Delphine Lechardeur

Intracellular Barriers to Non-Viral Gene Transfer

Non-viral vector mediated gene transfer, compared to viral vector mediated one, is a promising tool for the safe delivery of therapeutic DNA in genetic and acquired human diseases. Although the lack of specific immune response favor the clinical application of non-viral vectors, comprising of an expression cassette complexed to cationic liposome or cationic polymer, the limited efficacy and short duration of transgene expression impose major hurdles in the widespread application of non-viral gene therapy. The trafficking of transgene, complexed with chemical vectors, has been the subject of intensive investigations to improve our understanding of cellular and extracellular barriers impeding gene delivery. Here, we review those physical and metabolic impediments that account, at least in part, for the inefficient translocation of transgene into the nucleus of target cells. Following the internalization of the DNA-polycation complex by endocytosis, a large fraction is targeted to the lysosomal compartment by default. Since the cytosolic release of heterelogous DNA is a prerequisite for nuclear translocation, entrapment and degradation of plasmid DNA in endo-lysosomes constitute a major impediment to efficient gene transfer. Only a small fraction of internalized plasmid DNA penetrates the cytoplasm. Plasmid DNA encounters the diffusional and metabolic barriers of the cytoplasm, further decreasing the number of intact plasmid molecules reaching the nuclear pore complex (NPC), the gateway of nucleosol. Nuclear translocation of DNA requires either the disassembly of the nuclear envelope or active nuclear transport via the NPC. Comparison of viral and plasmid DNA cellular trafficking should reveal strategies that viruses have developed to overcome those cellular barriers that impede non-viral DNA delivery in gene therapy attempts.

C-terminal Truncations Destabilize the Cystic Fibrosis Transmembrane Conductance Regulator without Impairing Its Biogenesis A NOVEL CLASS OF MUTATION

Defective cAMP-stimulated chloride conductance of the plasma membrane of epithelial cell is the hallmark of cystic fibrosis (CF) and results from mutations in the cystic fibrosis transmembrane conductance regulator, CFTR. In the majority of CF patients, mutations in the CFTR lead to its misfolding and premature degradation at the endoplasmic reticulum (ER). Other mutations impair the cAMP-dependent activation or the ion conductance of CFTR chloride channel. In the present work we identify a novel mechanism leading to reduced expression of CFTR at the cell surface, caused by C-terminal truncations. The phenotype of C-terminally truncated CFTR, representing naturally occurring premature termination and frameshift mutations, were examined in transient and stable heterologous expression systems. Whereas the biosynthesis, processing, and macroscopic chloride channel function of truncated CFTRs are essentially normal, the degradation rate of the mature, complex-glycosylated form is 5- to 6-fold faster than the wild type CFTR. These experiments suggest that the C terminus has a central role in maintaining the metabolic stability of the complex-glycosylated CFTR following its exit from the ER and provide a plausible explanation for the severe phenotype of CF patients harboring C-terminal truncations.

Molecular Basis of Oligoubiquitin‐Dependent Internalization of Membrane Proteins in Mammalian Cells

Ubiquitination induced down‐regulation of cell surface proteins by internalization and lysosomal targeting plays a fundamental role in cell physiology and pathogenesis of diseases. The molecular basis of a single ubiquitin (Ub) as an autonomous endocytic signal, the widely accepted mechanism, however, remains elusive in higher eukaryotes. Using Ub containing reporter proteins without signalling abilities, we present evidence that only multiple Ub moieties, linked either covalently or assembled as oligomers with an intact interface for recognition by Ub‐interacting motifs (UIMs), are recognized by the endocytic machinery in vivo and associate with a subset of Ub‐binding clathrin adaptors in vitro. Genetic and pharmacological approaches show that internalization of plasma membrane proteins harbouring multiple Ub moieties is clathrin‐dependent, but caveolin‐independent. Functional assays demonstrate the cargo‐dependent involvement of eps15/15R and epsin, UIM containing clathrin adaptors, in the endocytosis of model proteins, CD4 and the activated β2‐adrenergic receptor complex, containing polymeric or oligomeric Ub. These results provide a paradigm for the clathrin‐mediated uptake of ubiquitinated membrane proteins in mammalian cells, requiring the assembly of multiple UIM–Ub interactions to overcome the low affinity binding of mono‐Ub to UIM.

