effects of ECM in regulating fibroblast quiescence and activation.