Proteolysis Efficiency Research Articles

Folate-PEG-PROTAC Micelles for Enhancing Tumor-Specific Targeting Proteolysis In Vivo.

Proteolysis targeting chimeras (PROTACs) technology is rapidly developed as a novel and selective medicinal strategy for the degradation of cellular proteins in cancer therapy. However, the applications of PROTACs as heterobifunctional molecules are largely limited by high molecular weight, low bioavailability, poor permeability, insufficient targeting, and low efficacy in vivo. Herein, self-assembling micelles of FA-PEG-PROTAC are designed for cancer cell selective targeting and reductive-response proteolysis in tumor-bearing mice. FA-PEG-PROTAC is prepared by conjugating folic acid (FA)-PEG with EGFR-targeting PROTAC via a disulfide bond. The FA-PEG-PROTAC micelles, formed by self-assembling, are demonstrated to significantly improve tumor targeting efficacy and exhibit excellent anti-tumor efficacy in the mouse xenograft model compared to the traditional PROTACs. The strategy of applying self-assembled FA-PEG-PROTAC micelles in tumor therapy can not only improve targeted proteolysis efficiency but also broaden applications in the development of PROTAC-based drugs.

A strategy to enhance the insecticidal potency of Vip3Aa by introducing additional cleavage sites to increase its proteolytic activation efficiency

Microbially derived, protein-based biopesticides have become a vital element in pest management strategies. Vip3 family proteins from Bacillus thuringiensis have distinct characteristics from known insecticidal Cry toxins and show efficient insecticidal activity against several detrimental lepidopteran pests. They are considered to be a promising toxic candidate for the management of various detrimental pests. In this study, we found that in addition to the preliminary digestion sites lysine, there are multiple cleavage activation sites in the linker region between domain I (DI) and DII of Vip3Aa. We further demonstrated that by adding more cleavage sites between DI and DII of Vip3Aa, its proteolysis efficiency by midgut proteases can be significantly increased, and correspondingly enhance its insecticidal activity against Spodoptera frugiperda and Helicoverpa armigera larvae. Our study promotes the understanding of the insecticidal mechanism of Vip3 proteins and illustrates an easily implementable strategy to increase the insecticidal potency of Vip3Aa. This facilitates their potential future development and efficient application for sustainable agriculture.

Fluorinated Human Serum Albumin as Potential 19F Magnetic Resonance Imaging Probe.

Fluorinated human serum albumin conjugates were prepared and tested as potential metal-free probes for 19F magnetic resonance imaging (MRI). Each protein molecule was modified by several fluorine-containing compounds via the N-substituted natural acylating reagent homocysteine thiolactone. Albumin conjugates retain the protein's physical and biological properties, such as its 3D dimensional structure, aggregation ability, good solubility, proteolysis efficiency, biocompatibility, and low cytotoxicity. A dual-labeled with cyanine 7 fluorescence dye and fluorine reporter group albumin were synthesized for simultaneous fluorescence imaging and 19F MRI. The preliminary in vitro studies show the prospects of albumin carriers for multimodal imaging.

Quantitative Cell Proteomic Atlas: Pathway-Scale Targeted Mass Spectrometry for High-Resolution Functional Profiling of Cell Signaling.

In spite of extensive studies of cellular signaling, many fundamental processes such as pathway integration, cross-talk, and feedback remain poorly understood. To enable integrated and quantitative measurements of cellular biochemical activities, we have developed the Quantitative Cell Proteomics Atlas (QCPA). QCPA consists of panels of targeted mass spectrometry assays to determine the abundance and stoichiometry of regulatory post-translational modifications of sentinel proteins from most known physiologic and pathogenic signaling pathways in human cells. QCPA currently profiles 1 913 peptides from 469 effectors of cell surface signaling, apoptosis, stress response, gene expression, quiescence, and proliferation. For each protein, QCPA includes triplets of isotopically labeled peptides covering known post-translational regulatory sites to determine their stoichiometries and unmodified protein regions to measure total protein abundance. The QCPA framework incorporates analytes to control for technical variability of sample preparation and mass spectrometric analysis, including TrypQuant, a synthetic substrate for accurate quantification of proteolysis efficiency for proteins containing chemically modified residues. The ability to precisely and accurately quantify most known signaling pathways should enable improved chemoproteomic approaches for the comprehensive analysis of cell signaling and clinical proteomics of diagnostic specimens. QCPA is openly available at https://qcpa.mskcc.org.

