Protein Cross-linking Research Articles

Exploring the relationship between the interfacial properties and emulsion properties of ultrasound-assisted cross-linked myofibrillar protein

This work aimed to illustrate the correlation between the dynamic adsorption process of ultrasound-assisted cross-linked MP on the oil-water interface and the stability of the fabricated emulsion. The interfacial pressure, adsorption kinetic coefficient, dynamic rheological properties of crosslinked MP (E-MP5), sonication before MP crosslinking (E-UMP5), and sonication after MP crosslinking (E-MPU5) and the corresponding emulsion properties, adsorbed proteins, microrheological properties, and multiple light scattering were determined. The results showed that the emulsion properties were positively correlated with the interfacial properties of MP. Ultrasound treatment could more effectively improve the interfacial properties and emulsion stability of the MP molecules than the Native treatment. Among the ultrasonic groups, the E-MPU5 group exhibited the highest interfacial pressure (15.81 mN/m), diffusion rate (Kdiff), penetration rate (KP), rearrangement rate (KR), dilatational modulus, elastic modulus, and E-π slope (2.65). Correspondingly, based on the change of Kdiff, KP, and KR, E-MPU5-fabricated emulsion had the lowest droplet size (1.56 μm), the maximum adsorbed protein content (93.08%), and the highest absolute zeta potential value. Additionally, the decreased viscosity, elastic index, macroscopic viscosity index, and turbiscan stability index in the E-MPU5-fabricated emulsion indicated the formation of a more stable emulsion. Therefore, this work suggests that sonication after crosslinking could greatly improve the interfacial adsorption behavior of MP, and the improvement of interfacial properties is conducive to the enhancement of emulsion stability.

Optimization of food-grade colloidal delivery systems for thermal processing applications: a review.

Colloidal delivery systems are widely used in the food industry to enhance the dispersibility, stability, efficacy, or bioavailability. However, when exposed to the high temperature, delivery systems are often prone to degradation, which limits its application in thermal processing. In this paper, the effects of thermal processing on the performance of traditional protein-based or starch-based delivery systems are firstly described, including the molecular structure changes of proteins, starches or lipids, and the degradation of embedded substances. These effects are unfavorable to the application of the delivery system in thermal processing. Then, strategies of improving the heat resistance of food grade colloid delivery system and their use in frying, baking and cooking food are mainly introduced. The heat resistance of the delivery system can be improved by a variety of strategies, including the development of new heat-resistant materials, the addition of heat-resistant coatings to the surface of delivery systems, the cross-linking of proteins or starches using cross-linking agents, the design of particle structures, the use of physical means such as ultrasound, or the optimization of the ingredient formula. These strategies will help to expand the application of heat-resistant delivery systems so that they can be used in real thermal processing.

Disassembly of bundled F-actin and cellular remodeling via an interplay of Mical, cofilin, and F-actin crosslinkers

Cellular form and function are controlled by the assembly and stability of actin cytoskeletal structures-but disassembling/pruning these structures is equally essential for the plasticity and remodeling that underlie behavioral adaptations. Importantly, the mechanisms of actin assembly have been well-defined-including that it is driven by actin's polymerization into filaments (F-actin) and then often bundling by crosslinking proteins into stable higher-order structures. In contrast, it remains less clear how these stable bundled F-actin structures are rapidly disassembled. We now uncover mechanisms that rapidly and extensively disassemble bundled F-actin. Using biochemical, structural, and imaging assays with purified proteins, we show that F-actin bundled with one of the most prominent crosslinkers, fascin, is extensively disassembled by Mical, the F-actin disassembly enzyme. Furthermore, the product of this Mical effect, Mical-oxidized actin, is poorly bundled by fascin, thereby further amplifying Mical's disassembly effects on bundled F-actin. Moreover, another critical F-actin regulator, cofilin, also affects fascin-bundled filaments, but we find herein that it synergizes with Mical to dramatically amplify its disassembly of bundled F-actin compared to the sum of their individual effects. Genetic and high-resolution cellular assays reveal that Mical also counteracts crosslinking proteins/bundled F-actin in vivo to control cellular extension, axon guidance, and Semaphorin/Plexin cell-cell repulsion. Yet, our results also support the idea that fascin-bundling serves to dampen Mical's F-actin disassembly in vitro and in vivo-and that physiologically relevant cellular remodeling requires a fine-tuned interplay between the factors that build bundled F-actin networks and those that disassemble them.

