Secondary structure of rhBMP-2 in a protective biopolymeric carrier material

Efficient delivery of growth factors is one of the great challenges of tissue engineering. Polyelectrolyte multilayer films (PEM) made of biopolymers have recently emerged as an interesting carrier for delivering recombinant human bone morphogenetic protein 2 (rhBMP-2 noted here BMP-2) to cells in a matrix-bound manner. We recently showed that PEM made of poly(l-lysine) and hyaluronan (PLL/HA) can retain high and tunable quantities of BMP-2 and can deliver it to cells to induce their differentiation in osteoblasts. Here, we investigate quantitatively by Fourier transform infrared spectroscopy (FTIR) the secondary structure of BMP-2 in solution as well as trapped in a biopolymeric thin film. We reveal that the major structural elements of BMP-2 in solution are intramolecular β-sheets and unordered structures as well as α-helices. Furthermore, we studied the secondary structure of rhBMP-2 trapped in hydrated films and in dry films since drying is an important step for future applications of these bioactive films onto orthopedic biomaterials. We demonstrate that the structural elements were preserved when BMP-2 was trapped in the biopolymeric film in hydrated conditions and, to a lesser extent, in dry state. Importantly, its bioactivity was maintained after drying of the film. Our results appear highly promising for future applications of these films as coatings of biomedical materials, to deliver bioactive proteins while preserving their bioactivity upon storage in dry state.This work was supported by the French Ministry of Research through an ANR-EmergenceBIO grant (ANR-09-EBIO-012-01), by the European Commission (FP7 program) via a European Research Council starting grant (BIOMIM, GA 259370), and by GRAVIT (081012_FIBIOS). C.P. is grafetul to IUF for financial support

Coupling changes in cell shape to chromosome segregation

Animal cells undergo dramatic changes in shape, mechanics and polarity as they progress through the different stages of cell division. These changes begin at mitotic entry, with cell–substrate adhesion remodelling, assembly of a cortical actomyosin network and osmotic swelling, which together enable cells to adopt a near spherical form even when growing in a crowded tissue environment. These shape changes, which probably aid spindle assembly and positioning, are then reversed at mitotic exit to restore the interphase cell morphology. Here, we discuss the dynamics, regulation and function of these processes, and how cell shape changes and sister chromatid segregation are coupled to ensure that the daughter cells generated through division receive their fair inheritance

New insights into lipid-Nucleoside Diphosphate Kinase-D interaction mechanism: Protein structural changes and membrane reorganisation

AbstractNucleoside Diphosphate Kinases (NDPKs) have long been considered merely as housekeeping enzymes. The discovery of the NME1 gene, an anti-metastatic gene coding for NDPK-A, led the scientific community to re-evaluate their role in the cell. It is now well established that the NDPK family is more complex than what was first thought, and despite the increasing amount of evidence suggesting the multifunctional role of nm23/NDPKs, the specific functions of each family member are still elusive. Among these isoforms, NDPK-D is the only one to present a mitochondria-targeting sequence. It has recently been shown that this protein is able to bind and cross-link with mitochondrial membranes, suggesting that NDPK-D can mediate contact sites and contributes to the mitochondrial intermembrane space structuring. To better understand the influence of NDPK-D on mitochondrial lipid organisation, we analysed its behaviour in different lipid environments. We found that NDPK-D not only interacts with CL or anionic lipids, but is also able to bind in a non negligible manner to zwitterionic PC. NDPK-D alters membrane organisation in terms of fluidity, hydration and lipid clustering, effects which depend on lipid structure. Changes in the protein structure after lipid binding were evidenced, both by fluorescence and infrared spectroscopy, regardless of membrane composition. Taking into account all these elements, a putative mechanism of interaction between NDPK-D and zwitterionic or anionic lipids was proposed

Mitochondrial creatine kinase interaction with cardiolipin-containing biomimetic membranes is a two step process involving adsorption and insertion

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Interaction of mitochondrial creatine kinase with phospholipids. A langmuir monolayer study.

