The Importance of Therapeutically Targeting the Binary Toxin from Clostridioides difficile

Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell’s cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.     

Integration of Data from Liquid–Liquid Phase Separation Databases Highlights Concentration and Dosage Sensitivity of LLPS Drivers

Liquid–liquid phase separation (LLPS) is a molecular process that leads to the formation of membraneless organelles, representing functionally specialized liquid-like cellular condensates formed by proteins and nucleic acids. Integrating the data on LLPS-associated proteins from dedicated databases revealed only modest agreement between them and yielded a high-confidence dataset of 89 human LLPS drivers. Analysis of the supporting evidence for our dataset uncovered a systematic and potentially concerning difference between protein concentrations used in a good fraction of the in vitro LLPS experiments, a key parameter that governs the phase behavior, and the proteomics-derived cellular abundance levels of the corresponding proteins. Closer scrutiny of the underlying experimental data enabled us to offer a sound rationale for this systematic difference, which draws on our current understanding of the cellular organization of the proteome and the LLPS process. In support of this rationale, we find that genes coding for our human LLPS drivers tend to be dosage-sensitive, suggesting that their cellular availability is tightly regulated to preserve their functional role in direct or indirect relation to condensate formation. Our analysis offers guideposts for increasing agreement between in vitro and in vivo studies, probing the roles of proteins in LLPS.     

Transglycosylation Activity of Engineered Bifidobacterium Lacto-N-Biosidase Mutants at Donor Subsites for Lacto-N-Tetraose Synthesis

The health benefits of human milk oligosaccharides (HMOs) make them attractive targets as supplements for infant formula milks. However, HMO synthesis is still challenging and only two HMOs have been marketed. Engineering glycoside hydrolases into transglycosylases may provide biocatalytic routes to the synthesis of complex oligosaccharides. Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hydrolyzes lacto-N-tetraose (LNT), the core structure of the most abundant type I HMOs. Here we report a mutational study in the donor subsites of the substrate binding cleft with the aim of reducing hydrolytic activity and conferring transglycosylation activity for the synthesis of LNT from p-nitrophenyl β-lacto-N-bioside and lactose. As compared with the wt enzyme with negligible transglycosylation activity, mutants with residual hydrolase activity within 0.05% to 1.6% of the wild-type enzyme result in transglycosylating enzymes with LNT yields in the range of 10–30%. Mutations of Trp394, located in subsite -1 next to the catalytic residues, have a large impact on the transglycosylation/hydrolysis ratio, with W394F being the best mutant as a biocatalyst producing LNT at 32% yield. It is the first reported transglycosylating LnbB enzyme variant, amenable to further engineering for practical enzymatic synthesis of LNT.     

Effects of Edible Insect Tenebrio molitor Larva Fermentation Extract as a Substitute Protein on Hepatosteatogenesis and Proteomic Changes in Obese Mice Induced by High-Fat Diet

Mealworms (Tenebrio molitor larva) are an edible insect and a protein-rich food; however, research on mealworms as a substitute protein is insufficient. In this study, mealworm fermentation extract (TMP) was assessed as a replacement for soy protein (SP) in a control diet (CON) or a high-fat diet (HFD) of mice for 12 weeks. TMP substitution reduced body weight, body weight gain, body fat mass (perirenal and mesenteric), fat size, glucose intolerance, and insulin resistance compared to the HFD-SP group. TMP alleviated hepatic steatosis (lipid contents and lipid droplets) in high-fat-fed mice and down-regulated the PPARγ, CD36, and DGAT2 gene levels. Proteomic analysis showed that a HFD for 12 weeks up-regulated 20 proteins and down-regulated 17 proteins in mice fed SP. On the other hand, TMP reversed the protein profiles. TMP significantly down-regulated KHK, GLO1, ATP5H, SOD, and DDAH1 and up-regulated DLD, Mup1, CPS1, Ces3b, PDI, and HYOU1 compared to the HFD-SP group. These proteins are involved in the glucose, lipid, and amino acid metabolism, as well as in oxidative stress and endoplasmic reticulum stress. Thus, substituting SP for TMP helped improve HFD-induced obesity, steatosis, and insulin resistance in mice. These results suggest that TMP is a potential substitute for commonly used protein sources.     

Role of Receptor Protein Tyrosine Phosphatases (RPTPs) in Insulin Signaling and Secretion

Changes in lifestyle in developed countries have triggered the prevalence of obesity and type 2 diabetes mellitus (T2DM) in the latest years. Consequently, these metabolic diseases associated to insulin resistance, and the morbidity associated with them, accounts for enormous costs for the health systems. The best way to face this problem is to identify potential therapeutic targets and/or early biomarkers to help in the treatment and in the early detection. In the insulin receptor signaling cascade, the activities of protein tyrosine kinases and phosphatases are coordinated, thus, protein tyrosine kinases amplify the insulin signaling response, whereas phosphatases are required for the regulation of the rate and duration of that response. The focus of this review is to summarize the impact of transmembrane receptor protein tyrosine phosphatase (RPTPs) in the insulin signaling cascade and secretion, and their implication in metabolic diseases such as obesity and T2DM.     

