Julia H. White

Suppression of inflammation by a synthetic histone mimic

Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, although vital for immune defence, is frequently deleterious to the host due to the exaggerated production of inflammatory proteins. The scope of inflammatory responses reflects the activation state of signalling proteins upstream of inflammatory genes as well as signal-induced assembly of nuclear chromatin complexes that support mRNA expression. Recognition of post-translationally modified histones by nuclear proteins that initiate mRNA transcription and support mRNA elongation is a critical step in the regulation of gene expression. Here we present a novel pharmacological approach that targets inflammatory gene expression by interfering with the recognition of acetylated histones by the bromodomain and extra terminal domain (BET) family of proteins. We describe a synthetic compound (I-BET) that by ‘mimicking’ acetylated histones disrupts chromatin complexes responsible for the expression of key inflammatory genes in activated macrophages, and confers protection against lipopolysaccharide-induced endotoxic shock and bacteria-induced sepsis. Our findings suggest that synthetic compounds specifically targeting proteins that recognize post-translationally modified histones can serve as a new generation of immunomodulatory drugs.

Discovery and Characterization of Small Molecule Inhibitors of the Bet Family Bromodomains.

Epigenetic mechanisms of gene regulation have a profound role in normal development and disease processes. An integral part of this mechanism occurs through lysine acetylation of histone tails which are recognized by bromodomains. While the biological and structural characterization of many bromodomain containing proteins has advanced considerably, the therapeutic tractability of this protein family is only now becoming understood. This paper describes the discovery and molecular characterization of potent (nM) small molecule inhibitors that disrupt the function of the BET family of bromodomains (Brd2, Brd3, and Brd4). By using a combination of phenotypic screening, chemoproteomics, and biophysical studies, we have discovered that the protein-protein interactions between bromodomains and acetylated histones can be antagonized by selective small molecules that bind at the acetylated lysine recognition pocket. X-ray crystal structures of compounds bound into bromodomains of Brd2 and Brd4 elucidate the molecular interactions of binding and explain the precisely defined stereochemistry required for activity.

Protein–protein interactions at G-protein-coupled receptors

The basic module of signal transduction that involves G-protein-coupled receptors is usually portrayed as comprising a receptor, a heterotrimeric G protein and an effector. It is now well established that regulated interactions between receptors and arrestins, and between G proteins and regulators of G-protein signalling alter the effectiveness and kinetics of information transfer. However, more recent studies have begun to identify a host of other proteins that interact selectively with individual receptors at both the intracellular and extracellular face of the membrane. Although the functional relevance of many of these interactions is only beginning to be understood, current information indicates that these interactions might determine receptor properties, such as cellular compartmentalization or signal selection, and can promote protein scaffolding into complexes that integrate function.

The γ-aminobutyric acid receptor B, but not the metabotropic glutamate receptor type-1, associates with lipid rafts in the rat cerebellum

Recent evidence suggests that specialized microdomains, called lipid rafts, exist within plasma membranes. These domains are enriched in cholesterol and sphingolipids and are resistant to non‐ionic detergent‐extraction at 4°C. They contain specific populations of membrane proteins, and can change their size and composition in response to cellular signals, resulting in activation of signalling cascades. Here, we demonstrate that both the metabotropic γ‐aminobutyric acid receptor B (GABAB receptor) and the metabotropic glutamate receptor‐1 from rat cerebellum are insoluble in the non‐ionic detergent Triton X‐100. However, only the GABAB receptor associates with raft fractions isolated from rat brain by sucrose gradient centrifugation. Moreover, increasing the stringency of isolation by decreasing the protein : detergent ratio caused an enrichment of the GABAB receptor in raft fractions. In contrast, depletion of cholesterol from cerebellar membranes by either saponin or methyl‐β‐cyclodextrin treatment, which solubilize known raft markers, also increased the solubility of the GABAB receptor. These properties are all consistent with an association of the GABAB receptor with lipid raft microdomains.

