Protease Cleavage Site Research Articles

Purification of recombinant bacterial collagens containing structural perturbations.

Streptococcus pyogenes-derived recombinant bacterial collagen-like proteins (CLPs) are emerging as a potential biomaterial for biomedical research and applications. Bacterial CLPs form stable triple helices and lack specific interactions with human cell surface receptors, thus enabling the design of novel biomaterials with specific functional attributes. Bacterial collagens have been instrumental in understanding collagen structure and function in normal and pathological conditions. These proteins can be readily produced in E. coli, purified using affinity chromatography, and subsequently isolated after cleavage of the affinity tag. Trypsin is a widely used protease during this purification step since the triple helix structure is resistant to trypsin digestion. However, the introduction of Gly→X mutations or natural interruptions within CLPs can perturb the triple helix structure, making them susceptible to trypsin digestion. Consequently, removing the affinity tag and isolating collagen-like (CL) domains containing mutations is impossible without degradation of the product. We present an alternative method to isolate CL domains containing Gly→X mutations utilizing a TEV protease cleavage site. Protein expression and purification conditions were optimized for designed protein constructs to achieve high yield and purity. Enzymatic digestion assays demonstrated that CL domains from wild-type CLPs could be isolated by digestion with either trypsin or TEV protease. In contrast, CLPs containing Gly→Arg mutations are readily digested by trypsin while digestion with TEV protease cleaved the His6-tag, enabling the isolation of mutant CL domains. The developed method can be adapted to CLPs containing various new biological sequences to develop multifunctional biomaterials for tissue engineering applications.

Comparison of methods generating antibody-epitope conjugates for targeting cancer with virus-specific T cells.

Therapeutic antibody-epitope conjugates (AECs) are promising new modalities to deliver immunogenic epitopes and redirect virus-specific T-cell activity to cancer cells. Nevertheless, many aspects of these antibody conjugates require optimization to increase their efficacy. Here we evaluated different strategies to conjugate an EBV epitope (YVL/A2) preceded by a protease cleavage site to the antibodies cetuximab and trastuzumab. Three approaches were taken: chemical conjugation (i.e. a thiol-maleimide reaction) to reduced cysteine side chains, heavy chain C-terminal enzymatic conjugation using sortase A, and genetic fusions, to the heavy chain (HC) C-terminus. All three conjugates were capable of T-cell activation and target-cell killing via proteolytic release of the EBV epitope and expression of the antibody target was a requirement for T-cell activation. Moreover, AECs generated with a second immunogenic epitope derived from CMV (NLV/A2) were able to deliver and redirect CMV specific T-cells, in which the amino sequence of the attached peptide appeared to influence the efficiency of epitope delivery. Therefore, screening of multiple protease cleavage sites and epitopes attached to the antibody is necessary. Taken together, our data demonstrated that multiple AECs could sensitize cancer cells to virus-specific T cells.

Mutations in α-synuclein, TDP-43 and tau prolong protein half-life through diminished degradation by lysosomal proteases

BackgroundAutosomal dominant mutations in α-synuclein, TDP-43 and tau are thought to predispose to neurodegeneration by enhancing protein aggregation. While a subset of α-synuclein, TDP-43 and tau mutations has been shown to increase the structural propensity of these proteins toward self-association, rates of aggregation are also highly dependent on protein steady state concentrations, which are in large part regulated by their rates of lysosomal degradation. Previous studies have shown that lysosomal proteases operate precisely and not indiscriminately, cleaving their substrates at very specific linear amino acid sequences. With this knowledge, we hypothesized that certain coding mutations in α-synuclein, TDP-43 and tau may lead to increased protein steady state concentrations and eventual aggregation by an alternative mechanism, that is, through disrupting lysosomal protease cleavage recognition motifs and subsequently conferring protease resistance to these proteins.ResultsTo test this possibility, we first generated comprehensive proteolysis maps containing all of the potential lysosomal protease cleavage sites for α-synuclein, TDP-43 and tau. In silico analyses of these maps indicated that certain mutations would diminish cathepsin cleavage, a prediction we confirmed utilizing in vitro protease assays. We then validated these findings in cell models and induced neurons, demonstrating that mutant forms of α-synuclein, TDP-43 and tau are degraded less efficiently than wild type despite being imported into lysosomes at similar rates.ConclusionsTogether, this study provides evidence that pathogenic mutations in the N-terminal domain of α-synuclein (G51D, A53T), low complexity domain of TDP-43 (A315T, Q331K, M337V) and R1 and R2 domains of tau (K257T, N279K, S305N) directly impair their own lysosomal degradation, altering protein homeostasis and increasing cellular protein concentrations by extending the degradation half-lives of these proteins. These results also point to novel, shared, alternative mechanism by which different forms of neurodegeneration, including synucleinopathies, TDP-43 proteinopathies and tauopathies, may arise. Importantly, they also provide a roadmap for how the upregulation of particular lysosomal proteases could be targeted as potential therapeutics for human neurodegenerative disease.

