The chemoreceptors of E. coli and S. typhimurium form stable oligomers that associate with the coupling protein CheW and the histidine kinase CheA to form an ultra-sensitive, ultra-stable signaling lattice. Attractant binding to the periplasmic domain of a given receptor dimer triggers a transmembrane conformational change transmitted through the receptor to its cytoplasmic kinase control module -a long 4-helix bundle that binds and regulates CheA kinase. The kinase control module comprises three functional regions: the adaptation region possessing the receptor adaptation sites; a coupling region that transmits signals between other regions; and the protein interaction region possessing contact sites for receptor oligomerization and for CheA/CheW binding. Based on the spatial clustering of known signal-locking Cys-substitutions and engineered disulfide bonds, the present study develops the yin-yang hypothesis for signal transmission through the kinase control module. This hypothesis proposes that signals are transmitted through the 4-helix bundle via changes in helix-helix packing, and that the helix packing changes in the adaptation and protein interaction regions are tightly and anti-symmetrically coupled. Specifically, strong helix packing in the adaptation region stabilizes the receptor on-state, while strong helix packing in the protein interaction region stabilizes the off-state.To test the yin-yang hypothesis, conserved sockets likely to strengthen specific helix-helix contacts via knob-in-hole packing interactions were identified in the adaptation, coupling and protein interaction regions. For 32 sockets, the knob side chain was truncated to Ala to weaken the knob-inhole packing and thereby destabilize the local helix-helix interaction provided by that socket. We term this approach a "knob truncation scan". Of the 32 knob truncations, 28 yielded stable receptors. Functional analysis of the signaling state of these receptors revealed 7 lock-off knob truncations, all located in the adaptation region, that trap the receptor in its "off" signaling state (low kinase activity, high methylation activity). Also revealed were 5 lock-on knob truncations, all located in the protein interaction region, that trap the "on" state (high kinase activity, low methylation activity). These findings provide strong evidence that a yin-yang coupling mechanism generates concerted, antisymmetric helix-helix packing changes within the adaptation and protein interaction regions during receptor on-off switching. Conserved sockets that stabilize local helix-helix interactions play a central role in this mechanism: in the on-state, sockets are formed in the adaptation region and disrupted in the protein interaction region, while the opposite is true in the off-state.*To whom correspondence should be addressed: falke@colorado.edu, Tel (303) Fax (303) The homodimeric chemoreceptors of bacterial chemotaxis span the inner cell membrane. These receptors bind attractants in the periplasmic space and generate a transmembrane co...

The HAMP domain is a conserved motif widely distributed in prokaryotic and lower eukaryotic organisms, where it is often found in transmembrane receptors that regulate two-component signaling pathways. The motif links receptor input and output modules and is essential to receptor structure and signal transduction. Recently, a structure was determined for a HAMP domain isolated from an unusual archeal membrane protein of unknown function [Hulko, M., et al. (2006) Cell 126, 929-940]. This study uses cysteine and disulfide chemistry to test this archeal HAMP model in the full-length, membrane-bound aspartate receptor of bacterial chemotaxis. The chemical reactivities of engineered Cys residues scanned throughout the aspartate receptor HAMP region are highly correlated with the degrees of solvent exposure of corresponding positions in the archeal HAMP structure. Both domains are homodimeric, and the individual subunits of both domains share the same helix-connector-helix organization with the same helical packing faces. Moreover, disulfide mapping reveals that the four helices of the aspartate receptor HAMP domain are arranged in the same parallel, four-helix bundle architecture observed in the archeal HAMP structure. One detectable difference is the packing of the extended connector between helices, which is not conserved. Finally, activity studies of the aspartate receptor indicate that contacts between HAMP helices 1 and 2' at the subunit interface play a critical role in modulating receptor on-off switching. Disulfide bonds linking this interface trap the receptor in its kinase-activating on-state, or its kinase inactivating off-state, depending on their location. Overall, the evidence suggests that the archeal HAMP structure accurately depicts the architecture of the conserved HAMP motif in transmembrane chemoreceptors. Both the on- and off-states of the aspartate receptor HAMP domain closely resemble the archeal HAMP structure, and only a small structural rearrangement occurs upon on-off switching. A model incorporating HAMP into the full receptor structure is proposed.

