Inhibition of complex glycosylation increases the formation of PrPsc

N-linked glycans with complex structure have a major role in the biological activity of a wide variety of cell surface and secreted glycoproteins. Here, we show that geldanamycin, an inhibitor of Hsp90, interferes with the formation of complex glycosylated mammalian prion protein (PrP^C). Similarly to inhibitors of α-mannosidases, geldanamycin stabilized a high mannose PrP^C glycoform and prevented the subsequent processing into complex structures. Moreover, a PrP/Grp94 complex could be isolated from geldanamycin-treated cells, suggesting that Grp94 might play a role in the processing of PrP^C in the endoplasmic reticulum. Inhibition of complex glycosylation did not interfere with the glycosylphosphatidylinositol (GPI) anchor attachment and cellular trafficking of high mannose PrP^C to the outer leaflet of the plasma membrane. In scrapie-infected neuroblastoma cells, however, high mannose PrP^C glycoforms were preferred substrates for the formation of PrP-scrapie (PrP^Sc). Our study reveals that complex glycosylation is dispensable for the cellular trafficking of PrP^C, but modulates the formation of PrP^Sc .     

Lithium induces clearance of protease resistant prion protein in prion-infected cells by induction of autophagy

Lithium is used for several decades to treat manic-depressive illness (bipolar affective disorder). Recently, it was found that lithium induces autophagy, thereby promoting the clearance of mutant huntingtin and α-synucleins in experimental systems. We show here for the first time that lithium significantly reduces the amount of pathological prion protein (PrP^Sc) in prion-infected neuronal and non-neuronal cultured cells by inducing autophagy. Treatment of prion-infected cells with 3-methyladenine, a potent inhibitor of autophagy, counteracted the anti-prion effect of lithium, demonstrating that induction of autophagy mediates degradation of PrP^Sc. Co-treatment with lithium and rapamycin, a drug widely used to induce autophagy, had an additive effect on PrP^Sc clearance compared to treatment with either drug alone. In addition, we provide evidence that the ability to reduce PrP^Sc and to induce autophagy is common for diverse lithium compounds, not only for the drug lithium chloride, usually administered in clinical therapy. Furthermore, we show here that besides reduction of PrP^Sc-aggregates, lithium-induced autophagy also slightly reduces the levels of cellular prion protein. Limiting the substrate available for conversion of cellular prion protein into PrP^Sc may provide an additional mechanism for reduction of PrP^Sc by lithium-induced autophagy.     

Characterization of cell-surface prion protein relative to its recombinant analogue: insights from molecular dynamics simulations of diglycosylated, membrane-bound human prion protein

The prion protein (PrP) is responsible for several fatal neurodegenerative diseases via conversion from its normal to disease-related isoform. The recombinant form of the protein is typically studied to investigate the conversion process. This constructs lacks the co- and post-translational modifications present in vivo , there the protein has two N-linked glycans and is bound to the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. The inherent flexibility and heterogeneity of the glycans, the plasticity of the GPI anchor, and the localization of the protein in a membrane make experimental structural characterization of biological constructs of cellular prion protein (PrP^C) challenging. Yet this characterization is central in determining not only the suitability of recombinant (rec)-PrP^C as a model for biological forms of the protein but also the potential role of co- and post-translational modifications on the disease process. Here, we present molecular dynamics simulations of three human prion protein constructs: (i) a protein-only construct modeling the recombinant form, (ii) a diglycosylated and soluble construct, and (iii) a diglycosylated and GPI-anchored construct bound to a lipid bilayer. We found that glycosylation and membrane anchoring do not significantly alter the structure or dynamics of PrP^C, but they do appreciably modify the accessibility of the polypeptide surface PrP^C. In addition, the simulations of membrane-bound PrP^C revealed likely recognition domains for the disease-initiating PrP^C:PrP^Sc (infectious and/or misfolded form of the prion protein) binding event and a potential mechanism for the observed inefficiency of conversion associated with differentially glycosylated PrP species.     