Aerobic Respiration Metabolism in Lactic Acid Bacteria and Uses in Biotechnology

The lactic acid bacteria (LAB) are essential for food fermentations and their impact on gut physiology and health is under active exploration. In addition to their well-studied fermentation metabolism, many species belonging to this heterogeneous group are genetically equipped for respiration metabolism. In LAB, respiration is activated by exogenous heme, and for some species, heme and menaquinone. Respiration metabolism increases growth yield and improves fitness. In this review, we aim to present the basics of respiration metabolism in LAB, its genetic requirements, and the dramatic physiological changes it engenders. We address the question of how LAB acquired the genetic equipment for respiration. We present at length how respiration can be used advantageously in an industrial setting, both in the context of food-related technologies and in novel potential applications.

Discovery of Intracellular Heme-binding Protein HrtR, Which Controls Heme Efflux by the Conserved HrtB-HrtA Transporter in Lactococcus lactis

Background: Heme is an essential cofactor yet toxic in free form, necessitating strict intracellular control. Results: A heme sensor regulates the conserved hrtBA genes in Lactococcus lactis, whose products mediate heme efflux. Conclusion: L. lactis controls heme homeostasis by sensing intracellular heme and activating heme efflux. Significance: The use of an intracellular heme sensor to control heme efflux constitutes a novel paradigm for bacterial heme homeostasis. Most commensal and food bacteria lack heme biosynthesis genes. For several of these, the capture of environmental heme is a means of activating aerobic respiration metabolism. Our previous studies in the Gram-positive bacterium Lactococcus lactis showed that heme exposure strongly induced expression of a single operon, called here hrtRBA, encoding an ortholog of the conserved membrane hrt (heme-regulated transporter) and a unique transcriptional regulator that we named HrtR. We show that HrtR expressed as a fusion protein is a heme-binding protein. Heme iron interaction with HrtR is non-covalent, hexacoordinated, and involves two histidines, His-72 and His-149. HrtR specifically binds a 15-nt palindromic sequence in the hrtRBA promoter region, which is needed for hrtRBA repression. HrtR-DNA binding is abolished by heme addition, which activates expression of the HrtB-HrtA (HrtBA) transporter in vitro and in vivo. The use of HrtR as an intracellular heme sensor appears to be conserved among numerous commensal bacteria, in contrast with numerous Gram-positive pathogens that use an extracellular heme-sensing system, HssRS, to regulate hrt. Finally, we show for the first time that HrtBA permease controls heme toxicity by its direct and specific efflux. The use of an intracellular heme sensor to control heme efflux constitutes a novel paradigm for bacterial heme homeostasis.

The 2-Cys peroxiredoxin, Alkyl hydroperoxide reductase C binds heme and participates in its intracellular availability in Streptococcus agalactiae

Heme is a redox-reactive molecule with vital and complex roles in bacterial metabolism, survival, and virulence. However, few intracellular heme partners were identified to date and are not well conserved in bacteria. The opportunistic pathogen Streptococcus agalactiae (group B Streptococcus) is a heme auxotroph, which acquires exogenous heme to activate an aerobic respiratory chain. We identified the alkyl hydroperoxide reductase AhpC, a member of the highly conserved thiol-dependent 2-Cys peroxiredoxins, as a heme-binding protein. AhpC binds hemin with a Kd of 0.5 μm and a 1:1 stoichiometry. Mutagenesis of cysteines revealed that hemin binding is dissociable from catalytic activity and multimerization. AhpC reductase activity was unchanged upon interaction with heme in vitro and in vivo. A group B Streptococcus ahpC mutant displayed attenuation of two heme-dependent functions, respiration and activity of a heterologous catalase, suggesting a role for AhpC in heme intracellular fate. In support of this hypothesis, AhpC-bound hemin was protected from chemical degradation in vitro. Our results reveal for the first time a role for AhpC as a heme-binding protein.

