Selective Proteolysis Research Articles

Superactivity of Bimetallic CuFeO2 Submicron Particles towards Selective Proteolysis, Depending on their Surface Hydroxyls.

Nano bimetallic oxides as nanoproteases have the great advantages in the heterogeneous hydrolysis of proteins. Here, we report that bimetallic delafossite CuFeO2 submicron particles (CuFeO2 SMPs) display a high protease activity towards selective cleavage of peptide bond involving hydrophobic residue at 25 °C. CuFeO2 SMPs have excellent regeneration performance with high structural stability. The strong Lewis acidity of Fe3+ and the strong nucleophilicity of Cu+ bound hydroxyl groups are both necessary for the high protease activity of CuFeO2 SMPs. Low-valent metal ion has a great advantage in that low-valent Cu+ bound hydroxyl has strong nucleophilicity, resulting in promotion of protein hydrolysis via high-efficient bimetallic catalysis. This study provides evidence that the protease activity of CuFeO2 SMPs depends on metal ion-bound hydroxyls on their surface. Our findings highlight that the valence of metal ions in artificial protease and their surface hydroxyls are two important factors that determine their catalytic efficiency.

Enhancer reprogramming underlies therapeutic utility of a SMARCA2 degrader in SMARCA4 mutant cancer.

Genomic studies have identified frequent mutations in subunits of the SWI/SNF (switch/sucrose non-fermenting) chromatin remodeling complex including SMARCA4 and ARID1A in non-small cell lung cancer (NSCLC). Genetic evidence indicates that the paralog SMARCA2 is synthetic lethal to SMARCA4 suggesting SMARCA2 is a valuable therapeutic target. However, the discovery of selective inhibitors of SMARCA2 has been challenging. Here, we utilized structure-activity relationship (SAR) studies to develop YD23, a potent and selective proteolysis targeting chimera (PROTAC) targeting SMARCA2. Mechanistically, we show that SMARCA2 degradation induces reprogramming of the enhancer landscape in SMARCA4-mutant cells with loss of chromatin accessibility at enhancers of genes involved in cell proliferation. Furthermore, we identified YAP/TEADas key partners to SMARCA2 in driving growth of SMARCA4-mutant cells. Finally, we show that YD23 has potent tumor growth inhibitory activity in SMARCA4-mutant xenografts. These findings provide the mechanistic basis for development of SMARCA2 degraders as synthetic lethal therapeutics against SMARCA4-mutant lung cancers.

Formation of thermal-induced microgels from soy protein hydrolysates: Effects of selective proteolysis on glycinin/β-conglycinin

This study explored the impact of selective proteolysis on the formation of thermally induced soy protein microgels. Glycinin hydrolysate (GH) and β-conglycinin hydrolysate (CH) were obtained by subjecting soy protein isolate to selective proteolysis for different hydrolysis time (10–90 min), as confirmed by SDS-PAGE. In the early stages of hydrolysis, free sulfhydryl, surface hydrophobicity, storage modulus (G′) and loss modulus (G″) of GH and CH increased, which enhanced their gelling potential. However, as hydrolysis time increased, the gel properties of the hydrolysates progressively weakened. Structural characterization of microgels revealed that GH yielded microgels with smaller particle sizes and coarser and relatively dispersed granular structures, while CH resulted in microgels with lower potential values, smoother surfaces, and lumps resembling strand-like formations. Analysis of the structure and intermolecular force of microgels showed that the microgel formed by the GH gradually tended to be disordered, whereas the secondary structure of microgels formed by CH showed lower random coil content, resulting in a dense gel network aggregated through disulfide bonding, hydrophobic interactions and hydrogen bonding as demonstrated by frequency-dependent storage moduli measurements. Overall, this study presents a thorough characterization of microgels and shows that they can be tailored by selective proteolysis, which enables controlling the β-conglycinin/glycinin ratio of soy protein.