A substrate for a cell free in vitro assay system to screen drugs targeting trypsin like protease-based cleavage of SARS-CoV-2 spike glycoprotein and viral entry.

Host proteases trypsin and trypsin-like proteases have been reported to facilitate the entry of coronavirus SARS-CoV-2 in its host cells. These protease enzymes cleave the viral surface glycoprotein, spike, leading to successful cell surface receptor attachment, fusion and entry of the virus in its host cell. The spike protein has protease cleavage sites between the two domains S1 and S2. Since the cleavage site is recognized by the host proteases, it can be a potential antiviral therapeutic target. Trypsin-like proteases play an important role in virus infectivity and the property of spike protein cleavage by trypsin and trypsin-like proteases can be used to design assays for screening of antiviral candidates against spike protein cleavage. Here, we have documented the development of a proof-of-concept assay system for screening drugs against trypsin/trypsin-like proteases that cleave spike protein between its S1 and S2 domains. The assay system developed uses a fusion substrate protein containing a NanoLuc luciferase reporter protein, the protease cleavage site between S1 and S2 domains of SARS-CoV-2 spike protein and a cellulose binding domain. The substrate protein can be immobilized on cellulose via the cellulose binding domain of the substrate. When trypsin and trypsin-like proteases cleave the substrate, the cellulose binding domain remain bound to the cellulose and the reporter protein is dislodged. Reporter assay using the released reporter protein is theread out of the protease activity. We have demonstrated the proof-of-concept using multiple proteases like trypsin, TMPRSS2, furin, cathepsin B, human airway trypsin and cathepsin L. A significant increment in fold change was observed with increasing enzyme concentration and incubation time. Introduction of increasing amounts of enzyme inhibitors in the reaction reduced the luminescent signal, thus validating the assay. Furthermore, we used SDS-PAGE and immunoblot analyses to study the cleavage band pattern and re-confirm the cleavage for enzymes tested in the assay. Taken together, we have tested an in-vitro assay system using the proposed substrate for screening drugs against trypsin like protease-based cleavage of SARS-CoV-2 spike glycoprotein. The assay system can also be potentially used for antiviral drug screening against any other enzyme that might cleave the used cleavage site.

The antigen presentation of MCMV specific Qa-1-restricted CD8 +T cells

Abstract Murine cytomegalovirus (MCMV) is a well-established model system for human CMV (HCMV), a ubiquitous herpesvirus that affects over 70% of people worldwide. Although the classical CD8 +T cell response to MCMV has been well characterized, little is known about the non-classical CD8 +T cell response. Our laboratory recently demonstrated that Qa-1-restricted CD8 +T cells are sufficient to protect mice from MCMV induced lethality. Our recent data shows that this protective T cell population has limited αβ TCR diversity. To identify the MCMV peptide(s) presented by Qa-1, we utilized bioinformatic tools that predict MHC binding, including NetMHC 4.0 and T Cell Epitope Prediction tools available on the Immune Epitope Database and Analysis Resource. Using these tools, we queried the entire MCMV coding sequence to identify potential nonamers predicted to bind to Qa-1. Furthermore, these candidates were also queried to identify protease cleavage sites to ensure that they are predicted to exist in vivo. Approximately 40 potential peptides were selected and tested in an in vitroscreen using Qa-1 expressing cell lines. Our results suggest that several candidate peptides bind to Qa-1 as strongly as well-defined canonical binding partners. Taken together, these data suggest that an MCMV-responsive T cell population with limited αβ TCR diversity recognizes MCMV-derived peptides presented by Qa-1. This population is likely to be of importance for viral protection when classical CD8 +T cell immunity is compromised. This work is supported by the NIH Research Grant AI046709. Shanelle Reilly is supported by research supplement R01 3AI046709-18S1 to promote diversity.

