The Catalytic Aspartate Is Protonated in the Michaelis Complex Formed between Trypsin and an in Vitro Evolved Substrate-like Inhibitor: A REFINED MECHANISM OF SERINE PROTEASE ACTION.

The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 A) x-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism. The electron density indicates the protonation state of all catalytic residues where the catalytic histidine is, as expected, in its neutral state prior to the acylation step by the catalytic serine. The carboxyl group of the catalytic aspartate displays an asymmetric electron density so that the O(delta2)-C(gamma) bond appears to be a double bond, with O(delta2) involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on O(delta1) atom could a density functional theory simulation reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mF(obs) - DF(calc) density above 2.5 sigma next to O(delta1). As a possible functional role for the neutral aspartate in the active site, we propose that in the substrate-bound form, the neutral aspartate residue helps to keep the pK(a) of the histidine sufficiently low, in the active neutral form. When the histidine receives a proton during the catalytic cycle, the aspartate becomes simultaneously negatively charged, providing additional stabilization for the protonated histidine and indirectly to the tetrahedral intermediate. This novel proposal unifies the seemingly conflicting experimental observations, which were previously seen as either supporting the charge relay mechanism or the neutral pK(a) histidine theory

Inhibition of DNA methyltransferase leads to increased genomic 5-hydroxymethylcytosine levels in hematopoietic cells.

5-Hydroxymethylcytosine (5hmC) is produced from 5-methylcytosine (5mC) by Ten-eleven translocation (TET) dioxygenases. The epigenetic modification 5hmC has crucial roles in both cellular development and differentiation. The 5hmC level is particularly high in the brain. While 5mC is generally associated with gene silencing/reduced expression, 5hmC is a more permissive epigenetic mark. To understand its physiological function, an easy and accurate quantification method is required. Here, we have developed a novel LC-MS/MS-based approach to quantify both genomic 5mC and 5hmC contents. The method is based on the liberation of nucleobases by formic acid. Applying this method, we characterized the levels of DNA methylation and hydroxymethylation in mouse brain and liver, primary hepatocytes, and various cell lines. Using this approach, we confirm that the treatment of different cell lines with the DNA methyltransferase inhibitor 5-aza-2\u27-deoxycytidine leads to a decrease in 5mC content. This decrease was accompanied by an increase in 5hmC levels in cell lines of hematopoietic origin. Finally, we showed that ascorbate elevates the levels of 5hmC and augments the effect of 5-aza-2\u27-deoxycytidine without significantly influencing 5mC levels

Evidence, detailed characterization and clinical context of complement activation in acute multisystem inflammatory syndrome in children

Multisystem inflammatory syndrome in children (MIS-C) is a rare, life-threatening complication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. MIS-C develops with high fever, marked inflammation and shock-like picture several weeks after exposure to, or mild infection with SARS-CoV-2. Deep immune profiling identified activated macrophages, neutrophils, B-plasmablasts and CD8 + T cells as key determinants of pathogenesis together with multiple inflammatory markers. The disease rapidly responds to intravenous immunoglobulin (IVIG) treatment with clear changes of immune features. Here we present the results of a comprehensive analysis of the complement system in the context of MIS-C activity and describe characteristic changes during IVIG treatment. We show that activation markers of the classical, alternative and terminal pathways are highly elevated, that the activation is largely independent of anti-SARS-CoV-2 humoral immune response, but is strongly associated with markers of macrophage activation. Decrease of complement activation is closely associated with rapid improvement of MIS-C after IVIG treatment