Thoracic aortic aneurysm/dissection (TAAD) is characterized by excessive smooth muscle cell (SMC) loss, extracellular matrix (ECM) degradation and inflammation. In response to certain stimuli, endoplasmic reticulum (ER) stress is activated and regulates apoptosis and inflammation. Excessive apoptosis promotes aortic inflammation and degeneration, leading to TAAD. Therefore, we studied the role of ER stress in TAAD formation. A lysyl oxidase inhibitor, 3‐aminopropionitrile fumarate (BAPN), was administrated to induce TAAD formation in mice, which showed significant SMC loss (α‐SMA level). Excessive apoptosis (TUNEL staining) and ER stress (ATF4 and CHOP), along with inflammation, were present in TAAD samples from both mouse and human. Transcriptional profiling of SMCs after mechanical stress demonstrated the expression of genes for ER stress and inflammation. To explore the causal role of ER stress in initiating degenerative signalling events and TAAD, we treated wild‐type (CHOP +/+) or CHOP −/− mice with BAPN and found that CHOP deficiency protected against TAAD formation and rupture, as well as reduction in α‐SMA level. Both SMC apoptosis and inflammation were significantly reduced in CHOP −/− mice. Moreover, SMCs isolated from CHOP −/− mice were resistant to mechanical stress‐induced apoptosis. Taken together, our results demonstrated that mechanical stress‐induced ER stress promotes SMCs apoptosis, inflammation and degeneration, providing insight into TAAD formation and progression. © 2015 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Abstract-Previous studies show that chemical regulation of connexin43 (Cx43) gap junction channels depends on the integrity of the carboxyl terminal (CT) domain. Experiments using Xenopus oocytes show that truncation of the CT domain alters the time course for current inactivation; however, correlation with the behavior of single Cx43 channels has been lacking. Furthermore, whereas chemical gating is associated with a "ball-and-chain" mechanism, there is no evidence whether transjunctional voltage regulation for Cx43 follows a similar model. We provide data on the properties of transjunctional currents from voltage-clamped pairs of mammalian tumor cells expressing either wild-type Cx43 or a mutant of Cx43 lacking the carboxyl terminal domain (Cx43M257

Enzymes function by stabilizing reaction transition states; therefore, comparison of the transition states of enzymatic and nonenzymatic model reactions can provide insight into biological catalysis. Catalysis of RNA 2′-O-transphosphorylation by ribonuclease A is proposed to involve electrostatic stabilization and acid/ base catalysis, although the structure of the rate-limiting transition state is uncertain. Here, we describe coordinated kinetic isotope effect (KIE) analyses, molecular dynamics simulations, and quantum mechanical calculations to model the transition state and mechanism of RNase A. Comparison of the 18 O KIEs on the 2′O nucleophile, 5′O leaving group, and nonbridging phosphoryl oxygens for RNase A to values observed for hydronium-or hydroxide-catalyzed reactions indicate a late anionic transition state. Molecular dynamics simulations using an anionic phosphorane transition state mimic suggest that H-bonding by protonated His12 and Lys41 stabilizes the transition state by neutralizing the negative charge on the nonbridging phosphoryl oxygens. Quantum mechanical calculations consistent with the experimental KIEs indicate that expulsion of the 5′O remains an integral feature of the rate-limiting step both on and off the enzyme. Electrostatic interactions with positively charged amino acid site chains (His12/ Lys41), together with proton transfer from His119, render departure of the 5′O less advanced compared with the solution reaction and stabilize charge buildup in the transition state. The ability to obtain a chemically detailed description of 2′-O-transphosphorylation transition states provides an opportunity to advance our understanding of biological catalysis significantly by determining how the catalytic modes and active site environments of phosphoryl transferases influence transition state structure. E nzymes achieve powerful rate enhancements by providing multiple catalytic modes to stabilize reaction transition states, including electrostatic interactions and proton transfer (1). For RNA 2′-O-transphosphorylation reactions, interactions with acid, base, or metal ion catalysts in solution can influence transition state structure (2). Understanding biological catalysis therefore requires knowledge of chemical mechanisms, transition state structure, and transition state interactions for both enzymatic and nonenzymatic RNA cleavage reactions. Comparisons of enzymatic and nonenzymatic catalysis can provide information on which catalytic modes are used by enzymes and whether unique features of the active site environment may alter the transition state charge distribution (3).RNA undergoes two competing transesterification reactions in solution: isomerization to a 2′,5′-phosphodiester and 2′-Otransphosphorylation to yield a cyclic 2′,3′-phosphodiester with concomitant release of the 5′O-nucleoside (4, 5). Reactions catalyzed by acid and by buffers yield both isomerization and cleavage products and proceed via a pentacoordinated phosphorane intermediate formed by an attack of the 2′OH at the adja...

