Structural insight into the Staphylococcus aureus ATP-driven exporter of virulent peptide toxins.

Staphylococcus aureus is a major human pathogen that has acquired alarming broad-spectrum antibiotic resistance. One group of secreted toxins with key roles during infection is the phenol-soluble modulins (PSMs). PSMs are amphipathic, membrane-destructive cytolytic peptides that are exported to the host-cell environment by a designated adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter, the PSM transporter (PmtABCD). Here, we demonstrate that the minimal Pmt unit necessary for PSM export is PmtCD and provide its first atomic characterization by single-particle cryo-EM and x-ray crystallography. We have captured the transporter in the ATP-bound state at near atomic resolution, revealing a type II ABC exporter fold, with an additional cytosolic domain. Comparison to a lower-resolution nucleotide-free map displaying an "open" conformation and putative hydrophobic inner chamber of a size able to accommodate the binding of two PSM peptides provides mechanistic insight and sets the foundation for therapeutic design.

Correlation Between Sarcopenia and Growth Rate of the Future Liver Remnant After Portal Vein Embolization in Patients with Colorectal Liver Metastases.

PURPOSE: To investigate whether sarcopenia and myosteatosis correlate with the degree of hypertrophy (DH) and kinetic growth rate (KiGR) of the future liver remnant (FLR) in patients with colorectal liver metastases undergoing portal vein embolization (PVE) in preparation for right hepatectomy. MATERIALS AND METHODS: Forty-two patients were included. Total liver volume and FLR volume were measured before and 2-4 weeks after PVE. KiGR of the FLR was calculated. Sarcopenia was assessed using the total psoas muscle volume (PMV), the psoas muscle cross-sectional area (PMCS) and the total skeletal muscle index (L3SMI) at the level of 3rd lumbar vertebra. Degree of myosteatosis was assessed by mean muscle attenuation at L3 (L3MA). Correlations between muscle indices and DH and KiGR were assessed using simple linear regression analyses. RESULTS: Mean DH was 8.9 ± 5.7%, and mean KiGR was 3.6 ± 2.3. Mean PMV was 55.56 ± 14.19 cm3/m3, mean PMCS was 8.76 ± 2.3 cm2/m2, mean L3SMI was 45.6 ± 9.89 cm2/m2, and mean L3MA was 27.9 ± 18.6 HU. There was a strong positive correlation between PMV and DH (R = 0.503, p = 0.001) and PMV and KiGR (R = 0.545, p < 0.001). Furthermore, there was a moderate correlation between PMCS and KiGR (R = 0.389, p = 0.014). L3SMI and L3MA were neither associated with DH (p = 0.390 and p = 0.768, respectively) nor with KiGR (p = 0.188 and p = 0.929, respectively). CONCLUSION: We identified a positive correlation between PMV and PMCS, as markers for sarcopenia, and the KiGR of the FLR after PVE. PMV and PMCS might therefore aid to identify patients who are poor candidates for FLR augmentation using PVE alone.

In vivo assembly of a truncated H subunit mutant of the Rhodobacter sphaeroides photosynthetic reaction centre and direct electron transfer from the QA quinone to an electrode.

We address a challenge in the engineering of proteins to redirect electron transfer pathways, using the bacterial photosynthetic reaction centre (RC) pigment-protein complex. Direct electron transfer is shown to occur from the QA quinone of the Rhodobacter sphaeroides RC containing a truncated H protein and bound on the quinone side to a gold electrode. In previous reports of binding to the quinone side of the RC, electron transfer has relied on the use of a soluble mediator between the RC and an electrode, in part because the probability of QB quinone reduction is much greater than that of direct electron transfer through the large cytoplasmic domain of the H subunit, presenting a ~ 25 Å barrier. A series of C-terminal truncations of the H subunit were created to expose the quinone region of the RC L and M proteins, and all truncated RC H mutants assembled in vivo. The 45M mutant was designed to contain only the N-terminal 45 amino acid residues of the H subunit including the membrane-spanning α-helix; the mutant RC was stable when purified using the detergent N-dodecyl-ß-D-maltoside, contained a near-native ratio of bacteriochlorophylls to bacteriopheophytins, and showed a charge-separated state of [Formula: see text]. The 45M-M229 mutant RC had a Cys residue introduced in the vicinity of the QA quinone on the newly exposed protein surface for electrode attachment, decreasing the distance between the quinone and electrode to ~ 12 Å. Steady-state photocurrents of up to around 200 nA/cm2 were generated in the presence of 20 mM hydroquinone as the electron donor to the RC. This novel configuration yielded photocurrents orders of magnitude greater than previous reports of electron transfer from the quinone region of RCs bound in this orientation to an electrode.

