Protein Cross-linking Research Articles

The prognostic value of bioinformatics analysis of ECM receptor signaling pathways and LAMB1 identification as a promising prognostic biomarker of lung adenocarcinoma.

The extracellular matrix (ECM) is a complex and dynamic network of cross-linked proteins and a fundamental building block in multicellular organisms. Our study investigates the impact of genes related to the ECM receptor interaction pathway on immune-targeted therapy and lung adenocarcinoma (LUAD) prognosis. This study obtained LUAD chip data (GSE68465, GSE31210, and GSE116959) from NCBI GEO. Moreover, the gene data associated with the ECM receptor interaction pathway was downloaded from the Molecular Signature Database. Differentially expressed genes were identified using GEO2R, followed by analyzing their correlation with immune cell infiltration. Univariate Cox regression analysis screened out ECM-related genes significantly related to the survival prognosis of LUAD patients. Additionally, Lasso regression and multivariate Cox regression analysis helped construct a prognostic model. Patients were stratified by risk score and survival analyses. The prognostic models were evaluated using receiver operating characteristic curves, and risk scores and prognosis associations were analyzed using univariate and multivariate Cox regression analyses. A core gene was selected for gene set enrichment analysis and CIBERSORT analysis to determine its function and tumor-infiltrating immune cell proportion, respectively. The results revealed that the most abundant pathways among differentially expressed genes in LUAD primarily involved the cell cycle, ECM receptor interaction, protein digestion and absorption, p53 signaling pathway, complement and coagulation cascade, and tyrosine metabolism. Two ECM-associated subtypes were identified by consensus clustering. Besides, an ECM-related prognostic model was validated to predict LUAD survival, and it was associated with the tumor immune microenvironment. Additional cross-analysis screened laminin subunit beta 1 (LAMB1) for further research. The survival time of LUAD patients with elevated LAMB1 expression was longer than those with low LAMB1 expression. Gene set enrichment analysis and CIBERSORT analyses revealed that LAMB1 expression correlated with tumor immune microenvironment. In conclusion, a prognostic model of LUAD patients depending on the ECM receptor interaction pathway was constructed. Screening out LAMB1 can become a prognostic risk factor for LUAD patients or a potential target during LUAD treatment.

Evaluation of the water state and protein characteristics of Tibetan pork under the storage conditions of modified atmosphere packaging: Effect of oxygen concentration

To explore the changes in water status and protein characteristics of Tibetan pork (TP) under modified atmosphere packaging (MAP) with different oxygen concentrations compared to Duroc×Landrace×Yorkshire pork (DLY), the water holding capacity (WHC), water distribution, protein oxidation, and conformation of both types were determined. Results indicate that under MAP, TP pork and DLY pork exhibited higher water retention and lower protein oxidation compared to air packaging. However, with increased oxygen concentration in the MAP, protein oxidation intensified, leading to reduced WHC in the pork. Compared to DLY pork, TP pork in different packaging conditions maintained the integrity of protein secondary and tertiary structures, reducing protein cross-linking aggregation. The lower content of P3 in the two-dimensional relaxation spectra, shorter T1 and T2 relaxation times, and higher proton density suggest better water retention properties in Tibetan pork. These findings support the development of long-distance preservation and transportation technologies for TP pork.

Enhanced tofu quality through calcium sulfate and transglutaminase treatment: physicochemical properties and digestibility analysis

SummaryTofu, a traditional soy‐based food, is widely consumed globally and serves as a significant source of plant protein. The quality of tofu can vary greatly depending on the coagulation process and the type of coagulants used. This study investigated the impact of a compound coagulant, consisting of transglutaminase (TGase) and calcium sulfate (CaSO4), on tofu quality, specifically focusing on pH, yield, water holding capacity (WHC), cooking loss, protein content, and in vitro digestibility. Soymilk was pre‐crosslinked with TGase for different durations (0, 1, 2, and 3 h) before the addition of CaSO4. Results indicated that TGase pre‐treatment significantly improved tofu yield and WHC, with optimal outcomes at 3 and 2 h, respectively. Cooking loss decreased with TGase treatment but increased at the 3‐h mark, suggesting over‐crosslinking. Electrophoresis revealed substantial protein crosslinking with TGase treatment, while in vitro digestion showed enhanced protein digestibility and higher peptide content in TGase‐treated samples. These findings underscore the potential of TGase treatment to enhance tofu's structural integrity and digestibility, offering valuable insights for optimising plant‐based protein processing to improve nutritional quality.

