P1 Residue Research Articles

Design of novel and highly selective SARS-CoV-2 main protease inhibitors.

We have synthesized a novel and highly selective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease peptide mimetic inhibitor mimicking the replicase 1ab recognition sequence -Val-Leu-Gln- and utilizing a cysteine selective acyloxymethyl ketone as the electrophilic warhead to target the active site Cys145. Utilizing a constrained cyclic peptide that locks the conformation between the P3 (Val) and P2 (Leu) residues, we identified a highly selective inhibitor that fills the P2 pocket occupied by the leucine residue sidechain of PF-00835231 and the dimethyl-3-azabicyclo-hexane motif in nirmatrelvir (PF-07321332). This strategy resulted in potent and highly selective Mpro inhibitors without inhibiting essential host cathepsin cysteine or serine proteases. The lead prototype compound 1 (MPro IC50 = 230 ± 18 nM) also inhibits the replication of multiple SARS-CoV-2 variants in vitro, including SARS-CoV-2 variants of concern, and can synergize at lower concentrations with the viral RNA polymerase inhibitor, remdesivir, to inhibit replication. It also reduces SARS-CoV-2 replication in SARS-CoV-2 Omicron-infected Syrian golden hamsters without obvious toxicities, demonstrating in vivo efficacy. This novel lead structure provides the basis for optimization of improved agents targeting evolving SARS-CoV-2 drug resistance that can selectively act on Mpro versus host proteases and are less likely to have off-target effects due to non-specific targeting. Developing inhibitors against the active site of the main protease (Mpro), which is highly conserved across coronaviruses, is expected to impart a higher genetic barrier to evolving SARS-CoV-2 drug resistance. Drugs that selectively inhibit the viral Mpro are less likely to have off-target effects warranting efforts to improve this therapy.

Water-medicated specifically targeting the S1 pockets among serine proteases using an arginine analogue

Because of the high similarity in structure and sequence, it is challenging to distinguish the S1 pocket among serine proteases, primarily due to the only variability at residue 190 (A190 and S190). Peptide or protein-based inhibitors typically target the negatively charged S1 pocket using lysine or arginine as the P1 residue, yet neither discriminates between the two S1 pocket variants. This study introduces two arginine analogues, L-4-guanidinophenylalanine (12) and L-3-(N-amidino-4-piperidyl)alanine (16), as novel P1 residues in peptide inhibitors. 16 notably enhances affinities across all tested proteases, whereas 12 specifically improved affinities towards proteases possessing S190 in the S1 pocket. By crystallography and molecular dynamics simulations, we discovered a novel mechanism involving a water exchange channel at the bottom of the S1 pocket, modulated by the variation of residue 190. Additionally, the specificity of 12 towards the S190-presenting S1 pocket is dependent on this water channel. This study not only introduces novel P1 residues to engineer inhibitory potency and specificity of peptide inhibitors targeting serine proteases, but also unveils a water-mediated molecular mechanism of targeting serine proteases.

A structural basis of T cell cross-reactivity to native and spliced self-antigens presented by HLA-DQ8

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that has a strong HLA association, where a number of self-epitopes have been implicated in disease pathogenesis. Human pancreatic islet-infiltrating CD4+ T cell clones not only respond to proinsulin C-peptide (PI40-54; GQVELGGGPGAGSLQ) but also cross-react with a hybrid insulin peptide (HIP; PI40-47-IAPP74-80; GQVELGGG-NAVEVLK) presented by HLA-DQ8. How T cell receptors recognise self-peptide and cross-react to HIPs is unclear. We investigated the cross-reactivity of the CD4+ T cell clones reactive to native PI40-54 epitope and multiple HIPs fused at the same N-terminus (PI40-54) to the degradation products of two highly expressed pancreatic islet proteins, Neuropeptide Y (NPY68-74) and amyloid polypeptide (IAPP23-29 and IAPP74-80). We observed that five out of the selected SKW3 T cell lines expressing TCRs isolated from CD4+ T cells of people with T1D responded to multiple HIPs. Despite shared TRAV26-1-TRBV5-1 gene usage in some T cells, these clones cross-reacted to varying degrees with the PI40-54 and HIP epitopes. Crystal structures of two TRAV26-1+-TRBV5-1+ T cell receptors (TCRs) in complex with PI40-54 and HIPs bound to HLA-DQ8 revealed that the two TCRs had distinct mechanisms responsible for their differential recognition of the PI40-54 and HIP epitopes. Alanine scanning mutagenesis of the PI40-54 and HIPs determined that the P2, P7 and P8 residues in these epitopes were key determinants of TCR specificity. Accordingly, we provide a molecular basis for cross-reactivity towards native insulin and HIP epitopes presented by HLA-DQ8.

