Control of steroid receptor dynamics and function by genomic actions of the cochaperones p23 and Bag-1L

Molecular chaperones encompass a group of unrelated proteins that facilitate the correct assembly and disassembly of other macromolecular structures, which they themselves do not remain a part of. They associate with a large and diverse set of coregulators termed cochaperones that regulate their function and specificity. Amongst others, chaperones and cochaperones regulate the activity of several signaling molecules including steroid receptors, which upon ligand binding interact with discrete nucleotide sequences within the nucleus to control the expression of diverse physiological and developmental genes. Molecular chaperones and cochaperones are typically known to provide the correct conformation for ligand binding by the steroid receptors. While this contribution is widely accepted, recent studies have reported that they further modulate steroid receptor action outside ligand binding. They are thought to contribute to receptor turnover, transport of the receptor to different subcellular localizations, recycling of the receptor on chromatin and even stabilization of the DNA-binding properties of the receptor. In addition to these combined effects with molecular chaperones, cochaperones are reported to have additional functions that are independent of molecular chaperones. Some of these functions also impact on steroid receptor action. Two well-studied examples are the cochaperones p23 and Bag-1L, which have been identified as modulators of steroid receptor activity in nuclei. Understanding details of their regulatory action will provide new therapeutic opportunities of controlling steroid receptor action independent of the widespread effects of molecular chaperones

DNA methylation in BHK-21 cells

The level of 5-methyl cytosine residues is higher in the DNA of polyoma-virus-transformed BHK-21/PyY cells than in the DNA of the non-transformed BHK-21/C13 cells. The 5-methyl cytosine residues arise as a result of the transfer of methyl groups from S-adenosyl-L-methionine to DNA cytosine moieties by DNA methylases located in nuclei of these cells. DNA methylases have been isolated and partially purified from nuclei of BHK-21/C13 and BHK-21/PyY cells and most of the properties of these two enzymes appear to be identical. However slight differences exist in the sequences these enzymes methylate in substrate E. coli DNA as shown by studies of the level of methylation of pyrimidine isostiohs fractionated from the in vitro methylated substrate. These differences can neither account for the differences in the m vivo level of DNA methylation in the two hamster cell lines nor can they provide any evidence to suggest that the higher level of methylation in the BHK-21/PyY DNA may be due to the presence of a virus-coded DNA methylase(s) in these cells. It seems that the two BHK-21 cell genomes may be organised differently. Further studies have shown differences in the arrangements of the two genomes with respect to 5-methyl cytosine residues. The highly repetitive region of the BHK-21/PyY cell DNA is twice as methylation as the corresponding region of the BHK-21/C13 cell DNA. The higher level of methylation of the BHK-21/PyY cell DNA may therefore be due to possible reiteration of certain sequences containing 5-methyl cytosine which may simply occur less frequently in the BHK-21/C13 cell genome. The role of eukaryotic DNA methylation is as yet uncertain. It has been shown in this work that nuclei of BHK-21/C13 cells contain a SAM-dependent nuclease which appears to degrade unmethylated DNA but Inactive towards DNA previously methylated by the BHK-21/PyY DNA methylase. The biological significance of this enzyme is unknown and it is not yet certain whether or not this enzyme is analogous to any of the bacterial restriction enzymes

Glucocorticoids synergize with IL-1β to induce TLR2 expression via MAP Kinase Phosphatase-1-dependent dual Inhibition of MAPK JNK and p38 in epithelial cells

BACKGROUND: Despite the importance of glucocorticoids in suppressing immune and inflammatory responses, their role in enhancing host immune and defense response against invading bacteria is poorly understood. Toll-like receptor 2 (TLR2) has recently gained importance as one of the major host defense receptors. The increased expression of TLR2 in response to bacteria-induced cytokines has been thought to be crucial for the accelerated immune response and resensitization of epithelial cells to invading pathogens. RESULTS: We show that IL-1β, a key proinflammatory cytokine, greatly up-regulates TLR2 expression in human epithelial cells via a positive IKKβ-IκBα-dependent NF-κB pathway and negative MEKK1-MKK4/7-JNK1/2 and MKK3/6-p38 α/β pathways. Glucocorticoids synergistically enhance IL-1β-induced TLR2 expression via specific up-regulation of the MAP kinase phosphatase-1 that, in turn, leads to dephosphorylation and inactivation of both MAPK JNK and p38, the negative regulators for TLR2 induction. CONCLUSION: These results indicate that glucocorticoids not only suppress immune and inflammatory response, but also enhance the expression of the host defense receptor, TLR2. Thus, our studies may bring new insights into the novel role of glucocorticoids in orchestrating and optimizing host immune and defense responses during bacterial infections and enhance our understanding of the signaling mechanisms underlying the glucocorticoid-mediated attenuation of MAPK

Highly disordered histone H1-DNA model complexes and their condensates.

Disordered proteins play an essential role in a wide variety of biological processes, and are often posttranslationally modified. One such protein is histone H1; its highly disordered C-terminal tail (CH1) condenses internucleosomal linker DNA in chromatin in a way that is still poorly understood. Moreover, CH1 is phosphorylated in a cell cycle-dependent manner that correlates with changes in the chromatin condensation level. Here we present a model system that recapitulates key aspects of the in vivo process, and also allows a detailed structural and biophysical analysis of the stages before and after condensation. CH1 remains disordered in the DNA-bound state, despite its nanomolar affinity. Phase-separated droplets (coacervates) form, containing higher-order assemblies of CH1/DNA complexes. Phosphorylation at three serine residues, spaced along the length of the tail, has little effect on the local properties of the condensate. However, it dramatically alters higher-order structure in the coacervate and reduces partitioning to the coacervate phase. These observations show that disordered proteins can bind tightly to DNA without a disorder-to-order transition. Importantly, they also provide mechanistic insights into how higher-order structures can be exquisitely sensitive to perturbation by posttranslational modifications, thus broadening the repertoire of mechanisms that might regulate chromatin and other macromolecular assemblies

Dual specificity phosphatase 1 (DUSP1) regulates a subset of LPS-induced genes and protects mice from lethal endotoxin shock

Activation of the mitogen-activated protein kinase (MAPK) cascade after Toll-like receptor stimulation enables innate immune cells to rapidly activate cytokine gene expression. A balanced response to signals of infectious danger requires that cellular activation is transient. Here, we identify the MAPK phosphatase dual specificity phosphatase 1 (DUSP1) as an essential endogenous regulator of the inflammatory response to lipopolysaccharide (LPS). DUSP1-deficient (DUSP1−/−) bone marrow–derived macrophages showed selectively prolonged activation of p38 MAPK and increased cytokine production. Intraperitoneal challenge of DUSP1−/− mice with LPS caused increased lethality and overshooting production of interleukin (IL)-6 and tumor necrosis factor α. Transcriptional profiling revealed that DUSP1 controls a significant fraction of LPS-induced genes, which includes IL-6 and IL-10 as well as the chemokines CCL3, CCL4, and CXCL2. In contrast, the expression of the important mediators of endotoxin lethality, interferon γ and IL-12, was not significantly altered by the absence of DUSP1. These data together demonstrate a specific regulatory role of DUSP1 in controlling a subset of LPS-induced genes that determines the outcome of endotoxin shock