Cleavage Of Peptide Substrate Research Articles

Differential specificity of SARS-CoV-2 main protease variants on peptide versus protein-based substrates.

The SARS-CoV-2 main protease (Mpro ) holds significant importance as a biological target in combating coronaviruses due to its importance in virus replication. Considering the emergence of novel SARS-CoV-2 variants and the mutations observed in the Mpro sequence, we hypothesized that these mutations may have a potential impact on the protease's specificity. To test this, we expressed Mpro corresponding to the original strain and variants Beta1, Beta2, and Omicron and analyzed their activity on protein-based and peptide substrates. Although we observed differential activity on the protein-based substrate, there was very little difference when analyzed on the peptide substrate. We conclude that mutations on the Mpro sequence, despite having a minor effect on a peptide substrate cleavage, did not change the catalytic site environment enough to build resistance to inhibition. Therefore, we propose that inhibitors initially designed for the Mpro of the original strain will be effective in all the variants. Thus, Mpro is likely to continue to be a target of therapeutic interest as mutations in its sequence are rare and, as we show here, have a minor effect on the protease's recognition of peptide-based molecules.

Co-folding and RNA activation of poliovirus 3Cpro polyprotein precursors

Positive-strand RNA viruses use long open reading frames to express large polyproteins that are processed into individual proteins by viral proteases. Polyprotein processing is highly regulated and yields intermediate species with different functions than the fully processed proteins, increasing the biochemical diversity of the compact viral genome while also presenting challenges in that proteins must remain stably folded in multiple contexts. We have used circular dichroism spectroscopy and single molecule microscopy to examine the solution structure and self-association of the poliovirus P3 region protein composed of membrane binding 3A, RNA priming 3B (VPg), 3Cpro protease, and 3Dpol RNA-dependent RNA polymerase proteins. Our data indicate that co-folding interactions within the 3ABC segment stabilize the conformational state of the 3C protease region, and this stabilization requires the full-length 3A and 3B proteins. Enzymatic activity assays show that 3ABC is also an active protease, and it cleaves peptide substrates at rates comparable to 3Cpro. The cleavage of a larger polyprotein substrate is stimulated by the addition of RNA, and 3ABCpro becomes 20-fold more active than 3Cpro in the presence of stoichiometric amounts of viral cre RNA. The data suggest that co-folding within the 3ABC region results in a protease that can be highly activated toward certain cleavage sites by localization to specific RNA elements within the viral replication center, providing a mechanism for regulating viral polyprotein processing.

Characterization of the enzymatic properties of human RNPEPL1/aminopeptidase Z.

It is now evident that the M1 family of aminopeptidases play important roles in many pathophysiological processes. Among them, the enzymatic properties of arginyl aminopeptidase-like 1 (RNPEPL1) are characterized only by its truncated form. No peptide substrate has been identified. To characterize the enzymatic properties of RNPEPL1 in more detail, the full-length protein was expressed in Escherichia coli and purified to homogeneity. The full-length RNPEPL1 showed rather restricted substrate specificity and basic amino acid preference towards synthetic substrates, which was different from the previously reported specificity characterized by the truncated form. Searching for peptide substrates, we found that several peptides, such as Met-enkephalin and kallidin, were cleaved. RNPEPL1 cleaved bradykinin to de-[Arg]-bradykinin despite the presence of proline at the P2'-position. The enzyme cleaved Met-enkephalin but not dynorphin A1-17. Similar to aminopeptidase B, the full-length RNPEPL1 showed basic amino acid preference towards both synthetic and peptide substrates. In addition to the unusual cleavage of bradykinin, this enzyme shows chain length-dependent cleavage of peptide substrates sharing N-terminal amino acid sequence. This is the first study to report the enzymatic properties of the full-length human RNPEPL1 as an aminopeptidase enzyme.

