Protease Cleavage Site Research Articles

Abstract MP10: Furin-Mediated Modification Is Required For Epithelial Sodium Channel-Activating Activity Of Soluble (Pro)Renin Receptor In Cultured Collecting Duct Cells

(Pro)renin receptor (PRR) contains overlapping cleavage site for site-1 protease (S1P) and furin for generation of soluble PRR (sPRR). Although S1P-mediated cleavage mediates the release of sPRR, the functional implication of furin-mediated cleavage is unclear. Here we tested whether furin-mediated cleavage was required for the activity of sPRR in activating ENaC in cultured M1 cells. M1 cells were transfected with pcDNA3.4 containing full-length PRR with (Furin Mut) or without (WT) mutagenesis of furin cleavage site; empty vector (EM) served a control. As compared with EM, overexpression of WT and Furin Mut vectors induced a more than 16-fold comparable increase in the release of sPRR. Amiloride-sensitive short circuit current as assessed by Ussing chamber technique was elevated by overexpression of WT PRR which was reduced by 37% by Furin Mut (ENaC activity: 1.00 + 0.06 μA/cm 2 in EM, 1.67 + 0.05 μA/cm 2 in WT, and 1.04 + 0.07 μA/cm 2 in Furin Mut, p < 0.05). In parallel, the expression of α-ENaC but not β or γ subunit as assessed by immunoblotting and qRT-PCR analysis was elevated by WT PRR and this increase was blunted by Furin Mut. In a separate experiment, M1 cells were transfected with pcDNA3.4 containing cDNA for sPRR with S1P cleavage (AA 1-282) (sPRR-S1P) or with furin cleavage (AA 1-279) (sPRR-furin); empty vector was used as a control. Overexpression of cDNA for the two types of sPRR induced a significant and comparable increase in the release of sPRR. By Ussing chamber technique, ENaC activity was 1.00 + 0.09 μA/cm 2 in EM, 1.03 + 0.10 μA/cm 2 in sPRR-S1P, and 1.39 + 0.14 μA/cm 2 in sPRR-furin, p < 0.05. Lastly, 293 cells were pretreated for 1 h with furin inhibitor α1-antitrypsin Portland followed by transfection with empty vector, WT PRR, or Furin Mut vectors. sPRR in the condition medium was enriched by using protein centrifugal filter devices and applied to M1 cells for 10 min followed by measurement of ENaC activity. Pretreatment with furin inhibition attenuated ENaC-acting activity induced by overexpression of WT PRR. Overall, the three independent approaches consistently demonstrated that furin-mediated modification is required for the activity of sPRR to increase ENaC-mediated Na + transport in the CD cells.

Identification of novel ADAMTS1, ADAMTS4 and ADAMTS5 cleavage sites in versican using a label-free quantitative proteomics approach

The chondroitin sulfate proteoglycan versican is important for embryonic development and several human disorders. The versican V1 splice isoform is widely expressed and cleaved by ADAMTS proteases at a well-characterized site, Glu441-Ala442. Since ADAMTS proteases cleave the homologous proteoglycan aggrecan at multiple sites, we hypothesized that additional cleavage sites existed within versican. We report a quantitative label-free approach that ranks abundance of liquid chromatography-tandem mass spectrometry (LC-MS/MS)-identified semi-tryptic peptides after versican digestion by ADAMTS1, ADAMTS4 and ADAMTS5 to identify site-specific cleavages. Recombinant purified versican V1 constructs were digested with the recombinant full-length proteases, using catalytically inactive mutant proteases in control digests. Semi-tryptic peptide abundance ratios determined by LC-MS/MS in ADAMTS:control digests were compared to the mean of all identified peptides to obtain a z-score by which outlier peptides were ranked, using semi-tryptic peptides identifying Glu441 -Ala442 cleavage as the benchmark. Tryptic peptides with higher abundance in control digests supported cleavage site identification. We identified several novel cleavage sites supporting the ADAMTS1/4/5 cleavage site preference for a P1-Glu residue in proteoglycan substrates. Digestion of proteins in vitro and application of this z-score approach is potentially widely applicable for mapping protease cleavage sites using label-free proteomics. SignificanceVersican abundance and turnover are relevant to the pathogenesis of several human disorders. Versican is cleaved by A Disintegrin-like And Metalloprotease with Thrombospondin type 1 motifs (ADAMTS) family members at Glu441-Ala442, generating a bioactive proteoform called versikine, but additional cleavage sites and the site-specificity of individual ADAMTS proteases is unexplored. Here, we used a label-free proteomics strategy to identify versican cleavage sites for 3 ADAMTS proteases, applying a novel z-score-based statistical approach to compare the protease digests of versican to controls (digests with inactive protease) using the known protease cleavage site as a benchmark. We identified 21 novel cleavage sites that had a comparable z-score to the benchmark. Given the functional significance of versikine, they represent potentially significant cleavages and helped to refine a substrate site preference for each protease.The z-score approach is potentially widely applicable for discovery of site-specific cleavages within an purified protein or small ensemble of proteins using any protease.

