The Proteasome-associated Protein Ecm29 Inhibits Proteasomal ATPase Activity and in Vivo Protein Degradation by the Proteasome

Several proteasome-associated proteins regulate degradation by the 26 S proteasome using the ubiquitin chains that mark most substrates for degradation. The proteasome-associated protein Ecm29, however, has no ubiquitin-binding or modifying activity, and its direct effect on substrate degradation is unclear. Here, we show that Ecm29 acts as a proteasome inhibitor. Besides inhibiting the proteolytic cleavage of peptide substrates in vitro, it inhibits the degradation of ubiquitin-dependent and -independent substrates in vivo. Binding of Ecm29 to the proteasome induces a closed conformation of the substrate entry channel of the core particle. Furthermore, Ecm29 inhibits proteasomal ATPase activity, suggesting that the mechanism of inhibition and gate regulation by Ecm29 is through regulation of the proteasomal ATPases. Consistent with this, we identified through chemical cross-linking that Ecm29 binds to, or in close proximity to, the proteasomal ATPase subunit Rpt5. Additionally, we show that Ecm29 preferentially associates with both mutant and nucleotide depleted proteasomes. We propose that the inhibitory ability of Ecm29 is important for its function as a proteasome quality control factor by ensuring that aberrant proteasomes recognized by Ecm29 are inactive.     

Zinc ion coordination as a modulating factor of the ZnuA histidine-rich loop flexibility: A molecular modeling and fluorescence spectroscopy study

ZnuA is the soluble component of the high-affinity ZnuABC zinc transporter belonging to the ATP-binding cassette-type periplasmic Zn-binding proteins. The zinc transporter ZnuABC is composed by three proteins: ZnuB, the membrane permease, ZnuC, the ATPase component and ZnuA, the soluble periplasmic metal-binding protein which captures Zn and delivers it to ZnuB.The ZnuA protein contains a charged flexible loop, rich in histidines and acidic residues, showing significant species-specific differences. Various studies have established that this loop contributes to the formation of a secondary zinc binding site, which has been proposed to be important in the acquisition of periplasmic Zn for its delivery to ZnuB or for regulation of zinc uptake. Due to its high mobility the structure of the histidine-rich loop has never been solved by X-ray diffraction studies. In this paper, through a combined use of molecular modeling, mutagenesis and fluorescence spectroscopy, we confirm the presence of two zinc binding sites characterized by different affinities for the metal ion and show that the flexibility of the loop is modulated by the binding of the zinc ions to the protein. The data obtained by fluorescence spectroscopy have then be used to validate a 3D model including the unsolved histidine-rich loop.     

The effect of a digital learning environment on children’s conceptions about the protection of endemic plants

This study presents the results of a pilot learning intervention for improving children’s ideas about plant protection. The research was executed in two phases. The first phase aimed at exploring children’s ideas about plant protection. These ideas were taken into account for the design and development of a digital learning environment. The second phase concerned the development, implementation and evaluation of the learning environment. In the first phase, 262 participants (10–12 years old) were asked to write a short text about ‘Environmental protection’. During the second phase, twenty-six participants (10–12 years old) were asked to write a text on ‘How we can protect the endemic plants’, before and after the implementation of the learning activities. Responses were content analysed and their meaning was classified according to the coding scheme of Blanchet-Cohen, Ragan, and Amsden (Children, Youth and Environments 2003; 13: 1–7). The results of the first phase showed that participants mentioned individual lifestyle actions of environmental protection and, therefore, the learning environment should emphasise the importance of other types of environmental protection (eg political actions, legislation, social actions). The proposed learning activities enriched participants’ awareness about the importance of social and legal actions for plant protection.     

