Protein Cross-linking Research Articles

Immuno-Sensing at Ultra-Low Concentration of TG2 Protein by Organic Electrochemical Transistors.

Transglutaminase 2 (TG2) is a ubiquitously expressed member of the transglutaminase family with Ca2+-dependent protein crosslinking activity. Its subcellular localization is crucial in determining its function, and indeed, TG2 is found in the extracellular matrix, mitochondria, recycling endosomes, plasma membrane, cytosol, and nucleus because it is associated with cell growth, differentiation, and apoptosis. It is involved in several pathologies, such as celiac disease, cardiovascular, hepatic, renal, and fibrosis diseases, carrying out opposite functions of up and down regulation in the progression of the same pathology. Therefore, this fine regulation requires a very sensitive and specific method of identification of TG2, which is to be detected in very small quantities in a deregulated condition. Here, we demonstrate the possibility of detecting TG2 down to attomolar concentration by using organic electrochemical transistors driven by gold electrodes functionalized with anti-TG2 antibodies. In particular, a direct correlation between the TG2 concentration and the transistor transconductance values, as extracted from typical transfer curves, was found. Overall, our findings highlight the potentialities of this new biosensing approach for the detection of TG2 in the context of pathological diseases, offering a rapid and cost-effective alternative to traditional methods.

Bioinformatics Analysis of Molecular Interactions between Endoplasmic Reticulum Stress and Ferroptosis under Stress Exposure.

Stress has become a universal biological phenomenon in the body, which leads to pathophysiological changes. However, the molecular network interactions between endoplasmic reticulum (ER) stress and ferroptosis under stressful conditions are not clear. For this purpose, we screened the gene expression profile of GSE173795 for intersection with ferroptosis genes and screened 68 differentially expressed genes (DEGs) (63 up-regulated, 5 down-regulated), mainly related to lipid and atherosclerosis, autophagy-animal, mitophagy-animal, focal adhesion, DNA replication, proteasome, oocyte meiosis, toll-like receptor signaling pathway, cell cycle, etc. Immune infiltration analysis revealed that stress resulted in decreased B cells memory, T cells CD8 and T cells CD4 memory resting, monocytes, macrophages M2, and increased B cells naive, T cells follicular helper, and macrophages M1. 19 core-DEGs (ASNS, TRIB3, ATF4, EIF2S1, CEBPG, RELA, HSPA5, DDIT3, STAT3, MAP3K5, HIF1A, HNF4A, MAPK14, HMOX1, CDKN1A, KRAS, SP1, SIRT1, EGFR) were screened, all of which were up-regulated DEGs. These biological processes and pathways were mainly involved in responding to ER stress, lipid and atherosclerosis, cellular response to stress, cellular response to chemical stress, and regulation of DNA-templated transcription in response to stress, etc. Spearman analysis did not find MAPK14 to be significantly associated with immune cells. Other core-DEGs were associated with immune cells, including B cells naive, T cells follicular helper, and monocytes. Based on core-DEGs, 283 miRNAs were predicted. Among the 22 miRNAs with highly cross-linked DEGs, 11 had upstream lncRNA, mainly targeting STAT3, SP1, CDKN1A, and SIRT1, and a total of 39 lncRNA were obtained. 85 potential drugs targeting 11 core-DEGs were identified and were expected to be potential immunotherapeutic agents for stress injury. Our experiments also confirmed that Liproxstatin-1 alleviates common cross-linked proteins between ER stress and ferroptosis. In conclusion, our study explored the molecular mechanisms and network interactions among stress-ER stress-ferroptosis from a novel perspective, which provides new research ideas for studying stressful injury.

Covalent polyphenol with soybean 11S protein to develop hypoallergenic conjugates for potential immunotherapy

Soybean 11S globulin is one of the most important allergens in soybean. This study investigated the effects of 11S covalent that conjugated covalently with (−)-Epigallo-catechin 3-gallate (EGCG) and chlorogenic acid (CHA) on protein structure, digestibility, and allergenicity. 11S conjugation with EGCG and CHA yielded covalent conjugates and formed cross-linked protein polymers, which changed the structures and increased the digestibility of the protein. In vitro experiments showed that the ability of IgE binding, and stimulation mast cell degranulation were lower for11S-EGCG and 11S-CHA conjugates compared to 11S. In vivo experiments showed that conjugation of EGCG and CHA reduced the allergenicity of 11S. The oral tolerance model showed that hypoallergenic 11S-EGCG conjugates successfully relieved allergic symptoms and induced oral tolerance by affecting antigen presentation, activating Treg cells to produce IL-10, and promoting Th1/Th2 immune balance. This study presented an innovative strategy to develop hypoallergenic food production that induce tolerance for allergen-specific immunotherapy.

