The Protein Journal最新文献

Considering the limited treatment options for acute lung injury (ALI) and pulmonary fibrosis (PF), ozone treatment may be promising as a new immunological agent with its ability to modulate cytokines and interferons. We aimed to investigate the effects of inhaled ozone therapy on both ALI and PF in rat models. A total of 48 albino Wistar male rats were included in the study. Lipopolysaccharide (LPS) was used to induce the ALI model, and bleomycin was used for the PF model. The effects of inhaled ozone (O₃) were investigated using the ELISA method. Hematoxylin&eosin staining, Masson’s trichrome staining, and immunohistochemical methods were used for histopathological evaluation. The Interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α), and Nuclear Factor kappa B subunit p65 (NF-κB p65) levels in the ALI + 0.08 ppm O₃, ALI + 0.12 ppm O₃, PF + 0.08 ppm O₃, and PF + 0.12 ppm O₃ groups statistically decreased to the same extent and approached the levels of control animals. It was observed that IL-1β, IL-6, TNF-α, and NF-κB p65 levels in lung tissues were significantly and dose-dependently decreased compared to the untreated PF and ALI groups, respectively. While fibrosis was severe in the PF + 0.08 ppm O₃ group, it decreased to more moderate levels in the PF + 0.12 ppm O₃ group. The cytokine levels confirmed that inhaled ozone protected the lungs from both ALI and the development of PF.

Butyrylcholinesterase (BChE; EC 3.1.1.8) is an enzyme found in blood plasma and various tissues, playing a key role in metabolizing esters and detoxifying various substances. In this study, we developed a modified purification protocol for BChE from human serum, achieving a higher purification yield (38.3%) and specific activity (15.09 U/mg protein) compared to previous reports. The method employed a single round of acid dialysis, Sephadex G50 gel filtration chromatography, and procainamide Sepharose 4 fast flow affinity chromatography. Our new approach excludes the commonly used DEAE Trisacryl M chromatography. The goal was to compare this method with our previously employed purification protocols. This study demonstrates that optimizing chromatography steps can enhance enzyme recovery and activity, though further refinement may be needed for higher purification folds. This improved methodology offers a valuable approach for efficient BChE purification with potential for broader applications.

Cocoonase is a naturally secreted protease responsible for facilitating moth emergence from inside of cocoon. This protease is considered as of prime importance for all the cocooning lepidopteron. It specifically degrades sericin, the glue protein of the cocoon without damaging the fibroin and makes an escape hatch for adult emergence. Owing to this property cocoonase was characterized and explored for its prospective utilization in eco-friendly enzyme-based silk degumming. However, the applicability of cocoonase has not been explored much other than in silk degumming. Moreover, being a serine protease, and because of its similarity to trypsin, there is, tremendous potential for this enzyme to have biomedical applications, as well as numerous other uses that need to be investigated. This review article presents the comprehensive physicochemical properties of the cocoonase and its possible scope of applications in the near future.

The Ferguson plot is a simple method for determining the molecular weight of native proteins and their complexes. In this study, we tested the validity of the Ferguson plot based on agarose native gel electrophoresis using multimeric chaperone protein, ClpB, derived from a moderate halophile that forms a native hexamer. The Ferguson plot showed a single band with a molecular weight of 1,500 kDa, approximately twice the size of the native hexamer. This result is consistent with the structure of other chaperons that form a double ring assembly comprising two hexameric units, i.e., a dodecamer. Supporting this, dynamic light scattering experiment showed two peaks, which likely correspond to the hexamer and dodecamer structures.

Trypsin inhibitor from the root-tuber of underutilized legume Winged bean (Psophocarpus tetragonolobus (L.) DC.) (WbT-TI) was purified using ion exchange chromatography followed by size-exclusion chromatography. The purified WbT-TI showed a molecular mass of 20,609 Da and an isoelectric point of 5.10. Ultraviolet circular dichroism (UV-CD) and intrinsic fluorescence reported, that WbT-TI interacts with trypsin. Domain-wise analysis of WbT-TI revealed it to belong to the Kunitz-type soybean trypsin inhibitor (STI) family with a specific β-trefoil fold. The sequence of WbT-TI showed 44% sequence coverage to acidic trypsin inhibitor from the seed of the same plant. Protein interaction similarity analysis (PIPSA) evaluated the electrostatic properties of WbT-TI and provided information about the interacting partners of trypsin inhibitors. The purified protein was quantified and tested for in vitro anticancer activity using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay against the human osteosarcoma (MG-63) cell line. At 5 µg/ml of WbT-TI, the highest inhibition was seen. These studies may lead to the development of winged bean protease inhibitor-based preventive and therapeutic strategies for different kinds of cancers.