Stimulus-responsive assembly of nonviral nucleocapsids

Controlled assembly of a protein shell around a viral genome is a key step in the life cycle of many viruses. Here we report a strategy for regulating the co-assembly of nonviral proteins and nucleic acids into highly ordered nucleocapsids in vitro. By fusing maltose binding protein to the subunits of NC-4, an engineered protein cage that encapsulates its own encoding mRNA, we successfully blocked spontaneous capsid assembly, allowing isolation of the individual monomers in soluble form. To initiate RNA-templated nucleocapsid formation, the steric block can be simply removed by selective proteolysis. Analyses by transmission and cryo-electron microscopy confirmed that the resulting assemblies are structurally identical to their RNA-containing counterparts produced in vivo. Enzymatically triggered cage formation broadens the range of RNA molecules that can be encapsulated by NC-4, provides unique opportunities to study the co-assembly of capsid and cargo, and could be useful for studying other nonviral and viral assemblies.

Targeted Degradation of Signal Transduction and Activator of Transcription 3 by Chaperone-Mediated Autophagy Targeting Chimeric Nanoplatform.

Chaperone-mediated autophagy (CMA) is a lysosomal-dependent proteolysis pathway for the degradation of cytosolic proteins. However, exploiting CMA-mediated proteolysis to degrade proteins of interest in cancer therapy has not been widely applied. In this study, we develop a CMA-targeting chimera (CMATAC) to efficiently and specifically degrade signal transduction and activator of transcription 3 (STAT3) in tumor cells. CMATAC consists of STAT3 and heat shock cognate 70 kDa protein (HSC70) targeting peptides connected by a linker. To efficiently deliver CMATACs into tumor cells, lipid nanoparticles (LNPs) are used to encapsulate CMATACs (nCMATACs) and decorated with an insulin-like growth factor 2 receptor (IGF2R) targeting peptide (InCMATACs) to achieve tumor targeting and precise delivery. The CMA pathway is activated in tumor cells by a fasting-mimicking diet (FMD). Furthermore, FMD treatment strongly enhances the cellular uptake and tumor accumulation of InCMATACs by upregulating the IGF2R expression. As a result, InCMATACs efficiently degrade STAT3 protein in both A549 and HCC827 tumor cells and inhibit tumor growths in vivo. This study demonstrates that InCMATACs can be used for selective proteolysis in cancer therapy.

Tethering ATG16L1 or LC3 induces targeted autophagic degradation of protein aggregates and mitochondria

ABSTRACT Proteolysis-targeting chimeras (PROTACs) based on the ubiquitin-proteasome system have made great progress in the field of drug discovery. There is mounting evidence that the accumulation of aggregation-prone proteins or malfunctioning organelles is associated with the occurrence of various age-related neurodegenerative disorders and cancers. However, PROTACs are inefficient for the degradation of such large targets due to the narrow entrance channel of the proteasome. Macroautophagy (hereafter referred to as autophagy) is known as a self-degradative process involved in the degradation of bulk cytoplasmic components or specific cargoes that are sequestered into autophagosomes. In the present study, we report the development of a generalizable strategy for the targeted degradation of large targets. Our results suggested that tethering large target models to phagophore-associated ATG16L1 or LC3 induced targeted autophagic degradation of the large target models. Furthermore, we successfully applied this autophagy-targeting degradation strategy to the targeted degradation of HTT65Q aggregates and mitochondria. Specifically, chimeras consisting of polyQ-binding peptide 1 (QBP) and ATG16L1-binding peptide (ABP) or LC3-interacting region (LIR) induced targeted autophagic degradation of pathogenic HTT65Q aggregates; and the chimeras consisting of mitochondria-targeting sequence (MTS) and ABP or LIR promoted targeted autophagic degradation of dysfunctional mitochondria, hence ameliorating mitochondrial dysfunction in a Parkinson disease cell model and protecting cells from apoptosis induced by the mitochondrial stress agent FCCP. Therefore, this study provides a new strategy for the selective proteolysis of large targets and enrich the toolkit for autophagy-targeting degradation.

Selective Proteolysis of Activated Transcriptional Factor by NIR-Responsive Palindromic DNA Thalidomide Conjugate Inhibits the Canonical Smad Pathway.

