A pharmacoproteomic approach implicates eukaryotic elongation factor 2 kinase in ER stress-induced cell death.

Apoptosis triggered by endoplasmic reticulum (ER) stress has been implicated in many diseases but its cellular regulation remains poorly understood. Previously, we identified salubrinal (sal), a small molecule that protects cells from ER stress-induced apoptosis by selectively activating a subset of endogenous ER stress-signaling events. Here, we use sal as a probe in a proteomic approach to discover new information about the endogenous cellular response to ER stress. We show that sal induces phosphorylation of the translation elongation factor eukaryotic translation elongation factor 2 (eEF-2), an event that depends on eEF-2 kinase (eEF-2K). ER stress itself also induces eEF-2K-dependent eEF-2 phosphorylation, and this pathway promotes translational arrest and cell death in this context, identifying eEF-2K as a hitherto unknown regulator of ER stress-induced apoptosis. Finally, we use both sal and ER stress models to show that eEF-2 phosphorylation can be activated by at least two signaling mechanisms. Our work identifies eEF-2K as a new component of the ER stress response and underlines the utility of novel small molecules in discovering new cell biology.

[Interleukin-1, inflammasome and autoinflammatory diseases]. / Interleukine-1, inflammasome et maladies auto-inflammatoires.

Interleukin-1 is a major cytokine of innate immunity and inflammation. It exerts various systemic effects during the inflammatory response, such as fever induction, thrombopoiesis and granulopoiesis, or leukocyte recruitment. Its involvement has been demonstrated in many inflammatory-mediated diseases, such as diabetes or gout. Moreover, interleukin-1 plays a pivotal role in some autoinflammatory diseases, such as cryopyrinopathies or familial Mediterranean fever. In these diseases, a constitutional defect of the inflammasome, a protein complex responsible for the activation of interleukin-1, explains the hypersecretion of interleukin-1. Other autoinflammatory diseases have a more complex pathophysiology involving deregulation of the interleukin-1 pathway, upstream or downstream of the inflammasome, or through more complex mechanisms. In this review, we are detailing the synthesis, the activation, the signalling, and the regulation of interleukin-1. We then describe the autoinflammatory diseases or related-diseases where the pathological role of interleukin-1 has been demonstrated.

The concept of a waiting period for preoperative patient consent: Prospective study of 51 shoulder arthroscopy cases.

INTRODUCTION: The French Code of Public Health (CSP) does not explicitly require that patients should be given a certain amount of time to think about a procedure, except for cosmetic surgery, where 15 days is required (Art. L 6322-2 CSP). We hypothesized that patients require a waiting period during their decision-making process for scheduled shoulder arthroscopy procedure. MATERIALS AND METHODS: This prospective observational study of 51 patients analysed the concept of a waiting period based on a 10-item questionnaire. A comparative statistical approach was used and the P values were calculated using a paired Wilcoxon rank-sum test. RESULTS: Of the 51 patients, 42 (82%) rejected the concept of a waiting period before the procedure and 37 patients (73%) did not want a mandatory waiting period imposed by law. DISCUSSION: This study looked at the decision-making process during scheduled orthopaedic surgery and differentiated between the conscious and unconscious approach corresponding to an active and passive waiting period. A waiting period does not allow patients to make a conceptually deliberative decision that conforms to the criteria defined by the French Health Authority. This study rejects the need for a mandatory waiting period imposed on surgeons and patients as it does not integrate itself into the informative model of ethical decision-making for scheduled shoulder arthroscopy. TYPE OF STUDY: Prospective, observational; level of evidence IV.

Assembly of the type II secretion machinery of Erwinia chrysanthemi: direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL.