To better understand the interactions between catalysts and transition states during RNA strand cleavage, primary 18 O kinetic isotope effects and solvent D 2 O isotope effects were measured to probe the mechanism of base-catalyzed 2'-O-transphosphorylation of the RNA dinucleotide 5'-UpG-3'. The observed 18 O KIEs for the nucleophilic 2'-O and in the 5'-O leaving group at pH 14 are both large relative to reactions of phosphodiesters with good leaving groups, indicating that the reaction catalyzed by hydroxide has a transition state (TS) with advanced phosphorus-oxygen bond fission to the leaving group ( 18 k LG = 1.034 ± 0.004) and phosphorous-nucleophile bond formation ( 18 k NUC = 0.984 ± 0.004). A breakpoint in the pH dependence of the 2'-Otransphosphorylation rate to a pH independent phase above pH 13 has been attributed to the pK a of the 2'-OH nucleophile. A smaller nucleophile KIE is observed at pH 12 ( 18 k NUC = 0.995 ± 0.004) that is interpreted as the combined effect of the equilibrium isotope effect (~1.02) on deprotonation of the 2′-hydroxyl nucleophile and the intrinsic KIE on the nucleophilic addition step (ca. 0.981). An alternative mechanism in which the hydroxide ion acts as a general base is considered unlikely given the lack of a solvent deuterium isotope effect above the breakpoint in the pH versus rate profile. These results represent the first direct analysis of the transition state for RNA strand cleavage. The primary 18 O KIE results and the lack of a kinetic solvent deuterium isotope effect together provide strong evidence for a late transition state and 2'-O nucleophile activation by specific base catalysis.

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Erythroid spectrin is the predominant component of the twodimensional protein network called the membrane skeleton, underlying the lipid bilayer of red cells (for recent reviews, seeRefs. 1-3). Formation of the membrane skeleton involves multiple protein-protein interactions among integral membrane proteins. Interactions of spectrin with other membrane proteins such as ankyrin, protein 4.1, and adducin provide a linkage of spectrin either to the plasma membrane or among spectrin tetramers. Many hereditary anemia mutations affect interactions of these integral membrane proteins, resulting in increased fragility and shortened lifespan of erythrocytes. In hereditary elliptocytosis and pyropoikilocytosis, the mutations have been localized in the ␣-and ␤-subunits of spectrin (reviewed in Refs. 4 and 5). Many of these proteins, including spectrin, which were first identified in red cells, have isoforms expressed in nonerythroid cells, but the structure and regulatory processes of the nonerythroid membrane skeleton are less well understood (reviewed in Refs. 1-3, 6, and 7). Functional differences between the membranes of erythroid and nonerythroid cells argue against the simple erythrocyte model of the membrane skeleton. Major differences between the erythroid model and other cells include differences in the expression of spectrin (8 -11) and ankyrin isoforms (12-15) (reviewed in Ref.16), interactions of spectrin and ankyrin with additional proteins (17-21), localization of spectrin in the cytoplasm as well as in the plasma membrane (10,11,22), and the potential for dramatic rearrangements of spectrin's cellular location (23, 24) (reviewed in Refs. 2 and 7).Several studies have demonstrated that both erythroid and nonerythroid spectrins are expressed in brain tissue (8 -11, 25). Neuronal compartmentalization of brain spectrin isoforms into axons and presynaptic terminals (nonerythroid spectrin) and into cell bodies and dendrites (erythroid spectrin) (10, 25) suggests that brain spectrin isoforms may perform related but distinct functions in neuronal cells. It has been suggested that nonerythroid spectrin performs a more general, constitutive role, while erythroid spectrin takes part in more specialized activities of differentiated cells (26). The ␣-subunit of erythroid spectrin, ␣I (27), 1 and the ␣-subunit of nonerythroid spectrin, ␣II (28, 29), each contains a unique SH3 2 domain. Distinct protein interactions are likely to involve these domains, and they may be important for specific distribution and specialized roles of brain spectrin isoforms.