Dysfunctional transcription factors that activate abnormal expressions of specific proteins are often associated with the progression of various diseases. Despite being attractive drug targets, the lack of druggable sites has dramatically hindered their drug development. The emergence of proteolysis targeting chimeras (PROTACs) has revitalized the drug development of many conventional hard-to-drug protein targets. Here, the use of a palindromic double-strand DNA thalidomide conjugate (PASTE) to selectively bind and induce proteolysis of targeted activated transcription factor (PROTAF) is reported. The selective proteolysis of the dimerized phosphorylated receptor-regulated Smad2/3 and inhibition of the canonical Smad pathway validates PASTE-mediated PROTAF. Further aptamer-guided active delivery of PASTE and near-infrared light-triggered PROTAF are demonstrated. Great potential in using PASTE for the selective degradation of the activated transcription factor is seen, providing a powerful tool for studying signaling pathways and developing precision medicines.

CuCoO2 Nanoparticles as a Nanoprotease for Selective Proteolysis with High Efficiency at Room Temperature

AbstractMany nanoproteases contain tetravalent metal ions and catalyze peptide‐bond hydrolysis only at high temperature (60 °C). Here, we report a new and effective strategy to explore nanoproteases from nanoparticles containing low valent metal ions. We found that flower‐like CuCoO2 nanoparticles (CuCoO2 NPs) containing low valent Cu+ possessed excellent catalytic activity towards selective cleavage of peptide bonds with hydrophobic residues in bovine serum albumin (BSA) at room temperature. CuCoO2 NPs exhibited excellent stability and had great reusability. CuCoO2 NPs also hydrolyzed heat‐denatured and surfactant‐denatured BSA. Mechanism analysis revealed that the high Lewis acidity of Co3+ and the low valence of Cu+ were both essential for the high protease activity of CuCoO2 NPs. The flower‐like structure of CuCoO2 NPs and the strong nucleophilicity of Cu+‐bound hydroxyl endow them with excellent catalytic performance. The findings open a new way for the design and discovery of high‐efficiency nanoproteases.

CuCoO2 Nanoparticles as a Nanoprotease for Selective Proteolysis with High Efficiency at Room Temperature.

Many nanoproteases contain tetravalent metal ions and catalyze peptide-bond hydrolysis only at high temperature (60 °C). Here, we report a new and effective strategy to explore nanoproteases from the nanoparticles containing low valent metal ions. We found that flower-like CuCoO2 nanoparticles (CuCoO2 NPs) containing low valent Cu+ possessed excellent catalytic activity towards selective cleavage of peptide bonds with hydrophobic residues in bovine serum albumin (BSA) at room temperature. CuCoO2 NPs exhibited excellent stability and had great reuse performance. CuCoO2 NPs also hydrolyzed heat-denatured and surfactant-denatured BSA. Mechanism analysis revealed that the high Lewis acidity of Co3+ and the low valence of Cu+ were both essential for the high protease activity of CuCoO2 NPs. The flower-like structure of CuCoO2 NPs and the strong nucleophilicity of Cu+-bound hydroxyl endue them with excellent catalytic performance. The findings open a new way for design and discovery of high-efficiency nanoproteases.

Preparation and Characterization of a Multicomponent Arthrospira platensis Biomass Hydrolysate with Superior Anti-Hypertensive, Anti-Hyperlipidemic and Antioxidant Activities via Selective Proteolysis.

Arthrospira platensis biomass is a sustainable source of bioactive products for the food, cosmetic, and medicine industries. As well as primary metabolites, different secondary metabolites can be obtained via distinct enzymatic degradation of biomass. In this work, different hydrophilic extracts were obtained after treating the biomass with: (i) a serine endo-peptidase (Alcalase®), (ii) a mixture of amino-, dipeptidyl-, and endo-peptidases (Flavourzyme®), (iii) a mixture of endo-1,3(4)-β-glucanase and an endo-1,4-xylanase, and β-glucanase (Ultraflo®), and (iv) an exo-1,3-glucanase (Vinoflow®) (all the enzymes from Novozymes A/S (bagsvaerd, Denmark)); with subsequent extraction of the biocomponents with an isopropanol/hexane mixture. The composition of each aqueous phase extract (in terms of amino acids, peptides, oligo-elements, carbohydrates, and phenols) and their in vitro functional properties were compared. The conditions described in this work using the enzyme Alcalase® permits the extraction of eight distinctive peptides. This extract is 7.3 times more anti-hypertensive, 106 times more anti-hypertriglyceridemic, 26 times more hypocholesterolemic, has 4.4 times more antioxidant activities, and has 2.3 times more phenols, than the extract obtained without any prior enzyme biomass digestion. Alcalase® extract is an advantageous product with potential application in functional food, pharmaceutics, and cosmetics.

