Quantitative Proteomic Approach Reveals Altered Metabolic Pathways in Response to the Inhibition of Lysine Deacetylases in A549 Cells under Normoxia and Hypoxia

Growing evidence is showing that acetylation plays an essential role in cancer, but studies on the impact of KDAC inhibition (KDACi) on the metabolic profile are still in their infancy. Here, we analyzed, by using an iTRAQ-based quantitative proteomics approach, the changes in the proteome of KRAS-mutated non-small cell lung cancer (NSCLC) A549 cells in response to trichostatin-A (TSA) and nicotinamide (NAM) under normoxia and hypoxia. Part of this response was further validated by molecular and biochemical analyses and correlated with the proliferation rates, apoptotic cell death, and activation of ROS scavenging mechanisms in opposition to the ROS production. Despite the differences among the KDAC inhibitors, up-regulation of glycolysis, TCA cycle, oxidative phosphorylation and fatty acid synthesis emerged as a common metabolic response underlying KDACi. We also observed that some of the KDACi effects at metabolic levels are enhanced under hypoxia. Furthermore, we used a drug repositioning machine learning approach to list candidate metabolic therapeutic agents for KRAS mutated NSCLC. Together, these results allow us to better understand the metabolic regulations underlying KDACi in NSCLC, taking into account the microenvironment of tumors related to hypoxia, and bring new insights for the future rational design of new therapies.     

Structural Cues for Understanding eEF1A2 Moonlighting

Spontaneous mutations in the EEF1A2 gene cause epilepsy and severe neurological disabilities in children. The crystal structure of eEF1A2 protein purified from rabbit skeletal muscle reveals a post-translationally modified dimer that provides information about the sites of interaction with numerous binding partners, including itself, and maps these mutations onto the dimer and tetramer interfaces. The spatial locations of the side chain carboxylates of Glu301 and Glu374, to which phosphatidylethanolamine is uniquely attached via an amide bond, define the anchoring points of eEF1A2 to cellular membranes and interorganellar membrane contact sites. Additional bioinformatic and molecular modeling results provide novel structural insight into the demonstrated binding of eEF1A2 to SH3 domains, the common MAPK docking groove, filamentous actin, and phosphatidylinositol-4 kinase IIIβ. In this new light, the role of eEF1A2 as an ancient, multifaceted, and articulated G protein at the crossroads of autophagy, oncogenesis and viral replication appears very distant from the “canonical” one of delivering aminoacyl-tRNAs to the ribosome that has dominated the scene and much of the thinking for many decades.     

The generalized discrete ‐centroid problem

The ‐centroid problem or leader–follower problem is generalized considering different customer choice rules where a customer may use facilities belonging to different firms, if the difference in travel distance (or time) is small enough. Assuming essential goods, some particular customer choice rules are analyzed. Linear programming formulations for the generalized ‐medianoid and ‐centroid problems are presented and an exact solution approach is applied. Some computational examples are included.     

Mast Cell Chymase and Kidney Disease

A sizable part (~2%) of the human genome encodes for proteases. They are involved in many physiological processes, such as development, reproduction and inflammation, but also play a role in pathology. Mast cells (MC) contain a variety of MC specific proteases, the expression of which may differ between various MC subtypes. Amongst these proteases, chymase represents up to 25% of the total proteins in the MC and is released from cytoplasmic granules upon activation. Once secreted, it cleaves the targets in the local tissue environment, but may also act in lymph nodes infiltrated by MC, or systemically, when reaching the circulation during an inflammatory response. MC have been recognized as important components in the development of kidney disease. Based on this observation, MC chymase has gained interest following the discovery that it contributes to the angiotensin-converting enzyme’s independent generation of angiotensin II, an important inflammatory mediator in the development of kidney disease. Hence, progress regarding its role has been made based on studies using inhibitors but also on mice deficient in MC protease 4 (mMCP-4), the functional murine counterpart of human chymase. In this review, we discuss the role and actions of chymase in kidney disease. While initially believed to contribute to pathogenesis, the accumulated data favor a more subtle view, indicating that chymase may also have beneficial actions.     

Nonhydrolyzable Heptose Bis- and Monophosphate Analogues Modulate Pro-inflammatory TIFA-NF-κB Signaling

d -Glycero-d -manno-heptose-1β,7-bisphosphate (HBP) and d -glycero-d -manno-heptose-1β-phosphate (H1P) are bacterial metabolites that were recently shown to stimulate inflammatory responses in host cells through the activation of the TIFA-dependent NF-κB pathway. To better understand structure-based activity in relation to this process, a family of nonhydrolyzable phosphonate analogues of HBP and H1P was synthesized. The inflammation modulation by which these molecules induce the TIFA-NF-κB signal axis was evaluated in vivo at a low-nanomolar concentration (6 nM) and compared to that of the natural metabolites. Our data showed that three phosphonate analogues had similar stimulatory activity to HBP, whereas two phosphonates antagonized HBP-induced TIFA-NF-κB signaling. These results open new horizons for the design of pro-inflammatory and innate immune modulators that could be used as vaccine adjuvant.     

Ga-doped IZO films obtained by magnetron sputtering as transparent conductors for visible and solar applications

C-axis textured thin films of gallium-doped indium zinc oxide (GIZO) with a 2% ratio of Ga/Zn, were obtained via RF-magnetron sputtering with high transparency and electrical conductivity. A Box-Behnken response surface design was used to evaluate the effects of the deposition parameters (In₂O₃ target power, deposition time, and substrate temperature) on the chemical composition, optical, electrical, and structural properties of the GIZO films. The optical constants and the electrical properties were obtained using optical models. The GIZO stoichiometry, and therefore the In/Zn atomic ratio, affected the crystallinity, crystalline parameters, band gap, and charge carrier mobility of the GIZO films. The charge carrier density was related to the change in the crystalline parameters of the hexagonal structure and the In/Zn atomic ratio. The best electrical conductivity values (1.75?×?10³ Ω^?1 cm^?1) were obtained for GIZO films with In/Zn ratio ≥?1. Several figures of merit (FOM) defined for the visible and solar regions were comparatively used to select the optimal In/Zn atomic ratio that provided the best balance between the conductivity and the transparency. The optimal In/Zn ratio was in a range of 0.85–0.90 for the GIZO films.     

Real‐time monitoring by proton relaxometry of radical polymerization reactions of acrylamide in aqueous solution

The potential of time‐domain nuclear magnetic resonance (TD‐NMR) for the real‐time monitoring of solution radical polymerizations is demonstrated. A model system composed of a redox‐pair initiator system, acrylamide as monomer and water as solvent was investigated. A second‐generation continuous wave free precession technique was employed to measure the longitudinal relaxation time constant (T₁) of the samples throughout the polymerization reactions. This parameter was shown to be sensitive to the reactant feed free‐radical enhancement of the water molecule relaxation time, making it a good probe to monitor monomer conversion in real time in an automated, non‐destructive fashion. It was found that the T₁ value was better than the transverse relaxation time constant (T₂) for describing the evolution of the polymerization reactions, due to its greater sensitivity to paramagnetic effects. The TD‐NMR signal variation observed was linked to the formation, propagation and termination steps of the radical polymerization kinetics scheme. These first results may contribute to the application of real‐time monitoring of radical polymerization reactions employing low‐cost and robust TD‐NMR spectrometers. © 2018 Society of Chemical Industry     

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.