Inhibition of steroid sulphatase activity by tricyclic coumarin sulphamates

The identification of the active pharmacophore required for potent inhibition of steroid sulphatase activity, i.e. an aryl-O-sulphamate structure, has led to the synthesis and testing of a large number of 1–4 ring-based inhibitors. 4-Methylcoumarin-7-O-sulphamate (COUMATE) was one of the first non-steroid based inhibitors identified. In an attempt to increase the potency of this class of inhibitor a series of tricyclic COUMATEs (665–6615 COUMATEs) have been synthesised and evaluated. Using placental microsomes as a source of oestrone sulphatase (E1-STS) the size of the third ring of the tricyclic COUMATEs was found to have a marked effect on inhibitor potency. Whereas 665- and 6615-COUMATEs had IC₅₀s of 200 and 370 nM, respectively, the most potent inhibitor in vitro in this series was 6610 COUMATE with an IC₅₀ of 1 nM. Selected inhibitors were tested for their in vivo potency by administration of a single dose (0.1 or 1 mg/kg, p.o.) to female rats. Surprisingly, in vivo 6615 COUMATE proved to be the most active drug, inhibiting rat liver E1-STS activity by 23 and 94% when assayed 24 h after administration of the 0.1 and 1 mg/kg doses. E1-STS activity in brain tissue and white blood cells was also found to be inhibited when selected drugs were tested. These studies have identified a number of tricyclic COUMATEs with therapeutic potential.     

Analyses of Conformational States of the Transporter Associated with Antigen Processing (TAP) Protein in a Native Cellular Membrane Environment

The transporter associated with antigen processing (TAP) plays a critical role in the MHC class I antigen presentation pathway. TAP translocates cellular peptides across the endoplasmic reticulum membrane in an ATP hydrolysis-dependent manner. We used FRET spectroscopy in permeabilized cells to delineate different conformational states of TAP in a native subcellular membrane environment. For these studies, we tagged the TAP1 and TAP2 subunits with enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, respectively, C-terminally to their nucleotide binding domains (NBDs), and measured FRET efficiencies under different conditions. Our data indicate that both ATP and ADP enhance the FRET efficiencies but that neither induces a maximally closed NBD conformation. Additionally, peptide binding induces a large and significant increase in NBD proximity with a concentration dependence that is reflective of individual peptide affinities for TAP, revealing the underlying mechanism of peptide-stimulated ATPase activity of TAP. Maximal NBD closure is induced by the combination of peptide and non-hydrolysable ATP analogs. Thus, TAP1-TAP2 NBD dimers are not fully stabilized by nucleotides alone, and substrate binding plays a key role in inducing the transition state conformations of the NBD. Taken together, these findings show that at least three steps are involved in the transport of peptides across the endoplasmic reticulum membrane for antigen presentation, corresponding to three dynamically and structurally distinct conformational states of TAP. Our studies elucidate structural changes in the TAP NBD in response to nucleotides and substrate, providing new insights into the mechanism of ATP-binding cassette transporter function.     

Is reduced SERCA2a expression detrimental or beneficial to postischemic cardiac function and injury? Evidence from heterozygous SERCA2a knockout mice

Recent studies have demonstrated that increased expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2a improves myocardial contractility and Ca2+ handling at baseline and in disease conditions, including myocardial ischemia-reperfusion (I/R). Conversely, it has also been reported that pharmacological inhibition of SERCA might improve postischemic function in stunned hearts or in isolated myocardium following I/R. The goal of this study was to test how decreases in SERCA pump level/activity affect cardiac function following I/R. To address this question, we used a heterozygous SERCA2a knockout (SERCA2a+/-) mouse model with decreased SERCA pump levels and studied the effect of myocardial stunning (20-min ischemia followed by reperfusion) and infarction (30-min ischemia followed by reperfusion) following 60-min reperfusion. Our results demonstrate that postischemic myocardial relaxation was significantly impaired in SERCA2a+/- hearts with both stunning and infarction protocols. Interestingly, postischemic recovery of contractile function was comparable in SERCA2a+/- and wild-type hearts subjected to stunning. In contrast, following 30-min ischemia, postischemic contractile function was reduced in SERCA2a+/- hearts with significantly larger infarction. Rhod-2 spectrofluorometry revealed significantly higher diastolic intracellular Ca2+ in SERCA2a+/- hearts compared with wild-type hearts. Both at 30-min ischemia and 2-min reperfusion, intracellular Ca2+ levels were significantly higher in SERCA2a+/- hearts. Electron paramagnetic resonance spin trapping showed a similar extent of postischemic free-radical generation in both strains. These data provide direct evidence that functional SERCA2a level, independent of oxidative stress, is crucial for postischemic myocardial function and salvage during I/R.     