Selective proteolysis of β-conglycinin as a tool to increase air-water interface and foam stabilising properties of soy proteins

Proteolysis of food proteins changes their conformation and often generates bioactive peptides, leading to improved nutritional value and techno-functional properties. Protein isolates that originated from plants are generally a mixture of several proteins. Enzymatic hydrolysis of such mixtures has a drawback, as it is often unclear which proteins in the mixture are hydrolysed, leading to poorly reproducible outcomes. In the present study, we applied a controlled methodology, called selective proteolysis, and investigated its effects on the air-water interface and foam stabilising properties of soy protein isolate (SPI). We selectively hydrolysed 7S β-conglycinin of SPI, while 11S glycinin remained relatively intact. Hydrolysates were obtained at two different degrees of hydrolysis (DH). Selective hydrolysis resulted in increased surface activity, and a higher foamability, which nearly doubled. The size of the peptides seemed to significantly affect the mechanical properties of the layer at the air-water interface, which was studied by removing peptides smaller than 14 kDa via dialysis. Peptides smaller than 14 kDa were found to hinder the interfacial network formation, while peptides larger than 14 kDa increased the interfacial stiffness. This was also reflected in the foam stability, as the hydrolysate with a low DH resulted in significantly higher foam stability than the hydrolysates with higher DH. We demonstrated that the selective proteolysis of β-conglycinin can increase the soy protein interface and foam stabilising properties, which could lead to new healthy and more sustainable food products.

(Invited, Digital Presentation) Development of Multiplex Electrode Array Sensors for Proteases Activity Profiling Toward Cancer Diagnosis

Proteases are a large family of enzymes involved in many important biological processes. Quantitative detection of the activity profile of specific target proteases is in high demand for the diagnosis and treatment monitoring of diseases such as cancers. The overexpression and enhanced activity of proteases were found to correlate well with cancer development. Protease inhibitors are also a class of major anti-cancer drug candidates. However, due to the presence of a large number (over 600) of proteases in human body, their complex interactions with each other, and large influence by the surrounding factors (such as buffer composition, pH value and temperature), it is challenging to accurately detect the activity of specific proteases in complex media. It is even more difficult to measure the activity profiles of a large set of proteases that are relevant to cancers. Here we summarize our studies on the development of a multiplex electrode array biosensor platform toward cancer diagnosis based on rapid profiling of multiple protease activities. Both nano- and micro-electrode arrays have been demonstrated for electrochemical detection of protease activities. The individually addressed 3x3 gold thin-film microelectrode array is particularly attractive for rapid profiling of multiple protease activities.The general sensor scheme involves attaching short peptides with specific sequences of 4 to 8 amino acids on the electrode surface of the electrochemical sensor chip, which serve as the substrates for selective proteolysis by the cognate proteases. The far end is covalently attached with a ferrocene (Fc) group as the redox tag for electrochemical measurements. The quantity of the intact peptides on the electrode surface is measured with an AC voltammetry (ACV) method, which is proportional to the peak current at the specific electrode potential when Fc oxidation is oxidized into ferrocenium (Fc+). As the peptide is cleaved by the cognate protease, the peak current decreases continuously in the continuously repeated ACV curves. The recorded kinetic proteolytic curve can be quantitatively described by the heterogeneous Michaelis-Menten model following an exponential decay. The inverse of exponential decay time constant, i.e., 1/τ, is found to represent the protease activity which equals to [E](kcat /KM), where [E] is the protease concentration and kcat /KM is the specificity constant defined by the kinetic proteolytic reaction constant kcat and the Michaelis equilibrium constant KM. The kcat /KM values have been investigated versus the substrate peptide sequence and length, temperature, and buffer composition to optimize the detection for activity of cathepsin B, a potential cancer biomarker. The limit of detection (LOD) for activated cathepsin B can reach down to 0.57 pM, sufficient to directly detect cathepsin B in diluted human serum.This electrochemical method has been compared and validated with the commonly used affinity assay, i.e., enzyme-linked immunosorbent assay (ELISA). The ELISA measurements were found to be more sensitive to the concentration of proenzyme (zymogen), while the electrochemical method detects the activity of the proteases which reflects the intrinsic biological function. This provides more critical information than the concentration derived from ELISA.Currently, two sets of peptides have been synthesized which are selective for detection of cathepsin B and ADAM-17, respectively. They are functionalized to the 3x3 gold thin-film microelectrode array and used to simultaneously detect these two types of proteases in serum samples from breast cancer patients at different stages. The activity profiles will be correlated to the cancer development. Figure 1

