Isopeptide Research Articles

A novel cell-permeable peptide prevents protein SUMOylation and supports the mislocalization and aggregation of TDP-43

SUMOylation is a post-translational modification (PTM) that exerts a regulatory role in different cellular processes, including protein localization, aggregation, and biological activities.It consists of the dynamic formation of covalent isopeptide bonds between a family member of the Small Ubiquitin Like Modifiers (SUMOs) and the target proteins. Interestingly, it is a cellular mechanism implicated in several neurodegenerative pathologies and potentially it could become a new therapeutic target; however, there are very few pharmacological tools to modulate the SUMOylation process.In this study, we have designed and tested the activity of a novel small cell-permeable peptide, COV-1, in a neuroblastoma cell line that specifically prevents protein SUMOylation.COV-1 inhibits UBC9-protein target interaction and efficiently decreases global SUMO-1ylation. Moreover, it can perturb RanGAP-1 perinuclear localization by inducing the downregulation of UBC9. In parallel, we found that COV-1 causes an increase in the ubiquitin degradation system up to its engulfment while enhancing the autophagic flux.Surprisingly, COV-1 modifies protein aggregation, and specifically it mislocalizes TDP-43 within cells, inducing its aggregation and co-localization with SUMO-1.These data suggest that COV-1 could be taken into future consideration as an interesting pharmacological tool to study the cellular cascade effects of SUMOylation prevention.

Transglutaminase-induced soybean protein isolate cold-set gels treated with combination of ultrasound and high pressure: Physicochemical properties and structural characterization

Soybean protein isolate (SPI) was treated by the combined exposure to ultrasound and high pressure and then subjected to transglutaminase (TGase)-catalyzed cross-linking to prepare SPI cold-set gels. The effects of combined treatments on physicochemical and structural properties of TGase-induced SPI cold-set gels were investigated. The combination of ultrasound and high pressure promoted the covalent disulfide bonds and ε-(γ-glutaminyl) lysine isopeptide bonds as well as non-covalent hydrophobic interactions, which further improved the gelation properties of SPI compared to ultrasound or high pressure alone. In particular, the 480 W ultrasound followed by high pressure treatment of gels led to higher strength (120.53 g), water holding capacity (95.39 %), immobilized water (93.92 %), lightness (42.18), whiteness (51.03), and elasticity (G′ = 407 Pa), as well as more uniform and compact microstructure, thus resulting in the improved gel network structure. The combination of two treatments produced more flexible secondary structure, tighter tertiary conformation and higher denaturation degree of protein in the gels, leading to more stable gel structure. The structural modifications of SPI contributed to the improvement of its gelation properties. Therefore, the combined application of ultrasound and high pressure can be an effective method for improving the structure and properties of TGase-induced SPI cold-set gels.

High-Affinity Fluorogenic Substrate for Tissue Transglutaminase Reveals Enzymatic Hysteresis.

Transglutaminases (TGases) are a family of calcium-dependent enzymes primarily known for their ability to cross-link proteins. Transglutaminase 2 (TG2) is one isozyme in this family whose role is multifaceted. TG2 can act not only as a typical transamidase through its catalytic core but also as a G-protein via its GTP binding site. These two discrete activities are tightly regulated by both environmental stimuli and redox reactions. Ubiquitously expressed in humans, TG2 has been implicated in numerous disease pathologies that require extensive investigation. The catalytic activity of TG2 can be monitored through various mechanisms, including hydrolysis, transamidation, or cleavage of isopeptide bonds. Activity assays are required to monitor the activity of this isozyme not only for studying its transamidation reaction but also for validation of therapeutics designed to abolish this activity. Herein, we present the design, synthesis, and evaluation of a new TG2 activity substrate based on a previously optimized inhibitor scaffold. The substrate APH7 exhibits excellent affinity, selectivity, and reactivity with TG2 (KM = 3.0 μM). Furthermore, its application also allowed the discovery of unique hysteresis at play within the catalytic activity and inhibition reactivity of TG2.

