Protease Substrates Research Articles

From Peptides to Receptors

—In the 1960s and 1970s, the Institute of Chemistry of Natural Compounds developed a topochemical approach for designing new biologically active peptide compounds, the applicability of which to the creation of inhibitors and effective substrates of proteolytic enzymes was shown by the author of this review under the direct supervision of V.T. Ivanov. The next task was to establish the conformation of protein neurotoxins from snake venoms and to study the topography of their binding to the target, the nicotinic acetylcholine receptor (nAChR) from the electric organ of the Torpedo marmorata ray. With selectively labeled derivatives containing one fluorescent or spin label on established amino acid residues, neurotoxin residues in contact with nAChR were identified for the first time. Later, in collaboration with the laboratory of V.T. Ivanov, new analogs of α-conotoxins (peptide neurotoxins from venomous Conus mollusks), were synthesized including their photoactivated derivatives, which showed the participation of all Torpedo nAChR subunits in the binding of α-conotoxins. In the final part, the review briefly presents the recent achievements of the Department of Molecular Neuroimmune Signaling (headed by V.I. Tsetlin) concerning the isolation and synthesis of new peptide and protein neurotoxins and the study of how they work.

Proteome-Derived Peptide Libraries for Deep Specificity Profiling of N-terminal Modification Reagents.

Protein and peptide N termini are important targets for selective modification with chemoproteomics reagents and bioconjugation tools. The N-terminal ⍺-amine occurs only once in each polypeptide chain, making it an attractive target for protein bioconjugation. In cells, new N termini can be generated by proteolytic cleavage and captured by N-terminal modification reagents that enable proteome-wide identification of protease substrates through tandem mass spectrometry (LC-MS/MS). An understanding of the N-terminal sequence specificity of the modification reagents is critical for each of these applications. Proteome-derived peptide libraries in combination with LC-MS/MS are powerful tools for profiling the sequence specificity of N-terminal modification reagents. These libraries are highly diverse, and LC-MS/MS enables analysis of the modification efficiencies of tens of thousands of sequences in a single experiment. Proteome-derived peptide libraries are a powerful tool for profiling the sequence specificities of enzymatic and chemical peptide labeling reagents. Subtiligase, an enzymatic modification reagent, and 2-pyridinecarboxaldehyde (2PCA), a chemical modification reagent, are two reagents that have been developed for selective N-terminal peptide modification and can be studied using proteome-derived peptide libraries. This protocol outlines the steps for generating N-terminally diverse proteome-derived peptide libraries and for applying these libraries to profile the specificity of N-terminal modification reagents. Although we detail the steps for profiling the specificity of 2PCA and subtiligase in Escherichia coli and human cells, these protocols can easily be adapted to alternative proteome sources and other N-terminal peptide labeling reagents. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of N-terminally diverse proteome-derived peptide libraries from E. coli Alternate Protocol: Generation of N-terminally diverse proteome-derived peptide libraries from human cells Basic Protocol 2: Characterizing the specificity of 2-pyridinecarboxaldehyde using proteome-derived peptide libraries Basic Protocol 3: Characterizing the specificity of subtiligase using proteome-derived peptide libraries.

Degron Pathways and Leishmaniasis: Debating Potential Roles of Leishmania spp. Proteases Activity on Guiding Hosts Immune Response and Their Relevance to the Development of Vaccines.

Degrons are short peptide sequences that signalize target sites for protein degradation by proteases. Herein, we bring forth the discussion on degrons present in proteins related to the immune system of Mus musculus that are potential targets for cysteine and serine proteases of Leishmania spp. and their possible roles on host immune regulation by parasites. The Merops database was used to identify protease substrates and proteases sequence motifs, while MAST/MEME Suite was applied to find degron motifs in murine cytokines (IFN-y, IL-4, IL-5, IL-13, IL-17) and transcription factors (NF-kappaB, STAT-1, AP-1, CREB, and BACH2). STRING tool was used to construct an interaction network for the immune factors and SWISS-MODEL server to generate three-dimensional models of proteins. In silico assays confirm the occurrence of degrons in the selected immune response factors. Further analyses were conducted only in those with resolved three-dimensional structures. The predicted interaction network of degron-containing M. musculus proteins shows the possibility that the specific activity of parasite proteases could interfere with the trend of Th1/Th2 immune responses. Data suggest that degrons may play a role in the immune responses in leishmaniases as targets for parasite proteases activity, directing the degradation of specific immune-related factors.