Structure and Dynamics of Zika Virus Protease and Its Insights into Inhibitor Design.

Zika virus (ZIKV)—a member of the Flaviviridae family—is an important human pathogen. Its genome encodes a polyprotein that can be further processed into structural and non-structural proteins. ZIKV protease is an important target for antiviral development due to its role in cleaving the polyprotein to release functional viral proteins. The viral protease is a two-component protein complex formed by NS2B and NS3. Structural studies using different approaches demonstrate that conformational changes exist in the protease. The structures and dynamics of this protease in the absence and presence of inhibitors were explored to provide insights into the inhibitor design. The dynamic nature of residues binding to the enzyme cleavage site might be important for the function of the protease. Due to the charges at the protease cleavage site, it is challenging to develop small-molecule compounds acting as substrate competitors. Developing small-molecule compounds to inhibit protease activity through an allosteric mechanism is a feasible strategy because conformational changes are observed in the protease. Herein, structures and dynamics of ZIKV protease are summarized. The conformational changes of ZIKV protease and other proteases in the same family are discussed. The progress in developing allosteric inhibitors is also described. Understanding the structures and dynamics of the proteases are important for designing potent inhibitors.

The Phage T4 Antiholin RI Has a Cleavable Signal Peptide, Not a SAR Domain.

Holin/endolysin-mediated lysis of phage T4 of Escherichia coli is tightly regulated by the antiholins RI and RIII. While regulation by the cytoplasmic RIII plays a minor role, the periplasmic antiholin RI binds tightly to the holin T and is believed to directly sense periplasmic phage DNA from superinfections as a trigger for the inhibition of lysis. RI has been reported to contain a non-cleavable signal peptide that anchors the protein to the membrane. Lysis is believed to be induced at some stage by a membrane depolarization that causes a release of RI into the periplasm without cleavage of the signal anchor. For the current model of phage lysis induction, it is thus a fundamental assumption that the N-terminal trans-membrane domain (TMD) of RI is such a signal anchor release (SAR) domain. Here we show that, in contrast to previous reports, this domain of RI is a cleavable signal peptide. RI is processed and released into the periplasm as a mature protein, and inactivation of its signal peptidase cleavage site blocks processing and membrane release. The signal peptide of RI can also mediate the normal translocation of a well-characterized Sec substrate, PhoA, into the periplasm. This simplifies the current view of phage lysis regulation and suggests a fundamentally different interpretation of the recently published structure of the soluble domains of the RI–T complex.

Predicting and characterising protein damage in the extracellular matrix

Chronic UVR exposure of human skin can result in photo-ageing which manifests both externally and internally (as remodelling of skin layers including the extracellular matrix-rich dermis). However, the intermittent nature of UVR exposure over a timescale of decades combined with the longevity of many structural dermal proteins makes the identification of dermal photo-ageing targets and mechanisms challenging. Over the past ten years work in our group has demonstrated that: (i) proteins which are rich in amino acid chromophores are susceptible to physiologically relevant doses of solar simulated radiation, (ii) this protein degradation is mediated primarily by the photodynamic production of reaction oxygen species and (iii) UVR-chromophore rich proteins are located in tissue regions where they may act as endogenous sunscreens. We have also shown that ECM proteases selectively degrade UVR-damaged assemblies in vitro and have developed new machine learning-based models to predict protease cleavage sites and hence relative protease susceptibilities in the human skin proteome. The recent development of peptide location fingerprinting applied to conventional mass spectrometry datasets has allowed the identification of novel candidate biomarkers of UVR-induced damage as a consequence of photo-ageing. For example, this approach is able to identify structure-associated modifications in collagen VI alpha chains, although collagen VI remodeling is not evident by conventional histological analysis. These methods and resources (skin proteome database, protein susceptibility prediction and peptide location fingerprinting analysis) are available for use at www.manchesterproteome.manchester.ac.uk. Key areas for future research include using peptide location fingerprinting to: (i) characterise the structural hallmarks of ageing and photo-ageing mechanisms across different phototypes and (ii) evaluate the efficacy of rejuvenating treatments against novel protein biomarkers.

