A Bioassay-Guided Fractionation of Rosemary Leaf Extract Identifies Carnosol as a Major Hypertrophy Inducer in Human Skeletal Muscle Cells.

A good quality of life requires maintaining adequate skeletal muscle mass and strength, but therapeutic agents are lacking for this. We developed a bioassay-guided fractionation approach to identify molecules with hypertrophy-promoting effect in human skeletal muscle cells. We found that extracts from rosemary leaves induce muscle cell hypertrophy. By bioassay-guided purification we identified the phenolic diterpene carnosol as the compound responsible for the hypertrophy-promoting activity of rosemary leaf extracts. We then evaluated the impact of carnosol on the different signaling pathways involved in the control of muscle cell size. We found that activation of the NRF2 signaling pathway by carnosol is not sufficient to mediate its hypertrophy-promoting effect. Moreover, carnosol inhibits the expression of the ubiquitin ligase E3 Muscle RING Finger protein-1 that plays an important role in muscle remodeling, but has no effect on the protein synthesis pathway controlled by the protein kinase B/mechanistic target of rapamycin pathway. By measuring the chymotrypsin-like activity of the proteasome, we found that proteasome activity was significantly decreased by carnosol and Muscle RING Finger 1 inactivation. These results strongly suggest that carnosol can induce skeletal muscle hypertrophy by repressing the ubiquitin-proteasome system-dependent protein degradation pathway through inhibition of the E3 ubiquitin ligase Muscle RING Finger protein-1.

Identification of a Sesquiterpene Lactone from Arctium lappa Leaves with Antioxidant Activity in Primary Human Muscle Cells.

Many pathologies affecting muscles (muscular dystrophies, sarcopenia, cachexia, renal insufficiency, obesity, diabetes type 2, etc.) are now clearly linked to mechanisms involving oxidative stress. In this context, there is a growing interest in exploring plants to find new natural antioxidants to prevent the appearance and the development of these muscle disorders. In this study, we investigated the antioxidant properties of Arctium lappa leaves in a model of primary human muscle cells exposed to H2O2 oxidative stress. We identified using bioassay-guided purification, onopordopicrin, a sesquiterpene lactone as the main molecule responsible for the antioxidant activity of A. lappa leaf extract. According to our findings, onopordopicrin inhibited the H2O2-mediated loss of muscle cell viability, by limiting the production of free radicals and abolishing DNA cellular damages. Moreover, we showed that onopordopicrin promoted the expression of the nuclear factor-erythroid-2-related factor 2 (Nrf2) downstream target protein heme oxygenase-1 (HO-1) in muscle cells. By using siRNA, we demonstrated that the inhibition of the expression of Nrf2 reduced the protective effect of onopordopicrin, indicating that the activation of the Nrf2/HO-1 signaling pathway mediates the antioxidant effect of onopordopicrin in primary human muscle cells. Therefore, our results suggest that onopordopicrin may be a potential therapeutic molecule to fight against oxidative stress in pathological specific muscle disorders.

Antioxidant and Anti-Inflammatory Potential of Shiitake Culinary-Medicinal Mushroom, Lentinus edodes (Agaricomycetes), Sporophores from Various Culture Conditions.

Lentinus edodes (= Lentinula edodes) is an edible mushroom grown and marketed for centuries due to its nutritional and medicinal properties. L. edodes has multiple pharmacological activities as an antioxidant and anti-inflammatory. Few studies were performed taking into account the influence of culture conditions to optimize the biological properties of L. edodes on human health. Our work focused on the comparison of antioxidant capacity and anti-inflammatory activity of L. edodes fruit bodies cultivated by three mushroom producers in the French Occitanie region using the same strain in various growing conditions (organic and nonorganic). Sequential extraction was performed on freeze-dried fungal materials. All extracts have a quantifiable but moderate antioxidant activity measured via DPPH and ORAC tests. The anti-inflammatory activity of the ethanol and aqueous extracts was evaluated on a model of inflammatory macrophages. The ethanol extracts inhibit NO production in a dose-dependent manner when the cells are pretreated for 4 h with a 24 h stimulation time.