Effect of transglutaminase on the structure, properties and oil absorption of wheat flour

The structure, properties, as well as the oil absorption characteristics of wheat flour (WF) treated with varying concentrations of transglutaminase (TG) (0 U/g ∼ 50 U/g) were characterized. The content of free amino groups in WF modified by TG (TG-WF) decreased and protein aggregated. The isopeptide bonds and disulfide bonds played important roles in protein crosslinking. The thermal stability, the peak viscosity after gelatinization and protein secondary structure stability of TG-WF were improved. In addition, the oil absorption and surface oil content of TG-WF after frying were reduced. TG enhanced the protein-protein interactions in WF, so that protein played barrier roles in the process of high-temperature frying, protecting the starch particles covered by them from the infiltration of oil, thus reducing the oil absorption of TG-WF during frying. Among them, the oil content of TG-WF-30 U/g after frying was the lowest, which decreased by 10.73 % compared with the control group.

Insight into protein cross-linking and casein polymerization in pre- and post-hydrolyzed lactose-free UHT milk during long-term storage

During processing and storage of both conventional and lactose-hydrolyzed UHT milk (LHM), aggregation of milk proteins occurs. Protein aggregation can inter alia occur via non-reducible covalent cross-links derived from either Maillard or dehydroalanine (DHA) pathways. To study this further in relation to processing method and lactase enzyme purity, LHM was produced using 3 different lactase preparations, with lactase enzymes added in a dairy setting either before (pre-hydrolysis) or after (post-hydrolysis) UHT treatment. The prepared LHM types were subsequently stored at either 25°C or 35°C for up to one year. Mass spectrometry was used to absolutely quantify the level of furosine, N-ɛ-(carboxymethyl)lysine (CML) and N-ɛ-(carboxyethyl)lysine (CEL), lanthionine (LAN) and lysinoalanine (LAL) in these products using a newly developed method on Triple Q for these processing-induced markers. The results showed higher levels of Maillard related processing markers in pre-hydrolyzed LHM compared with post-hydrolyzed LHM and conventional UHT milk which, on the other hand, contained higher concentrations of DHA-derived cross-links. Proteomics of collected particles from asymmetrical flow field-flow fractionation (AsFlFFF) in combination with gel electrophoresis indicated presence of intra-micellar cross-links during storage, for especially larger particles involving αS1- and αS2-caseins.

Targeting PYK2 with heterobifunctional T6BP helps mitigate MASLD and MASH-HCC progression

Background & AimsThe mechanisms underlying the regulation of hepatocyte non-receptor tyrosine kinases in metabolic dysfunction-associated steatohepatitis (MASH) remain largely unclear. MethodsHepatocyte-specific overexpression or deletion and anti-protein tyrosine kinase 2 beta (PYK2) or anti-TRAF6-binding protein (T6BP) crosslinking were utilised to study fatty liver protection by T6BP. P-PTC, a peptide-proteolysis targeting chimaera, degrades PYK2 to block MASH progression. ResultsSince PYK2 activation is promoter signalling in steatohepatitis development, we find that T6BP is a novel and critical suppressor of PYK2 that reduces hepatic lipid accumulation, pro-inflammatory factor release, and pro-fibrosis production by ubiquitin ligase CBL to degrade PYK2. Mechanistic evidence suggests that T6BP directly targets PYK2 and prevents its N-terminal FERM domain-triggered dimerization, disrupting downstream PYK2-JNK signalling hyperactivation. Additionally, T6BP favourably recruits CBL, a particular E3 ubiquitin ligase targeting PYK2, to form a complex and degrade PYK2. T6BP (F1), a core fragment of T6BP, directly blocks N-terminal FERM domain-associated dimerization of PYK2, followed by T6BP-recruiting CBL-mediated PYK2 degradation in a typical T6BP-dependent manner when the tiny fragment is specifically expressed using thyroxine binding globulin (TBG)-ground vectors. This inhibits the progression of MASH, metabolic dysfunction-associated steatotic liver disease (MASLD)-related HCC (MASH-HCC), and metabolic syndrome in dietary rodent models. First-ever peptide-proteolysis targeting chimaera (P-PTC) based on the core segment of T6BP as a ligand for targeted recruitment of CBL targeting metabolic disorders like MASH has been devised and validated in animal models. ConclusionsOur study revealed a previously unknown mechanism: identification of T6BP as a key eliminator of fatty liver strongly contributes to the development of promising therapeutic targets, and the discovery of crucial fragments of T6BP-based pharmacon that interrupt PYK2 dimerization are novel and viable treatments for fatty liver and its advanced symptoms and complications. Impact and implicationsExcessive high-energy diet ingestion is critical in driving steatohepatitis via regulation of hepatocyte non-receptor tyrosine kinases. The mechanisms under lying the regulation of hepatocyte PYK2 in metabolic dysfunction-associated steatohepatitis (MASH) remain largely unclear. Here, we found that T6BP as a critical fatty liver eliminator has a significant impact on the development of promising therapeutic targets. Additionally, vital T6BP-based pharmacon fragments that impede PYK2 dimerization have been found, offering new and effective treatments for advanced fatty liver symptoms and complications.