Comparative cellular distribution of GABAA and GABAB receptors in the human basal ganglia: Immunohistochemical colocalization of the α1 subunit of the GABAA receptor, and the GABABR1 and GABABR2 receptor subunits

The GABAB receptor is a G‐protein linked metabotropic receptor that is comprised of two major subunits, GABABR1 and GABABR2. In this study, the cellular distribution of the GABABR1 and GABABR2 subunits was investigated in the normal human basal ganglia using single and double immunohistochemical labeling techniques on fixed human brain tissue. The results showed that the GABAB receptor subunits GABABR1 and GABABR2 were both found on the same neurons and followed the same distribution patterns. In the striatum, these subunits were found on the five major types of interneurons based on morphology and neurochemical labeling (types 1, 2, 3, 5, 6) and showed weak labeling on the projection neurons (type 4). In the globus pallidus, intense GABABR1 and GABABR2 subunit labeling was found in large pallidal neurons, and in the substantia nigra, both pars compacta and pars reticulata neurons were labeled for both receptor subunits. Studies investigating the colocalization of the GABAA α1 subunit and GABAB receptor subunits showed that the GABAA receptor α1 subunit and the GABABR1 subunit were found together on GABAergic striatal interneurons (type 1 parvalbumin, type 2 calretinin, and type 3 GAD neurons) and on neurons in the globus pallidus and substantia nigra pars reticulata. GABABR1 and GABABR2 were found on substantia nigra pars compacta neurons but the GABAA receptor α1 subunit was absent from these neurons. The results of this study provide the morphological basis for GABAergic transmission within the human basal ganglia and provides evidence that GABA acts through both GABAA and GABAB receptors. That is, GABA acts through GABAB receptors, which are located on most of the cell types of the striatum, globus pallidus, and substantia nigra. GABA also acts through GABAA receptors containing the α1 subunit on specific striatal GABAergic interneurons and on output neurons of the globus pallidus and substantia nigra pars reticulata. J. Comp. Neurol. 470:339–356, 2004.

Prolyl oligopeptidase binds to GAP-43 and functions without its peptidase activity

Inhibitors of the enzyme prolyl oligopeptidase (PO) improve performance in rodent learning and memory tasks. PO inhibitors are also implicated in the action of drugs used to treat bipolar disorder: they reverse the effects of three mood stabilizers on the dynamic behaviour of neuronal growth cones. PO cleaves prolyl bonds in short peptides, suggesting that neuropeptides might be its brain substrates. PO is located in the cytosol, however, where it would not contact neuropeptides. Here, we show that mice with a targeted PO null-mutation have altered growth cone dynamics. The wild-type phenotype is restored by PO cDNAs encoding either native or a catalytically-dead enzyme. In addition, we show that PO binds to the growth-associated protein GAP-43, which is a key regulator of synaptic plasticity. Taken together, our results show that peptidase activity is not required for PO function in neurons and suggest that PO instead acts by binding to cytosolic proteins that control growth cone and synaptic function.

Coordinated action of NSF and PKC regulates GABAB receptor signaling efficacy.

The obligatory heterodimerization of the GABAB receptor (GBR) raises fundamental questions about molecular mechanisms controlling its signaling efficacy. Here, we show that NEM sensitive fusion (NSF) protein interacts directly with the GBR heterodimer both in rat brain synaptosomes and in CHO cells, forming a ternary complex that can be regulated by agonist stimulation. Inhibition of NSF binding with a peptide derived from GBR2 (TAT‐Pep‐27) did not affect basal signaling activity but almost completely abolished agonist‐promoted GBR desensitization in both CHO cells and hippocampal slices. Taken with the role of PKC in the desensitization process, our observation that TAT‐Pep‐27 prevented both agonist‐promoted recruitment of PKC and receptor phosphorylation suggests that NSF is a priming factor required for GBR desensitization. Given that GBR desensitization does not involve receptor internalization, the NSF/PKC coordinated action revealed herein suggests that NSF can regulate GPCR signalling efficacy independently of its role in membrane trafficking. The functional interaction between three bona fide regulators of neurotransmitter release, such as GBR, NSF and PKC, could shed new light on the modulation of presynaptic GBR action.

