NACHO Engages N-Glycosylation ER Chaperone Pathways for α7 Nicotinic Receptor Assembly.

The α7 nicotinic acetylcholine receptor participates in diverse aspects of brain physiology and disease. Neurons tightly control α7 assembly, which relies upon NACHO, an endoplasmic reticulum (ER)-localized integral membrane protein. By constructing α7 chimeras and mutants, we find that NACHO requires the α7 ectodomain to promote receptor assembly and surface trafficking. Also critical are two amino acids in the α7 second transmembrane domain. NACHO-mediated assembly is independent and separable from that induced by cholinergic ligands or RIC-3 protein, the latter of which acts on the large α7 intracellular loop. Proteomics indicates that NACHO associates with the ER oligosaccharyltransferase machinery and with calnexin. Accordingly, NACHO-mediated effects on α7 assembly and channel function require N-glycosylation and calnexin chaperone activity. These studies identify ER pathways that mediate α7 assembly by NACHO and provide insights into novel pharmacological strategies for these crucial nicotinic receptors.

CX3CL1 homo-oligomerization drives cell-to-cell adherence.

During inflammatory response, blood leukocytes adhere to the endothelium. This process involves numerous adhesion molecules, including a transmembrane chemokine, CX3CL1, which behaves as a molecular cluster. How this cluster assembles and whether this association has a functional role remain unknown. The analysis of CX3CL1 clusters using native electrophoresis and single molecule fluorescence kinetics shows that CX3CL1 is a homo-oligomer of 3 to 7 monomers. Fluorescence recovery after photobleaching assays reveal that the CX3CL1-transmembrane domain peptide self-associates in both cellular and acellular lipid environments, while its random counterpart (i.e. peptide with the same residues in a different order) does not. This strongly indicates that CX3CL1 oligomerization is driven by its intrinsic properties. According to the molecular modeling, CX3CL1 does not associate in compact bundles but rather with monomers linearly assembled side by side. Finally, the CX3CL1 transmembrane peptide inhibits both the CX3CL1 oligomerization and the adhesive function, while its random counterpart does not. This demonstrates that CX3CL1 oligomerization is mandatory for its adhesive potency. Our results provide a new direction to control CX3CL1-dependent cellular adherence in key immune processes.

Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection.

Influenza A viruses cause major morbidity and represent a severe global health problem. Current influenza vaccines are mainly egg-based products requiring the split of whole viruses using classical detergents such as Triton X-100, which implies certain limitations. Here, we report the use of the novel calixarene-based surfactant CALX133ACE as an alternative to classical detergents for influenza inactivated split vaccine preparation. We confirmed that CALX133ACE-based split HA antigens are fully functional and quantifiable by the "gold standard" method SRID. Additionally, as in the case of the Triton X-100-based split, the CALX133ACE-based split antigens are stable for at least 6 months at 4 °C. Moreover, immunization of mice with CALX133ACE-based split NYMC X-179A (H1N1) antigens harboring 10 to 30-fold less antigen than the commercialized trivalent inactivated vaccines Vaxigrip® or Fluviral® induced comparable efficient protection and neutralizing antibody responses against A(H1N1)pdm09 infection. Taken together, our results demonstrate for the first time the use of a calixarene-based detergent as an efficient splitting agent for the production of optimized influenza split antigens, paving the way for significant improvement in the vaccine manufacturing process, notably with regard to the current regulation on the prohibition of endocrine disruptors, such as Triton X-100.

Biochemical and biophysical characterization of purified native CD20 alone and in complex with rituximab and obinutuzumab.

CD20 is a B-lymphocyte specific integral membrane protein, an activated-glycosylated phosphoprotein expressed on the surface of B-cells and a clinically validated target of monoclonal antibodies such as rituximab, ocrelizumab, ofatumumab and obinutuzumab in the treatment of all B cell lymphomas and leukemias as well as autoimmune diseases. Here, we report the extraction and purification of native CD20 from SUDHL4 and RAMOS cell lines. To improve the protein yield, we applied a calixarene-based detergent approach to solubilize, stabilize and purify native CD20 from HEK293 cells. Size Exclusion Chromatography (SEC) and Analytical Ultracentrifugation show that purified CD20 was non-aggregated and that CD20 oligomerization is concentration dependent. Negative stain electron microscopy and atomic force microscopy revealed homogenous populations of CD20. However, no defined structure could be observed. Interestingly, micellar solubilized and purified CD20 particles adopt uniformly confined nanodroplets which do not fuse and aggregate. Finally, purified CD20 could bind to rituximab and obinutuzumab as demonstrated by SEC, and Surface Plasmon Resonance (SPR). Specificity of binding was confirmed using CD20 antibody mutants to human B-cell lymphoma cells. The strategy described in this work will help investigate CD20 binding with newly developed antibodies and eventually help to optimize them. This approach may also be applicable to other challenging membrane proteins.

Externalized Keratin 8: A Target at the Interface of Microenvironment and Intracellular Signaling in Colorectal Cancer Cells.

Accumulating evidence supports the remarkable presence at the membrane surface of cancer cells of proteins, which are normally expressed in the intracellular compartment. Although these proteins, referred to as externalized proteins, represent a highly promising source of accessible and druggable targets for cancer therapy, the mechanisms via which they impact cancer biology remain largely unexplored. The aim of this study was to expose an externalized form of cytokeratin 8 (eK8) as a key player of colorectal tumorigenesis and characterize its mode of action. To achieve this, we generated a unique antagonist monoclonal antibody (D-A10 MAb) targeting an eight-amino-acid-long domain of eK8, which enabled us to ascertain the pro-tumoral activity of eK8 in both KRAS-mutant and wild-type colorectal cancers (CRC). We showed that this pro-tumoral activity involves a bidirectional eK8-dependent control of caspase-mediated apoptosis in vivo and of the plasminogen-induced invasion process in cellulo. Furthermore, we demonstrated that eK8 is anchored at the plasma membrane supporting this dual function. We, therefore, identified eK8 as an innovative therapeutic target in CRC and provided a unique MAb targeting eK8 that displays anti-neoplastic activities that could be useful to treat CRC, including those harboring KRAS mutations.