Erwinia chrysanthemi secretes, by the type II secretory pathway, a large number of enzymes, including cellulases and pectinases. This process requires the products of the out genes, which are widely conserved in Gram-negative bacteria. The Out proteins are thought to form a membrane-associated multiprotein complex. Here, we investigated interaction between OutE, the putative ATP binding component, and OutL, an inner membrane protein. We showed, by limited proteolysis, genetic suppression and the yeast two-hybrid system, that OutE and OutL interact directly. Analysis of truncated forms of OutE demonstrated that the N terminus of OutE (residues 1-97) is important for the OutE/OutL interaction. Moreover, results from the yeast two-hybrid system suggested that OutE and OutL are each able to form homomultimers. The region required for homomultimerisation of OutE is located in its C terminus. Limited proteolysis assay indicated that OutE induces a conformational change in OutL, in both its cytoplasmic and periplasmic domains. Moreover, the secretion process requires a conformational change in OutE which depends on both the interaction with OutL and on the presence of an intact Walker A motif in OutE. Our results support the view that interaction occurring on the cytoplasmic side influences the events occurring in the outer membrane. We discuss a model in which OutE uses ATP to control the assembly of the type II secretion machinery.

Type II protein secretion in gram-negative pathogenic bacteria: the study of the structure/secretion relationships of the cellulase Cel5 (formerly EGZ) from Erwinia chrysanthemi.

Erwinia chrysanthemi, a Gram-negative plant pathogen, secretes the cellulase Cel5 (formerly EGZ) via the type II secretion pathway (referred to as Out). Cel5 is composed of two domains, a large N-terminal catalytic domain (390 amino acid residues) and a small C-terminal cellulose-binding domain (62 amino acid residues) separated by a linker region. A combination of mutagenesis and structural analysis permitted us to investigate the structure/secretion relationships with respect to the catalytic domain of Cel5. The 3D structure of the catalytic domain was solved by molecular replacement at 2.3 A resolution. Cel5 exhibits the (beta/alpha)8 structural fold and two extra-barrel features. Our previous genetic study based upon tRNA-mediated suppression allowed us to predict positions of importance in the molecule in relation to structure and catalysis. Remarkably, all of the predictions proved to be correct when compared with the present structural information. Mutations of Arg57, which is located at the heart of the catalytic domain, allowed us to test the consequences of structural modifications on the secretion efficiency. The results revealed that secretability imposes remarkably strong constraints upon folding. In particular, an Arg-to-His mutation yielded a species that folded to a stable conformation close to, but distinct from the wild-type, which however was not secretable. We discuss the relationships between folding of a protein in the periplasm, en route to the cell exterior, and presentation of secretion information. We propose that different solutions have been selected for type II secreted exoproteins in order to meet the constraints imposed by their interaction with their respective secretion machineries. We propose that evolutionary pressure has led to the adaptation of different secretion motifs for different type II exoproteins.

[Influence of individual cultural life on the penal system: the role of the medical expert]. / L'influence du vécu culturel individuel sur la procédure pénale: le rôle du médecin-expert.

A French Melanesian citizen who killed his uncle attributed his act to local beliefs involving witchcraft. The medical expert called on to give his psychiatric assessment ponders if he was able to fulfill his role of mediator. Since examination of the defendant revealed no mental abnormalities, the only possible explanation for the crime was the one implicating the local culture that made the defendant say that he had not killed a relative but rather a sorcerer. The psychiatrist asked the lawyer for a complete legal opinion. From the penal standpoint establishing responsibility depends on the defendant's powers of discrimination. Criminal law distinguishes between the intent, motive, and objectives of an act on the basis of one criteria which is damage in most cases. In the present case the act was intentional, the motive could not be taken into account, and the objective could not be considered to have social value. This case forces the conclusion that the juridical concept of "justifying fact" is based on social choices that rarely coincide with the culture of individuals in a minority group. By finding the defendant guilty the judicial system achieved social redress with respect to one culture but denied moral redress according to the values of another.

[3000 interviews before elective interruption of pregnancy]. / Trois mille entretiens avant interruption volontaire de grossesse (I.V.G.).