Prebound State Discovered in the Unbinding Pathway of Fluorinated Variants of the Trypsin-BPTI Complex Using Random Acceleration Molecular Dynamics Simulations.

The serine protease trypsin forms a tightly bound inhibitor complex with the bovine pancreatic trypsin inhibitor (BPTI). The complex is stabilized by the P1 residue Lys15, which interacts with negatively charged amino acids at the bottom of the S1 pocket. Truncating the P1 residue of wildtype BPTI to α-aminobutyric acid (Abu) leaves a complex with moderate inhibitor strength, which is held in place by additional hydrogen bonds at the protein-protein interface. Fluorination of the Abu residue partially restores the inhibitor strength. The mechanism with which fluorination can restore the inhibitor strength is unknown, and accurate computational investigation requires knowledge of the binding and unbinding pathways. The preferred unbinding pathway is likely to be complex, as encounter states have been described before, and unrestrained umbrella sampling simulations of these complexes suggest additional energetic minima. Here, we use random acceleration molecular dynamics to find a new metastable state in the unbinding pathway of Abu-BPTI variants and wildtype BPTI from trypsin, which we call the prebound state. The prebound state and the fully bound state differ by a substantial shift in the position, a slight shift in the orientation of the BPTI variants, and changes in the interaction pattern. Particularly important is the breaking of three hydrogen bonds around Arg17. Fluorination of the P1 residue lowers the energy barrier of the transition between the fully bound state and prebound state and also lowers the energy minimum of the prebound state. While the effect of fluorination is in general difficult to quantify, here, it is in part caused by favorable stabilization of a hydrogen bond between Gln194 and Cys14. The interaction pattern of the prebound state offers insights into the inhibitory mechanism of BPTI and might add valuable information for the design of serine protease inhibitors.

Fragment-Based Design, Synthesis, and Characterization of Aminoisoindole-Derived Furin Inhibitors.

A 1H-isoindol-3-amine was identified as suitable P1 group for the proprotein convertase furin using a crystallographic screening with a set of 20 fragments known to occupy the S1 pocket of trypsin-like serine proteases. Its binding mode is very similar to that observed for the P1 group of benzamidine-derived peptidic furin inhibitors suggesting an aminomethyl substitution of this fragment to obtain a couplable P1 residue for the synthesis of substrate-analogue furin inhibitors. The obtained inhibitors possess a slightly improved picomolar inhibitory potency compared to their benzamidine-derived analogues. The crystal structures of two inhibitors in complex with furin revealed that the new P1 group is perfectly suited for incorporation in peptidic furin inhibitors. Selected inhibitors were tested for antiviral activity against respiratory syncytial virus (RSV) and a furin-dependent influenza A virus (SC35M/H7N7) in A549 human lung cells and demonstrated an efficient inhibition of virus activation and replication at low micromolar or even submicromolar concentrations. First results suggest that the Mas-related G-protein coupled receptor GPCR-X2 could be a potential off-target for certain benzamidine-derived furin inhibitors.

Synthesis and biological evaluation of selective Pepstatin based trifluoromethylated inhibitors of Cathepsin D

Cathepsin D (CD) is overexpressed in several types of cancer and constitutes an important biological target. Pepstatin A, a pentapeptide incorporating two non-proteinogenic statin residues, is among the most potent inhibitor of CD but lacks selectivity and suffers from poor bioavailability. Eight analogues of Pepstatin A, were synthesized, replacing residues in P3 or P1 position by non-canonical (S)- and (R)-α-Trifluoromethyl Alanine (TfmAla), (S)- and (R)-Trifluoromethionine (TFM) or non-natural d-Valine. The biological activities of those analogues were quantified on isolated CD and Pepsin by fluorescence-based assay (FRET) and cytotoxicity of the best fluorinated inhibitors was evaluated on SKOV3 ovarian cancer cell line. (R)-TFM based analog of Pepstatin A (compound 6) returned a sub-nanomolar IC50 against CD and an increased selectivity. Molecular Docking experiments could partially rationalize these results. Stabilized inhibitor 6 in the catalytic pocket of CD showed strong hydrophobic interactions of the long and flexible TFM side chain with lipophilic residues of S1 and S3 sub-pockets of the catalytic pocket. The newly synthesized inhibitors returned no cytotoxicity at IC50 concentrations on SKOV3 cancer cells, however the compounds derived from (S)-TfmAla and (R)-TFM led to modifications of cells morphologies, associated with altered organization of F-actin and extracellular Fibronectin.