Information Fusion for Biological Prediction

Information fusion has become a powerful tool for challenging applications such as biological prediction problems. In this paper, we apply a new information-theoretical fusion technique to HIV-1 protease cleavage site prediction, which is a problem that has been in the focus of much interest and investigation of the machine learning community recently. It poses a difficult classification task due to its high dimensional feature space and a relatively small set of available training patterns. We also apply a new set of biophysical features to this problem and present experiments with neural networks, support vector machines, and decision trees. Application of our feature set results in high recognition rates and concise decision trees, producing manageable rule sets that can guide future experiments. In particular, we found a combination of neural networks and support vector machines to be beneficial for this problem.

Abstract 1824: HER2-XPAT, a novel protease-activatable T-cell engager (TCE) with potent T-cell activation and efficacy in solid tumors and large safety margins in non-human primate (NHP)

Abstract Background: TCEs are effective in leukemias but have been challenging in solid tumors due to on-target, off-tumor toxicity. Attempts to circumvent cytokine release syndrome (CRS) including step-up dosing and/or complex molecular designs have been unsuccessful due to toxicity and/or enhanced immunogenicity. HER2-XPAT, or XTENylated Protease-Activated bispecific T-Cell Engager, is a prodrug TCE that exploits the protease activity present in tumors vs healthy tissue to expand the therapeutic index (TI). The core of the HER2-XPAT (PAT) consists of 2 tandem scFvs targeting CD3 and HER2. Attached to the core, two unstructured polypeptide masks (XTEN) sterically reduce target engagement and extend T1/2. Protease cleavage sites at the base of the XTEN masks enable proteolytic activation of XPATs in the tumor microenvironment, unleashing a potent TCE with short T1/2, further improving the TI. Methods: Preclinical studies were conducted to characterize the activity of HER2-XPAT, HER2-PAT (cleaved XPAT), and HER2-NonClv (a non-cleavable XPAT) for cytotoxicity in vitro, anti-tumor efficacy and cleavage in xenograft models, stability in human plasma, and safety in NHPs. Results: The unmasked HER2-PAT demonstrated potent in vitro T-cell cytotoxicity (EC50 1-2pM) and target-dependent T-cell activation and cytokine production by hPBMCs. HER2-XPAT provided up to 14,000-fold protection against killing of HER2 tumor cells and exhibited only minimal cytotoxicity against cardiomyocytes at 1μM. In vivo, HER2-XPAT induced complete tumor regressions (CRs) in BT-474 tumors with equimolar dosing as HER2-PAT, whereas HER2-NonClv lacked efficacy, supporting a requirement of protease cleavage. HER2-XPAT was also highly efficacious in the HER2 low-expressing HT-55 colorectal model. Preferential proteolytic cleavage of fluorescent-labeled HER2-XPAT in tumors vs healthy organs was demonstrated in vivo. In NHP, HER2-XPAT has been dose-escalated safely up to 42mg/kg (MTD). HER2-XPAT demonstrated early T-cell margination at 2mg/kg but largely spared CRS, cytokines, and major tissue toxicity up to 42mg/kg. In vivo, the PK of HER2-XPAT was comparable to its non-cleavable counterpart, consistent with its ex vivo stability for cleavage in cancer pts plasma for 7 days at 37°C. Continuous infusion of HER2-PAT induced lethal CRS and cytokine spikes at 0.3mg/kg/d but was tolerated at 0.25mg/kg/d, providing HER2-XPAT with 500-fold protection in tolerated Cmax vs HER2-PAT. Conclusions: HER2-XPAT is a potent, prodrug TCE exhibiting protease-dependent tumor efficacy in mouse xenografts and a wide TI with no CRS in NHPs. XTEN's low immunogenicity has already been demonstrated in humans (growth hormone and FVIII). HER2-XPAT represents a promising solution for HER2-high tumors with potential expansion to HER2 low expressing tumors. IND studies are ongoing. Citation Format: Fiore Cattaruzza, Ayesha Nazeer, Zachary Lange, Caitlin Koski, Mikhail Hammond, Milton To, Pete Yeung, Sharon Lam, Mika K. Derynck, Bryan Irving, Volker Schellenberger. HER2-XPAT, a novel protease-activatable T-cell engager (TCE) with potent T-cell activation and efficacy in solid tumors and large safety margins in non-human primate (NHP) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1824.

PNPLA3 and TLL-1 Polymorphisms as Potential Predictors of Disease Severity in Patients With COVID-19.

