Joël Chopineau

Glycogen synthase kinase 3‐mediated voltage‐dependent anion channel phosphorylation controls outer mitochondrial membrane permeability during lipid accumulation

Nonalcoholic steatosis is a liver pathology characterized by fat accumulation and severe metabolic alterations involving early mitochondrial impairment and late hepatocyte cell death. However, mitochondrial dysfunction mechanisms remain elusive. Using four models of nonalcoholic steatosis, i.e., livers from patients with fatty liver disease, ob/ob mice, mice fed a high‐fat diet, and in vitro models of lipotoxicity, we show that outer mitochondrial membrane permeability is altered and identified a posttranslational modification of voltage‐dependent anion channel (VDAC), a membrane channel and NADH oxidase, as a cause of early mitochondrial dysfunction. Thus, in nonalcoholic steatosis VDAC exhibits reduced threonine phosphorylation, which increases the influx of water and calcium into mitochondria, sensitizes the organelle to matrix swelling, depolarization, and cytochrome c release without inducing cell death. This also amplifies VDAC enzymatic and channel activities regulation by calcium and modifies its interaction with proteic partners. Moreover, lipid accumulation triggers a rapid lack of VDAC phosphorylation by glycogen synthase kinase 3 (GSK3). Pharmacological and genetic manipulations proved GSK3 to be responsible for VDAC phosphorylation in normal cells. Notably, VDAC phosphorylation level correlated with steatosis severity in patients. Conclusion: VDAC acts as an early sensor of lipid toxicity and its GSK3‐mediated phosphorylation status controls outer mitochondrial membrane permeabilization in hepatosteatosis. (HEPATOLOGY 2013)

Characterization of a Membrane-active Peptide from the Bordetella pertussis CyaA Toxin

Background: The translocation of the Bordetella pertussis CyaA toxin across membrane is still poorly understood. Results: A membrane-active peptide isolated from the CyaA toxin is characterized by biophysical approaches. Conclusion: The α-helical peptide is inserted in plane and induces membrane permeabilization. Significance: The membrane-destabilizing activity of this peptide may assist the initial steps of the CyaA translocation process. Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, secretes several virulence factors, among which is the adenylate cyclase toxin (CyaA) that plays a crucial role in the early stages of human respiratory tract colonization. CyaA invades target cells by translocating its catalytic domain directly across the plasma membrane and overproduces cAMP, leading to cell death. The molecular process leading to the translocation of the catalytic domain remains largely unknown. We have previously shown that the catalytic domain per se, AC384, encompassing residues 1–384 of CyaA, did not interact with lipid bilayer, whereas a longer polypeptide, AC489, spanning residues 1–489, binds to membranes and permeabilizes vesicles. Moreover, deletion of residues 375–485 within CyaA abrogated the translocation of the catalytic domain into target cells. Here, we further identified within this region a peptidic segment that exhibits membrane interaction properties. A synthetic peptide, P454, corresponding to this sequence (residues 454–485 of CyaA) was characterized by various biophysical approaches. We found that P454 (i) binds to membranes containing anionic lipids, (ii) adopts an α-helical structure oriented in plane with respect to the lipid bilayer, and (iii) permeabilizes vesicles. We propose that the region encompassing the helix 454–485 of CyaA may insert into target cell membrane and induce a local destabilization of the lipid bilayer, thus favoring the translocation of the catalytic domain across the plasma membrane.

Monoacylation of ribonuclease A enables its transport across an in vitro model of the blood–brain barrier

A major challenge in correcting disorders affecting the central nervous system is to induce blood-brain barrier (BBB) crossing of exogenous biological compounds such as proteins or specific nucleic acid sequences. Fatty acids, due to their high membrane affinity and low toxicity, are good potential candidates to promote this barrier crossing when covalently bound to proteins. In this paper, we report that regiospecific monoacylation of ribonuclease A (RNase A) enables its transport across an in vitro model of the BBB. Myristoylated, palmitoylated and stearoylated RNases A were prepared using reversed micelles as microreactors. All the purified acylated RNases A kept their original enzymatic activity. A single fatty acid moiety was linked to RNase A through the alpha-amino group of its N-terminal lysine as shown by powerful analytical techniques. The ability of monoacylated RNases A to cross an in vitro model of the BBB is strictly dependent on the acyl chain length, which must be at least 16 carbon atoms long.