Singlet oxygen-mediated photochemical cross-linking of an engineered fluorescent flavoprotein iLOV

Genetically-encoded photoactive proteins are integral tools in modern biochemical and molecular biological research. Within this tool box, truncated variants of the phototropin 2 light-oxygen-voltage (LOV) flavoprotein have been developed to photochemically generate singlet oxygen (1O2) in vitro and in vivo, yet the effect of 1O2 on these genetically encoded photosensitizers remains underexplored. In this study, we demonstrate that the "improved" LOV (iLOV) flavoprotein is capable of photochemical 1O2 generation. Once generated, 1O2 induces protein oligomerization via covalent cross-linking. The molecular targets of protein oligomerization by cross-linking are not endogenous tryptophans or tyrosines, but rather primarily histidines. Substitution of surface-exposed histidines for serine or glycine residues effectively eliminates protein cross-linking. When used in biochemical applications, such protein-protein cross-links may interfere with native biological responses to 1O2, which can be ameliorated by substitution of the surface exposed histidines of iLOV or other 1O2-generating flavoproteins.

Ouroboros: cross-linking protein expression perturbations and cancer histology imaging with generative-predictive modeling.

Imagine if we could simultaneously predict spatial protein expression in tissues from their routine Hematoxylin and Eosin (H&E) stained images, and create tissue images given protein expression profiles thus enabling virtual simulations of how protein expression alterations impact histology in complex diseases like cancer. Such an approach could lead to more informed diagnostic and therapeutic decisions for precision medicine at lower costs and shorter turnaround times, more detailed insights into underlying disease pathology as well as improvement in predictive and generative performance. In this study, we investigate the intricate correlation between protein expressions obtained from Hyperion mass cytometry and histopathological microstructures in conventional H&E stained glioblastoma (GBM) samples, unveiling morphological patterns and cellular-level spatial alterations associated with protein expression changes. To model these complex relationships, we propose a novel generative-predictive framework called Ouroboros for producing H&E images from protein expressions and simultaneously predicting protein expressions from H&E images. Our comprehensive sample-independent validation over 9920 tissue spots from 4 GBM samples encompassing visual image analysis, quantitative analysis, subspace alignment and perturbation experiments shows that the proposed generative-predictive approach offers significant improvements in predicting protein expression from images in comparison to baseline methods as well as accurate generation of virtual GBM sample images. This proof of concept study can contribute to advancing our understanding of histological responses to protein expression perturbations and lays the foundations for further developments in this area. Implementation and associated data for the proposed approach are available at the URL: https://github.com/Srijay/Ouroboros.

Reducing Filamin A Restores Cortical Synaptic Connectivity and Early Social Communication Following Cellular Mosaicism in Autism Spectrum Disorder Pathways.