Identification of SARS-CoV-2 PLpro and 3CLpro human proteome substrates using substrate phage display coupled with protein network analysis

Viral proteases play key roles in viral replication, and they also facilitate immune escape by proteolyzing diverse target proteins. Deep profiling of viral protease substrates in host cells is beneficial for understanding viral pathogenesis and for antiviral drug discovery. Here, we utilized substrate phage display coupled with protein network analysis to identify human proteome substrates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteases, including papain-like protease (PLpro) and 3C-like protease (3CLpro). We first performed peptide substrates selection of PLpro and 3CLpro, and we then used the top 24 preferred substrate sequences to identify a total of 290 putative protein substrates. Protein network analysis revealed that the top clusters of PLpro and 3CLpro substrate proteins contain ubiquitin-related proteins and cadherin-related proteins, respectively. We verified that cadherin-6 and cadherin-12 are novel substrates of 3CLpro, and CD177 is a novel substrate of PLpro using invitro cleavage assays. We thus demonstrated that substrate phage display coupled with protein network analysis is a simple and high throughput method to identify human proteome substrates of SARS-CoV-2 viral proteases for further understanding of virus-host interactions.

From Peptides to Receptors

In the 1960s and 1970s, the Institute of Chemistry of Natural Compounds developed a topochemical approach for designing new biologically active peptide compounds, the applicability of which to the creation of inhibitors and effective substrates of proteolytic enzymes was shown by the author of this review under the direct supervision of V.T. Ivanov. The next task was to establish the conformation of protein neurotoxins from snake venoms and to study the topography of their binding to the target, the nicotinic acetylcholine receptor (nAChR) from the electric organ of the Torpedo marmorata ray. With selectively labeled derivatives containing one fluorescent or spin label on established amino acid residues, neurotoxin residues in contact with nAChR were identified for the first time. Later, in collaboration with the laboratory of V.T. Ivanov, new analogues of α-conotoxins (peptide neurotoxins from poisonous mollusks Conus), were synthesized including their photoactivated derivatives, which showed the participation of all Torpedo nAChR subunits in the binding of α‑conotoxins. The final part of the review briefly presents the recent achievements of the Department of Molecular Neuroimmune Signaling (headed by V.I. Tsetlin) concerning the isolation and synthesis of new peptide and protein neurotoxins and the study of their mechanism of action.

Switch-on Fluorescence Analysis of Protease Activity with the Assistance of a Nickel Ion-Nitrilotriacetic Acid-Conjugated Magnetic Nanoparticle.

Heterogeneous protease biosensors show high sensitivity and selectivity but usually require the immobilization of peptide substrates on a solid interface. Such methods exhibit the disadvantages of complex immobilization steps and low enzymatic efficiency induced by steric hindrance. In this work, we proposed an immobilization-free strategy for protease detection with high simplicity, sensitivity and selectivity. Specifically, a single-labeled peptide with oligohistidine-tag (His-tag) was designed as the protease substrate, which can be captured by a nickel ion-nitrilotriacetic acid (Ni-NTA)-conjugated magnetic nanoparticle (MNP) through the coordination interaction between His-tag and Ni-NTA. When the peptide was digested by protease in a homogeneous solution, the signal-labeled segment was released from the substrate. The unreacted peptide substrates could be removed by Ni-NTA-MNP, and the released segments remained in solution to emit strong fluorescence. The method was used to determine protease of caspase-3 with a low detection limit (4 pg/mL). By changing the peptide sequence and signal reporters, the proposal could be used to develop novel homogeneous biosensors for the detection of other proteases.