Discovery of a potent and selective proteolysis targeting chimera (PROTAC) degrader of NSD3 histone methyltransferase

Nuclear receptor binding SET domain protein 3 (NSD3) is an attractive potential target in the therapy for human cancers. Herein, we report the discovery of a series of small-molecule NSD3 degraders based on the proteolysis targeting chimera (PROTAC) strategy. The represented compound 8 induces NSD3 degradation with DC50 values of 1.43 and 0.94 μM in NCI–H1703 and A549 lung cancer cells, respectively, and shows selectivity over two other NSD proteins. 8 reduces histone H3 lysine 36 methylation and induces apoptosis and cell cycle arrest in lung cancer cells. Moreover, the RNA sequencing and immunohistochemistry assays showed that 8 downregulates NSD3-associated gene expression. Significantly, 8, but not 1 (a reported NSD3-PWWP antagonist) could inhibit the cell growth of NCI–H1703 and A549 cells. A single administration of 8 effectively decreases the NSD3 protein level in lung cancer xenograft models. Therefore, this study demonstrated that inducing NSD3 degradation is a more effective approach inhibiting the function of NSD3 than blocking the NSD3-PWWP domain, which may provide a potential therapeutic approach for lung cancer.

The AUTOTAC chemical biology platform for targeted protein degradation via the autophagy-lysosome system

Targeted protein degradation allows targeting undruggable proteins for therapeutic applications as well as eliminating proteins of interest for research purposes. While several degraders that harness the proteasome or the lysosome have been developed, a technology that simultaneously degrades targets and accelerates cellular autophagic flux is still missing. In this study, we develop a general chemical tool and platform technology termed AUTOphagy-TArgeting Chimera (AUTOTAC), which employs bifunctional molecules composed of target-binding ligands linked to autophagy-targeting ligands. AUTOTACs bind the ZZ domain of the otherwise dormant autophagy receptor p62/Sequestosome-1/SQSTM1, which is activated into oligomeric bodies in complex with targets for their sequestration and degradation. We use AUTOTACs to degrade various oncoproteins and degradation-resistant aggregates in neurodegeneration at nanomolar DC50 values in vitro and in vivo. AUTOTAC provides a platform for selective proteolysis in basic research and drug development.

A Selective Small-Molecule c-Myc Degrader Potently Regresses Lethal c-Myc Overexpressing Tumors.

MYC oncogene is involved in the majority of human cancers and is often associated with poor outcomes, rendering it an extraordinarily desirable target, but therapeutic targeting of c‐Myc protein has been a challenge for >30 years. Here, WBC100, a novel oral active molecule glue that selectively degrades c‐Myc protein over other proteins and potently kills c‐Myc overexpressing cancer cells is reported. WBC100 targets the nuclear localization signal 1 (NLS1)–Basic–nuclear localization signal 2 (NLS2) region of c‐Myc and induces c‐Myc protein degradation through ubiquitin E3 ligase CHIP mediated 26S proteasome pathway, leading to apoptosis of cancer cells. In vivo, WBC100 potently regresses multiple lethal c‐Myc overexpressing tumors such as acute myeloid leukemia, pancreatic, and gastric cancers with good tolerability in multiple xenograft mouse models. Identification of the NLS1–Basic–NLS2 region as a druggable pocket for targeting the “undruggable” c‐Myc protein and that single‐agent WBC100 potently regresses c‐Myc overexpressing tumors through selective c‐Myc proteolysis opens new perspectives for pharmacologically intervening c‐Myc in human cancers.