Molecular Detection and Characterization of Hemagglutinin-neuraminidase (HN) and Fusion (F) Genes of Newcastle Disease Virus (NDV) from Duck in NER India

Background: Newcastle disease is one of the major highly contagious disease of birds only next to the highly pathogenic avian influenza virus infection. Ducks are found to be natural reservoirs transmitting the virus to other birds-in-contact. The present study was conducted with the aim to isolate the NDV virus from free ranging ducks and detection of the genotype and pathotype based on HN and F genes. Methods: The virus was isolated in chicken embryo fibroblast (CEF) primary cell culture and confirmed by cytopathic effects as well as by hemagglutination and hemagglutination inhibition tests. Molecular detection was performed using primers specific for HN and F genes. Amplified partial HN and F genes were cloned and sequenced. Phylogenetic tree was prepared based on both partial HN and F genes. Deduced amino acid alignment of protease cleavage site of F protein was performed to determine the strain. Result: The isolated virus could be confirmed by CPE as well as molecular detection by PCR. The target genes, partial HN and F genes exhibited an amplicon of 652 bp and 534 bp, respectively by PCR. On phylogenetic analysis, the present isolate was found to be genotype Xlll and on cleavage site analysis of fusion protein, which was found to be similar in pattern with velogenic pathotype of NDV.

New Perspectives on Escherichia coli Signal Peptidase I Substrate Specificity: Investigating Why the TasA Cleavage Site Is Incompatible with LepB Cleavage.

Escherichia coli signal peptidase I (LepB) has been shown to inefficiently cleave secreted proteins with aromatic amino acids at the second position after the signal peptidase cleavage site (P2'). The Bacillus subtilis exported protein TasA contains a phenylalanine at P2', which in B. subtilis is cleaved by a dedicated archaeal-organism-like signal peptidase, SipW. We have previously shown that when the TasA signal peptide is fused to maltose binding protein (MBP) up to the P2' position, the TasA-MBP fusion protein is cleaved very inefficiently by LepB. However, the precise reason why the TasA signal peptide hinders cleavage by LepB is not known. In this study, a set of 11 peptides were designed to mimic the inefficiently cleaved secreted proteins, wild-type TasA and TasA-MBP fusions, to determine whether the peptides interact with and inhibit the function of LepB. The binding affinity and inhibitory potential of the peptides against LepB were assessed by surface plasmon resonance (SPR) and a LepB enzyme activity assay. Molecular modeling of the interaction between TasA signal peptide and LepB indicated that the tryptophan residue at P2 (two amino acids before the cleavage site) inhibited the active site serine-90 residue on LepB from accessing the cleavage site. Replacing the P2 tryptophan with alanine (W26A) allowed for more efficient processing of the signal peptide when the TasA-MBP fusion was expressed in E. coli. The importance of this residue to inhibit signal peptide cleavage and the potential to design LepB inhibitors based on the TasA signal peptide are discussed. IMPORTANCE Signal peptidase I is an important drug target, and understanding its substrate is critically important to develop new bacterium-specific drugs. To that end, we have a unique signal peptide that we have shown is refractory to processing by LepB, the essential signal peptidase I in E. coli, but previously has been shown to be processed by a more human-like signal peptidase found in some bacteria. In this study, we demonstrate how the signal peptide can bind but is unable to be processed by LepB, using a variety of methods. This can inform the field on how to better design drugs that can target LepB and understand the differences between bacterial and human-like signal peptidases.

Epithelial sodium channel (ENaC) in GtoPdb v.2023.1

Epithelial sodium channel (ENaC) in GtoPdb v.2023.1

A Versatile Platform for the Tumor‐Targeted Intracellular Delivery of Peptides, Proteins, and siRNA

AbstractThe efficient delivery of biologics into cells provide unique opportunities to modulate intracellular targets not druggable by conventional small molecules. The supercharged polypeptide (SCP) has become a novel intracellular delivery system due to their special advantages, including enhanced delivery efficiency and serum tolerance. However, owing to their cationic charge and non‐specificity characteristics, the in vivo application of SCP is limited. Here, an activatable SCP (ASCP) with a pH‐sensitive charge shielding sequence (CSS), a protease cleavage site, and SCP are engineered. This system shows the potential to reduce the non‐specific binding and effectively deliver various cargo (peptide, protein, small molecule, and siRNA) into the cytosol not only in vitro but also in vivo. Furthermore, an ASCP fusion protein is designed to co‐delivery of peptide (KLA)/siRNA (IKBKE) with different tumorigenesis pathways to triple negative breast cancer (TNBC) for optimal therapeutic outcomes. It is believed that ASCP delivery system will facilitate the development of bioactive molecules for use against intracellular targets. This simple yet versatile delivery system can also pave the way for the co‐delivery of multiple therapeutic cargos to address the emerging needs of combination cancer therapy.

