Murine guanylate-binding protein: incomplete geranylgeranyl isoprenoid modification of an interferon-gamma-inducible guanosine triphosphate-binding protein

Farnesylation of Ras proteins is necessary for transforming activity. Although farnesyl transferase inhibitors show promise as anticancer agents, prenylation of the most commonly mutated Ras isoform, K-Ras4B, is difficult to prevent because K-Ras4B can be alternatively modified with geranylgeranyl (C20). Little is known of the mechanisms that produce incomplete or inappropriate prenylation. Among non-Ras proteins with CaaX motifs, murine guanylate-binding protein (mGBP1) was conspicuous for its unusually low incorporation of [(3)H]mevalonate. Possible problems in cellular isoprenoid metabolism or prenyl transferase activity were investigated, but none that caused this defect was identified, implying that the poor labeling actually represented incomplete prenylation of mGBP1 itself. Mutagenesis indicated that the last 18 residues of mGBP1 severely limited C20 incorporation but, surprisingly, were compatible with farnesyl modification. Features leading to the expression of mutant GBPs with partial isoprenoid modification were identified. The results demonstrate that it is possible to alter a protein's prenylation state in a living cell so that graded effects of isoprenoid on function can be studied. The C20-selective impairment in prenylation also identifies mGBP1 as an important model for the study of substrate/geranylgeranyl transferase I interactions.     

Protein farnesyl and geranylgeranyl transferases]

Posttranslational prenylation of proteins synthesized as soluble precursors enhances their hydrophobicity and enables them to bind biological membranes. These modifications consist in the attachment of a C15 farnesyl or a C20 geranylgeranyl moiety to the cysteine residue(s) of proteins bearing CAAX, CC or CXC C-terminal sequences (where C = cysteine, A = aliphatic residue and X = any amino-acid), such as proteins of the ras superfamily, gamma subunits of heterotrimetric G proteins, lamin B as well as yeast mating factor a. A farnesyl transferase (FTase) and two distinct geranylgeranyl transferases (GGTases I and II) have been recently identified. FTase and GGTase I modify proteins containing a C-terminal CAAX motif; such a sequence is necessary and sufficient for recognition by the enzymes. The nature of the fourth residue determines the nature of the modification: when X is a serine, a methionine or a phenylalanine, the protein is farnesylated, whereas the presence of a leucine residue results in the attachment of a geranylgeranyl group. Both these enzymes are alpha beta heterodimers; their purification, molecular cloning of their coding sequences as well as mutational studies in yeast have shown that they share a common alpha subunit, and that their beta subunits exhibit a significant level of sequence similarity. GGTase II modifies ras-related proteins exhibiting CC and CXC C-terminal sequences; the enzyme as well as its recognition motif are yet largely uncharacterized.     

Protein isoprenylation in suspension-cultured tobacco cells.

Many mammalian and yeast proteins, including small ras-like GTP binding proteins, heterotrimeric G protein gamma subunits, and nuclear lamins, have been shown to be covalently linked to isoprenoid derivatives of mevalonic acid. Isoprenylation of these proteins is required for their assembly into membranes and, hence, for their biological activity. In this report, it is shown that cultured tobacco cells, when pretreated with an inhibitor of endogenous mevalonic acid synthesis (lovastatin), incorporate radioactivity from 14C-mevalonic acid into proteins. Most of these proteins are membrane associated, and many are similar in mass to mammalian ras-like GTP binding proteins and nuclear lamins. Furthermore, it is shown that tobacco cell extracts catalyze the transfer of radioactivity from 3H-farnesyl pyrophosphate and 3H-geranylgeranyl pyrophosphate to protein substrates in vitro. These studies indicate the presence of at least two distinct prenyl:protein transferases in tobacco extracts: one that utilizes farnesyl pyrophosphate and preferentially modifies a substrate protein with a CAIM carboxy terminus (farnesyl:protein transferase) and one that utilizes geranylgeranyl pyrophosphate and preferentially modifies a substrate protein with a CAIL carboxy terminus (geranylgeranyl:protein transferase type I). This work provides a basis for future work on the role of protein isoprenylation in plant cell growth, signal transduction, and membrane biogenesis.     

Changes in Protein Isoprenylation during the Growth of Suspension-Cultured Tobacco Cells

Isoprenylation facilitates the association of proteins with intracellular membranes and/or other proteins. In mammalian and yeast cells, isoprenylated proteins are involved in signal transduction, cell division, organization of the cytoskeleton, and vesicular transport. Recently, protein isoprenylation has been demonstrated in higher plants, but little is currently known about the functions of isoprenylated plant proteins. We report that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (lovastatin) or prenyl:protein transferases (perilly alcohol) severely impair the growth of cultured tobacco (Nicotiana tabacum) cells but only when added within the first 2 d following transfer to fresh medium, before any increase in culture volume is detectable. This "window" of sensitivity to inhibitors of protein isoprenylation correlates temporally with an increase in [14C]mevalonate incorporation into tobacco cell proteins in vitro. We have also observed a marked increase in farnesyl:protein transferase activity at this early time in the growth of tobacco cultures. In contrast, type I geranylgeranyl:protein transferase activity does not change significantly during culture growth. Although these events coincide with the replication of DNA, I [mu]M lovastatin-treated cells are capable of DNA synthesis, suggesting that lovastatin-induced cell growth arrest is not due to inhibition of DNA replication. Together, these data support the hypothesis that protein isoprenylation is necessary for the early stages of growth of tobacco cultures.     