Albeit the pathogenesis of COVID-19 remains unclear, host’s genetic polymorphisms in genes involved in infection and reinfection, inflammation, or immune stimulation could play a role in determining the course and outcome. We studied in the early phase of pandemic consecutive patients (N = 383) with SARS-CoV-2 infection, whose subsequent clinical course was classified as mild or severe, the latter being characterized by admission to intensive therapy unit or death. Five host gene polymorphisms (MERTK rs4374383, PNPLA3 rs738409, TLL-1 rs17047200, IFNL3 rs1297860, and INFL4 rs368234815) were assessed by using whole nucleic acids extracted from nasopharyngeal swabs. Specific protease cleavage sites of TLL-1 on the SARS-CoV-2 Spike protein were predicted in silico. Male subjects and older patients were significantly at higher risk for a severe outcome (p = 0.02 and p < 0.001, respectively). By considering patients ≤65 years, after adjusting for potential confounding due to sex, an increased risk of severe outcome was found in subjects with the GG genotype of PNPLA3 (adj-OR: 4.69; 95% CI = 1.01–22.04) or TT genotype of TLL-1 (adj-OR=9.1; 95% CI = 1.45–57.3). In silico evaluation showed that TLL-1 is potentially involved in the Spike protein cleavage which is essential for viral binding and entry into the host cells using the host receptor angiotensin-converting enzyme 2 (ACE2). Subjects carrying a GG genotype in PNPLA3 gene might have a constitutive upregulation of the NLRP3 inflammasome and be more prone to tissue damage when infected by SARS-CoV-2. The TT genotype in TLL-1 gene might affect its protease activity on the SARS-CoV-2 Spike protein, enhancing the ability to infect or re-infect host’s cells. The untoward effect of these variants on disease course is evident in younger patients due to the relative absence of comorbidities as determinants of prognosis. In the unresolved pathogenetic scenery of COVID-19, the identification of genetic variants associates with more prolonged course or with a severe outcome of infection would support the development of predictive tools useful to stratify subjects by risk class at presentation. Moreover, the individuation of key genes could contribute to a better understanding of the pathways involved in the pathogenesis, giving the basis for rational therapeutic approaches.

The SARS-CoV-2 SSHHPS Recognized by the Papain-like Protease.

Viral proteases are highly specific and recognize conserved cleavage site sequences of ∼6–8 amino acids. Short stretches of homologous host–pathogen sequences (SSHHPS) can be found spanning the viral protease cleavage sites. We hypothesized that these sequences corresponded to specific host protein targets since >40 host proteins have been shown to be cleaved by Group IV viral proteases and one Group VI viral protease. Using PHI-BLAST and the viral protease cleavage site sequences, we searched the human proteome for host targets and analyzed the hit results. Although the polyprotein and host proteins related to the suppression of the innate immune responses may be the primary targets of these viral proteases, we identified other cleavable host proteins. These proteins appear to be related to the virus-induced phenotype associated with Group IV viruses, suggesting that information about viral pathogenesis may be extractable directly from the viral genome sequence. Here we identify sequences cleaved by the SARS-CoV-2 papain-like protease (PLpro) in vitro within human MYH7 and MYH6 (two cardiac myosins linked to several cardiomyopathies), FOXP3 (an X-linked Treg cell transcription factor), ErbB4 (HER4), and vitamin-K-dependent plasma protein S (PROS1), an anticoagulation protein that prevents blood clots. Zinc inhibited the cleavage of these host sequences in vitro. Other patterns emerged from multispecies sequence alignments of the cleavage sites, which may have implications for the selection of animal models and zoonosis. SSHHPS/nsP is an example of a sequence-specific post-translational silencing mechanism.

The COVID-19 Menace.

Coronavirus disease 2019 (COVID‐19) is caused by the new severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), which binds to ectoenzyme angiotensin‐converting enzyme 2. It is very contagious and is spreading rapidly around the world. Until now, coronaviruses have mainly been associated with the aerodigestive tract due to the presence of a monobasic cleavage site for the resident transmembrane serine protease 2. Notably, SARS‐CoV‐2 is equipped with a second, polybasic cleavage site for the ubiquitous furin protease, which may determine the widespread tissue tropism. Furthermore, the terminal sequence of the furin‐cleaved spike protein also binds to neuropilin receptors. Clinically, there is enormous variability in the severity of the disease. Severe consequences are seen in a relatively small number of patients, most show moderate symptoms, but asymptomatic cases, especially among young people, drive disease spread. Unfortunately, the number of local infections can quickly build up, causing disease outbreaks suddenly exhausting health services’ capacity. Therefore, COVID‐19 is dangerous and unpredictable and has become the most serious threat for generations. Here, the latest research on COVID‐19 is summarized, including its spread, testing methods, organ‐specific complications, the role of comorbidities, long‐term consequences, mortality, as well as a new hope for immunity, drugs, and vaccines.

