Search

Introduction: Blood group antigens of the RH system (formerly known as “Rhesus”) play an important role in transfusion medicine because of the severe haemolytic consequences of antibodies to these antigens. No crystal structure is available for RhD proteins with its partner RhAG, and the precise stoichiometry of the trimer complex remains unknown.Methods: To analyse their structural properties, the trimers formed by RhD and/or RhAG subunits were generated by protein modelling and molecular dynamics simulations were performed.Results: No major differences in structural behaviour were found between trimers of different compositions. The conformation of the subunits is relatively constant during molecular dynamics simulations, except for three large disordered loops.Discussion: This work makes it possible to propose a reasonable stoichiometry and demonstrates the potential of studying the structural behaviour of these proteins to investigate the hundreds of genetic variants relevant to transfusion medicine.

Lipid-bilayer nanodiscs and liposomes have been developed to stabilize membrane proteins in order to study their structures and functions. Nanodiscs are detergent-free, water-soluble, and size-controlled planar phospholipid-bilayer platforms. On the other hand, liposomes are curved phospholipid-bilayer spheres with an aqueous core used as drug delivery systems and model membrane platforms for studying cellular activities. A long-standing challenge is the generation of a homogenous and monodispersed lipid-bilayer system with a very wide range of dimensions and curvatures (elongation, bending, and twisting). A DNA-origami template provides a way to control the shapes, sizes, and arrangements of lipid bilayers via enforcing the assembly of lipid bilayers within the cavities created by DNA nanostructures. Here, we provide a concise overview and discuss how to design planar and curved lipid-bilayer membranes by using DNA-origami nanostructures as templates. Finally, we will discuss the potential applications of DNA-origami nanostructures in the structural and functional studies of large membrane proteins and their complexes.

Antimicrobial peptides (AMPs) are a unique and diverse group of molecules endowed with a broad spectrum of antibiotics properties that are being considered as new alternative therapeutic agents. Most of these peptides are membrane-active molecules, killing bacteria by membrane disruption. However, recently an increasing number of AMPs was shown to enter bacterial cells and target intracellular processes fundamental for bacterial life. In this paper we investigated the mechanism of action of Maganin-2 (Mag-2), a well-known antimicrobial peptide isolated from the African clawed frog Xenopus laevis, by functional proteomic approaches. Several proteins belonging to E. coli macromolecular membrane complexes were identified as Mag-2 putative interactors. Among these, we focused our attention on BamA a membrane protein belonging to the BAM complex responsible for the folding and insertion of nascent β-barrel Outer Membrane Proteins (OMPs) in the outer membrane. In silico predictions by molecular modelling, in vitro fluorescence binding and Light Scattering experiments carried out using a recombinant form of BamA confirmed the formation of a stable Mag-2/BamA complex and indicated a high affinity of the peptide for BamA. Functional implications of this interactions were investigated by two alternative and complementary approaches. The amount of outer membrane proteins OmpA and OmpF produced in E. coli following Mag-2 incubation were evaluated by both western blot analysis and quantitative tandem mass spectrometry in Multiple Reaction Monitoring scan mode. In both experiments a gradual decrease in outer membrane proteins production with time was observed as a consequence of Mag-2 treatment. These results suggested BamA as a possible good target for the rational design of new antibiotics since this protein is responsible for a crucial biological event of bacterial life and is absent in humans.

Human zinc transporter 1 (hZnT1) belongs to the cation diffusion facilitator (CDF) family. It plays a major role in transporting zinc (Zn2+) from the cytoplasm across the plasma membrane and into the extracellular space thereby protecting cells from Zn2+ toxicity. Through homology with other CDF family members, ZnT1 is predicted to contain a transmembrane region and a soluble C-terminal domain though little is known about its biochemistry. Here, we demonstrate that human ZnT1 and a variant can be produced by heterologous expression in Saccharomyces cerevisiae cells and purified in the presence of detergent and cholesteryl hemisuccinate. We show that the purified hZnT1 variant has Zn2+/H+ antiporter activity. Furthermore, we expressed, purified and characterized the soluble C-terminal domain of hZnT1 (hZnT1-CTD) in a bacterial expression system. We found that the hZnT1-CTD melting temperature increases at acidic pH, thus, we used an acetate buffer at pH 4.5 for purifications and concentration of the protein up to 12 mg/mL. Small-angle X-ray scattering analysis of hZnT1-CTD is consistent with the formation of a dimer in solution with a V-shaped core.