Prolylcarboxypeptidase promotes IGF1R/HER3 signaling and is a potential target to improve endocrine therapy response in estrogen receptor positive breast cancer

ABSTRACT Prolylcarboxypeptidase (PRCP) is a lysosomal serine protease that cleaves peptide substrates when the penultimate amino acid is proline. Previous studies have linked PRCP to blood-pressure and appetite control through its ability to cleave peptide substrates such as angiotensin II and α-MSH. A potential role for PRCP in cancer has to date not been widely appreciated. Endocrine therapy resistance in breast cancer is an enduring clinical problem mediated in part by aberrant receptor tyrosine kinase (RTK) signaling. We previously found PRCP overexpression promoted 4-hydroxytamoxifen (4-OHT) resistance in estrogen receptor-positive (ER+) breast cancer cells. Currently, we tested the potential association between PRCP with breast cancer patient outcome and RTK signaling, and tumor responsiveness to endocrine therapy. We found high PRCP protein levels in ER+ breast tumors associates with worse outcome and earlier recurrence in breast cancer patients, including patients treated with TAM. We found a PRCP specific inhibitor (PRCPi) enhanced the response of ER+ PDX tumors and MCF7 tumors to endoxifen, an active metabolite of TAM in mice. We found PRCP increased IGF1R/HER3 signaling and AKT activation in ER+ breast cancer cells that was blocked by PRCPi. Thus, PRCP is an adverse prognostic marker in breast cancer and a potential target to improve endocrine therapy in ER+ breast cancers.

A peptide-DNA hybrid bio-nanomicelle and its application for detection of caspase-3 activity.

Bio-nanomicelles based on biomaterials such as nucleic acids, peptides, glycans, and lipids have developed rapidly in the field of bioanalysis. Although DNA and peptides have unique advantages, unfortunately, there are few bio-nanomicelles integrating DNA with peptides. Here, we designed a peptide-DNA hybrid bio-nanomicelle for the activity detection of caspase-3. The detection mechanism is based on caspase-3 specific recognition and cleavage of peptide substrates, which owns high sensitivity and selectivity. Under optimal conditions, the detection of caspase-3 activity can be achieved using our designed bio-nanomicelles and the detection limit is 0.72 nM. Furthermore, the proposed method was also successfully applied for the detection of caspase-3 in cell lysate samples after apoptosis-inducing.

A General, Label-Free and Homogeneous Electrochemical Strategy for Probing of Protease Activity and Screening of Inhibitor.

Proteases play a critical role in regulating various physiological processes from protein digestion to wound healing. Monitoring the activity of proteases and screening their inhibitors as potential drug molecules are of great importance for the early diagnosis and treatment of many diseases. In this work, we reported a general, label-free and homogeneous electrochemical method for monitoring protease activity based on the peptide–copper interaction. Cleavage of peptide substrate results in the generation of a copper-binding chelator peptide with a histidine residue in the first or third position (His1 or His3) at the N-terminal. The redox potential and current of copper coordinated with the product are different from the free copper or the copper complex with the substrate, thus allowing for the detection of protease activity. Angiotensin-converting enzyme (ACE) and thrombin were determined as the model analytes. The label-free and homogeneous electrochemical method can be used for screening protease inhibitors with high simplicity and sensitivity.

Prolyl Carboxypeptidase Maintains Receptor Tyrosine Kinase Signaling and Is a Potential Therapeutic Target in Triple Negative Breast Cancer.

Simple SummaryTriple negative breast cancer (TNBC) is an aggressive cancer type with limited treatment options and poor prognosis. Our research has revealed that a protein called prolylcarboxypeptidase (PRCP) is a potential therapy target for TNBC. We found that high levels of PRCP in tumors coincides with worse prognosis in TNBC patients. Inhibition of PRCP with a small molecule inhibitor blocked TNBC cell and tumor growth and inhibited the activity of several receptor tyrosine kinases (RTKs), proteins that are located on the surface of cells and that are important for cancer development and progression. Our findings suggest that PRCP is a novel prognostic factor for TNBC and that specific inhibitors of PRCP could be developed for TNBC treatment.TNBC is an aggressive cancer sub-type with limited treatment options and poor prognosis. New therapeutic targets are needed to improve outcomes in TNBC patients. PRCP is a lysosomal serine protease that cleaves peptide substrates when the penultimate amino acid is proline. A role for PRCP in TNBC or other cancers, and its potential as a therapy target has not yet been tested. In the current study, we found high tumor expression of PRCP associates with worse outcome and earlier recurrence in TNBC patients. Knockdown of PRCP or treatment with a small molecule PRCP inhibitor blocked proliferation and survival in TNBC cell lines and inhibited growth of TNBC tumors in mice. Mechanistically, we found PRCP maintains signaling from multiple receptor tyrosine kinases (RTKs), potentially by promoting crosstalk between RTKs and G-protein coupled receptors (GPCRs). Lastly, we found that the PRCP inhibitor caused synergistic killing of TNBC cells when combined with the EGFR and ErbB2 inhibitor lapatinib. Our results suggest that PRCP is potential prognostic marker for TNBC patient outcome and a novel therapeutic target for TNBC treatment.