Prion diseases: contribution of high-resolution immunomorphology

The transmisible spongiform encephalopathies or prion diseases are fatal neurological diseases that occur in animals and humans. They are characterized by the accumulation in the cerebral tissue of the abnormal form of prion protein (PrP^sc) produced by a post-translational event involving conformational change of its normal cellular counterpart (PrP^c). In this short review, we present some results on the biology of prion proteins which have benefited from morphological approaches combining the electron microscopy techniques and the immunodetection methods. We discuss data concerning in particular the physiological function of the normal cellular prion prion (PrP^c) which have allowed to open up new vistas on prion diseases, the biogenesis of amyloid plaque and the cellular site involved in the prion protein conversion process.     

Gene expression profile of quinacrine-cured prion-infected mouse neuronal cells

Prion diseases are transmissible fatal neurodegenerative diseases of humans and animals, characterised by the presence of an abnormal isoform (scrapie prion protein; PrP^Sc) of the endogenous cellular prion protein (PrP^C). The pathological mechanisms at the basis of prion diseases remain elusive, although the accumulation of PrP^Sc has been linked to neurodegeneration. Different genomic approaches have been applied to carry out large-scale expression analysis in prion-infected brains and cell lines, in order to define factors potentially involved in pathogenesis. However, the general lack of overlap between the genes found in these studies prompted us to carry an analysis of gene expression using an alternative approach. Specifically, in order to avoid the complexities of shifting gene expression in a heterogeneous cell population, we used a single clone of GT1 cells that was de novo infected with mouse prion-infected brain homogenate and then treated with quinacrine to clear PrP^Sc. By comparing the gene expression profiles of about 15 000 genes in quinacrine-cured and not cured prion-infected GT1 cells, we investigated the influence of the presence or the absence of PrP^Sc. By real-time PCR, we confirmed that the gene encoding for laminin was down-regulated as a consequence of the elimination of PrP^Sc by the quinacrine treatment. Thus, we speculate that this protein could be a specific candidate for further analysis of its role in prion infection and pathogenesis.     

Non-infectious aggregates of the prion protein react with several PrPSc-directed antibodies

The key event in the pathogenesis of prion diseases is the conformational conversion of the normal prion protein (PrP) (PrP^C) into an infectious, aggregated isoform (PrP^Sc) that has a high content of β-sheet. Historically, a great deal of effort has been devoted to developing antibodies that specifically recognize PrP^Sc but not PrP^C, as such antibodies would have enormous diagnostic and experimental value. A mouse monoclonal IgM antibody (designated 15B3) and three PrP motif-grafted monoclonal antibodies (referred to as IgG 19–33, 89–112, and 136–158) have been previously reported to react specifically with infectious PrP^Sc but not PrP^C. In this study, we extend the characterization of these four antibodies by testing their ability to immunoprecipitate and immunostain infectious and non-infectious aggregates of wild-type, mutant, and recombinant PrP. We find that 15B3 as well as the motif-grafted antibodies recognize multiple types of aggregated PrP, both infectious and non-infectious, including forms found in brain, in transfected cells, and induced in vitro from purified recombinant protein. These antibodies are exquisitely selective for aggregated PrP, and do not react with soluble PrP even when present in vast excess. Our results suggest that 15B3 and the motif-grafted antibodies recognize structural features common to both infectious and non-infectious aggregates of PrP. Our study extends the utility of these antibodies for diagnostic and experimental purposes, and it provides new insight into the structural changes that accompany PrP oligomerization and prion propagation.     

Evidence for an association of prion protein and sphingolipid-mediated signaling

The physiological function of the cellular prion protein (PrP^c) is unclear. PrP^c associates with lipid rafts, highly glycolipid-rich membrane domains containing a large variety of signaling molecules, e.g., sphingolipids (SL). In this study, we investigated possible connections between PrP^c and sphingolipid-associated signaling pathways. Using PrP^c-wt and PrP^c-k.o. hippocampal cell lines and mouse brains we showed higher activity of neutral and acid sphingomyelinase (SMase) in PrP^c-k.o.-groups, while ceramide and sphingomyelin-levels were unchanged. Furthermore, despite lower basal expression levels of sphingosine kinase (SphK) in PrP^c-k.o.-groups, the levels of its metabolite sphingosine-1-phosphate were increased, whereas S1P₃-receptor expression was higher in PrP^c-wt-groups again. In addition, we detected enhanced activity of phospholipase D1, an enzyme that seems to be suitable to act as a connector between the S1P₃ receptor and continuative signaling. Finally, evidence for an impact on downstream signaling cascades, especially activation of the PI3K/Akt pathway, was found. In summary, our data suggest that PrP^c is involved in sphingolipid-associated signaling, modulating pathways that exert anti-apoptotic functions, hence indicating that PrP^c plays a role in neuroprotection.     