Tissue transglutaminase: a multifunctional and multisite regulator in health and disease.

Tissue transglutaminase (TG2) is a widely distributed multifunctional protein involved in a broad range of cellular and metabolic functions carried out in a variety of cellular compartments. In addition to transamidation, TG2 also functions as a Gα signaling protein, a protein disulfide isomerase (PDI), a protein kinase, and a scaffolding protein. In the nucleus, TG2 modifies histones and transcription factors. The PDI function catalyzes the trimerization and activation of heat shock factor-1 in the nucleus and regulates the oxidation state of several mitochondrial complexes. Cytosolic TG2 modifies proteins by the addition of serotonin or other primary amines and in this way affects cell signaling. Modification of protein-bound glutamines reduces ubiquitin-dependent proteasomal degradation. At the cell membrane, TG2 is associated with G protein-coupled receptors (GPCRs), where it functions in transmembrane signaling. TG2 is also found in the extracellular space, where it functions in protein cross-linking and extracellular matrix stabilization. Of particular importance in transglutaminase research are recent findings concerning the role of TG2 in gene expression, protein homeostasis, cell signaling, autoimmunity, inflammation, and hypoxia. Thus, TG2 performs a multitude of functions in multiple cellular compartments, making it one of the most versatile cellular proteins. Additional evidence links TG2 with multiple human diseases including preeclampsia, hypertension, cardiovascular disease, organ fibrosis, cancer, neurodegenerative diseases, and celiac disease. In conclusion, TG2 provides a multifunctional and multisite response to physiological stress.

Discovery and Characterization of an Acid-Labile Serine-Lysine Cross-Link in Antibody High-Molecular-Weight Species Using a Multipronged Mass Spectrometry Approach.

Characterizing the cross-links responsible for the covalent high-molecular-weight (HMW) species in therapeutic monoclonal antibodies (mAbs) is of great importance as it not only provides a framework for risk assessment but also offers insights for process improvement. However, owing to the complexity and low abundance, identification of novel and unknown cross-links in mAb products can be very challenging. Here, applying a multipronged MS-based approach, we report the discovery of a novel covalent cross-link formed via an imine bond between lysine and serine residues. In particular, this Ser-Lys cross-link was found to be acid-labile and can be easily overlooked by conventional LC-MS techniques operated at low pH. It is worth noting that although imine-based cross-link has been previously reported in collagen protein cross-linking, this is the first time that a Ser-Lys cross-link has been found in a mAb product that contributes to covalent HMW species formation.

Acne Scar Management: Minoxidil as a Promising Approach or a Mirage?

Atrophic and hypertrophic scars can result from various conditions, such as acne, trauma, and surgery. Minoxidil, a medication used for the treatment of severe hypertension and hair loss, has been explored as a potential treatment for scars. This review aims to evaluate the current evidence regarding the role of minoxidil in the treatment of scars. Previously published reviews have primarily focused on the use of minoxidil in hair loss and have only briefly mentioned its potential use for scars. However, minoxidil may have a beneficial effect as an antifibrotic agent. Several studies have reported reduced collagen accumulation and fibrosis after treatment with minoxidil. The proposed mechanism of action is inhibition of the production of lysyl hydroxylases (LHs), which modify and cross-link proteins by converting lysine to hydroxylysine, making collagen more resistant to degradation. Minoxidil, as an LH inhibitor, has been shown to potentially benefit wound healing and regeneration in vitro by inhibiting the proliferation and migration of fibroblasts. To date, direct studies of the efficacy of minoxidil in treating acne scars have not been conducted; however, its inhibitory effects on fibroblast function and antifibrotic outcomes in some in vivo studies suggest that such use may be considered.