(Invited) Toward Rapid Profiling of Proteases Activities for Cancer Diagnosis Based on Multiplex Microelectrode Array Sensors

We report the development of an electrochemical biosensor platform based on multiplex micro-/nano- electrode arrays toward cancer diagnosis based on rapid profiling of protease activities. Proteases are a large family of enzymes involved in many important biological processes. Quantitative detection of the activity profile of specific target proteases is in high demand for the diagnosis and treatment monitoring of diseases such as cancers. This study demonstrates the fabrication and characterization of a nanoelectrode array platform based on embedded vertically aligned carbon nanofibers for electrochemical detection of protease activities and further development into an individually addressed 3x3 gold thin-film microelectrode array for rapid profiling of multiple protease activities.Peptides with specific sequences of 4 to 8 amino acids are designed and synthesized as the substrates for selective proteolysis by the cognate proteases, which are attached to the electrode surface as the specific probes. The far end is covalently attached with a ferrocene (Fc) group as the redox moiety for electrochemical measurements. The quantity of the intact peptides on the electrode surface can be sensitively detected with an AC voltammetry method and presents as the peak current at the specific potential for Fc oxidation into ferrocenium (Fc+). As the peptide is cleaved by the cognate protease, the peak current decreases exponentially in the continuously repeated AC voltammetry measurements. The recorded kinetic proteolytic curve can be quantitatively described by a surface-based heterogeneous Michaelis-Menten model. The inverse of exponential decay time constant, 1/t, is found to represent the protease activity which equals to [E](kcat /KM), where [E] is the protease concentration and kcat /KM is the specificity constant defined by the kinetic proteolytic reaction constant kcat and the Michaelis equilibrium constant KM. We have systematically investigated the factors that affect the value of kcat /KM, including the peptide sequence, peptide length, temperature and buffer composition, and optimized the conditions for highly sensitive detection of protease activity of cathepsin B, a potential cancer biomarker.Toward rapid profiling of multiple proteases, we have developed a multiplex electrochemical sensor chip based on a 3x3 gold microelectrode array. The nine individual gold microelectrodes are partially buried underneath the surrounding SiO2 thin film, which show highly consistent cyclic voltammetric signals in gold surface cleaning experiments and detecting benchmark redox species in solution. Upon selectively functionalizing the individual gold microelectrodes with the specific ferrocene-labeled peptide probes, simultaneous detection of the proteolytic curves of the target proteases can be obtained over 9 channels by monitoring the decay of the AC voltammetry signal of the ferrocene-labeled peptide molecules. So far, the algorithm for fitting the kinetic proteolytic curves to accurately derive the activity of cathepsin B has been established. Simultaneous detection of the proteolysis of cathepsin B on the microelectrode array functionalized with three different hexapeptides has been demonstrated, showing the potential of this sensor platform for rapid detection of the activity profiles of multiple proteases.Interestingly, the above-discussed electrochemical method detects the activity of the proteases, which reflects the true biological function and is fundamentally different from the concentration derived from commonly used affinity biosensors or assays such as enzyme-linked immunosorbent assay (ELISA). The direct comparison has shown that ELISA measurements will give the same results no matter the original proenzyme form (zymogen) in the sample is activated or not, while the electrochemically measured activity shows dramatic increase after being chemically activated. In addition, in the current practice, each protease is measured separately in its optimal buffer with the pH value varying from 5 to 9. It is not possible to measure them in a common buffer simultaneously. We have demonstrated that the electrochemical method can be applied for protease activity profiling in a buffer at pH = 7.4 that is compatible to the physiology conditions. This enables measuring the activity of multiple proteases in human serum, which is a critical step toward protease activity for cancer diagnosis.The figure shows the optical images of (a) the whole 3x3 Au microelectrode array chip and (b) the active electrode area, and the schematic illustration of (c) the mechanism of proteolysis of the peptide probes, (d) the change of the AC voltammetry signal before and after proteolysis and (e) the kinetic proteolytic curves, i.e. the exponential peak current decay over time, at different protease concentration [E]. Figure 1

Linear and non-linear rheology of heat-set soy protein gels: Effects of selective proteolysis of β-conglycinin and glycinin