Analysis of F12 gene variants and molecular mechanisms in patients with coagulation factor Ⅻ deficiency

To analyze the sequence of the F12 gene and molecular mechanism for 20 patients with coagulation factor Ⅻ (FⅫ) deficiency. The patients were selected from the outpatient department of the Second Hospital of Shanxi Medical University from July 2020 to January 2022. The activity of coagulation factor Ⅷ (FⅧ:C), factor Ⅸ (FⅨ:C), factor Ⅺ (FⅪ:C) and factor Ⅻ (FⅫ:C) were determined by using a one-stage clotting assay. All exons and 5' and 3' UTR of the F12 gene were analyzed by Sanger sequencing to detect the potential variants. Bioinformatic software was used to predict the pathogenicity of the variants, conservation of amino acids, and protein models. The FⅫ:C of the 20 patients has ranged from 0.07% to 20.10%, which was far below the reference values, whilst the other coagulation indexes were all normal. Sanger sequencing has identified genetic variants in 10 patients, including 4 with missense variants [c.820C>T (p.Arg274Cys), c.1561G>A (p.Glu521Lys), c.181T>C (p.Cys61Arg) and c.566.G>C (p.Cys189Ser)], 4 deletional variants c.303_304delCA(p.His101GlnfsX36), 1 insertional variant c.1093_1094insC (p.Lys365GlnfsX69) and 1 nonsense variant c.1763C>A (p.Ser588*). The remaining 10 patients only harbored the 46C/T variant. The heterozygous c.820C>T(p.Arg274Cys) missense variant in patient 1 and the homozygous c.1763C>A (p.Ser588*) nonsense variant in patient 2 were not included in the ClinVar and the Human Gene Mutation Database. Bioinformatic analysis predicted that both variants were pathogenic, and the corresponding amino acids are highly conserved. The protein prediction models suggested that the c.820C>T (p.Arg274Cys) variant may affect the stability of the secondary structure of FⅫ protein by disrupting the original hydrogen bonding force and truncating the side chain, leading to changes in the vital domain. c.1763C>A (p.Ser588*) may produce a truncated C-terminus which may alter the spatial conformation of the protein domain and affect the serine protease cleavage site, resulting in extremely reduced FⅫ:C. Among individuals with low low FⅫ:C detected by one-stage clotting assay, 50% have harbored variants of the F12 gene, among which the c.820C>T and c.1763C>A were novel variants underlying the reduced coagulating factor FⅫ.

Comparison and monitoring of antibody response in convalescent and healthy vaccinated individuals against RBD and PCS of SARS-CoV-2 spike protein

The prevalence of SARS-CoV-2 as a global health threat has called for population-wide vaccination to curb COVID-19. Hence, the World Health Organization (WHO) has approved several platforms of SARS-CoV-2 vaccines for emergency use. Therefore, a more comprehensive study on the immune response induced by vaccines in diverse individuals is still required. Here, we expressed a local variant of SARS-CoV-2 receptor-binding domain (RBD) and protease cleavage site (PCS), playing a vital role in binding and fusion in Rosetta (DE3). We then characterized it through SDS-PAGE analysis and western blotting. Moreover, we compared and monitored ChAdOx1 nCoV-19 vaccination-induced antibody response in convalescent and healthy vaccinated individuals after the first and second vaccine doses through serologic assay against RBD and PCS, which have not yet been compared. We investigated a cohort of 100 sera samples; based on our parameters, 25 serum samples were selected as convalescent samples and 25 serum samples as healthy samples for comparison. These findings demonstrate that most of the convalescent sera show more reactivity with PCS (80%) than with RBD (56%). Interestingly, IgG antibody response against PCS was more significant in both pre- and post-vaccination in convalescent individuals than in healthy individuals. Indeed, anti-RBD antibody titers were most significant in pre-vaccination and post–first vaccination in convalescent individuals than in healthy individuals and not in pre-vaccination and post–second vaccination. Besides monitoring IgG antibody response against COVID-19, these findings could shed light on the progress, assessment, and efficacy evaluation of SARS-CoV-2 vaccines.Communicated by Ramaswamy H. Sarma

Urinary endogenous peptides as biomarkers for prostate cancer.

