Hydration Structures of the Human Protein Kinase CK2α Clarified by Joint Neutron and X-ray Crystallography

Casein kinase 2 (CK2) has broad phosphorylation activity against various regulatory proteins, which are important survival factors in eukaryotic cells. To clarify the hydration structure and catalytic mechanism of CK2, we determined the crystal structure of the alpha subunit of human CK2 containing hydrogen and deuterium atoms using joint neutron (1.9 Å resolution) and X-ray (1.1 Å resolution) crystallography. The analysis revealed the structure of conserved water molecules at the active site and a long potential hydrogen bonding network originating from the catalytic Asp156 that is well known to enhance the nucleophilicity of the substrate OH group to the γ-phospho group of ATP by proton elimination. His148 and Asp214 conserved in the protein kinase family are located in the middle of the network. The water molecule forming a hydrogen bond with Asp214 appears to be deformed. In addition, mutational analysis of His148 in CK2 showed significant reductions by 40%–75% in the catalytic efficiency with similar affinity for ATP. Likewise, remarkable reductions to less than 5% were shown by corresponding mutations on His131 in death-associated protein kinase 1, which belongs to a group different from that of CK2. These findings shed new light on the catalytic mechanism of protein kinases in which the hydrogen bond network through the C-terminal domain may assist the general base catalyst to extract a proton with a link to the bulk solvent via intermediates of a pair of residues.     

Protein‐engineering of chitosanase from Bacillus sp. MN to alter its substrate specificity

Partially acetylated chitosan oligosaccharides (paCOS) have various potential applications in agriculture, biomedicine, and pharmaceutics due to their suitable bioactivities. One method to produce paCOS is partial chemical hydrolysis of chitosan polymers, but that leads to poorly defined mixtures of oligosaccharides. However, the effective production of defined paCOS is crucial for fundamental research and for developing applications. A more promising approach is enzymatic depolymerization of chitosan using chitinases or chitosanases, as the substrate specificity of the enzyme determines the composition of the oligomeric products. Protein‐engineering of these enzymes to alter their substrate specificity can overcome the limitations associated with naturally occurring enzymes and expand the spectrum of specific paCOS that can be produced. Here, engineering the substrate specificity of Bacillus sp. MN chitosanase is described for the first time. Two muteins with active site substitutions can accept N‐acetyl‐D‐glucosamine units at their subsite (?2), which is impossible for the wildtype enzyme.     

Glyoxal as an alternative fixative to formaldehyde in immunostaining and super‐resolution microscopy

Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA. Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA‐based protocols. Glyoxal acted faster than PFA, cross‐linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA‐based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.     

Selenium Utilization by GPX4 Is Required to Prevent Hydroperoxide-Induced Ferroptosis

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A three‐component system incorporating Ppd‐D1, copy number variation at Ppd‐B1, and numerous small‐effect quantitative trait loci facilitates adaptation of heading time in winter wheat cultivars of worldwide origin

The broad adaptability of heading time has contributed to the global success of wheat in a diverse array of climatic conditions. Here, we investigated the genetic architecture underlying heading time in a large panel of 1,110 winter wheat cultivars of worldwide origin. Genome‐wide association mapping, in combination with the analysis of major phenology loci, revealed a three‐component system that facilitates the adaptation of heading time in winter wheat. The photoperiod sensitivity locus Ppd‐D1 was found to account for almost half of the genotypic variance in this panel and can advance or delay heading by many days. In addition, copy number variation at Ppd‐B1 was the second most important source of variation in heading, explaining 8.3% of the genotypic variance. Results from association mapping and genomic prediction indicated that the remaining variation is attributed to numerous small‐effect quantitative trait loci that facilitate fine‐tuning of heading to the local climatic conditions. Collectively, our results underpin the importance of the two Ppd‐1 loci for the adaptation of heading time in winter wheat and illustrate how the three components have been exploited for wheat breeding globally.     

Burning Forests,Rising Power: Towards a Constitutionality Process in Mount Carmel Biosphere Reserve

Powerful states and elites frequently manage protected areas with little or no concern for historic land uses, people, or governance practices, justified by ideologies that portray these areas as “pure nature” to be protected from humans. New international participatory platforms, such as the UNESCO Man and Biosphere Program, coupled with strategic active agency, have provided an opportunity for challenging the fortress model of conservation in Israel. We examine the change in Israel’s government ecological policies following its failure in managing the Carmel forests, whereby its bargaining power with the local Druze-Arab minority was significantly reduced, opening a window of opportunity for the Druze to take advantage of new UNESCO rules on local participation to create management institutions for the local forest commons.