Specific Binding Proteins Research Articles

Integrins in the kidney - beyond the matrix.

The development and proper functioning of the kidney is dependent on the interaction of kidney cells with the surrounding extracellular matrix (ECM). These interactions are mediated by heterodimeric membrane-bound receptors called integrins, which bind to the ECM via their extracellular domain and via their cytoplasmic tail to intracellular adaptor proteins, to assemble large macromolecular adhesion complexes. These interactions enable integrins to control cellular functions such as intracellular signalling and organization of the actin cytoskeleton and are therefore crucial to organ function. The different nephron segments and the collecting duct system have unique morphologies, functions and ECM environments and are thus equipped with unique sets of integrins with distinct specificities for the ECM with which they interact. These cell-type-specific functions are facilitated by specific intracellular integrin binding proteins, which are critical in determining the integrin activation status, ligand-binding affinity and the type of ECM signals that are relayed to the intracellular structures. The spatiotemporal expression of integrins and their specific interactions with binding partners underlie the proper development, function and repair processes of the kidney. This Review summarizes our current understanding of how integrins, their binding partners and the actin cytoskeleton regulate kidney development, physiology and pathology.

Cyclic vinyl sulfones activate NRF2 to protect from oxidative stress-induced programmed necrosis

The NRF2 transcriptional factor is a member of cellular stress response machinery and is activated in response to oxidative stress caused either by cellular homeostasis imbalance or by environmental challenges. NRF2 levels are stringently controlled by rapid and continuous proteasomal degradation. KEAP1 is a specific NRF2 binding protein that acts as a bridge between NRF2 and the E3 ligase Cullin-3. In this study, we examine model cyclic vinyl sulfone derivatives as potential NRF2 activating probes. Previously, we and other authors have found anti-inflammatory properties of these compounds in in vivo models; however, the mechanism of action remained unknown. Here, we show that the naphthohydroquinone derivative LCB1353 efficiently stabilizes NRF2 protein levels and upregulates its target genes. At low 5–10 µM concentrations LCB1353 protects non-small cell lung cancer H1299 cells from ferroptotic death induced by cytotoxic concentrations of RSL3, reducing cell death from 90 % to 5 %. Thus, we suggest that cyclic vinyl sulfones are promising scaffolds for the design of protective molecules for conditions associated with toxic and inflammatory levels of oxidative stress.

Fast-response and stable weak measurement system for protein-antibody specific detection

The probing technique for the specific binding of proteins and antibodies plays a crucial role in the domains of biotechnology, medicine, and pharmacology. The development of rapid and highly sensitive probing technique for specific interactions remains an enduring focus. This paper presents a quantum weak measurement technique for rapid and highly sensitive detection of protein-antibody specific binding, effectively reducing noise and achieving measurement accuracy approaching the standard quantum limit. The speed of our detection technology is determined by the response time of the dual-channel detection, which can reach nanoseconds. The results show that the detection limit can reach 26 ng/ml, and the detection range is 0∼3.44μg/ml. The quantum weak measurement technique developed in this study offers a heuristic approach for protein-antibody interaction detection with broad application prospects.

Post-mortem utility of Neuron Specific Enolase (NSE) and Calcium Binding Protein B (S100B) for differentiating traumatic brain injury from other causes of death.

In forensic pathology, identifying causes of death in traumatic brain injuries (TBIs) devoid of observable signs presents a significant challenge. Post-mortem biochemistry plays a crucial role in forensic medicine, particularly in determining causes of death in TBIs that lack macroscopic or histopathological evidence. This study aimed to evaluate the utility of Neuron Specific Enolase (NSE) and S100 Calcium Binding Protein B (S100B) in post-mortem serum and cerebrospinal fluid (CSF) as markers for TBI. The relationship of these biochemical markers with survival time and post-mortem interval was also studied. The study sample consisted of 63 cases each from the TBI and the Non-TBI (NTBI) group. The NTBI group comprised of deaths due to mechanical asphyxia, myocardial infarction and isolated trunk trauma. While serum S100B and CSF NSE emerged as a promising marker for TBI, CSF S100B failed to differentiate TBI from the other causes of death. The absence of an association between the level of markers and survival time or post-mortem interval in TBIs highlights the limitations of these biomarkers in such contexts. This study underscores the potential of biochemical markers like serum S100B and CSF NSE in identifying TBI deaths, aiding forensic diagnoses where there are evidentiary limitations in traditional methods. Further research exploring additional markers and body fluids could enhance diagnostic precision in forensic neuropathology.