Identification of Human Host Substrates of the SARS-CoV-2 Mpro and PLpro Using Subtiligase N-Terminomics.

The recent emergence of SARS-CoV-2 in the human population has caused a global pandemic. The virus encodes two proteases, Mpro and PLpro, that are thought to play key roles in the suppression of host protein synthesis and immune response evasion during infection. To identify the specific host cell substrates of these proteases, active recombinant SARS-CoV-2 Mpro and PLpro were added to A549 and Jurkat human cell lysates, and subtiligase-mediated N-terminomics was used to capture and enrich protease substrate fragments. The precise location of each cleavage site was identified using mass spectrometry. Here, we report the identification of over 200 human host proteins that are potential substrates for SARS-CoV-2 Mpro and PLpro and provide a global mapping of proteolysis for these two viral proteases in vitro. Modulating proteolysis of these substrates will increase our understanding of SARS-CoV-2 pathobiology and COVID-19.

Conditionally activated affibody-based prodrug targeting EGFR demonstrates improved tumour selectivity

Safety and efficacy of cancer-targeting treatments can be improved by conditional activation enabled by the distinct milieu of the tumour microenvironment. Proteases are intricately involved in tumourigenesis and commonly dysregulated with elevated expression and activity. Design of prodrug molecules with protease-dependent activation has the potential to increase tumour-selective targeting while decreasing exposure to healthy tissues, thus improving the safety profile for patients. Higher selectivity could also allow for administration of higher doses or use of more aggressive treatment options, leading to higher therapeutic efficacy. We have previously developed an affibody-based prodrug with conditional targeting of EGFR conferred by an anti-idiotypic affibody masking domain (ZB05). We could show that binding to endogenous EGFR on cancer cells in vitro was restored following proteolytic removal of ZB05. In this study we evaluate a novel affibody-based prodrug design, which incorporates a protease substrate sequence recognized by cancer-associated proteases and demonstrate the potential of this approach for selective tumour-targeting and shielded uptake in healthy tissues in vivo using tumour-bearing mice. This may widen the therapeutic index of cytotoxic EGFR-targeted therapeutics by decreasing side effects, improving selectivity of drug delivery, and enabling the use of more potent cytotoxic drugs.

Degradation mechanism of AtrA mediated by ClpXP and its application in daptomycin production in Streptomyces roseosporus.

The efficiency of drug biosynthesis depends on different transcriptional regulatory pathways in Streptomyces, and the protein degradation system adds another layer of complexity to the regulatory processes. AtrA, a transcriptional regulator in the A-factor regulatory cascade, stimulates the production of daptomycin by binding to the dptE promoter in Streptomyces roseosporus. Using pull-down assays, bacterial two-hybrid system and knockout verification, we demonstrated that AtrA is a substrate for ClpP protease. Furthermore, we showed that ClpX is necessary for AtrA recognition and subsequent degradation. Bioinformatics analysis, truncating mutation, and overexpression proved that the AAA motifs of AtrA were essential for initial recognition in the degradation process. Finally, overexpression of mutated atrA (AAA-QQQ) in S. roseosporus increased the yield of daptomycin by 225% in shake flask and by 164% in the 15 L bioreactor. Thus, improving the stability of key regulators is an effective method to promote the ability of antibiotic synthesis.

Cloning of BHV-UL25 Conserved Fragment in a Lentiviral Transfer Plasmid for the Preparation of a Monitoring Cell Line

: Bovine herpesvirus-1 (BHV-1) is the causative agent of a domestic and wild cow disease namely infectious bovine rhinotracheitis (IBR). Severe economic damage due to IBR is possible to occur and vaccines are not completely effective against the disease. Therefore, over the recent years, the development of effective and genetic-based therapies to control viral infections, such as IBR has been remarkable. A common way to evaluate such therapeutic strategies is cloning of the viral target sequence into appropriate vectors for the preparation of cell lines expressing viral subgenomic replicons. Due to the required duties of UL25 gene, serine protease substrate domain of this gene was cloned in pCDH-CMV-MCS-EF1-cGFP-T2A-Puro lentiviral vector at the upstream of GFP gene. The cloning accuracy was verified by restriction of enzyme digestion and sequencing. Thus, this recombinant plasmid will be available to produce lentiviral vectors with the desired gene; after infection of eukaryotic cells with such lentiviral vectors the target gene will be expressed.