Prostate cancer (PCa) is one of the most prevalent types of cancer in men worldwide; however, the main diagnostic tests available for PCa have limitations and a biopsy is required for histopathological confirmation of the disease. Prostate-specific antigen (PSA) is the main biomarker used for the early detection of PCa, but an elevated serum concentration is not cancer-specific. Therefore, there is a need for the discovery of new non-invasive biomarkers that can accurately diagnose PCa. The present study used trichloroacetic acid-induced protein precipitation and liquid chromatography-mass spectrometry to profile endogenous peptides in urine samples from patients with PCa (n=33), benign prostatic hyperplasia (n=25) and healthy individuals (n=28). Receiver operating characteristic curve analysis was performed to evaluate the diagnostic performance of urinary peptides. In addition, Proteasix tool was used for in silico prediction of protease cleavage sites. Five urinary peptides derived from uromodulin were revealed to be significantly altered between the study groups, all of which were less abundant in the PCa group. This peptide panel showed a high potential to discriminate between the study groups, resulting in area under the curve (AUC) values between 0.788 and 0.951. In addition, urinary peptides outperformed PSA in discriminating between malignant and benign prostate conditions (AUC=0.847), showing high sensitivity (81.82%) and specificity (88%). From in silico analyses, the proteases HTRA2, KLK3, KLK4, KLK14 and MMP25 were identified as potentially involved in the degradation of uromodulin peptides in the urine of patients with PCa. In conclusion, the present study allowed the identification of urinary peptides with potential for use as non-invasive biomarkers in PCa diagnosis.

Mass Spectral Analyses of Salmonella Myovirus SPN3US Reveal Conserved and Divergent Themes in Proteolytic Maturation of Large Icosahedral Capsids.

Salmonella myovirus SPN3US has a T = 27 capsid composed of >50 different gene products, including many that are packaged along with the 240 kb genome and ejected into the host cell. Recently, we showed that an essential phage-encoded prohead protease gp245 is responsible for cleavage of proteins during SPN3US head assembly. This proteolytic maturation step induces major changes in precursor head particles, enabling them to expand and undergo genome packaging. To comprehensively define the composition of the mature SPN3US head and elucidate how it is modified by proteolysis during assembly, we conducted tandem mass spectrometry analysis of purified virions and tailless heads. Fourteen protease cleavage sites were identified in nine proteins, including eight sites not previously identified in head proteins in vivo. Among these was the maturation cleavage site of gp245 which was identical to the autocleavage site we had previously identified in purified recombinant gp245. Our findings underscore the value of employing multiple mass spectrometry-based experimental strategies as a way to enhance the detection of head protein cleavage sites in tailed phages. In addition, our results have identified a conserved set of head proteins in related giant phages that are similarly cleaved by their respective prohead proteases, suggesting that these proteins have important roles in governing the formation and function of large icosahedral capsids.

Manipulation of hydrophobic motifs and optimization of sequence patterns to design high stability peptides against piglet bacterial infections

The risk of resistance to traditional antibiotics has led to a shift in attention to novel antimicrobial functional biomaterials. Antimicrobial peptides (AMPs) have been recognized since the date of birth as a powerful candidate for antibiotic replacement materials. However, immature salt and protease stability, as well as systemic toxicity, hinder the translation of AMPs from the bench to the bedside. Herein, the original motif (KP)3(HYXP)n(KP)3-NH2 (X = Trp or Phe, Y=Ile or Leu, n = 2, 3, 4, 5, or 6) was designed based on the principles of manipulating hydrophobic motifs, optimizing sequence patterns, as well as circumventing cleavage sites of proteases. Original motif was derived a 20-member library for obtaining AMPs with excellent antimicrobial activity, high salt, and protease stability. Results indicated that the target peptides HLFP-5 and HLWP-4 obtained using a comprehensive and integrated screening process exhibited excellent antibacterial ability, salts stability, and protease stability. Membrane cleavage and cell cycle interference-dominated mechanisms of action make target peptides less susceptible to drug resistance than antibiotics. Additionally, advanced target peptides HLFP-5 and HLWP-4 were able to exert direct antimicrobial efficacy in vivo to treat piglets with a systemic bacterial infection. Collectively, these findings may provide novel insights for future design principles, and help drive the application of antibacterial biomaterials in animal husbandry for replacing antibiotics.