Peanut allergen characterization and allergenicity throughout development.

Peanut allergy (PA) in children is a major concern. There is a need for better biological material for both diagnosis and oral immunotherapy (OIT) treatments. The unique state of seeds at early reproductive stages may affect the allergenicity of storage proteins, and impact clinical diagnostic and OIT protocols. The objective of this study was to evaluate the major allergen content in sequential seed developmental stages and monitor allergenicity via specific IgE binding quantification and skin prick testing. Seeds were collected from peanut plants and sorted into five developmental stages: initial (S1), developing (S2), full-size without coloration (S3), full-size with coloration (S4), and fully mature (S5) seeds. Samples were characterized by RNA-Seq, ELISA, and immunohistochemistry. Lyophilized, ground preparations were used for evaluation of skin test responses in sixty challenge-proven PA children. Gene expression, protein content, and specific IgE binding of allergenic proteins increased throughout seed maturation and development. An expression bias towards the less allergenic A-genome copy of the major allergen Ara h 2 was found in earlier stages, especially in stage S2. Immunohistochemical staining showed that Ara h 2 is more dispersed in the cell and less accumulated within organized bodies at stage S2 versus stage S4. Significant differences were found in mean wheal responses between the commercial peanut extract (equivalent to stage S5) and stages S1 and S2, but not with stage S4, upon skin prick testing in subjects with PA. The observed decrease in peanut-specific IgE binding of immature peanut seeds may be a result not only of decreased amounts of allergenic proteins, but also of profound changes in seed composition and conformation. This may be significant for developing a safer and more effective peanut OIT protocol.

Research Progress on Saccharide Molecule Detection Based on Nanopores.

Saccharides, being one of the fundamental molecules of life, play essential roles in the physiological and pathological functions of cells. However, their intricate structures pose challenges for detection. Nanopore technology, with its high sensitivity and capability for single-molecule-level analysis, has revolutionized the identification and structural analysis of saccharide molecules. This review focuses on recent advancements in nanopore technology for carbohydrate detection, presenting an array of methods that leverage the molecular complexity of saccharides. Biological nanopore techniques utilize specific protein binding or pore modifications to trigger typical resistive pulses, enabling the high-sensitivity detection of monosaccharides and oligosaccharides. In solid-state nanopore sensing, boronic acid modification and pH gating mechanisms are employed for the specific recognition and quantitative analysis of polysaccharides. The integration of artificial intelligence algorithms can further enhance the accuracy and reliability of analyses. Serving as a crucial tool in carbohydrate detection, we foresee significant potential in the application of nanopore technology for the detection of carbohydrate molecules in disease diagnosis, drug screening, and biosensing, fostering innovative progress in related research domains.

Integrated CRISPR/Cas12a with terminal protection strategy for homogeneous protein assay

Developing appropriate protein assay methods is necessary, as protein assays play an important role in molecular diagnosis, drug screening, and biomedical research. This article presents a universal homogeneous method based on the CRISPR/Cas12a system and the terminal protection strategy for protein assays. Initially, a single-stranded DNA (ssDNA) labeled with a small molecule at its 3’-end was designed to respond to the target protein and serve as an activator of Cas12a. In the absence of the target protein, small-molecule-linked ssDNA was effectively cleaved using exonuclease I (Exo I). The lack of the activator resulted in the trans-cleavage inactivity of Cas12a, leading to low fluorescence in the detection solution. However, in the presence of the target protein, the specific binding of the target protein to the small molecules on the activator protected the activator against digestion by Exo I. The remaining intact activator triggered the trans-cleavage activation of Cas12a, producing strong fluorescence. Based on this principle, our method achieved fluorescence enhanced detection of the target protein. After experimental condition optimization, our method achieved highly sensitive and specific protein detection in two models: folate receptor and streptavidin. The excellent performance of the fluorescence method highlights the applicability and effectiveness of the CRISPR/Cas12a system in protein assays, and broadening its application scope.

