Bacterial transferase MraY inhibitors: Synthesis and biological evaluation

New inhibitors of the bacterial transferase MraY are described. Their structure is based on an aminoribosyl-O-uridine like scaffold, readily obtained in two key steps. The amino group can be coupled with proline or guanylated. Alternatively, these amino, prolinyl or guanidinyl groups can be introduced through a triazole linker. Biological evaluation of these compounds on MraY from Bacillus subtilis revealed interesting inhibitory activity for both amino compounds.     

Does probabilistic modelling of linkage disequilibrium evolution improve the accuracy of QTL location in animal pedigree?

Background

Since 2001, the use of more and more dense maps has made researchers aware that combining linkage and linkage disequilibrium enhances the feasibility of fine-mapping genes of interest. So, various method types have been derived to include concepts of population genetics in the analyses. One major drawback of many of these methods is their computational cost, which is very significant when many markers are considered. Recent advances in technology, such as SNP genotyping, have made it possible to deal with huge amount of data. Thus the challenge that remains is to find accurate and efficient methods that are not too time consuming. The study reported here specifically focuses on the half-sib family animal design. Our objective was to determine whether modelling of linkage disequilibrium evolution improved the mapping accuracy of a quantitative trait locus of agricultural interest in these populations. We compared two methods of fine-mapping. The first one was an association analysis. In this method, we did not model linkage disequilibrium evolution. Therefore, the modelling of the evolution of linkage disequilibrium was a deterministic process; it was complete at time 0 and remained complete during the following generations. In the second method, the modelling of the evolution of population allele frequencies was derived from a Wright-Fisher model. We simulated a wide range of scenarios adapted to animal populations and compared these two methods for each scenario.

Results

Our results indicated that the improvement produced by probabilistic modelling of linkage disequilibrium evolution was not significant. Both methods led to similar results concerning the location accuracy of quantitative trait loci which appeared to be mainly improved by using four flanking markers instead of two.

Conclusions

Therefore, in animal half-sib designs, modelling linkage disequilibrium evolution using a Wright-Fisher model does not significantly improve the accuracy of the QTL location when compared to a simpler method assuming complete and constant linkage between the QTL and the marker alleles. Finally, given the high marker density available nowadays, the simpler method should be preferred as it gives accurate results in a reasonable computing time.     

Purified E255L Mutant SERCA1a and Purified PfATP6 Are Sensitive to SERCA-type Inhibitors but Insensitive to Artemisinins

The antimalarial drugs artemisinins have been described as inhibiting Ca²⁺-ATPase activity of PfATP6 (Plasmodium falciparum ATP6) after expression in Xenopus oocytes. Mutation of an amino acid residue in mammalian SERCA1 (Glu²⁵⁵) to the equivalent one predicted in PfATP6 (Leu) was reported to induce sensitivity to artemisinin in the oocyte system. However, in the present experiments, we found that artemisinin did not inhibit mammalian SERCA1a E255L either when expressed in COS cells or after purification of the mutant expressed in Saccharomyces cerevisiae. Moreover, we found that PfATP6 after expression and purification from S. cerevisiae was insensitive to artemisinin and significantly less sensitive to thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone than rabbit SERCA1 but retained higher sensitivity to cyclopiazonic acid, another type of SERCA1 inhibitor. Although mammalian SERCA and purified PfATP6 appear to have different pharmacological profiles, their insensitivity to artemisinins suggests that the mechanism of action of this class of drugs on the calcium metabolism in the intact cell is complex and cannot be ascribed to direct inhibition of PfATP6. Furthermore, the successful purification of PfATP6 affords the opportunity to develop new antimalarials by screening for inhibitors against PfATP6.     

Prolonged blockade of VEGF receptors does not damage retinal photoreceptors or ganglion cells

It has recently been reported that relatively short‐term inhibition of vascular endothelial growth factor (VEGF) signaling can cause photoreceptor cell death, a potentially clinically important finding since VEGF blockade has become an important modality of treatment of ocular neovascularization and macular edema. However, in a set of studies in which we achieved extended and complete blockage of VEGF‐induced vascular leakage through retinal expression of a VEGF binding protein, we did not observe any toxicity to retinal neurons. To follow‐up on these apparently discrepant findings, we designed a set of experiments with the kinase inhibitor SU4312, which blocks phosphorylation of VEGF receptors, to look directly for evidence of VEGF inhibition‐related retinal toxicity. Using transgenic mice with sustained expression of VEGF in photoreceptors, we determined that periocular injection of 3 µg of SU4312 every 5 days markedly suppressed subretinal neovascularization, indicating effective blockade of VEGF signaling. Wild‐type mice given periocular injections of 5 µg of SU4312 every 5 days for up to 12 weeks showed normal scotopic and photopic electroretinograms (ERGs), no TUNEL stained cells in the retina, and no reduction in outer nuclear layer thickness. Incubation of cultured ganglion cells or retinal cultures containing photoreceptors with high doses of SU4312 did not reduce cell viability. These data suggest that blocking VEGF signaling in the retina for up to 12 weeks does not damage photoreceptors nor alter ERG function and should reassure patients who are receiving frequent injections of VEGF antagonists for choroidal and retinal vascular diseases. J. Cell. Physiol. 224:262–272, 2010 © 2010 Wiley‐Liss, Inc.     