Xaliproden (SR57746A) induces 5-HT1A receptor-mediated MAP kinase activation in PC12 cells.

Neurotrophic growth factors are involved in cell survival. However, natural growth factors have a very limited therapeutic use because of their short half-life. In the present study, we investigated the mechanism of action of a non-peptidic neurotrophic drug, Xaliproden, a potential molecule for the treatment of motoneuron diseases, since the transduction pathways of this synthetic 5-HT1A agonist are very poorly understood. Xaliproden does not activate the Trk receptor but causes a rapid increase in the activities of the ERK1 and ERK2 isoforms of MAP kinase, which then rapidly decrease to the basal level. We demonstrate that isoforms of the SHC adapter protein are phosphorylated independently of each other and are probably not the source of the Xaliproden-induced MAP kinases activation. The inhibitor of Ras farnesylation, FPT-1, and the protein kinase C inhibitors, GF 109203X and chelerythrine, inhibited the Xaliproden-induced MAP kinase activation, suggesting p21Ras and PKC involvement. Moreover, the observations that the 5-HT1A antagonist, pindobind, and pertussis toxin abolished the Xaliproden-induced ERK stimulation suggested that Xaliproden activates the MAP kinase pathways by stimulating the G protein-coupled receptor, 5-HT1A. These results demonstrate clearly that the non-peptidic compound, Xaliproden, exerts its neurotrophic effects through a mechanism of action differing from that of neurotrophins. These findings suggest that this compound does not involve MAPK activation by TrkA receptor stimulation but acts by MAP kinase pathway by a pertussis toxin-sensitive mechanism involving 5-HT1A receptors, p21 Ras and MEK-1 and by PKC and Akt pathways.

MAPK activation via 5-hydroxytryptamine 1A receptor is involved in the neuroprotective effects of xaliproden.

Motoneurons require neurotrophic factors for their survival and their differentiation. Xaliproden (SR57746A) is a synthetic compound that exhibits in vivo and in vitro neurotrophic effects in several experimental studies. Here we demonstrate that neuroprotective effects of Xaliproden on motoneuron cultures are mediated by the activation of the mitogen activated protein kinase pathway. It is inhibited by PD98059, a selective and irreversible inhibitor of MEK1. The activation of this pathway seems to involve two different proteins, the protein kinase C and the Ras. Indeed, we show that Xaliproden is able to activate the MAP kinases ERK1/2 and PKC in motoneurons. In addition, the use of a 5-hydroxytryptamine 1A receptor antagonist, Pindobind and pertussis toxin, inhibits the effect of Xaliproden on motoneuron survival, suggesting the involvement of this G-protein coupled receptor. Morever, 8-OH-DPAT, an agonist of 5-hydroxytryptamine 1A receptor, increases the survival of mouse motoneurons but not by the same extent as BDNF or xaliproden. Since 8-OH-DPAT does not act synergistically with Xaliproden, it is likely that their neuroprotective properties involve a similar pathway. Taken together, these results indicate that neuroprotective effects of Xaliproden on mouse motoneurons are dependent on the mitogen-activated protein kinase activation via 5-hydroxytryptamine 1A receptor.

Quantification of neurotrophin mRNA expression in PMN mouse: modulation by xaliproden.

Compounds possessing neurotrophic properties may represent a possible treatment for neurodegenerative disorders such as amyotrophic lateral sclerosis. Xaliproden (SR57746A), an orally-active non-peptide compound, which has been found to exhibit neurotrophic effects in vitro and in vivo, increased the lifespan and delayed the progression of the motor neuron degeneration in PMN mice. We have used a quantitative reverse transcription/polymerase chain reaction amplification technique to study the regulation of neurotrophin mRNA and trk mRNA expression in PMN mice. NGF and NT-3 mRNA are downregulated in PMN mice. These deficiencies can be overcome by a treatment with xaliproden. Such an effect could contribute to neurotrophic effects of xaliproden in vivo and in vitro.

Projection structure and oligomeric properties of a bacterial core protein translocase.

