This review describes different synthetic strategies towards sequence‐defined, monodisperse macromolecules, which are built up by iterative approaches and lead to linear non‐natural polymer ...structures. The review is divided in three parts: solution phase‐, solid phase‐, and fluorous‐ and polymer‐tethered approaches. Moreover, synthesis procedures leading to conjugated and non‐conjugated macromolecules are considered and discussed in the respective sections. A major focus in the evaluation is the applicability of the different approaches in polymer chemistry. In this context, simple procedures for monomer and oligomer synthesis, overall yields, scalability, purity of the oligomers, and the achievable level of control (side‐chains, backbone, stereochemistry) are important benchmarks.
A review of synthesis procedures leading to monodisperse and sequence‐defined linear macromolecules, including conjugated and non‐conjugated oligomers, is presented. The approaches are summarized and analyzed in terms of applicability in polymer science. Simple synthesis procedures, scales, overall yields, achievable level of control, and the purity of the obtained macromolecules are important benchmarks.
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•First cryo-EM structures of CRAF2/14-3-32 (3.4 Å) and CRAF2/14-3-32/MEK12 (4.2 Å)•EM density visible for ATP-site inhibitor GDC-0879 in CRAF2/14-3-32/MEK12 structure.•Overall similar ...architecture to corresponding BRAF structures.
RAF protein kinases are essential effectors in the MAPK pathway and are important cancer drug targets. Structural understanding of RAF activation is so far based on cryo-electron microscopy (cryo-EM) and X-ray structures of BRAF in different conformational states as inactive or active complexes with KRAS, 14-3-3 and MEK1. In this study, we have solved the first cryo-EM structures of CRAF2/14-3-32 at 3.4 Å resolution and CRAF2/14-3-32/MEK12 at 4.2 Å resolution using CRAF kinase domain expressed as constitutively active Y340D/Y341D mutant in insect cells. The overall architecture of our CRAF2/14-3-32 and CRAF2/14-3-32/MEK12 cryo-EM structures is highly similar to corresponding BRAF structures in complex with 14-3-3 or 14-3-3/MEK1 and represent the activated dimeric RAF conformation. Our CRAF cryo-EM structures provide additional insights into structural understanding of the activated CRAF2/14-3-32/MEK12 complex.
Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by chronic inflammation and progressive fibrosis of the biliary tree. The majority of PSC patients suffer ...from concomitant inflammatory bowel disease (IBD), which has been suggested to promote disease development and progression. However, the molecular mechanisms by which intestinal inflammation may aggravate cholestatic liver disease remain incompletely understood. Here, we employ an IBD-PSC mouse model to investigate the impact of colitis on bile acid metabolism and cholestatic liver injury. Unexpectedly, intestinal inflammation and barrier impairment improve acute cholestatic liver injury and result in reduced liver fibrosis in a chronic colitis model. This phenotype is independent of colitis-induced alterations of microbial bile acid metabolism but mediated via hepatocellular NF-κB activation by lipopolysaccharide (LPS), which suppresses bile acid metabolism in-vitro and in-vivo. This study identifies a colitis-triggered protective circuit suppressing cholestatic liver disease and encourages multi-organ treatment strategies for PSC.
Methane and carbon dioxide were measured with an autonomous and continuous running system on a ferry line crossing the Baltic Sea on a 2-3 day interval from the Mecklenburg Bight to the Gulf of ...Finland in 2010. Surface methane saturations show great seasonal differences in shallow regions like the Mecklenburg Bight (103-507%) compared to deeper regions like the Gotland Basin (96-161%). The influence of controlling parameters like temperature, wind, mixing depth and processes like upwelling, mixing of the water column and sedimentary methane emissions on methane oversaturation and emission to the atmosphere are investigated. Upwelling was found to influence methane surface concentrations in the area of Gotland significantly during the summer period. In February 2010, an event of elevated methane concentrations in the surface water and water column of the Arkona Basin was observed, which could be linked to a wind-derived water level change as a potential triggering mechanism. The Baltic Sea is a source of methane to the atmosphere throughout the year, with highest fluxes occurring during the winter season. Stratification was found to promote the formation of a methane reservoir in deeper regions like Gulf of Finland or Bornholm Basin, which leads to long lasting elevated methane concentrations and enhanced methane fluxes, when mixed to the surface during mixed layer deepening in autumn and winter. Methane concentrations and fluxes from shallow regions like the Mecklenburg Bight are predominantly controlled by sedimentary production and consumption of methane, wind events and the change in temperature-dependent solubility of methane in the surface water. Methane fluxes vary significantly in shallow regions (e.g. Mecklenburg Bight) and regions with a temporal stratification (e.g. Bornholm Basin, Gulf of Finland). On the contrary, areas with a permanent stratification like the Gotland Basin show only small seasonal fluctuations in methane fluxes.
Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors and chromatin regulators, drive high expression of genes ...implicated in cell identity and disease, such as lineage-controlling transcription factors and oncogenes. BRD4 and CDK7 are positive regulators of SE-mediated transcription. By contrast, negative regulators of SE-associated genes have not been well described. Here we show that the Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We report that the natural product cortistatin A (CA) selectively inhibits Mediator kinases, has anti-leukaemic activity in vitro and in vivo, and disproportionately induces upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the transcription factors CEBPA, IRF8, IRF1 and ETV6 (refs 6-8). The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has anti-leukaemic activity. Individually increasing or decreasing the expression of these transcription factors suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to the dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types, and can be pharmacologically targeted as a therapeutic approach to AML.
