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 in vitro 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.

Exosite binding modulates the specificity of the immunomodulatory enzyme ScpA, a C5a inactivating bacterial protease.

The C5a peptidase from Streptococcus pyogenes (ScpA) is a highly specific enzyme with potential therapeutic value. ScpA is a good model for studying determinants of specificity in the multidomain immunomodulatory enzymes (IMEs), which comprise a large family of bacterial surface proteases. The surface exposed region of ScpA has 5 main domains which includes 3 C-terminal Fn3-like domains (Fn1, Fn2 and Fn3) (Kagawa et al. 2009). Progressive deletion of the Fn3-like domains from the C-ter resulted in loss of enzyme activity and showed an important role for the Fn2 domain in enzyme function. Functional investigation of specific acidic residues on the Fn2 domain identified 3 residues 30-50 Å from the catalytic site (D783, E864 and D889) which impacted to differing degrees on binding and on catalysis, supporting the presence of an exosite on the Fn2. In particular, residue D783 was observed to impact on both substrate binding affinity and the activity of ScpA. A double mutant cycle analysis showed energetic coupling between the targeted ScpA residues and residues in the core portion (residues 1-67) of the C5a substrate. The data supports the presence of a communication network between the active site and the exosite on Fn2. These findings provide a basis for rational engineering of this important enzyme family to enhance stability, activity and/or specificity.

An intermolecular salt bridge linking substrate binding and P1 substrate specificity switch of arterivirus 3C-like proteases.

Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) represent two members of the family Arteriviridae and pose a major threat to the equine- and swine-breeding industries throughout the world. Previously, we and others demonstrated that PRRSV 3C-like protease (3CLpro) had very high glutamic acid (Glu)-specificity at the P1 position (P1-Glu). Comparably, EAV 3CLpro exhibited recognition of both Glu and glutamine (Gln) at the P1 position. However, the underlying mechanisms of the P1 substrate specificity shift of arterivirus 3CLpro remain unclear. We systematically screened the specific amino acids in the S1 subsite of arterivirus 3CLpro using a cyclized luciferase-based biosensor and identified Gly116, His133 and Ser136 (using PRRSV 3CLpro numbering) are important for recognition of P1-Glu, whereas Ser136 is nonessential for recognition of P1-Gln. Molecular dynamics simulations and biochemical experiments highlighted that the PRRSV 3CLpro and EAV 3CLpro formed distinct S1 subsites for the P1 substrate specificity switch. Mechanistically, a specific intermolecular salt bridge between PRRSV 3CLpro and substrate P1-Glu (Lys138/P1-Glu) are invaluable for high Glu-specificity at the P1 position, and the exchange of K138T (salt bridge interruption, from PRRSV to EAV) shifted the specificity of PRRSV 3CLpro toward P1-Gln. In turn, the T139K exchange of EAV 3CLpro showed a noticeable shift in substrate specificity, such that substrates containing P1-Glu are likely to be recognized more efficiently. These findings identify an evolutionarily accessible mechanism for disrupting or reorganizing salt bridge with only a single mutation of arterivirus 3CLpro to trigger a substrate specificity switch.

Characterization of Self-Processing Activities and Substrate Specificities of Porcine Torovirus 3C-Like Protease.

The 3C-like protease (3CLpro) of nidovirus plays an important role in viral replication and manipulation of host antiviral innate immunity, which makes it an ideal antiviral target. Here, we characterized that porcine torovirus (PToV; family Tobaniviridae, order Nidovirales) 3CLpro autocatalytically releases itself from the viral precursor protein by self-cleavage. Site-directed mutagenesis suggested that PToV 3CLpro, as a serine protease, employed His53 and Ser160 as the active-site residues. Interestingly, unlike most nidovirus 3CLpro, the P1 residue plays a less essential role in N-terminal self-cleavage of PToV 3CLpro Substituting either P1 or P4 residue of substrate alone has little discernible effect on N-terminal cleavage. Notably, replacement of the two residues together completely blocks N-terminal cleavage, suggesting that N-terminal self-cleavage of PToV 3CLpro is synergistically affected by both P1 and P4 residues. Using a cyclized luciferase-based biosensor, we systematically scanned the polyproteins for cleavage sites and identified (FXXQ↓A/S) as the main consensus sequences. Subsequent homology modeling and biochemical experiments suggested that the protease formed putative pockets S1 and S4 between the substrate. Indeed, mutants of both predicted S1 (D159A, H174A) and S4 (P62G/L185G) pockets completely lost the ability of cleavage activity of PToV 3CLpro In conclusion, the characterization of self-processing activities and substrate specificities of PToV 3CLpro will offer helpful information for the mechanism of nidovirus 3C-like proteinase's substrate specificities and the rational development of the antinidovirus drugs.IMPORTANCE Currently, the active-site residues and substrate specificities of 3C-like protease (3CLpro) differ among nidoviruses, and the detailed catalytic mechanism remains largely unknown. Here, porcine torovirus (PToV) 3CLpro cleaves 12 sites in the polyproteins, including its N- and C-terminal self-processing sites. Unlike coronaviruses and arteriviruses, PToV 3CLpro employed His53 and Ser160 as the active-site residues that recognize a glutamine (Gln) at the P1 position. Surprisingly, mutations of P1-Gln impaired the C-terminal self-processing but did not affect N-terminal self-processing. The "noncanonical" substrate specificity for its N-terminal self-processing was attributed to the phenylalanine (Phe) residue at the P4 position in the N-terminal site. Furthermore, a double glycine (neutral) substitution at the putative P4-Phe-binding residues (P62G/L185G) abolished the cleavage activity of PToV 3CLpro suggested the potential hydrophobic force between the PToV 3CLpro and P4-Phe side chains.