Structural Insights into the Human Astrovirus Capsid.

Astroviruses (AstVs) are non-enveloped, positive single-stranded RNA viruses that cause a wide range of inflammatory diseases in mammalian and avian hosts. The T = 3 viral capsid is unique in its ability to infect host cells in a process driven by host proteases. Intercellular protease cleavages allow for viral egress from a cell, while extracellular cleavages allow for the virus to enter a new host cell to initiate infection. High-resolution models of the capsid core indicate a large, exposed region enriched with protease cleavage sites. The virus spike protein allows for binding to target cells and is the major target for naturally occurring and engineered neutralizing antibodies. During maturation, the capsid goes through significant structural changes including the loss of many surface spikes. The capsid interacts with host membranes during the virus life cycle at multiple stages such as assembly, host cell entry and exit. This review will cover recent findings and insights related to the structure of the capsid and its function. Further understanding of the viral capsid structure and maturation process can contribute to new vaccines, gastric therapeutics, and viral engineering applications.

Tau Protein as a Fusion Tag for Recombinant Peptide/Protein Expression

The microtubule-associated protein tau plays an important role in regulating the assembly and spatial organization of microtubule in the central nervous system. Although hyperphosphorylated tau forms amyloid filaments in a number of neurodegenerative diseases known as tauopathies, such as Alzheimer's disease, unmodified tau is stable and resistant to aggregation even in boiling water. Tau protein is highly expressed in E. coli and can be easily purified from cell lysis by utilizing its thermo stability. So for, widely used tags for recombinant protein expression are folded proteins, such as glutathione S-transferase, maltose binding protein, and ubiquitin. In this work, we designed a novel recombinant protein/peptide expression system based on the microtubule-binding domain of intrinsically disordered protein tau. A His-tag was arranged at the N-terminal of tau protein to facilitate affinity purification. A TEV protease cleavage site was introduced between tau and target protein/peptide, enabling release of target protein/peptide from the tagged protein (Figure 1). To demonstrate the usability of this tau fusion expression system, we tested the expression of three proteins/peptides, i.e., the N-terminal p53 binding domain of MdmX, the azurin derived anticancer peptide p28, and the C-terminal transactivation domain of HIF-1α. Compared to GST-fused MdmX, the expression level and solubility of tau-fused MdmX was higher under identical expression temperature and IPTG concentration. p28 and HIF-1a are disordered peptides with 28 and 51 residues, respectively. Both tau-fused p28 and tau-fused HIF-1α were mainly expressed as soluble proteins at high level. Furthermore, most bacterial proteins were separated from tau-fused p28 and tau-fused HIF-1a by boiling the cell lyses. Finally, the tau-fused MdmX, p28, and HIF-1α could be efficiently purified by using Ni-NTA and the purity was higher than 90% based on SDS-PAGE (Figure 2). Therefore, the highly expressed and thermo stable disordered protein tau is expected to be applicable to prepare various recombinant proteins.

Potential 3-chymotrypsin-like cysteine protease cleavage sites in the coronavirus polyproteins pp1a and pp1ab and their possible relevance to COVID-19 vaccine and drug development.

Coronavirus (CoV) 3‐chymotrypsin (C)‐like cysteine protease (3CLpro) is a target for anti‐CoV drug development and drug repurposing because along with papain‐like protease, it cleaves CoV‐encoded polyproteins (pp1a and pp1ab) into nonstructural proteins (nsps) for viral replication. However, the cleavage sites of 3CLpro and their relevant nsps remain unclear, which is the subject of this perspective. Here, we address the subject from three standpoints. First, we explore the inconsistency in the cleavage sites and relevant nsps across CoVs, and investigate the function of nsp11. Second, we consider the nsp16 mRNA overlapping of the spike protein mRNA, and analyze the effect of this overlapping on mRNA vaccines. Finally, we study nsp12, whose existence depends on ribosomal frameshifting, and investigate whether 3CLpro requires a large number of inhibitors to achieve full inhibition. This perspective helps us to clarify viral replication and is useful for developing anti‐CoV drugs with 3CLpro as a target in the current coronavirus disease 2019 (COVID‐19) pandemic.

