Protein kinase A (PKA), the main effector of cAMP in eukaryotes, is a paradigm for the mechanisms of ligand-dependent and allosteric regulation in signalling. Here we report the orthologous but ...cAMP-independent PKA of the protozoan Trypanosoma and identify 7-deaza-nucleosides as potent activators (EC
≥ 6.5 nM) and high affinity ligands (K
≥ 8 nM). A co-crystal structure of trypanosome PKA with 7-cyano-7-deazainosine and molecular docking show how substitution of key amino acids in both CNB domains of the regulatory subunit and its unique C-terminal αD helix account for this ligand swap between trypanosome PKA and canonical cAMP-dependent PKAs. We propose nucleoside-related endogenous activators of Trypanosoma brucei PKA (TbPKA). The existence of eukaryotic CNB domains not associated with binding of cyclic nucleotides suggests that orphan CNB domains in other eukaryotes may bind undiscovered signalling molecules. Phosphoproteome analysis validates 7-cyano-7-deazainosine as powerful cell-permeable inducer to explore cAMP-independent PKA signalling in medically important neglected pathogens.
The sympathetic nervous system is a major mediator of heart function. Intercalated discs composed of desmosomes, adherens junctions, and gap junctions provide the structural backbone for coordinated ...contraction of cardiac myocytes.
Gap junctions dynamically remodel to adapt to sympathetic signaling. However, it is unknown whether such rapid adaption also occurs for the adhesive function provided by desmosomes and adherens junctions.
Atomic force microscopy revealed that β-adrenergic signaling enhances both the number of desmoglein 2-specific interactions along cell junctions and the mean desmoglein 2-mediated binding forces, whereas N-cadherin-mediated interactions were not affected. This was accompanied by increased cell cohesion in cardiac myocyte cultures and murine heart slices. Enhanced desmoglein 2-positive contacts and increased junction length as revealed by immunofluorescence and electron microscopy reflected cAMP-induced reorganization of intercellular contacts. The mechanism underlying cAMP-mediated strengthening of desmoglein 2 binding was dependent on expression of the intercalated disc plaque protein plakoglobin (Pg) and direct phosphorylation at S665 by protein kinase A: Pg deficiency as well as overexpression of the phospho-deficient Pg-mutant S665A abrogated both cAMP-mediated junctional remodeling and increase of cohesion. Moreover, Pg knockout hearts failed to functionally adapt to adrenergic stimulation.
Taken together, we provide first evidence for positive adhesiotropy as a new cardiac function of sympathetic signaling. Positive adhesiotropy is dependent on Pg phosphorylation at S665 by protein kinase A. This mechanism may be of high medical relevance because loss of junctional Pg is a hallmark of arrhythmogenic cardiomyopathy.
The
Ash1 protein is a trithorax-group (trxG) regulator that antagonizes Polycomb repression at HOX genes. Ash1 di-methylates lysine 36 in histone H3 (H3K36me2) but how this activity is controlled and ...at which genes it functions is not well understood. We show that Ash1 protein purified from
exists in a complex with MRG15 and Caf1 that we named AMC. In
and human AMC, MRG15 binds a conserved FxLP motif near the Ash1 SET domain and stimulates H3K36 di-methylation on nucleosomes.
-null and
catalytic mutants show remarkably specific trxG phenotypes: stochastic loss of HOX gene expression and homeotic transformations in adults. In mutants lacking AMC, H3K36me2 bulk levels appear undiminished but H3K36me2 is reduced in the chromatin of HOX and other AMC-regulated genes. AMC therefore appears to act on top of the H3K36me2/me3 landscape generated by the major H3K36 methyltransferases NSD and Set2. Our analyses suggest that H3K36 di-methylation at HOX genes is the crucial physiological function of AMC and the mechanism by which the complex antagonizes Polycomb repression at these genes.
Histone methylation plays a key role in establishing and maintaining stable gene expression patterns during cellular differentiation and embryonic development. Here, we report the characterization of ...the fungal metabolite chaetocin as the first inhibitor of a lysine-specific histone methyltransferase. Chaetocin is specific for the methyltransferase SU(VAR)3-9 both in vitro and in vivo and may therefore be used to study heterochromatin-mediated gene repression.