Nucleotide Variability and Translation Efficiency of the 5′ Untranslated Region of Hepatitis A Virus: Update from Clinical Isolates Associated with Mild and Severe Hepatitis

Mutations in the internal ribosome entry site (IRES) of hepatitis A virus (HAV) have been associated with enhanced in vitro replication and viral attenuation in animal models. To address the possible role of IRES variability in clinical presentation, IRES sequences were obtained from HAV isolates associated with benign (n = 8) or severe (n = 4) hepatitis. IRES activity was assessed using a bicistronic dual-luciferase expression system in adenocarcinoma (HeLa) and hepatoma (HuH7) cell lines. Activity was higher in HuH7 than in HeLa cells, except for an infrequently isolated genotype IIA strain. Though globally low, significant variation in IRES-dependent translation efficiency was observed between field isolates, reflecting the low but significant genetic variability of this region (94.2% ± 0.5% nucleotide identity). No mutation was exclusive of benign or severe hepatitis, and variations in IRES activity were not associated with a clinical phenotype, indirectly supporting the preponderance of host factors in determining the clinical presentation.Hepatitis A virus (HAV) is a nonenveloped RNA virus of the Picornaviridae family. The viral genome consists of an approximately 7,500-nucleotide (nt)-long, positive-stranded RNA divided in three parts: a 5′ untranslated region (5′ UTR), a single open reading frame that encodes both structural and nonstructural proteins, and a 3′ UTR with a short poly(A) tail. By sequencing of the VP1-2A junction and the VP1 gene, 3 genotypes (I, II, and III) divided into A and B subtypes have been described in humans (7, 27). HAV is the main cause of acute viral hepatitis worldwide. The majority of cases follow a benign course, but some may be present with fulminant forms, characterized by acute liver failure (factor V levels of <50% and encephalopathy). HAV-induced liver disease appears to result primarily from immunologic mechanisms, chiefly on the basis of in vitro studies. Most HAV strains have no detectable cytopathic effect in cell culture and no apparent effect on cell growth or metabolism (16), and HAV-infected cells are lysed by cytotoxic T cells isolated from the liver of acutely infected patients (30, 31). Clinical studies have suggested that host factors such as age and underlying liver disease were involved in the severity of liver diseases (32, 33) and that the host immune response also played a role in the fulminant forms of hepatitis A, as evidenced by markedly low viral loads (26).Nevertheless, the existence of viral determinants of hepatitis A severity is suggested by both experimental and clinical studies. Indeed, mutations within the VP1-2A and 2C genes have been shown to enhance virulence in tamarins (9). It has also been suggested that 5′ UTR mutations associated with viral adaptation to cell culture were also responsible for viral attenuation in vivo (15). The 5′ UTR of HAV is about 735 nucleotides long and is considered the most conserved region of the genome. The 5′ UTR is involved in genome replication and translation initiation. Folding predictions and biochemical probing showed that this region forms a highly ordered secondary structure containing a pyrimidine-rich tract (PRT) and an internal ribosomal entry site (IRES) with 10 to 12 AUG triplets upstream of the initiator codon (18). The IRES allows the initiation of the cap-independent translation of the viral genome. Most knowledge of HAV IRES activity is derived from studies of the HM-175 reference strain and its cell culture-adapted variants (4, 5, 36). These experiments have shown that HAV presents the lowest IRES-dependent translation initiation activity among picornaviruses both in reticulocyte lysates and in a variety of cell lines, including the human hepatoma cell line HepG2 (type III IRES) (3, 6). These features have been attributed to a lower affinity of the HAV 5′ UTR for translation factors (6). The hypothesis that the slow growth of HAV in cell culture could be related to this inefficient translation is supported by the emergence of 5′ UTR mutations in cell culture-adapted variants with enhanced viral replication (8). The finding that these mutations were associated with viral attenuation in vivo supports the hypothesis of viral determinants of virulence in the 5′ UTR (15). Among the few clinical studies which have addressed this question, Fujiwara et al., by comparing full-length HAV genomes obtained from Japanese patients with benign or fulminant hepatitis, found less nucleotide variation in the 5′ UTRs from patients with fulminant hepatitis (12, 13) and suggested that two IRES mutations (G324A and C372G/T) might influence the course of HAV infection (14).The aim of the present study was to further examine the genetic variability of 5′ UTR sequences from field isolates, to assess the potential impact of nucleotide variations on IRES activity by using validated techniques, and to search for a relationship with disease severity by comparing isolates obtained from patients with benign or fulminant forms of hepatitis A.     

