Scavenging Bacterial Siderophores with Engineered Lipocalin Proteins as an Alternative Antimicrobial Strategy

Iron acquisition mediated by siderophores, high-affinity chelators for which bacteria have evolved specific synthesis and uptake mechanisms, plays a crucial role in microbiology and in host–pathogen interactions. In the ongoing fight against bacterial infections, this area has attracted biomedical interest. Beyond several approaches to interfere with siderophore-mediated iron uptake from medicinal and immunochemistry, the development of high-affinity protein scavengers that tightly complex the siderophores produced by pathogenic bacteria has appeared as a novel strategy. Such binding proteins have been engineered based on siderocalin—also known as lipocalin 2—an endogenous human scavenger of enterobactin and bacillibactin that controls the systemic spreading of commensal bacteria such as Escherichia coli. By using combinatorial protein design, siderocalin was reshaped to bind several siderophores from Pseudomonas aeruginosa and, in particular, petrobactin from Bacillus anthracis, none of which is recognized by the natural protein. Such engineered versions of siderocalin effectively suppress the growth of corresponding pathogenic bacteria by depriving them of their iron supply and offer the potential to complement antibiotic therapy in situations of acute or persistent infection.     

Identification of Host Cellular Protein Substrates of SARS-COV-2 Main Protease

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease-19 (COVID-19) being associated with severe pneumonia. Like with other viruses, the interaction of SARS-CoV-2 with host cell proteins is necessary for successful replication, and cleavage of cellular targets by the viral protease also may contribute to the pathogenesis, but knowledge about the human proteins that are processed by the main protease (3CLpro) of SARS-CoV-2 is still limited. We tested the prediction potentials of two different in silico methods for the identification of SARS-CoV-2 3CLpro cleavage sites in human proteins. Short stretches of homologous host-pathogen protein sequences (SSHHPS) that are present in SARS-CoV-2 polyprotein and human proteins were identified using BLAST analysis, and the NetCorona 1.0 webserver was used to successfully predict cleavage sites, although this method was primarily developed for SARS-CoV. Human C-terminal-binding protein 1 (CTBP1) was found to be cleaved in vitro by SARS-CoV-2 3CLpro, the existence of the cleavage site was proved experimentally by using a His₆-MBP-mEYFP recombinant substrate containing the predicted target sequence. Our results highlight both potentials and limitations of the tested algorithms. The identification of candidate host substrates of 3CLpro may help better develop an understanding of the molecular mechanisms behind the replication and pathogenesis of SARS-CoV-2.     

非常规蛋白基木材胶黏剂研究现状及发展前景

介绍了动物和植物型非常规蛋白资源的种类及其制备蛋白基木材胶黏剂的研究现状和存在的问题,展望了非常规蛋白胶黏剂的发展前景。     

Understanding the metabolic burden of recombinant antibody production in Saccharomyces cerevisiae using a quantitative metabolomics approach

The cellular changes induced by heterologous protein expression in the yeast Saccharomyces cerevisiae have been analysed on many levels and found to be significant. However, even though high‐level protein production poses a metabolic burden, evaluation of the expression host at the level of the metabolome has often been neglected. We present a comparison of metabolite profiles of a wild‐type strain with those of three strains producing recombinant antibody variants of increasing size and complexity: an scFv fragment, an scFv–Fc fusion protein and a full‐length IgG molecule. Under producing conditions, all three recombinant strains showed a clear decrease in growth rate compared with the wild‐type strain and the severity of the growth phenotype increased with size of the protein. The levels of 76 intracellular metabolites were determined using a targeted (semi) quantitative mass spectrometry based approach. Based on unsupervised and supervised multivariate analysis of metabolite profiles, together with pathway activity profiling, the recombinant strains were found to be significantly different from each other and from the wild‐type strain. We observed the most prominent changes in metabolite levels for metabolites involved in amino acid and redox metabolism. Induction of the unfolded protein response was detected in all producing strains and is considered to be a contributing factor to the overall metabolic burden on the cells.     