Difluoromethylornithine (DFMO), an Inhibitor of Polyamine Biosynthesis,and Antioxidant N-Acetylcysteine Potentiate Immune Response in Mice to the Recombinant Hepatitis C Virus NS5B Protein

Hepatitis C virus (HCV) is one of the main triggers of chronic liver disease. Despite tremendous progress in the HCV field, there is still no vaccine against this virus. Potential vaccines can be based on its recombinant proteins. To increase the humoral and, especially, cellular immune response to them, more effective adjuvants are needed. Here, we evaluated a panel of compounds as potential adjuvants using the HCV NS5B protein as an immunogen. These compounds included inhibitors of polyamine biosynthesis and urea cycle, the mTOR pathway, antioxidants, and cellular receptors. A pronounced stimulation of cell proliferation and interferon-γ (IFN-γ) secretion in response to concanavalin A was shown for antioxidant N-acetylcysteine (NAC), polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO), and TLR9 agonist CpG ODN 1826 (CpG). Their usage during the immunization of mice with the recombinant NS5B protein significantly increased antibody titers, enhanced lymphocyte proliferation and IFN-γ production. NAC and CpG decreased relative Treg numbers; CpG increased the number of myeloid-derived suppressor cells (MDSCs), whereas neither NAC nor DFMO affected MDSC counts. NAC and DFMO suppressed NO and interleukin 10 (IL-10) production by splenocytes, while DFMO increased the levels of IL-12. This is the first evidence of immunomodulatory activity of NAC and DFMO during prophylactic immunization against infectious diseases.     

The Autophagy-Related Organelle Autophagoproteasome Is Suppressed within Ischemic Penumbra

The peri-infarct region, which surrounds the irreversible ischemic stroke area is named ischemic penumbra. This term emphasizes the borderline conditions for neurons placed within such a critical region. Area penumbra separates the ischemic core, where frank cell loss occurs, from the surrounding healthy brain tissue. Within such a brain region, nervous matter, and mostly neurons are impaired concerning metabolic conditions. The classic biochemical marker, which reliably marks area penumbra is the over-expression of the heat shock protein 70 (HSP70). However, other proteins related to cell clearing pathways are modified within area penumbra. Among these, autophagy proteins like LC3 increase in a way, which recapitulates Hsp70. In contrast, components, such as P20S, markedly decrease. Despite apparent discrepancies, the present study indicates remarkable overlapping between LC3 and P20S redistribution within area penumbra. In fact, the amount of both proteins is markedly reduced within vacuoles. Specifically, a massive loss of LC3 + P20S immuno-positive vacuoles (autophagoproteasomes) is reported here. This represents the most relevant sub-cellular alteration here described in cell clearing pathways within area penumbra. The functional significance of these findings remains to be determined and it will take a novel experimental stream to decipher the fine-tuning of such a phenomenon.     

Constitutive Photomorphogenic 1 Enhances ER Stress Tolerance in Arabidopsis

Interaction between light signaling and stress response has been recently reported in plants. Here, we investigated the role of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a key regulator of light signaling, in endoplasmic reticulum (ER) stress response in Arabidopsis. The cop1-4 mutant Arabidopsis plants were highly sensitive to ER stress induced by treatment with tunicarmycin (Tm). Interestingly, the abundance of nuclear-localized COP1 increased under ER stress conditions. Complementation of cop1-4 mutant plants with the wild-type or variant types of COP1 revealed that the nuclear localization and dimerization of COP1 are essential for its function in plant ER stress response. Moreover, the protein amount of ELONGATED HYPOCOTYL 5 (HY5), which inhibits bZIP28 to activate the unfolded protein response (UPR), decreased under ER stress conditions in a COP1-dependent manner. Accordingly, the binding of bZIP28 to the BIP3 promoter was reduced in cop1-4 plants and increased in hy5 plants compared with the wild type. Furthermore, introduction of the hy5 mutant locus into the cop1-4 mutant background rescued its ER stress-sensitive phenotype. Altogether, our results suggest that COP1, a negative regulator of light signaling, positively controls ER stress response by partially degrading HY5 in the nucleus.     

防治畜类寄生线虫的又一阿维菌素类化合物--道拉菌素

道拉菌素是利用生物突变合成方法得到的新型阿维菌素族广谱杭寄生虫药。它的化学结构和作用机制与依维菌素相似。试验证实道拉菌素在低剂量下对多种体内外寄生虫具有良好的防治效果。