Functional consequences of stably expressing a mutant calsequestrin (CASQ2D307H) in the CASQ2 null background

The role of calsequestrin (CASQ2) in cardiac sarcoplasmic reticulum (SR) calcium (Ca(2+)) transport has gained significant attention since point mutations in CASQ2 were reported to cause ventricular arrhythmia. In the present study, we have critically evaluated the functional consequences of expressing the CASQ2(D307H) mutant protein in the CASQ2 null mouse. We recently reported that the mutant CASQ2(D307H) protein can be stably expressed in CASQ2 null hearts, and it targets appropriately to the junctional SR (Kalyanasundaram A, Bal NC, Franzini-Armstrong C, Knollmann BC, Periasamy M. J Biol Chem 285: 3076-3083, 2010). In this study, we found that introduction of CASQ2(D307H) protein in the CASQ2 null background partially restored triadin 1 levels, which were decreased in the CASQ2 null mice. Despite twofold expression (relative to wild-type CASQ2), the mutant protein failed to increase SR Ca(2+) load. We also found that the Ca(2+) transient decays slower in the CASQ2 null and CASQ2(D307H) cells. CASQ2(D307H) myocytes, when rhythmically paced and challenged with isoproterenol, exhibit spontaneous Ca(2+) waves similar to CASQ2 null myocytes; however, the stability of Ca(2+) cycling was increased in the CASQ2(D307H) myocytes. In the presence of isoproterenol, Ca(2+)-transient amplitude in CASQ2(D307H) myocytes was significantly decreased, possibly indicating an inherent defect in Ca(2+) buffering capacity and release from the mutant CASQ2 at high Ca(2+) concentrations. We also observed polymorphic ventricular tachycardia in the CASQ2(D307H) mice, although lesser than in the CASQ2 null mice. These data suggest that CASQ2(D307H) point mutation may affect Ca(2+) buffering capacity and Ca(2+) release. We propose that poor interaction between CASQ2(D307H) and triadin 1 could affect ryanodine receptor 2 stability, thereby increasing susceptibility to delayed afterdepolarizations and triggered arrhythmic activity.     

Mycobacterium tuberculosis oriC sequestration by MtrA response regulator

The regulators of Mycobacterium tuberculosis DNA replication are largely unknown. Here, we demonstrate that in synchronously replicating M. tuberculosis, MtrA access to origin of replication (oriC) is enriched in the post‐replication (D) period. The increased oriC binding results from elevated MtrA phosphorylation (MtrA～P) as evidenced by reduced expression of dnaN, dnaA and increased expression of select cell division targets. Overproduction of gain‐of‐function MtrA_Y102C advanced the MtrA oriC access to the C period, reduced dnaA and dnaN expression, interfered with replication synchrony and compromised cell division. Overproduction of wild‐type (MtrA+) or phosphorylation‐defective MtrA_D56N did not promote oriC access in the C period, nor affected cell cycle progression. MtrA interacts with DnaA signaling a possibility that DnaA helps load MtrA on oriC. Therefore, oriC sequestration by MtrA～P in the D period may normally serve to prevent untimely initiations and that DnaA–MtrA interactions may facilitate regulated oriC replication. Finally, despite the near sequence identity of MtrA in M. smegmatis and M. tuberculosis, the M. smegmatis oriC is not MtrA‐target. We conclude that M. tuberculosis oriC has evolved to be regulated by MtrA and that cell cycle progression in this organisms are governed, at least in part, by oscillations in the MtrA～P levels.     

Patterns of co-existence between humans and mammals in Yemen: some species thrive while others are nearly extinct

This paper describes the results of a camera-trapping study in Hawf, eastern Yemen, of the relationships between human presence and the richness, diversity and distribution of wild medium-sized and large mammals. The study comprised 3,351 trap-nights over 225.3 km² during which 963 captures of 14 mammalian species and 985 captures of humans were obtained. Human presence was positively linked with species richness and diversity, but only when the number of human captures was ≤100 captures/site. At higher numbers of human captures, the positive trend reversed and only three species (wolf, fox and porcupine) succeeded in dominating their communities. Human presence was also strongly and positively associated with colonization and occupancy of some species, but decreased their detection probabilities. Contrary to expectations, human presence did not affect activity patterns of species. Co-existence between human presence and mammals caused by resource sharing, viz. establishment and use of same trails and water sources, attraction to livestock, garbage and food leftovers, is discussed. In contrast, the leopard, wild cat, Nubian ibex and mountain gazelle are severely depleted. Co-existence between humans and mammals has significant implications for proper management and wildlife conservation in unprotected human-dominated landscapes, protected areas and buffer zones of developing countries such as Yemen.     