SARS-CoV-2 Main Protease Targets Host Selenoproteins and Glutathione Biosynthesis for Knockdown via Proteolysis, Potentially Disrupting the Thioredoxin and Glutaredoxin Redox Cycles

Associations between dietary selenium status and the clinical outcome of many viral infections, including SARS-CoV-2, are well established. Multiple independent studies have documented a significant inverse correlation between selenium status and the incidence and mortality of COVID-19. At the molecular level, SARS-CoV-2 infection has been shown to decrease the expression of certain selenoproteins, both in vitro and in COVID-19 patients. Using computational methods, our group previously identified a set of six host proteins that contain potential SARS-CoV-2 main protease (Mpro) cleavage sites. Here we show experimentally that Mpro can cleave four of the six predicted target sites, including those from three selenoproteins: thioredoxin reductase 1 (TXNRD1), selenoprotein F, and selenoprotein P, as well as the rate-limiting enzyme in glutathione synthesis, glutamate-cysteine ligase catalytic subunit (GCLC). Cleavage was assessed by incubating recombinant SARS-CoV-2 Mpro with synthetic peptides spanning the proposed cleavage sites, and analyzing the products via UPLC-MS. Furthermore, upon incubation of a recombinant Sec498Ser mutant of the full TXNRD1 protein with SARS-CoV-2 Mpro, the predicted cleavage was observed, destroying the TXNRD1 C-terminal redox center. Mechanistically, proteolytic knockdown of both TXNRD1 and GCLC is consistent with a viral strategy to inhibit DNA synthesis, conserving the pool of ribonucleotides for increased virion production. Viral infectivity could also be enhanced by GCLC knockdown, given the ability of glutathione to disrupt the structure of the viral spike protein via disulfide bond reduction. These findings shed new light on the importance of dietary factors like selenium and glutathione in COVID-19 prevention and treatment.

Automated SSHHPS Analysis Predicts a Potential Host Protein Target Common to Several Neuroinvasive (+)ssRNA Viruses.

Within the viral genome, short stretches of homologous host pathogen sequences (SSHHPS) span the protease cleavage sites. To identify host proteins that may be cleaved during infection, we searched the human proteome for viral protease cleavage sites (~20 amino acids). We developed a sequence-to-symptom tool, automating the search and pairing process. We used the viral protein sequence, PHI-BLAST, and UniProt database for gene ontologies and disease relationships. We applied the tool to nine neuroinvasive viruses: Venezuelan and Eastern Equine encephalitis virus (VEEV, EEEV); severe acute respiratory syndrome (SARS, SARS-CoV-2); Middle East respiratory syndrome (MERS); EV-71; Japanese encephalitis virus (JEV); West Nile (WNV); and Zika (ZIKV). A comparison of the hits identified a protein common to all nine viruses called ADGRA2 (GPR124). ADGRA2 was a predicted hit of the 3CL main protease and papain-like protease (PLpro) of SARS-CoV-2. ADGRA2 is an adhesion G protein-coupled receptor and a key endothelial regulator of brain-specific angiogenesis. It is a Wnt7A/Wnt7B specific coactivator of beta-catenin signaling and is essential for blood-brain barrier (BBB) integrity in central nervous system (CNS) diseases. We show the cleavage of the predicted sequences in MYOM1, VWF by the SARS-CoV-2 PLpro; DNAH8 (dynein) by the MERS PLpro; ADGRA2 by the alphaviral VEEV nsP2 protease; and POT1 by the SARS-CoV-2 and MERS PLpro.

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.

In Vitro Properties and Pharmacokinetics of Temporarily PEGylated Onc72 Prodrugs.