Assessment of the population coverage of an HIV-1 vaccine targeting sequences surrounding the viral protease cleavage sites in Gag, Pol, or all 12 protease cleavage sites

Development of an effective HIV-1 vaccine has been a great challenge faced by the research community. Recently a novel strategy targeting the viral protease cleavage sites (PCSs) has been tested and shown promising results. This T cell-based vaccine strategy depends on individuals expressing certain HLA class I molecules and since each population has unique distributions of HLA class I alleles, population coverage analysis is required to assess feasibility. Utilizing the validated CD8 T cell epitope data from previous studies we conducted coverage analysis of an HIV-1 vaccine targeting the sequences surrounding all 12-PCSs, Gag-PCSs, and Pol-PCSs. The population coverage, average epitope hit, and minimum number of epitopes recognized by 90% of the population (PC90) was compiled for 66 countries and 16 geographical regions using the web tool provided by “Immune Epitope Database and Analysis Resource”. Our analysis shows that the coverage for an HIV-1 vaccine targeting sequences surrounding all 12 PCSs, 5 PCSs in Gag or 6 PCSs in Pol can cover ~ 70% to ~ 100% of the populations analyzed. There was no statistical difference in population coverages for the majority of populations examined when comparing the CD8 T cell epitope sets (12-PCSs, Gag-PCSs, and Pol-PCSs). As expected, vaccines targeting more sequences will have more CD8 T cell epitopes, as the mean average epitope hit for the 12-PCSs, Gag-PCSs, and Pol-PCSs across all countries studied was 9.45, 4.76, and 4.74, respectively, and across all geographical regions was 9.76, 4.99, and 4.92, respectively. The average PC90 for the 12-PCSs, Gag-PCSs, and Pol-PCSs across all countries studied was 2.53, 1.31, and 1.41, respectively, and across all geographical regions was 2.24, 1.23, and 1.29, respectively. Thus, vaccines targeting sequences surrounding the HIV-1 PCSs can cover broad populations; however, whether targeting a subset of the PCSs is sufficient to prevent acquisition requires further preclinical investigation.

DeepDigest: Prediction of Protein Proteolytic Digestion with Deep Learning.

Proteolytic digestion of proteins by one or more proteases is a key step in shotgun proteomics, in which the proteolytic products, i.e., peptides, are taken as the surrogates of their parent proteins for further qualitative or quantitative analysis. The proteases generally cleave proteins at specific amino acid residue sites, but digestion is hardly complete (wide existence of missed cleavage sites). Therefore, it would be of great help to improve the prior experimental design and the posterior data analysis if the digestion behaviors of proteases can be accurately modeled and predicted. At present, systematic studies about the commonly used proteases in proteomics are insufficient, and there is a lack of easy-to-use tools to predict the cleavage sites of different proteases. Here, we propose a novel sequence-based deep learning algorithm-DeepDigest, which integrates convolutional neural networks and long short-term memory networks for protein digestion prediction. DeepDigest can predict the cleavage probability of each potential cleavage site on the protein sequences for eight popular proteases including trypsin, ArgC, chymotrypsin, GluC, LysC, AspN, LysN, and LysargiNase. We compared DeepDigest with three traditional machine learning algorithms, i.e., logistic regression, random forest, and support vector machine. On the eight training data sets, the 10-fold cross-validation accuracies (AUCs) of DeepDigest were 0.956-0.982, significantly higher than those of the three traditional algorithms. On the 11 independent test data sets, DeepDigest achieved AUCs between 0.849 and 0.978, outperforming the other traditional algorithms in most cases. Transfer learning then further improved the prediction accuracy. Besides, some interesting characteristics of different proteases were revealed and discussed. Ultimately, as an application, we used DeepDigest to predict the digestibilities of peptides and demonstrated that peptide digestibility is an informative new feature to discriminate between correct and incorrect peptide identifications.

Predicting HIV-1 Protease Cleavage Sites With Positive-Unlabeled Learning.

Understanding the substrate specificity of HIV-1 protease plays an essential role in the prevention of HIV infection. A variety of computational models have thus been developed to predict substrate sites that are cleaved by HIV-1 protease, but most of them normally follow a supervised learning scheme to build classifiers by considering experimentally verified cleavable sites as positive samples and unknown sites as negative samples. However, certain noisy can be contained in the negative set, as false negative samples are possibly existed. Hence, the performance of the classifiers is not as accurate as they could be due to the biased prediction results. In this work, unknown substrate sites are regarded as unlabeled samples instead of negative ones. We propose a novel positive-unlabeled learning algorithm, namely PU-HIV, for an effective prediction of HIV-1 protease cleavage sites. Features used by PU-HIV are encoded from different perspectives of substrate sequences, including amino acid identities, coevolutionary patterns and chemical properties. By adjusting the weights of errors generated by positive and unlabeled samples, a biased support vector machine classifier can be built to complete the prediction task. In comparison with state-of-the-art prediction models, benchmarking experiments using cross-validation and independent tests demonstrated the superior performance of PU-HIV in terms of AUC, PR-AUC, and F-measure. Thus, with PU-HIV, it is possible to identify previously unknown, but physiologically existed substrate sites that are able to be cleaved by HIV-1 protease, thus providing valuable insights into designing novel HIV-1 protease inhibitors for HIV treatment.