Membrane proteins require lipid bilayers for function. While lipid compositions reach enormous complexities, high‐resolution structures are usually obtained in artificial detergents. To understand whether and how lipids guide membrane protein function, we use single‐molecule FRET to probe the dynamics of DtpA, a member of the proton‐coupled oligopeptide transporter (POT) family, in various lipid environments. We show that detergents trap DtpA in a dynamic ensemble with cytoplasmic opening. Only reconstitutions in more native environments restore cooperativity, allowing an opening to the extracellular side and a sampling of all relevant states. Bilayer compositions tune the abundance of these states. A novel state with an extreme cytoplasmic opening is accessible in bilayers with anionic head groups. Hence, chemical diversity of membranes translates into structural diversity, with the current POT structures only sampling a portion of the full structural space. [ABSTRACT FROM AUTHOR]

Kinugasa reactions hold potential for bioorthogonal chemistry in that the reagents can be biocompatible. Unlike other bioorthogonal reaction products, β-lactams are potentially reactive, which can be useful for synthesizing new biomaterials. A limiting factor for applications consists of slow reaction rates. Herein, we report an optimized aqueous copper(I)-catalyzed alkyne–nitrone cycloaddition involving rearrangement (CuANCR) with rate accelerations made possible by the use of surfactant micelles. We have investigated the factors that accelerate the aqueous CuANCR reaction and demonstrate enhanced modification of a model membrane-associated peptide. We discovered that lipids/surfactants and alkyne structure have a significant impact on the reaction rate, with biological lipids and electron-poor alkynes showing greater reactivity. These new findings have implications for the use of CuANCR for modifying integral membrane proteins as well as live cell labelling and other bioorthogonal applications. [ABSTRACT FROM AUTHOR]

Unlike cytosolic proteins, membrane proteins (MPs) are embedded within the plasma membrane and the lipid bilayer of intracellular organelles. MPs serve in various cellular processes and account for over 65% of the current drug targets. The development of membrane mimetic systems such as bicelles, short synthetic polymers or amphipols, and membrane scaffold proteins (MSP)-based nanodiscs has facilitated the accommodation of synthetic lipids to stabilize MPs, yet the preparation of these membrane mimetics remains detergent-dependent. Bio-inspired synthetic polymers present an invaluable tool for excision and liberation of superstructures of MPs and their surrounding annular lipid bilayer in the nanometric discoidal assemblies. In this article, we discuss the significance of self-assembling process in design of biomimetic systems, review development of multiple series of amphipathic polymers and the significance of these polymeric “belts” in biomedical research in particular in unraveling the structures, dynamics and functions of several high-value membrane protein targets.

Membrane proteins (MPs) are essential for cellular functions. Understanding the functions of MPs is crucial as they constitute an important class of drug targets. However, MPs are a challenging class of biomolecules to analyze because they cannot be studied outside their native environment. Their structure, function and activity are highly dependent on the local lipid environment, and these properties are compromised when the protein does not reside in the cell membrane. Mammalian cell membranes are complex and composed of different lipid species. Model membranes have been developed to provide an adequate environment to envisage MP reconstitution. Among them, tethered-Bilayer Lipid Membranes (tBLMs) appear as the best model because they allow the lipid bilayer to be decoupled from the support. Thus, they provide a sufficient aqueous space to envisage the proper accommodation of large extra-membranous domains of MPs, extending outside. Additionally, as the bilayer remains attached to tethers covalently fixed to the solid support, they can be investigated by a wide variety of surface-sensitive analytical techniques. This review provides an overview of the different approaches developed over the last two decades to achieve sophisticated tBLMs, with a more and more complex lipid composition and adapted for functional MP reconstitution.

The functional mechanisms of membrane proteins are extensively investigated with cysteine mutagenesis. To complement cysteine-based approaches, we engineered a membrane protein with thiol-independent crosslinkable groups using azidohomoalanine (AHA), a non-canonical methionine analogue containing an azide group that can selectively react with cycloalkynes through a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. We demonstrate that AHA can be readily incorporated into the Shaker Kv channel in place of methionine residues and modified with azide-reactive alkyne probes in Xenopus oocytes. Using voltage-clamp fluorometry, we show that AHA incorporation permits site-specific fluorescent labeling to track voltage-dependent conformational changes similar to cysteine-based methods. By combining AHA incorporation and cysteine mutagenesis in an orthogonal manner, we were able to site-specifically label the Shaker Kv channel with two different fluorophores simultaneously. Our results identify a facile and straightforward approach for chemical modification of membrane proteins with bioorthogonal chemistry to explore their structure-function relationships in live cells. [ABSTRACT FROM AUTHOR]