Changes in glycated myofibrillar proteins conformation on the formation of Nε-carboxymethyllysine under gradient thermal conditions

Nε-carboxymethyllysine (CML), a frequently used marker of advanced glycation end products (AGEs) in food, was generated in food processing easily and caused changes in myofibrillar proteins (MPs) characterization. The relevance between glycosylated MPs structure alternation and CML formation under thermal conditions have been reported. However, the correlation mechanism was not clear yet. In this work, the influence of gradient heating (50℃, 60℃, 70℃, 80℃, and 90℃) on the different degrees of glycated MPs, which determined the correlation with CML formation in protein structural changes of MPs. In the rising stage of the CML level, glycation accelerated the fibrillation and aggregation behavior of MPs during heating and increased surface hydrophobicity and particle size. The protein cross-linking affected the protein modification caused by heating and glycation. This work highlights the substantial influences of glycosylation and thermal treatments on MPs, which transformed the MPs structural characteristics and CML level.

Protein interactions during dry and wet heat pre-treatment of skim milk powder (dispersions) and their effect on the heat stability of recombined filled evaporated milk

Skim milk powder (SMP) as well as aqueous dispersions were subjected to dry and wet heat pre-treatment, respectively, to improve the heat stability of recombined filled evaporated milk (RFEM) derived therefrom. However, microrheological analysis revealed that prolonged incubation caused detrimental effects on the heat stability of RFEM, which were thought to be due to protein interactions. SDS-PAGE results indicated that protein aggregation via non-disulfide covalent bonds occurred upon long-time dry or wet heat incubation. This was probably related to some Maillard reaction products, which is sustained by the increase in lactulose and protein carbonyl content. Considerable protein aggregation via disulfide bonds in the serum was found upon wet heat incubation at temperatures of at least 80 °C. Principal component analysis (PCA) revealed that the negative effects of overprocessing on the heat stability of RFEM were predominantly related to protein cross-linking via non-disulfide covalent bonds related to protein carbonylation.

Estrogen-related receptor γ (ERRγ) is a key regulator of lysyl oxidase gene expression in mouse hepatocytes

Lysyl oxidase (LOX), the copper-dependent extracellular enzyme, plays a critical role in the regulation of protein cross-linking in the extracellular matrix (ECM). It is also involved in liver regeneration and liver fibrosis. However, the mechanism of LOX regulation in mouse hepatocytes is still unclear. Here, we identify a molecular mechanism showing that orphan nuclear receptor estrogen-related receptor γ (ERRγ) regulates LOX gene expression in the presence of the pro-inflammatory cytokine, interleukin 6 (IL6). IL6 significantly stimulated the expression of ERRγ and LOX in mouse hepatocytes. Overexpression of ERRγ increased LOX mRNA and protein levels. Moreover, knockdown of ERRγ attenuated IL6-mediated LOX gene expression at mRNA and protein levels. Overexpression of ERRγ or IL6 treatment upregulated LOX gene promoter activity, while knockdown of ERRγ decreased the IL6-induced LOX promoter activity. Furthermore, GSK5182, a specific ERRγ inverse agonist, inhibited the induction effect of IL6 on LOX promoter activity and gene expression in mouse hepatocytes. Overall, our study elucidates the mechanism involved in the LOX gene regulation by nuclear receptor ERRγ in response to IL6 in mouse hepatocytes, suggesting that, in conditions such as chronic inflammation, IL6 may contribute to liver fibrosis via inducing LOX gene expression. Thus, LOX gene regulation by the inverse agonist of ERRγ can be applied to improve liver fibrosis.

Getting cells into shape by calcium-dependent actin cross-linking proteins.