Nanotechnology: R & D challenges and opportunities for application in biotechnology

Physical methods of observing and manipulating individual molecules will prove useful, not only as research tools for investigating biomolecular structure and behaviour, but also for the creation of nanostructures through microelectronics, self-assembly and template-directed assembly. Nanostructures can also be used as nanoscale bioreactors for synthesizing or tailoring molecules, and it is becoming possible to follow and drive biochemical reactions at the molecular level. Nanobiotechnology is likely to become a growth area for research and development over the next few years, with the commercial development of new analytical devices and preparative bioreactors.

The Adenine Nucleotide Translocase 2, a Mitochondrial Target for Anticancer Biotherapy

Apoptosis or programmed cell death is one of the most important signaling pathways, which controls the cell fate and is frequently impaired in cancer cells. The major consequences of apoptosis inhibition are the accumulation of mutated cells and their enhanced resistance to chemotherapeutic agents. More generally, intrinsic or acquired apoptosis resistance may favor tumor growth and dissemination of mutated cells, and this resistance can be responsible of treatment failure. Mitochondria are central organelles in the signaling pathway of apoptosis and have been proposed as favorite candidates for anticancer biotherapy because they accommodate potential biological targets. Indeed, although cancer cells are highly glycolytic and become energetically independent of oxidative phosphorylation. Mitochondrial proteins involved in the so-called mitochondrial membrane permeabilization (MMP), such as the adenine nucleotide translocase (ANT) can be instrumental to elicit cancer cell death. Thus, multiple pharmacological and molecular studies revealed ANT could be a promising therapeutic target for the following reasons: (i) ANT is a bi-functional protein, it mediates the vital exchange of cytosolic ADP and mitochondrial ATP and participates to MMP via its capacity to become a lethal pore in the mitochondrial inner membrane; (ii) both ANT functions are under the control of the (anti)-oncogenes from the Bax/Bcl-2 family, (iii) several chemotherapeutic agents directly modulate the pore-forming activity of ANT and (iv) ANT2 isoform, which is anti-apoptotic, can be overexpressed in human cancers and its invalidation sensitize cells to apoptosis. In this review, we will introduce the knowledge of the role of ANT in MMP, illustrate the modulation of ANT by several strategies and propose the possibility to target preferentially the ANT2 isoform for induction of cancer cell apoptosis.

Study of hydrophobic interactions between acylated proteins and phospholipid bilayers using BIACORE

Intracellular proteins of eukaryotic cells are frequently covalently modified by the addition of long chain fatty acids. These modifications are thought to allow otherwise soluble proteins to associate with membranes by lipid–lipid based hydrophobic interactions. The purpose of this work was to quantify the effect of acyl chain length on hydrophobic interactions between acylated proteins and phospholipid monolayers. The binding of an artificially acylated model protein to electrically neutral phospholipids was studied by surface plasmon resonance, using BIACORE. Kinetic rates for the binding of bovine pancreatic ribonuclease A (RNase A), monoacylated on its N‐terminal lysine with fatty acids of 10, 12, 14, 16 or 18 carbon atoms, to phospholipids on hydrophobic sensor chips, were measured. Unlike unmodified ribonuclease, acylated RNase A bound to the phospholipids, and the association level increased with the acyl chain length to reach a maximum for C16. Reproducible kinetics were obtained which did not fit a 1:1 Langmuir model but rather a two‐step binding profile. Copyright

ANT-VDAC1 interaction is direct and depends on ANT isoform conformation in vitro.

The voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT) have central roles in mitochondrial functions such as nucleotides transport and cell death. The interaction between VDAC, an outer mitochondrial membrane protein and ANT, an inner membrane protein, was studied in isolated mitochondria and in vitro. Both proteins were isolated from various mitochondrial sources and reconstituted in vitro using a biomimetic system composed of recombinant human VDAC isoform 1 (rhVDAC1) immobilized on a surface plasmon resonance (SPR) sensor chip surface. Two enriched-preparations of (H)ANT (ANT from heart, mainly ANT1) and (L)ANT (ANT from liver, mainly ANT2) isoforms interacted differently with rhVDAC1. Moreover, the pharmacological ANT inhibitors atractyloside and bongkrekic acid modulated this interaction. Thus, ANT-VDAC interaction depends both on ANT isoform identity and on the conformation of ANT.