The yin and yang of solubilization and stabilization for wild-type and full-length membrane protein.

Membrane proteins (MP) are stable in their native lipid environment. To enable structural and functional investigations, MP need to be extracted from the membrane. This is a critical step that represents the main obstacle for MP biochemistry and structural biology. General guidelines and rules for membrane protein solubilization remain difficult to establish. This review aims to provide the reader with a comprehensive overview of the general concepts of MP solubilization and stabilization as well as recent advances in detergents innovation. Understanding how solubilization and stabilization are intimately linked is key to facilitate MP isolation toward fundamental structural and functional research as well as drug discovery applications. How to manage the tour de force of destabilizing the lipid bilayer and stabilizing MP at the same time is the holy grail of successful isolation and investigation of such a delicate and fascinating class of proteins.

Novel systematic detergent screening method for membrane proteins solubilization.

Membrane proteins play crucial role in many cellular processes including cell adhesion, cell-cell communication, signal transduction and transport. To better understand the molecular basis of such central biological machines and in order to specifically study their biological and medical role, it is necessary to extract them from their membrane environment. To do so, it is challenging to find the best solubilization condition. Here we describe, a systematic screening method called BMSS (Biotinylated Membranes Solubilization & Separation) that allow screening 96 conditions at once. Streptavidine magnetic beads are used to separate solubilized proteins from remaining biotinylated membranes after solubilization. Relative quantification of dot blots help to select the best conditions to be confirmed by classical ultra-centrifugation and western blot. Classical detergents with different physical-chemical characteristics, novel calixarene based detergents and combination of both, were used for solubilization trials to obtain broad spectrum of conditions. Here, we show the application of BMSS to discover solubilization conditions of a GPCR target (MP-A) and a transporter (MP-B). The selected conditions allowed the solubilization and purification of non-aggregated and homogenous native membrane proteins A and B. Taken together, BMSS represent a rapid, reproducible and high throughput assessment of solubilization toward biochemical/functional characterization, biophysical screening and structural investigations of membrane proteins of high biological and medical relevance.

Expression and purification of native and functional influenza A virus matrix 2 proton selective ion channel.

Influenza A virus displays one of the highest infection rates of all human viruses and therefore represents a severe human health threat associated with an important economical challenge. Influenza matrix protein 2 (M2) is a membrane protein of the viral envelope that forms a proton selective ion channel. Here we report the expression and native isolation of full length active M2 without mutations or fusions. The ability of the influenza virus to efficiently infect MDCK cells was used to express native M2 protein. Using a Calixarene detergents/surfactants based approach; we were able to solubilize most of M2 from the plasma membrane and purify it. The tetrameric form of native M2 was maintained during the protein preparation. Mass spectrometry shows that M2 was phosphorylated in its cytoplasmic tail (serine 64) and newly identifies an acetylation of the highly conserved Lysine 60. ELISA shows that solubilized and purified M2 was specifically recognized by M2 antibody MAB65 and was able to displace the antibody from M2 MDCK membranes. Using a bilayer voltage clamp measurement assay, we demonstrate a pH dependent proton selective ion channel activity. The addition of the M2 ion channel blocker amantadine allows a total inhibition of the channel activity, illustrating therefore the specificity of purified M2 activity. Taken together, this work shows the production and isolation of a tetrameric and functional native M2 ion channel that will pave the way to structural and functional characterization of native M2, conformational antibody development, small molecules compounds screening towards vaccine treatment.

Structuring detergents for extracting and stabilizing functional membrane proteins.

BACKGROUND: Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. METHODOLOGY/PRINCIPAL FINDINGS: Anionic calix[4]arene based detergents (C4Cn, n=1-12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. CONCLUSION/SIGNIFICANCE: These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may contribute to the membrane protein crystallization field.

ABCG2 transports and transfers heme to albumin through its large extracellular loop.

ABCG2 is an ATP-binding cassette (ABC) transporter preferentially expressed by immature human hematopoietic progenitors. Due to its role in drug resistance, its expression has been correlated with a protection role against protoporhyrin IX (PPIX) accumulation in stem cells under hypoxic conditions. We show here that zinc mesoporphyrin, a validated fluorescent heme analog, is transported by ABCG2. We also show that the ABCG2 large extracellular loop ECL3 constitutes a porphyrin-binding domain, which strongly interacts with heme, hemin, PPIX, ZnPPIX, CoPPIX, and much less efficiently with pheophorbide a, but not with vitamin B12. K(d) values are in the range 0.5-3.5 µm, with heme displaying the highest affinity. Nonporphyrin substrates of ABCG2, such as mitoxantrone, doxo/daunorubicin, and riboflavin, do not bind to ECL3. Single-point mutations H583A and C603A inside ECL3 prevent the binding of hemin but hardly affect that of iron-free PPIX. The extracellular location of ECL3 downstream from the transport sites suggests that, after membrane translocation, hemin is transferred to ECL3, which is strategically positioned to release the bound porphyrin to extracellular partners. We show here that human serum albumin could be one of these possible partners as it removes hemin bound to ECL3 and interacts with ABCG2, with a K(d) of about 3 µm.