The major route for protein export or membrane integration in bacteria occurs via the Sec-dependent transport apparatus. The core complex in the inner membrane, consisting of SecYEG, forms a protein-conducting channel, while the ATPase SecA drives translocation of substrate across the membrane. The SecYEG complex from Escherichia coli was overexpressed, purified and crystallized in two dimensions. A 9 A projection structure was calculated using electron cryo-microscopy. The structure exhibits P12(1) symmetry, having two asymmetric units inverted with respect to one another in the unit cell. The map shows elements of secondary structure that appear to be transmembrane helices. The crystallized form of SecYEG is too small to comprise the translocation channel and does not contain a large pore seen in other studies. In detergent solution, the SecYEG complex displays an equilibrium between monomeric and tetrameric forms. Our results therefore indicate that, unlike other known channels, the SecYEG complex can exist as both an assembled channel and an unassembled smaller unit, suggesting that transitions between the two states occur during a functional cycle.

The AprX protein of Pseudomonas aeruginosa: a new substrate for the Apr type I secretion system.

Protein secretion in Pseudomonas aeruginosa involves different mechanisms. The type II and type III secretory pathways control the extracellular release of a wide range of substrates. The type I secretion process, or ABC transporter, was believed to be exclusively involved in alkaline protease secretion. Recently, it was discovered that a P. aeruginosa heme binding protein, HasAp, is also secreted by a type I process. We present here the identification of a third putative type I-dependent protein of P. aeruginosa, AprX. The function of this protein has not yet been elucidated but very interestingly it appears to be linked to the apr cluster, and organized in one single operon together with the aprD, -E and -F genes.

Effect of the nonpeptide neurotrophic compound SR 57746A on the phenotypic survival of purified mouse motoneurons.

1. Neurotrophic factors have been used for the treatment of several neurodegenerative diseases. However, their use is limited by their inability to cross the blood-brain barrier, their short half life and their side effects. SR 57746A is a new orally active compound that exhibits in vivo and in vitro neurotrophic effects in several experimental models. 2. We show here that SR 57746A (1 microM) increases the phenotypic survival of embryonic purified mouse motoneurons in vitro to the same extent as brain-derived neurotrophic factor (100 ng ml-1), and increases the outgrowth and number of their neurites. It acts in a dose-dependent manner up to 1 microM which is the optimal concentration. Above this concentration, its neurotrophic effect decreases. 3. Genistein (10 microM), a protein tyrosine kinase inhibitor, also increases the phenotypic survival and differentiation of mouse motoneurons. It does not act in a synergistic or additive manner with SR 57746A. However, at concentrations equal or superior to 25 microM, it decreases the survival of motoneurons. This suggests that the neurotrophic effect of genistein is due to a favourable alteration of equilibrium between phosphorylated and dephosphorylated states of proteins involved in survival and differentiation of motoneurons. 4. Like genistein, SR 57746A should be used at a critical concentration (1 microM) to exert its optimal effects. Since SR 57746A does not act synergistically with genistein, it is likely that its mechanism of action involves a pathway similar to that affected by this tyrosine kinase inhibitor. 5. At the present time, SR 57746A is the only orally active compound and the only synthetic compound shown to be active on motoneurons in vitro. It should thus be considered as a good candidate for the treatment of motoneuron diseases.

The PrlA and PrlG phenotypes are caused by a loosened association among the translocase SecYEG subunits.

prlA mutations in the gene encoding the SecY subunit of the membrane domain of the Escherichia coli preprotein translocase confer many phenotypes: enhanced translocation rates, increased affinity for SecA, diminished requirement for functional leader sequences, reduced proton-motive force (PMF) dependence of preprotein translocation and facilitated translocation of preproteins with folded domains. We now report that both prlA and prlG mutations weaken the associations between the SecY, SecE and SecG subunits of the translocase. This loosened association increases the initiation of translocation by facilitating the insertion of SecA with its bound preprotein but reduces the stimulatory effect of the PMF during the initial step of translocation. Furthermore, the originally isolated prlA4 mutant, which possesses a particularly labile SecYEG complex, acquired a secondary mutation that restored the stability while conserving the flexibility of the complex. Combinations of certain prlA and prlG mutations, known to cause synthetic lethality in vivo, dramatically loosen subunit association and lead to complete disassembly of SecYEG. These findings underscore the importance of the loosened SecYEG association for the Prl phenotypes. We propose a model in which each of the PrlA and PrlG phenotypes derive from this enhanced SecYEG conformational flexibility.

The effect of the nonpeptide neurotrophic compound SR 57746A on the progression of the disease state of the pmn mouse.