PrPc activation induces neurite outgrowth and differentiation in PC12 cells: role for caveolin-1 in the signal transduction pathway

Cellular prion protein (PrP^c) is a ubiquitous glycoprotein, whose physiological role is poorly characterized. It has been suggested that PrP^c participates in neuritogenesis, neuroprotection, copper metabolism, and signal transduction. In this study we detailed the intracellular events induced by PrP^c antibody-mediated cross-linking in PC12 cells. We found a Fyn-dependent activation of the Ras-Raf pathway, which leads to a rapid and transient phosphorylation of extracellular regulated kinases. In addition, this activation cascade relies on the engagement of integrins, and involves focal adhesion kinase activation. We demonstrated the tyrosine phosphorylation of caveolin-1 as a consequence of PrP^c stimulation, and showed that phosphocaveolin-1 scaffolds and coordinates protein complexes involved in PrP^c-dependent signaling. Moreover, we found that caveolin-1 phosphorylation, is a mechanism for recruiting the C-terminal Src kinase and inactivating Fyn, so as to terminate cell signaling. Furthermore our data support a significant role for PrP^c as a response mediator in neuritogenesis and cell differentiation.     

Different structural stability and toxicity of PrP(ARR) and PrP(ARQ) sheep prion protein variants

The polymorphisms at amino acid residues 136, 154, and 171 in ovine prion protein (PrP) have been associated with different susceptibility to scrapie: animals expressing PrP^ARQ [PrP(Ala136/Arg154/Gln171)] show vulnerability, whereas those that express PrP^ARR [PrP(Ala136/Arg154/Arg171)] are resistant to scrapie. The aim of this study was to evaluate the in vitro toxic effects of PrP^ARR and PrP^ARQ variants in relation with their structural characteristics. We show that both peptides cause cell death inducing apoptosis but, unexpectedly, the scrapie resistant PrP^ARR form was more toxic than the scrapie susceptible PrP^ARQ variant. Moreover, the α-helical conformation of PrP^ARR was less stable than that of PrP^ARQ and the structural determinants responsible of these different conformational stabilities were characterized by spectroscopic analysis. We observed that PrP toxicity was inversely related to protein structural stability, being the unfolded conformation more toxic than the native one. However, the PrP^ARQ variant displays a higher propensity to form large aggregates than PrP^ARR. Interestingly, in the presence of small amounts of PrP^ARR, PrP^ARQ aggregability was reduced to levels similar to that of PrP^ARR. Thus, in contrast to PrP^ARR toxicity, scrapie transmissibility seems to reside in the more stable conformation of PrP^ARQ that allows the formation of large amyloid fibrils.     

Involvement of mitochondria in endoplasmic reticulum stress-induced apoptotic cell death pathway triggered by the prion peptide PrP(106-126)

Prion disorders are progressive neurodegenerative diseases characterized by extensive neuronal loss and by the accumulation of the pathogenic form of prion protein, designated PrP^Sc. Recently, we have shown that PrP_106–126 induces endoplasmic reticulum (ER) stress, leading to mitochondrial cytochrome c release, caspase 3 activation and apoptotic death. In order to further clarify the role of mitochondria in ER stress-mediated apoptotic pathway triggered by the PrP peptide, we investigated the effects of PrP_106–126 on the Ntera2 human teratocarcinoma cell line that had been depleted of their mitochondrial DNA, termed NT2 ρ0 cells, characterized by the absence of functional mitochondria, as well as on the parental NT2 ρ+ cells. In this study, we show that PrP_106–126 induces ER stress in both cell lines, given that ER Ca²⁺ content is low, glucose-regulated protein 78 levels are increased and caspase 4 is activated. Furthermore, in parental NT2 ρ+ cells, PrP_106–126-activated caspase 9 and 3, induced poly (ADP-ribose) polymerase cleavage and increased the number of apoptotic cells. Dantrolene was shown to protect NT2 ρ+ from PrP_106–126-induced cell death, demonstrating the involvement of Ca²⁺ release through ER ryanodine receptors. However, in PrP_106–126-treated NT2 ρ0 cells, apoptosis was not able to proceed. These results demonstrate that functional mitochondria are required for cell death as a result of ER stress triggered by the PrP peptide, and further elucidate the molecular mechanisms involved in the neuronal loss that occurs in prion disorders.     