Biphenyl substituted lysine derivatives as recognition elements for the matrix metalloproteinases MMP-2 and MMP-9

Matrix metalloproteinases (MMPs) are an important factor in cancer progression and metastasis, especially gelatinases MMP-2 and MMP-9. A simple methodology for their detection and monitoring is highly desirable. Molecular probes have been very widely and successfully applied to study the activity of MMPs in cellular processes in vitro. We thus synthesized a small compound library of MMP-2 and MMP-9 binding probes based on drug molecules and endowed with free amine groups for the functionalization of transducer surfaces. In this study, we combined experimental results obtained by a kinetic fluorogenic peptide substrate cleavage assay with molecular modeling studies in order to assess the ability of the probe to bind to their target enzymes. The synthesized biphenyl substituted lysine derivatives showed IC50-values in the low nanomolar concentration range against MMP-2 (ligands 3a-d: 3 nM to 8 µM, ligands 4a-d: 45 nM to 350 µM) and low micromolar range against MMP-9 (ligands 3a-d: 350 nM to 60 µM, ligands 4a-d: 5 µM to 600 µM), with a selectivity up to more than 160-fold for MMP-2. The experimental results correlated well with molecular modelling with FleXAID and X-score functions. We showed that in our compound series, the side chain remained far away from the S1′ cavity and the ligand for all the docked minima. Ligands 4a-d with their free amine group on the side chain may thus be bound to transducer surfaces for the fabrication of sensors, while retaining their activity against their target enzymes.

Coenzyme-Mediated Electro-RAFT Polymerization for Amplified Electrochemical Interrogation of Trypsin Activity.

Trypsin is a key proteolytic enzyme in the digestive system and its abnormal levels are indicative of some pancreatic diseases. Taking advantage of the coenzyme-mediated electrografting of ferrocenyl polymers as a novel strategy for signal amplification, herein, a signal-on cleavage-based electrochemical biosensor is reported for the highly selective interrogation of trypsin activity at ultralow levels. The construction of the trypsin biosensor involves (i) the immobilization of peptide substrates (without free carboxyl groups) via the N-terminus, (ii) the tryptic cleavage of peptide substrates, (iii) the site-specific labeling of the reversible addition-fragmentation chain transfer (RAFT) agents, and (iv) the grafting of ferrocenyl polymers through the electro-RAFT (eRAFT) polymerization, which is mediated by potentiostatic reduction of nicotinamide adenine dinucleotide (NAD+) coenzymes. Through the NAD+-mediated eRAFT (NAD+-eRAFT) polymerization of ferrocenylmethyl methacrylate (FcMMA), the presence of a few tryptic cleavage events can eventually result in the recruitment of a considerable amount of ferrocene redox tags. Obviously, the NAD+-eRAFT polymerization is low-cost and easy to operate as a highly efficient strategy for signal amplification. As expected, the as-constructed biosensor is highly selective and sensitive toward the signal-on interrogation of trypsin activity. Under optimal conditions, the detection limit can be as low as 18.2 μU/mL (∼72.8 pg/mL). The results also demonstrate that the as-constructed electrochemical trypsin biosensor is applicable to inhibitor screening and the interrogation of enzyme activity in the presence of complex sample matrices. Moreover, it is low-cost, less susceptible to false-positive results, and relatively easy to fabricate, thus holding great potential in diagnostic and therapeutic applications.

Profiling of multiple matrix metalloproteinases activities in the progression of osteosarcoma by peptide microarray-based fluorescence assay on polymer brush coated zinc oxide nanorod substrate