Importance of the Choice of a Recombinant System to Produce Large Amounts of Functional Membrane Protein hERG.

Human ether-a-gogo related gene (hERG) product is the membrane potassium channel Kv11.1, which is involved in the electrical activity of the heart. As such, it is a key player in the toxicity of many drug candidates. Therefore, having this protein at hand during earlier stages of drug discovery is important for preventing later toxicity. Furthermore, having a fair quantity of functional channels may help in the development of the necessary techniques for gaining insight in this channel structure. Thus, we performed a comparative study of methods for over-expressing a mutated but functional, hERG in different orthologous hosts, such as yeast, bacteria, insect and human cell lines. We also engineered the protein to test various constructs of a functional channel. We obtained a significant amount of a functional mutant channel from HEK cells that we thoroughly characterized. The present work paves the way for the expression of large amounts of this protein, with which protein crystallization or cryo-electronic microscopy will be attempted. This will be a way to gain information on the structure of the hERG active site and its modelization to obtain data on the pauses of various reference compounds from the pharmacopeia, as well as to gain information about the thermodynamics of the hERG/ligand relationship.

Concomitant systolic and diastolic alterations during chronic hypertension in pig.

The mechanical and cellular relationships between systole and diastole during left ventricular (LV) dysfunction remain to be established. LV contraction-relaxation coupling was examined during LV hypertrophy induced by chronic hypertension. Chronically instrumented pigs received angiotensin II infusion for4weeks to induce chronic hypertension (133 ±â€¯7 mmHg vs 98 ±â€¯5 mmHg for mean arterial pressure at Day 28 vs 0, respectively) and LV hypertrophy. LV function was investigated with the instrumentation and echocardiography for LV twist-untwist assessment before and after dobutamine infusion. The cellular mechanisms were investigated by exploring the intracellular Ca2+ handling. At Day 28, pigs exhibited LV hypertrophy with LV diastolic dysfunction (impaired LV isovolumic relaxation, increased LV end-diastolic pressure, decreased and delayed LV untwisting rate) and LV systolic dysfunction (impaired LV isovolumic contraction and twist) although LV ejection fraction was preserved. Isolated cardiomyocytes exhibited altered shortening and lengthening. Interestingly, contraction-relaxation coupling remained preserved both in vivo and in vitro during LV hypertrophy. LV systolic and diastolic dysfunctions were associated to post-translational remodeling and dysfunction of the type 2 cardiac ryanodine receptor/Ca2+ release channel (RyR2), i.e., PKA hyperphosphorylation of RyR2, depletion of calstabin 2 (FKBP12.6), RyR2 leak and hypersensitivity of RyR2 to cytosolic Ca2+ during both contraction and relaxation phases. In conclusion, LV contraction-relaxation coupling remained preserved during chronic hypertension despite LV systolic and diastolic dysfunctions. This implies that LV diastolic dysfunction is accompanied by LV systolic dysfunction. At the cellular level, this is linked to sarcoplasmic reticulum Ca2+ leak through PKA-mediated RyR2 hyperphosphorylation and depletion of its stabilizing partner.

Post-translational remodeling of ryanodine receptor induces calcium leak leading to Alzheimer's disease-like pathologies and cognitive deficits.

The mechanisms underlying ryanodine receptor (RyR) dysfunction associated with Alzheimer disease (AD) are still not well understood. Here, we show that neuronal RyR2 channels undergo post-translational remodeling (PKA phosphorylation, oxidation, and nitrosylation) in brains of AD patients, and in two murine models of AD (3 × Tg-AD, APP +/- /PS1 +/-). RyR2 is depleted of calstabin2 (KFBP12.6) in the channel complex, resulting in endoplasmic reticular (ER) calcium (Ca2+) leak. RyR-mediated ER Ca2+ leak activates Ca2+-dependent signaling pathways, contributing to AD pathogenesis. Pharmacological (using a novel RyR stabilizing drug Rycal) or genetic rescue of the RyR2-mediated intracellular Ca2+ leak improved synaptic plasticity, normalized behavioral and cognitive functions and reduced Aß load. Genetically altered mice with congenitally leaky RyR2 exhibited premature and severe defects in synaptic plasticity, behavior and cognitive function. These data provide a mechanism underlying leaky RyR2 channels, which could be considered as potential AD therapeutic targets.