Communication in the form of nonverbal, social vocalization, or crying is evolutionary conserved in mammals and is impaired early in human infants that are later diagnosed with autism spectrum disorder (ASD). Defects in infant vocalization have been proposed as an early sign of ASD that may exacerbate ASD development. However, the neural mechanisms associated with early communicative deficits in ASD are not known. Here, we expressed a constitutively active mutant of Rheb (RhebS16H), which is known to upregulate two ASD core pathways, mTOR complex 1 (mTORC1) and ERK1/2, in Layer (L) 2/3 pyramidal neurons of the neocortex of mice of either sex. We found that cellular mosaic expression of RhebS16H in L2/3 pyramidal neurons altered the production of isolation calls from neonatal mice. This was accompanied by an expected misplacement of neurons and dendrite overgrowth, along with an unexpected increase in spine density and length, which was associated with increased excitatory synaptic activity. This contrasted with the known decrease in spine density in RhebS16H neurons of 1-month-old mice. Reducing the levels of the actin cross-linking and adaptor protein filamin A (FLNA), known to be increased downstream of ERK1/2, attenuated dendrite overgrowth and fully restored spine properties, synaptic connectivity, and the production of pup isolation calls. These findings suggest that upper-layer cortical pyramidal neurons contribute to communicative deficits in a condition known to affect two core ASD pathways and that these mechanisms are regulated by FLNA.

From the TOP: Formation, recognition and resolution of topoisomerase DNA protein crosslinks

Since the report of “DNA untwisting” activity in 1972, ∼50 years of research has revealed seven topoisomerases in humans (TOP1, TOP1mt, TOP2α, TOP2β, TOP3α, TOP3β and Spo11). These conserved regulators of DNA topology catalyze controlled breakage to the DNA backbone to relieve the torsional stress that accumulates during essential DNA transactions including DNA replication, transcription, and DNA repair. Each topoisomerase-catalyzed reaction involves the formation of a topoisomerase cleavage complex (TOPcc), a covalent protein-DNA reaction intermediate formed between the DNA phosphodiester backbone and a topoisomerase catalytic tyrosine residue. A variety of perturbations to topoisomerase reaction cycles can trigger failure of the enzyme to re-ligate the broken DNA strand(s), thereby generating topoisomerase DNA-protein crosslinks (TOP-DPC). TOP-DPCs pose unique threats to genomic integrity. These complex lesions are comprised of structurally diverse protein components covalently linked to genomic DNA, which are bulky DNA adducts that can directly impact progression of the transcription and DNA replication apparatus. A variety of genome maintenance pathways have evolved to recognize and resolve TOP-DPCs. Eukaryotic cells harbor tyrosyl DNA phosphodiesterases (TDPs) that directly reverse 3′-phosphotyrosyl (TDP1) and 5′-phoshotyrosyl (TDP2) protein-DNA linkages. The broad specificity Mre11-Rad50-Nbs1 and APE2 nucleases are also critical for mitigating topoisomerase-generated DNA damage. These DNA-protein crosslink metabolizing enzymes are further enabled by proteolytic degradation, with the proteasome, Spartan, GCNA, Ddi2, and FAM111A proteases implicated thus far. Strategies to target, unfold, and degrade the protein component of TOP-DPCs have evolved as well. Here we survey mechanisms for addressing Topoisomerase 1 (TOP1) and Topoisomerase 2 (TOP2) DPCs, highlighting systems for which molecular structure information has illuminated function of these critical DNA damage response pathways.

The Application of Lophatherum Gracile Brongn Flavonoids in Wheat Flour Products: Effects on the Structural and Functional Characteristics of Wheat Dough.

The effects of lophatherum gracile brongn flavonoids on the multiscale structure and functional properties of wheat dough were investigated. Wheat dough samples with varying contents of lophatherum gracile brongn flavonoids were analyzed to assess changes in thermal-mechanical rheological properties, microstructure, chemical interactions, water distribution, and macropolymer formation by Mixolab mixer, fluorescence microscopy, and low-field nuclear magnetic resonance (LF-NMR). The findings revealed that lophatherum gracile brongn flavonoids disrupted the three-dimensional network of gluten proteins in the wheat dough, leading to decreased water-binding capacity and reduced gluten protein crosslinking while enhancing thermal stability and inhibiting the starch retrogradation of the dough. This study provided important insights into the interaction mechanisms between lophatherum gracile brongn flavonoids and the proteins/starch in wheat dough, offering theoretical guidance for the development of novel wheat-based products for industrialization and practical production.

Nuclear α-actinin-4 regulates breast cancer invasiveness and EMT.