Oligomerization state of the DNA fragmentation factor in normal and apoptotic cells

The caspase-activated DNase (CAD) is the primary nuclease responsible for oligonucleosomal DNA fragmentation during apoptosis. The DNA fragmentation factor (DFF) is composed of the 40-kDa CAD (DFF40) in complex with its cognate 45-kDa inhibitor (inhibitor of CAD: ICAD or DFF45). The association of ICAD with CAD not only inhibits the DNase activity but is also essential for the co-translational folding of CAD. Activation of CAD requires caspase-3-dependent proteolysis of ICAD. The tertiary structures of neither the inactive nor the activated DFF have been conclusively established. Whereas the inactive DFF is thought to consist of the CAD/ICAD heterodimer, activated CAD has been isolated as a large (>MDa) multimer, as well as a monomer. To establish the subunit stoichiometry of DFF and some of its structural determinants in normal and apoptotic cells, we utilized size-exclusion chromatography in combination with co-immunoprecipitation and mutagenesis techniques. Both endogenous and heterologously expressed DFF have an apparent molecular mass of 160-190 kDa and contain 2 CAD and 2 ICAD molecules (CAD/ICAD)2 in HeLa cells. Although the N-terminal (CIDE-N) domain of CAD is not required for ICAD binding, it is necessary but not sufficient for ICAD homodimerization in the DFF. In contrast, the CIDE-N domain of ICAD is required for CAD/ICAD association. Using bioluminescence resonance energy transfer (BRET), dimerization of ICAD in DFF was confirmed in live cells. In apoptotic cells, endogenous and exogenous CAD forms limited oligomers, representing the active nuclease. A model is proposed for the rearrangement of the DFF subunit stoichiometry in cells undergoing programmed cell death.

Contrasting nuclear dynamics of the caspase-activated DNase (CAD) in dividing and apoptotic cells.

Although compelling evidence supports the central role of caspase-activated DNase (CAD) in oligonucleosomal DNA fragmentation in apoptotic nuclei, the regulation of CAD activity remains elusive in vivo. We used fluorescence photobleaching and biochemical techniques to investigate the molecular dynamics of CAD. The CAD-GFP fusion protein complexed with its inhibitor (ICAD) was as mobile as nuclear GFP in the nucleosol of dividing cells. Upon induction of caspase-3–dependent apoptosis, activated CAD underwent progressive immobilization, paralleled by its attenuated extractability from the nucleus. CAD immobilization was mediated by its NH2 terminus independently of its DNA-binding activity and correlated with its association to the interchromosomal space. Preventing the nuclear attachment of CAD provoked its extracellular release from apoptotic cells. We propose a novel paradigm for the regulation of CAD in the nucleus, involving unrestricted accessibility of chromosomal DNA at the initial phase of apoptosis, followed by its nuclear immobilization that may prevent the release of the active nuclease into the extracellular environment.

Visualization of the role of host heme on the virulence of the heme auxotroph Streptococcus agalactiae.

Heme is essential for several cellular key functions but is also toxic. Whereas most bacterial pathogens utilize heme as a metabolic cofactor and iron source, the impact of host heme during bacterial infection remains elusive. The opportunist pathogen Streptococcus agalactiae does not synthesize heme but still uses it to activate a respiration metabolism. Concomitantly, heme toxicity is mainly controlled by the HrtBA efflux transporter. Here we investigate how S. agalactiae manages heme toxicity versus benefits in the living host. Using bioluminescent bacteria and heme-responsive reporters for in vivo imaging, we show that the capacity of S. agalactiae to overcome heme toxicity is required for successful infection, particularly in blood-rich organs. Host heme is simultaneously required, as visualized by a generalized infection defect of a respiration-negative mutant. In S. agalactiae, HrtBA expression responds to an intracellular heme signal via activation of the two-component system HssRS. A hssRS promoter-driven intracellular luminescent heme sensor was designed to identify host compartments that supply S. agalactiae with heme. S. agalactiae acquires heme in heart, kidneys, and liver, but not in the brain. We conclude that S. agalactiae response to heme is organ-dependent, and its efflux may be particularly relevant in late stages of infection.