Discovery of a Potent Deubiquitinase (DUB) Small‐Molecule Activity‐Based Probe Enables Broad Spectrum DUB Activity Profiling in Living Cells**

AbstractDeubiquitinases (DUBs) are a family of >100 proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates, often leading to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been implicated in many diseases, including cancer, neurodegeneration and auto‐inflammation, and several have been recognized as attractive targets for therapeutic intervention. Ubiquitin‐derived covalent activity‐based probes (ABPs) provide a powerful tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which can account for regulation of DUB activity in intact cells or organisms. Here, through comprehensive chemoproteomic warhead profiling, we identify cyanopyrrolidine (CNPy) probe IMP‐2373 (12) as a small molecule pan‐DUB ABP to monitor DUB activity in physiologically relevant live cells. Through proteomics and targeted assays, we demonstrate that IMP‐2373 quantitatively engages more than 35 DUBs across a range of non‐toxic concentrations in diverse cell lines. We further demonstrate its application to quantification of changes in intracellular DUB activity during pharmacological inhibition and during MYC deregulation in a model of B cell lymphoma. IMP‐2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the context of disease‐relevant phenotypes.

Discovery ofaPotent Deubiquitinase (DUB)Small Molecule Activity-based Probe Enables Broad Spectrum DUBActivity Profiling in Living Cells.

Deubiquitinases (DUBs) are a family of >100 proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates. This leads to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been implicated in many diseases, including cancer, neurodegeneration and auto-inflammation, and several have been recognized as attractive targets for therapeutic intervention. Ubiquitin-derived covalent activity-based probes (ABPs) provide a powerful tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which can account for regulation of DUB activity in intact cells or organisms. Here, through comprehensive chemoproteomic warhead profiling, we identify cyanopyrrolidine (CNPy) probe IMP-2373 (12) as a small molecule pan-DUB ABP to monitor DUB activity in physiologically relevant live cells. Through proteomics and targeted assays, we demonstrate that IMP-2373 quantitatively engages more than 35 DUBs across a range of non-toxic concentrations in diverse cell lines. We further demonstrate its application to quantification of changes in intracellular DUB activity during pharmacological inhibition and during MYC deregulation in a model of B cell lymphoma. IMP-2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the context of disease-relevant phenotypes.

Quantifying the Kinetics of Pilus-Specific Sortase-Catalyzed Crosslinking Using High-Performance Liquid Chromatography.

Gram-positive bacteria display pili whose protein components (pilins) are covalently crosslinked by pilus-specific sortase enzymes. These cysteine transpeptidase enzymes catalyze a transpeptidation reaction that joins the pilins together via lysine isopeptide bonds. The crosslinking reaction that builds the SpaA pilus in Corynebacterium diphtheriae is mediated by the SrtA sortase (CdSrtA) and has been reconstituted in vitro. Here, we present a protocol that can be used to measure the kinetics of CdSrtA-catalyzed crosslinking using high-performance liquid chromatography (HPLC). In principle, this biochemical procedure can be used to measure the in vitro crosslinking activity of any pilus-specific sortase.

Cardoon seed oil cake proteins as substrate for microbial transglutaminase: Their application as matrix for bio-based packaging to extend the shelf-life of peanuts

In this study, microbial transglutaminase (mTGase), an enzyme that can alter the biological properties of various proteins by creating isopeptide bonds between glutamine and lysine residues, was tested for the first time on proteins from cardoon oil cakes (CPs). mTGase crosslinked CPs were used as matrix for preparing bio-based materials further evaluated for their technological characteristics, such as mechanical and hydrophilicity behavior, gas barrier and thermal properties, color and opacity. All the performed analyses demonstrated that the microbial enzyme affected the film features by making them more resistant (Young's modulus and tensile strength were 52 MPa and 1.7 MPa for the film manufactured in the absence of enzyme and 98 MPa and 3.3 MPa for the crosslinked film, respectively), less hydrophilic and permeable to gases likely due to the formation of a more compact structure due to isopeptide bonds formed by mTGase as demonstrated by scanning electron microscope analyses. For example, the water vapor permeability of neat films was 8.37 g mm m−2 d−1 kPa−1, whereas that performed by mTGase-crosslinked material was equal to 5.16 g mm m−2 d−1 kPa-1. Bio-disintegration test proved that the materials were able to be decomposed under soil-like conditions. Thermal analyses also showed that the crosslinked materials had a matrix more stable over different temperatures, as a function of enzyme concentrations. Finally, the prepared bioplastics were used as sachets to protect peanuts chosen as an example of a high-oil content snack. The findings revealed that during a 30-day period, peroxidase levels and water content of peanuts packed in sachets manufactured from mTGase-crosslinked CP were lower than those packed in neat film and unpacked, and similar to those packed in LDPE, indicating that the novel bioplastics may be employed as biodegradable materials to prolong the shelf-life of such food goods.