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Protein "amyloid-like" networks at the phospholipid membrane formed by 4-hydroxy-2-nonenal-modified mitochondrial creatine kinase

International audience4-Hydroxy-2-nonenal (4-HNE) is a reactive aldehyde and a lipid peroxidation product formed in biological tissues under physiological and pathological conditions. Its concentration increases with oxidative stress and induces deleterious modifications of proteins and membranes. Mitochondrial and cytosolic isoforms of creatine kinase were previously shown to be affected by 4-HNE. In the present study, we analyzed the effect of 4-HNE on mitochondrial creatine kinase, an abundant protein from the mitochondrial intermembrane space with a key role in mitochondrial physiology. We show that this effect is double: 4-HNE induces a step-wise loss of creatine kinase activity together with a fast protein aggregation. Protein-membrane interaction is affected and amyloid-like networks formed on the biomimetic membrane. These fibrils may disturb mitochondrial organisation both at the membrane and in the inter membrane space

Carrier-inside-carrier: polyelectrolyte microcapsules as reservoir for drug-loaded liposomes

International audienceConventional liposomes have a short life-time in blood, unless they are protected by a polymer envelope, most often polyethylene glycol. However, these stabilizing polymers frequently interfere with cellular uptake, impede liposome-membrane fusion and inhibit escape of liposome content from endosomes. To overcome such drawbacks, polymer-based systems as carriers for liposomes are currently developed. Conforming to this approach, we propose a new and convenient method for embedding small size liposomes, 30–100 nm, inside porous calcium carbonate microparticles. These microparticles served as templates for deposition of various polyelectrolytes to form a protective shell. The carbonate particles were then dissolved to yield hollow polyelectrolyte microcapsules. The main advantage of using this method for liposome encapsulation is that carbonate particles can serve as a sacrificial template for deposition of virtually any polyelectrolyte. By carefully choosing the shell composition, bioavailability of the liposomes and of the encapsulated drug can be modulated to respond to biological requirements and to improve drug delivery to the cytoplasm and avoid endosomal escape

Two-Step Membrane Binding of NDPK‑B Induces Membrane Fluidity Decrease and Changes in Lipid Lateral Organization and Protein Cluster Formation

Nucleoside diphosphate kinases (NDPKs) are crucial elements in a wide array of cellular physiological or pathophysiological processes such as apoptosis, proliferation, or metastasis formation. Among the NDPK isoenzymes, NDPK-B, a cytoplasmic protein, was reported to be associated with several biological membranes such as plasma or endoplasmic reticulum membranes. Using several membrane models (liposomes, lipid monolayers, and supported lipid bilayers) associated with biophysical approaches, we show that lipid membrane binding occurs in a two-step process: first, initiation by a strong electrostatic adsorption process and followed by shallow penetration of the protein within the membrane. The NDPK-B binding leads to a decrease in membrane fluidity and formation of protein patches. The ability of NDPK-B to form microdomains at the membrane level may be related to protein–protein interactions triggered by its association with anionic phospholipids. Such accumulation of NDPK-B would amplify its effects in functional platform formation and protein recruitment at the membrane

Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker

International audienceMany cellular processes depend on ERM (ezrin, moesin, and radixin) proteins mediating regulated linkage between plasma membrane and actin cytoskeleton. Although conformational activation of the ERM protein is mediated by the membrane PIP2, the known properties of the two described PIP2-binding sites do not explain activation. To elucidate the structural basis of possible mechanisms, we generated informative moesin mutations and tested three attributes: membrane localization of the expressed moesin, moesin binding to PIP2, and PIP2-induced release of moesin autoinhibition. The results demonstrate for the first time that the POCKET containing inositol 1,4,5-trisphosphate on crystal structure (the "POCKET" Lys-63, Lys-278 residues) mediates all three functions. Furthermore the second described PIP2-binding site (the "PATCH," Lys253/Lys-254, Lys-262/Lys-263) is also essential for all three functions. In native autoinhibited ERM proteins, the POCKET is a cavity masked by an acidic linker, which we designate the "FLAP." Analysis of three mutant moesin constructs predicted to influence FLAP function demonstrated that the FLAP is a functional autoinhibitory region. Moreover, analysis of the cooperativity and stoichiometry demonstrate that the PATCH and POCKET do not bind PIP2 simultaneously. Based on our data and supporting published data, we propose a model of progressive activation of autoinhibited moesin by a single PIP2 molecule in the membrane. Initial transient binding of PIP2 to the PATCH initiates release of the FLAP, which enables transition of the same PIP2 molecule into the newly exposed POCKET where it binds stably and completes the conformational activation