Silencing of the proteasome and oxidative stress impair endoplasmic reticulum targeting and signal cleavage of a prostate carcinoma antigen

The endoplasmic reticulum (ER) is an organelle with high protein density and therefore prone to be damaged by protein aggregates. One proposed preventive measure is a pre-emptive quality control pathway that attenuates ER import during protein folding stress. ER resident proteins are targeted into the ER via signal peptides cleaved rapidly upon ER insertion by the ER signal peptidase. Here we show that the ER insertion and cleavage of the ER-targeting peptide of the prostate carcinoma antigen prostate stem cell antigen (PSCA) is retarded and strongly reduced when the proteasome is inhibited or genetically silenced. Also overexpression of the C-terminally extended ubiquitin variant Ub2-UBB+1 or oxidative stress attenuated signal peptide processing. Proteasome inhibition likewise protracted ER signal processing of the ER targeted hormone leptin and the MHC class I molecule H–2Dd. These findings, which are consistent with a pre-emptive ER quality control pathway, may explain why an immunodominant MHC class I peptide ligand of PSCA spanning its ER signal peptidase cleavage site is efficiently generated in the cytoplasm from PSCA precursors that fail to reach the ER.

Predicting Proteolysis in Complex Proteomes Using Deep Learning.

Both protease- and reactive oxygen species (ROS)-mediated proteolysis are thought to be key effectors of tissue remodeling. We have previously shown that comparison of amino acid composition can predict the differential susceptibilities of proteins to photo-oxidation. However, predicting protein susceptibility to endogenous proteases remains challenging. Here, we aim to develop bioinformatics tools to (i) predict cleavage site locations (and hence putative protein susceptibilities) and (ii) compare the predicted vulnerabilities of skin proteins to protease- and ROS-mediated proteolysis. The first goal of this study was to experimentally evaluate the ability of existing protease cleavage site prediction models (PROSPER and DeepCleave) to identify experimentally determined MMP9 cleavage sites in two purified proteins and in a complex human dermal fibroblast-derived extracellular matrix (ECM) proteome. We subsequently developed deep bidirectional recurrent neural network (BRNN) models to predict cleavage sites for 14 tissue proteases. The predictions of the new models were tested against experimental datasets and combined with amino acid composition analysis (to predict ultraviolet radiation (UVR)/ROS susceptibility) in a new web app: the Manchester proteome susceptibility calculator (MPSC). The BRNN models performed better in predicting cleavage sites in native dermal ECM proteins than existing models (DeepCleave and PROSPER), and application of MPSC to the skin proteome suggests that: compared with the elastic fiber network, fibrillar collagens may be susceptible primarily to protease-mediated proteolysis. We also identify additional putative targets of oxidative damage (dermatopontin, fibulins and defensins) and protease action (laminins and nidogen). MPSC has the potential to identify potential targets of proteolysis in disparate tissues and disease states.

Molecular adaptive evolution of SARS-COV-2 spike protein in Saudi Arabia.

The sequences of SARS-CoV-2 spike (S) from Saudi Arabia along with SARS-CoV and bat SARS-like CoVs were obtained. Positive selection analysis and secondary structure investigation of spike sequences were performed. Adaptive molecular evolution was observed in SARS-CoV-2 displayed by positive selection pressure at N-terminal domain (NTD; codons 41, 163, 174 and 218), Receptor binding domain (RBD; codons 378 and 404) and S1/S2 Cleavage site (codon 690). Furthermore, the spike protein secondary structure depicted by the homo-trimer structure showed a high similarity between Saudi SARS-CoV-2 isolate and the parental strain (bat SL-COVZC45). Despite the high similarity depicted in the spike sequence model alignment, it displayed a significant difference when each chain was treated solely owing to 7 motif differences in the three composing chains. In addition, SARS-CoV-2 S trimer model uncovered the presence of N-acetyl glucosamine ligands. Eventually, 3C-like proteinase cleavage site was observed in S2 domain could be used as a site for drug discovery. Genetics and molecular evolutionary facts are useful for assessment of evolution, host adaptation and epidemic patterns ultimately helpful for adaptation of control strategies.