The favorable properties of antimicrobial peptides (AMPs) to rapidly kill pathogens are often limited by unfavorable pharmacokinetics due to fast degradation and renal clearance rates. Here, a prodrug strategy linking proline-rich AMP Onc72 to polyethylene glycol (PEGs) with average molecular weights of 5 and 20kDa via a peptide linker containing a protease cleavage site is tested for the first time in vivo. Onc72 is released from these 5k- and 20k-prodrugs in mouse serum with half-life times (t1/2 ) of 8 and 14h, respectively. Importantly, PEGylation protects Onc72 from proteolytic degradation providing a prolonged release of Onc72, balancing the degradation of free Onc72, and leading to relatively stable Onc72 concentrations and high antibacterial activities. The prodrugs are not hemolytic on human erythrocytes and show only slight cytotoxic effects on human cell lines indicating promising safety margins. When administered subcutaneously to female CD-1 mice, the prodrugs elimination t1/2 are 66min and ≈5.5h, respectively, compared to 43min of free Onc72. The maximal Onc72 plasma levels are obtained ≈1 and ≈8h postadministration, respectively. In conclusion, the prodrugs provide extended elimination t1/2 and a constant release of Onc72 in mice, potentially limiting adverse effects and increasing efficacy.

High-level production of keratinocyte growth factor 2 in Escherichia coli

Recombinant human keratinocyte growth factor 2 (KGF-2), also known as repifermin, is used in various therapeutic applications. However, KGF-2 production has not been optimized for facilitating large-scale production. Therefore, we attempted to attain high-level production of bioactive KGF-2. KGF-2 was fused with 6HFh8 (6HFh8-KGF-2) at the tobacco etch virus protease cleavage site. The 6HFh8-KGF-2 was expressed in Escherichia coli with high expression levels of approximately 33% and 20% of soluble protein in flask culture and 5 L fermentation, respectively. 6HFh8-KGF-2 was purified via nickel affinity chromatography. To maintain a stable form of KGF-2, the conditions of the cleavage reaction were optimized based on the isoelectric point. KGF-2 was purified via ion-exchange chromatography to high purity (>99%) with an optimal purification yield (91%). Circular dichroism spectroscopy demonstrated that purified KGF-2 had a secondary structure and thermal stability similar to that of commercial KGF-2. Bioactivity assays indicated that purified KGF-2 could induce MCF-7 cell proliferation in the same manner as commercial KGF-2. These results demonstrate that bioactive KGF-2 was overexpressed in E. coli and purified to high quality. Our findings indicated that bioactive KGF-2 can be produced in large quantities in E. coli.

A Bioengineering Strategy to Control ADAM10 Activity in Living Cells.

A Disintegrin and Metalloprotease 10, also known as ADAM10, is a cell surface protease ubiquitously expressed in mammalian cells where it cuts several membrane proteins implicated in multiple physiological processes. The dysregulation of ADAM10 expression and function has been implicated in pathological conditions, including Alzheimer's disease (AD). Although it has been suggested that ADAM10 is expressed as a zymogen and the removal of the prodomain results in its activation, other potential mechanisms for the ADAM10 proteolytic function and activation remain unclear. Another suggested mechanism is post-translational modification of the cytoplasmic domain, which regulates ADAM10-dependent protein ectodomain shedding. Therefore, the precise and temporal activation of ADAM10 is highly desirable to reveal the fine details of ADAM10-mediated cleavage mechanisms and protease-dependent therapeutic applications. Here, we present a strategy to control prodomain and cytosolic tail cleavage to regulate ADAM10 shedding activity without the intervention of small endogenous molecule signaling pathways. We generated a series of engineered ADAM10 analogs containing Tobacco Etch Virus protease (TEV) cleavage site (TEVcs), rendering ADAM10 cleavable by TEV. This strategy revealed that, in the absence of other stimuli, the TEV-mediated removal of the prodomain could not activate ADAM10. However, the TEV-mediated cleavage of the cytosolic domain significantly increased ADAM10 activity. Then, we generated ADAM10 with a minimal constitutively catalytic activity that increased significantly in the presence of TEV or after activating a chemically activatable TEV. Our results revealed a bioengineering strategy for controlling the ADAM10 activity in living cells, paving the way to obtain spatiotemporal control of ADAM10. Finally, we proved that our approach of controlling ADAM10 promoted α-secretase activity and the non-amyloidogenic cleavage of amyloid-β precursor protein (APP), thereby increasing the production of the neuroprotective soluble ectodomain (sAPPα). Our bioengineering strategy has the potential to be exploited as a next-generation gene therapy for AD.