Respiratory muscle contractile inactivity induced by mechanical ventilation in piglets leads to leaky ryanodine receptors and diaphragm weakness.

Respiratory muscle contractile inactivity during mechanical ventilation (MV) induces diaphragm muscle weakness, a condition referred to as ventilator-induced diaphragmatic dysfunction (VIDD). Although VIDD pathophysiological mechanisms are still not fully understood, it has been recently suggested that remodeling of the sarcoplasmic reticulum (SR) calcium release channel/ryanodine receptors (RyR1) in the diaphragm is a proximal mechanism of VIDD. Here, we used piglets, a large animal model of VIDD that is more relevant to human pathophysiology, to determine whether RyR1 alterations are observed in the presence of diaphragm weakness. In piglets, diaphragm weakness induced by 72 h of respiratory muscle unloading was associated with SR RyR1 remodeling and abnormal resting SR Ca2+ leak in the diaphragm. Specifically, following controlled mechanical ventilation, diaphragm contractile function was reduced. Moreover, RyR1 macromolecular complexes were more oxidized, S-nitrosylated and phosphorylated at Ser-2844 and depleted of the stabilizing subunit calstabin1 compared with controls on adaptive support ventilation that maintains diaphragmatic contractile activity. Our study strongly supports the hypothesis that RyR1 is a potential therapeutic target in VIDD and the interest of using small molecule drugs to prevent RyR1-mediated SR Ca2+ leak induced by respiratory muscle unloading in patients who require controlled mechanical ventilation.

Leaky ryanodine receptors contribute to diaphragmatic weakness during mechanical ventilation.

Ventilator-induced diaphragmatic dysfunction (VIDD) refers to the diaphragm muscle weakness that occurs following prolonged controlled mechanical ventilation (MV). The presence of VIDD impedes recovery from respiratory failure. However, the pathophysiological mechanisms accounting for VIDD are still not fully understood. Here, we show in human subjects and a mouse model of VIDD that MV is associated with rapid remodeling of the sarcoplasmic reticulum (SR) Ca(2+) release channel/ryanodine receptor (RyR1) in the diaphragm. The RyR1 macromolecular complex was oxidized, S-nitrosylated, Ser-2844 phosphorylated, and depleted of the stabilizing subunit calstabin1, following MV. These posttranslational modifications of RyR1 were mediated by both oxidative stress mediated by MV and stimulation of adrenergic signaling resulting from the anesthesia. We demonstrate in the murine model that such abnormal resting SR Ca(2+) leak resulted in reduced contractile function and muscle fiber atrophy for longer duration of MV. Treatment with ß-adrenergic antagonists or with S107, a small molecule drug that stabilizes the RyR1-calstabin1 interaction, prevented VIDD. Diaphragmatic dysfunction is common in MV patients and is a major cause of failure to wean patients from ventilator support. This study provides the first evidence to our knowledge of RyR1 alterations as a proximal mechanism underlying VIDD (i.e., loss of function, muscle atrophy) and identifies RyR1 as a potential target for therapeutic intervention.

Dynamic interplay of membrane-proximal POTRA domain and conserved loop L6 in Omp85 transporter FhaC.

Omp85 transporters mediate protein insertion into, or translocation across, membranes. They have a conserved architecture, with POTRA domains that interact with substrate proteins, a 16-stranded transmembrane ß barrel, and an extracellular loop, L6, folded back in the barrel pore. Here using electrophysiology, in vivo biochemical approaches and electron paramagnetic resonance, we show that the L6 loop of the Omp85 transporter FhaC changes conformation and modulates channel opening. Those conformational changes involve breaking the conserved interaction between the tip of L6 and the inner ß-barrel wall. The membrane-proximal POTRA domain also exchanges between several conformations, and the binding of FHA displaces this equilibrium. We further demonstrate a dynamic, physical communication between the POTRA domains and L6, which must take place via the ß barrel. Our findings thus link all three essential components of Omp85 transporters and indicate that they operate in a concerted fashion in the transport cycle.