Prion protein—Semisynthetic prion protein (PrP) variants with posttranslational modifications

Deciphering the pathophysiologic events in prion diseases is challenging, and the role of posttranslational modifications (PTMs) such as glypidation and glycosylation remains elusive due to the lack of homogeneous protein preparations. So far, experimental studies have been limited in directly analyzing the earliest events of the conformational change of cellular prion protein (PrP^C) into scrapie prion protein (PrP^Sc) that further propagates PrP^C misfolding and aggregation at the cellular membrane, the initial site of prion infection, and PrP misfolding, by a lack of suitably modified PrP variants. PTMs of PrP, especially attachment of the glycosylphosphatidylinositol (GPI) anchor, have been shown to be crucially involved in the PrP^Sc formation. To this end, semisynthesis offers a unique possibility to understand PrP behavior invitro and invivo as it provides access to defined site‐selectively modified PrP variants. This approach relies on the production and chemoselective linkage of peptide segments, amenable to chemical modifications, with recombinantly produced protein segments. In this article, advances in understanding PrP conversion using semisynthesis as a tool to obtain homogeneous posttranslationally modified PrP will be discussed.     

The Fate of PrP GPI‐Anchor Signal Peptide is Modulated by P238S Pathogenic Mutation

Glycosylphosphatidylinositol (GPI)‐anchored proteins are localized to the plasma membrane via a C‐terminally linked GPI anchor. The GPI anchor is added concomitantly to the cleavage of the carboxy‐terminal GPI‐anchor signal sequence, thereby causing the release of a C‐terminal hydrophobic peptide, whose fate has not yet been investigated. Here we followed the fate of the GPI‐attachment signal of the prion protein (PrP), a protein implicated in various types of transmissible neurodegenerative spongiform encephalopathies (TSE). The PrP GPI‐anchor signal sequence shows a remarkable and unusual degree of conservation across the species and contains two point mutations (M232R/T and P238S) that are responsible for genetic forms of prion disorders. We show that the PrP GPI‐anchor signal peptide (SP), but not the one from an unrelated GPI‐anchored protein (folate receptor), undergoes degradation via the proteasome. Moreover, the P238S point mutation partially protects the PrP GPI‐anchor SP from degradation. Our data provide the first attempt to address the fate of a GPI‐anchor SP and identify a role for the P238S mutation, suggesting the possibility that the PrP GPI‐anchor SP could play a role in neurodegenerative prion diseases.      

Trafficking of prion proteins through a caveolae-mediated endosomal pathway

To understand the posttranslational conversion of the cellular prion protein (PrPC) to its pathologic conformation, it is important to define the intracellular trafficking pathway of PrPC within the endomembrane system. We studied the localization and internalization of PrPC in CHO cells using cryoimmunogold electron microscopy. At steady state, PrPC was enriched in caveolae both at the TGN and plasma membrane and in interconnecting chains of endocytic caveolae. Protein A-gold particles bound specifically to PrPC on live cells. These complexes were delivered via caveolae to the pericentriolar region and via nonclassical, caveolae-containing early endocytic structures to late endosomes/lysosomes, thereby bypassing the internalization pathway mediated by clathrin-coated vesicles. Endocytosed PrPC-containing caveolae were not directed to the ER and Golgi complex. Uptake of caveolae and degradation of PrPC was slow and sensitive to filipin. This caveolae-dependent endocytic pathway was not observed for several other glycosylphosphatidyl inositol (GPI)-anchored proteins. We propose that this nonclassical endocytic pathway is likely to determine the subcellular location of PrPC conversion.     

