Measuring the activity of proteases is essential for investigating both the physiological functions and commercial applications of these enzymes. In contrast to the numerous protease assays that are based on chromogenic or fluorogenic peptide substrates, there is a lack of approaches to monitor degradation of proteins in real time. Here we report a protease assay where SYPRO Orange is employed as a fluorogenic probe to follow proteolysis. The functionality of the assay was demonstrated with the two subtilases of varying thermostability, using four different protein substrates. The assay is compatible with a real-time PCR instrument which allows continuous fluorescence measurements in low-volume samples even at high temperatures. This makes the assay especially suitable for high-throughput characterization of thermostable proteases.

The global impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its companion disease, COVID-19, has reminded us of the importance of basic coronaviral research. In this study, a comprehensive approach using molecular docking, in vitro assays, and molecular dynamics simulations was applied to identify potential inhibitors for SARS-CoV-2 papain-like protease (PL^pro), a key and underexplored viral enzyme target. A focused protease inhibitor library was initially created and molecular docking was performed using CmDock software (v0.2.0), resulting in the selection of hit compounds for in vitro testing on the isolated enzyme. Among them, compound 372 exhibited promising inhibitory properties against PL^pro, with an IC50 value of 82 ± 34 μM. The compound also displayed a new triazolopyrimidinyl scaffold not yet represented within protease inhibitors. Molecular dynamics simulations demonstrated the favorable binding properties of compound 372. Structural analysis highlighted its key interactions with PL^pro, and we stress its potential for further optimization. Moreover, besides compound 372 as a candidate for PL^pro inhibitor development, this study elaborates on the PL^pro binding site dynamics and provides a valuable contribution for further efforts in pan-coronaviral PL^pro inhibitor development.

2-Hydroxyquinoline forms a highly emissive ternary complex with rePON1 : Tb ³⁺ .

Microbial extracellular subtilases are highly active proteolytic enzymes commonly used in commercial applications. These subtilases are synthesized in their inactive proform, which matures into the active protease under the control of the propeptide domain. In mesophilic bacterial prosubtilases, the propeptide functions as both an obligatory chaperone and an inhibitor of the subtilase catalytic domain. In contrast, the propeptides of hyperthermophilic archaeal prosubtilases act mainly as tight inhibitors and are not essential for subtilase folding. It is unclear whether this stronger inhibitory activity of hyperthermophilic propeptides results in their higher selectivity toward their cognate subtilases, in contrast to promiscuous mesophilic propeptides. Here, we showed that the propeptide of pernisine, a hyperthermostable archaeal subtilase, strongly interacts with and inhibits pernisine, but not the homologous subtilisin Carlsberg and proteinase K. Instead, the pernisine propeptide was readily degraded by subtilisin Carlsberg and proteinase K. In addition, the catalytic domain of unprocessed propernisine was also susceptible to degradation but became proteolytically stable after autoprocessing of propernisine into the inactive, noncovalent complex propeptide:pernisine. This allowed efficient transactivation of the autoprocessed complex propeptide:pernisine through degradation of pernisine propeptide by subtilisin Carlsberg and proteinase K at mesophilic temperature. Moreover, we demonstrated that active pernisine molecules are inhibited by the propeptide that is released after pernisine-catalyzed degradation of the unprocessed propernisine catalytic domain. This highlights the high inhibitory potency of the hyperthermophilic propeptide toward its cognate subtilase and its importance in regulating subtilase maturation, to prevent the degradation of the unprocessed subtilase precursors by the prematurely activated molecules. IMPORTANCE: Many microorganisms secrete proteases into their environment to degrade protein substrates for their growth. The important group of these extracellular enzymes are subtilases, which are also widely used in practical applications. These subtilases are inhibited by their propeptide domain, which is degraded during the prosubtilase maturation process. Here, we showed that the propeptide of pernisine, a prion-degrading subtilase from the hyperthermophilic archaeon, strongly inhibits pernisine with extraordinarily high binding affinity. This interaction proved to be highly selective, as pernisine propeptide was rapidly degraded by mesophilic pernisine homologs. This in turn allowed rapid transactivation of propernisine by mesophilic subtilases at lower temperatures, which might simplify the procedures for preparation of active pernisine for commercial use. The results reported in this study suggest that the hyperthermophilic subtilase propeptide evolved to function as tight and selective regulator of maturation of the associated prosubtilase to prevent its premature activation under high temperatures.

Chaperones ensure protein homeostasis and are conserved across species. The ATP-dependent chaperone Hsp90 is present from bacteria to eukaryotes, where it stabilizes and activates a wide range of substrate proteins called clients. However, what determines whether a protein depends on Hsp90 remains an open question. Here, we focused on the bacterial chaperone Hsp90 and its obligate client TilS (referred to as TilS_So) in the bacterium Shewanella oneidensis. Although Hsp90 is indispensable in S. oneidensis under heat stress by protecting the essential protein TilS_So from degradation by the protease HslUV, Hsp90 is dispensable in Escherichia coli, suggesting that E. coli TilS (TilS_Ec) is Hsp90 independent. We therefore compared the TilS orthologs with respect to in vitro stability, in vivo degradation, and interaction with Hsp90 to identify determinants of Hsp90 dependence. We found that in contrast to TilS_So, TilS_Ec was more stable, was not degraded by protease in the absence of Hsp90, and did not interact with Hsp90, indicating that TilS_Ec is not a client of Hsp90. Chimeras between TilS_So and TilS_Ec as well as directed mutagenesis revealed a region of TilS_So that is key for protease degradation and Hsp90 protection. Consistent with these results, the growth of S. oneidensis producing TilS_Ec was no longer dependent on Hsp90 under heat stress. Conversely, Hsp90 became essential for the growth of E. coli that produced TilS_So instead of TilS_Ec. Altogether, our work reveals that protein-specific features, such as stability and degradation sensitivity, can determine whether orthologous proteins require the bacterial Hsp90 chaperone in vivo.

Erylysin A (EryA), an aegerolysin protein produced by the edible king oyster mushroom ( Pleurotus eryngii ), interacts strongly with an invertebrate‐specific membrane sphingolipid ceramide phosphoethanolamine. Recently, a fluorescently fused variant of EryA was shown to bind to artificial and bacterial lipid membranes containing cardiolipin (CL). This tetra‐acylated glycerophospholipid, present in bacteria and in inner mitochondrial membranes of eukaryotic cells, was shown to be externalized to the plasma membrane surface during the process of apoptosis. In this work, we evaluated the interaction of EryA‐mCherry with CL‐containing artificial lipid vesicles and with mammalian cells undergoing apoptosis and compared its binding affinity and specificity to that of the well‐established apoptosis marker, annexin V‐FITC. Our results show that, in contrast to annexin V‐FITC, which binds several negatively charged glycerophospholipids, EryA‐mCherry specifically recognizes and binds CL in artificial membrane systems. However, this binding of EryA‐mCherry to CL‐supplemented membranes is less effective ( K _D = 4.7 ± 1.6 μ m ) than that of annexin V‐FITC, whose binding is observed at nanomolar concentrations. Experiments using mammalian cells showed the ability of EryA‐mCherry to selectively label the membranes of apoptotic cells, binding to the same membrane regions as anti‐CL antibodies and annexin V‐FITC. Our data suggest that EryA‐mCherry might be used as a marker of early apoptosis, as well as a marker of CL in biological and artificial lipid membranes.