PIP: A statistical study has been conducted in France on 3004 interviews preceding abortion, to objectively try to evaluate the consequences of the law of January 17, 1975, legalizing abortion. It was possible to conclude: 1) that almost 50% of abortions are not the result of a free decision, but that the woman was subjected to hard pressures to terminate her pregnancy; 2) that if serious problems will result as consequence of the pregnancy in about 25% of cases, abortion for pure decency's sake or for convenience's sake is practiced in 25% of cases; 3) that the percentage of unterminated pregnancies diminishes steeply along the years. These data show that abortion in France has become a trivial and almost matter of fact habit.^ieng

[Patient information duties in radiation oncology]. / Devoir d'information au patient en radiothérapie.

Patient information duties are a basic task of radiation oncologists in their daily practice. This article is essentially a factsheet on legal obligations, the value of written informed consent and information documents that ought to be given to patient.

Caspase-1 activity affects AIM2 speck formation/stability through a negative feedback loop.

The inflammasome is an innate immune signaling platform leading to caspase-1 activation, maturation of pro-inflammatory cytokines and cell death. Recognition of DNA within the host cytosol induces the formation of a large complex composed of the AIM2 receptor, the ASC adaptor and the caspase-1 effector. Francisella tularensis, the agent of tularemia, replicates within the host cytosol. The macrophage cytosolic surveillance system detects Francisella through the AIM2 inflammasome. Upon Francisella novicida infection, we observed a faster kinetics of AIM2 speck formation in ASC(KO) and Casp1(KO) as compared to WT macrophages. This observation was validated by a biochemical approach thus demonstrating for the first time the existence of a negative feedback loop controlled by ASC/caspase-1 that regulates AIM2 complex formation/stability. This regulatory mechanism acted before pyroptosis and required caspase-1 catalytic activity. Our data suggest that sublytic caspase-1 activity could delay the formation of stable AIM2 speck, an inflammasome complex associated with cell death.

Mechanisms of iron-sulfur cluster assembly: the SUF machinery.

Biosynthesis of iron-sulfur clusters is a cellular process which depends on complex protein machineries. Escherichia coli contains two such biosynthetic systems, ISC and SUF. In this review article we specifically make a presentation of the various Suf proteins and discuss the molecular mechanisms by which these proteins work together to assemble Fe and S atoms within a scaffold and to transfer the resulting cluster to target proteins.

Mutagenesis of cellulase EGZ for studying the general protein secretory pathway in Erwinia chrysanthemi.

Extracellular secretion of endoglucanase Z (EGZ) from Erwinia chrysanthemi is mediated by the so-called Out general secretion pathway and, presumably, involves recognition of EGZ-carried structural information by one or more of the Out proteins. Investigating the relationships between structure and secretability of EGZ was the purpose of the present work. EGZ is made of two independent domains, located at the N- and C-proximal sides, separated by a Ser/Thr-rich region, which are responsible for catalysis and cellulose-binding, respectively. The existence of a secretion region ('targeting signal') was investigated by studying the secretability of modified EGZ derivatives. These resulted from deletion or peptide insertion and were designed by using the domain organization cited above as a guide. Catalytic and/or cellulose-binding tests showed that all proteins exhibited at least a functional EGZ domain while immunoblot analyses confirmed that neither the insertions nor the deletions led to grossly misfolded proteins. In contrast, all of the proteins lost their secretability in E. chrysanthemi. This suggested that at least two secretion motifs existed, one lying within each functional domain. The role of the Ser/Thr-rich linker region was subsequently tested. Accordingly, two proteins containing a linker region whose length was increased by the addition of 8 and 18 additional residues and one protein lacking the linker region were studied. All three exhibited endoglucanase activity and cellulose-binding ability, confirming the independence of the domains within the context of EGZ/polysaccharide interaction. In contrast, none was secreted by E. chrysanthemi.(ABSTRACT TRUNCATED AT 250 WORDS)

An inner membrane platform in the type II secretion machinery of Gram-negative bacteria.