The actin cytoskeleton represents a highly dynamic filament system providing cell structure and mechanical forces to drive a variety of cellular processes. The dynamics of the actin cytoskeleton are controlled by a number of conserved proteins that maintain the pool of actin monomers, promote actin nucleation, restrict the length of actin filaments and cross-link filaments into networks or bundles. Previous work has been established that cytoplasmic calcium is an important signal to rapidly relay information to the actin cytoskeleton, but the underlying mechanisms remain poorly understood. Here, we summarize new recent perspectives on how calcium fluxes are transduced to the actin cytoskeleton in a physiological context. In this mini-review we will focus on three calcium-binding EF-hand-containing actin cross-linking proteins, α-actinin, plastin and EFHD2/Swiprosin-1, and how these conserved proteins affect the cell's actin reorganization in the context of cell migration and wound closure in response to calcium.

Spt6 directly interacts with Cdc73 and is required for Paf1 complex occupancy at active genes in Saccharomyces cerevisiae.

The Paf1 complex (Paf1C) is a conserved transcription elongation factor that regulates transcription elongation efficiency, facilitates co-transcriptional histone modifications, and impacts molecular processes linked to RNA synthesis, such as polyA site selection. Coupling of the activities of Paf1C to transcription elongation requires its association with RNA polymerase II (Pol II). Mutational studies in yeast identified Paf1C subunits Cdc73 and Rtf1 as important mediators of Paf1C association with Pol II on active genes. While the interaction between Rtf1 and the general elongation factor Spt5 is relatively well-understood, the interactions involving Cdc73 have not been fully elucidated. Using a site-specific protein cross-linking strategy in yeast cells, we identified direct interactions between Cdc73 and two components of the Pol II elongation complex, the elongation factor Spt6 and the largest subunit of Pol II. Both of these interactions require the tandem SH2 domain of Spt6. We also show that Cdc73 and Spt6 can interact in vitro and that rapid depletion of Spt6 dissociates Paf1 from chromatin, altering patterns of Paf1C-dependent histone modifications genome-wide. These results reveal interactions between Cdc73 and the Pol II elongation complex and identify Spt6 as a key factor contributing to the occupancy of Paf1C at active genes in Saccharomyces cerevisiae.

The force-dependent filamin A-G3BP1 interaction regulates phase-separated stress granule formation.

Filamin A (FLNA) is an actin crosslinking protein that mediates mechanotransduction. External and internal mechanical forces, through the actin cytoskeleton, can induce conformational changes of the FLNA molecule to expose cryptic binding sites for its binding partners. Here, we identified G3BP1 (Ras-GTPase activating protein SH3 domain-binding protein 1) as a new FLNA mechanobinding partner. Unlike other FLNA-binding partners to the mechanosensing domain repeat 21 (R21), G3BP1 requires an additional neighboring R22 to interact. We demonstrated that their interaction occurs in the cytosol of living cells in an actin polymerization-dependent manner. We also mapped the FLNA-binding site on G3BP1 and found that a F360A point mutation in the RNA recognition motif disrupts the interaction. RNA interferes with the FLNA-G3BP1 interaction and FLNA does not localize in RNA-rich stress granules (SGs). Disruption of the interaction is sufficient to promote phase-separated SG formation and arsenite treatment further stimulates formation of SGs. Taken together, these data identify G3BP1 as a new mechanobinding protein that interacts with the FLNA mechanosensing domain R21 and suggest that SG formation is partially regulated by mechanical force.

Structural characterization of soy protein hydrolysates and their transglutaminase-induced gelation properties

Soy protein hydrolates (SPHs) were produced by hydrolyzing soy protein isolate (SPI) for different times (5–180 min) using Alcalase, followed by cross-linking with transglutaminase (TGase) and their gelation capacity were then evaluated. The hydrolysis degree (DH) of SPHs increased from 1.37% to 6.61% as the increasing hydrolysis time from 5 min to 180 min. Compared to SPI, enzymatic hydrolysis enhanced the surface hydrophobicity and random coil structure, but decreased the free sulfydryl content, α-helix and β-turn structures of SPHs. Notably, the low hydrolysis (DH: 1.37–2.17%) improved the protein solubility, disulfide bond content and β-sheet structure, decreased the turbidity of SPHs solutions in comparison with those hydrolyzed highly (DH: 3.12–6.61%). In addition, the TGase-pretreated SPHs displayed typical weak gel-forming capacities upon acyl transfer and protein cross-linking. In contrast, low-hydrolyzed SPHs (DH: 2.17%) exhibited superior gelation behaviors, as confirmed by uniform networks with small cavities, larger relaxation time distribution, higher viscoelasticity and water-holding capacity of 54%. In general, TGase indeed promoted the gelation capacity of SPHs subjected to limited hydrolysis, which facilitates the application of SPHs in food formulation.