Soy protein is widely used in the food industry as a gelling agent in many food products. Selectively hydrolyzing one of the two main components (β-conglycinin and glycinin) of soy protein is expected to modify its gelling ability in a novel way. To explore these modifications, selective proteolysis was applied on native soy protein isolate (SPI). Degraded β-conglycinin hydrolysate (DβH) and degraded glycinin hydrolysate (DGH) were obtained with a similar degree of hydrolysis (DH), as verified by SDS-PAGE and pH-stat. Heat-set gels were formed by SPI, hydrolysates, and dialyzed hydrolysates at different protein concentrations (6%, 10%, 14%). Rheological properties of these gels in the linear regime were determined by small amplitude oscillatory shear (SAOS) tests, while their non-linear rheology was studied by large amplitude oscillatory shear (LAOS) tests. Scaling theory, CLSM, and SEM images were used to link the rheological behavior to differences in the gel microstructure.Our results showed that hydrolysates had a shorter gelling time and lower gelling temperature, but the non-network peptides made hydrolysate gels less elastic and resulted in a lower critical linear strain than SPI gels. At low concentration and DH, DβH not only had a shorter gelling time but also formed stiffer gels than SPI, which could be due to increased hydrophobic interactions and disulphide bonds, as well as the more homogeneous gel network. On the other hand, DGH formed weaker gels than SPI at every concentration, and their gel structure was coarse, consisting of small and branched flocs. With increased strain amplitude, all gels showed a transition from predominantly elastic to plastic behavior. DβH displayed intercycle strain softening, while DGH and SPI displayed a weak and strong strain overshoot, respectively. In the medium strain range, SPI showed stronger intracycle shear stiffening and DβH gels showed stronger intracycle shear thinning. At the large strain regime, SPI and DβH gels showed abrupt yielding behavior while DGH gels yielded more gradually. Overall, the “rheological fingerprint” of soy protein heat-set gels was established, and these rheological properties can be tailored by selectively hydrolyzing different components of soy protein, and varying the DH.

Engineering subtilisin proteases that specifically degrade active RAS

We describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit.

APC/C-mediated proteolysis of cyclin B and securin promotes anaphase entry, inactivating CDK1 and permitting chromosome segregation, respectively. Reduction of CDK1 activity relieves inhibition of the CDK1-counteracting phosphatases PP1 and PP2A-B55, allowing wide-spread dephosphorylation of substrates. Meanwhile, continued APC/C activity promotes proteolysis of other mitotic regulators. Together, these activities orchestrate a complex series of events during mitotic exit. However, the relative importance of regulated proteolysis and dephosphorylation in dictating the order and timing of these events remains unclear. Using high temporal-resolution proteomics, we compare the relative extent of proteolysis and protein dephosphorylation. This reveals highly-selective rapid proteolysis of cyclin B, securin and geminin at the metaphase-anaphase transition, followed by slow proteolysis of other substrates. Dephosphorylation requires APC/C-dependent destruction of cyclin B and was resolved into PP1-dependent categories with unique sequence motifs. We conclude that dephosphorylation initiated by selective proteolysis of cyclin B drives the bulk of changes observed during mitotic exit.

An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins

Densely O-glycosylated mucin domains are found in a broad range of cell surface and secreted proteins, where they play key physiological roles. In addition, alterations in mucin expression and glycosylation are common in a variety of human diseases, such as cancer, cystic fibrosis, and inflammatory bowel diseases. These correlations have been challenging to uncover and establish because tools that specifically probe mucin domains are lacking. Here, we present a panel of bacterial proteases that cleave mucin domains via distinct peptide- and glycan-based motifs, generating a diverse enzymatic toolkit for mucin-selective proteolysis. By mutating catalytic residues of two such enzymes, we engineered mucin-selective binding agents with retained glycoform preferences. StcEE447D is a pan-mucin stain derived from enterohemorrhagic Escherichia coli that is tolerant to a wide range of glycoforms. BT4244E575A derived from Bacteroides thetaiotaomicron is selective for truncated, asialylated core 1 structures commonly associated with malignant and premalignant tissues. We demonstrated that these catalytically inactive point mutants enable robust detection and visualization of mucin-domain glycoproteins by flow cytometry, Western blot, and immunohistochemistry. Application of our enzymatic toolkit to ascites fluid and tissue slices from patients with ovarian cancer facilitated characterization of patients based on differences in mucin cleavage and expression patterns.