Inhibitors of HIV-1 attachment. Part 9: An assessment of oral prodrug approaches to improve the plasma exposure of a tetrazole-containing derivative

7-(2H-Tetrazol-5-yl)-1H-indole 3 was found to be a potent inhibitor of HIV-1 attachment but the compound lacked oral bioavailability in rats. The cause of the low exposure was believed to be poor absorption attributed to the acidic nature of the tetrazole moiety and, in an effort to address this liability, three more lipohilic tetrazole analogs, N-acetoxymethyl 4, N-pivaloyloxymethyl 5, and N-methyl 6, were evaluated as potential oral prodrugs in rats. Prodrug 5 was ineffective in improving the plasma concentration of 3 in vivo but compound 4 provided a 15-fold enhancement of the plasma concentration of 3. Most interestingly, oral dosing of analog 6 afforded a substantial increase in the plasma concentration of the parent in rats when compared to dosing of parent. This represents a novel example of a methyl tetrazole that acts as a prodrug for a free NH tetrazole-containing compound.     

The Lack of Toxic Effect of High-Power Short-Pulse 101 GHz Millimeter Waves on Healthy Mice

Irradiation of cancer cells by non-ionizing millimeter waves (MMW) causes increased cell mortality. We examined if MMW have toxic effects on healthy mice. To that end, the skin of healthy C57BL/6 mice was irradiated locally at the right flank with 101 GHz MMW in a pulsed (5–10 µs) regime using a free electron laser. Irradiation was performed in a dose-dependent manner, with 20–50 pulses and a power range of 0.5–1.5 kW. Physical, physiological, and pathological parameters as well as behavior were examined before and after irradiation. Our results showed that all parameters were within normal range for all experimental mice groups and for the control group. No significant changes were noted in the physical, physiological, or behavioral status of the mice following irradiation as compared with the control group. In addition, no significant changes were found in locomotor, exploratory behavior, or anxiety of the irradiated mice and no pathological changes were noted following the hematological and biochemical blood analysis. Our results indicate that irradiation of healthy mice with MMW does not cause any general toxic effects. Bioelectromagnetics. © 2020 Bioelectromagnetics Society.     

Antioxidant defense system responses and role of nitrate reductase in the redox balance maintenance in Bradyrhizobium japonicum strains exposed to cadmium

In this work, we evaluated the effects of cadmium (Cd) on the antioxidant defense system responses and the role of nitrate reductase (NR) in the redox balance maintenance in Bradyrhizobium japonicum strains. For that, B. japonicum USDA110 and its NR defective mutant strain (GRPA1) were used. Results showed that the addition of 10 μM Cd did not modify the aerobic growth of the wild type strain while the mutant strain was strongly affected. Anaerobic growth revealed that only the parental strain was able to grow under this condition. Cd reduced drastically the NR activity in B. japonicum USDA110 and increased lipid peroxide content in both strains. Cd decreased reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio in B. japonicum USDA110 although, a significant increased was observed in the mutant GRPA1. GSH-related enzymes were induced by Cd, being more evident the increase in the mutant strain. This different behavior observed between strains suggests that NR enzyme plays an important role in the redox balance maintenance in B. japonicum USDA 110 exposed to Cd.     

Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

Background

The pathogenesis of HIV-associated dementia (HAD) is poorly understood. To date, detailed proteomic fingerprinting directly from autopsied brain tissues of HAD and HIV non-dementia patients has not been performed.

Result

Here, we have analyzed total proteins from the frontal cortex of 9 HAD and 5 HIV non-dementia patients. Using 2-Dimensional differential in-gel electrophoresis (2-DIGE) to analyze the brain tissue proteome, 76 differentially expressed proteins (p < 0.05; fold change>1.25) were identified between HAD and HIV non-dementia patients, of which 36 protein spots (based on 3D appearance of spots on the images) were chosen for the mass spectrometry analysis. The large majority of identified proteins were represented in the energy metabolic (mitochondria) and signal transduction pathways. Furthermore, over 90% of the protein candidates are common to both HAD and other non-viral neurodegenerative disease, such as Alzheimer's disease. The data was further validated using specific antibodies to 4 proteins (CA2, GS, CKMT and CRMP2) by western blot (WB) in the same samples used for 2D-DIGE, with additional confirmation by immunohistochemitsry (IHC) using frontal lobe tissue from different HAD and HIV+ non-dementia patients. The validation for all 4 antibodies by WB and IHC was in concordance with the DIGE results, lending further credence to the current findings.