Magnetic amyloid-based biocatalyst for the hydrolysis of urea

The presence of urea in wines and other alcoholic beverages represents a critical problem since it can chemically react with ethanol, which leads to the formation of ethyl carbamate, a carcinogenic agent according to the World Health Organization. Here we report the creation of a biocatalyst for the hydrolysis of urea, which could potentially be used before bottling alcoholic drinks. For this, the effective surface area of streptavidin-labeled magnetic microparticles was amplified by functionalization with biotin-labeled hen egg lysozyme amyloid fibers. Subsequently, by using copper and hydrogen peroxide induced cross-linking of unmodified proteins (CHICUP), soybean urease was immobilized to the fibers. This gave rise to a magnetic biocatalyst with remarkable urease activity, which was maintained even after 10 reuses. We propose that this strategy could be used as a platform for immobilizing other molecules to design and develop a myriad of biocatalysts for the food industry.

Anti-glycation and phytochemical properties of cinnamon stem-bark water extract

The study evaluated the ability of cinnamon stem-bark water extract (CWE) to break established cross-links formed between proteins and advanced glycation end-products (AGEs) as well as its anti-glycation effect. The extract showed a dose-dependent anti-glycation effect against total fluorescent AGEs (FAGEs) derived from both glucose and fructose. CWE recorded a uniform IC50 value of 0.13 mg/mL for both glucose and fructose-derived FAGEs while aminoguanidine, a well-known synthetic anti-glycative agent gave IC50 values of 0.14 mg/mL for the glucose and 0.17 mg/mL for the fructose derived FAGEs. The anti-glycative effect of CWE was also significantly higher than aminoguanidine against total immunogenic AGE (TIAGEs) in both sugar models (p < 0.001). While CWE and aminoguanidine showed weak protein cross-link breaking activity selectively on fructose-derived protein cross-links already formed, none whatsoever was detected exerted on the established glucose-derived protein cross-links. Phytochemical screening revealed the presence of several important secondary metabolites which may have contributed to the anti-glycative effect of CWE. Gas chromatography mass spectrometry enabled the identification of (+)-alpha-tocopherol acetate, a chain breaker, in the methanol fraction of CWE. Although CWE showed great potential for inhibition of formation of major types of AGEs, it appears to be poor in breaking established cross-links formed between proteins and AGEs.

Effect of egg white protein on the protein structure of highland barley noodles during processing

The effect of egg white protein on the protein structure of highland barely noodles during processing was investigated, and the underlying mechanism was examined. Egg white protein significantly influenced the stress relaxation of highland barley dough. 1% and 2% egg white protein improved the cooking and textural properties of highland barely noodles. During mixing and sheeting, it improved the structure of the protein network by promoting protein aggregation and cross-linking, whereas its effect on non-covalent interactions was quite different. During cooking, egg white protein promoted protein aggregation and cross-linking via heat-induced polymerization, and the distribution regularity of the protein network was improved as its flexibility diminished. The protein structure of highland barely noodles during processing was closely related to the addition amount of egg white protein, and the cooking, textural, and chemical interactions of highland barely noodles during processing changed considerably when more than 3% egg white protein was added.

Cross-Linked α-Synuclein as Inhibitor of Amyloid Formation.

The aggregation and amyloid formation of α-synuclein is associated with Parkinson's disease and other synucleinopathies. In its native, monomeric form α-synuclein is an intrinsically disordered protein represented by highly dynamic conformational ensembles. Inhibition of α-synuclein aggregation using small molecules, peptides, or proteins has been at the center of interest in recent years. Our aim was to explore the effects of cross-linking on the structure and aggregation/amyloid formation properties of α-synuclein. Comparative analysis of available high-resolution amyloid structures and representative structural models and MD trajectory of monomeric α-synuclein revealed that potential cross-links in the monomeric protein are mostly incompatible with the amyloid forms and thus might inhibit fibrillation. Monomeric α-synuclein has been intramolecularly chemically cross-linked under various conditions using different cross-linkers. We determined the location of cross-links and their frequency using mass spectrometry and found that most of them cannot be realized in the amyloid structures. The inhibitory potential of cross-linked proteins has been experimentally investigated using various methods, including thioflavin-T fluorescence and transmission electron microscopy. We found that conformational constraints applied by cross-linking fully blocked α-synuclein amyloid formation. Moreover, DTSSP-cross-linked molecules exhibited an inhibitory effect on the aggregation of unmodified α-synuclein as well.

Transglutaminases in fibrosis-overview and recent advances.