Rescaling protein-protein interactions improves Martini 3 for flexible proteins in solution

Multidomain proteins with flexible linkers and disordered regions play important roles in many cellular processes, but characterizing their conformational ensembles is difficult. We have previously shown that the coarse-grained model, Martini 3, produces too compact ensembles in solution, that may in part be remedied by strengthening protein–water interactions. Here, we show that decreasing the strength of protein–protein interactions leads to improved agreement with experimental data on a wide set of systems. We show that the ‘symmetry’ between rescaling protein–water and protein–protein interactions breaks down when studying interactions with or within membranes; rescaling protein-protein interactions better preserves the binding specificity of proteins with lipid membranes, whereas rescaling protein-water interactions preserves oligomerization of transmembrane helices. We conclude that decreasing the strength of protein–protein interactions improves the accuracy of Martini 3 for IDPs and multidomain proteins, both in solution and in the presence of a lipid membrane.

Association of R-loop binding proteins with prognosis and anti-tumor drug sensitivity in lung adenocarcinoma: a bioinfor-matic study.

To investigate the association of R-loop binding proteins with prognosis and chemotherapy efficacy in lung adenocarcinoma. The data related to R-loop regulatory genes were obtained from literature of R-loop proteomics and relevant databases. We used 403 cases of lung adenocarcinoma in the Cancer Genome Atlas as training set, and two datasets GSE14814 and GSE31210 in Gene Expression Omnibus as validation sets. The weighted gene co-expression network analysis (WGCNA) was employed to identify R-loop genes with a significant impact on the clinical phenotype of lung adenocarcinoma. Least absolute shrinkage and selection operator (LASSO) regression analysis was utilized to eliminate genes exhibiting multicollinearity. A multivariate Cox regression analysis was employed to scrutinize clinical variables and R-loop characteristic genes that exert independent prognostic effects on patient survival. Subsequently, a risk score model was constructed. The predictive capacity of this model for the prognosis of patients was analyzed and validated. Additionally, the performance of risk model on the anti-tumor drug sensitivity was assessed. The mutations of R-loop genes were analyzed by maftools. The effect of PLEC expression on anti-tumor drug sensitivity was tested on non-small cell lung adenocarcinoma H1299 and A549 cells in vitro. A collection of 1551 R-loop genes were obtained, and 78 genes exhibited significant effects on the clinical phenotype shown on WGCNA. The LASSO regression analysis retained fourteen R-loop genes. A multivariate Cox regression analysis further identified three R-loop genes (HEXIM1, GLI2, PLEC) and a clinical variable (tumor grading) that were associated with patient prognosis. Risk prediction model was established according to the regression coefficients of each parameter. Kaplan-Meier survival analysis showed that the prognosis of high-risk group was significantly worse than that of low-risk group (P<0.01). The time-dependent ROC curve showed that the risk model had good predictive ability in both training and validation sets. Predictive analyses of anti-neoplastic drug sensitivity indicated a diminished responsiveness to both chemotherapy and targeted treatment drugs among high-risk patients. The expression of PLEC was strongly correlated with sensitivity to gefitinib, a classical EGFR inhibitor. R-loop binding proteins have been identified as significant determinants in the prognosis and therapeutic strategies for lung adenocarcinoma, which indicates that therapeutic interventions targeting these specific R-loop binding proteins might contribute to a better survival of the patients.

Dynamic RNA methylation modifications and their regulatory role in mammalian development and diseases.

Among over 170 different types of chemical modifications on RNA nucleobases identified so far, RNA methylation is the major type of epitranscriptomic modifications existing on almost all types of RNAs, and has been demonstrated to participate in the entire process of RNA metabolism, including transcription, pre-mRNA alternative splicing and maturation, mRNA nucleus export, mRNA degradation and stabilization, mRNA translation. Attributing to the development of high-throughput detection technologies and the identification of both dynamic regulators and recognition proteins, mechanisms of RNA methylation modification in regulating the normal development of the organism as well as various disease occurrence and developmental abnormalities upon RNA methylation dysregulation have become increasingly clear. Here, we particularly focus on three types of RNA methylations: N6-methylcytosine (m6A), 5-methylcytosine (m5C), and N7-methyladenosine (m7G). We summarize the elements related to their dynamic installment and removal, specific binding proteins, and the development of high-throughput detection technologies. Then, for a comprehensive understanding of their biological significance, we also overview the latest knowledge on the underlying mechanisms and key roles of these three mRNA methylation modifications in gametogenesis, embryonic development, immune system development, as well as disease and tumor progression.