NMR structure of emfourin, a novel protein metalloprotease inhibitor: Insights into the mechanism of action

Emfourin (M4in) is a protein metalloprotease inhibitor recently discovered in the bacterium Serratia proteamaculans and the prototype of a new family of protein protease inhibitors with an unknown mechanism of action. Protealysin-like proteases (PLPs) of the thermolysin family are natural targets of emfourin-like inhibitors widespread in bacteria and known in archaea. The available data indicate the involvement of PLPs in interbacterial interaction as well as bacterial interaction with other organisms and likely in pathogenesis. Arguably, emfourin-like inhibitors participate in the regulation of bacterial pathogenesis by controlling PLP activity. Here, we determined the 3D structure of M4in using solution NMR spectroscopy. The obtained structure demonstrated no significant similarity to known protein structures. This structure was used to model the M4in-enzyme complex and the complex model was verified by small-angle X-ray scattering. Based on the model analysis, we propose a molecular mechanism for the inhibitor, which was confirmed by site-directed mutagenesis. We show that two spatially close flexible loop regions are critical for the inhibitor-protease interaction. One region includes aspartic acid forming a coordination bond with catalytic Zn2+ of the enzyme and the second region carries hydrophobic amino acids interacting with protease substrate binding sites. Such an active site structure corresponds to the noncanonical inhibition mechanism. This is the first demonstration of such a mechanism for protein inhibitors of thermolysin family metalloproteases, which puts forward M4in as a new basis for the development of antibacterial agents relying on selective inhibition of prominent factors of bacterial pathogenesis belonging to this family.

Display of a naïve affibody library on staphylococci for selection of binders by means of flow cytometry sorting

Within the field of combinatorial protein engineering there is a great demand for robust high-throughput selection platforms that allow for unbiased protein library display, affinity-based screening, and amplification of selected clones. We have previously described the development of a staphylococcal display system used for displaying both alternative-scaffolds and antibody-derived proteins. In this study, the objective was to generate an improved expression vector for displaying and screening a high-complexity naïve affibody library, and to facilitate downstream validation of isolated clones. A high-affinity normalization tag, consisting of two ABD-moieties, was introduced to simplify off-rate screening procedures. In addition, the vector was furnished with a TEV protease substrate recognition sequence upstream of the protein library which enables proteolytic processing of the displayed construct for improved binding signal. In the library design, 13 of the 58 surface-exposed amino acid positions were selected for full randomization (except proline and cysteine) using trinucleotide technology. The genetic library was successfully transformed to Staphylococcus carnosus cells, generating a protein library exceeding 109 members. De novo selections against three target proteins (CD14, MAPK9 and the affibody ZEGFR:2377) were successfully performed using magnetic bead-based capture followed by flow-cytometric sorting, yielding affibody molecules binding their respective target with nanomolar affinity. Taken together, the results demonstrate the feasibility of the staphylococcal display system and the proposed selection procedure to generate new affibody molecules with high affinity.