Single-Molecule Correlated Chemical Probing: A Revolution in RNA Structure Analysis.

RNA molecules convey biological information both in their linear sequence and in their base-paired secondary and tertiary structures. Chemical probing experiments, which involve treating an RNA with a reagent that modifies conformationally dynamic nucleotides, have broadly enabled examination of short- and long-range RNA structure in diverse contexts, including in living cells. For decades, chemical probing experiments have been interpreted in a per-nucleotide way, such that the reactivity measured at each nucleotide reports the average structure at a position over all RNA molecules within a sample. However, there are numerous important cases where per-nucleotide chemical probing falls short, including for RNAs that are bound by proteins, RNAs that form complex higher order structures, and RNAs that sample multiple conformations.Recent experimental and computational innovations have started a revolution in RNA structure analysis by transforming chemical probing into a massively parallel, single-molecule experiment. Enabled by a specialized reverse transcription strategy called mutational profiling (MaP), multiple chemical modification events can be measured within individual RNA molecules. Nucleotides that communicate structurally through direct base pairing or large-scale folding-unfolding transitions will react with chemical probes in a correlated manner, thereby revealing structural complexity hidden to conventional approaches. These single-molecule correlated chemical probing (smCCP) experiments can be interpreted to directly identify nucleotides that base pair (the PAIR-MaP strategy) and to reveal long-range, through-space structural communication (RING-MaP). Correlated probing can also define the thermodynamic populations of complex RNA ensembles (DANCE-MaP). Complex RNA-protein networks can be interrogated by cross-linking proteins to RNA and measuring correlations between cross-linked positions (RNP-MaP).smCCP thus visualizes RNA secondary and higher-order structure with unprecedented accuracy, defining novel structures, RNA-protein interaction networks, time-resolved dynamics, and allosteric structural switches. These strategies are not mutually exclusive; in favorable cases, multiple levels of RNA structure ─ base pairing, through-space structural communication, and equilibrium ensembles ─ can be resolved concurrently. The physical experimentation required for smCCP is profoundly simple, and experiments are readily performed in cells on RNAs of any size, including large noncoding RNAs and mRNAs. Single-molecule correlated chemical probing is paving the way for a new generation of biophysical studies on RNA in living systems.

Effect of Black Tea Powder on Antioxidant Activity and Gel Characteristics of Silver Carp Fish Balls.

The effect of black tea powder on the antioxidant activity and gel characteristics of fish balls from silver carp were investigated after freezing storage for 7 days. The results show that black tea powder with different concentrations of 0.1%, 0.2% and 0.3% (w/w) could significantly increase the antioxidant activity of fish balls (p < 0.05). In particular, at the concentration of 0.3%, the antioxidant activity was the strongest among these samples, where the reducing power, DPPH, ABTS and OH free radical scavenging rate were up to 0.33, 57.93%, 89.24% and 50.64%, respectively. In addition, black tea powder at the level of 0.3% significantly increased the gel strength, hardness and chewiness while greatly reducing the whiteness of the fish balls (p < 0.05). ESEM observation found that the addition of black tea powder could promote the crosslinking of proteins and reduced the pore size of the gel network structure of the fish balls. The results suggest that black tea powder could be used as a natural antioxidant and gel texture enhancer in fish balls, which we found to be much related to the phenolic compounds of black tea powder.

Regulation of 3' splice site selection after step 1 of splicing by spliceosomal C* proteins.

Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3' splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA-mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3'ss. Cryo-electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3' region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3'ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3'ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3'ss choices.

Cellular FXIII in Human Macrophage-Derived Foam Cells.

Macrophages express the A subunit of coagulation factor XIII (FXIII-A), a transglutaminase which cross-links proteins through Nε-(γ-L-glutamyl)-L-lysyl iso-peptide bonds. Macrophages are major cellular constituents of the atherosclerotic plaque; they may stabilize the plaque by cross-linking structural proteins and they may become transformed into foam cells by accumulating oxidized LDL (oxLDL). The combination of oxLDL staining by Oil Red O and immunofluorescent staining for FXIII-A demonstrated that FXIII-A is retained during the transformation of cultured human macrophages into foam cells. ELISA and Western blotting techniques revealed that the transformation of macrophages into foam cells elevated the intracellular FXIII-A content. This phenomenon seems specific for macrophage-derived foam cells; the transformation of vascular smooth muscle cells into foam cells fails to induce a similar effect. FXIII-A containing macrophages are abundant in the atherosclerotic plaque and FXIII-A is also present in the extracellular compartment. The protein cross-linking activity of FXIII-A in the plaque was demonstrated using an antibody labeling the iso-peptide bonds. Cells showing combined staining for FXIII-A and oxLDL in tissue sections demonstrated that FXIII-A-containing macrophages within the atherosclerotic plaque are also transformed into foam cells. Such cells may contribute to the formation of lipid core and the plaque structurization.

