Impact of aeration and heme-activated respiration on Lactococcus lactis gene expression: identification of a heme-responsive operon

Lactococcus lactis is a widely used food bacterium mainly characterized for its fermentation metabolism. However, this species undergoes a metabolic shift to respiration when heme is added to an aerobic medium. Respiration results in markedly improved biomass and survival compared to fermentation. Whole-genome microarrays were used to assess changes in L. lactis expression under aerobic and respiratory conditions compared to static growth, i.e., nonaerated. We observed the following. (i) Stress response genes were affected mainly by aerobic fermentation. This result underscores the differences between aerobic fermentation and respiration environments and confirms that respiration growth alleviates oxidative stress. (ii) Functions essential for respiratory metabolism, e.g., genes encoding cytochrome bd oxidase, menaquinone biosynthesis, and heme uptake, are similarly expressed under the three conditions. This indicates that cells are prepared for respiration once O(2) and heme become available. (iii) Expression of only 11 genes distinguishes respiration from both aerobic and static fermentation cultures. Among them, the genes comprising the putative ygfCBA operon are strongly induced by heme regardless of respiration, thus identifying the first heme-responsive operon in lactococci. We give experimental evidence that the ygfCBA genes are involved in heme homeostasis.     

Dexamethasone downregulates the expression of parathyroid hormone-related protein (PTHrP) in mesenchymal stem cells

Parathyroid hormone-related protein (PTHrP) has been shown to have anabolic effects in women with postmenopausal osteoporosis. PTHrP promotes the recruitment of osteogenic cells and prevents apoptotic death of osteoblasts and osteocytes. The receptor responsible for the effects of PTHrP is the common PTH/PTHrP receptor (PTH1R). Glucocorticoids (GC) are commonly used as drugs to treat inflammatory diseases. Long-term GC treatments are often associated with bone loss which can lead to GC-induced osteoporosis. The aim of this work was to study the effects of the glucocorticoid dexamethasone (Dex) on the expression of PTHrP and PTH1R in adult human mesenchymal stem cells, the progenitor cells of osteoblasts.Adult human mesenchymal stem cells (hMSC) were cultured and differentiated by standard methods. The expression of PTHrP and PTH1R mRNA was assayed by real-time qPCR. The PTHrP release into the culture media was measured by an immunoradiometric assay.Treatment with Dex (10 nM) resulted in an 80% drop in the PTHrP release within 6 h. A 24 h Dex treatment also reduced the expression of PTHrP mRNA by up to 90%. The expression of PTH1R receptor mRNA was simultaneously increased up to 20-fold by 10 nM Dex. The effects of Dex on PTHrP and PTH1R were dose-dependent and experiments with the GC-receptor antagonist mifepristone showed an involvement of GC-receptors in these effects. In addition to the Dex-induced effects on PTHrP and PTH1R, Dex also increased mineralization and the expression of the osteoblast markers Runx2 and alkaline phosphatase. In our studies, we show that dexamethasone decreases the expression of PTHrP and increases the expression of the PTH1R receptor. This could have an impact on PTHrP-mediated anabolic actions on bone and could also affect the responsiveness of circulating PTH. The results indicate that glucocorticoids affect the signalling pathway of PTHrP by regulating both PTHrP and PTH1R expression and these mechanisms could be involved in glucocorticoid-induced osteoporosis.     

γ-Tubulin ring complexes regulate microtubule plus end dynamics

γ-Tubulin is critical for the initiation and regulation of microtubule (MT) assembly. In Drosophila melanogaster, it acts within two main complexes: the γ-tubulin small complex (γ-TuSC) and the γ-tubulin ring complex (γ-TuRC). Proteins specific of the γ-TuRC, although nonessential for viability, are required for efficient mitotic progression. Until now, their role during interphase remained poorly understood. Using RNA interference in Drosophila S2 cells, we show that the γ-TuRC is not critical for overall MT organization. However, depletion of any component of this complex results in an increase of MT dynamics. Combined immunofluorescence and live imaging analysis allows us to reveal that the γ-TuRC localizes along interphase MTs and that distal γ-tubulin spots match with sites of pause or rescue events. We propose that, in addition to its role in nucleation, the γ-TuRC associated to MTs may regulate their dynamics by limiting catastrophes.     

