Low copy numbers of complement C4 and C4A deficiency are risk factors for myositis, its subgroups and autoantibodies.

BACKGROUND: Idiopathic inflammatory myopathies (IIM) are a group of autoimmune diseases characterised by myositis-related autoantibodies plus infiltration of leucocytes into muscles and/or the skin, leading to the destruction of blood vessels and muscle fibres, chronic weakness and fatigue. While complement-mediated destruction of capillary endothelia is implicated in paediatric and adult dermatomyositis, the complex diversity of complement C4 in IIM pathology was unknown. METHODS: We elucidated the gene copy number (GCN) variations of total C4, C4A and C4B, long and short genes in 1644 Caucasian patients with IIM, plus 3526 matched healthy controls using real-time PCR or Southern blot analyses. Plasma complement levels were determined by single radial immunodiffusion. RESULTS: The large study populations helped establish the distribution patterns of various C4 GCN groups. Low GCNs of C4T (C4T=2+3) and C4A deficiency (C4A=0+1) were strongly correlated with increased risk of IIM with OR equalled to 2.58 (2.28-2.91), p=5.0×10-53 for C4T, and 2.82 (2.48-3.21), p=7.0×10-57 for C4A deficiency. Contingency and regression analyses showed that among patients with C4A deficiency, the presence of HLA-DR3 became insignificant as a risk factor in IIM except for inclusion body myositis (IBM), by which 98.2% had HLA-DR3 with an OR of 11.02 (1.44-84.4). Intragroup analyses of patients with IIM for C4 protein levels and IIM-related autoantibodies showed that those with anti-Jo-1 or with anti-PM/Scl had significantly lower C4 plasma concentrations than those without these autoantibodies. CONCLUSIONS: C4A deficiency is relevant in dermatomyositis, HLA-DRB1*03 is important in IBM and both C4A deficiency and HLA-DRB1*03 contribute interactively to risk of polymyositis.

Lipid and Protein Transfer between Nanolipoprotein Particles and Supported Lipid Bilayers.

A nanolipoprotein particle (NLP) is a lipid bilayer disc stabilized by two amphipathic "scaffold" apolipoproteins. It has been most notably utilized as a tool for solubilizing a variety of membrane proteins while preserving structural and functional properties. Transfer of functional proteins from NLPs into model membrane systems such as supported lipid bilayers (SLBs) would enable new opportunities, for example, two-dimensional protein crystallization and studies on protein-protein interactions. This work used fluorescence microscopy and atomic force microscopy to investigate the interaction between NLPs and SLBs. When incubated with SLBs, NLPs were found to spontaneously deliver lipid and protein cargo. The impact of membrane composition on lipid exchange was explored, revealing a positive correlation between the magnitude of lipid transfer and concentration of defects in the target SLB. Incorporation of lipids capable of binding specifically to polyhistidine tags encoded into the apolipoproteins also boosted transfer of NLP cargo. Optimal conditions for lipid and protein delivery from NLPs to SLBs are proposed based on interaction mechanisms.

Cell-Free Co-Translational Approaches for Producing Mammalian Receptors: Expanding the Cell-Free Expression Toolbox Using Nanolipoproteins.

Membranes proteins make up more than 60% of current drug targets and account for approximately 30% or more of the cellular proteome. Access to this important class of proteins has been difficult due to their inherent insolubility and tendency to aggregate in aqueous solutions. Understanding membrane protein structure and function demands novel means of membrane protein production that preserve both their native conformational state as well as function. Over the last decade, cell-free expression systems have emerged as an important complement to cell-based expression of membrane proteins due to their simple and customizable experimental parameters. One approach to overcome the solubility and stability limitations of purified membrane proteins is to support them in stable, native-like states within nanolipoprotein particles (NLPs), aka nanodiscs. This has become common practice to facilitate biochemical and biophysical characterization of proteins of interest. NLP technology can be easily coupled with cell-free systems to achieve functional membrane protein production for this purpose. Our approach involves utilizing cell-free expression systems in the presence of NLPs or using co-translation techniques to perform one-pot expression and self-assembly of membrane protein/NLP complexes. We describe how cell-free reactions can be modified to render control over nanoparticle size and monodispersity in support of membrane protein production. These modifications have been exploited to facilitate co-expression of full-length functional membrane proteins such as G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). In particular, we summarize the state of the art in NLP-assisted cell-free coexpression of these important classes of membrane proteins as well as evaluate the advances in and prospects for this technology that will drive drug discovery against these targets. We conclude with a prospective on the use of NLPs to produce as well as deliver functional mammalian membrane-bound proteins for a range of applications.

Mechanism of Acid-Triggered Cargo Release from Lipid Bilayer-Coated Mesoporous Silica Particles.

Lipid bilayer-coated mesoporous silica nanoparticles are unique core-shell nanomaterials currently being developed as drug delivery vehicles. To improve cargo loading and biocirculation, the pore structure and surface chemistry of the particle have been modified and well characterized. However, an understanding of cargo release mechanisms from cellular uptake pathways remains largely unexplored. Here, we present a study of the release mechanism of lipid bilayer-coated silica particles induced by endosomal-like pH change from 7.4 to 5.0. We found that this relatively small pH change produces rapid deformation of the supported lipid bilayer that ultimately results in holes in the membrane. Using a combination of dye release studies, wide-field and confocal fluorescence microscopies, and surface area modeling analysis, we determined that small blister-like structures are formed, which lead to lateral membrane displacement and hole formation. Possible mechanisms for the blister formation, which include curvature effects and interfacial interactions, are discussed.

Preparation and Characterization of Solid-Supported Lipid Bilayers Formed by Langmuir-Blodgett Deposition: A Tutorial.

The structure, phase behavior, and properties of cellular membranes are derived from their composition, which includes phospholipids, sphingolipids, sterols, and proteins with various levels of glycosylation. Because of the intricate nature of cellular membranes, a plethora of in vitro studies have been carried out with model membrane systems that capture particular properties such as fluidity, permeability, and protein binding but vastly simplify the membrane composition in order to focus in detail on a specialized property or function. Supported lipid bilayers (SLB) are widely used as archetypes for cellular membranes, and this instructional review primarily focuses on the preparation and characterization of SLB systems formed by Langmuir deposition methods. Typical characterization methods, which take advantage of the planar orientation of SLBs, are illustrated, and references that go into more depth are included. This invited instructional review is written so that nonexperts can quickly gain in-depth knowledge regarding the preparation and characterization of SLBs. In addition, this work goes beyond traditional instructional reviews to provide expert readers with new results that cover a wider range of SLB systems than those previously reported in the literature. The quality of an SLB is frequently not well described, and details such as topological defects can influence the results and conclusions of an individual study. This article quantifies and compares the quality of SLBs fabricated from a variety of gel and fluid compositions, in correlation with preparation techniques and parameters, to generate general rules of thumb to guide the construction of designed SLB systems.