Investigation of the enhancement mechanism of ethanol addition on the gel performance of heat-induced surimi

This study investigated the mechanism of ethanol enhancing the gel properties of heat-induced silver carp (Hypophthalmichthys molitrix) surimi gels. The presence of ethanol reduced the thermal stability of surimi protein, and the denaturation temperature of myosin decreased by approximately 7 °C in the presence of 5% ethanol. The gel strength, textural characteristics, and storage modulus (G′) of the gels improved depending on ethanol levels, while the water holding capacity (WHC) remained steady. Adding ethanol stabilized the α-helix and β-sheet in the protein structures of the gels, reducing the content of the random coil. Appropriate heat denaturation rate induced by ethanol increased protein cross-linking, which may relate to forming more disulfide bonds. In addition, ethanol incorporation promoted the formation of more isopeptide bonds catalyzed by transglutaminase (TGase) and weakened the hydrolysis of surimi proteins by cathepsins, further enhancing the formation of a dense and uniform gel network.

Microbiological transglutaminase: Biotechnological application in the food industry.

Microbial transglutaminases (mTGs) belong to the family of global TGs, isolated and characterised by various bacterial strains, with the first being Streptomyces mobaraensis. This literature review also discusses TGs of animal and plant origin. TGs catalyse the formation of an isopeptide bond, cross-linking the amino and acyl groups. Due to its broad enzymatic activity, TG is extensively utilised in the food industry. The annual net growth in the utilisation of enzymes in the food processing industry is estimated to be 21.9%. As of 2020, the global food enzymes market was valued at around $2.3 billion USD (mTG market was estimated to be around $200 million USD). Much of this growth is attributed to the applications of mTG, benefiting both producers and consumers. In the food industry, TG enhances gelation and modifies emulsification, foaming, viscosity, and water-holding capacity. Research on TG, mainly mTG, provides increasing insights into the wide range of applications of this enzyme in various industrial sectors and promotes enzymatic processing. This work presents the characteristics of TGs, their properties, and the rationale for their utilisation. The review aims to provide theoretical foundations that will assist researchers worldwide in building a methodological framework and furthering the advancement of biotechnology research.

Synergistic effect of YOD1 and USP21 on the Hippo signaling pathway

BackgroundDeubiquitinating enzymes (DUBs) comprise a family of proteases responsible for cleaving the peptide or isopeptide bond between ubiquitin and its substrate proteins. Ubiquitin is essential for regulating diverse cellular functions by attaching to target proteins. The Hippo signaling pathway plays a crucial role in controlling tissue size, cell proliferation, and apoptosis. In a previous study, we discovered that YOD1 regulates the Hippo signaling pathway by deubiquitinating the neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4), an E3 ligase of large tumor suppressor kinase 1 (LATS1). Here, our aim was to investigate potential substrates of YOD1 implicated in the Hippo signaling pathway.MethodsWe employed various bioinformatics tools (BioGRID, STRING, and Cytoscape) to identify novel potential substrates of YOD1. Furthermore, we used western blotting, co-immunoprecipitation (co-IP), glutathione S-transferase (GST) pull-down, immunocytochemistry (ICC) assays to investigate cellular interactions. To evaluate cell proliferation, we performed cell counting kit-8 (CCK-8), wound healing, colony forming, and flow cytometry assays using A549, HEK293T, and HeLa cells. Additionally, we assessed the expression levels of YAP and p-YAP in A549, HEK293T, and HeLa cells through western blotting.ResultsOur investigations revealed that YOD1 interacts with ubiquitin-specific proteases 21 (USP21), a DUB involved in the Hippo signaling pathway, and deubiquitinates the microtubule-affinity regulating kinase (MARK). Intriguingly, YOD1 and USP21 mutually deubiquitinate each other; while YOD1 regulates the protein stability of USP21, USP21 does not exert a regulatory effect on YOD1. Moreover, we observed the synergistic effect of YOD1 and USP21 on cell proliferation through the modulation of the Hippo signaling pathway.ConclusionsOur study revealed multiple cellular interactions between YOD1 and USP21. Moreover, our findings suggest that the combined activities of YOD1 and USP21 synergistically influence cell proliferation in A549 cells by regulating the Hippo signaling pathway.