Species Difference? Bovine, Trout, and Human Plasma Protein Binding of Per- and Polyfluoroalkyl Substances.

Per- and polyfluoroalkyl substances (PFAS) strongly bind to proteins and lipids in blood, which govern their accumulation and distribution in organisms. Understanding the plasma binding mechanism and species differences will facilitate the quantitative in vitro-to-in vivo extrapolation and improve risk assessment of PFAS. We studied the binding mechanism of 16 PFAS to bovine serum albumin (BSA), trout, and human plasma using solid-phase microextraction. Binding of anionic PFAS to BSA and human plasma was found to be highly concentration-dependent, while trout plasma binding was linear for the majority of the tested PFAS. At a molar ratio of PFAS to protein ν < 0.1 molPFAS/molprotein, the specific protein binding of anionic PFAS dominated their human plasma binding. This would be the scenario for physiological conditions (ν < 0.01), whereas in in vitro assays, PFAS are often dosed in excess (ν > 1) and nonspecific binding becomes dominant. BSA was shown to serve as a good surrogate for human plasma. As trout plasma contains more lipids, the nonspecific binding to lipids affected the affinities of PFAS for trout plasma. Mass balance models that are parameterized with the protein-water and lipid-water partitioning constants (chemical characteristics), as well as the protein and lipid contents of the plasma (species characteristics), were successfully used to predict the binding to human and trout plasma.

Molecular Mimicry of Transposable Elements in Plants.

Transposable elements (TEs) are mobile DNA elements that are particularly abundant in the plant genomes. They have long been considered as junk DNA; however, a growing body of evidence suggests that TE insertions promote genetic diversity that is essential for the adaptive evolution of a species. Thus far, studies have mainly investigated the cis-acting regulatory roles of TEs generated by their insertions nearby or within the host genes. However, the trans-acting effects of TE-derived RNA and DNA remained obscure to date. TEs contain various regulatory elements within their sequences that can accommodate the binding of specific RNAs and proteins. Recently, it was suggested that some of these cellular regulators are shared between TEs and the host genes, and the competition for the common host factors underlies the fine-tuned developmental reprogramming. In this review, we will highlight and discuss the latest discoveries on the biological functions of plant TEs, with a particular focus on their competitive binding with specific developmental regulators.

Synthetic Sialosides Terminated with 8-N-Substituted Sialic Acid as Selective Substrates for Sialidases from Bacteria and Influenza Viruses.

Sialosides containing C8-modified sialic acids are challenging synthetic targets but potentially useful probes for diagnostic substrate profiling of sialidases and elucidating the binding specificity of sialic acid-interacting proteins. Here, we demonstrate efficient chemoenzymatic methods for synthesizing para-nitrophenol-tagged α2-3- and α2-6-linked sialyl galactosides containing C8-acetamido, C8-azido, or C8-amino derivatized N-acetylneuraminic acid (Neu5Ac). High-throughput substrate specificity studies showed that the C8-modification of sialic acid significantly changes its recognition by sialidases from humans, various bacteria, and different influenza A and B viruses. Sialosides carrying Neu5Ac with a C8-azido modification were generally well tolerated by all the sialidases we tested, whereas sialosides containing C8-acetamido-modified Neu5Ac were only cleaved by selective bacterial sialidases. In contrast, sialosides with C8-amino-modified Neu5Ac were cleaved by a combination of selective bacterial and influenza A virus sialidases. These results indicate that sialosides terminated with a C8-amino or C8-acetamido-modified sialic acid can be used with other sialosides for diagnostic profiling of disease-causing sialidase-producing pathogens.

Porphysome Engineered With Specific Protein Binding Sites as a Multimodal Theranostic Nanocarrier for Targeted Protein Delivery.