Abstract PS17-11: Her2-xpat, a novel protease-activatable prodrug t-cell engager (tce), exhibits potent t-cell activation and efficacy in her2 tumors, yielding large predicted safety margins based on non-human primate (nhp)

Abstract Background TCEs are effective at inducing remissions in hematologic cancers but have been challenging in solid tumors due to on-target, off-tumor toxicity. Attempts to circumvent CRS include fractionated or step-up dosing and/or complex molecular designs, but these have been unsuccessful due to toxicity and/or enhanced immunogenicity. Amunix has developed a conditionally active TCE, XPAT or XTENylated Protease-Activated bispecific T-Cell Engager, that exploits the protease activity present in tumors vs. healthy tissue to expand the therapeutic index (TI). The core of the HER2-XPAT (PAT) consists of 2 tandem scFVs targeting CD3 and HER2. Two unstructured polypeptide masks (XTEN) are attached to the core that sterically reduce target engagement and extend T1/2. Protease cleavage sites at the base of the XTEN masks enable proteolytic activation of XPATs in the tumor microenvironment, unleashing a highly potent TCE with a short T1/2 and further improving the TI. Although checkpoint inhibitors have been ineffective in HER2+ or HR+ breast cancer, TCEs can co-opt T-cells regardless of their antigenic specificity, providing the potential to introduce effective T-cell immunity against tumors expressing HER2. HER2-XPAT, as a tumor protease-activatable prodrug with a wide safety margin, offers the potential to induce effective T-cell cytotoxicity against HER2hi tumors and HR+/HER2 1-2+ tumors. Methods Preclinical studies were conducted to characterize the activity of HER2-XPAT, HER2-PAT (cleaved XPAT), and HER2-NonClv (a non-cleavable XPAT) for cytolytic activity in vitro, for anti-tumor efficacy in xenograft models, and for stability and safety in NHPs. Results HER2-PAT (cleaved XPAT) demonstrated potent in vitro tumor-directed T-cell cytotoxicity (EC50 1-2pM) and target-dependent T-cell activation and production of cytokines by PBMCs. HER2-PAT also exhibited cytotoxicity in HR+, HER2 1+ MCF7s at higher doses (EC50 0.13nM). HER2-XPAT provided up to 14,000-fold protection against killing of HER2 tumor cells and exhibited no cytotoxicity against cardiomyocytes at concentrations up to 1uM. In vivo, HER2-XPAT induced complete tumor regressions in BT-474 tumor models with equimolar dosing to HER2-PAT, whereas non-cleavable HER2-NonClv had no efficacy, supporting tumor protease cleavage requirement for T-cell activity. In NHP, HER2-XPAT has been dose-escalated safely up to 42mg/kg (MTD) but was not tolerated at 50mg/kg. HER2-XPAT demonstrated early T-cell margination at 2mg/kg but largely spared CRS, cytokine production, and tissue toxicity at doses up to 42mg/kg. PK profiles of HER2-XPAT and HER2-NonClv were comparable in NHP, indicating minimal systemic cleavage of XPAT, consistent with its ex vivo stability when incubated in the plasma of cancer pts for 7 days at 37°C. Given by continuous infusion, HER2-PAT induced lethal CRS and cytokine spikes at 0.3mg/kg/d, but was tolerated at 0.2mg/kg/d, providing HER2-XPAT with &amp;gt;3000-fold protection in tolerated Cmax versus HER2-PAT, &amp;gt;4 logs over cytotoxicity EC50s for HER2 and HR+ cell lines, and a 20-fold margin of safety over the dose required for pharmacodynamic activity. Conclusions HER2-XPAT is a potent prodrug T-cell engager with promising evidence of activity at low doses while exhibiting minimal CRS and a potentially wide TI based on NHP tolerability at doses up to 42mg/kg. With XTEN’s prior clinical data demonstrating low immunogenicity, the XPAT TCEs provide a promising solution. IND studies are currently ongoing. Additional PK, PD, cytokines, safety, and efficacy data will be presented. Citation Format: Fiore Cattaruzza, Ayesha Nazeer, Zachary Lange, Caitlin Koski, Mikhail Hammond, Angela Henkensiefken, Trang Dao-Pick, Mika K Derynck, Bryan Irving, Volker Schellenberger. Her2-xpat, a novel protease-activatable prodrug t-cell engager (tce), exhibits potent t-cell activation and efficacy in her2 tumors, yielding large predicted safety margins based on non-human primate (nhp) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-11.