Epithelial-to-mesenchymal transition (EMT) is a key process where cells lose their adhesion properties and augment their invasive properties. α-Actinin4 (ACTN4) is an actin crosslinking protein that responds to mechanical stimuli and is found to be elevated in breast cancer patients. While ACTN4 has been implicated in regulating cancer invasiveness by modulating cytoskeletal organization, its nuclear functions remain much less explored. Here we address this question by first establishing a correlation between nuclear localization and invasiveness in breast cancer cells. Using cancer databases, we then establish a correlation between ACTN4 expression and EMT in breast cancer. Interestingly, TGFβ-induced EMT induction in MCF10A normal mammary epithelial cells leads to increased ACTN4 expression and nuclear enrichment. We then show that ACTN4 knockdown in MDA-MB-231 breast cancer cells, which harbor sizeable fraction of nuclear ACTN4, leads to reduced invasiveness and loss of mesenchymal traits. Similar behavior was observed in knockdown cells expressing K255E ACTN4, which is primarily localized to the cytosol. Together, our findings establish a role for nuclear ACTN4 in regulating invasiveness via modulation of EMT.

Local photo-crosslinking of native tissue matrix regulates cell function.

Within most tissues, the extracellular microenvironment provides mechanical cues that guide cell fate and function. Changes in the extracellular matrix such as aberrant deposition, densification and increased crosslinking are hallmarks of late-stage fibrotic diseases that often lead to organ dysfunction. Biomaterials have been widely used to mimic the mechanical properties of the fibrotic matrix and study cell function. However, the initiation of fibrosis has largely been overlooked, due to the challenges in recapitulating early fibrotic lesions within the native extracellular microenvironment. Using visible light mediated photochemistry, we induced local crosslinking and stiffening of extracellular matrix proteins within ex vivo murine and human tissue. In ex vivo lung tissue of epithelial cell lineage-traced mice, local matrix crosslinking mimicked early fibrotic lesions that increased alveolar epithelial cell spreading, differentiation and extracellular matrix remodeling. However, inhibition of cytoskeletal tension or integrin engagement reduced epithelial cell spreading and differentiation, resulting in alveolar epithelial cell dedifferentiation and reduced extracellular matrix deposition. Our findings emphasize the role of local extracellular matrix crosslinking and remodeling in early-stage tissue fibrosis and have implications for ex vivo disease modeling and applications to other tissues.

Abstract Mo047: DNA METHYLATION AND TRANSCRIPTIONAL MARKERS OF MYOCARDIAL REVERSE REMODELING IN HF PATIENTS ON LVAD SUPPORT

Heart failure (HF) involves global genome modifications. Adverse structural and function remodeling that occurs in HF was initially considered to be irreversible. However, ~15% of HF patients on left ventricular assist device (LVAD) support show significant structural and functional reverse remodeling, distinguishing them as responders (R), while ~85% are non-responders (NR). DNA methylation and transcriptional changes that exert myocardial recovery is not well understood. To address this, we performed whole-genome bisulphite sequencing and RNA sequencing on paired myocardial biopsies from HF patients (n=84) before (pre-LVAD) and after LVAD unloading (post-LVAD). Post-LVAD samples showed activation of pathways including TGFβ signaling, PTEN and Fatty acid oxidation compared to pre. Integrating DNA methylation and transcriptomics data revealed hypomethylation (upregulation) of genes including Scaper, Frat1 and Magi1 and hypermethylation (downregulation) of Tpp3 and Icam. Overall, suggesting a unique ECM-specific stabilization pathway post-LVAD unloading. Segregating samples based on their response to LVAD unloading, pathways such as inflammation and fibrotic signaling were activated in postNR compared to preNR. Hypomethylation of Magi1 and hypermethylation of Tppp3 was observed in postNR suggesting cross-linking proteins and destabilization of microtubule formation as potential mechanisms of functional regression in NR. Conversly, postR showed activation of deubiquitination, oxidative phosphorylation and NLR signaling compared to preR. Finally, we observed integrin signaling, syndecan interaction, collagen degradation and wound healing pathways were activated in postR compared to postNR. Hypomethylation of actin cytoskeleton ( Myl12a ), microtubule/motor proteins ( Kif5b ) and tropomyosin ( Tpm3 ) were observed in postR, suggesting a plausible reverse remodeling mechanism mediated by ECM degradation and stabilization of myocardium in these patients. Western blot analysis also showed a trend towards an increase in collagen in postNR compared to postR (n=4, p=0.06). Finally, a mouse model of HF and recovery was used and pathways including ECM degradation and cardioprotective signaling were activated in mice that recovered from HF compared to mice that did not. Overall, our data suggests a unique ECM regulation pathway to contribute towards myocardial reverse remodeling in HF patients.