Proteolysis efficiency and structural traits of corn gluten meal: Impact of different frequency modes of a low-power density ultrasound

The influence of varying frequency modes of a low-power density ultrasound (LPDU) on the enzymolysis efficacy and structural property of corn gluten meal (CGM) was investigated. Sonication pretreatment (of CGM) with sequential and simultaneous duple-frequency modes enhanced notably the relative enzymolysis efficiency, compared to other LPDU frequency modes. With a sequential duple-frequency of 20/40 kHz showing the most significant effect, the maximum value of enzymolysis efficiency and protein dissolution rate were 15.99% and 61.69%, respectively. Changes in the surface hydrophobicity, secondary structure and microstructure revealed alterations of conformation of CGM by ultrasound-induced effect. Furthermore, the molecular weight distribution CGM hydrolysates primarily distributed in 200–500 Da following ultrasonication. Sonication efficaciously enhanced the susceptibility of CGM to alcalase proteolysis. Thus, the use of various LPDU frequency modes in pretreating target proteins (CGM) may be considered as a practical approach to improve protein-enzyme reactions (proteolysis).

Data regarding the sensibility to proteolysis of a natural apolipoprotein A-I mutant.

The article shows dataset of the proteolysis of a natural variant of apolipoprotein A-I (apoA-I) with a substitution of a leucine by and arginine in position 60 (L60R), in comparison with the protein with the native sequence (Wt). This information demonstrates the potential of in vitro partial proteolysis experiments as it may be applicable to different approaches in the biophysical field. We have analyzed by different electrophoresis techniques apoA-I variants, quantified the degree of proteolysis after staining and compared the proteolysis efficiency with the computed cleavage patterns. The data shown here clearly strengthen the usefulness of this approach to test protein flexibility, as it may be attained with enzymes which are not expected to modify in vivo this protein but have a well-known digestion pattern. In addition it is appropriate for evaluating protein catabolism, as it is exemplified here by the evidence with metalloproteinase 12 (MMP-12), which is a physiological protease that may elicit the pro-inflammatory processing of this variant within the lesions. We support the work “Structural analysis of a natural apolipoprotein A-I variant (L60R) associated with amyloidosis” (Gaddi, et al., 2020), gaining insights on protein folding from a characterization by proteolysis analysis [1].

A Genotypic Comparison Reveals That the Improvement in Nitrogen Remobilization Efficiency in Oilseed Rape Leaves Is Related to Specific Patterns of Senescence-Associated Protease Activities and Phytohormones.

Oilseed rape (Brassica napus L.) is an oleoproteaginous crop characterized by low N use efficiency (NUE) that is mainly related to a weak Nitrogen Remobilization Efficiency (NRE) during the sequential leaf senescence of the vegetative stages. Based on the hypothesis that proteolysis efficiency is crucial for the improvement of leafNRE, our objective was to characterize key senescence-associated proteolytic mechanisms of two genotypes (Ténor and Samouraï) previously identified with contrasting NREs. To reach this goal, biochemical changes, protease activities and phytohormone patterns were studied in mature leaves undergoing senescence in two genotypes with contrasting NRE cultivated in a greenhouse under limiting or ample nitrate supply. The genotype with the higher NRE (Ténor) possessed enhanced senescence processes in response to nitrate limitation, and this led to greater degradation of soluble proteins compared to the other genotype (Samouraï). This efficient proteolysis is associated with (i) an increase in serine and cysteine protease (CP) activities and (ii) the appearance of new CP activities (RD21-like, SAG12-like, RD19-like, cathepsin-B, XBCP3-like and aleurain-like proteases) during senescence induced by N limitation. Compared to Samouraï, Ténor has a higher hormonal ratio ([salicylic acid] + [abscisic acid])/([cytokinins]) that promotes senescence, particularly under low N conditions, and this is correlated with the stronger protein degradation and serine/CP activities observed during senescence. Short statement: The improvement in N recycling during leaf senescence in a genotype of Brassica napus L. characterized by a high nitrogen remobilization efficiency is related to a high phytohormonal ratio ([salicylic acid] + [abscisic acid])/([cytokinins]) that promotes leaf senescence and is correlated with an increase or the induction of specific serine and cysteine protease activities.

