“Click Peptide“: pH‐Triggered in Situ Production and Aggregation of Monomer Aβ1–42

Into neutral : We demonstrate the unique features of a pH click peptide based on an O‐acyl isopeptide method. Under acidic conditions, the click peptide remains in a monomeric form. Upon increase of the pH to 7.4, the click peptide is quickly able to convert into Aβ1–42 through an O‐to‐N intramolecular acyl migration. Further study using this pH click peptide would elucidate the pathological role of Aβ1–42 in Alzheimer's disease.

     

TRANSGLUTAMINASE IN DAIRY PRODUCTS: CHEMISTRY, PHYSICS, APPLICATIONS

Literature on the effects of microbial transglutaminase on various dairy‐based systems is discussed. Beginning with a short synopsis on the development of microbial transglutaminase as a functional tool for modifying foods, the principles of reactions catalyzed by transglutaminase and their structural implications, as well as the mechanisms of formation and cleavage of isopeptide bonds are reviewed. After summarizing the present knowledge on the specificity of microbial transglutaminase towards milk proteins, including reactions determined by individual lysine and glutamine residues, emphasis is placed on the effects of enzymatic cross‐linking on physicochemical properties in foods and, particularly, dairy‐based systems. Discussed are implications of cross‐linking on acidified milk gels including yogurt and effects on single milk protein fractions, with respect to several physicochemical properties including rheology and mechanical properties of these systems, but also syneresis, and emulsification behaviour.     

mTGase交联蛋白质的安全性评价及相关检测研究进展

微生物谷氨酰胺转氨酶(mTGase)作为一种性能优良的交联酶,用于提高蛋白质的加工性能。由于异肽键的生成,mTGase交联能够引起蛋白质消化率和免疫原性的变化;mTGase可能会作为乳糜泻患者的自身抗原,引发自身免疫疾病。为此,作者主要分析了mTGase交联对健康的影响和相应的检测手段,为mTGase的安全应用提供参考。     

Controlled Production of Amyloid β Peptide from a Photo‐Triggered,Water‐Soluble Precursor “Click Peptide“

In biological experiments, poor solubility and uncontrolled assembly of amyloid β peptide (Aβ) 1–42 pose significant obstacles to establish an experiment system that clarifies the function of Aβ1–42 in Alzheimer's disease (AD). Herein, as an experimental tool to overcome these problems, we developed a water‐soluble photo‐“click peptide” with a coumarin‐derived photocleavable protective group that is based on an O‐acyl isopeptide method. The click peptide had nearly 100‐fold higher water solubility than Aβ1–42 and did not self‐assemble, as the isomerized structure in its peptide backbone drastically changed the conformation that was derived from Aβ1–42. Moreover, the click peptide afforded Aβ1–42 quickly under physiological conditions (pH 7.4, 37 °C) by photoirradiation followed by an O–N intramolecular acyl migration. Because the in situ production of intact Aβ1–42 from the click peptide could improve the difficulties in handling Aβ1–42 caused by its poor solubility and highly aggregative nature, this click peptide strategy would provide a reliable experiment system for investigating the pathological function of Aβ1–42 in AD.     

Genetically Directed Production of Recombinant,Isosteric and Nonhydrolysable Ubiquitin Conjugates

We describe the genetically directed incorporation of aminooxy functionality into recombinant proteins by using a mutant Methanosarcina barkeri pyrrolysyl‐tRNA synthetase/tRNA_CUA pair. This allows the general production of nonhydrolysable ubiquitin conjugates of recombinant origin by bioorthogonal oxime ligation. This was exemplified by the preparation of nonhydrolysable versions of diubiquitin, polymeric ubiquitin chains and ubiquitylated SUMO. The conjugates exhibited unrivalled isostery with the native isopeptide bond, as inferred from structural and biophysical characterisation. Furthermore, the conjugates functioned as nanomolar inhibitors of deubiquitylating enzymes and were recognised by linkage‐specific antibodies. This technology should provide a versatile platform for the development of powerful tools for studying deubiquitylating enzymes and for elucidating the cellular roles of diverse polyubiquitin linkages.     

Amine-binding capacities of food proteins in transglutaminase reaction and digestibility of wheat gliadin with ϵ-attached lysine

Several food proteins were examined for their capacity as substrate in transglutaminase reaction by using a fluorescent amine, monodansyl cadaverine, as donor substrate and following the increase in fluorescence of the reaction mixture. Wheat gluten, or one of its component proteins, gliadin, was the best acceptor among the proteins tested. Compared with intact protein, a preparation of wheat gliadin, modified by transglutaminase-mediated incorporation of [¹⁴C]lysine, showed a marked decrease in in-vitro digestibility. In rat feeding tests, however, the luminal leavings and excreta collected 24 h after administration of gliadin with ?-attached [¹⁴C]lysine contained less than one-tenth of the radioactivity originally administered. The results presented support the previous notion that occurrence of the ?-γ isopeptide linkage in protein does not significantly impair the in-vivo digestibility of the protein and nutritional availability of isopeptide bound lysine moieties.     

Rounding Out the Understanding of ACD Toxicity with the Discovery of Cyclic Forms of Actin Oligomers

Actin is an essential element of both innate and adaptive immune systems and can aid in motility and translocation of bacterial pathogens, making it an attractive target for bacterial toxins. Pathogenic Vibrio and Aeromonas genera deliver actin cross-linking domain (ACD) toxin into the cytoplasm of the host cell to poison actin regulation and promptly induce cell rounding. At early stages of toxicity, ACD covalently cross-links actin monomers into oligomers (AOs) that bind through multivalent interactions and potently inhibit several families of actin assembly proteins. At advanced toxicity stages, we found that the terminal protomers of linear AOs can get linked together by ACD to produce cyclic AOs. When tested against formins and Ena/VASP, linear and cyclic AOs exhibit similar inhibitory potential, which for the cyclic AOs is reduced in the presence of profilin. In coarse-grained molecular dynamics simulations, profilin and WH2-motif binding sites on actin subunits remain exposed in modeled AOs of both geometries. We speculate, therefore, that the reduced toxicity of cyclic AOs is due to their reduced configurational entropy. A characteristic feature of cyclic AOs is that, in contrast to the linear forms, they cannot be straightened to form filaments (e.g., through stabilization by cofilin), which makes them less susceptible to neutralization by the host cell.