Coeliac disease (CeD) is characterized by gliadin-induced intestinal inflammation appearing in genetically susceptible individuals, such as HLA-DQ2.5 carriers. CeD, as well as other chronic ...intestinal disorders, such as Crohn's disease (CD) and ulcerative colitis, has been associated with increased morbidity and mortality, but the causes are unknown. We systematically analysed CeD-associated diagnoses and compared them to conditions enriched in subjects with CD/UC as well as in HLA-DQ2.5 carriers without CeD. We compared the overall and cause-specific mortality and morbidity of 3,001 patients with CeD, 2,020 with CD, 4,399 with UC and 492,200 controls in the community-based UK Biobank. Disease-specific phenotypes were assessed with the multivariable Phenome Wide Association Study (PheWAS) method. Associations were adjusted for age, sex and body mass index. All disease groups displayed higher overall mortality than controls (CD: aHR = 1.911.70-2.17; UC: aHR = 1.32 1.20-1.46; CeD: aHR = 1.38 1.22-1.55). Cardiovascular and cancer-related deaths were responsible for the majority of fatalities. PheWAS analysis revealed 166 Phecodes overrepresented in all three disorders, whereas only ~ 20% of enriched Phecodes were disease specific. Seven of the 58 identified CeD-specific Phecodes were enriched in individuals homozygous for HLA-DQ2.5 without diagnosed CeD. Four out of these seven Phecodes and eight out of 19 HLA-DQ2.5 specific Phecodes were more common in homozygous HLA-DQ2.5 subjects with vs. without CeD, highlighting the interplay between genetics and diagnosis-related factors. Our study illustrates that the morbidity and mortality in CeD share similarities with CD/UC, while the CeD-restricted conditions might be driven by both inherited and acquired factors.
In contrast with the very well explored concept of structure–activity relationship, similar studies are missing for the dependency between binding kinetics and compound structure of a protein ligand ...complex, the structure–kinetic relationship. Here, we present a structure–kinetic relationship study of the cyclin-dependent kinase 8 (CDK8)/cyclin C (CycC) complex. The scaffold moiety of the compounds is anchored in the kinase deep pocket and extended with diverse functional groups toward the hinge region and the front pocket. These variations can cause the compounds to change from fast to slow binding kinetics, resulting in an improved residence time. The flip of the DFG motif (“DMG” in CDK8) to the inactive DFG-out conformation appears to have relatively little influence on the velocity of binding. Hydrogen bonding with the kinase hinge region contributes to the residence time but has less impact than hydrophobic complementarities within the kinase front pocket.
The Mediator kinase module regulates eukaryotic transcription by phosphorylating transcription-related targets and by modulating the association ofMediator and RNA polymerase II. The activity of its ...catalytic core, cyclin-dependent kinase 8 (CDK8), is controlled by Cyclin C and regulatory subunit MED12, with its deregulation contributing to numerous malignancies. Here, we combine in vitro biochemistry, cross-linking coupled to mass spectrometry, and in vivo studies to describe the binding location of the N-terminal segment of MED12 on the CDK8/Cyclin C complex and to gain mechanistic insights into the activation of CDK8 by MED12. Our data demonstrate that the N-terminal portion of MED12 wraps around CDK8, whereby it positions an “activation helix” close to the T-loop of CDK8 for its activation. Intriguingly, mutations in the activation helix that are frequently found in cancers do not diminish the affinity of MED12 for CDK8, yet likely alter the exact positioning of the activation helix. Furthermore, we find the transcriptome-wide gene-expression changes in human cells that result from a mutation in the MED12 activation helix to correlate with deregulated genes in breast and colon cancer. Finally, functional assays in the presence of kinase inhibitors reveal that binding of MED12 remodels the active site of CDK8 and thereby precludes the inhibition of ternary CDK8 complexes by type II kinase inhibitors. Taken together, our results not only allowus to propose a revised model of how CDK8 activity is regulated by MED12, but also offer a path forward in developing small molecules that target CDK8 in its MED12-bound form.
Genetic variants at the SORT1 locus in humans, which cause increased SORT1 expression in the liver, are significantly associated with reduced plasma levels of LDL cholesterol and apolipoprotein B ...(apoB). However, the role of hepatic sortilin remains controversial, as genetic deletion of sortilin in mice has resulted in variable and conflicting effects on apoB secretion. Here, we found that Sort1-KO mice on a chow diet and several Sort1-deficient hepatocyte lines displayed no difference in apoB secretion. When these models were challenged with high-fat diet or ER stress, the loss of Sort1 expression resulted in a significant increase in apoB-100 secretion. Sort1-overexpression studies yielded reciprocal results. Importantly, carriers of SORT1 variant with diabetes had larger decreases in plasma apoB, TG, and VLDL and LDL particle number as compared with people without diabetes with the same variants. We conclude that, under basal nonstressed conditions, loss of sortilin has little effect on hepatocyte apoB secretion, whereas, in the setting of lipid loading or ER stress, sortilin deficiency leads to increased apoB secretion. These results are consistent with the directionality of effect in human genetics studies and suggest that, under stress conditions, hepatic sortilin directs apoB toward lysosomal degradation rather than secretion, potentially serving as a quality control step in the apoB secretion pathway in hepatocytes.
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