Epigenetic mechanisms, including DNA methylation and histone acetylation, regulate gene expression in idiopathic pulmonary arterial hypertension (IPAH). These mechanisms can modulate expression of extracellular superoxide dismutase (SOD3 or EC-SOD), a key vascular antioxidant enzyme, and loss of vascular SOD3 worsens outcomes in animal models of pulmonary arterial hypertension. We hypothesized that SOD3 gene expression is decreased in patients with IPAH due to aberrant DNA methylation and/or histone deacetylation. We used lung tissue and pulmonary artery smooth muscle cells (PASMC) from subjects with IPAH at transplantation and from failed donors (FD). Lung SOD3 mRNA expression and activity was decreased in IPAH vs. FD. In contrast, mitochondrial SOD (Mn-SOD or SOD2) protein expression was unchanged and intracellular SOD activity was unchanged. Using bisulfite sequencing in genomic lung or PASMC DNA, we found the methylation status of the SOD3 promoter was similar between FD and IPAH. Furthermore, treatment with 5-aza-2'-deoxycytidine did not increase PASMC SOD3 mRNA, suggesting DNA methylation was not responsible for PASMC SOD3 expression. Though total histone deacetylase (HDAC) activity, histone acetyltransferase (HAT) activity, acetylated histones, and acetylated SP1 were similar between IPAH and FD, treatment with two selective class I HDAC inhibitors increased SOD3 only in IPAH PASMC. Class I HDAC3 siRNA also increased SOD3 expression. Trichostatin A, a pan-HDAC inhibitor, decreased proliferation in IPAH, but not in FD PASMC. These data indicate that histone deacetylation, specifically via class I HDAC3, decreases SOD3 expression in PASMC and HDAC inhibitors may protect IPAH in part by increasing PASMC SOD3 expression.

Urothelial carcinoma accounts for most of the bladder cancer cases. Using next-generation sequencing (NGS) technology, we found that a significant percentage (83%) of tumors had mutations in chromatin-remodeling genes. Here, we examined the functional relevance of mutations in two chromatin-remodeling genes, EP300 and its paralog, CREBBP, which are mutated in almost one-third of patients. Interestingly, almost half of missense mutations cluster in the histone-acetyltransferase (HAT) domain of EP300/CREBBP. This domain catalyzes the transfer of an acetyl group to target molecules such as histones, thereby regulating chromatin dynamics. Thus, patients with EP300 or CREBBP mutations may have alterations in the ability of the corresponding proteins to modify histone proteins and control transcriptional profiles. In fact, it was determined that many of the missense HAT mutations in EP300 (64%) and CREBBP (78%) were HAT-inactivating. These inactivating mutations also correlated with invasive disease in patients. Strikingly, the prediction software Mutation Assessor accurately predicted the functional consequences of each HAT missense mutation. Finally, a gene expression signature was developed that associated with loss of HAT activity and that this signature was associated with more aggressive cancer in four patient datasets. Further supporting the notion that this score accurately reflects HAT activity, we found it is responsive to treatment of cancer cells to mocetinostat, a histone deacetylase (HDAC) inhibitor. Implication This study provides a rationale for targeted sequencing of EP300 and CREBBP and use of a gene profiling signature for predicting therapeutic response in patients.

Extracellular superoxide dismutase (EC-SOD), one of three mammalian SOD isoforms, is the sole extracellular enzymatic defense against superoxide. A known human single nucleotide polymorphism (SNP) in the matrix-binding domain of EC-SOD characterized by an arginine-to-glycine substitution at position 213 (R213G) redistributes EC-SOD from the matrix into extracellular fluids. We previously reported that knock-in mice harboring the human R213G SNP (R213G mice) exhibited enhanced resolution of inflammation with subsequent protection against fibrosis following bleomycin treatment compared with wild-type (WT) littermates. Herein we set out to determine the underlying pathways with RNA-Seq analysis of WT and R213G lungs 7 days post-PBS and bleomycin. RNA-Seq analysis uncovered significant differential gene expression changes induced in WT and R213G strains in response to bleomycin. Ingenuity Pathways Analysis was used to predict differentially regulated up- and downstream processes based on transcriptional changes. Most prominent was the induction of inflammatory and immune responses in WT mice, which were suppressed in the R213G mice. Specifically, PKC signaling in T lymphocytes, IL-6, and NFΚB signaling were opposed in WT mice when compared with R213G. Several upstream regulators such as IFNγ, IRF3, and IKBKG were implicated in the divergent responses between WT and R213G mice. Our data suggest that the redistributed EC-SOD due to the R213G SNP attenuates the dysregulated inflammatory responses observed in WT mice. We speculate that redistributed EC-SOD protects against dysregulated alveolar inflammation via reprogramming of recruited immune cells toward a proresolving state.