Transglutaminases (TGs) are a family of protein cross-linking enzymes that are capable of stiffening and insolubilizing proteins and creating protein networks, and thereby altering biological functions of proteins. Their role in fibrosis progression has been widely investigated with a focus on kidney, lung, liver, and heart where activity is triggered by various stimuli including hypoxia, inflammation, and hyperglycemia. TG2 has been considered one of the key enzymes in the pathogenesis of fibrosis mainly through transforming growth factor beta (TGF-beta) signaling and matrix cross-linking mechanisms. Although TG2 has been most widely studied in this context, the involvement of other TGs, TG1 and Factor XIII-A (FXIII-A), is beginning to emerge. This mini-review highlights the major steps taken in the TG and fibrosis research and summarizes the most recent advances and contributions of TG2, TG1, and FXIII-A to the progression of fibrosis in various animal models. Also, their mechanisms of action as well as therapeutic prospects are discussed.

Photo-Induced Cross-Linking of Unmodified α-Synuclein Oligomers.

Photo-induced cross-linking of unmodified proteins (PICUP) has been used in the past to study size distributions of protein assemblies. PICUP may, for example, overcome the significant experimental challenges related to the transient nature, heterogeneity, and low concentration of amyloid protein oligomers relative to monomeric and fibrillar species. In the current study, a reaction chamber was designed, produced, and used for PICUP reaction optimization in terms of reaction conditions and lighting time from ms to s. These efforts make the method more reproducible and accessible and enable the use of shorter reaction times compared to previous studies. We applied the optimized method to an α-synuclein aggregation time course to monitor the relative concentration and size distribution of oligomers over time. The data are compared to the time evolution of the fibril mass concentration, as monitored by thioflavin T fluorescence. At all time points, the smaller the oligomer, the higher its concentration observed after PICUP. Moreover, the total oligomer concentration is highest at short aggregation times, and the decline over time follows the disappearance of monomers. We can therefore conclude that these oligomers form from monomers.

Phosphorylation controls spatial and temporalactivities of motor-PRC1 complexes to complete mitosis.

During mitosis, spindle architecture alters as chromosomes segregate into daughter cells. The microtubule crosslinker protein regulator of cytokinesis 1 (PRC1) is essential for spindle stability, chromosome segregation and completion of cytokinesis, but how it recruits motors to the central spindle to coordinate the segregation of chromosomes is unknown. Here, we combine structural and cell biology approaches to show that the human CENP-E motor, which is essential for chromosome capture and alignment by microtubules, binds to PRC1 through a conserved hydrophobic motif. This binding mechanism is also used by Kinesin-4 Kif4A:PRC1. Using in vitro reconstitution, we demonstrate that CENP-E slides antiparallel PRC1-crosslinked microtubules. We find that the regulation of CENP-E -PRC1 interaction is spatially and temporally coupled with relocalization to overlapping microtubules in anaphase. Finally, we demonstrate that the PRC1-microtubule motor interaction is essential in anaphase to control chromosome partitioning, retain central spindle integrity and ensure cytokinesis. Taken together our findings reveal the molecular basis for the cell cycle regulation of motor-PRC1 complexes to couple chromosome segregation and cytokinesis.

All-in-One Pseudo-MS3 Method for the Analysis of Gas-Phase Cleavable Protein Crosslinking Reactions.

Crosslinking mass spectrometry (XL-MS) supports structure analysis of individual proteins and highly complex whole-cell interactomes. The identification of crosslinked peptides from enzymatic digests remains challenging, especially at the cell level. Empirical methods that use gas-phase cleavable crosslinkers can simplify the identification process by enabling an MS3-based strategy that turns crosslink identification into a simpler problem of detecting two separable peptides. However, the method is limited to select instrument platforms and is challenged by duty cycle constraints. Here, we revisit a pseudo-MS3 concept that incorporates in-source fragmentation, where a fast switch between gentle high-transmission source conditions and harsher in-source fragmentation settings liberates peptides for standard MS2-based peptide identification. We present an all-in-one method where retention time matches between the crosslink precursor and the liberated peptides establish linkage, and MS2 sequencing identifies the source-liberated peptides. We demonstrate that DC4, a very labile cleavable crosslinker, generates high-intensity peptides in-source. Crosslinks can be identified from these liberated peptides, as they are chromatographically well-resolved from monolinks. Using bovine serum albumin (BSA) as a crosslinking test case, we detect 27% more crosslinks with pseudo-MS3 over a best-in-class MS3 method. While performance is slightly lower for whole-cell lysates (generating two-thirds of the identifications of a standard method), we find that 60% of these hits are unique, highlighting the complementarity of the method.