Conformational dynamics of protein transporter FhaC: large-scale motions of plug helix.

FhaC is an integral outer membrane protein of the whooping cough agent Bordetella pertussis that mediates the transport to the cell surface of a major virulence factor, the filamentous haemagglutinin adhesin FHA. The FHA/FhaC pair is a prototypic TpsA/TpsB system of the widespread 'Two-Partner Secretion' pathway, dedicated to the transport of long extracellular proteins in various pathogenic and environmental Gram-negative bacteria. FhaC belongs to the ubiquitous Omp85 superfamily of protein transporters. The X-ray structure of FhaC shows that the transmembrane ß-barrel channel hypothesized to serve as the FHA-conducting pore is obstructed by two structural elements conserved among TpsB transporters, an N-terminal α helix and an extracellular loop. Here, we provide evidence for conformational dynamics of FhaC related to the secretion mechanism. Using paramagnetic electron resonance, electrophysiology and in vivo approaches, we showed that FhaC exchanges between open and closed conformations. The interaction with its secretory partner FHA alters this distribution of conformations. The open conformation of FhaC implies a large displacement from the channel of the N-terminal 'plug' helix, which remains in the periplasm during FHA secretion. The membrane environment favours the dynamics of the TpsB transporter.

Type 2 ryanodine receptor: a novel therapeutic target in myocardial ischemia/reperfusion.

Cardiac pathologies remain the main cause of mortality worldwide. Among them the most common cause is cardiac ischemia. The rapid reperfusion after coronary occlusion has considerably improved the cardiac outcome, however reperfusion per se has deleterious effect also called reperfusion injuries. Cytosolic calcium overload is now well admitted as an essential pathophysiological mechanism involved in reperfusion injuries although the source and origin of calcium remain to be determined. Recent works have pointed out the potential defect of sarcoplasmic reticulum calcium release channels (ryanodine receptor, RyR) as a primary cause of calcium overload during ischemia-reperfusion. This finding opens new pharmacological perspectives in limiting reperfusion injuries since allosteric modulators able to restore and prevents RyR dysfunction have been developed during the last decade.

ANT-VDAC1 interaction is direct and depends on ANT isoform conformation in vitro.

The voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT) have central roles in mitochondrial functions such as nucleotides transport and cell death. The interaction between VDAC, an outer mitochondrial membrane protein and ANT, an inner membrane protein, was studied in isolated mitochondria and in vitro. Both proteins were isolated from various mitochondrial sources and reconstituted in vitro using a biomimetic system composed of recombinant human VDAC isoform 1 (rhVDAC1) immobilized on a surface plasmon resonance (SPR) sensor chip surface. Two enriched-preparations of (H)ANT (ANT from heart, mainly ANT1) and (L)ANT (ANT from liver, mainly ANT2) isoforms interacted differently with rhVDAC1. Moreover, the pharmacological ANT inhibitors atractyloside and bongkrekic acid modulated this interaction. Thus, ANT-VDAC interaction depends both on ANT isoform identity and on the conformation of ANT.

Substrate recognition by the POTRA domains of TpsB transporter FhaC.