The immobilization of proteins on the surface of carriers is challenging due to the loss of protein structure and function in this process. Here, we report the development of the protein immobilization on the surface of the metallated-porphyrin complex in the porphysome nanocarrier. The conjugated Ni-porphyrin to fatty acid (as a tail) has been synthesized and independently placed at the depth of the bilayer center of Dipalmitoylphosphatidylcholine (DPPC) in which the Ni-porphyrin was at the polar region of the membrane and is thus superficial. This porphysome (DPPC: Ni-porphyrin, 4 : 1 mole ratio) was formed by supramolecular self-assembly with a diameter of 173±7 nm and zeta potential -8.5±3.4 mv, which exhibited no significant toxicity at the experimental concentrations and acceptable cellular uptake on MCF-7 cells. The physicochemical properties and specific protein binding sites of the firefly luciferase as a model protein into the porphysome (1 : 2 mole ratio) show the conjugation efficiency about 80 % and the conformation of protein was completely maintained. Furthermore, bioluminescence assay and SDS-PAGE confirmed the preservation of protein function. The stabilized platform of porphyrin-lipid structure can potentially improve the efficacy of protein functionality for a particular display, shifting porphysomes from a simple carrier to a therapeutic agent.

Protecting the piglet gut microbiota against ETEC-mediated post-weaning diarrhoea using specific binding proteins

Post-weaning diarrhoea (PWD) in piglets presents a widespread problem in industrial pig production and is often caused by enterotoxigenic E. coli (ETEC) strains. Current solutions, such as antibiotics and medicinal zinc oxide, are unsustainable and are increasingly being prohibited, resulting in a dire need for novel solutions. Thus, in this study, we propose and evaluate a protein-based feed additive, comprising two bivalent heavy chain variable domain (VHH) constructs (VHH-(GGGGS)3-VHH, BL1.2 and BL2.2) as an alternative solution to manage PWD. We demonstrate in vitro that these constructs bind to ETEC toxins and fimbriae, whilst they do no affect bacterial growth rate. Furthermore, in a pig study, we show that oral administration of these constructs after ETEC challenge reduced ETEC proliferation when compared to challenged control piglets (1-2 log10 units difference in gene copies and bacterial count/g faeces across day 2–7) and resulted in week 1 enrichment of three bacterial families (Prevotellaceae (estimate: 1.12 ± 0.25, q = 0.0054), Lactobacillaceae (estimate: 2.86 ± 0.52, q = 0.0012), and Ruminococcaceae (estimate: 0.66 ± 0.18, q = 0.049)) within the gut microbiota that appeared later in challenged control piglets, thus pointing to an earlier transition towards a more mature gut microbiota. These data suggest that such VHH constructs may find utility in industrial pig production as a feed additive for tackling ETEC and reducing the risk of PWD in piglet populations.

Molecular Imprinting-Based SERS Detection Strategy for the Large-Size Protein Quantitation and Curbing Non-Specific Recognition.

Molecular imprinting-based surface-enhanced Raman scattering (MI-SERS) sensors have shown remarkable potential from an academic standpoint. However, their practical applications, especially in the detection of large-size protein (≥10 nm), face challenges due to the lack of versatile sensing strategies and nonspecific fouling of matrix species. Herein, we propose a Raman reporter inspector mechanism (RRIM) implemented on a protein-imprinted polydopamine (PDA) layer coated on the SERS active substrate. In the RRIM, after large-size protein recognition, the permeability of the PDA imprinted cavities undergoes changes that are scrutinized by Raman reporter molecules. Target proteins can specifically bind and fully occupy the imprinted cavities, whereas matrix species cannot. Then, Raman reporter molecules with suitable size are introduced to serve as both inspectors of the recognition status and inducers of the SERS signal, which can only penetrate through the vacant and nonspecifically filled cavities. Consequently, changes in the SERS signal exclusively originate from the specific binding of target proteins, while the nonspecific recognition of matrix species is curbed. The RRIM enables reproducible quantitation of the large-size cyanobacteria-specific protein model (≥10 nm), phycocyanin, at the level down to 2.6 × 10-3 μg L-1. Finally, the practical applicability of the RRIM is confirmed by accurately analyzing crude urban waterway samples over 21 min without any pretreatment.