DNA encoded peptide library for SARS-CoV-2 3CL protease covalent inhibitor discovery and profiling.

Covalent protease inhibitors serve as valuable tools for modulating protease activity and are essential for investigating the functions of protease targets. These inhibitors typically consist of a recognition motif and a covalently reactive electrophile. Substrate peptides, featuring residues capable of fitting into the substrate pockets of proteases, undergo chemical modification at the carbonyl carbon of the P1 residue with an electrophile and have been widely applied in the development of covalent inhibitors. In this study, we utilized a DNA-encoded peptide library to replicate peptide binder sequences and introduced a vinyl sulfone warhead at the C-termini to construct the DNA-encoded peptide covalent inhibitor library (DEPCIL) for targeting cysteine proteases. Screening results toward 3CL protease demonstrated the efficacy of this library, not only in identifying protease inhibitors, but also in discovering amino acids that can conform to aligned protease pockets. The identified peptide sequences provide valuable insight into the amino acid preferences within substrate binding pockets, and our novel technology is indicative of the potential for similar strategies to discover covalent inhibitors and profile binding preferences of other proteases.

Solid-Phase Synthesis of Caged Luminescent Peptides via Side Chain Anchoring.

The synthesis of caged luminescent peptide substrates remains challenging, especially when libraries of the substrates are required. Most currently available synthetic methods rely on a solution-phase approach, which is less suited for parallel synthesis purposes. We herein present a solid-phase peptide synthesis (SPPS) method for the synthesis of caged aminoluciferin peptides via side chain anchoring of the P1 residue. After the synthesis of a preliminary test library consisting of 40 compounds, the synthetic method was validated and optimized for up to >100 g of resin. Subsequently, two separate larger peptide libraries were synthesized either having a P1 = lysine or arginine residue containing in total 719 novel peptide substrates. The use of a more stable caged nitrile precursor instead of caged aminoluciferin rendered our parallel synthetic approach completely suitable for SPPS and serine protease profiling was demonstrated using late-stage aminoluciferin generation.

AlphaFold modeling of nepovirus 3C-like proteinases provides new insights into their diverse substrate specificities

The majority of picornaviral 3C proteinases (3Cpro) cleavage sites possess glutamine at the P1 position. Plant nepovirus 3C-like proteinases (3CLpro) show however much broader specificity, cleaving not only after glutamine, but also after several basic and hydrophobic residues. To investigate this difference, we employed AlphaFold to generate structural models of twelve selected 3CLpro, representing six substrate specificities. Generally, we observed favorable correlations between the architecture and charge of nepovirus proteinase S1 subsites and their ability to accept or restrict larger residues. The models identified a conserved aspartate residue close to the P1 residue in the S1 subsites of all nepovirus proteinases examined, consistent with the observed strong bias against negatively-charged residues at the P1 position of nepovirus cleavage sites. Finally, a cramped S4 subsite along with the presence of two unique histidine and serine residues explains the strict requirement of the grapevine fanleaf virus proteinase for serine at the P4 position.

Structure-Based Optimization and Biological Evaluation of Potent and Selective MMP-7 Inhibitors for Kidney Fibrosis.

Matrix metalloproteinase-7 (MMP-7) has been shown to play important roles in pathophysiological processes involved in the development/progression of diseases such as cancer and fibrosis. We discovered selective MMP-7 inhibitors composed of arylsulfonamide, carboxylate, and short peptides by a molecular hybridization approach. These compounds interacted with MMP-7 via multiple hydrogen bonds in the cocrystal structures. To obtain compounds for in vivo evaluation, we attempted structural optimization, particularly targeting Tyr167 at the S3 subsite through structure-based drug design, and identified compound 15 as showing improved MMP-7 potency and MMP subtype selectivity. A novel π-π stacking interaction with Tyr167 was achieved when 4-pyridylalanine was introduced as the P3 residue. Compound 15 suppressed the progression of kidney fibrosis in a dose-dependent manner in a mouse model of unilateral ureteral obstruction. Thus, we demonstrated, for the first time, that potent and selective MMP-7 inhibitors could prevent the progression of kidney fibrosis.

A Systematic Survey of Reversibly Covalent Dipeptidyl Inhibitors of the SARS-CoV-2 Main Protease.