Osteosarcoma (OS) is a primary malignant bone tumor with high rate of recurrence and lung metastasis. Matrix metalloproteinases (MMPs) are demonstrated as important biomarkers of tumor invasion and metastasis. Herein, a peptide microarray-based fluorescence assay is proposed to profile multiple MMPs (MMP-1, -2, -3, -7, -9 and -13) activities in the progression of OS by using the mouse-bearing xenograft U-2OS and Saos-2 human OSs. The peptide microarray containing different biotinylated peptide substrate spots is fabricated on the poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) brush coated zinc oxide nanorod ([email protected](GMA-HEMA) decorated glass slides, and labeled by cyanine 3 (Cy3) modified avidin (Avidin-Cy3) to produce fluorescence signal. Special cleavage of peptide substrate by MMP resulted the decrease of fluorescence signal. The MMPs activities are positively correlated with the change of fluorescence intensity. This method has excellent selectivity and sensitivity, which enables to detect the activities of cellular secreted MMP-1, -2, -3, -7, -9, and -13 with limit of detection downs to 10 pmol L−1, 30 pmol L−1, 113 pmol L−1, 13 pmol L−1, 93 pmol L−1 and 12 pmol L−1, respectively. Furthermore, it is demonstrated that the activity pattern of MMPs in serum closely relevant to the disease progression and type of tumor.

Waveguide-based surface-enhanced Raman spectroscopy detection of protease activity using non-natural aromatic amino acids.

Surface enhanced Raman spectroscopy (SERS) is a selective and sensitive technique, which allows for the detection of protease activity by monitoring the cleavage of peptide substrates. Commonly used free-space based SERS substrates, however, require the use of bulky and expensive instrumentation, limiting their use to laboratory environments. An integrated photonics approach aims to implement various free-space optical components to a reliable, mass-reproducible and cheap photonic chip. We here demonstrate integrated SERS detection of trypsin activity using a nanoplasmonic slot waveguide as a waveguide-based SERS substrate. Despite the continuously improving SERS performance of the waveguide-based SERS substrates, they currently still do not reach the SERS enhancements of free-space substrates. To mitigate this, we developed an improved peptide substrate in which we incorporated the non-natural aromatic amino acid 4-cyano-phenylalanine, which provides a high intrinsic SERS signal. The use of non-natural aromatics is expected to extend the possibilities for multiplexing measurements, where the activity of several proteases can be detected simultaneously.

Electrochemically controlled grafting of polymers for ultrasensitive electrochemical assay of trypsin activity

As one of the most important proteolytic enzymes, trypsin is useful as a reliable and specific biomarker for the diagnosis of pancreatitis and other pathological conditions. In this paper, a novel signal-on electrochemical biosensor based on the use of electrochemically controlled grafting of polymers as an amplification strategy is described for the ultrasensitive assay of trypsin activity. The carboxyl-group-free peptide, serving as the substrate for the recognition of trypsin, is first immobilized via its N-terminus. The tryptic cleavage of peptide substrate can generate a free carboxyl group at the C-terminus of the truncated peptide, to which through the carboxylate-Zr(IV)-carboxylate linkage the carboxyl-group-containing initiator for atom transfer radical polymerization (ATRP) can be conjugated. The subsequent surface-initiated grafting of polymers (SI-GOP) based on electrochemically controlled ATRP (eATRP), with ferrocenylmethyl methacrylate (FcMMA) as the monomer, can bring a large amount of Fc tags to electrode surface, resulting in the generation of a very high detection signal. The eATRP-based SI-GOP is easy to operate and low-cost as an amplification strategy. Under optimal conditions, the detection limit for trypsin activity can be down to 0.016 mU mL−1 (~2.68 pM or ~0.064 ng mL−1). As the current signal increases with trypsin activity, this trypsin biosensor is less susceptible to false positives due to the signal-on mode. Moreover, it is highly selective and applicable to inhibitor screening and the assay of trypsin activity in the presence of complex biological matrices. Taking together, this electrochemical trypsin biosensor may hold great potential in diagnostic applications.

Importance of Tyr409 and Tyr414 in constructing the substrate pocket of human aminopeptidase B.

Aminopeptidase B (APB, EC 3.4.11.6) preferentially hydrolyzes basic amino acids of synthetic substrates and requires a physiological concentration of chloride anions for optimal activity. Several amino acid residues of APB responsible for its enzymatic activity have been elucidated. In this study, we further searched for residues critical to its enzymatic activity, especially toward peptide substrates. APB residues Tyr409 (Y409) and Tyr414 (Y414), both of which were critical to its hydrolytic activity toward synthetic substrates, were predicted by molecular modeling to be involved in cleaving peptide substrates via its interaction with amino acids in the P1' cleavage site. Using site-directed mutagenesis, several mutant APBs were prepared. In contrast to synthetic substrates, wild-type and Y409F/Y414F double mutant enzymes showed P1'-dependent cleavage of peptide substrates, indicating that both tyrosine residues were not indispensable for hydrolytic activity toward peptide substrates. Moreover, the Y409F/Y414F double mutant enzyme cleaved peptides with a Pro residue at the P1' site, which is uncommon among the M1 family of aminopeptidases. These results suggested that Tyr409 and Tyr414 of APB play important roles in enzymatic function and characteristic properties of APB via proper formation of the S1' site.