In Situ Caging of Biomolecules in Graphene Hybrids for Light Modulated Bioactivity.

Remote and noninvasive modulation of protein activity is essential for applications in biotechnology and medicine. Optical control has emerged as the most attractive approach owing to its high spatial and temporal resolutions; however, it is challenging to engineer light responsive proteins. In this work, a near-infrared (NIR) light-responsive graphene-silica-trypsin (GST) nanoreactor is developed for modulating the bioactivity of trypsin molecules. Biomolecules are spatially confined and protected in the rationally designed compartment architecture, which not only reduces the possible interference but also boosts the bioreaction efficiency. Upon NIR irradiation, the photothermal effect of the GST nanoreactor enables the ultrafast in situ heating for remote activation and tuning of the bioactivity. We apply the GST nanoreactor for remote and ultrafast proteolysis of proteins, which remarkably enhances the proteolysis efficiency and reduces the bioreaction time from the overnight of using free trypsin to seconds. We envision that this work not only provides a promising tool of ultrafast and remotely controllable proteolysis for in vivo proteomics in study of tissue microenvironment and other biomedical applications but also paves the way for exploring smart artificial nanoreactors in biomolecular modulation to gain insight in dynamic biological transformation.

Knock-in human GDF5 proregion L373R mutation as a mouse model for proximal symphalangism.

Proximal symphalangism (SYM1) is an autosomal dominant disorder, mainly characterized by bony fusions of the proximal phalanges of the hands and feet. GDF5 and NOG were identified to be responsible for SYM1. We have previously reported on a p.Leu373Arg mutation in the GDF5 proregion present in a Chinese family with SYM1. Here, we investigated the effects of the GDF-L373R mutation. The variant caused proteolysis efficiency of GDF5 increased in ATDC5 cells. The variant also caused upregulation of SMAD1/5/8 phosphorylation and increased expression of target genes SMURF1, along with COL2A1 and SOX9 which are factors associated with chondrosis. Furthermore, we developed a human-relevant SYM1 mouse model by making a Gdf5L367R (the orthologous position for L373R in humans) knock-in mouse. Gdf5L367R/+ and Gdf5L367R/L367R mice displayed stiffness and adhesions across the proximal phalanx joint which were in complete accord with SYM1. It was also confirmed the joint formation and development was abnormal in Gdf5L367R/+ and Gdf5L367R/L367R mice, including the failure to develop the primary ossification center and be hypertrophic chondrocytes during embryonic development. This knock-in mouse model offers a tool for assessing the pathogenesis of SYM1 and the function of the GDF5 proregion.

Human Prolactin Point Mutations and Their Projected Effect on Vasoinhibin Generation and Vasoinhibin-Related Diseases.

A dysregulation of the generation of vasoinhibin hormones by proteolytic cleavage of prolactin (PRL) has been brought into context with diabetic retinopathy, retinopathy of prematurity, preeclampsia, pregnancy-induced hypertension, and peripartum cardiomyopathy. Factors governing vasoinhibin generation are incompletely characterized, and the composition of vasoinhibin isoforms in human tissues or compartments, such as the circulation, is unknown. The aim of this study was to determine the possible contribution of PRL point mutations to the generation of vasoinhibins as well as to project their role in vasoinhibin-related diseases. Prolactin sequences, point mutations, and substrate specificity information about the PRL cleaving enzymes cathepsin D, matrix metalloproteinases 8 and 13, and bone-morphogenetic protein 1 were retrieved from public databases. The consequences of point mutations in regard to their possible effect on vasoinhibin levels were projected on the basis of a score indicating the suitability of a particular sequence for enzymatic cleavage that result in vasoinhibin generation. The relative abundance and type of vasoinhibin isoforms were estimated by comparing the relative cleavage efficiency of vasoinhibin-generating enzymes. Six point mutations leading to amino acid substitutions in vasoinhibin-generating cleavage sites were found and projected to either facilitate or inhibit vasoinhibin generation. Four mutations affecting vasoinhibin generation in cancer tissues were found. The most likely composition of the relative abundance of vasoinhibin isoforms is projected to be 15 > 17.2 > 16.8 > 17.7 > 18 kDa vasoinhibin. Prolactin point mutations are likely to influence vasoinhibin levels by affecting the proteolysis efficiency of vasoinhibin-generating enzymes and should be monitored in patients with vasoinhibin-related diseases. Attempts to characterize vasoinhibin-related diseases should include the 15, 17.2, 16.8, 17.7, and 18 kDa vasoinhibin isoforms.