Identification of SARS-CoV-2 Main Protease (Mpro) Cleavage Sites Using Two-Dimensional Electrophoresis and In Silico Cleavage Site Prediction

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a crucial role in its life cycle. The Mpro-mediated limited proteolysis of the viral polyproteins is necessary for the replication of the virus, and cleavage of the host proteins of the infected cells may also contribute to viral pathogenesis, such as evading the immune responses or triggering cell toxicity. Therefore, the identification of host substrates of the viral protease is of special interest. To identify cleavage sites in cellular substrates of SARS-CoV-2 Mpro, we determined changes in the HEK293T cellular proteome upon expression of the Mpro using two-dimensional gel electrophoresis. The candidate cellular substrates of Mpro were identified by mass spectrometry, and then potential cleavage sites were predicted in silico using NetCorona 1.0 and 3CLP web servers. The existence of the predicted cleavage sites was investigated by in vitro cleavage reactions using recombinant protein substrates containing the candidate target sequences, followed by the determination of cleavage positions using mass spectrometry. Unknown and previously described SARS-CoV-2 Mpro cleavage sites and cellular substrates were also identified. Identification of target sequences is important to understand the specificity of the enzyme, as well as aiding the improvement and development of computational methods for cleavage site prediction.

The conformational space of the SARS-CoV-2 main protease active site loops is determined by ligand binding and interprotomer allostery.

In this study, we characterize two SARS-CoV-2 main protease (Mpro) active site loops that play a role in catalysis using large scale all-atom molecular dynamics simulations. Machine-learning methods are used to identify open and closed states in both the upper and lower loops, as well as an additional intermediate state in the lower loop. In the closed and intermediate states, the positions of the catalytic Cys-His dyad are stabilized, bringing Asp187 into position to mediate charge from a proton transfer during catalysis. Simulations of a protease substrate mimetic complex exhibit an increased frequency of closed and intermediate states and reveals that a substrate binding to one protomer of the homodimer causes its apo partner to more closely resemble the structural ensemble of the bound state. We use dynamic network analysis to reveal the optimal allosteric path between active sites, which travels through a pathway bridged by the N-terminus, providing useful information for the development of potential allosteric inhibitors. This study offers insight into relationships between the flexible loops and the substrate binding in a prime drug target.

Ecotin: A versatile protease inhibitor of bacteria and eukaryotes.

Serine protease inhibitors are a large family of proteins involved in important pathways and processes, such as inflammatory responses and blood clotting. Most are characterized by a precise mode of action, thereby targeting a narrow range of protease substrates. However, the serine-protease inhibitor ecotin is able to inhibit a broad range of serine proteases that display a wide range of specificities. This specificity is driven by special structural features which allow unique flexibility upon binding to targets. Although frequently observed in many human/animal-associated bacteria, ecotin homologs may also be found in plant-associated taxa and environmental species. The purpose of this review is to provide an update on the biological importance, role in host-microbe interactions, and evolutionary relationship between ecotin orthologs isolated from Eukaryotic and Prokaryotic species across the Tree of Life.

Identification of Substrates of Secreted Bacterial Protease by APEX2-Based Proximity Labeling.

The interactions between bacterial virulence factors and host receptors play a critical role during bacterial infection. Therefore, the identification of interactions between host receptor and bacterial virulence factors can not only elucidate the molecular mechanisms of bacterial infection but also provide a framework for new therapeutic and prevention strategies. Herein, we report an APEX2-based live cell proximity labeling strategy in combination with high-resolution quantitative mass spectrometry to profile the substrates of Helicobacter pylori HtrA protease on the membrane of human stomach epithelial cells. This strategy can be further applied to identify other interactions between secreted bacterial virulence factors and host receptors on live cells.

Sensitive and High-Throughput Exploration of Protein N-Termini by TMT-TAILS N-Terminomics.

Terminal amine isotopic labeling of substrates (TAILS) is a sensitive and robust quantitative mass spectrometry (MS)-based proteomics method used for the characterization of physiological or proteolytically processed protein N-termini, as well as other N-terminal posttranslational modifications (PTMs). TAILS is a well-established, high-throughput, negative enrichment workflow that enables system-wide exploration of N-terminomes independent of sample complexity. TAILS makes use of amine reactivity of free N-termini and a highly efficient aldehyde-functionalized polymer to deplete internal peptides generated after proteolytic digestion during sample preparation. Thereby, it enriches for natural N-termini, allowing for unbiased and complete investigation of differential proteolysis, protease substrate discovery, and analysis of N-terminal PTMs. In this chapter, we provide a state-of-the-art protocol, with detailed steps in all parts of the TAILS sample preparation, MS analysis, and post-processing of acquired data.

