Inhibition of immune-mediated meningoencephalitis in persistently Borna disease virus-infected rats by cyclosporine A

In rats persistently infected with Borna disease virus (BDV), severe neurologic disorders and occasional death are the consequences of a T cell-mediated immunopathologic reaction in the brain. It is shown here that the pathologic alterations in the brain and as a result, Borna Disease (BD) can be prevented if animals are treated with the immunosuppressive drug cyclosporine A (CSA) under the following optimal conditions: greater than or equal to 25 mg/kg/day of CSA, started before infection and given for 4 wk. Rats treated with lower doses of CSA, for shorter periods or after infection displayed encephalitic lesions and developed BD. When CSA treatment was begun even as early as 1 day after infection, encephalitis and disease were not influenced. Immune spleen cells passively transferred into CSA-treated rats induced the disease in the recipients, whereas lymphoid cells from CSA-treated rats did not induce BD in infected cyclophosphamide-treated recipients. Antibodies were not involved in BD because rats treated with CSA revealed an inhibition of the synthesis of virus-specific antibodies for all regimens of treatment used (whether successful in preventing BD or not). After i.v. challenge of CSA-treated healthy rats with BDV, antiviral antibodies at low titers could be induced in some animals; however, no encephalitis or disease symptoms could be observed at any time after infection. The same was true for rats reinfected intracerebrally with BDV after discontinuation of CSA. These results support the hypothesis that unresponsiveness and even tolerance can be induced by CSA in the presence of the foreign Ag, demonstrating the beneficial effect of this immunosuppressive drug during a persistent viral infection.     

Oncogenic function for the Dlg1 mammalian homolog of the Drosophila discs-large tumor suppressor

The fact that several different human virus oncoproteins, including adenovirus type 9 E4-ORF1, evolved to target the Dlg1 mammalian homolog of the membrane-associated Drosophila discs-large tumor suppressor has implicated this cellular factor in human cancer. Despite a general belief that such interactions function solely to inactivate this suspected human tumor suppressor protein, we demonstrate here that E4-ORF1 specifically requires endogenous Dlg1 to provoke oncogenic activation of phosphatidylinositol 3-kinase (PI3K) in cells. Based on our results, we propose a model wherein E4-ORF1 binding to Dlg1 triggers the resulting complex to translocate to the plasma membrane and, at this site, to promote Ras-mediated PI3K activation. These findings establish the first known function for Dlg1 in virus-mediated cellular transformation and also surprisingly expose a previously unrecognized oncogenic activity encoded by this suspected cellular tumor suppressor gene.     

Protein shape and crowding drive domain formation and curvature in biological membranes

Folding, curvature, and domain formation are characteristics of many biological membranes. Yet the mechanisms that drive both curvature and the formation of specialized domains enriched in particular protein complexes are unknown. For this reason, studies in membranes whose shape and organization are known under physiological conditions are of great value. We therefore conducted atomic force microscopy and polarized spectroscopy experiments on membranes of the photosynthetic bacterium Rhodobacter sphaeroides. These membranes are densely populated with peripheral light harvesting (LH2) complexes, physically and functionally connected to dimeric reaction center-light harvesting (RC-LH1-PufX) complexes. Here, we show that even when converting the dimeric RC-LH1-PufX complex into RC-LH1 monomers by deleting the gene encoding PufX, both the appearance of protein domains and the associated membrane curvature are retained. This suggests that a general mechanism may govern membrane organization and shape. Monte Carlo simulations of a membrane model accounting for crowding and protein geometry alone confirm that these features are sufficient to induce domain formation and membrane curvature. Our results suggest that coexisting ordered and fluid domains of like proteins can arise solely from asymmetries in protein size and shape, without the need to invoke specific interactions. Functionally, coexisting domains of different fluidity are of enormous importance to allow for diffusive processes to occur in crowded conditions.     

Jumping mode atomic force microscopy on grana membranes from spinach

The thylakoid membrane system is a complex membrane system that organizes and reorganizes itself to provide plants optimal chemical energy from sunlight under different and varying environmental conditions. Grana membranes are part of this system and contain the light-driven water-splitting enzyme Photosystem II (PSII) and light-harvesting antenna complexes. Here, we present a direct visualization of PSII complexes within grana membranes from spinach. By means of jumping mode atomic force microscopy in liquid, minimal forces were applied between the scanning tip and membrane or protein, allowing complexes to be imaged with high detail. We observed four different packing arrangements of PSII complexes, which occur primarily as dimers: co-linear crystalline rows, nanometric domains of straight or skewed rows, and disordered domains. Upon storing surface-adhered membranes at low temperature prior to imaging, large-scale reorganizations of supercomplexes between PSII and light-harvesting complex II could be induced. The highest resolution images show the existence of membrane domains without obvious topography extending beyond supercomplexes. These observations illustrate the possibility for diffusion of proteins and smaller molecules within these densely packed membranes.     