Bacterial division ring stabilizing ZapA versus destabilizing SlmA modulate FtsZ switching between biomolecular condensates and polymers.

Cytokinesis is a fundamental process for bacterial survival and proliferation, involving the formation of a ring by filaments of the GTPase FtsZ, spatio-temporally regulated through the coordinated action of several factors. The mechanisms of this regulation remain largely unsolved, but the inhibition of FtsZ polymerization by the nucleoid occlusion factor SlmA and filament stabilization by the widely conserved cross-linking protein ZapA are known to play key roles. It was recently described that FtsZ, SlmA and its target DNA sequences (SlmA-binding sequence (SBS)) form phase-separated biomolecular condensates, a type of structure associated with cellular compartmentalization and resistance to stress. Using biochemical reconstitution and orthogonal biophysical approaches, we show that FtsZ-SlmA-SBS condensates captured ZapA in crowding conditions and when encapsulated inside cell-like microfluidics microdroplets. We found that, through non-competitive binding, the nucleotide-dependent FtsZ condensate/polymer interconversion was regulated by the ZapA/SlmA ratio. This suggests a highly concentration-responsive tuning of the interconversion that favours FtsZ polymer stabilization by ZapA under conditions mimicking intracellular crowding. These results highlight the importance of biomolecular condensates as concentration hubs for bacterial division factors, which can provide clues to their role in cell function and bacterial survival of stress conditions, such as those generated by antibiotic treatment.

Stepwise Optimization of Inducible Expression System for the Functional Secretion of Horseradish Peroxidase in Saccharomyces cerevisiae.

Horseradish peroxidase (HRP) is a plant-derived glycoprotein that can be developed as a food additive to cross-link proteins or biopolymers. Although Saccharomyces cerevisiae has advantages in the production of food-grade HRP, the low expressional level and inefficient secretion hindered its application values. After comparing the effects of constitutive and inducible expression on cell growth, the strength of HRP expression was roughly tuned by replacing core regions of the promoter in the GAL80-knockout strain and further finely tuned by terminator screening. Additionally, the most suitable signal peptide was selected, and the pre-peptide with pro-peptides was modified to balance the transport of HRP in the endoplasmic reticulum. The extracellular HRP activity of the best strain reached 13 506 U/L at the fermenter level, 330-fold higher than the previous result of 41 U/L in S. cerevisiae. The strategy can be applied to alleviate the inhibition of cell growth caused by the expression of toxic proteins and improve their secretion.

Effect of pH-Shifting Process on the Cathepsin Activity, Muddy Off-Odor Compounds' Content and Gelling Properties of Isolated Protein from Silver Carp.

Silver carp (Hypophthalmichthys molitrix) is a potential source for making surimi products. However, it has the disadvantages of bony structures, high level of cathepsines and muddy off-odor which is mainly caused by geosmin (GEO) and 2-methylisoborneol (MIB). These disadvantages make the conventional water washing process of surimi inefficient (low protein recovery rate, and high residual muddy off-odor). Thus, the effect of the pH-shifting process (acid-isolating process and alkali-isolating process) on the cathepsins activity, GEO content, MIB content, and gelling properties of the isolated proteins (IPs) was investigated, comparing it with surimi obtained through the conventional cold water washing process (WM). The alkali-isolating process greatly boosted the protein recovery rate from 28.8% to 40.9% (p < 0.05). In addition, it removed 84% GEO and 90% MIB. The acid-isolating process removed about 77% GEO and 83% MIB. The acid-isolated protein (AC) displayed the lowest elastic modulus (G'), the highest TCA-peptide content (90.89 ± 4.65 mg/g) and the highest cathepsin L activity (65.43 ± 4.91 U/g). The AC modori (60 °C for 30 min) gel also demonstrated the lowest breaking force (226.2 ± 19.5 g) and breaking deformation (8.3 ± 0.4 mm), indicating that proteolysis caused by the cathepsin deteriorated the gel quality of AC. The setting (40 °C for 30 min) considerably increased the breaking force (386.4 ± 15.7 g) and breaking deformation (11.6 ± 0.2 mm) of the gel made from the alkali-isolated protein (AK) (p < 0.05). In AC and AK gel, a clearly visible cross-linking protein band with a molecular weight greater than MHC was seen, demonstrating the presence of endogenous trans-glutaminase (TGase) activity, that improved the gel quality of AK. In conclusion, the alkali-isolating process was an effective alternative method for making water-washed surimi from silver carp.