Lab-on-a-chip for the easy and visual detection of SARS-CoV-2 in saliva based on sensory polymers

The initial stages of the pandemic caused by SARS-CoV-2 showed that early detection of the virus in a simple way is the best tool until the development of vaccines. Many different tests are invasive or need the patient to cough up or even drag a sample of mucus from the throat area. Besides, the manufacturing time has proven insufficient in pandemic conditions since they were out of stock in many countries. Here we show a new method of manufacturing virus sensors and a proof of concept with SARS-CoV-2. We found that a fluorogenic peptide substrate of the main protease of the virus (Mpro) can be covalently immobilized in a polymer, with which a cellulose-based material can be coated. These sensory labels fluoresce with a single saliva sample of a positive COVID-19 patient. The results matched with that of the antigen tests in 22 of 26 studied cases (85% success rate).

Interaction models between peptide substrate and Alphavirus Protease nsP2 of Chikungunya and Mayaro and implications to the mechanism of action

The Arbovirus (Arthropod-borne virus) is a group which comprises viruses whose transmission is carried out by arthropod vectors infecting vertebrates. Some arboviruses related to human diseases have been given considerable relevance as Chikungunya and Mayaro of the family Togaviridae, genus Alphavirus. The lack of proper specific treatment has prompted the requirement for deeper structural studies that could unveil leads to new drugs. Among possible targets, viral proteases are recognized as proteins with big potential. These proteins, termed nsP2 in Alphavirus, have the function of cleaving certain regions of the viral polyprotein, being vital to the viral cycle. In this research, we used docking and molecular dynamics to analyze the contact between the protease nsP2 of Alphavirus Chikungunya and Mayaro and substrates formed by peptides with ten amino acid residues. A model of the Mayaro nsP2 was constructed based on homologous proteases. Our study suggests that the glycine specificity motif, a region where a highly conserved glycine residue in position P2 of the protease substrate is positioned, facilitates the nucleophilic attack by assisting in placing the P1 carbonyl group carbon. Stabilization of different substrate regions maybe explained by relevant contacts with the enzyme. Besides that, the phi and psi angles in the outlier region of the Ramachandran plot found for the P2 glycine of the Chikungunya substrate seems to indicate the necessity of this residue that can accommodate angles not allowed to other residues. Communicated by Ramaswamy H. Sarma

A Type I and a Type II Metacaspase Are Differentially Regulated during Corolla Development and in Response to Abiotic and Biotic Stresses in Petunia × hybrida

Metacaspases are structural homologs of the metazoan caspases that are found in plants, fungi, and protozoans. They are cysteine proteases that function during programmed cell death, stress, and cell proliferation. A putative metacaspase designated PhMC2 was cloned from Petunia × hybrida, and sequence alignment and phylogenetic analysis revealed that it encodes a type II metacaspase. PhMC2 cleaved protease substrates with an arginine residue at the P1 site and cysteine (iodoacetamide) and arginal (leupeptin) protease inhibitors nearly abolished this activity. The activity of PhMC2 was highest at pH 8, and the putative catalytic site cysteine residue was required for optimal activity. Quantitative PCR showed that PhMC2 transcripts were detectable in petunia corollas, styles, and ovaries. Expression patterns were not upregulated during petal senescence but were higher at the middle stages of development when flower corollas were fully open but not yet starting to wilt. PhMC1, a type I metacaspase previously identified in petunia, and PhMC2 were differentially regulated in vegetative tissues in response to biotic and abiotic stresses. PhMC2 expression was upregulated to a greater extent than PhMC1 following Botrytis cinerea infection, while PhMC1 was upregulated more by drought, salinity, and low nutrient stress. These results suggest that petunia metacaspases are involved in flower development, senescence, and stress responses.