Graphical Abstract

Thyroglobulin is a major autoantigen to which autoimmune response, destroying the thyroid gland in Hashimoto’s thyroiditis, is directed. To detect a pathological autoimmune response to thyroglobulin, as well as the successful induction of experimental autoimmune thyroiditis, thyroglobulin carrying thyroiditogenic epitopes is necessary. It is not known which features of thyroglobulin structure determine the presence of thyroiditogenic epitopes and can serve as markers of their presence. We compared structure of thyroglobulin bearing thyroiditogenic epitopes (freshly isolated thyroglobulin) and thyroglobulin which had lost thyroiditogenic epitopes (lyophilized thyroglobulin). Fourier-transform infrared (FTIR) spectroscopy was used to elucidate the structure of thyroglobulin. The markers indicating the presence of thyroiditogenic epitopes on thyroglobulin are the vibrations of diiodotyrosine, monoiodotyrosine/diiodotyrosine relation in the range of 0.24–0.43 (95% confidence interval) and relatively high (> 32%) α-helix content. The loss of thyroiditogenic epitopes on thyroglobulin is associated with a weakening or complete disappearance of diiodotyrosine oscillations and a decrease in the proportion of α-helices in secondary structure. Thyroglobulin extracted with phenylmethylsulfonyl fluoride (PMSF) added is characterized by the same relatively high monoiodotyrosine/diiodotyrosine relation and low proportion of alpha helices as thyroglobulin without thyroiditogenic epitopes. Therefore, serine protease inhibitor PMSF is not suitable for extraction of native thyroglobulin bearing thyroiditogenic epitopes. FTIR spectroscopy can be used to detect thyroiditogenic epitopes on thyroglobulin.

Over the past decades, the incidence of Parkinson’s disease (PD) cases has doubled in industrialized countries. While patients over 70 years old still represent more than half of the cases, the disease is increasingly affecting younger individuals. Environmental factors have been implicated, such as the effects of certain pesticides or chemicals on neurons, such as rotenone or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Researchers have also demonstrated the influence of genetic mutations in younger patients. A-synuclein is a protein encoded by the SNCA gene, known to undergo various mutations in hereditary cases of PD. These mutations alter the composition and spatial arrangements of α-synuclein. The proteins, originally of linear shape, aggregate during the progression of PD, forming fibrillary structures that propagate through brain tissues. Among the physical therapies investigated for treating α-synuclein aggregation, ultrasonic waves, capable of altering protein and cell behaviors, have recently been used to disrupt α-synuclein fibrils within tissues in cellular and animal models, with the hope of developing treatments based on ultrasound properties. However, detecting fibrils typically requires invasive and non-biocompatible chemical compounds or cumbersome machinery. In this study, our acoustic experimental setup allowed us to investigate the response of α-synuclein to ultrasound perturbations. By capturing the transmitted wave across proteins over a frequency range 10 kHz to 10 MHz, no ultrasound signature indicating the presence of proteins was observed.

Significance Statement: The results report there is no ultrasound signature of the presence of α-synuclein fibrils, from 10 kHz to 10 MHz.

Due to the large size and rapid growth of the global therapeutic antibody market, there is major interest in understanding the aggregation of protein products as it can compromise efficacy, concentration, and safety. Various production and storage conditions have been identified as capable of inducing aggregation of polyclonal and monoclonal antibody (mAb) therapies such as low pH, freezing, light exposure, lyophilisation and increased ionic strength. The addition of stabilising excipients to these therapeutics helps to combat the formation of aggregates with future aggregation inhibition mechanisms involving the introduction of point mutations and glycoengineering within aggregation prone regions (APRs). Antibody aggregation also plays an integral role in the pathogenesis of a condition known as amyloid light chain (AL) amyloidosis which is characterised by the production of improperly folded and amyloidogenic immunoglobulin light chains (LCs). Current diagnostic tools rely heavily on histological staining with their future moving towards amyloid component identification and proteomic analysis. For many years, treatment options designed for multiple myeloma (MM) have been applied to AL amyloidosis patients by depleting plasma cell numbers. More recently, treatment strategies more specific to this condition have been developed with many designed to recognize amyloid fibrils and trigger their degradation without causing systemic plasma cell cytotoxicity. Amyloid fibrils in AL disease and aggregates in antibody therapeutics are both formed through the oligomerisation of misfolded / modified proteins attempting to reach a thermodynamically stable, free energy minimum that is lower than the respective monomers themselves. Although the final morphologies are different, by understanding the principles underlying such aggregation, we expect to find common insights that may contribute to the development of new and effective methods of antibody aggregation and/or amyloidosis management. We envision that this area of research will continue to be very relevant in both industry and clinical settings.

Previous studies reported that _D-amino acid oxidase (DAAO) activity was closely associated with neuropathic pain, cognitive characteristics of schizophrenia and so on. To determine DAAO mutant sites in different strains of mice and their effects on enzyme activity, we successfully constructed a prokaryotic expression system for heterologous expression of DAAO in vitro. There were total five nucleotide mutations distributed in exons 2, 8, 9, 10 of C57 mice. Three mutations located on exons 8 and 9 were synonymous mutations and had no variation on the encoded amino acid. The remaining two mutations in exons 2 (V64A) and 10 (R295H) were non-synonymous mutations, which might affect enzymatic activity and protein structure of mDAAO. Based on the determination of the kinetic constants and IC₅₀ of mDAAO mutants in vitro, the differences in amino acid levels at these two sites (V64A, R295H) increased the affinity of C57 DAAO with substrate and enhanced its catalytic efficiency. Besides, the IC₅₀ value of C57 DAAO was less than that of Balb/c and other DAAO mutants (SUN: reducted by about 11.9%; CBIO: reducted by about 26.5%), which meant that the affinity of C57 DAAO with CBIO was higher.