Alternative Translation Initiation Generates Cytoplasmic Sheep Prion Protein

Cytoplasmic localization of the prion protein (PrP) has been observed in different species and cell types. We have investigated this poorly understood phenomenon by expressing fusion proteins of sheep prion protein and green fluorescent protein (^GFPPrP) in N2a cells, with variable sequence context surrounding the start codon Met¹. ^GFPPrP expressed with the wild-type sequence was transported normally through the secretory pathway to the cell surface with acquisition of N-glycan groups, but two N-terminal fragments of ^GFPPrP were detected intracellularly, starting in frame from Met¹⁷. When ^GFPPrP was expressed with a compromised Kozak sequence (^GFPPrP*), dispersed intracellular fluorescence was observed. A similar switch from pericellular to intracellular PrP localization was seen when analogous constructs of sheep PrP, without inserted GFP, were expressed, showing that this phenomenon is not caused by the GFP tag. Western blotting revealed a reduction in glycosylated forms of ^GFPPrP*, whereas the N-terminal fragments starting from Met¹⁷ were still present. Formation of these N-terminal fragments was completely abolished when Met¹⁷ was replaced by Thr, indicating that leaky ribosomal scanning occurs for normal sheep PrP and that translation from Met¹⁷ is the cause of the aberrant cytoplasmic localization observed for a fraction of the protein. In contrast, the same phenomenon was not detected upon expression of similar constructs for mouse PrP. Analysis of samples from sheep brain allowed immunological detection of N-terminal PrP fragments, indicating that sheep PrP is subject to similar processing mechanisms in vivo.PrP^{C 2} is a cell surface glycoprotein with an essential role in the pathogenesis of transmissible neurodegenerative prion diseases (1, 2). According to the prion hypothesis, a misfolded, pathogenic form of the protein (PrP^Sc) is the sole constituent of transmissible prions (3, 4), but the molecular details and required environs for the misfolding are incompletely understood. As would be expected for a glycosylphosphatidylinositol-anchored protein with N-linked glycans, PrP^C is observed at the outer leaflet of the plasma membrane, the end point of the secretory route. The half-time at the plasma membrane is fairly short, because the protein may undergo shedding or endocytic internalization (5–9). Thus, PrP^C can be encountered throughout the secretory and endocytic routes and is also able to leave cells via exosomes derived from multivesicular endosomes (10). In agreement with this, studies of the subcellular distribution of PrP^C in mammalian brain have identified localization to the outer cell membrane, in the Golgi apparatus, and in endosomal vesicles (11, 12). However, others have found that PrP^C is not solely associated with membranes, but, in some subpopulations of neurons, is localized to the cytoplasm (13, 14). In line with the latter observations, transgenic mice expressing PrP carrying a C-terminal GFP tag demonstrated intense cytoplasmic fluorescence from a limited number (approximately 1%) of the neurons in certain brain areas, such as the hippocampus (15). Immunohistochemical detection of intracellular, possibly cytoplasmic, PrP has also been reported from large mononuclear cells in the gut wall of sheep (16) and from enteric neurons in mice (17). The recent observations of pronounced cytoplasmic aggregation of PrP in pancreatic β-cells of rats prone to development of diabetes mellitus provide a perplexing example of nonstandard PrP localization in non-neuronal cells (18).The flexibility observed in the subcellular localization of PrP^C has been suggested to be a requirement for normal functions of the protein (14, 19, 20), but how cytoplasmic and nuclear variants arise has not been established. Cytoplasmic PrP could be a result of retro-translocation from the endoplasmic reticulum (ER), as part of an unfolded protein response (21–23) or from attenuated ER import of PrP under conditions of lumenal stress in the ER (24, 25). The finding of intact ER-targeting signal sequences on cytoplasmic PrPs (25, 26) favors the latter mechanism, namely a reduced ER import of PrP, possibly caused by saturation of the ER translocation machinery or an overload of unfolded proteins within the ER. However, no signs of stress or pathology could be detected in neurons of wild-type mice expressing cytoplasmic PrP (14), which led to the suggestion that the cytoplasmic appearance of PrP could constitute a physiologically relevant, but minor, pathway for the protein.Forced cytoplasmic expression of PrP in transgenic mice (22) and in the nematode Caenorhabditis elegans (27) resulted in neurodegenerative disease, suggesting that toxic mislocalization of PrP could be part of the pathogenic mechanism in prion diseases (28). However, transgenic mice expressing cytoplasmic PrP, on a PrP-null background, developed cerebellar atrophy but were resistant to experimental prion infection (29), suggesting that cytoplasmic PrP is unlikely to serve as substrate for prion replication. Furthermore, data obtained from transgenic mice expressing an anchorless secretory PrP show that, although these mice accumulate PrP-containing amyloid plaques upon challenge with PrP^Sc, they fail to develop clinical prion disease (30). Thus, membrane-attached PrP appears to be a prerequisite for development of prion-derived neurodegeneration.In eukaryotes, ribosomes bind specifically to linear mRNAs carrying a 7-methylguanosine 5′-end cap and slide along the mRNA in the 5′ → 3′ direction until they encounter the first start codon (AUG), from which the protein translation starts exclusively. Therefore, eukaryotic mRNAs are generally monocistronic. However, deviations from this standard principle have been reported, in which protein translation is initiated at alternative start codons either up or downstream from the primary AUG. The best characterized mechanism is known as context-dependent leaky ribosomal scanning (LRS) (31). This cap-dependent mechanism is particularly operative when the optimal (5′-GCCRCCaugG-3′) sequence context surrounding the first AUG codon is compromised, most notably at positions R⁻³ (where R= purine, A or G, but optimally G) and G⁺⁴ (32, 33).In this work, we report that in a cell culture system, sheep PrP mRNA displays a tendency to allow alternative translation initiation through LRS. Met¹⁷ serves as an internal in-frame alternative start codon giving rise to PrP with a severely shortened ER-targeting peptide.Although the LRS mechanism is active in sheep PrP, it appears to occur much less in mouse PrP (34). The molecular explanation and possible pathophysiological relevance of these observations in relation to PrP function await further studies. Interestingly, during the review process of this paper, observations of cytoplasmic PrP similar to some of those described herein were reported for human and hamster PrP (35).     