SARS-CoV-2, the COVID-19 pathogen, relies on its main protease (MPro) for replication and pathogenesis. MPro is a demonstrated target for the development of antivirals for SARS-CoV-2. Past studies have systematically explored tripeptidyl inhibitors such as nirmatrelvir as MPro inhibitors. However, dipeptidyl inhibitors especially those with a spiro residue at their P2 position have not been systematically investigated. In this work, we synthesized about 30 dipeptidyl MPro inhibitors and characterized them on enzymatic inhibition potency, structures of their complexes with MPro, cellular MPro inhibition potency, antiviral potency, cytotoxicity, and in vitro metabolic stability. Our results indicated that MPro has a flexible S2 pocket to accommodate inhibitors with a large P2 residue and revealed that dipeptidyl inhibitors with a large P2 spiro residue such as (S)-2-azaspiro [4,4]nonane-3-carboxylate and (S)-2-azaspiro[4,5]decane-3-carboxylate have favorable characteristics. One compound, MPI60, containing a P2 (S)-2-azaspiro[4,4]nonane-3-carboxylate displayed high antiviral potency, low cellular cytotoxicity, and high in vitro metabolic stability.

Computational identification of a multi-peptide vaccine candidate in E2 glycoprotein against diverse Hepatitis C virus genotypes

Hepatitis C Virus (HCV) is estimated to affect nearly 180 million people worldwide, culminating in ∼0.7 million yearly casualties. However, a safe vaccine against HCV is not yet available. This study endeavored to identify a multi-genotypic, multi-epitopic, safe, and globally competent HCV vaccine candidate. We employed a consensus epitope prediction strategy to identify multi-epitopic peptides in all known envelope glycoprotein (E2) sequences, belonging to diverse HCV genotypes. The obtained peptides were screened for toxicity, allergenicity, autoimmunity and antigenicity, resulting in two favorable peptides viz., P2 (VYCFTPSPVVVG) and P3 (YRLWHYPCTV). Evolutionary conservation analysis indicated that P2 and P3 are highly conserved, supporting their use as part of a designed multi-genotypic vaccine. Population coverage analysis revealed that P2 and P3 are likely to be presented by >89% Human Leukocyte Antigen (HLA) molecules from six geographical regions. Indeed, molecular docking predicted the physical binding of P2 and P3 to various representative HLAs. We designed a vaccine construct using these peptides and assessed its binding to toll-like receptor 4 (TLR-4) by molecular docking and simulation. Subsequent analysis by energy-based and machine learning tools predicted high binding affinity and pinpointed the key binding residues (i.e. hotspots) in P2 and P3. Also, a favorable immunogenic profile of the construct was predicted by immune simulations. We encourage the scientific community to validate our vaccine construct in vitro and in vivo. Communicated by Ramaswamy H. Sarma

Broad-spectrum coronavirus 3C-like protease peptidomimetic inhibitors effectively block SARS-CoV-2 replication in cells: Design, synthesis, biological evaluation, and X-ray structure determination

Despite the approval of vaccines, monoclonal antibodies and restrictions during the pandemic, the demand for new efficacious and safe antivirals is compelling to boost the therapeutic arsenal against the COVID-19. The viral 3-chymotrypsin-like protease (3CLpro) is an essential enzyme for replication with high homology in the active site across CoVs and variants showing an almost unique specificity for Leu-Gln as P2–P1 residues, allowing the development of broad-spectrum inhibitors.The design, synthesis, biological activity, and cocrystal structural information of newly conceived peptidomimetic covalent reversible inhibitors are herein described. The inhibitors display an aldehyde warhead, a Gln mimetic at P1 and modified P2–P3 residues. Particularly, functionalized proline residues were inserted at P2 to stabilize the β-turn like bioactive conformation, modulating the affinity. The most potent compounds displayed low/sub-nM potency against the 3CLpro of SARS-CoV-2 and MERS-CoV and inhibited viral replication of three human CoVs, i.e. SARS-CoV-2, MERS-CoV, and HCoV 229 in different cell lines. Particularly, derivative 12 exhibited nM-low μM antiviral activity depending on the virus, and the highest selectivity index. Some compounds were co-crystallized with SARS-CoV-2 3CLpro validating our design. Altogether, these results foster future work toward broad-spectrum 3CLpro inhibitors to challenge CoVs related pandemics.