Inhibition of MARCKS phosphorylation attenuates of dendritic cell migration in a murine model of acute asthma

BackgroundMARCKS (myristoylated alanine-rich C kinase substrates) serves as a substrate for protein kinase C, residing in the plasma membrane while acts as an actin filament crosslinking protein. This investigation aims to elucidate phosphorylated MARCKS (p-MARCKS) levels and activity in allergic asthma patients and explore the therapeutic potential of peptide inhibitors targeting p-MARCKS in an acute mouse model of allergic asthma. MethodsImmunohistochemistry and histology staining were employed on lung tissue slides to evaluate p-MARCKS expression and allergic asthma symptoms. Airway resistance was measured using invasive whole-body plethysmography. Flow cytometry detected lung dendritic cell migration, and migration/maturation assays were conducted on isolated murine bone marrow-derived dendritic cells (BM-DCs). ResultsElevated p-MARCKS expression was observed in both human asthmatic tissues and animal models immunized with ovalbumin or Alternaria alternata. Remarkably, asthmatic individuals showed elevated high p-MARCKS expression in lung tissues. Intraperitoneal injection of the peptide MPS, targeting the MARCKS phosphorylation site domain, before allergen challenged, effectively suppressed MARCKS phosphorylation in murine lung tissues. MPS inhibited both in vivo and in vitro migration and maturation of dendritic cells (BM-DCs) and reduced Th2-related lymphocyte activation in bronchoalveolar lavage fluid (BALF). MPS pretreatment additionally suppressed all symptoms associated with allergic airway asthma, including a reduction in inflammatory cell influx, airway mucous cell metaplasia, and airway hyperreactivity. ConclusionThese findings suggest that phosphorylated MARCKS occurs in asthmatic lung tissue, and the inhibition of MARCKS phosphorylation by the MPS peptide reduces dendritic cell migration and Th2-related lymphocytes in the lungs in a murine model of acute asthma.

Intravenous injection of nattokinase-heparin electrostatic complex improves the therapeutic effect of advanced tumors by dissolving cancer-related thrombosis

AimsCancer-related thrombosis (CAT) is a common complication in cancer patients, significantly impacting their quality of life and survival prospects. Nattokinase (NK) has potent thrombolytic properties, however, its efficacy is limited by low oral bioavailability and the risk of severe allergic reactions with intravenous use. Heparin (HP) is a widely used anticoagulant in clinical settings. This study aimed to overcome the intravenous toxicity of NK and explore its effect on CAT in advanced tumors. Main methodsIn this study, NK-HP electrostatic complexes were constructed, and their safety and thrombolytic efficacy were verified through guinea pig allergy tests, mouse tail vein tests, and both in vivo and in vitro thrombolysis experiments. Additionally, an S180 advanced tumor model was developed and combined with sialic acid-modified doxorubicin liposomes (DOX-SAL) to investigate the impact of NK-HP on CAT and its antitumor effects in advanced tumors. Key findingsWe observed that NK-HP can eliminate the intravenous injection toxicity of NK, has strong thrombolytic performance, and can prevent thrombosis formation. Intravenous injection of NK-HP can enhance the antitumor effect of DOX-SAL by reducing the fibrin content in advanced tumors and increasing the levels of the cross-linked protein degradation product D-dimer. SignificanceThis study developed a method to eliminate the intravenous injection toxicity of NK, proposing a promising therapeutic strategy for CAT treatment, particularly for CAT in advanced tumors, and improving the efficacy of nano-formulations in anti-tumor therapy.

Single-electron transfer between sulfonium and tryptophan enables site-selective photo crosslinking of methyllysine reader proteins.

The identification of readers, an important class of proteins that recognize modified residues at specific sites, is essential to uncover the biological roles of post-translational modifications. Photoreactive crosslinkers are powerful tools for investigating readers. However, existing methods usually employ synthetically challenging photoreactive warheads, and their high-energy intermediates generated upon irradiation, such as nitrene and carbene, may cause substantial non-specific crosslinking. Here we report dimethylsulfonium as a methyllysine mimic that binds to specific readers and subsequently crosslinks to a conserved tryptophan inside the binding pocket through single-electron transfer under ultraviolet irradiation. The crosslinking relies on a protein-templated σ-π electron donor-acceptor interaction between sulfonium and indole, ensuring excellent site selectivity for tryptophan in the active site and orthogonality to other methyllysine readers. This method could escalate the discovery of methyllysine readers from complex cell samples. Furthermore, this photo crosslinking strategy could be extended to develop other types of microenvironment-dependent conjugations to site-specific tryptophan.