1. The progressive motor neuronopathy (pmn) mouse is an autosomal recessive mutant, in which the homozygotes suffer caudio-cranial degeneration of motor axons and die several weeks after birth. This strain provides the opportunity of testing potential therapeutic strategies for the treatment of motor neurone diseases such as amyotrophic lateral sclerosis. We have performed a study of the effects on the pmn mouse of SR 57746A, an orally-active, non-peptide compound which has been found to exhibit neurotrophic effects in vitro and in vivo. In order to treat the affected mice from birth, the mothers were administered 2.5 mg kg(-1). p.o., SR 57746A every two days until the weaning of the offspring (at day 20); then the offspring were given every two days a dose of 30 microg kg(-1), p.o., until their death. 2. Affected mice treated with SR 57746A had a lifespan 50% longer than that of the vehicle-treated mice (P=0.01). Compared to vehicle-treated pmn mice, SR 57746A improved the performance of the pmn mice in three different behavioural tasks. SR 57746A also maintained the amplitude of the motor evoked response of the gastrocnemius muscle, reduced the distal motor latency, and delayed the occurrence of the spontaneous denervation activity in this muscle. Histological studies indicated that at 20 days of age the mean surface areas of the fibres of the sciatic nerve were higher in SR 57746A-treated than in vehicle-treated mice. 3. At present, SR 57746A is the only orally active, nonpeptide compound known to be capable of delaying the progression of the motor neurone degeneration in pmn mice.

Sec-dependent membrane protein biogenesis: SecYEG, preprotein hydrophobicity and translocation kinetics control the stop-transfer function.

Preprotein translocase catalyzes membrane protein integration as well as complete translocation. Membrane proteins must interrupt their translocation and be laterally released from the translocase into the lipid bilayer. We have analyzed the translocation arrest and lateral release activities of Escherichia coli preprotein translocase with an in vitro reaction and the preprotein proOmpA carrying a synthetic stop-transfer sequence. Membrane protein integration is catalytic, occurs with kinetics similar to those of proOmpA itself and only requires the functions of SecYEG and SecA. Though a strongly hydrophobic segment will direct the protein to leave the translocase and enter the lipid bilayer, a protein with a segment of intermediate hydrophobicity partitions equally between the translocated and membrane-integrated states. Analysis of the effects of PMF, varied ATP concentrations or synthetic translocation arrest show that the stop-translocation efficiency of a mildly hydrophobic segment depends on the translocation kinetics. In contrast, the lateral partitioning from translocase to lipids depends solely on temperature and does not require SecA ATP hydrolysis or SecA membrane cycling. Thus translocation arrest is controlled by the SecYEG translocase activity while lateral release and membrane integration are directed by the hydrophobicity of the segment itself. Our results suggest that a greater hydrophobicity is required for efficient translocation arrest than for lateral release into the membrane.

The SecDFyajC domain of preprotein translocase controls preprotein movement by regulating SecA membrane cycling.

Escherichia coli preprotein translocase comprises a membrane-embedded hexameric complex of SecY, SecE, SecG, SecD, SecF and YajC (SecYEGDFyajC) and the peripheral ATPase SecA. The energy of ATP binding and hydrolysis promotes cycles of membrane insertion and deinsertion of SecA and catalyzes the movement of the preprotein across the membrane. The proton motive force (PMF), though not essential, greatly accelerates late stages of translocation. We now report that the SecDFyajC domain of translocase slows the movement of preprotein in transit against both reverse and forward translocation and exerts this control through stabilization of the inserted form of SecA. This mechanism allows the accumulation of specific translocation intermediates which can then complete translocation under the driving force of the PMF. These findings establish a functional relationship between SecA membrane insertion and preprotein translocation and show that SecDFyajC controls SecA membrane cycling to regulate the movement of the translocating preprotein.

Distinct catalytic roles of the SecYE, SecG and SecDFyajC subunits of preprotein translocase holoenzyme.

Escherichia coli preprotein translocase contains a membrane-embedded trimeric complex of SecY, SecE and SecG (SecYEG) and the peripheral SecA protein. SecYE is the conserved functional 'core' of the SecYEG complex. Although sufficient to provide sites for high-affinity binding and membrane insertion of SecA, and for its activation as a preprotein-dependent ATPase, SecYE has only very low capacity to support translocation. The proteins encoded by the secD operon--SecD, SecF and YajC--also form an integral membrane heterotrimeric complex (SecDFyajC). Physical and functional studies show that these two trimeric complexes are associated to form SecYEGDFyajC, the hexameric integral membrane domain of the preprotein translocase 'holoenzyme'. Either SecG or SecDFyajC can support the translocation activity of SecYE by facilitating the ATP-driven cycle of SecA membrane insertion and de-insertion at different stages of the translocation reaction. Our findings show that each of the prokaryote-specific subunits (SecA, SecG and SecDFyajC) function together to promote preprotein movement at the SecYE core of the translocase.