Biological Fate of Fe3O4 Core-Shell Mesoporous Silica Nanoparticles Depending on Particle Surface Chemistry

The biological fate of nanoparticles (NPs) for biomedical applications is highly dependent of their size and charge, their aggregation state and their surface chemistry. The chemical composition of the NPs surface influences their stability in biological fluids, their interaction with proteins, and their attraction to the cell membranes. In this work, core-shell magnetic mesoporous silica nanoparticles (Fe3O4@MSN), that are considered as potential theranostic candidates, are coated with polyethylene glycol (PEG) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayer. Their biological fate is studied in comparison to the native NPs. The physicochemical properties of these three types of NPs and their suspension behavior in different media are investigated. The attraction to a membrane model is also evaluated using a supported lipid bilayer. The surface composition of NPs strongly influences their dispersion in biological fluids mimics, protein binding and their interaction with cell membrane. While none of these types of NPs is found to be toxic on mice four days after intravenous injection of a dose of 40 mg kg−1 of NPs, their surface coating nature influences the in vivo biodistribution. Importantly, NP coated with DMPC exhibit a strong accumulation in liver and a very low accumulation in lung in comparison with nude or PEG ones.

The species origin of the serum in the culture medium influences the in vitro toxicity of silica nanoparticles to HepG2 cells

The formation of a protein corona around nanoparticles can influence their toxicity, triggering cellular responses that may be totally different from those elicited by pristine nanoparticles. The main objective of this study was to investigate whether the species origin of the serum proteins forming the corona influences the in vitro toxicity assessment of silica nanoparticles. Coronas were preformed around nanoparticles before cell exposures by incubation in fetal bovine (FBS) or human (HS) serum. The compositions of these protein coronas were assessed by nano-LC MS/MS. The effects of these protein-coated nanoparticles on HepG2 cells were monitored using real-time cell impedance technology. The nanoparticle coronas formed in human or fetal bovine serum comprised many homologous proteins. Using human compared with fetal bovine serum, nanoparticle toxicity in HepG2 cells decreased by 4-fold and 1.5-fold, when used at 50 and 10μg/mL, respectively. It is likely that “markers of self” are present in the serum and are recognized by human cell receptors. Preforming a corona with human serum seems to be more appropriate for in vitro toxicity testing of potential nanocarriers using human cells. In vitro cytotoxicity assays must reflect in vivo conditions as closely as possible to provide solid and useful results.

Kinetics of Interaction between ADP-ribosylation Factor-1 (Arf1) and the Sec7 Domain of Arno Guanine Nucleotide Exchange Factor, Modulation by Allosteric Factors, and the Uncompetitive Inhibitor Brefeldin A

Background: Kinetic modulations of Arf1-Sec7 domain complex, by the uncompetitive inhibitor brefeldin A and allosteric factors, are not established. Results: Brefeldin A reorients the binary Arf1-Sec7 domain complex to an abortive one with reduced association and dissociation rates. Conclusion: Kinetic hallmarks allow distinguishing the level, nature, and fate of interacting species. Significance: Similar approach will solve the inhibitory mechanism of new inhibitor families of sec7 domains. The GDP/GTP nucleotide exchange of Arf1 is catalyzed by nucleotide exchange factors (GEF), such as Arno, which act through their catalytic Sec7 domain. This exchange is a complex mechanism that undergoes conformational changes and intermediate complex species involving several allosteric partners such as nucleotides, Mg2+, and Sec7 domains. Using a surface plasmon resonance approach, we characterized the kinetic binding parameters for various intermediate complexes. We first confirmed that both GDP and GTP counteract equivalently to the free-nucleotide binary Arf1-Arno complex stability and revealed that Mg2+ potentiates by a factor of 2 the allosteric effect of GDP. Then we explored the uncompetitive inhibitory mechanism of brefeldin A (BFA) that conducts to an abortive pentameric Arf1-Mg2+-GDP-BFA-Sec7 complex. With BFA, the association rate of the abortive complex is drastically reduced by a factor of 42, and by contrast, the 15-fold decrease of the dissociation rate concurs to stabilize the pentameric complex. These specific kinetic signatures have allowed distinguishing the level and nature as well as the fate in real time of formed complexes according to experimental conditions. Thus, we showed that in the presence of GDP, the BFA-resistant Sec7 domain of Arno can also associate to form a pentameric complex, which suggests that the uncompetitive inhibition by BFA and the nucleotide allosteric effect combine to stabilize such abortive complex.