Conclusion

These results suggest not only convergent pathogenetic pathways for the two diseases but also the possibility of increased Alzheimer's disease (AD) susceptibility in HAD patients whose life expectancy has been significantly increased by highly active antiretroviral therapy.     

Citrobacter rodentium infection causes iNOS-independent intestinal epithelial dysfunction in mice

Attaching-effacing bacteria are major causes of infectious diarrhea in humans worldwide. Citrobacter rodentium is an attaching-effacing enteric pathogen that causes transmissible murine colonic mucosal hyperplasia. We characterized colonic inflammation and ion transport at 3, 7, 10, 30, and 60 d after infection of C57Bl/6 mice with C. rodentium. Macroscopic damage score was significantly increased 7 and 10 d after infection. Colonic wall thickness was increased at 7, 10, 30, and 60 d. Myeloperoxidase (MPO) activity was significantly increased at 3, 7, and 10 d and returned to control levels by days 30 and 60. The expressions of inducible nitric oxide synthase and cyclooxygenase-2 were increased by C. rodentium infection. Significant reductions in the epithelial secretory response to carbachol, but not to electrical field stimulation or forskolin, were observed at 3 and 10 d of infection. Translocation of enteric bacteria into the mesenteric lymph nodes was observed 10 d following infection. There was no difference in response to infection between animals deficient in inducible nitric oxide synthase and wild-type controls. The COX-2 inhibitor rofecoxib caused decreased wall thickness and MPO activity at day 10. However, COX-2 inhibition did not alter infection-induced changes in ion transport. Citrobacter rodentium infection causes colonic inflammation, mucosal hyperplasia, and nitric-oxide-independent epithelial dysfunction in association with increased permeability to luminal bacteria.     

Acetylcarnitine and cellular stress response: roles in nutritional redox homeostasis and regulation of longevity genes

Aging is associated with a reduced ability to cope with physiological challenges. Although the mechanisms underlying age-related alterations in stress tolerance are not well defined, many studies support the validity of the oxidative stress hypothesis, which suggests that lowered functional capacity in aged organisms is the result of an increased generation of reactive oxygen and nitrogen species. Increased production of oxidants in vivo can cause damage to intracellular macromolecules, which can translate into oxidative injury, impaired function and cell death in vulnerable tissues such as the brain. To survive different types of injuries, brain cells have evolved networks of responses, which detect and control diverse forms of stress. This is accomplished by a complex network of the so-called longevity assurance processes, which are composed of several genes termed vitagenes. Among these, heat shock proteins form a highly conserved system responsible for the preservation and repair of the correct protein conformation. The heat shock response contributes to establishing a cytoprotective state in a wide variety of human diseases, including inflammation, cancer, aging and neurodegenerative disorders. Given the broad cytoprotective properties of the heat shock response, there is now a strong interest in discovering and developing pharmacological agents capable of inducing the heat shock response. Acetylcarnitine is proposed as a therapeutic agent for several neurodegenerative disorders, and there is now evidence that it may play a critical role as modulator of cellular stress response in health and disease states. In the present review, we first discuss the role of nutrition in carnitine metabolism, followed by a discussion of carnitine and acetyl-l-carnitine in mitochondrial dysfunction, in aging, and in age-related disorders. We then review the evidence for the role of acetylcarnitine in modulating redox-dependent mechanisms leading to up-regulation of vitagenes in brain, and we also discuss new approaches for investigating the mechanisms of lifetime survival and longevity.