Hybrid insulin peptide isomers spontaneously form in pancreatic beta-cells from an aspartic anhydride intermediate

Hybrid insulin peptides (HIPs) form in beta-cells when insulin fragments link to other peptides through a peptide bond. HIPs contain nongenomic amino acid sequences and have been identified as targets for autoreactive T cells in type 1 diabetes. A subgroup of HIPs, in which N-terminal amine groups of various peptides are linked to aspartic acid residues of insulin C-peptide, was detected through mass spectrometry in pancreatic islets. Here, we investigate a novel mechanism that leads to the formation of these HIPs in human and murine islets. Our research herein shows that these HIPs form spontaneously in beta-cells through a mechanism involving an aspartic anhydride intermediate. This mechanism leads to the formation of a regular HIP containing a standard peptide bond as well as a HIP-isomer containing an isopeptide bond by linkage to the carboxylic acid side chain of the aspartic acid residue. We used mass spectrometric analyses to confirm the presence of both HIP isomers in islets, thereby validating the occurrence of this novel reaction mechanism in beta-cells. The spontaneous formation of new peptide bonds within cells may lead to the development of neoepitopes that contribute to the pathogenesis of type 1 diabetes as well as other autoimmune diseases.

Investigation of High Molecular Weight Size Variant Formation in Antibody-Drug Conjugates: Microbial Transglutaminase-Mediated Crosslinking

Microbial transglutaminase (mTG) has become a powerful tool for manufacturing antibody-drug conjugates (ADCs). It enables site-specific conjugation by catalyzing formation of stable isopeptide bond between glutamine (Q) side chain and primary amine. However, the downstream impact of mTG-mediated conjugation on ADC product quality, especially on high molecular weight (HMW) size variant formation has not been studied in a systematic manner. This study investigates the mechanisms underlying the formation of HMW size variants in mTG-mediated ADCs using size exclusion chromatography (SEC) and liquid chromatography-mass spectrometry (LC-MS). Our findings revealed that the mTG-mediated glutamine and lysine (K) crosslinking is the primary source of the increased level of HMW size variants in the ADCs. In the study, two monoclonal antibodies (mAbs) with glutamine engineered for site-specific conjugation were used as model systems. Based on the LC-MS analysis, a single lysine (K56) in the heavy chain (HC) was identified as the major Q-K crosslinking site in one of the two mAbs. The HC C-terminal K was observed to crosslink to the target Q in both mAbs. Quantitative correlation was established between the percentage of HMW size variants determined by SEC and the percentage of crosslinked peptides quantified by MS peptide mapping. Importantly, it was demonstrated that the level of HMW size variants in the second ADC was substantially reduced by the complete removal of HC C-terminal K before conjugation. The current work demonstrates that crosslinking and other side reactions during mTG-mediated conjugation needs to be carefully monitored and controlled to ensure process consistency and high product quality of the final ADC drug product.

Compact and modular bioprobe: Integrating SpyCatcher/SpyTag recombinant proteins with zwitterionic polymer-coated quantum dots

The development of quantum dot (QD)-based modular bioprobe that has a compact size and enable a facile conjugation of various biofunctional groups is in high demand. To address this, we surface engineered QDs with zwitterion polymer ligands to have an inherent compact size and derivatized them sequentially with the recombinant proteins SpyCatcher/SpyTag (SC/ST) to use their protein ligation system. SC/ST spontaneously form one complex through the isopeptide bond between them. SC-conjugated QDs (QD–SC) were used as base building blocks. Then, ST–biomolecules were added for modular biofunctionalization. We synthesized compact sized (∼15 nm) QD–SC–ST–affibody (antibody-mimicking small protein for tumor detection) conjugates, which showed successful cell-receptor targeting. The target cell-receptor could be easily tuned by changing the type of ST-affibody. We also demonstrated that anti-human-chorionic-gonadotropin mouse IgG1 antibodies can be labeled on the QD surface by mixing QD–SC with the ST–MG1Nb (mouse-IgG1-specific protein). The immunoassay performance of the antibody-labeled QDs was evaluated using a pregnancy test kit, displaying equivalent detection sensitivity to a commercially available kit. This study proposed an innovative strategy for QD biofunctionalization in a modular manner, which can be expanded to a diverse range of colloidal particle derivatization.

Synergistic improvement of quinoa protein heat-induced gel properties treated by high-intensity ultrasound combined with transglutaminase.