Tight junctions (TJs) are cellular junctions within the mammalian epithelial cell sheet that function as a physical barrier to molecular transport within the intercellular space. Dysregulation of TJs leads to various diseases. Tricellular TJs (tTJs), specialized structural variants of TJs, are formed by multiple transmembrane proteins (e.g., lipolysis-stimulated lipoprotein receptor [LSR] and tricellulin) within tricellular contacts in the mammalian epithelial cell sheet. However, the mechanism for recruiting LSR and tricellulin to tTJs is largely unknown. Previous studies have identified that tyrphostin 9, the dual inhibitor of Pyk2 (a nonreceptor tyrosine kinase) and receptor tyrosine kinase platelet-derived growth factor receptor (PDGFR), suppresses LSR and tricellulin recruitment to tTJs in EpH4 (a mouse mammary epithelial cell line) cells. In this study, we investigated the effect of Pyk2 inhibition on LSR and tricellulin localization to tTJs. Pyk2 inactivation by its specific inhibitor or repression by RNAi inhibited the localization of LSR and downstream tricellulin to tTJs without changing their expression level in EpH4 cells. Pyk2-dependent changes in subcellular LSR and tricellulin localization were independent of c-Jun N-terminal kinase (JNK) activation and expression. Additionally, Pyk2-dependent LSR phosphorylation at Tyr-237 was required for LSR and tricellulin localization to tTJs and decreased epithelial barrier function. Our findings indicated a novel mechanism by which Pyk2 regulates tTJ assembly and epithelial barrier function in the mammalian epithelial cell sheet. [ABSTRACT FROM AUTHOR]

Variation in steroid hormone levels has wide implications for health and disease. The genes encoding the proteins involved in steroid disposition represent key determinants of interindividual variation in steroid levels and ultimately, their effects. Beginning with metabolomic data from genome-wide association studies (GWAS), we observed that genetic variants in the orphan transporter, SLC22A24 were significantly associated with levels of androsterone glucuronide and etiocholanolone glucuronide (sentinel SNPs p-value <1x10-30). In cells over-expressing human or various mammalian orthologs of SLC22A24, we showed that steroid conjugates and bile acids were substrates of the transporter. Phylogenetic, genomic, and transcriptomic analyses suggested that SLC22A24 has a specialized role in the kidney and appears to function in the reabsorption of organic anions, and in particular, anionic steroids. Phenome-wide analysis showed that functional variants of SLC22A24 are associated with human disease such as cardiovascular diseases and acne, which have been linked to dysregulated steroid metabolism. Collectively, these functional genomic studies reveal a previously uncharacterized protein involved in steroid homeostasis, opening up new possibilities for SLC22A24 as a pharmacological target for regulating steroid levels. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis is an obligate intracellular bacterium with a distinctive biphasic developmental cycle that alternates between two distinct cell types; the extracellular infectious elementary body (EB) and the intracellular replicating reticulate body (RB). Members of the genus Chlamydia are dependent on the formation and degradation of protein disulfide bonds. Moreover, disulfide cross-linking of EB envelope proteins is critical for the infection phase of the developmental cycle. We have identified in C. trachomatis a homologue of the Disulfide Bond forming membrane protein Escherichia coli (E. coli) DsbB (hereafter named CtDsbB) and—using recombinant purified proteins—demonstrated that it is the redox partner of the previously characterised periplasmic oxidase C. trachomatis Disulfide Bond protein A (CtDsbA). CtDsbA protein was detected in C. trachomatis inclusion vacuoles at 20 h post infection, with more detected at 32 and similar levels at 44 h post infection as the developmental cycle proceeds. As a redox pair, CtDsbA and CtDsbB largely resemble their homologous counterparts in E. coli; CtDsbA is directly oxidised by CtDsbB, in a reaction in which both periplasmic cysteine pairs of CtDsbB are required for complete activity. In our hands, this reaction is slow relative to that observed for E. coli equivalents, although this may reflect a non-native expression system and use of a surrogate quinone cofactor. CtDsbA has a second non-catalytic disulfide bond, which has a small stabilising effect on the protein’s thermal stability, but which does not appear to influence the interaction of CtDsbA with its partner protein CtDsbB. Expression of CtDsbA during the RB replicative phase and during RB to EB differentiation coincided with the oxidation of the chlamydial outer membrane complex (COMC). Together with our demonstration of an active redox pairing, our findings suggest a potential role for CtDsbA and CtDsbB in the critical disulfide bond formation step in the highly regulated development cycle. [ABSTRACT FROM AUTHOR]