Identification of a novel asthma susceptibility gene on chromosome 1qter and its functional evaluation

Asthma is a multifactorial disease, in which the intricate interplay between genetic and environmental factors underlies the overall phenotype of the disease. Using a genome-wide scan for linkage in a population comprising of Danish families, we identified a novel linked locus on chromosome 1qter (LOD 3.6, asthma) and supporting evidence for this locus was identified for both asthma and atopic-asthma phenotypes in the GAIN (Genetics of Asthma International Network) families. The putative susceptibility gene was progressively localized to a 4.5 Mb region on chromosome 1q adjacent to the telomere, through a series of genotyping screens. Further screening using the pedigree-based association test (PBAT) identified polymorphisms in the OPN3 and CHML genes as being associated with asthma and atopic asthma after correcting for multiple comparisons. We observed that polymorphisms flanking the OPN3 and CHML genes wholly accounted for the original linkage in the Danish population and the genetic association was also confirmed in two separate studies involving the GAIN families. OPN3 and CHML are unique genes with no known function that are related to the pathophysiology of asthma. Significantly, analysis of gene expression at both RNA and protein levels, clearly demonstrated OPN3 expression in lung bronchial epithelia as well as immune cells, while CHML expression appeared minimal. Moreover, OPN3 down-regulation by siRNA knock-down in Jurkat cells suggested a possible role for OPN3 in modulation of T-cell responses. Collectively, these data suggest that OPN3 is an asthma susceptibility gene on 1qter, which unexpectedly may play a role in immune modulation.

GABAB receptor subunits, R1 and R2, in brainstem catecholamine and serotonin neurons.

GABA(B) receptors have been implicated in the GABAergic modulation of catecholaminergic and serotonergic pathways in the central nervous system. The GABA(B) receptor may require two subunits, GABA(B)R1 and GABA(B)R2, for functional activity. Using dual immunofluorescent labelling on adjacent cryostat sections, we investigated the presence of immunoreactivity for the GABA(B)R1 and GABA(B)R2 subunits in brainstem catecholamine (tyrosine hydroxylase-immunoreactive) and serotonin (tryptophan hydroxylase-immunoreactive) neurons. All neurons (>98%) examined in catecholamine groups A1, A2, A5, A6, C1, and serotonin groups B1-3 and B6-8 were immunoreactive for the GABA(B)R1 subunit. All A5 and A6 neurons (>97%) and at least 86% of A1, A2, C1, B2, B3, B7 and B8 neurons examined were GABA(B)R2-immunoreactive. The proportion of neurons with immunoreactivity for the GABA(B)R2 subunit varied between 0% and 99% for B1 neurons, and between 35% and 93% for B6 neurons. Statistical analysis showed that similar proportions of sampled neurons were immunoreactive for GABA(B)R1 and GABA(B)R2 in the A1, A5, A6, C1, B2 and B7 cell groups, whereas a smaller proportion of A2, B1, B3, B6 and B8 neurons were GABA(B)R2-immunoreactive than GABA(B)R1-immunoreactive. In general, our results suggest that GABA(B)R1 and GABA(B)R2 co-exist in the great majority of brainstem catecholamine and serotonin neurons. In the neurons that lack GABA(B)R2, the GABA(B)R1 subunit may act alone or with another protein.

IkappaB kinase epsilon interacts with p52 and promotes transactivation via p65.

The members of the NF-κB transcription factor family are key regulators of gene expression in the immune response. Different combinations of NF-κB subunits not only diverge in timing to induce transcription but also recognize varying sequences of the NF-κB-binding site of their target genes. The p52 subunit is generated as a result of processing of NF-κB2 p100. Here, we demonstrate that the non-canonical IκB kinase ϵ (IKKϵ) directly interacts with p100. In a transactivation assay, IKKϵ promoted the ability of p52 to transactivate gene expression. This effect was indirect, requiring p65, which was shown to be part of the IKKϵ-p52 complex and to be phosphorylated by IKKϵ. These novel interactions reveal a hitherto unknown function of IKKϵ in the regulation of the alternative NF-κB activation pathway involving p52 and p65.