The detection of the proteasomal proteolysis efficiency in C2C12 myoblast

BackgroundThe loss of skeletal muscle mass in older adults, which is called sarcopenia, is a rapidly growing global health issue. The balance of muscle protein synthesis and degradation is the key point of the regulation of skeletal muscle mass. Previous reports showed that muscle‐specific deletion of a crucial proteasomal gene, Rpt3 (also known as Psmc4), resulted in sever skeletal muscle atrophy (Kitajima et al., 2014). Bortezomib and MG132, which are proteasome inhibitor induced a decrease in cellular amino acid levels (Suraweera et al., 2012, Zhang et al., 2014). We assume that proteasomal proteolysis play an important role in maintaining myocellular amino acid level. However, the effective detection of the proteasomal proteolysis method is not well established. One possibility of amino acids detection is click chemistry technology by using Azidohomoalanine (AHA). We therefore investigate whether click chemistry was able to detect the proteasomal proteolysis effectively. To examine this hypothesis, we used the AHA, which is methionine‐like chemical.MethodsMouse C2C12 myoblasts were cultured under proliferate conditions (37 °C under a humidified atmosphere containing 5% CO2) in high glucose DMEM supplemented with 10% fetal bovine serum and 100mg/ml penicillin streptomycin solution. C2C12 myoblasts stained using click chemistry technology. The medium was replaced with DMEM containing AHA without methionine for 3 hours. Next, We exposed proteasome inhibitor which is MG132 to C2C12 for 3h, 6h, 9h, 18h, 24h. Following fixation and permeabilization, cells were stained with green fluorescent Alexa Fluor 488 alkyne to detected the intracellular AHA intensity (arbitrary units).ResultsC2C12 myoblasts could be stained implied that click chemistry technology was available for C2C12 myoblasts. The intracellular AHA of normal C2C12 myoblasts decreased sequentially; on the other hand, the intracellular AHA level of proteasome inhibited C2C12 myoblasts did not change. Additionally the inhibition of proteasome induced cell death in C2C12 myoblasts. In summary, these data suggested that the AHA might be able to detect the proteasomal proteolysis effectively. This procedure may contribute to clarify more detail of the role of proteasomal proteolysis.ConclusionsWe concluded that the click chemistry technology is visualized sequential proteasomal proteolysis on C2C12 myoblasts.

Effects of high pressure processing (hydrostatic high pressure and ultra-high pressure homogenisation) on whey protein native state and susceptibility to tryptic hydrolysis at atmospheric pressure

Dispersion of whey protein isolate (WPI) prepared at 10% proteins (w/w) and pH6.5, was pressure-processed using high hydrostatic pressure (HHP) at 300MPa and 25°C for 15min, or ultra-high pressure homogenisation (UHPH) at 300MPa and initial fluid temperature (Tin) of 24°C. UHPH-processing was followed (or not) by rapid cooling of the processed fluid at the immediate HP-valve outlet. Short-time thermal treatment (STTT) at 75°C for 10s, 43s or 110s was studied for comparison. Processing-induced effects were investigated in terms of (i) protein denaturation by differential scanning calorimetry (DSC) and ammonium sulphate (AS) precipitation, (ii) susceptibility to tryptic proteolysis at atmospheric pressure after processing, (iii) protein particle sizes from light scattering measurement during the hydrolysis process and, (iv) residual proteins by SDS-PAGE after 135min of hydrolysis. UHPH followed by efficient cooling at the HP-valve outlet, limited significantly (p=0.05) protein denaturation as assessed by DSC and AS precipitation, while increasing notably protein susceptibility to tryptic attack, comparing with the non-processed sample. In opposite, neglecting the cooling step at the HP-valve outlet, led to significantly (p=0.05) lower residual protein native state, then tryptic hydrolysis efficiency. HHP treatment led to intermediate results. The slight protein unfolding that pre-processing could induced, favoured thus the further trypsin attack. Generally speaking, high pressure (avoiding overheating) offers this potential. Process-induced protein aggregation, but also reassembly of protein fragments and peptides released during hydrolysis may affect the proteolysis efficiency.