Breast cancers that express epidermal growth factor (EGF) receptors (EGFRs) are associated with poor prognosis. Our group recently showed in breast cancer patients that EGFR expression is strongly correlated with high tumor uptake of the glucose analogue, 18F-fluorodeoxyglucose (FDG). Here, we explored the cellular mechanism and signaling pathways that can explain the relation between EGFR and breast cancer cell glucose metabolism. FDG uptake, lactate production and hexokinase (HK) activity were measured, and proliferation assays and western blots were performed. EGF stimulated an increase of FDG uptake in EGFR-positive T47D and MDA-MB-468 cells, but not in MCF-7 cells. In T47D cells, the effect was dose-dependent and was accompanied by increased lactate production, indicating a shift toward glycolytic flux. This metabolic response occurred through enhanced HK activity and upregulated glucose transporter 1 (GLUT1) expression. EGFR stimulation also increased T47D cell proliferation. Blocking EGFR activation with BIBX1382 or gefitinib completely abolished both FDG uptake and proliferation effects. EGFR stimulation induced MAP kinase (MAPK) and PI3 kinase (PI3K) activation. Increased cell proliferation by EGFR stimulation was completely abolished by MAPK inhibition with PD98059 or by PI3K inhibition with LY294002. Increased FDG uptake was also completely abrogated by PI3K inhibition but was uninfluenced by MAPK inhibition. These findings suggest that the association between breast tumor EGFR expression and high FDG uptake might be contributed by stimulation of the PI3K pathway downstream of EGFR activation. This was in contrast to EGFR-mediated cell proliferation that required MAPK as well as PI3K signaling. [ABSTRACT FROM AUTHOR]

Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are secreted by several phytopathogenic microorganisms. They trigger necrosis in various eudicot plants upon binding to plant sphingolipid glycosylinositol phosphorylceramides (GIPC). Interestingly, HaNLP3 from the obligate biotroph oomycete Hyaloperonospora arabidopsidis does not induce necrosis. We determined the crystal structure of HaNLP3 and showed that it adopts the NLP fold. However, the conformations of the loops surrounding the GIPC headgroup-binding cavity differ from those of cytotoxic Pythium aphanidermatum NLPPya. Essential dynamics extracted from μs-long molecular dynamics (MD) simulations reveals a limited conformational plasticity of the GIPC-binding cavity in HaNLP3 relative to toxic NLPs. This likely precludes HaNLP3 binding to GIPCs, which is the underlying reason for the lack of toxicity. This study reveals that mutations at key protein regions cause a switch between non-toxic and toxic phenotypes within the same protein scaffold. Altogether, these data provide evidence that protein flexibility is a distinguishing trait of toxic NLPs and highlight structural determinants for a potential functional diversification of non-toxic NLPs utilized by biotrophic plant pathogens. [ABSTRACT FROM AUTHOR]

Flavodoxins are small electron transfer proteins containing flavin mononucleotide (FMN) as a prosthetic group, which play an important role during oxidative stress or iron limitation. The aims of this study were the identification and characterization of flavodoxins in the model aromatic-degrader Paraburkholderia xenovorans LB400 and the analyses of their protective effects during oxidative stress induced by paraquat and H2O2. Two genes (BxeA0278 and BxeB0391) encoding flavodoxins (hereafter referred to as fldX for avooxin from P. enovorans), were identified at the LB400 major and minor chromosome. Genomic context of the flavodoxin-encoding genes showed genes encoding membrane proteins, transporters, and proteins involved in redox processes and biosynthesis of macromolecules. A secondary structure prediction of both LB400 flavodoxins showed the characteristic flavodoxin structure of five ß-sheets intercalated with five α-helices. FldX1 contains a loop intercalated in the fifth β-strand, which indicates that it belongs to the long-chain flavodoxins, whereas FldX2 is a short-chain flavodoxin. A phylogenetic analysis of 73 flavodoxins from 43 bacterial genera revealed eight clusters (I-VIII), while FldX1 and FldX2 grouped separately within a long-chain and a short-chain flavodoxin clades. FldX1 and FldX2 were overexpressed in P. xenovorans. Interestingly, the strain overexpressing the long-chain flavodoxin FldX1 (p2-fldX1) showed a faster growth in glucose than the control strain. The recombinant strain overexpressing the long-chain flavodoxin FldX1 (p2-fldx1) exposed to paraquat (20 mM) possessed lower susceptibility to growth inhibition on plates and higher survival in liquid medium than the control strain. The strains overexpressing the flavodoxins FldX1 and FldX2 showed higher survival during exposure to 1 mM paraquat (>95%) than the control strain (68%). Compared to the control strain, strains overexpressing FldX1 and FldX2 showed lower lipid peroxidation (>20%) after exposure to 1 mM paraquat and a lower protein carbonylation (~30%) after exposure to 1 mM H2O2 was observed. During exposure to paraquat, strain p2-fldx1 downregulated the katG4, hpf, trxB1 and ohr genes (> 2-fold), whereas strain p2-fldx2 upregulated the oxyR and ahpC1 genes (> 2-fold). In conclusion, the flavodoxins FldX1 and FldX2 of P. xenovorans LB400 conferred protection to cells exposed to the oxidizing agents paraquat and H2O2. [ABSTRACT FROM AUTHOR]