Amino Acid Substitutions at P1 Position Change the Inhibitory Activity and Specificity of Protease Inhibitors BmSPI38 and BmSPI39 from Bombyx mori

It was found that silkworm serine protease inhibitors BmSPI38 and BmSPI39 were very different from typical TIL-type protease inhibitors in sequence, structure, and activity. BmSPI38 and BmSPI39 with unique structure and activity may be good models for studying the relationship between the structure and function of small-molecule TIL-type protease inhibitors. In this study, site-directed saturation mutagenesis at the P1 position was conducted to investigate the effect of P1 sites on the inhibitory activity and specificity of BmSPI38 and BmSPI39. In-gel activity staining and protease inhibition experiments confirmed that BmSPI38 and BmSPI39 could strongly inhibit elastase activity. Almost all mutant proteins of BmSPI38 and BmSPI39 retained the inhibitory activities against subtilisin and elastase, but the replacement of P1 residues greatly affected their intrinsic inhibitory activities. Overall, the substitution of Gly54 in BmSPI38 and Ala56 in BmSPI39 with Gln, Ser, or Thr was able to significantly enhance their inhibitory activities against subtilisin and elastase. However, replacing P1 residues in BmSPI38 and BmSPI39 with Ile, Trp, Pro, or Val could seriously weaken their inhibitory activity against subtilisin and elastase. The replacement of P1 residues with Arg or Lys not only reduced the intrinsic activities of BmSPI38 and BmSPI39, but also resulted in the acquisition of stronger trypsin inhibitory activities and weaker chymotrypsin inhibitory activities. The activity staining results showed that BmSPI38(G54K), BmSPI39(A56R), and BmSPI39(A56K) had extremely high acid-base and thermal stability. In conclusion, this study not only confirmed that BmSPI38 and BmSPI39 had strong elastase inhibitory activity, but also confirmed that P1 residue replacement could change their activity and inhibitory specificity. This not only provides a new perspective and idea for the exploitation and utilization of BmSPI38 and BmSPI39 in biomedicine and pest control, but also provides a basis or reference for the activity and specificity modification of TIL-type protease inhibitors.

Dickkopf-1 inhibits the secretion of MUC5AC induced by Mycoplasma pneumoniae P1-C in mouse lung epithelial cells

Mycoplasma pneumoniae is the most common pathogen of respiratory tract infection in children and adults. Clinical observation shows that M. pneumoniae infection can cause massive mucus secretion in the respiratory tract, which makes the breathing of patients difficult. Studies have shown that M. pneumoniae infection can cause massive secretion of mucin 5AC (MUC5AC). Adhesin P1 plays an important role in the pathogenesis of M. pneumoniae infection by mediating the adhesion of pathogens to host cells, and the C-terminal residues of P1 (P1-C) are immunogenic. This study investigated the molecular mechanism of Wnt/β-catenin signaling pathway inhibitor Dickkopf-1 (DKK1) in the secretion of MUC5AC in mouse airway epithelial cells (MAECs) induced by P1-C. Scanning electron microscope and hematoxylin-eosin staining were used to observe the effect of P1-C on mucus secretion of MAECs. Protein chip was used to detect the secretion of cytokines and analyse the enrichment of related signaling pathways induced by P1-C in MAECs. Periodic acid schiff stain (PAS) staining, Tunel staining and Masson staining were used to detect the damage of the lungs of mouse exposed to P1-C. Immunohistochemistry was used to detect the secretion of MUC5AC expression, and Western blotting was used to reveal the molecular mechanism of DKK1-regulated secretion of MUC5AC induced by P1-C protein in MACES. The results showed that P1-C induced the massive secretion of mucus and inflammatory factors in MAECs. During P1-C infection, DKK1 down-regulated janus kinase 2 (JAK2), phosphorylation signaling and transcription activator 1 (p-STAT1) and phosphorylation signaling and activator of transcription 3 (p-STAT3) expression. Overexpression of DKK1 significantly up-regulated the expression of MUC5AC repressor transcription factor fork-head box protein A2 (FOXA2). At the same time, the expression of MUC5AC induced by P1-C was inhibited significantly. It is speculated that DKK1 can effectively reduce the secretion of MUC5AC in MAECs induced by P1-C by inhibiting the JAK/STAT1-STAT3 signaling pathway and up-regulating the expression of FOXA2.

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

Boroleucine-Derived Covalent Inhibitors of the ZIKV Protease.