c-Type Cytochrome Assembly in Saccharomyces cerevisiae: A Key Residue for Apocytochrome c1/Lyase Interaction

The electron transport chains in the membranes of bacteria and organelles generate proton-motive force essential for ATP production. The c-type cytochromes, defined by the covalent attachment of heme to a CXXCH motif, are key electron carriers in these energy-transducing membranes. In mitochondria, cytochromes c and c₁ are assembled by the cytochrome c heme lyases (CCHL and CC₁HL) and by Cyc2p, a putative redox protein. A cytochrome c₁ mutant with a CAPCH heme-binding site instead of the wild-type CAACH is strictly dependent upon Cyc2p for assembly. In this context, we found that overexpression of CC₁HL, as well as mutations of the proline in the CAPCH site to H, L, S, or T residues, can bypass the absence of Cyc2p. The P mutation was postulated to shift the CXXCH motif to an oxidized form, which must be reduced in a Cyc2p-dependent reaction before heme ligation. However, measurement of the redox midpoint potential of apocytochrome c₁ indicates that neither the P nor the T residues impact the thermodynamic propensity of the CXXCH motif to occur in a disulfide vs. dithiol form. We show instead that the identity of the second intervening residue in the CXXCH motif is key in determining the CCHL-dependent vs. CC₁HL-dependent assembly of holocytochrome c₁. We also provide evidence that Cyc2p is dedicated to the CCHL pathway and is not required for the CC₁HL-dependent assembly of cytochrome c₁.THE c-type cytochromes, also referred to as cytochrome c, represent a universal class of heme-containing proteins that function as electron carriers in the energy-transducing pathways of bacteria, plastids, and mitochondria (Thöny-Meyer 1997; Nakamoto et al. 2000; Bonnard et al. 2010). Because cytochromes c carry a heme covalently attached to a CXXCH motif, they constitute an attractive object of study to address the question of cofactor protein assembly. The biochemical requirements for cytochrome c assembly were deduced from in vivo and in vitro studies, and the conclusion is that both apocytochromes c and heme are transported independently across at least one biological membrane and maintained as reduced prior to catalysis of the heme attachment reaction (Allen et al. 2003; Hamel et al. 2009; Kranz et al. 2009; Sanders et al. 2010). Bacterial cytochromes c are assembled in the periplasmic space, a compartment where cysteine pairs in proteins form disulfide bonds in reactions catalyzed by dedicated enzymes (Inaba 2009; Kadokura and Beckwith 2010). The current thinking holds that a c-type apocytochrome is a substrate of the disulfide bond-forming pathway, which introduces an intramolecular disulfide between the two cysteines of the CXXCH sequence (Allen et al. 2003; Sanders et al. 2010). This disulfide needs to be reduced to a dithiol to provide free sulfhydryls for the heme ligation. Consistent with this view is the fact that groups of specific oxido-reductases that constitute a transmembrane dithiol-disulfide relay from the cytosol to the periplasmic space have been shown to function as c-type cytochrome assembly factors (Allen et al. 2003; Kadokura et al. 2003; Mapller and Hederstedt 2006; Sanders et al. 2010). The proposal that the components of this pathway control the in vivo redox status of the CXXCH sulfhydryls has been inferred from the presence of motifs in their protein sequences that are consistent with a function in redox chemistry and also from the demonstration that their recombinant forms participate in dithiol–disulfide exchange reactions (Monika et al. 1997; Setterdahl et al. 2000). Moreover, the ability of exogenous thiol compounds to bypass the lack of these factors in vivo substantiates the view that the redox components have a disulfide-reducing activity in the pathway (e.g., Sambongi and Ferguson 1994; Fabianek et al. 1998; Beckett et al. 2000; Deshmukh et al. 2000; Bardischewsky and Friedrich 2001; Erlendsson and Hederstedt 2002; Erlendsson et al. 2003; Feissner et al. 2005; Turkarslan et al. 2008).While the role of these pathways is well established in bacteria, much less is known about the components that catalyze thiol/disulfide chemistry in the mitochondrial intermembrane space (IMS), which is topologically equivalent to the bacterial periplasm. By analogy with the bacterial pathways, the participation of redox-active factors that catalyze thiol formation is expected, as the mitochondrial IMS houses two c-type cytochromes, the soluble cytochrome c and the membrane-bound cytochrome c₁, both of which function in respiration. In fungi, heme attachment to apocytochromes c and c₁ is dependent