The type II secretion machinery allows most Gram-negative bacteria to deliver virulence factors into their surroundings. We report that in Erwinia chrysanthemi, GspE (the putative NTPase), GspF, GspL and GspM constitute a complex in the inner membrane that is presumably used as a platform for assembling other parts of the secretion machinery. The GspE-GspF-GspL-GspM complex was demonstrated by two methods: (i) co-immunoprecipitation of GspE-GspF-GspL with antibodies raised against either GspE or GspF; (ii) interactions in the yeast two-hybrid system between GspF and GspE, GspF and GspL, GspL and GspM. GspL was found to have an essential role in complex formation. We propose a model in which the GspE-GspF-GspL-GspM proteins constitute a building block within the secretion machinery on top of which another building block, referred to as a pseudopilus, assembles. By analogy, we predict that a similar platform is required for the biogenesis of the type IV pilus.

mRNA degradation in Escherichia coli: a novel factor which impedes the exoribonucleolytic activity of PNPase at stem-loop structures.

Stem-loop structures can protect upstream mRNA from degradation by impeding the processive activities of 3'-5' exoribonucleases. The ability of such structures to impede exonuclease activity in vitro is insufficient to account for the stability they can confer on mRNA in vivo. In this study we identify a factor from Escherichia coli which specifically impedes the processive activity of the 3'-5' exonuclease PNPase at stem-loop structures in vitro. This factor can, potentially, reconcile the apparent discrepancy between the ability of 3' stem-loop structures to stabilize upstream mRNA in vitro and in vivo. Its mechanism of action, and possible role in regulating mRNA degradation, is discussed.

Periplasmic disulphide bond formation is essential for cellulase secretion by the plant pathogen Erwinia chrysanthemi.

Secretion to the cell exterior of cellulase EGZ and of at least six pectinases enables the Gram-negative Erwinia chrysanthemi to cause severe plant disease. The C-terminal cellulose-binding domain (CBD) of EGZ was found to contain a disulphide bond which forms, in the periplasm, between residues Cys-325 and Cys-382. Dithiothreitol (DTT)-treatment of native EGZ showed that the disulphide bond was dispensable, both for catalysis and cellulose binding. Adding DTT to E. chrysanthemi cultures led to immediate arrest of secretion of EGZ which accumulated in the periplasm where the CBD was eventually proteolysed. Site-directed mutagenesis that affected Cys residues involved in disulphide bond formation resulted in molecules that were catalytically active and able to bind to cellulose but were no longer secreted. Instead they accumulated in the periplasm. Interestingly, the region around EGZ Cys-325 is conserved in two pectinases secreted by the same pathway as EGZ. We conclude that the conserved Cys, and possibly adjacent residues, bear essential information for EGZ to be secreted and that periplasmic disulphide bond formation is an obligatory step which provides a pre-folded functional form of EGZ with secretion competence.

A protein complex mediating mRNA degradation in Escherichia coli.

mRNA degradation in Escherichia coli is mediated by a combination of exo- and endoribonucleases. We present evidence for a multiprotein complex which includes at least two enzymes that play important roles in mRNA degradation: the exoribonuclease polynucleotide phosphorylase (PNPase) and the endoribonuclease RNase E. An activity which impedes the processive activity of PNPase at stem-loop structures also appears to be associated with the complex. This complex is estimated to have a molecular mass of about 500 kDa and includes several additional polypeptides whose functions are unknown. The identification of a complex which includes several activities associated with mRNA degradation has implications for the mechanisms and co-ordinated control of mRNA degradation.

Polyphosphate kinase is a component of the Escherichia coli RNA degradosome.

The Escherichia coli degradosome is a multienzyme complex with four major protein components: the endoribonuclease RNase E, the exoribonuclease PNPase, the RNA helicase RhlB and enolase. The first three of these proteins are known to have important functions in mRNA processing and degradation. In this work, we identify an additional component of the degradosome, polyphosphate kinase (PPK), which catalyses the reversible polymerization of the gamma-phosphate of ATP into polyphosphate (poly(P)). An E. coli strain deleted for the ppk gene showed increased stability of the ompA mRNA. Purified His-tagged PPK was shown to bind RNA, and RNA binding was prevented by hydrolysable ATP. Chemical modification of RNA by PPK, for example the addition or removal of 3' or 5' terminal phosphates, could not be detected. However, polyphosphate was found to inhibit RNA degradation by the degradosome in vitro. This inhibition was overcome by the addition of ADP, required for the degradation of polyphosphate and for the regeneration of ATP by PPK in the degradosome. Thus, PPK in the degradosome appears to maintain an appropriate microenvironment, removing inhibitory polyphosphate and NDPs and regenerating ATP.