Polyaniline-based bovine serum albumin imprinted electrochemical sensor for ultra-trace-level detection in clinical and food safety applications

Monitoring bovine serum albumin (BSA) at ultra-low levels is crucial for clinical and food safety applications, as it plays a significant role in identifying various health conditions and potential risks, necessitating fast, trace-level detection of BSA. This study proposes an approach to address these challenges by employing molecularly imprinted polymer (MIP) to develop an ultra-trace-level and cost-effective BSA sensing platform. The MIP electrochemical sensor was developed using polyaniline (PANI) combined with the protein crosslinker glutaraldehyde (GA) to optimize BSA surface imprinting in the MIP. As a result, the sensor achieves a sensitivity of 1.24 μA/log(pg/mL), with a picomolar detectable limit of 2.3 pg/mL (0.035 pM) and a wide detection range from 20 pg/mL to 200,000 pg/mL (0.303 pM to 3030 pM), making it suitable for clinical and food safety applications. Additionally, the study explores the interaction between an acidic surfactant protein eluent (acetic acid with sodium dodecyl sulfate, AcOH-SDS) and BSA vacant sites, enhancing recognition and re-binding. The PANI-based MIP sensor demonstrates initial feasibility and practicality in commercial milk and real human serum, opening avenues for early disease detection and ensuring food safety in BSA-related immune responses.

The Structural Logic of Dynamic Signaling in the Escherichia coli Serine Chemoreceptor.

The experimental challenges posed by integral membrane proteins hinder molecular understanding of transmembrane signaling mechanisms. Here, we exploited protein crosslinking assays in living cells to follow conformational and dynamic stimulus signals in Tsr, the Escherichia coli serine chemoreceptor. Tsr mediates serine chemotaxis by integrating transmembrane serine-binding inputs with adaptational modifications of a methylation helix bundle to regulate a signaling kinase at the cytoplasmic tip of the receptor molecule. We created a series of cysteine replacements at Tsr residues adjacent to hydrophobic packing faces of the bundle helices and crosslinked them with a cell-permeable, bifunctional thiol-reagent. We identified an extensively crosslinked dynamic junction midway through the methylation helix bundle that seemed uniquely poised to respond to serine signals. We explored its role in mediating signaling shifts between different packing arrangements of the bundle helices by measuring crosslinking in receptor molecules with apposed pairs of cysteine reporters in each subunit and assessing their signaling behaviors with an in vivo kinase assay. In the absence of serine, the bundle helices evinced compact kinase-ON packing arrangements; in the presence of serine, the dynamic junction destabilized adjacent bundle segments and shifted the bundle to an expanded, less stable kinase-OFF helix-packing arrangement. An AlphaFold 3 model of kinase-active Tsr showed a prominent bulge and kink at the dynamic junction that might antagonize stable structure at the receptor tip. Serine stimuli probably inhibit kinase activity by shifting the bundle to a less stably-packed conformation that relaxes structural strain at the receptor tip, thereby freezing kinase activity.

Effects of sodium tripolyphosphate on the quality and digestion properties of PSE pork

This study aimed to examine the impact of sodium tripolyphosphate (STPP) on the quality and digestive characteristics of PSE pork. The results showed a notable decrease in cooking loss of PSE pork from 29.11% to 25.67% with increasing STPP concentration (P < 0.05). Additionally, the gastric digestibility of PSE pork decreased significantly from 52.01% to 45.81% (P < 0.05). The particle size of digesta decreased significantly after gastrointestinal digestion (P < 0.05). These changes were primarily due to the enhanced cross-linking of proteins through ionic interactions, hydrogen bonds and hydrophobic interactions, and resulted in the embedding of hydrophobic groups and endogenous fluorophores. Furthermore, denser network was formed. These findings give a new insight into considering the impact of STPP on meat nutrition when used to enhance texture and water holding capacity.