Abstract-Chemical regulation of connexin (Cx) 40 and Cx43 follows a ball-and-chain model, in which the carboxyl terminal (CT) domain acts as a gating particle that binds to a receptor affiliated with the pore. Moreover, Cx40 channels can be closed by a heterodomain interaction with the CT domain of Cx43 and vice versa. Here, we report similar interactions in the establishment of the unitary conductance and voltage-dependent profile of Cx40 in N2A cells. Two mean unitary conductance values ("lower conductance" and "main") were detected in wild-type Cx40. Truncation of the CT domain at amino acid 248 (Cx40tr248) caused the disappearance of the lower-conductance state. Coexpression of Cx40tr248 with the CT fragment of either Cx40 (homodomain interactions) or Cx43 (heterodomain interactions) rescued the unitary conductance profile of Cx40. In the N2A cells, the time course of macroscopic junctional current relaxation was best described by a biexponential function in the wild-type Cx40 channels, but it was reduced to a single-exponential function after truncation. However, macroscopic junctional currents recorded in the oocyte expression system were not significantly different between the wild-type and mutant channels. Concatenation of the CT domain of Cx43 to amino acids 1 to 248 of Cx40 yielded a chimeric channel with unitary conductance and voltage-gating profile indistinguishable from that of wild-type Cx40. We conclude that residence of Cx40 channels in the lower-conductance state involves a ball-and-chain type of interaction between the CT domain and the pore-forming region. This interaction can be either homologous (Cx40 truncation with Cx40CT) or heterologous (with the Cx43CT). These channels mediate vitally important processes such as impulse propagation, 1-3 regulation of cell growth, 4 and organ development. 5,6 Moreover, several hereditary human diseases are linked to mutations in a gap junction protein. 7 Clearly, a better understanding of the molecular mechanisms controlling channel function is warranted.In vertebrates, gap junctions are formed by oligomerization of a protein called connexin (Cx). Most cells express more than one connexin isotype. 7 In particular, Cx40 and Cx43 are both expressed in the atrium, 8,9 endothelium, 10 and smooth muscle cells, 10,11 and there is evidence that they heteromerize. [12][13][14][15] The various connexins also have a similar putative membrane topology, as follows: 4 membrane-spanning domains linked by 1 cytoplasmic and 2 extracellular loops and a cytoplasmic N-and C-terminus. 7 The cytoplasmic N-and C-termini of connexins differ in primary sequence and length, whereas the transmembrane and extracellular domains are highly conserved. 7Connexins are highly regulatable molecules, susceptible to association with a number of kinases and with other junctional 7,16 and nonjunctional proteins. 17 We have previously proposed a "ball-and-chain-like" model 18 for the chemical regulation of Cx40 and Cx43. According to this model, the carboxyl terminal (CT) domain acts as a ga...

We investigated the effects of substituting two of the four tryptophans (the “inner pair” Trp9,11 or the “outer pair” Trp13,15) in gramicidin A (gA) channels. The conformational preferences of the double-substituted gA analogues were assessed using circular dichroism spectroscopy and size-exclusion chromatography, which show that the inner tryptophans 9 and 11 are critical for the gA’s conformational preference in lipid bilayer membranes. [Phe13,15]gA largely retains the single-stranded helical channel structure, whereas of [Phe9,11]gA exists primarily as double-stranded conformers. Within this context, the 2H-NMR spectra from labeled tryptophans were used to examine the changes in average indole ring orientations, induced by the Phe substitutions and by the shift in conformational preference. Using a method for deuterium labeling of already synthesized gAs, we introduced deuterium selectively onto positions C2 and C5 of the remaining tryptophan indole rings in the substituted gA analogues for solid-state 2H-NMR spectroscopy. The (least possible) changes in orientation and overall motion of each indole ring were estimated from the experimental spectra. Regardless of the mixture of backbone folds, the indole ring orientations observed in the analogues are similar to those found previously for gA channels. Both Phe-substituted analogues form single-stranded channels, as judged from the formation of heterodimeric channels with the native gA. [Phe13,15]gA channels have Na+ currents that are ~50% and lifetimes ~80% those of native gA channels. The double-stranded conformer(s) of [Phe9,11]gA do not form detectable channels. The minor single-stranded population of [Phe9,11]gA forms channels with Na+ currents that are ~25% and single-channel lifetimes that are ~300% those of native gA channels. Our results suggest that Trp9 and Trp11, when “reaching” for the interface, tend to drive both monomer folding (to “open” a channel) and dimer dissociation (to “close” a channel). Furthermore, the dipoles of Trp9 and Trp11 are relatively more important for the single-channel conductance than are the dipoles of Trp13 and Trp15.