The Zika virus (ZIKV) remains a potential threat to the public health due to the lack of both an approved vaccination or a specific treatment. In this work, a series of peptidic inhibitors of the ZIKV protease with boroleucine as P1 residue was synthesized. The highest affinities with Ki values down to 8 nM were observed for compounds with basic residues in both P2 and P3 position and at the N-terminus. The low potency of reference compounds containing leucine, leucine-amide or isopentylamide as P1 residue suggested a covalent binding mode of the boroleucine-derived inhibitors. This was finally proven by crystal structure determination of the most potent inhibitor from this series in complex with the ZIKV protease.

Cloning and functional identification of pmKPI cDNA in Poecilobdella manillensis.

Kazal-type serine protease inhibitors play a role in physiological processes such as blood coagulation and fibrinolysis. The amino acid residues at the P1 site are different, and they inhibit different types of proteases. The inhibitory mechanism of the protease in the salivary glands of Poecilobdella manillensis is still unclear. Based on cloning, prokaryotic expression and bioinformatics analysis, we studied the role of Kazal-type serine protease inhibitors in P. manillensis and analyzed their expression by quantitative real-time PCR. The results suggested that the recombinant protein was successfully expressed in the supernatant when a prokaryotic expression vector was constructed and induced with 0.2 mmol/L IPTG at 37°C for 4h, and the enzymatic activity was determined. The mature protein encodes 91 amino acids and has a relative molecular weight of 9929.32 Da, and after removing the signal peptide, the theoretical isoelectric point was 8.79. It is an unstable protein without a transmembrane domain. The mature protein contains two Kazal-type domains, in which all P1 residues are Lys, consisting of an α helix and three antiparallel β sheets. The upregulated expression of the mRNA was induced after a meal was provided, and the results showed an increasing and then decreasing trend. Taken together, the results indicate that mature proteins from P. manillensis inhibit thrombin activity, laying the foundation for the subsequent in-depth study of the function of genes encoding Kazal-type serine protease inhibitors.

Profiling Substrate Specificity of the SUMO Protease Ulp1 by the YESS-PSSC System to Advance the Conserved Mechanism for Substrate Cleavage.

SUMO modification is a vital post-translational regulation process in eukaryotes, in which the SUMO protease is responsible for the maturation of the SUMO precursor and the deconjugation of the SUMO protein from modified proteins by accurately cleaving behind the C-terminal Gly–Gly motif. To promote the understanding of the high specificity of the SUMO protease against the SUMO protein as well as to clarify whether the conserved Gly–Gly motif is strictly required for the processing of the SUMO precursor, we systematically profiled the specificity of the S. cerevisiae SUMO protease (Ulp1) on Smt3 at the P2–P1↓P1’ (Gly–Gly↓Ala) position using the YESS–PSSC system. Our results demonstrated that Ulp1 was able to cleave Gly–Gly↓ motif-mutated substrates, indicating that the diglycine motif is not strictly required for Ulp1 cleavage. A structural-modeling analysis indicated that it is the special tapered active pocket of Ulp1 conferred the selectivity of small residues at the P1–P2 position of Smt3, such as Gly, Ala, Ser and Cys, and only which can smoothly deliver the scissile bond into the active site for cleavage. Meanwhile, the P1’ position Ala of Smt3 was found to play a vital role in maintaining Ulp1’s precise cleavage after the Gly–Gly motif and replacing Ala with Gly in this position could expand Ulp1 inclusivity against the P1 and P2 position residues of Smt3. All in all, our studies advanced the traditional knowledge of the SUMO protein, which may provide potential directions for the drug discovery of abnormal SUMOylation-related diseases.

Granzyme K initiates IL-6 and IL-8 release from epithelial cells by activating protease-activated receptor 2.

Granzyme K (GzmK) is a tryptic member of the granzyme family of chymotrypsin-like serine proteases produced by cells of the immune system. Previous studies have indicated that GzmK activates protease-activated receptor 1 (PAR1) enhancing activation of monocytes and wound healing in endothelial cells. Here, we show using peptides and full length proteins that GzmK and, to a lesser extent the related protease GzmA, are capable of activating PAR1 and PAR2. These cleavage events occur at the canonical arginine P1 residue and involve exosite interactions between protease and receptor. Despite cleaving PAR2 at the same point as trypsin, GzmK does not induce a classical Ca2+ flux but instead activates a distinct signalling cascade, involving recruitment of β-arrestin and phosphorylation of ERK. In epithelial A549 cells, PAR2 activation by GzmK results in the release of inflammatory cytokines IL-6 and IL-8. These data suggest that during an immune response GzmK acts as a pro-inflammatory regulator, rather than as a cytotoxin.