Reactivity of U-associated osteopontin with lactoferrin: a one-to-many complex.

Uranium is the heaviest natural element, mainly found in aqueous medium as the hexavalent uranyl ion (UO22+). Bones are the main organs in which uranium accumulates, depending on as yet unknown molecular and cellular mechanisms. Recently, it has been revealed that osteopontin (OPN), a protein involved in bio-mineralization processes, and its main naturally occurring cleaved form (fOPN), have nanomolar affinities for UO22+. The binding of UO22+ is due to both the phosphorylation sites and acidic residues of these proteins and is accompanied by a slight gain in secondary structure. OPN is an Intrinsically Disordered Protein (IDP), a family of proteins which play a crucial role in several interaction networks, where phosphorylations are thought to be key elements. OPN has been shown to bind lactoferrin (LF) and the two proteins have antagonist functions in the modulation of the bio-mineralization process. However, to date, there has been no evidence that UO22+ and LF compete in their binding to OPN or not. Based on a series of convergent experimental data, this study first addressed in detail the LF/fOPN interaction and proposed a LF:fOPN 4/1 maximal stoichiometry. Moreover the phosphorylations were demonstrated to be necessary for the stability of such complexes. The interaction of preformed UO22+/fOPN complexes with LF was also investigated and the occurrence of several entities involving the three partners was demonstrated. These complexes did not reveal any significant conformational changes compared to those obtained in the absence of UO22+. The results have shown not only that LF and UO22+ do not compete, but also that these complexes are likely to be more stable than LF/fOPN complexes, as indicated by their melting temperature (Tm) values. The potential impact of those uranyl-stabilized ternary complexes on some biological pathways now remains to be assessed. Nonetheless, this work has contributed to shedding light on the formation of stable ternary complexes involving a large structured protein, an IDP and an exogenous metal.

Dystrophin Hot-Spot Mutants Leading to Becker Muscular Dystrophy Insert More Deeply into Membrane Models than the Native Protein.

Dystrophin (DYS) is a membrane skeleton protein whose mutations lead to lethal Duchenne muscular dystrophy or to the milder Becker muscular dystrophy (BMD). One third of BMD "in-frame" exon deletions are located in the region that codes for spectrin-like repeats R16 to R21. We focused on four prevalent mutated proteins deleted in this area (called RΔ45-47, RΔ45-48, RΔ45-49, and RΔ45-51 according to the deleted exon numbers), analyzing protein/membrane interactions. Two of the mutants, RΔ45-48 and RΔ45-51, led to mild pathologies and displayed a similar triple coiled-coil structure as the full-length DYS R16-21, whereas the two others, RΔ45-47 and RΔ45-49, induced more severe pathologies and showed "fractional" structures unrelated to the normal one. To explore lipid packing, small unilamellar liposomes (SUVs) and planar monolayers were used at various initial surface pressures. The dissociation constants determined by microscale thermophoresis (MST) were much higher for the full-length DYS R161-21 than for the mutants; thus the wild type protein has weaker SUV binding. Comparing surface pressures after protein adsorption and analysis of atomic force microscopy images of mixed protein/lipid monolayers revealed that the mutants insert more into the lipid monolayer than the wild type does. In fact, in both models every deletion mutant showed more interactions with membranes than the full-length protein did. This means that mutations in the R16-21 part of dystrophin disturb the protein's molecular behavior as it relates to membranes, regardless of whether the accompanying pathology is mild or severe.

Becker muscular dystrophy severity is linked to the structure of dystrophin.

In-frame exon deletions of the Duchenne muscular dystrophy (DMD) gene produce internally truncated proteins that typically lead to Becker muscular dystrophy (BMD), a milder allelic disorder of DMD. We hypothesized that differences in the structure of mutant dystrophin may be responsible for the clinical heterogeneity observed in Becker patients and we studied four prevalent in-frame exon deletions, i.e. Δ45-47, Δ45-48, Δ45-49 and Δ45-51. Molecular homology modelling revealed that the proteins corresponding to deletions Δ45-48 and Δ45-51 displayed a similar structure (hybrid repeat) than the wild-type dystrophin, whereas deletions Δ45-47 and Δ45-49 lead to proteins with an unrelated structure (fractional repeat). All four proteins in vitro expressed in a fragment encoding repeats 16-21 were folded in α-helices and remained highly stable. Refolding dynamics were slowed and molecular surface hydrophobicity were higher in fractional repeat containing Δ45-47 and Δ45-49 deletions compared with hybrid repeat containing Δ45-48 and Δ45-51 deletions. By retrospectively collecting data for a series of French BMD patients, we showed that the age of dilated cardiomyopathy (DCM) onset was delayed by 11 and 14 years in Δ45-48 and Δ45-49 compared with Δ45-47 patients, respectively. A clear trend toward earlier wheelchair dependency (minimum of 11 years) was also observed in Δ45-47 and Δ45-49 patients compared with Δ45-48 patients. Muscle dystrophin levels were moderately reduced in most patients without clear correlation with the deletion type. Disease progression in BMD patients appears to be dependent on the deletion itself and associated with a specific structure of dystrophin at the deletion site.

Cholesterol favors the anchorage of human dystrophin repeats 16 to 21 in membrane at physiological surface pressure.

Dystrophin (DYS) is a filamentous protein that connects the cytoskeleton and the extracellular matrix via the sarcolemma, conferring resistance to muscular cells. In this study, interactions between the DYS R16-21 fragment and lipids were examined using Langmuir films made of anionic and zwitterionic lipids. The film fluidity was modified by the addition of 15% cholesterol. Whatever the lipid mixture examined, at low surface pressure (20 mN/m) few differences appeared on the protein insertion and the presence of cholesterol did not affect the protein/lipid interactions. At high surface pressure (30 mN/m), the protein insertion was very low and occurred only in zwitterionic films in the liquid-expanded phase. In anionic films, electrostatic interactions prevented the protein insertion outright, and caused accumulation of the protein on the hydrophilic part of the monolayer. Addition of cholesterol to both lipid mixtures drastically modified the protein-lipid interactions: the DYS R16-21 insertion increased and its organization in the monolayer appeared to be more homogeneous. The presence of accessible cholesterol recognition amino-acid consensus sequences in this fragment may enhance the protein/membrane binding at physiological lateral pressure. These results suggest that the anchorage of dystrophin to the membrane in vivo may be stabilized by cholesterol-rich nano-domains in the inner leaflet of sarcolemma.