Sporadic Infantile Epileptic Encephalopathy Caused by Mutations in PCDH19 Resembles Dravet Syndrome but Mainly Affects Females

Dravet syndrome (DS) is a genetically determined epileptic encephalopathy mainly caused by de novo mutations in the SCN1A gene. Since 2003, we have performed molecular analyses in a large series of patients with DS, 27% of whom were negative for mutations or rearrangements in SCN1A. In order to identify new genes responsible for the disorder in the SCN1A-negative patients, 41 probands were screened for micro-rearrangements with Illumina high-density SNP microarrays. A hemizygous deletion on chromosome Xq22.1, encompassing the PCDH19 gene, was found in one male patient. To confirm that PCDH19 is responsible for a Dravet-like syndrome, we sequenced its coding region in 73 additional SCN1A-negative patients. Nine different point mutations (four missense and five truncating mutations) were identified in 11 unrelated female patients. In addition, we demonstrated that the fibroblasts of our male patient were mosaic for the PCDH19 deletion. Patients with PCDH19 and SCN1A mutations had very similar clinical features including the association of early febrile and afebrile seizures, seizures occurring in clusters, developmental and language delays, behavioural disturbances, and cognitive regression. There were, however, slight but constant differences in the evolution of the patients, including fewer polymorphic seizures (in particular rare myoclonic jerks and atypical absences) in those with PCDH19 mutations. These results suggest that PCDH19 plays a major role in epileptic encephalopathies, with a clinical spectrum overlapping that of DS. This disorder mainly affects females. The identification of an affected mosaic male strongly supports the hypothesis that cellular interference is the pathogenic mechanism.     

Design and synthesis of APTCs (aminopyrrolidinetricarboxylic acids): identification of a new group III metabotropic glutamate receptor selective agonist

A new family of mGlu receptor orthosteric ligands called APTCs was designed and synthesized using a parallel chemistry approach. Amongst 65 molecules tested on mGlu4, mGlu6 and mGlu8 subtypes, (2S,4S)-4-amino-1-[(E)-3-carboxyacryloyl]pyrrolidine-2,4-dicarboxylic acid (8a06-FP0429) has been shown to be a full mGlu4 agonist and a partial mGlu8 agonist. In addition, 8a06 was shown to be selective versus group I and II mGlu subtypes. A possible explanation for this efficacy difference is proposed by docking experiment performed with molecular model of the receptor.     

Translation initiator EIF4G1 mutations in familial Parkinson disease

Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.