Widespread in Gram-negative bacteria, the two-partner secretion (TPS) pathway mediates the secretion of large, ß-helical 'TpsA' proteins with various functions. TpsA proteins harbour a conserved, N-proximal TPS domain essential for secretion. TpsB transporters specifically recognize their TpsA partners in the periplasm and mediate their translocation across the outer membrane through a hydrophilic channel. The FHA/FhaC pair of Bordetella pertussis represents a model TPS system. FhaC is composed of a ß barrel preceded by two periplasmic POTRA domains in tandem. Here we show that both POTRAs are involved in FHA recognition. Surface plasmon resonance analyses indicated an interaction of micromolar affinity between the POTRAs and the TPS domain with fast association and dissociation steps, consistent with the transient character of this interaction in vivo. Major interaction sites in POTRAs correspond to hydrophobic grooves formed by a ß sheet edge and the flanking α helix, well-suited to accommodate extended, amphipathic strands of the substrate and consistent with ß augmentation. The initial recruitment of the TPS domain to POTRAs appears to be facilitated by electrostatic attractions. A domain corresponding to the first part of the repeat-rich central region of FHA is also recognized by the POTRAs, suggesting successive interactions in the course of secretion.

Structure of the Mycobacterium tuberculosis OmpATb protein: a model of an oligomeric channel in the mycobacterial cell wall.

The pore-forming outer membrane protein OmpATb from Mycobacterium tuberculosis is a virulence factor required for acid resistance in host phagosomes. In this study, we determined the 3D structure of OmpATb by NMR in solution. We found that OmpATb is composed of two independent domains separated by a proline-rich hinge region. As expected, the high-resolution structure of the C-terminal domain (OmpATb(198-326)) revealed a module structurally related to other OmpA-like proteins from Gram-negative bacteria. The N-terminal domain of OmpATb (73-204), which is sufficient to form channels in planar lipid bilayers, exhibits a fold, which belongs to the α+ß sandwich class fold. Its peculiarity is to be composed of two overlapping subdomains linked via a BON (Bacterial OsmY and Nodulation) domain initially identified in bacterial proteins predicted to interact with phospholipids. Although OmpATb(73-204) is highly water soluble, current-voltage measurements demonstrate that it is able to form conducting pores in model membranes. A HADDOCK modeling of the NMR data gathered on the major monomeric form and on the minor oligomeric populations of OmpATb(73-204) suggest that OmpATb(73-204) can form oligomeric rings able to insert into phospholipid membrane, similar to related proteins from the Type III secretion systems, which form multisubunits membrane-associated rings at the basal body of the secretion machinery.

Functional importance of a conserved sequence motif in FhaC, a prototypic member of the TpsB/Omp85 superfamily.

In Gram-negative bacteria, the two-partner secretion pathway mediates the secretion of TpsA proteins with various functions. TpsB transporters specifically recognize their TpsA partners in the periplasm and mediate their transport through a hydrophilic channel. The filamentous haemagglutinin adhesin (FHA)/FhaC pair represents a model two-partner secretion system, with the structure of the TpsB transporter FhaC providing the bases to decipher the mechanism of action of these proteins. FhaC is composed of a ß-barrel preceded by two periplasmic polypeptide-transport-associated (POTRA) domains in tandem. The barrel is occluded by an N-terminal helix and an extracellular loop, L6, folded back into the FhaC channel. In this article, we describe a functionally important motif of FhaC. The VRGY tetrad is highly conserved in the TpsB family and, in FhaC, it is located at the tip of L6 reaching the periplasm. Replacement by Ala of the invariant Arg dramatically affects the secretion efficiency, although the structure of FhaC and its channel properties remain unaffected. This substitution affects the secretion mechanism at a step beyond the initial TpsA-TpsB interaction. Replacement of the conserved Tyr affects the channel properties, but not the secretion activity, suggesting that this residue stabilizes the loop in the resting conformation of FhaC. Thus, the conserved motif at the tip of L6 represents an important piece of two-partner secretion machinery. This motif is conserved in a predicted loop between two ß-barrel strands in more distant relatives of FhaC involved in protein transport across or assembly into the outer membranes of bacteria and organelles, suggesting a conserved function in the molecular mechanism of transport.

NMR structure and ion channel activity of the p7 protein from hepatitis C virus.

The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly α-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by (1)H and (13)C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal α-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism.

EtpB is a pore-forming outer membrane protein showing TpsB protein features involved in the two-partner secretion system.