Characterization and screening of IgG binding to the neonatal Fc receptor

The neonatal Fc receptor (FcRn) protects immunoglobulin G (IgG) from degradation and increases the serum half-life of IgG, thereby contributing to a higher concentration of IgG in the serum. Because altered FcRn binding may result in a reduced or prolonged half-life of IgG molecules, it is advisable to characterize Fc receptor binding of therapeutic antibody lead candidates prior to the start of pre-clinical and clinical studies. In this study, we characterized the interactions between FcRn of different species (human, cynomolgus monkey, mouse and rat) and nine IgG molecules from different species and isotypes with common variable heavy (VH) and variable light chain (VL) domains. Binding was analyzed at acidic and neutral pH using surface plasmon resonance (SPR) and biolayer interferometry (BLI). Furthermore, we transferred the well-accepted, but low throughput SPR-based method for FcRn binding characterization to the BLI-based Octet platform to enable a higher sample throughput allowing the characterization of FcRn binding already during early drug discovery phase. We showed that the BLI-based approach is fit-for-purpose and capable of discriminating between IgG molecules with significant differences in FcRn binding affinities. Using this high-throughput approach we investigated FcRn binding of 36 IgG molecules that represented all VH/VL region combinations available in the fully human, recombinant antibody library Ylanthia®. Our results clearly showed normal FcRn binding profiles for all samples. Hence, the variations among the framework parts, complementarity-determining region (CDR) 1 and CDR2 of the fragment antigen binding (Fab) domain did not significantly change FcRn binding.     

The Use of Contact Mode Atomic Force Microscopy in Aqueous Medium for Structural Analysis of Spinach Photosynthetic Complexes