Gelation properties of goose liver protein recovered by isoelectric solubilisation/precipitation process

Isoelectric solubilisation/precipitation (ISP) process was applied to goose liver (GL) for protein extraction. The gelation properties of proteins extracted by acid processes (ACP, pH 2.0, 2.5 and 3.0) and alkaline processes (ALP, pH 11.0, 11.5 and 12.0) were estimated, where the unextracted ground GL was set as the control. Nearly 58.39~79.00% of GL proteins were recovered by ISP treatments. High molecular weight (100~250 kDa) proteins were found to be partially hydrolysed by ACP, while few changes in proteins occurred during ALP. As evidenced by rheological and textural measurements, ALP proteins formed gels with high elasticity and superior texture, whereas ACP proteins had inferior gelation properties. Moreover, ALP proteins were able to form a highly interconnected and homogeneous three‐dimensional microstructure. Predominantly, gels produced by 11.0 had optimal texture and the lowest cooking loss (P < 0.05). These results suggested that the ISP process (ALP) is a potential method to improve the economic value of GL.     

Rationally Designed Polypharmacology: α-Helix Mimetics as Dual Inhibitors of the Oncoproteins Mcl-1 and HDM2

Protein–protein interactions (PPIs), many of which are dominated by α-helical recognition domains, play key roles in many essential cellular processes, and the dysregulation of these interactions can cause detrimental effects. For instance, aberrant PPIs involving the Bcl-2 protein family can lead to several diseases including cancer, neurodegenerative diseases, and diabetes. Interactions between Bcl-2 pro-life proteins, such as Mcl-1, and pro-death proteins, such as Bim, regulate the intrinsic pathway of apoptosis. p53, a tumor-suppressor protein, also has a pivotal role in apoptosis and is negatively regulated by its E3 ubiquitin ligase HDM2. Both Mcl-1 and HDM2 are upregulated in numerous cancers, and, interestingly, there is crosstalk between both protein pathways. Recently, synergy has been observed between Mcl-1 and HDM2 inhibitors. Towards the development of new anticancer drugs, we herein describe a polypharmacology approach for the dual inhibition of Mcl-1 and HDM2 by employing three densely functionalized isoxazoles, pyrazoles, and thiazoles as mimetics of key α-helical domains of their partner proteins.     

Enzymatic Responses to Low-Intensity Radiation of Tritium

The present study considers a possible role of enzymatic reactions in the adaptive response of cells to the beta-emitting radionuclide tritium under conditions of low-dose exposures. Effects of tritiated water (HTO) on the reactions of bacterial luciferase and NAD(P)H:FMN-oxidoreductase, as well as a coupled system of these two reactions, were studied at radioactivity concentrations ≤ 200 MBq/L. Additionally, one of the simplest enzymatic reactions, photobiochemical proton transfer in Coelenteramide-containing Fluorescent Protein (CLM-FP), was also investigated. We found that HTO increased the activity of NAD(P)H:FMN-oxidoreductase at the initial stage of its reaction (by up to 230%); however, a rise of luciferase activity was moderate (<20%). The CLM-FP samples did not show any increase in the rate of the photobiochemical proton transfer under the exposure to HTO. The responses of the enzyme systems were compared to the ‘hormetic’ response of luminous marine bacterial cells studied earlier. We conclude that (1) the oxidoreductase reaction contributes significantly to the activation of the coupled enzyme system and bacterial cells by tritium, and (2) an increase in the organization level of biological systems promotes the hormesis phenomenon.     

Design and Characterization of a New pVII Combinatorial Phage Display Peptide Library for Protease Substrate Mining Using Factor VII Activating Protease (FSAP) as Model

We describe a novel, easy and efficient combinatorial phage display peptide substrate-mining method to map the substrate specificity of proteases. The peptide library is displayed on the pVII capsid of the M13 bacteriophage, which renders pIII necessary for infectivity and efficient retrieval, in an unmodified state. As capture module, the 3XFLAG was chosen due to its very high binding efficiency to anti-FLAG mAbs and its independency of any post-translational modification. This library was tested with Factor-VII activating protease (WT-FSAP) and its single-nucleotide polymorphism variant Marburg-I (MI)-FSAP. The WT-FSAP results confirmed the previously reported Arg/Lys centered FSAP cleavage site consensus as dominant, as well as reinforcing MI-FSAP as a loss-of-function mutant. Surprisingly, rare substrate clones devoid of basic amino acids were also identified. Indeed one of these peptides was cleaved as free peptide, thus suggesting a broader range of WT-FSAP substrates than previously anticipated.