PrP的胞内运输与朊病毒病

朊病毒病,即传染性海绵状脑病（transmissible spongiform encephalopathies,TSEs）,是一类致死性的神经退行性疾病,存在散发性、感染性和遗传性3种形式。在朊病毒病的病理过程中,细胞正常朊蛋白PrPc（cellular PrP）转化为异常构象的PrP^Sc（scrapie PrP）是至关重要的,但是朊病毒的增殖如何导致神经元凋亡仍不清楚。PrPc的胞内运输在朊病毒病中发挥重要作用,朊病毒感染后PrP^C转化为PrP^Sc,及遗传性朊病毒病中PrP突变可能影响PrP的生物合成、亚细胞定位及转运过程,通过干扰PrP^C的正常功能或产生毒性中间体而导致神经系统病变。现对近年来关于PrP胞内运输在朊病毒病中的作用进行综述。     

Sorting Nexin 11 Regulates Lysosomal Degradation of Plasma Membrane TRPV3

The trafficking of ion channels to/from the plasma membrane is considered an important mechanism for cellular activity and an interesting approach for disease therapies. The transient receptor potential vanilloid 3 (TRPV3) ion channel is widely expressed in skin keratinocytes, and its trafficking mechanism to/from the plasma membrane is unknown. Here, we report that the vesicular trafficking protein sorting nexin 11 (SNX11) downregulates the level of the TRPV3 plasma membrane protein. Overexpression of SNX11 causes a decrease in the level of TRPV3 current and TRPV3 plasma membrane protein in TRPV3‐transfected HEK293T cells. Subcellular localizations and western blots indicate that SNX11 interacts with TRPV3 and targets it to lysosomes for degradation, which is blocked by the lysosomal inhibitors chloroquine and leupeptin. Both TRPV3 and SNX11 are highly expressed in HaCaT cells. We show that TRPV3 agonists‐activated Ca²⁺ influxes and the level of native TRPV3 total protein in HaCaT cells are decreased by overexpression of SNX11 and increased by knockdown of SNX11. Our findings reveal that SNX11 promotes the trafficking of TRPV3 from the plasma membrane to lysosomes for degradation via protein‐protein interactions, which demonstrates a previously unknown function of SNX11 as a regulator of TRPV3 trafficking from the plasma membrane to lysosomes.     

Green tea extracts interfere with the stress-protective activity of PrP and the formation of PrP

A hallmark in prion diseases is the conformational transition of the cellular prion protein (PrP(C)) into a pathogenic conformation, designated scrapie prion protein (PrP(Sc)), which is the essential constituent of infectious prions. Here, we show that epigallocatechin gallate (EGCG) and gallocatechin gallate, the main polyphenols in green tea, induce the transition of mature PrP(C) into a detergent-insoluble conformation distinct from PrP(Sc). The PrP conformer induced by EGCG was rapidly internalized from the plasma membrane and degraded in lysosomal compartments. Isothermal titration calorimetry studies revealed that EGCG directly interacts with PrP leading to the destabilizing of the native conformation and the formation of random coil structures. This activity was dependent on the gallate side chain and the three hydroxyl groups of the trihydroxyphenyl side chain. In scrapie-infected cells EGCG treatment was beneficial; formation of PrP(Sc) ceased. However, in uninfected cells EGCG interfered with the stress-protective activity of PrP(C). As a consequence, EGCG-treated cells showed enhanced vulnerability to stress conditions. Our study emphasizes the important role of PrP(C) to protect cells from stress and indicate efficient intracellular pathways to degrade non-native conformations of PrP(C).     

PrPc on the road: trafficking of the cellular prion protein

The glycosylphosphatidylinositol (GPI)-anchored cellular prion protein (PrPc) has a fundamental role in prion diseases. Intracellular trafficking of PrPc is important in the generation of protease resistant PrP species but little is known of how endocytosis affects PrPc function. Here, we discuss recent experiments that have illuminated how PrPc is internalized and what are the possible destinations taken by the protein. Contrary to what would be expected for a GPI-anchored protein there is increasing evidence that clathrin-mediated endocytosis and classical endocytic organelles participate in PrPc trafficking. Moreover, the N-terminal domain of PrPc may be involved in sorting events that can direct the protein during its intracellular journey. Indeed, the concept that the GPI-anchor determines PrPc trafficking has been challenged. Cellular signaling can be triggered or be regulated by PrPc and we suggest that endocytosis of PrPc may influence signaling in several ways. Definition of the processes that participate in PrPc endocytosis and intracellular trafficking can have a major impact on our understanding of the mechanisms involved in PrPc function and conversion to protease resistant conformations.     