A DEAD-box RNA helicase in the Escherichia coli RNA degradosome.

The Escherichia coli RNA degradosome is a multi-enzyme complex that contains the exoribonuclease polynucleotide phosphorylase (PNPase) and the endoribonuclease RNase E. Both enzymes are important in RNA processing and messenger RNA degradation. Here we report that enolase and RhlB are two other major components of the degradosome. Enolase is a glycolytic enzyme with an unknown role in RNA metabolism. RhlB is a member of the DEAD-box family of ATP-dependent RNA helicases, which are found in both prokaryotes and eukaryotes. We show that the degradosome has an ATP-dependent activity that aids the degradation of structured RNA by PNPase. Incubation of the degradosome with affinity-purified antibody against RhlB inhibited the ATP-stimulated RNA degradation. These results suggest that RhlB acts by unwinding RNA structures that impede the processive activity of PNPase. RhlB is thus an important enzyme in mRNA turnover.

Cellulase EGZ of Erwinia chrysanthemi: structural organization and importance of His98 and Glu133 residues for catalysis.

Biochemical, genetic and primary sequence analyses of the Erwinia chrysanthemi endoglucanase EGZ allowed us to identify two functional domains and to locate their boundaries. The catalytic domain extends from residue 1 to 288, while a domain required for EGZ to bind to microcrystalline cellulose lies from residues 324 to 385. Each domain was found capable of functioning in the absence of the other. A region rich in Pro, Thr, and Ser residues links both domains and appeared to be susceptible to proteolytic attack. Based upon predictions derived from a method developed to compare sequences sharing a low level of similarity, e.g. hydrophobic cluster analysis (HCA), we analysed the importance of either residue His98 or Glu133 in EGZ catalytic activity. Two EGZ-derived proteins were engineered in which either His98 or Glu133 amino acid was converted to an Ala residue. Characterization of the purified proteins showed that no enzymatic activity could be detected, by using carboxymethylcellulose (CMC) or paranitrophenyl-cellobioside (pNPC) as substrates, while both mutated proteins retained the capacity to bind to microcrystalline cellulose. These studies, which to date constitute the first experimental testing of HCA-derived predictions, allowed us to identify two particular amino acids involved in cellulolytic activity. By taking into account data from chemical modification studies of other cellulases, we speculate that the His98 residue is involved in the folding of the catalytic domain while the Glu133 residue intervenes directly in the beta, 1-4 glycosidic bond cleavage.

Ribonuclease E organizes the protein interactions in the Escherichia coli RNA degradosome.

The Escherichia coli RNA degradosome is the prototype of a recently discovered family of multiprotein machines involved in the processing and degradation of RNA. The interactions between the various protein components of the RNA degradosome were investigated by Far Western blotting, the yeast two-hybrid assay, and coimmunopurification experiments. Our results demonstrate that the carboxy-terminal half (CTH) of ribonuclease E (RNase E) contains the binding sites for the three other major degradosomal components, the DEAD-box RNA helicase RhlB, enolase, and polynucleotide phosphorylase (PNPase). The CTH of RNase E acts as the scaffold of the complex upon which the other degradosomal components are assembled. Regions for oligomerization were detected in the amino-terminal and central regions of RNase E. Furthermore, polypeptides derived from the highly charged region of RNase E, containing the RhlB binding site, stimulate RhlB activity at least 15-fold, saturating at one polypeptide per RhlB molecule. A model for the regulation of the RhlB RNA helicase activity is presented. The description of RNase E now emerging is that of a remarkably complex multidomain protein containing an amino-terminal catalytic domain, a central RNA-binding domain, and carboxy-terminal binding sites for the other major components of the RNA degradosome.