Attachment to host tissues is a critical step in the pathogenesis of most bacterial infections. Enterotoxigenic Escherichia coli (ETEC) remains one of the principal causes of infectious diarrhea in humans. The recent identification of additional ETEC surface molecules suggests that new targets may be exploited in vaccine development. The EtpA protein identified in ETEC H10407 is a large glycosylated adhesin secreted via the two-partner secretion system. EtpA requires its putative partner EtpB for translocation across the outer membrane (OM). We investigated the biochemical and electrophysiological properties of purified EtpB. We showed that EtpB is 65-kDa heat-modifiable protein localized to the OM. Electrophysiological experiments indicated that EtpB is able to form pores in planar lipid bilayer membranes with an asymmetric current, suggesting its functional asymmetry. The pore of EtpB frequently assumes an opened conformation and fluctuates between three well-defined conductance states. In silico analysis of the EtpB amino acid sequence and molecular modeling suggest that EtpB is similar to the well-known TpsB protein FhaC from Bordetella pertussis and has a C-terminal transmembrane beta-barrel domain that is occluded by an N-terminal alpha-helix, an extracellular loop, and two periplasmic polypeptide-transport-associated (POTRA) domains. Together, these data confirm that EtpB is a pore-forming protein mainly folded into a beta-barrel conformation and indicate that EtpB presents typical features of the OM TpsB proteins.

First structural insights into the TpsB/Omp85 superfamily.

Proteins of the TpsB/Omp85 superfamily are involved in protein transport across, or assembly into, the outer membrane of Gram-negative bacteria, and their distant eukaryotic relatives exert similar functions in chloroplasts and mitochondria. The X-ray structure of one TpsB transporter, FhaC, provides the bases to decipher the mechanisms of action of these proteins. With two POTRA domains in the periplasm, a transmembrane beta barrel and a large loop harboring a functionally important motif, FhaC epitomizes the conserved features of the super-family.

Structural and functional analysis of the HCV p7 protein.

The p7 membrane polypeptide from HCV is essential for virus infection. It exhibits ion-channel activity reported to be specifically blocked by various compounds. These properties make p7 an attractive candidate target for antiviral intervention to combat viral hepatitis C infection. In this context, in vitro functional analyses of isolated p7 coupled to structural characterization are critical for further understanding of the molecular mechanisms of p7 ion-channel activity and for the development of new antiviral drugs. We present here in vitro assays designed to purify synthetic p7 by RP-HPLC, to investigate its ion-channel properties by means of planar lipid-bilayer assays and patch-clamp recordings after reconstitution into liposomes, and to analyze its structural features by circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular dynamics (MD).

Influence of the passenger domain of a model autotransporter on the properties of its translocator domain.

Autotransporters are a superfamily of proteins secreted by Gram-negative bacteria including many virulence factors. They are modular proteins composed of an N-terminal signal peptide, a surface-exposed 'passenger' domain carrying the activity of the protein, and a C-terminal 'translocator' domain composed of an alpha-helical linker region and a transmembrane beta-barrel. The translocator domain plays an essential role for the secretion of the passenger domain across the outer membrane; however, the mechanism of autotransport remains poorly understood. The whooping cough agent Bordetella pertussis produces an autotransporter serine-protease, SphB1, which is involved in the maturation of an adhesin at the bacterial surface. SphB1 also mediates the proteolytic maturation of its own precursor. We used SphB1 as a model autotransporter and performed the first comparisons of the biochemical and biophysical properties of an isolated translocator domain with those of the same domain preceded by the C-terminal moiety of its natural passenger. By using cross-linking and dynamic light scattering, we provide evidence that the passenger domain promotes the auto-association of SphB1, although these interactions appear rather labile. Electrophysiological studies revealed that the passenger domain of the autotransporter appears to maintain the translocator channel in a low-conductance conformation, most likely by stabilizing the alpha-helix inside the pore. That the passenger may significantly influence AT physicochemical properties is likely to be relevant for the in vivo maturation and stability of AT proteins.