Empowering canonical biochemicals with cross-linked novelty: Recursions in applications of protein cross-links.

Diversity in the biochemical workhorses of the cell-that is, proteins-is achieved by the innumerable permutations offered primarily by the 20 canonical L-amino acids prevalent in all biological systems. Yet, proteins are known to additionally undergo unusual modifications for specialized functions. Of the various post-translational modifications known to occur in proteins, the recently identified non-disulfide cross-links are unique, residue-specific covalent modifications that confer additional structural stability and unique functional characteristics to these biomolecules. We review an exclusive class of amino acid cross-links encompassing aromatic and sulfur-containing side chains, which not only confer superior biochemical characteristics to the protein but also possess additional spectroscopic features that can be exploited as novel chromophores. Studies of their in vivo reaction mechanism have facilitated their specialized in vitro applications in hydrogels and protein anchoring in monolayer chips. Furthering the discovery of unique canonical cross-links through new chemical, structural, and bioinformatics tools will catalyze the development of protein-specific hyperstable nanostructures, superfoods, and biotherapeutics.

Contrasting effects of filamin A and B proteins in modulating filovirus entry.

Ebola (EBOV) and Marburg viruses (MARV) cause severe hemorrhagic fever associated with high mortality rates in humans. A better understanding of filovirus-host interactions that regulate the EBOV and MARV lifecycles can provide biological and mechanistic insight critical for therapeutic development. EBOV glycoprotein (eGP) and MARV glycoprotein (mGP) mediate entry into host cells primarily by actin-dependent macropinocytosis. Here, we identified actin-binding cytoskeletal crosslinking proteins filamin A (FLNa) and B (FLNb) as important regulators of both EBOV and MARV entry. We found that entry of pseudotype psVSV-RFP-eGP, infectious recombinant rVSV-eGP-mCherry, and live authentic EBOV and MARV was inhibited in filamin A knockdown (FLNaKD) cells, but was surprisingly enhanced in filamin B knockdown (FLNbKD) cells. Mechanistically, our findings suggest that differential regulation of macropinocytosis by FLNa and FLNb likely contributes to their specific effects on EBOV and MARV entry. This study is the first to identify the filamin family of proteins as regulators of EBOV and MARV entry. These findings may provide insight into the development of new countermeasures to prevent EBOV and MARV infections.

Advanced glycation end products: Key mediator and therapeutic target of cardiovascular complications in diabetes.

The incidence of type 2 diabetes mellitus is growing in epidemic proportions and has become one of the most critical public health concerns. Cardiovascular complications associated with diabetes are the leading cause of morbidity and mortality. The cardiovascular diseases that accompany diabetes include angina, myocardial infarction, stroke, peripheral artery disease, and congestive heart failure. Among the various risk factors generated secondary to hyperglycemic situations, advanced glycation end products (AGEs) are one of the important targets for future diagnosis and prevention of diabetes. In the last decade, AGEs have drawn a lot of attention due to their involvement in diabetic patho-physiology. AGEs can be derived exogenously and endogenously through various pathways. These are a non-homogeneous, chemically diverse group of compounds formed non-enzymatically by condensation between carbonyl groups of reducing sugars and free amino groups of protein, lipids, and nucleic acid. AGEs mediate their pathological effects at the cellular and extracellular levels by multiple pathways. At the cellular level, they activate signaling cascades via the receptor for AGEs and initiate a complex series of intracellular signaling resulting in reactive oxygen species generation, inflammation, cellular proliferation, and fibrosis that may possibly exacerbate the damaging effects on cardiac functions in diabetics. AGEs also cause covalent modifications and cross-linking of serum and extracellular matrix proteins; altering their structure, stability, and functions. Early diagnosis of diabetes may prevent its progression to complications and decrease its associated comorbidities. In the present review, we recapitulate the role of AGEs as a crucial mediator of hyperglycemia-mediated detrimental effects in diabetes-associated complications. Furthermore, this review presents an overview of future perspectives for new therapeutic interventions to ameliorate cardiovascular complications in diabetes.

