Structure of the Catalytic Domain of EZH2 Reveals Conformational Plasticity in Cofactor and Substrate Binding Sites and Explains Oncogenic Mutations

Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation.     

2-Bromopalmitate Reduces Protein Deacylation by Inhibition of Acyl-Protein Thioesterase Enzymatic Activities

S-acylation, the covalent attachment of palmitate and other fatty acids on cysteine residues, is a reversible post-translational modification that exerts diverse effects on protein functions. S-acylation is catalyzed by protein acyltransferases (PAT), while deacylation requires acyl-protein thioesterases (APT), with numerous inhibitors for these enzymes having already been developed and characterized. Among these inhibitors, the palmitate analog 2-brompalmitate (2-BP) is the most commonly used to inhibit palmitoylation in cells. Nevertheless, previous results from our laboratory have suggested that 2-BP could affect protein deacylation. Here, we further investigated in vivo and in vitro the effect of 2-BP on the acylation/deacylation protein machinery, with it being observed that 2-BP, in addition to inhibiting PAT activity in vivo, also perturbed the acylation cycle of GAP-43 at the level of depalmitoylation and consequently affected its kinetics of membrane association. Furthermore, 2-BP was able to inhibit in vitro the enzymatic activities of human APT1 and APT2, the only two thioesterases shown to mediate protein deacylation, through an uncompetitive mechanism of action. In fact, APT1 and APT2 hydrolyzed both the monomeric form as well as the micellar state of the substrate palmitoyl-CoA. On the basis of the obtained results, as APTs can mediate deacylation on membrane bound and unbound substrates, this suggests that the access of APTs to the membrane interface is not a necessary requisite for deacylation. Moreover, as the enzymatic activity of APTs was inhibited by 2-BP treatment, then the kinetics analysis of protein acylation using 2-BP should be carefully interpreted, as this drug also inhibits protein deacylation.     

Potential Application of Turnip Oil (Raphanus sativus L.) for Biodiesel Production: Physical–Chemical Properties of Neat Oil, Biofuels and their Blends with Ultra-Low Sulphur Diesel (ULSD)

Turnip oil (TO; Raphanus sativus L.) produces seeds that contain around 26 wt% of inedible base stock that are suitable as a potential feedstock for biodiesel production. A turnip oil methyl ester (TME) was prepared from acid-catalyzed pretreated TO in an effort to evaluate important fuel properties of turnip oil-based biodiesel, such as kinematic viscosity, cloud point, pour point (PP), cold filter plugging point, acid value, oxidative stability and lubricity. A comparison was made with soybean oil methyl esters (SME) as per biodiesel fuel standards such as ASTM D6751 and EN 14214. TME was characterized using FTIR, HPLC and ¹H NMR. Except PP property, SME displays superior fuel properties compared to TME. Blends (B5 and B20) of TME in ultra-low sulphur diesel fuel (ULSD) were also assessed for the aforesaid fuel properties and compared to an analogous set of blends of soybean oil methyl ester in ULSD as per petro diesel fuel standards such as ASTM D975 and D7467. TME B5 blends in ULSD displayed improved PP property in comparison to neat ULSD and blends of SME in ULSD. It was demonstrated that the B5 and B20 blends of TME in ULSD had acceptable fuel properties as per ASTM D975 (for B5 blend) and ASTM D7467 (for B20 blend). In summary, turnip oil has potential as an alternative, non-food feedstock for biodiesel production.     

Method for Quantitative Study of Airway Functional Microanatomy Using Micro-Optical Coherence Tomography

We demonstrate the use of a high resolution form of optical coherence tomography, termed micro-OCT (μOCT), for investigating the functional microanatomy of airway epithelia. μOCT captures several key parameters governing the function of the airway surface (airway surface liquid depth, periciliary liquid depth, ciliary function including beat frequency, and mucociliary transport rate) from the same series of images and without exogenous particles or labels, enabling non-invasive study of dynamic phenomena. Additionally, the high resolution of μOCT reveals distinguishable phases of the ciliary stroke pattern and glandular extrusion. Images and functional measurements from primary human bronchial epithelial cell cultures and excised tissue are presented and compared with measurements using existing gold standard methods. Active secretion from mucus glands in tissue, a key parameter of epithelial function, was also observed and quantified.     

Electrophysiological Heterogeneity of Fast-Spiking Interneurons: Chandelier versus Basket Cells

In the prefrontal cortex, parvalbumin-positive inhibitory neurons play a prominent role in the neural circuitry that subserves working memory, and alterations in these neurons contribute to the pathophysiology of schizophrenia. Two morphologically distinct classes of parvalbumin neurons that target the perisomatic region of pyramidal neurons, chandelier cells (ChCs) and basket cells (BCs), are generally thought to have the same “fast-spiking” phenotype, which is characterized by a short action potential and high frequency firing without adaptation. However, findings from studies in different species suggest that certain electrophysiological membrane properties might differ between these two cell classes. In this study, we assessed the physiological heterogeneity of fast-spiking interneurons as a function of two factors: species (macaque monkey vs. rat) and morphology (chandelier vs. basket). We showed previously that electrophysiological membrane properties of BCs differ between these two species. Here, for the first time, we report differences in ChCs membrane properties between monkey and rat. We also found that a number of membrane properties differentiate ChCs from BCs. Some of these differences were species-independent (e.g., fast and medium afterhyperpolarization, firing frequency, and depolarizing sag), whereas the differences in the first spike latency between ChCs and BCs were species-specific. Our findings indicate that different combinations of electrophysiological membrane properties distinguish ChCs from BCs in rodents and primates. Such electrophysiological differences between ChCs and BCs likely contribute to their distinctive roles in cortical circuitry in each species.     

Length-weight relationships of seven fish species from the laguna del cisne basin (Canelones,Uruguay)

This work provides new length-weight information for seven species of freshwater fishes. The analyzed specimens were collected monthly during a year (April 2018–March 2019) in a coastal lagoon system and its associated streams in southern Uruguay. During each sampling campaign, fishes were captured using gillnets in the lagoon and electrofishing in the streams. This study reports a new maximum size for four species and the first length-weight relationship report for Gymnogeophagus terrapurpura. Length-weight estimates and their confidence intervals are provided for all species.     

Polysome-associated lncRNAs during cardiomyogenesis of hESCs

Molecular and Cellular Biochemistry - In stress conditions, as neoplastic transformation, amino acids serve not only as nutrients to maintain the cell survival but also as mediators of several...     

Thermal ecology of two sympatric saxicolous lizards of the genus Phymaturus from the Payunia region (Argentina)

We evaluated the thermal biology of two sympatric saxicolous species of the genus Phymaturus, endemic from the Argentine Payunia region. Taking into account that the distributional range of Phymaturus roigorum (the largest species) is greater than the range of P. payuniae, we evaluated the habitat (type of rocks) used by these species. We recorded body temperature and operative temperatures in different habitats, and we determined the preferred body temperature in the laboratory. We compared the thermal quality of habitats occupied and not occupied by Phymaturus payuniae, and the accuracy and effectiveness of thermoregulation between species.