To investigate the dynamics of photosynthetic pigment-protein complexes in vascular plants at high resolution in an aqueous environment, membrane-protruding oxygen-evolving complexes (OECs) associated with photosystem II (PSII) on spinach (Spinacia oleracea) grana membranes were examined using contact mode atomic force microscopy. This study represents, to our knowledge, the first use of atomic force microscopy to distinguish the putative large extrinsic loop of Photosystem II CP47 reaction center protein (CP47) from the putative oxygen-evolving enhancer proteins 1, 2, and 3 (PsbO, PsbP, and PsbQ) and large extrinsic loop of Photosystem II CP43 reaction center protein (CP43) in the PSII-OEC extrinsic domains of grana membranes under conditions resulting in the disordered arrangement of PSII-OEC particles. Moreover, we observed uncharacterized membrane particles that, based on their physical characteristics and electrophoretic analysis of the polypeptides associated with the grana samples, are hypothesized to be a domain of photosystem I that protrudes from the stromal face of single thylakoid bilayers. Our results are interpreted in the context of the results of others that were obtained using cryo-electron microscopy (and single particle analysis), negative staining and freeze-fracture electron microscopy, as well as previous atomic force microscopy studies.Oxygenic photosynthesis supports most life on Earth through the absorption of solar energy, which powers the extraction of electrons from water and the subsequent use of those electrons to convert CO₂ into organic compounds (Nelson and Ben-Shem, 2004; Merchant and Sawaya, 2005; Nelson, 2011). The light-dependent reactions of photosynthesis occur within photosynthetic or thylakoid membranes and are catalyzed by two reaction centers, PSI and PSII. Both photosystems have associated light-harvesting complexes (LHCI and LHCII) that act as antenna to efficiently capture light energy. The oxygen-evolving complex (OEC) is an integral component of PSII, catalyzing the extraction of electrons from water. The two photosystems are connected through an intersystem electron transport chain that includes the hydrophobic electron carrier plastoquinone, the membrane-bound cytochrome b₆f complex (cyt b₆f), and the mobile electron carrier plastocyanin. The electrochemical gradient generated during light-driven electron flow is used in the synthesis of ATP by the ATP synthase complex. Components of the photosynthetic apparatus vary among photosynthetic organisms and under different environmental conditions, especially for proteins associated with light-harvesting complexes (Liu and Scheuring, 2013). However, investigations of the mechanisms associated with the dynamic acclimation of photosynthetic electron transport and light harvesting to environmental cues require real-time observations that are difficult to achieve because of limitations in our ability to view such changes (e.g. difficulties in tagging proteins with fluorophores and resolving fluorescent images; Zaks et al., 2013).In vascular plants, thylakoid membranes form a network of interconnected tubular structures enclosing a lumenal space. This membrane system can be divided into two morphologically distinct regions: the grana, which are formed by stacks of appressed membranes; and the stroma lamellae, which are unappressed membranes that form connections between grana stacks. These distinct thylakoid regions are enriched for specific photosynthetic complexes. The major complexes in grana are PSII and LHCII, which can interact and form a variety of PSII-LHCII supercomplexes (Dekker and Boekema, 2005; Kouřil et al., 2012), as well as cyt b₆f (Johnson et al., 2014). Grana stacks are also the site of water oxidation and oxygen evolution; the Mn₄CaO₅ cluster is the PSII cofactor that catalyzes this process (Umena et al., 2011). This cluster resides between the transmembrane subunits of the PSII core (formed by PSII proteins D1 and D2 and their associated pigment cofactors, with PSII reaction center proteins CP43 and CP47, α- and β-subunits of cytochrome b559, and PSII reaction center protein I [PsbI]) and the lumenal, peripheral membrane proteins of the OEC. The OEC is composed of extrinsic membrane polypeptides of 33 kD, 23 kD, and 17 kD, designated oxygen-evolving enhancer protein 1, 2, and 3 (PsbO, PsbP, and PsbQ) that protrude into the thylakoid lumen in vascular and nonvascular plants as well as in green algae. Cyanobacteria also have PsbO, with PsbV and PsbU serving as functional analogs of plant PsbP and PsbQ, respectively (Dekker and Boekema, 2005). Based on removal/reconstitution experiments, these subunits have been shown to be critical for PSII stability and oxygen evolution activity (Kuwabara and Murata, 1983; Ljungberg et al., 1983; Ghanotakis et al., 1984). They may also impact the association of Ca²⁺ and Cl⁻ with PSII, the polypeptide conformation around the manganese cluster, and the formation of channels within PSII that allow access of water to the catalytic site and the exit of protons from the complex as water is oxidized (Bricker et al., 2012).The x-ray crystal structures of purified PsbP (Kohoutová et al., 2009) and PsbQ (Balsera et al., 2005) from spinach (Spinacia oleracea) have been determined. For