Species-Specific Unbound Fraction Differences in Highly Bound PFAS: A Comparative Study across Human, Rat, and Mouse Plasma and Albumin.

Per- and polyfluoroalkyl substances (PFAS) are a diverse group of fluorinated compounds which have yet to undergo comprehensive investigation regarding potential adverse health effects and bioaccumulative properties. With long half-lives and accumulative properties, PFAS have been linked to several toxic effects in both non-clinical species such as rat and mouse as well as human. Although biological impacts and specific protein binding of PFAS have been examined, there is no study focusing on the species-specific fraction unbound (fu) in plasma and related toxicokinetics. Herein, a presaturation equilibrium dialysis method was used to measure and validate the binding of 14 individual PFAS with carbon chains containing 4 to 12 perfluorinated carbon atoms and several functional head-groups to albumin and plasma of mouse (C57BL/6 and CD-1), rat, and human. Equivalence testing between each species-matrix combination showed positive correlation between rat and human when comparing fu in plasma and binding to albumin. Similar trends in binding were also observed for mouse plasma and albumin. Relatively high Spearman correlations for all combinations indicate high concordance of PFAS binding regardless of matrix. Physiochemical properties of PFAS such as molecular weight, chain length, and lipophilicity were found to have important roles in plasma protein binding of PFAS.

Principles, functions, and biological implications of m6A in plants.

Over the past decade, N 6-methyladenosine (m6A) has emerged as a prevalent and dynamically regulated modification across the transcriptome; it has been reversibly installed, removed, and interpreted by specific binding proteins, and has played crucial roles in molecular and biological processes. Within this scope, we consolidate recent advancements of m6A research in plants regarding gene expression regulation, diverse physiologic and pathogenic processes, as well as crop trial implications, to guide discussions on challenges associated with and leveraging epitranscriptome editing for crop improvement.

In vitro and in silico studies of enterobactin-inspired Ciprofloxacin and Fosfomycin first generation conjugates on the antibiotic resistant E. coli OQ866153

BackgroundThe emergence of antimicrobial resistance in bacterial pathogens is a growing concern worldwide due to its impact on the treatment of bacterial infections. The "Trojan Horse" strategy has been proposed as a potential solution to overcome drug resistance caused by permeability issues.ObjectiveThe objective of our research was to investigate the bactericidal activity and mechanism of action of the "Trojan Horse" strategy using enterobactin conjugated with Ciprofloxacin and Fosfomycin against the antibiotic-resistant Escherichia coli strain OQ866153.MethodologyEnterobactin, a mixed ligand of E. coli OQ866153, was conjugated with Ciprofloxacin and Fosfomycin individually to aid active absorption via specific enterobactin binding proteins (FepABCDG). The effectiveness of the conjugates was assessed by measuring their bactericidal activity against E. coli OQ866153, as well as their ability to inhibit DNA gyrase enzyme and biofilm formation.ResultsThe Fe+3-enterobactin-Ciprofloxacin conjugate effectively inhibited the DNA gyrase enzyme (Docking score = -8.597 kcal/mol) and resulted in a lower concentration (25 μg/ml) required to eliminate supercoiled DNA plasmids compared to the parent drug (35 μg/ml; Docking score = -6.264 kcal/mol). The Fe+3-Enterobactin-Fosfomycin conjugate showed a higher inhibition percentage (100%) of biofilm formation compared to Fosfomycin (21.58%) at a concentration of 2 mg/ml, with docking scores of -5.481 and -3.756 kcal/mol against UDP-N acetylglucosamine 1-carboxyvinyltransferase MurA.ConclusionThe findings of this study suggest that the "Trojan Horse" strategy using enterobactin conjugated with Ciprofloxacin and Fosfomycin can effectively overcome permeability issues caused by efflux proteins and enhance the bactericidal activity of these drugs against antibiotic-resistant strains of E. coli.

Binding affinity and specificity of opacity-associated proteins and human carcinoembryonic antigen-related cell adhesion molecules

Binding affinity and specificity of opacity-associated proteins and human carcinoembryonic antigen-related cell adhesion molecules