In-situ generated Co2P nanoparticle-embedded porous carbon membrane as an advanced hybrid electrode for high-areal-capacitance supercapacitors

Hybridizing powdery carbons with transition metal phosphides (TMPs) can effectively address the issues of sluggish kinetics and volume expansion associated with TMPs in supercapacitors (SCs). However, the practical use of powdery TMPs/carbon materials in SCs is limited by tedious electrode preparation process, unfavorable performance, and inferior cycling stability. Herein, we develop carbon membrane-based hybrid electrodes (Co2P@CTF) by the direct carbonization of Co-containing tofu (TF) aerogel prepared by coagulating soybean proteins using Co2+ ions. During carbonization, the cross-linked protein frameworks were transformed into a porous, mechanically strong, and conductive carbon matrix, whereas the Co species were in situ self-phosphorized to be highly dispersed carbon-coated Co2P nanoparticles without using external phosphorus sources. The best Co2P@CTF electrode (Co2P@CTF-1000) demonstrates a high areal capacitance (Ca) of 2.44 F cm−2 at 1.0 mA cm−2. Moreover, a symmetric SC assembled with Co2P@CTF-1000 deliveries a Ca of 1.33 F cm−2 at 1.0 mA cm−2, achieves an energy density of 90 μWh cm−2 at a power density of 699.4 μW cm−2, and retains a 104.8% capacitance with a nearly unit coulomb efficiency at 10 mA cm−2 after 50000 cycles. This study offers an effective strategy towards carbon membrane-supported TMPs hybrid electrodes based on the direct self-phosphorization mechanism for the practical applications in SCs.

Cell-Impermeable Inhibitors Confirm That Intracellular Human Transglutaminase 2 Is Responsible for the Transglutaminase-Associated Cancer Phenotype.

Transglutaminase 2 (TG2) is a multifunctional enzyme primarily responsible for crosslinking proteins. Ubiquitously expressed in humans, TG2 can act either as a transamidase by crosslinking two substrates through formation of an Nε(ɣ-glutaminyl)lysine bond or as an intracellular G-protein. These discrete roles are tightly regulated by both allosteric and environmental stimuli and are associated with dramatic changes in the conformation of the enzyme. The pleiotropic nature of TG2 and multi-faceted activities have resulted in TG2 being implicated in numerous disease pathologies including celiac disease, fibrosis, and cancer. Targeted TG2 therapies have not been selective for subcellular localization, such that currently no tools exist to selectively target extracellular over intracellular TG2. Herein, we have designed novel TG2-selective inhibitors that are not only highly potent and irreversible, but also cell impermeable, targeting only extracellular TG2. We have also further derivatized the scaffold to develop probes that are intrinsically fluorescent or bear an alkyne handle, which target both intra- and extracellular TG2, in order to facilitate cellular labelling and pull-down assays. The fluorescent probes were internalized and imaged in cellulo, and provide the first implicit experimental evidence that by comparison with their cell-impermeable analogues, it is specifically intracellular TG2, and presumably its G-protein activity, that contributes to transglutaminase-associated cancer progression.

Silk-protein-based gradient hydrogels with multimode reprogrammable shape changes for biointegrated devices

Biocompatible and morphable hydrogels capable of multimode reprogrammable, and adaptive shape changes are potentially useful for diverse biomedical applications. However, existing morphable systems often rely on complicated structural designs involving cumbersome and energy-intensive fabrication processes. Here, we report a simple electric-field-activated protein network migration strategy to reversibly program silk-protein hydrogels with controllable and reprogrammable complex shape transformations. The application of a low electric field enables the convergence of net negatively charged protein cross-linking networks toward the anode (isoelectric point plane) due to the pH gradient generated in the process, facilitating the formation of a gradient network structure and systems suitable for three-dimensional shape change. These tunable protein networks can be reprogrammed or permanently fixed by control of the polymorphic transitions. We show that these morphing hydrogels are capable of conformally interfacing with biological tissues by programming the shape changes and a bimorph structure consisting of aligned carbon nanotube multilayers and the silk hydrogels was assembled to illustrate utility as an implantable bioelectronic device for localized low-voltage electrical stimulation of the sciatic nerve in a rabbit.