Peptide microarray-based fluorescence assay for quantitatively monitoring the tumor-associated matrix metalloproteinase-2 activity

Matrix metalloproteinases (MMPs) are important biomarkers of various tumors. Herein, a peptide microarray-based fluorescence assay is developed for quantitatively profiling of MMP-2 activity in different matrices through the binding of the immobilized biotinylated peptides on the microarray with the fluorescein isothiocyanate (FITC) modified neutravidins. In the presence of MMP-2, the biotin moiety is released from microarray by enzymatic cleavage of peptide substrate, resulting in the decrease of fluorescence signal. The change of fluorescence intensity is correlated with MMP-2 activity. The detection limit down to 14 pg mL−1 in buffer solution is obtained by a selected peptide substrate for MMP-2 from eleven peptide substrate candidates. The cellular secreted MMP-2 activity levels of different living cells are further successfully quantitatively evaluated by the selected peptide substrate. Using mouse-bearing MG-63 human osteosarcoma as a model system, we also demonstrate that the activity of MMP-2 in serum is closely related with the cancer progression.

A competitive thrombin-linked aptamer assay for small molecule: aflatoxin B1.

We described a competitive thrombin-linked aptamer assay for small molecule, using aflatoxin B1 (AFB1) as a model, taking advantage of aptamer affinity binding and enzymatic activity of thrombin. We designed a dual functional DNA probe that contained the aptamer sequence for thrombin and the aptamer sequence for AFB1. Thrombin was labeled on the DNA probe by affinity binding between thrombin and anti-thrombin aptamer. This thrombin-labeled DNA probe was attached on AFB1-bovine serum albumin conjugate (BSA-AFB1) coated on a microplate through the affinity interaction between AFB1 and anti-AFB1 aptamer. The thrombin attached on the microplate catalyzed the cleavage of peptide substrate into detectable product, generating signal for detection. In the presence of AFB1, free AFB1 competed with BSA-AFB1 in the binding to the limited amount of DNA probe, leading to signal decrease. Detection of AFB1 was achieved by measuring the signal change. Under optimized conditions, AFB1 was successfully detected in the range from 0.5nM to 1μM when fluorogenic peptide substrate of thrombin and fluorescence analysis were applied. The use of chromogenic peptide substrate in the assay allowed the detection of AFB1 ranging from 0.5 to 125nM by simple absorbance analysis. The thrombin-linked aptamer assay showed good selectivity towards AFB1, and enabled the detection of AFB1 spiked in diluted beer and corn flour. This TLAA strategy extends the analytical application of thrombin and aptamers in detection of small molecules. Graphical abstract.

Construction of cathepsin B-responsive fluorescent probe and photosensitizer using a ferrocenyl boron dipyrromethene dark quencher

A ferrocenyl boron dipyrromethene (BODIPY) has been developed and utilized as a dark quencher to construct a cathepsin B-responsive fluorescent probe and photosensitizer. The smart fluorescent probe and photosensitizer (Pc-FcQ) contains a zinc(II) phthalocyanine as the fluorescent and photosensitizing unit which is conjugated to the ferrocenyl BODIPY dark quencher via a cathepsin B-cleavable peptide substrate [Gly-Phe-Leu-Gly-Lys]. The photosensitizing properties of Pc-FcQ, including fluorescence and singlet oxygen generation, are significantly quenched through energy transfer to the BODIPY unit and subsequently by the photoinduced electron transfer from the nearby ferrocenyl moiety. Upon exposure of cathepsin B in human hepatocellular carcinoma HepG cells, the fluorescence emission of Pc-FcQ could be restored, indicating the cleavage of the peptide substrate and the separation of the phthalocyanine and ferrocenyl BODIPY unit. However, the intracellular fluorescence intensity of Pc-FcQ was largely diminished after the cells were pre-treated with cathepsin B inhibitor. Its intracellular fluorescence intensity was comparable to that of the control compound in which the peptide substrate was replaced by the non-cleavable one [Gly-Gly-Gly-Gly-Lys]. The singlet oxygen generation of Pc-FcQ was also examined in HepG2 cells as reflected by the cytotoxicity assay. The Pc-FcQ exhibited higher potency when compared with the non-cleavable analogue due to the cleavage of peptide substrate and the detachment of the BODIPY dark quencher from the phthalocyanine. The activation of the Pc-FcQ was also demonstrated in tumor-bearing nude mice. After intratumoral injection of Pc-FcQ, the fluorescence intensity at the tumor region increased gradually over 10 h as a result of the detachment of the dark quencher upon the action of cathepsin B. All the results suggest that this ferrocenyl BODIPY could serve as an efficient dark quencher and the resulting Pc-FcQ could act as the cathepsin B-responsive fluorescent probe and activatable photosensitizer.