upon the IMS resident cytochrome c and c₁ heme lyases, CCHL and CC₁HL, although the exact role of these lyases in the assembly process is still unclear (Dumont et al. 1987; Zollner et al. 1992). Conversion of apocytochrome to holocytochrome c depends only on CCHL, while apocytochrome c₁ can be acted upon by both CCHL and CC₁HL (Matner and Sherman 1982; Dumont et al. 1987; Stuart et al. 1990; Zollner et al. 1992; Bernard et al. 2003). In animals, apoforms of cytochromes c and c₁ are assembled by a unique heme lyase, HCCS, which carries both the CCHL and CC₁HL activities (Prakash et al. 2002; Schwarz and Cox 2002; Bernard et al. 2003).Cyc2p, a component first described as a mitochondrial biogenesis factor in yeast (Matner and Sherman 1982; Dumont et al. 1993; Pearce et al. 1998; Sanchez et al. 2001), was recently rediscovered in the context of cytochrome c₁ maturation (Bernard et al. 2003). Cyc2p is located at the mitochondrial inner membrane with its C-terminal domain containing a non-covalently bound FAD exposed to the IMS (Bernard et al. 2005). A redox function for Cyc2p is likely based on the finding that a recombinant form of the molecule exhibits a NAD(P)H-dependent reductase activity (Bernard et al. 2005). However, as Cyc2p activity is not essential for the maturation process, a functional redundancy was postulated based on the fact that a cyc2-null mutant still assembles holoforms of cytochromes c and c₁ (Bernard et al. 2005). The absolute requirement of Cyc2p was revealed via genetic analysis of the cyc2-null cyt1-34 combination that displays a synthetic respiratory-deficient phenotype with loss of holocytochrome c₁ assembly (Bernard et al. 2005). The cyt1-34 mutation maps to the gene encoding cytochrome c₁ and results in a CAPCH heme-binding site replacing the wild-type CAACH site (Bernard et al. 2005). The synthetic interaction is specific for the cyt1-34 allele carrying the A-to-P mutation and is not observed in a cyc2-null cyt1-48 strain carrying an A-to-D mutation at the heme-binding site of apocytochrome c₁ (Bernard et al. 2005). The fact that Cyc2p becomes essential when the cytochrome c₁ heme-binding site carries an A-to-P mutation suggests that the CXXCH motif could be the target of Cyc2p action in vivo. One possible interpretation for this observation is that the P residue alters the reactivity of the cysteinyl thiols to redox chemistry so that the apocytochrome c₁ CAPCH heme-binding site occurs in an oxidized (disulfide) form, which must be reduced in a Cyc2p-dependent reaction before heme attachment can occur.In this article, we have undertaken a genetic approach to elucidate this pathway and searched for suppressors that alleviate the respiratory deficiency of the cyc2-null cyt1-34 strain. Either overexpression of CC₁HL or replacement of the P mutation in the heme-binding site by H, L, S, or T residues restore the assembly of holocytochrome c₁. In vitro measurement of redox potential of apoforms of CA(A/P/T)CH cytochrome c₁ indicates that there is no change in the thermodynamic stability of the disulfide at the CXXCH motif that could account for the Cyc2p-dependent assembly of cytochrome c₁. Genetic studies reveal that the replacement of the second A residue at the CAACH motif by H, L, P, S, and T residues is key in determining the conversion of apocytochrome c₁ to its corresponding holoform via the CCHL and/or CC₁HL-dependent pathway. We also demonstrate that Cyc2p is a component dedicated to the CCHL pathway and is not required for the CC₁HL-dependent assembly of cytochrome c₁. We propose that the CAPCH cytochrome c₁ is strictly dependent upon CCHL and Cyc2p for its assembly but becomes a substrate of CC₁HL upon overexpression of CC₁HL or in the presence of H, L, S, or T mutations.     

Interaction between non-anionic phospholipids and cytochrome c induced by reactive oxygen species

The oxidative interaction of cytochrome c (Cyt c) with liposomes of Palmitoyl Linoleyl Phosphatidyl Choline (PLPC) initiated by radio-induced free radicals was investigated. Results showed that the peroxidation of PLPC is decreased in the presence of Cyt c, meaning that this latter is the preferential target of hydroxyl radicals. In addition, when Cyt c was incubated with peroxidized PLPC, it was found to be able to decompose hydroperoxides of PLPC into hydroxides. The peroxidase activity of Cyt c proceeded via the opening of the tertiary structure of Cyt c, as suggested by the loss of the sixth coordination bond of the heme-iron. Even if it is known to preferentially interact with cardiolipin, this work shows that Cyt c is also able to interact with hydroperoxide species of non-anionic phospholipids.