Formulation and characterization of insulin nanoclusters for a controlled release

The growing interest in biopharmaceuticals combined with the challenges regarding formulation and delivery continues to encourage the development of new and improved formulations of this class of therapeutics. Nanoclusters (NCs) represent a type of formulation strategy where the biopharmaceutical is clustered in a reversible manner to function as both the therapeutic and the vehicle. In this study, insulin NCs (INCs) were formulated by a new methodology of first crosslinking proteins followed by desolvation. Crosslinking of the protein with the reducible DTSSP crosslinker improved control of the INC synthesis process to give INCs with a mean size of 198 ± 7 nm and a mean zeta potential of −39 ± 1 mV. Crosslinking and clustering of insulin did not induce cytotoxicity or major differences in the biological activity compared to the free unmodified protein. The potency of the crosslinked insulin and the INCs appeared slightly lower than that of the unmodified protein, and significantly higher doses of the INCs compared to the free protein were applied to achieve similar blood sugar lowering effects in vivo. Interestingly, the INCs allowed for high doses to be subcutaneously delivered with prolonged efficacy without being lethal in rats.

Improvement of physicochemical and gel properties of chlorogenic acid-modified oxidized myofibrillar proteins by transglutaminase

Chlorogenic acid (CA), a natural polyphenolic antioxidant, is used in meat products to prevent spoilage due to oxidation. Transglutaminase (TGase) is an effective protein cross-linking agent applied to meat gel products to increase the elasticity of the protein gel. The present study illustrated the effect of CA at various additions (5, 20, and 100 μmol/g protein) and/or TGase (0.2 g/100g protein) on physicochemical and gel characteristics of myofibrillar protein (MP). Texture and rheological results demonstrated that the TGase improved the gel structures of oxidized MP treated with CA (5 and 20 μmol/g protein). Scanning electron microscope (SEM) result showed that TGase addition made the gel network orderly, and overcame the phenomenon induced by excessive aggregation of proteins by CA at a high dose (100 μmol/g protein). In addition, water-holding capability (WHC) and low-field NMR (LF-NMR) results demonstrated that the TGase dramatically enhanced the water-holding capability of oxidized MP gel treated with CA (P < 0.05), and the moisture distribution became uniform. FT-IR analysis showed that reduced α-helix structure contributed to improving gel properties. SDS-PAGE indicated that TGase enhanced cross-linking of proteins. Conclusionally, TGase improved the physicochemical and gel properties of CA-modified oxidized myofibrillar proteins.

Induction of filamin-C and its involvement in the regulation of cellular senescence and apoptosis in Huh-7 hepatoma cells during arsenic trioxide exposure

Arsenic trioxide (ATO) is one of the most toxic inorganic arsenic compounds. In this study, we examined the effects of long-term (7 days) exposure to low dose (5 μM) ATO on a human hepatocellular carcinoma cell line, Huh-7. Along with apoptosis accompanied by secondary necrosis though GSDME cleavage, we observed enlarged and flattened cells adhering to the culture dish and surviving even after exposure to ATO. An increase in cyclin-dependent kinase inhibitor p21 levels as well as positive staining for senescence-associated β-galactosidase activity were observed in ATO-treated cells, indicating cellular senescence. Screening for both ATO-inducible proteins by MALDI-TOF-MS analysis and ATO-inducible genes by DNA microarray analysis showed a marked increase in filamin-C (FLNC), an actin cross-linking protein. Interestingly, the increase in FLNC was observed in both dead and surviving cells, suggesting that the upregulation of FLNC by ATO occurs in both apoptotic and senescent cells. Small interference RNA-mediated knock down of FLNC resulted in not only a reduction of senescence-associated enlarged morphology of the cells, but also an exacerbation of cell death. Taken together, these results suggest a regulatory role of FLNC in the execution of senescence as well as apoptosis during ATO exposure.