Separable ATPase and membrane insertion domains of the SecA subunit of preprotein translocase.

The SecA subunit of preprotein translocase drives ATP-dependent translocation of preproteins across the bacterial inner membrane concomitant with cycles of membrane insertion and de-insertion (Economou, A., and Wickner, W. (1994) Cell 78, 835-843). We have identified the membrane-inserting region of SecA as a 30-kDa domain in the C-terminal third of the protein beginning at aminoacyl residue 610. Limited proteolysis in the absence of translocation ligands indicates that the SecA monomer is composed of two primary structural domains, the 30-kDa membrane-inserting domain and an N-terminal 65-kDa ATPase domain. This limited protease treatment of SecA results in constitutive ATPase activity, indicating that intramolecular constraints between the two domains may play a role in the regulation of ATP hydrolysis by SecA.

Protein secretion by heterologous bacterial ABC-transporters: the C-terminus secretion signal of the secreted protein confers high recognition specificity.

Pseudomonas aeruginosa releases several extracellular proteins which are secreted via two independent secretion pathways. Alkaline protease (AprA) Is released by its own specific secretion machinery which is an ABC-transporter. Despite sequence similarities between components of ABC-transporters in different bacteria, each transporter is dedicated to the secretion of a particular protein or a family of closely related proteins. Heterologous complementation between ABC-transporters for unrelated polypeptides can occur, but only at a very low level. We show that the 50 C-terminal amino acids of AprA constitute an autonomous secretion signal. By heterologous complementation experiments between the unrelated alpha-haemolysin (HlyA) and Apr secretion systems we demonstrated that it is only the recognition of the secretion signal by the translocator which confers specificity to the secretion process. Secretion was size-dependent. However inclusion of glycine-rich repeats from HlyA in AprA seems to overcome the size limitation exerted by the Apr secretion apparatus such that the machinery secreted a hybrid protein 20 kDa larger than the normal maximal size.

The Pseudomonas fluorescens lipase has a C-terminal secretion signal and is secreted by a three-component bacterial ABC-exporter system.

Both Pseudomonas aeruginosa and Pseudomonas fluorescens secrete a lipase into the extracellular medium. Unlike the lipase of P. aeruginosa, the lipase produced by P. fluorescens does not contain any N-terminal signal sequence. We show that the P. fluorescens lipase is secreted through the signal peptide-independent pathway of the alkaline protease that we previously identified in P. aeruginosa. Secretion of this protease (AprA) is dependent on the presence of three genes located adjacent to the aprA gene, aprD, aprE and aprF. The three secretion functions permit an efficient secretion of P. fluorescens lipase. Inactivation of one of them (AprE) prevented this secretion. In Escherichia coli, the three proteins AprD, AprE, AprF are necessary and sufficient for efficient secretion of lipase to the extracellular medium. The secretion signal is located within the C-terminal part of the lipase sequence and can promote efficient secretion of a passenger protein. Thus the P. fluorescens lipase secretion system belongs to the group of the three-component bacterial ABC-exporter systems.

Sequence of a cluster of genes controlling synthesis and secretion of alkaline protease in Pseudomonas aeruginosa: relationships to other secretory pathways.

A genetic locus implicated in the synthesis and secretion of alkaline protease (APR) in Pseudomonas aeruginosa has been previously described [Guzzo et al., J. Bacteriol. 172 (1990) 942-948]. The nucleotide sequence of the DNA fragment encoding these functions was determined and revealed the existence of five open reading frames: aprA, the structural gene encoding APR; aprI, which encodes a protease inhibitor; and aprD, aprE, aprF whose products are involved in protease secretion. The AprD, AprE and AprF proteins share significant homology with proteins implicated in secretion of Erwinia chrysanthemi proteases and Escherichia coli alpha-haemolysin. These results provide further evidence for the existence of a specialized secretory system widespread among Gram- bacteria.

Pseudomonas aeruginosa alkaline protease: evidence for secretion genes and study of secretion mechanism.

A 6.5-kb DNA fragment carrying the functions required for specific secretion of the extracellular alkaline protease produced by Pseudomonas aeruginosa was cloned. The whole 6.5-kb DNA fragment was transcribed in one direction and probably carried three genes involved in secretion. The expression in trans of these genes, together with the apr gene, in Escherichia coli allowed synthesis and secretion of the alkaline protease, which was extensively investigated by performing pulse-chase experiments under various conditions. We demonstrated the absence of a precursor form, as well as the independence of alkaline protease translocation from SecA. The absence of secretion genes impaired alkaline protease secretion; the protein then remained intracellular and was partially degraded.