Amino acid composition of enzymatically hydrolysed potato protein preparations

We determine the effects of the technology of obtaining potato protein preparation and of different variants of enzymatic hydrolysis on the chemical and amino acid compositions of the hydrolysates obtained. Potato protein concentrates obtained through their thermal coagulation in potato juice with calcium chloride, calcium lactate or without salt addition were subjected to enzymatic hydrolysis using two commercial hydrolytic enzymes: endopeptidase (Alcalase) and exopeptidase (Flavourzyme). Chemical (contents of ash, total and coagulable protein) and amino acid compositions of the hydrolysates obtained were determined. On the ground of the findings it was stated that the type of potato protein preparation used and conditions of enzymatic modification influenced on the properties of the hydrolysates obtained. Preparations obtained during the study were characterised by similar chemical and amino acid compositions, whereas the preparation obtained through thermal coagulation with the use of calcium lactate contained insignificantly more protein and essential amino acids. The least liable to enzymatic hydrolysis was the preparation obtained by using calcium chloride, particularly when only endopeptidase was used. The application of endopeptidase enzyme enabled to obtain 60% of proteolysis efficiency and the addition of the second enzyme (exopeptidase) to the protein solution insignificantly increased the proteolysis efficiency (to ca 70%), mainly when the preparation coagulated with the use of calcium chloride was hydrolysed. Proteolysis of the protein preparations obtained with the use of two enzymes was more favourable, particularly due to the quantity of free amino acids in and amino acids composition of the hydrolysates. 

Efficient proteolysis strategies based on microchip bioreactors

In proteome research, proteolysis is an important procedure prior to the mass spectrometric identification of proteins. The typical time of conventional in-solution proteolysis is as long as several hours to half a day. To enhance proteolysis efficiency, a variety of microchip bioreactors have been developed for the rapid digestion and identification of proteins in the past decade. This review mainly focuses on the recent advances and the key strategies of microchip bioreactors in protein digestion. The subjects covered include microchip proteolysis systems, the immobilization of proteases in microchannels, the applications of microchip bioreactors in highly efficient proteolysis, and future prospects. It is expected that microchip bioreactors will become powerful tools in protein analysis and will find a wide range of applications in high-throughput protein identification.

Development of hemoglobin A1c certified reference material by liquid chromatography isotope dilution mass spectrometry

We report the development of a National Institute of Metrology (NIM) hemoglobin A(1c) (HbA(1c)) certified reference material (CRM). Each CRM unit contains about 10 μL of hemoglobin. Both hemoglobin and glycated hemoglobin were quantitatively determined by high-performance liquid chromatography (HPLC)-isotope dilution mass spectrometry (IDMS) with synthesized VHLTPE and glycated VHLTPE as standards. The mass fraction of synthesized VHLTPE or glycated VHLTPE was also quantitatively determined by HPLC-IDMS with NIM amino acid CRMs as standards. The homogeneity and stability of the CRMs were examined with a commercial HbA(1c) analyzer based on the HPLC principle. Fifteen units were randomly selected for homogeneity examination, and statistical analysis showed there was no inhomogeneity. Examination of the stability showed that the CRM was stable for at least 6 months at -80 °C. Uncertainty components of the balance, amino acid purity, hydrolysis and proteolysis efficiency, method reproducibility, homogeneity, and stability were taken into consideration for uncertainty evaluation. The certified value of NIM HbA(1c) CRM was expressed as the ratio of HbA(1c) to total hemoglobin in moles, and was (9.6 ± 1.9)%. The CRM can be used as a calibration or validation standard for clinical diagnostics. It is expected to improve the comparability for HbA(1c) measurement in China.