A Photoacoustic Probe for the Imaging of Tumor Apoptosis by Caspase-Mediated Macrocyclization and Self-Assembly.

Photoacoustic (PA) imaging shows promise in the sensitive detection of caspase-3 activated in early tumor apoptosis in response to chemotherapy; smart PA probes are thus in high demand. Herein, we report the first smart PA probe (1-RGD) responsive to caspase-3, enabling real-time and high-resolution imaging of tumor apoptosis. 1-RGD is designed to leverage the synergetic effect of active delivery and caspase-3 activation. It is selectively recognized by active caspase-3 to trigger peptide substrate cleavage and biocompatible macrocyclization-mediated self-assembly, leading to an amplified PA imaging signal and prolonged retention in apoptotic tumor cells. Strong, high-resolution PA images are obtained in chemotherapy-induced apoptotic tumors in living mice after intravenous administration with 1-RGD, facilitating sensitive reporting of caspase-3 activity and distribution within tumor tissues.

A Photoacoustic Probe for the Imaging of Tumor Apoptosis by Caspase‐Mediated Macrocyclization and Self‐Assembly

AbstractPhotoacoustic (PA) imaging shows promise in the sensitive detection of caspase‐3 activated in early tumor apoptosis in response to chemotherapy; smart PA probes are thus in high demand. Herein, we report the first smart PA probe (1‐RGD) responsive to caspase‐3, enabling real‐time and high‐resolution imaging of tumor apoptosis. 1‐RGD is designed to leverage the synergetic effect of active delivery and caspase‐3 activation. It is selectively recognized by active caspase‐3 to trigger peptide substrate cleavage and biocompatible macrocyclization‐mediated self‐assembly, leading to an amplified PA imaging signal and prolonged retention in apoptotic tumor cells. Strong, high‐resolution PA images are obtained in chemotherapy‐induced apoptotic tumors in living mice after intravenous administration with 1‐RGD, facilitating sensitive reporting of caspase‐3 activity and distribution within tumor tissues.

Porcine Deltacoronavirus nsp5 Antagonizes Type I Interferon Signaling by Cleaving STAT2.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus. The first outbreak of PDCoV was announced from the United States in 2014, followed by reports in Asia. The nonstructural protein nsp5 is a 3C-like protease of coronavirus, and our previous study showed that PDCoV nsp5 inhibits type I interferon (IFN) production. In this study, we found that PDCoV nsp5 significantly inhibited IFN-stimulated response element (ISRE) promoter activity and transcription of IFN-stimulated genes (ISGs), suggesting that PDCoV nsp5 also suppresses IFN signaling. Detailed analysis showed that nsp5 cleaved signal transducer and activator of transcription 2 (STAT2) but not Janus kinase 1 (JAK1), tyrosine kinase 2 (TYK2), STAT1, and interferon regulatory factor 9 (IRF9), key molecules of the JAK-STAT pathway. STAT2 cleavage was dependent on the protease activity of nsp5. Interestingly, nsp5 cleaved STAT2 at two sites, glutamine 685 (Q685) and Q758, and similar cleavage was observed in PDCoV-infected cells. As expected, cleaved STAT2 impaired the ability to induce ISGs, demonstrating that STAT2 cleavage is an important mechanism utilized by PDCoV nsp5 to antagonize IFN signaling. We also discussed the substrate selection and binding mode of PDCoV nsp5 by homologous modeling of PDCoV nsp5 with the two cleaved peptide substrates. The results of our study demonstrate that PDCoV nsp5 antagonizes type I IFN signaling by cleaving STAT2 and provides structural insights for comprehending the cleavage mechanism of PDCoV nsp5, revealing a potential new function for PDCoV nsp5 in type I IFN signaling.IMPORTANCE The 3C-like protease encoded by nsp5 is a major protease of coronaviruses; thus, it is an attractive target for development of anticoronavirus drugs. Previous studies have revealed that the 3C-like protease of coronaviruses, including PDCoV and porcine epidemic diarrhea virus (PEDV), antagonizes type I IFN production by targeting the NF-κB essential modulator (NEMO). Here, for the first time, we demonstrate that overexpression of PDCoV nsp5 also antagonizes IFN signaling by cleaving STAT2, an essential component of transcription factor complex ISGF3, and that PDCoV infection reduces the levels of STAT2, which may affect the innate immune response.