Enzyme kinetic and binding studies identify determinants of specificity for the immunomodulatory enzyme ScpA, a C5a inactivating bacterial protease.

The Streptococcal C5a peptidase (ScpA) specifically inactivates the human complement factor hC5a, a potent anaphylatoxin recently identified as a therapeutic target for treatment of COVID-19 infections. Biologics used to modulate hC5a are predominantly monoclonal antibodies. Here we present data to support an alternative therapeutic approach based on the specific inactivation of hC5a by ScpA in studies using recombinant hC5a (rhC5a). Initial characterization of ScpA confirmed activity in human serum and against rhC5a desArg (rhC5adR), the predominant hC5a form in blood. A new FRET based enzyme assay showed that ScpA cleaved rhC5a at near physiological concentrations (K m 185 nM). Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC) studies established a high affinity ScpA-rhC5a interaction (K D 34 nM, K D ITC 30.8 nM). SPR analyses also showed that substrate binding is dominated (88% of ΔG°bind) by interactions with the bulky N-ter cleavage product (PN, 'core' residues 1-67) with interactions involving the C-ter R74 contributing most of the remaining ΔG°bind. Furthermore, reduced binding affinity following mutation of a subset of positively charged Arginine residues of PN and in the presence of higher salt concentrations, highlighted the importance of electrostatic interactions. These data provide the first in-depth study of the ScpA-C5a interaction and indicate that ScpA's ability to efficiently cleave physiological concentrations of C5a is driven by electrostatic interactions between an exosite on the enzyme and the 'core' of C5a. The results and methods described herein will facilitate engineering of ScpA to enhance its potential as a therapeutic for excessive immune response to infectious disease.

UL36 Encoded by Marek's Disease Virus Exhibits Linkage-Specific Deubiquitinase Activity.

(1) Background: Deubiquitinase (DUB) regulates various important cellular processes via reversing the protein ubiquitination. The N-terminal fragment of a giant tegument protein, UL36, encoded by the Marek's disease (MD) virus (MDV), encompasses a putative DUB (UL36-DUB) and shares no homology with any known DUBs. The N-terminus 75 kDa fragment of UL36 exists in MD T lymphoma cells at a high level and participates in MDV pathogenicity. (2) Methods: To characterize deubiquitinating activity and substrate specificity of UL36-DUB, the UL36 N-terminal fragments, UL36(323), UL36(480), and mutants were prepared using the Bac-to-Bac system. The deubiquitinating activity and substrate specificity of these recombinant UL36-DUBs were analyzed using various ubiquitin (Ub) or ubiquitin-like (UbL) substrates and activity-based deubiquitinating enzyme probes. (3) Results: The results indicated that wild type UL36-DUBs show a different hydrolysis ability against varied types of ubiquitin chains. These wild type UL36-DUBs presented the highest activity to K11, K48, and K63 linkage Ub chains, weak activity to K6, K29, and K33 Ub chains, and no activity to K27 linkage Ub chain. UL36 has higher cleavage efficiency for K48 and K63 poly-ubiquitin than linear ubiquitin chain (M1-Ub4), but no activity on various ubiquitin-like modifiers. The mutation of C98 and H234 residues eliminated the deubiquitinating activity of UL36-DUB. D232A mutation impacted, but did not eliminated UL36(480) activity. The Ub-Br probe can bind to wild type UL36-DUB and mutants UL36(480)H234A and UL36(480)D232A, but not C98 mutants. These in vitro results suggested that the C98 and H234 are essential catalytic residues of UL36-DUB. UL36-DUB exhibited a strict substrate specificity. Inhibition assay revealed that UL36-DUB exhibits resistance to the Roche protease inhibitor cocktail and serine protease inhibitor, but not to the Solarbio protease inhibitor cocktail. (4) Conclusions: UL36-DUB exhibited a strict substrate preference, and the protocol developed in the current study for obtaining active UL36-DUB protein should promote the high-throughput screening of UL36 inhibitors and the study on the function of MDV-encoded UL36.