Characterization of efficient proteolysis by trypsin loaded macroporous silica

Tryptic digestion of proteins in trypsin loaded porous silica has been shown to be highly efficient. Enzymatic silica-reactors were prepared by immobilizing trypsin into macroporous ordered siliceous foam (MOSF) and into mesoporous SBA-15 silica which has a smaller pore size. The tryptic products from the silica reactors were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and a higher proteolysis efficiency was obtained with MOSF. These results can be well interpreted by a sequential digestion model taking into account the confinement and concentration enrichment of both the substrates and enzymes within the silica pores. Proteins at low concentrations and proteins in urea and surfactant solutions were also successfully digested with the MOSF-based reactor and identified by MS. Considering that the immobilized trypsin could retain its enzymatic activity for weeks, this MOSF reactor provides many advantages compared to free enzyme proteolysis. As a proof-of-concept, the digest of a real complex sample extracted from the cytoplasm of mouse liver tissue using trypsin loaded MOSF yielded better results than the typical in-solution protocol.

Macroporous Materials as Novel Catalysts for Efficient and Controllable Proteolysis

A novel nanopore based digestion strategy has been developed by directly adding a macroporous material as catalyst to the conventional in-solution reaction system. Without increasing the enzyme or protein concentrations, this simple digestion approach exhibits high proteolysis efficiency and selectivity due to the in situ fast adsorption of both enzymes and proteins from bulk solution into the macropores of the catalysts, where the target substrates and enzymes are greatly concentrated and confined in the nanospace to realize a quick digestion. Based on the electrostatic interaction matching between the biomolecules and catalysts, selective extraction and digestion of proteins with different isoelectric points can be achieved by adjusting the surface charge of the catalysts. This nanoporous reaction system has been successfully applied to the analysis of a complex biological sample, where 293 proteins are identified, while only 100 proteins are obtained by the standard overnight in-solution digestion. The present nanospace confined digestion strategy will lead to promising advances not only in proteomics but also in other applications where enzymatic reactions are involved.

A role for plasmin in platelet aggregation: Differential regulation of IGF release from IGF–IGFBP complexes?

Objectives To determine if plasmin differentially augments platelet aggregation through variable efficiencies of IGF–IGFBP complex cleavage. Methods We utilized ADP-triggered platelet aggregation assays to test the effects of IGF-I versus IGF-II in complex with IGFBP-2 or IGFBP-3 upon the efficiency of plasmin (a known IGFBP protease) as a pro-aggregatory stimulus. In vitro proteolysis assays were performed as controls. Results We found that IGF-I complexes augmented platelet-mediated aggregation whereas IGF-II either had no effect (IGFBP-2) or inhibited platelet-mediated aggregation (IGFBP-3). In vitro proteolysis assays of IGFBP-2 and IGFBP-3 using plasmin revealed that three of the four aggregation findings were explained by the disparate efficiencies of IGFBP proteolysis associated with each IGF. Only IGF-II–IGFBP-2 complex resulted in a finding that could not be explained by the concept of differential regulation of plasmin’s proteolysis efficiency by the two IGF ligands. Conclusions Our findings demonstrate that the plasmin can differentially modulate platelet aggregation in response to intrinsic heterogeneities within the IGF axis.

Nanopore-Based Proteolytic Reactor for Sensitive and Comprehensive Proteomic Analyses

Various silica-based microreactors have been designed that use enzyme immobilization to address technical concerns in proteolysis including inefficient and incomplete protein digestion. Most of current designs for proteolytic reactors can improve either protease stability or proteolysis efficiency of individual protein(s). However, the desired features such as rapid digestion, larger sequence coverage, and high sensitivity have not been achieved by a single microreactor design for broad range proteins with diverse physical properties. Here, unlike conventional enzyme immobilization strategies, we describe a novel proteolytic nanoreactor based on the unique three-dimensional nanopore structure of our newly synthesized mesoporous silica (MPS), FDU-12, which integrates substrate enrichment, "reagent-free" protein denaturation, and efficient proteolytic digestion. In our design, protein substrates were first captured by MPS nanopore structure and were concentrated from the solution. Following the pH change and applying trypsin, the denaturation and concurrent proteolysis of broad-range proteins were efficiently achieved. In minutes, many more sample peptides from the in-nanopore digestion of protein mixtures were detected by mass spectrometry, resulting in the identifications of a broad range of diverse proteins with high sequence coverage. The unique features of FDU-12 nanostructure that allow rapid, complete proteolysis and resulting enhanced sequence coverage of individual proteins were investigated by using Raman spectroscopy and comparative studies with respect to other MPSs.