cyanobacteria, PSII crystals have been used to establish high-resolution structures for PsbO, PsbV, and PsbU (Umena et al., 2011), with more recent analyses at room temperature (Kern et al., 2013). To study the bound state of these peripheral proteins in spinach, electron density maps were established based on cryo-electron microscopy (cryo-EM) and single particle analysis of purified PSII-LHCII supercomplexes, with structural verification based on the removal of extrinsic polypeptides of the complexes from the membranes (Nield et al., 2002). In these structures, determined at less than 2 nm (or 20 Å) resolution, the PsbP and PsbQ subunits of OEC were assigned to a single membrane protrusion, with a second membrane protrusion assigned to PsbO (Boekema et al., 2000a); these topological structures are the most prominent protruding features of the reaction center-containing membrane protein complexes. Since two PSII reaction centers associate to form a dimeric PSII-LHCII supercomplex (Bumba and Vácha, 2003), the six OEC subunits (two PsbO, two PsbP, and two PsbQ) are visualized as four protrusions associated with each supercomplex. However, after the cyanobacterial PSII core structure was solved (including the positions of the extrinsic subunits) and aligned to the cryo-EM of PSII-LHCII supercomplexes (Nield and Barber, 2006), the structure was reevaluated. The PSII lumenal small protruding mass was assigned to the large extrinsic loop of CP47 (encoded by psbB), while the larger protrusion was assigned to PsbO, PsbP, PsbQ, and the large extrinsic loop associated with CP43 (encoded by psbC).Attempts have been made to visualize PSII complexes and proteins in their native membrane environment using transmission electron microscopy (TEM) in conjunction with freeze fracture (Johnson et al., 2011) and negative staining of grana membranes from both spinach (Boekema et al., 2000b) and Arabidopsis (Arabidopsis thaliana; Betterle et al., 2009; Wientjes et al., 2013). These techniques provide little information regarding the extent of the protrusion of the polypeptide subunits out of the plane of the membranes. Even though cryo-electron tomography of isolated chloroplasts and plunge-frozen thylakoid membranes (Daum et al., 2010) and grana stacks (Kouřil et al., 2011) can preserve sample hydration using a vitrification process during freezing, it is difficult to determine the height of the extrinsic thylakoid protein protrusions from the membrane surface. Furthermore, at the resolution obtained with these techniques, the small and large protrusions of each PSII monomer may appear merged into a single structure. This would result in the visualization of only two distinguishable topological entities for each PSII-OEC dimer.The generation of images by atomic force microscopy (AFM), which involves raster scanning by a sharp tip that is in contact with the sample, complements other structural determination methods. The vertical position of the tip is controlled in order to maintain a constant imaging force (balancing interaction forces between the tip and the scanned structure). Control is implemented by a feedback loop that continuously monitors the force with a highly sensitive force sensor that activates a high-precision actuator. Logging the vertical position of the piezoelectric actuator that controls the vertical position of the tip can provide particle height relative to the membrane at high vertical resolution; this is done concurrently with logging the lateral position of each pixel to generate the image (Bippes and Muller, 2011). Probing samples with AFM in air has been employed to image spinach grana membranes (Kirchhoff et al., 2008) to elucidate the arrangement of Arabidopsis PSII-LHCII supercomplexes associated with nonphotochemical quenching (Onoa et al., 2014) and to determine the areal density of Arabidopsis PSII-OEC during the PSII repair cycle (Puthiyaveetil et al., 2014).Most AFM studies of grana have been performed with membrane surfaces exposed to air. This raises issues concerning the extent to which membrane properties are altered during measurements in a nonaqueous environment (Zaks et al., 2013), where it may be impossible to maintain appropriate hydration and ionic conditions. However, AFM has also been used in aqueous medium to establish high-resolution topography images of membrane proteins (Bippes and Muller, 2011) and specifically to characterize the PSII-OEC, which was previously observed as an ordered array within spinach grana membranes (Sznee et al., 2011). In recent studies, a map of the lumenal surface of grana membranes was generated in aqueous medium that distinguishes cyt b₆f dimers from PSII-OEC (Johnson et al., 2014). However, the potential of AFM imaging in a liquid environment has not been realized for the high-resolution analysis of features associated with thylakoid membranes and the PSII-OEC dimer. We used contact mode atomic force microscopy (CM-AFM) to (1) image PSII-OEC topology in liquid medium at high resolution, (2) identify other features/particles associated with grana membranes, and (3) optimize the use of AFM for monitoring the dynamics of thylakoid membrane complexes as the conditions of the environment are modulated (e.g. light, specific ions, and temperature).     