DNA-protein crosslinks (DPCs) are pervasive DNA lesions that are induced by reactive metabolites and various chemotherapeutic agents. Here, we develop a technique for the Purification of x-linked ...Proteins (PxP), which allows identification and tracking of diverse DPCs in mammalian cells. Using PxP, we investigate DPC repair in cells genetically-engineered to express variants of the SPRTN protease that cause premature ageing and early-onset liver cancer in Ruijs-Aalfs syndrome patients. We find an unexpected role for SPRTN in global-genome DPC repair, that does not rely on replication-coupled detection of the lesion. Mechanistically, we demonstrate that replication-independent DPC cleavage by SPRTN requires SUMO-targeted ubiquitylation of the protein adduct and occurs in addition to proteasomal DPC degradation. Defective ubiquitin binding of SPRTN patient variants compromises global-genome DPC repair and causes synthetic lethality in combination with a reduction in proteasomal DPC repair capacity.
Histone lysine methylation is generally performed by SET domain methyltransferases and regulates chromatin structure and gene expression. Here, we identify human C21orf127 (HEMK2, N6AMT1, PrmC), a ...member of the seven-β-strand family of putative methyltransferases, as a novel histone lysine methyltransferase. C21orf127 functions as an obligate heterodimer with TRMT112, writing the methylation mark on lysine 12 of histone H4 (H4K12) in vitro and in vivo. We characterized H4K12 recognition by solving the crystal structure of human C21orf127-TRMT112, hereafter termed 'lysine methyltransferase 9' (KMT9), in complex with S-adenosyl-homocysteine and H4K12me1 peptide. Additional analyses revealed enrichment for KMT9 and H4K12me1 at the promoters of numerous genes encoding cell cycle regulators and control of cell cycle progression by KMT9. Importantly, KMT9 depletion severely affects the proliferation of androgen receptor-dependent, as well as that of castration- and enzalutamide-resistant prostate cancer cells and xenograft tumors. Our data link H4K12 methylation with KMT9-dependent regulation of androgen-independent prostate tumor cell proliferation, thereby providing a promising paradigm for the treatment of castration-resistant prostate cancer.
Loss of the p53-inducible
in p53-proficient CRC cell lines results in increased sensitivity to DNA-damaging chemotherapeutics. Here, we comprehensively analyze how
affects the p53-induced ...transcriptional program.
Using a CRISPR/Cas9-approach, we deleted the p53 binding site in the
promoter of SW480 colorectal cancer cells and subjected them to RNA-Seq analysis after the activation of ectopic p53. RNA affinity purification followed by mass spectrometry was used to identify proteins associated with
.
Loss of the p53-inducibility of
resulted in an ~1.8-fold increase in the number of significantly regulated mRNAs compared to
wild-type cells after ectopic activation of p53. A subset of direct p53 target genes, such as
and
displayed significantly stronger induction when the p53-inducibility of
was abrogated. Loss of the p53-inducibility of
resulted in alternative splicing of a small number of mRNAs, such as
,
, and
. Several RNA binding proteins involved in pre-mRNA splicing were identified as interaction partners of
by mass spectrometry.
Our results suggest that
may restrict the extent and strength of p53-mediated transcriptional changes via context-dependent regulation of the expression and splicing of a subset of p53-regulated genes.
Binge drinking and chronic exposure to ethanol contribute to alcoholic liver diseases (ALDs). A potential link between ALDs and circadian disruption has been observed, though how different patterns ...of alcohol consumption differentially impact hepatic circadian metabolism remains virtually unexplored. Using acute versus chronic ethanol feeding, we reveal differential reprogramming of the circadian transcriptome in the liver. Specifically, rewiring of diurnal SREBP transcriptional pathway leads to distinct hepatic signatures in acetyl-CoA metabolism that are translated into the subcellular patterns of protein acetylation. Thus, distinct drinking patterns of alcohol dictate differential adaptation of hepatic circadian metabolism.
Packaging of DNA into nucleosomes and the formation of higher-order chromatin structures determine DNA accessibility and activity of genome domains. We identified an RNA-dependent mechanism ...maintaining the open chromatin structure within euchromatic regions in Drosophila cells. The mechanism of reversible chromatin opening, reconstituted in vitro, depends on the Drosophila decondensation factor 31 (Df31) that specifically binds to RNA and localizes to euchromatic regions. Df31 is capable to tether a heterogeneous pool of short, single-stranded RNAs to chromatin. This class of chromatin-associated RNA (caRNA) is stably linked to chromatin and is largely composed of snoRNAs, which are preferentially bound by Df31. We suggest that the Df31-mediated linkage of snoRNAs and chromatin, forms a RNA-chromatin network resulting in the establishment of open chromatin domains. Analysis of caRNAs in human cells also reveals a strong enrichment of snoRNAs, implying a conserved role for these molecules in higher-order structures of chromatin.
► snoRNAs maintain higher-order structures of chromatin accessible ► A fraction of the snoRNAs is stably associated with chromatin ► Df31 protein is specifically binding to histones and snoRNAs ► Df31 tethers snoRNA to euchromatic regions in Drosophila
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