Quinoa protein is enriched with a wide range of amino acids, including all nine essential amino acids necessary for the human body, and in appropriate proportions. However, as the main ingredient of gluten-free food, it is difficult for quinoa to form a certain network structure for lack of gluten protein. The aim of this work was to enhance the gel properties of quinoa protein. Therefore, the texture characteristics of quinoa protein treated with different ultrasound intensities coupled with transglutaminase (TGase) were investigated. The gel strength of quinoa protein gel increased markedly by 94.12% with 600 W ultrasonic treatment, and the water holding capacity increased from 56.6% to 68.33%. The gel solubility was reduced and free amino content increased the apparent viscosity and the consistency index. Changes in the free sulfhydryl group and hydrophobicity indicated that ultrasound stretched protein molecules and exposed active sites. The enhanced intrinsic fluorescence intensity at 600 W demonstrated that ultrasonic treatment affected the conformation of quinoa protein. New bands emerged in sodium dodecylsulfate-polyacrylamide gel electrophoresis indicating that high-molecular-weight polymers were generated through TGase-mediated isopeptide bonds. Furthermore, scanning electron microscopy showed that the gel network structure of TGase-catalyzed quinoa protein was more uniform and denser, thereby improving the gel quality of quinoa protein. The results suggested that high-intensity ultrasound combined with TGase would be an effective way to develop higher-quality quinoa protein gel. © 2023 Society of Chemical Industry.

Structural Premise of Selective Deubiquitinase USP30 Inhibition by Small-Molecule Benzosulfonamides

Dampening functional levels of the mitochondrial deubiquitylating enzyme Ubiquitin-specific protease 30 (USP30) has been suggested as an effective therapeutic strategy against neurodegenerative disorders such as Parkinson’s Disease. USP30 inhibition may counteract the deleterious effects of impaired turnover of damaged mitochondria, which is inherent to both familial and sporadic forms of the disease. Small-molecule inhibitors targeting USP30 are currently in development, but little is known about their precise nature of binding to the protein. We have integrated biochemical and structural approaches to gain novel mechanistic insights into USP30 inhibition by a small-molecule benzosulfonamide-containing compound, USP30inh. Activity-based protein profiling mass spectrometry confirmed target engagement, high selectivity, and potency of USP30inh for USP30 against 49 other deubiquitylating enzymes in a neuroblastoma cell line. In vitro characterization of USP30inh enzyme kinetics inferred slow and tight binding behavior, which is comparable with features of covalent modification of USP30. Finally, we blended hydrogen–deuterium exchange mass spectrometry and computational docking to elucidate the molecular architecture and geometry of USP30 complex formation with USP30inh, identifying structural rearrangements at the cleft of the USP30 thumb and palm subdomains. These studies suggest that USP30inh binds to this thumb–palm cleft, which guides the ubiquitin C terminus into the active site, thereby preventing ubiquitin binding and isopeptide bond cleavage, and confirming its importance in the inhibitory process. Our data will pave the way for the design and development of next-generation inhibitors targeting USP30 and associated deubiquitinylases.

Recombinant Multi-l-Arginyl-Poly-l-Aspartate (Cyanophycin) as an Emerging Biomaterial.

Multi-l-arginyl-poly-l-aspartate (MAPA) is a non-ribosomal polypeptide which synthesis is directed by cyanophycin synthetase, and its production can be achieved using recombinant microorganisms carrying the cphA gene. Along its poly-aspartate backbone, arginine or lysine links to each aspartate via an isopeptide bond. MAPA is a zwitterionic polyelectrolyte full of charged carboxylic, amine, and guanidino groups. In aqueous solution, MAPA exhibits dual thermal and pH responses similar to those stimuli-responsive polymers. Being biocompatible, the films containing MAPA can support cell proliferation and elicits minimal immune response in macrophages. Dipeptides from MAPA after enzymatic treatments can provide nutritional benefits. In light of the increasing interest in MAPA, this article focuses on the recent discovery of the function of cyanophycin synthetase and the potentials of MAPA as a biomaterial.

Designed Multifunctional Isopeptide for Enhanced Annexin A1 Biosensing Based on Peptide-Protein Interactions in Human Blood.