New Hyperactive Factor IX Variants Bioengineered Based upon Biochemical Studies of Factor IX Padua

Recent clinical trial successes of AAV-based gene therapy for hemophilia B utilizing the hyperactive factor (F) IX variant, Padua (Arg338Leu), demonstrate the potential of this strategy to achieve disease ameliorating FIX activity levels at lower vectors doses. However, the underlying molecular mechanisms responsible for the 8-fold increase in specific activity remain unanswered. To address this question, we have undertaken a series of biochemical comparisons between recombinant FIX-Padua and FIX wild-type (WT). We observe FIX-Padua demonstrates increased aPTT-based clotting activity and increased thrombin generation compared to FIX-WT, as both a zymogen and activated (FIXa) enzyme. Furthermore, FIX-Padua and FIX-WT are activated by FXIa at similar rates. Combined, these results suggest that the hyperactivity of FIX-Padua is due to alterations of the activated enzyme and not due to differences in the zymogen or its activation. We therefore investigated the protease ability of FIXa-Padua and FIXa-WT. In the absence of FVIIIa, FIXa-Padua and FIXa-WT display similar rates of cleavage of peptide substrates. Similarly, FIXa-Padua and FIXa-WT are inactivated by the suicide substrate antithrombin at similar rates. However, when combined with FVIIIa within the intrinsic Xase complex, FIXa-Padua, demonstrates a 3-fold increased catalytic rate (kcat) of FX activation compared to FIXa-WT. Thus, the hyperactivity of FIX-Padua appears to be due to enhanced enzymatic activity with its incorporation into the intrinsic Xase during clotting, and not due to differences in activation or inactivation. The additional fold increase in plasma-based clotting activity may be due to a combination of amplification and feedback reactions occurring in plasma-based assays since the relative difference in clotting activity between FIX-Padua and FIX-WT increases with increasing FX concentration. These biochemical studies support the safety of FIX-Padua as a therapeutic since they suggest that FIX-Padua is regulated similarly to FIX-WT. As such, thrombotic complications would only be anticipated at supratherapeutic FIX activity levels and would be similar for FIX-Padua and FIX-WT at a given activity level. The implication of these results is that the active site of FIXa in the intrinsic Xase is improved by the Padua substitution. We also observe that most substitutions in a 20 amino acid screen at position 338 result in FIX variants with at least equal activity compared to WT. FIX-Padua is the most active variant, while surprisingly, FIX-WT is one the least functional that does not cause hemophilia. Since Arg-338 is highly conserved within FIX mammalian orthologs, these results suggest that FIX-WT may have evolved to have limited procoagulant activity; indeed, our biochemical studies suggest that the FIX-WT active site is sub-optimal for FX activation. We, therefore, examined if there were additional positions in FIX where amino acid substitutions would lead to variants with increased clotting activity. To direct our search, we focused on positions within the protease domain that were 1) likely structurally important for interactions with FVIIIa and 2) where hemophilia B causing mutations have not been reported. Using this strategy, we identified 3 additional positions where single amino acid substitutions resulted in FIX variants with 2 - 6 fold increased clotting activity compared to FIX-WT. Moreover, the most active novel substitution can be additively combined with the Padua substitution: recombinant FIX of this two amino-acid substituted variant displayed 15-fold increased specific activity compared to FIX-WT and almost 2-fold increased specific activity compared to FIX-Padua. Given the proven benefit of the enhanced activity of FIX-Padua now being employed in clinical trials, novel hyperactive substitutions of FIX could further enhance the safety and efficacy of these therapeutics. Disclosures Camire: Pfizer: Consultancy, Patents & Royalties, Research Funding; sparK: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Novo Nordisk: Research Funding; Bayer: Consultancy. Arruda:Pfizer: Patents & Royalties, Research Funding.