Activin and bone morphogenetic proteins are present in perinatal sensory neuron target tissues that induce neuropeptides

Previous studies have shown that sensory target tissues induce neuropeptides in na?ve sensory neurons, and that activin and bone morphogenetic proteins (BMPs) are capable of inducing neuropeptides associated with nociception in embryonic sensory neurons in vitro. The goal of the present study was to learn if these ligands were available in native sensory neuron target tissues at correct developmental periods to play this inductive role in vivo. Sensory neurons initially contact their peripheral target tissues and begin to express neuropeptides during late embryogenesis, and we demonstrate that activin and BMPs are present in the embryo and neonate to regulate sensory neuron differentiation. Native embryonic and neonatal target tissues were analyzed by immunoblot and immunohistochemical studies using ligand-specific antibodies. Although activin was easily solubilized, BMPs were detected only after high salt extraction, suggesting that BMPs were bound to extracellular moieties and were capable of acting only locally in native tissues. One inhibitor, noggin, was present in both embryonic skin and muscle. In combination, these data suggest that neuronal differentiation is unlikely to be regulated by simple expression of ligand, but that the functional availability of ligand is a critical component confering biological activity.     

Cell-free protein synthesis of membrane (1,3)-β-d-glucan (curdlan) synthase: Co-translational insertion in liposomes and reconstitution in nanodiscs

A membrane-embedded curdlan synthase (CrdS) from Agrobacterium is believed to catalyse a repetitive addition of glucosyl residues from UDP-glucose to produce the (1,3)-β-d-glucan (curdlan) polymer. We report wheat germ cell-free protein synthesis (WG-CFPS) of full-length CrdS containing a 6xHis affinity tag and either Factor Xa or Tobacco Etch Virus proteolytic sites, using a variety of hydrophobic membrane-mimicking environments. Full-length CrdS was synthesised with no variations in primary structure, following analysis of tryptic fragments by MALDI-TOF/TOF Mass Spectrometry. Preparative scale WG-CFPS in dialysis mode with Brij-58 yielded CrdS in mg/ml quantities. Analysis of structural and functional properties of CrdS during protein synthesis showed that CrdS was co-translationally inserted in DMPC liposomes during WG-CFPS, and these liposomes could be purified in a single step by density gradient floatation. Incorporated CrdS exhibited a random orientation topology. Following affinity purification of CrdS, the protein was reconstituted in nanodiscs with Escherichia coli lipids or POPC and a membrane scaffold protein MSP1E3D1. CrdS nanodiscs were characterised by small-angle X-ray scattering using synchrotron radiation and the data obtained were consistent with insertion of CrdS into bilayers. We found CrdS synthesised in the presence of the Ac-AAAAAAD surfactant peptide or co-translationally inserted in liposomes made from E. coli lipids to be catalytically competent. Conversely, CrdS synthesised with only Brij-58 was inactive. Our findings pave the way for future structural studies of this industrially important catalytic membrane protein.     

Temperature and pressure effects on structural and conformational properties of POPC/SM/cholesterol model raft mixtures--a FT-IR, SAXS, DSC, PPC and Laurdan fluorescence spectroscopy study

We report on the effects of temperature and pressure on the structure, conformation and phase behavior of aqueous dispersions of the model lipid "raft" mixture palmitoyloleoylphosphatidylcholine (POPC)/bovine brain sphingomyelin (SM)/cholesterol (Chol) (1:1:1). We investigated interchain interactions, hydrogen bonding, conformational and structural properties as well as phase transformations of this system using Fourier transform-infrared (FT-IR) spectroscopy, small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), and Laurdan fluorescence spectroscopy. The IR spectral parameters in combination with the scattering patterns from the SAXS measurements were used to detect structural and conformational transformations upon changes of pressure up to 7-9 kbar and temperature in the range from 1 to about 80 degrees C. The generalized polarization function (GP) values, obtained from the Laurdan fluorescence spectroscopy studies also reveal temperature and pressure dependent phase changes. DSC and PPC were used to detect thermodynamic properties accompanying the temperature-dependent phase changes. In combination with literature fluorescence spectroscopy and microscopy data, a tentative p,T stability diagram of the mixture has been established. The data reveal a broad liquid-order/solid-ordered (lo+so) two-phase coexistence region below 8+/-2 degrees C at ambient pressure. With increasing temperature, a lo+ld+so three-phase region is formed, which extends up to approximately 27 degrees C, where a liquid-ordered/liquid-disordered (lo+ld) immiscibility region is formed. Finally, above 48+/-2 degrees C, the POPC/SM/Chol (1:1:1) mixture becomes completely fluid-like (liquid-disordered, ld). With increasing pressure, all phase transition lines shift to higher temperatures. Notably, the lo+ld (+so) phase coexistence region, mimicking raft-like lateral phase separation in natural membranes, extends over a rather wide temperature range of about 40 degrees C, and a pressure range, which extends up to about 2 kbar for T=37 degrees C. Interestingly, in this pressure range, ceasing of membrane protein function in natural membrane environments has been observed for a variety of systems.