Specific peptide-protein interactions play an important role in biosensing systems based on functional peptides; however, the non-specific interactions with unrelated biomolecules and poor proteolytic stability restrict the clinical application of natural peptides. Here, we leveraged a self-designed multifunctional isopeptide (MISP) to construct an electrochemical biosensing platform for annexin A1 (ANXA1) detection in human blood. The MISP was designed to contain two parts: an antifouling cyclotide cyclo-C(EK)4 and a d-amino acid-containing carbohydrate-mimetic recognizing peptide IF-7 (D-IF7) connected by the isopeptide bond. We have discussed the properties of the cyclotide and illustrated its unique advantage over the natural linear antifouling peptides by molecular dynamics simulations, and the results were further confirmed by dissipative quartz crystal microbalance (QCM-D). In addition, through electrochemical experiments and fluorescence imaging experiments, we demonstrated that the MISP-based biosensor possessed excellent antifouling ability and proteinase hydrolysis stability. Interestingly, the assaying results of the MISP-biosensor were consistent with those of the commercial ANXA1 kits in a variety of healthy and ANXA1-upregulated clinical blood samples, and, more importantly, for the analysis of blood samples with lower ANXA1 expressions, the sensing capability of the biosensor was greatly superior to that of the kits because of the lower detection limit of the MISP-biosensor. This biosensing platform based on the designed MISP offers enormous potential for achieving accurate biomarker detection with robust operation in complex biological samples.

The basal and major pilins in the Corynebacterium diphtheriae SpaA pilus adopt similar structures that competitively react with the pilin polymerase.

Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the Cd SrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that Cd SrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA (N SpaA) that is also crosslinked by Cd SrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed "latch" mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting N SpaA for access to a shared thioester enzyme-substrate reaction intermediate.

Effect of High-Intensity Ultrasound Pretreatment on the Properties of the Transglutaminase (TGase)-Induced β-Conglycinin (7S) Gel.

In this study, we investigated the effects of different high-intensity ultrasound (HIU) pretreatment times (0-60 min) on the structure of β-conglycinin (7S) and the structural and functional properties of 7S gels induced by transglutaminase (TGase). Analysis of 7S conformation revealed that 30 min HIU pretreatment significantly induced the unfolding of the 7S structure, with the smallest particle size (97.59 nm), the highest surface hydrophobicity (51.42), and the lowering and raising of the content of the α-helix and β-sheet, respectively. Gel solubility showed that HIU facilitated the formation of ε-(γ-glutamyl)lysine isopeptide bonds, which maintain the stability and integrity of the gel network. The SEM revealed that the three-dimensional network structure of the gel at 30 min exhibited filamentous and homogeneous properties. Among them, the gel strength and water-holding capacity were approximately 1.54 and 1.23 times higher than those of the untreated 7S gels, respectively. The 7S gel obtained the highest thermal denaturation temperature (89.39 °C), G', and G″, and the lowest tan δ. Correlation analysis demonstrated that the gel functional properties were negatively correlated with particle size and the α-helix, while positively with Ho and β-sheet. By contrast, gels without sonication or with excessive pretreatment showed a large pore size and inhomogeneous gel network, and poor properties. These results will provide a theoretical basis for the optimization of HIU pretreatment conditions during TGase-induced 7S gel formation, to improve gelling properties.

Development of peptide-based biosensors for detecting cross-linking and deamidation activities of transglutaminases.

Transglutaminases (TGs) are a protein family that catalyzes isopeptide bond formation between glutamine and lysine residues of various proteins. There are eight TG isozymes in humans, and each is involved in diverse biological phenomena due to their characteristic distribution. Abnormal activity of TG1 and TG2, which are major TG isozymes, is believed to cause various diseases, such as ichthyosis and celiac disease. To elucidate TGs' mechanisms of action and develop new therapeutic strategies, it is essential to develop bioprobes that can specifically examine the activity of each TG isozyme, which has not been sufficiently studied. We previously have identified several substrate peptide sequences containing Gln residues for each isozyme and developed a method to detect isozyme-specific activities by incorporating a labeled substrate peptide into lysine residues of proteins. We prepared the fluorescein isothiocyanate (FITC)-labeled Gln substrate peptide (FITC-K5 and FITC-T26) and Rhodamine B-labeled Lys substrate peptide (RhoB-Kpep). Each TG reaction specifically cross-linked these probe pairs, and the proximity of FITC and Rhodamine B significantly decreased the fluorescence intensity of FITC depending on the concentration and reaction time of each TG. In this study, we developed a peptide-based biosensor that quickly and easily measures TG isozyme-specific activity. This probe is expected to be helpful in elucidating TG's physiological and pathological functions and in developing compounds that modulate TG activity.