Sialic Acid on the Glycosylphosphatidylinositol Anchor Regulates PrP-mediated Cell Signaling and Prion Formation

The prion diseases occur following the conversion of the cellular prion protein (PrP^C) into disease-related isoforms (PrP^Sc). In this study, the role of the glycosylphosphatidylinositol (GPI) anchor attached to PrP^C in prion formation was examined using a cell painting technique. PrP^Sc formation in two prion-infected neuronal cell lines (ScGT1 and ScN2a cells) and in scrapie-infected primary cortical neurons was increased following the introduction of PrP^C. In contrast, PrP^C containing a GPI anchor from which the sialic acid had been removed (desialylated PrP^C) was not converted to PrP^Sc. Furthermore, the presence of desialylated PrP^C inhibited the production of PrP^Sc within prion-infected cortical neurons and ScGT1 and ScN2a cells. The membrane rafts surrounding desialylated PrP^C contained greater amounts of sialylated gangliosides and cholesterol than membrane rafts surrounding PrP^C. Desialylated PrP^C was less sensitive to cholesterol depletion than PrP^C and was not released from cells by treatment with glimepiride. The presence of desialylated PrP^C in neurons caused the dissociation of cytoplasmic phospholipase A₂ from PrP-containing membrane rafts and reduced the activation of cytoplasmic phospholipase A₂. These findings show that the sialic acid moiety of the GPI attached to PrP^C modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP^Sc formation. These results suggest that pharmacological modification of GPI glycosylation might constitute a novel therapeutic approach to prion diseases.     

A novel sorting nexin modulates endocytic trafficking and alpha-secretase cleavage of the amyloid precursor protein

Ectodomain shedding of the amyloid precursor protein (APP) by the two proteases alpha- and beta-secretase is a key regulatory event in the generation of the Alzheimer disease amyloid beta peptide (Abeta). beta-Secretase catalyzes the first step in Abeta generation, whereas alpha-secretase cleaves within the Abeta domain, prevents Abeta generation, and generates a secreted form of APP with neuroprotective properties. At present, little is known about the cellular mechanisms that control APP alpha-secretase cleavage and Abeta generation. To explore the contributory pathways, we carried out an expression cloning screen. We identified a novel member of the sorting nexin (SNX) family of endosomal trafficking proteins, called SNX33, as a new activator of APP alpha-secretase cleavage. SNX33 is a homolog of SNX9 and was found to be a ubiquitously expressed phosphoprotein. Exogenous expression of SNX33 in cultured cells increased APP alpha-secretase cleavage 4-fold but surprisingly had little effect on beta-secretase cleavage. This effect was similar to the expression of the dominant negative dynamin-1 mutant K44A. SNX33 bound the endocytic GTPase dynamin and reduced the rate of APP endocytosis in a dynamin-dependent manner. This led to an increase of APP at the plasma membrane, where alpha-secretase cleavage mostly occurs. In summary, our study identifies SNX33 as a new endocytic protein, which modulates APP endocytosis and APP alpha-secretase cleavage, and demonstrates that the rate of APP endocytosis is a major control factor for APP alpha-secretase cleavage.     

Does the tail wag the dog? How the structure of a glycosylphosphatidylinositol anchor affects prion formation

There is increasing interest in the role of the glycosylphosphatidylinositol (GPI) anchor attached to the cellular prion protein (PrP^C). Since GPI anchors can alter protein targeting, trafficking and cell signaling, our recent study examined how the structure of the GPI anchor affected prion formation. PrP^C containing a GPI anchor from which the sialic acid had been removed (desialylated PrP^C) was not converted to PrP^Sc in prion-infected neuronal cell lines and in scrapie-infected primary cortical neurons. In uninfected neurons desialylated PrP^C was associated with greater concentrations of gangliosides and cholesterol than PrP^C. In addition, the targeting of desialylated PrP^C to lipid rafts showed greater resistance to cholesterol depletion than PrP^C. The presence of desialylated PrP^C caused the dissociation of cytoplasmic phospholipase A₂ (cPLA₂) from PrP-containing lipid rafts, reduced the activation of cPLA₂ and inhibited PrP^Sc production. We conclude that the sialic acid moiety of the GPI attached to PrP^C modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP^Sc formation.