Characterization of the Human Sulfatase Sulf1 and Its High Affinity Heparin/Heparan Sulfate Interaction Domain

The extracellular sulfatases Sulf1 and Sulf2 remodel the 6O-sulfation state of heparan sulfate proteoglycans on the cell surface, thereby modulating growth factor signaling. Different from all other sulfatases, the Sulfs contain a unique, positively charged hydrophilic domain (HD) of about 320 amino acid residues. Using various HD deletion mutants and glutathione S-transferase (GST)-HD fusion proteins, this study demonstrates that the HD is required for enzymatic activity and acts as a high affinity heparin/heparan sulfate interaction domain. Association of the HD with the cell surface is sensitive to heparinase treatment, underlining specificity toward heparan sulfate chains. Correspondingly, isolated GST-HD binds strongly to both heparin and heparan sulfate in vitro and also to living cells. Surface plasmon resonance studies indicate nanomolar affinity of GST-HD toward immobilized heparin. The comparison of different mutants reveals that especially the outer regions of the HD mediate heparan sulfate binding, probably involving “tandem” interactions. Interestingly, binding to heparan sulfate depends on the presence of 6O-sulfate substrate groups, suggesting that substrate turnover facilitates release of the enzyme from its substrate. Deletion of the inner, less conserved region of the HD drastically increases Sulf1 secretion without affecting enzymatic activity or substrate specificity, thus providing a tool for the in vitro modulation of HS-dependent signaling as demonstrated here for the signal transduction of fibroblast growth factor 2. Taken together, the present study shows that specific regions of the HD influence different aspects of HS binding, cellular localization, and enzyme function.The human sulfatases represent a family of 17 enzymes responsible for the turnover and remodeling of sulfate esters and sulfamates. Their reaction mechanism relies on a special amino acid residue, Cα-formylglycine, which is generated post-translationally via oxidation of a conserved cysteine residue in the active site (1–3). Besides the lysosomal sulfatases involved in the cellular degradation of various sulfated substrates (4), two extracellular sulfatases, Sulf1 and Sulf2 (the Sulfs), have been described (5, 6). The Sulfs are endosulfatases with restricted substrate specificity toward 6O-sulfate groups of heparan sulfate (HS),² an information-rich glycosaminoglycan (GAG) polymer attached to proteoglycans at the cell surface and in the extracellular matrix (6–8). HS proteoglycans (HSPGs) act as co-receptors in cell signaling pathways and provide binding sites for growth factors and morphogens via specific sulfation patterns on their HS chains. By enzymatically removing 6O-sulfate groups from HSPGs on the cell surface, Sulf1 and Sulf2 differentially regulate the activity of FGF, vascular endothelial growth factor, Wnt, and other HS ligands, thereby modulating important processes such as development, cell growth, and differentiation (9–12). Misregulation of the Sulfs has been linked with both tumor progression and suppression, depending on either activating or inhibitory effects upon cell signaling (13–16).To investigate the physiological role of Sulf1 and Sulf2, single and double knock-out mice were generated (17–21). Both Sulf1 and Sulf2 knock-out mice are characterized by increased embryonic lethality, impaired neurite outgrowth, and other neurological abnormalities in the developing and adult nervous system (22). The corresponding double knock-out mice display an obvious reduction in body weight and developmental malformations, including skeletal and renal defects (18, 19, 23). Together with biochemical analyses on the impact of Sulf loss on HS sulfation, the phenotypic observations suggest a functional cooperativity between Sulf1 and Sulf2 in modulating the 6O-sulfation of UA(2S)-GlcNS(6S) disaccharide units within the S-domains of HS chains (17, 24). Moreover, analyses of heparan sulfate disaccharide compositions from Sulf1 and Sulf2 knock-out mice cell lines have indicated dynamic influences of Sulf loss also on non-substrate N-, 2O-, and 6O-sulfate groups via modulation of sulfotransferase expression, which may contribute to the developmental defects associated with the Sulf knock-out mice (24).From the biochemical perspective, it is an important question how the Sulfs are able to recognize their HSPG substrates and how cell surface localization is achieved, despite a lack of transmembrane domains or lipid anchors. Classical GAG-binding proteins, such as antithrombin III (25) or FGF1 (26), interact with their negatively charged GAG partners via small clusters of positively charged amino acid residues. Although some consensus sequences for heparin binding have been identified (XBBXBX, XBBBXXBX, and XBBXXBBBXXBBX, where B is a basic residue and X a hydropathic) (27–29), they are neither required nor sufficient. Unlike these classical GAG-binding proteins, Sulf1 and Sulf2 contain a large hydrophilic domain (HD), located between the N-terminal catalytic domain and the C-terminal domain. The HD is a unique feature of the extracellular sulfatases that is neither found in other sulfatases nor shows any homology with other known protein domains. According to sequence alignments, the HD of human Sulf1 has a size of ∼320 amino acid residues, 27% of which are basic and 14% acidic, resulting in a strong positive charge at neutral pH and a high theoretical pI of 9.8. Remarkably, the C-terminal end of the HD is composed of a cluster of 12 basic amino acid residues. Whereas the outer regions of the HD are highly conserved between Sulf1 and Sulf2 as well as between human, murine, and avian orthologs, the inner region, encoded by exons 13 and 14 in the case of human Sulf1 (6), is significantly less conserved.The role of the HD has previously been investigated for the avian ortholog QSulf2 (30). Results from this study indicated that the HD binds to negatively charged ligands and might serve to anchor the enzyme on the cell surface. Sulfate release assays indicated the necessity of the avian HD for enzymatic activity. Moreover, a very recent analysis of the HD of human Sulf1/Sulf2 revealed the presence of two furin-type proteinase cleavage sites within the inner region, explaining their partial processing into disulfide-linked subunits of 75 and 50 kDa (31). Sulf1/2 mutants, in which these sites were deleted, retained enzymatic activity but failed to potentiate Wnt signaling when overexpressed in human embryonic kidney 293 cells.Due to the observed differences in enzyme secretion and detergent solubility between the human and avian orthologs (24, 30) and the likely importance of this domain for mammalian Sulf localization and activity, we analyzed the function of the HD of human Sulf1 in mediating enzyme activity, cell surface targeting, secretion, and substrate recognition. Using different Sulf1 deletion mutants and glutathione S-transferase (GST)-HD fusion proteins, this study demonstrates that specific regions of the HD, especially at the conserved N and C termini, are responsible for heparin/HS binding, cell surface localization, and enzymatic activity of human Sulf1. Interaction analyses show that binding of the HD to heparin is significantly stronger compared with other typical heparin-binding proteins, suggesting a new mode of GAG binding. The deletion of the inner region of the HD leads to significantly increased secretion of the enzyme, allowing the purification of an active variant that is able to modulate FGF signaling in cell culture experiments.