Structures of RabGGTase-substrate/product complexes provide insights into the evolution of protein prenylation

Post-translational isoprenylation of proteins is carried out by three related enzymes: farnesyltransferase, geranylgeranyl transferase-I, and Rab geranylgeranyl transferase (RabGGTase). Despite the fact that the last one is responsible for the largest number of individual protein prenylation events in the cell, no structural information is available on its interaction with substrates and products. Here, we present structural and biophysical analyses of RabGGTase in complex with phosphoisoprenoids as well as with the prenylated peptides that mimic the C terminus of Rab7 GTPase. The data demonstrate that, unlike other protein prenyl transferases, both RabGGTase and its substrate RabGTPases completely 'outsource' their specificity for each other to an accessory subunit, the Rab escort protein (REP). REP mediates the placement of the C terminus of RabGTPase into the active site of RabGGTase through a series protein-protein interactions of decreasing strength and selectivity. This arrangement enables RabGGTase to prenylate any cysteine-containing sequence. On the basis of our structural and thermodynamic data, we propose that RabGGTase has evolved from a GGTase-I-like molecule that 'learned' to interact with a recycling factor (GDI) that, in turn, eventually gave rise to REP.     

Thematic review series: lipid posttranslational modifications. geranylgeranylation of Rab GTPases

Rab GTPases require special machinery for protein prenylation, which include Rab escort protein (REP) and Rab geranylgeranyl transferase (RGGT). The current model of Rab geranylgeranylation proposes that REP binds Rab and presents it to RGGT. After geranylgeranylation of Rab C-terminal cysteines, REP delivers the prenylated protein to membranes. The REP-like protein Rab GDP dissociation inhibitor (RabGDI) then recycles the prenylated Rab between the membrane and the cytosol. The recent solution of crystal structures of the Rab prenylation machinery has helped to refine this model and provided further insights. The hydrophobic prenyl binding pocket of RGGT and geranylgeranyl transferase type-I (GGT-I) differs from that of farnesyl transferase (FT). A bulky tryptophan residue in FT restricts the size of the pocket, whereas in RGGT and GGT-I, this position is occupied by smaller residues. A highly conserved phenylalanine in REP, which is absent in RabGDI, is critical for the formation of the REP:RGGT complex. Finally, a geranylgeranyl binding site conserved in REP and RabGDI has been identified within helical domain II. The postprenylation events, including the specific targeting of Rabs to target membranes and the requirement for single versus double geranylgeranylation by different Rabs, remain obscure and should be the subject of future studies.     

A novel statin-mediated “prenylation block-and-release” assay provides insight into the membrane targeting mechanisms of small GTPases

Ras super-family small GTPases regulate diverse cellular processes such as vesicular transport and signal transduction. Critical to these activities is the ability of these proteins to target to specific intracellular membranes. To allow association with membranes Ras-related GTPases are post-translationally modified by covalent attachment of prenyl groups to conserved cysteine residues at or near their C-terminus. Here we used the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGCR) inhibitor mevastatin to develop a ‘prenylation block-and-release’ assay that allows membrane targeting of prenylated proteins to be visualized in living cells. Using this assay we investigated the cytosol to membrane targeting of several small GTPases to compartments of the secretory and endocytic pathways. We found that all Rabs tested were targeted directly to the membrane on which they reside at steady-state and not via an intermediate location as reported for Ras and Rho proteins. However, we observed that the kinetics of cytosol to membrane targeting differed for each Rab tested. Comparison of the mevastatin sensitivity and kinetics of membrane targeting of Rab23, Rab23 prenylation motif mutants and H-Ras revealed that these parameters are strongly dependent upon the prenyl transferase with Rab geranylgeranyl transferase substrates exhibiting higher sensitivity and requiring greater time to recover from mevastatin inhibition than farnesyl transferase substrates. We propose that this assay is a useful tool to investigate the kinetics, biological functions and the mechanisms of membrane targeting of prenylated proteins.     

Functional aspects of polyisoprenoid protein substituents: roles in protein-protein interaction and trafficking

There are now numerous examples of post-translational modification with geranylgeranyl or farnesyl substituents. Once thought of as solely a mechanism for association of proteins with membranes, other functional aspects of protein prenylation have come to be appreciated. Although, in almost all instances, such proteins are membrane associated, they are often found to also engage in protein-protein interactions. In some instances, such interactions are critical aspects of prenylated protein trafficking. In this review, the role of prenylation in mediating protein-protein interactions will be considered. The hypothesis will be developed that such interactions occur through recognition of the prenyl group and a second domain, on the prenylated protein, by a heterodimeric protein partner.     

Protein Prenylation and Synaptic Plasticity: Implications for Alzheimer’s Disease

Protein prenylation is an important lipid posttranslational modification of proteins. It includes protein farnesylation and geranylgeranylation, in which the 15-carbon farnesyl pyrophosphate or 20-carbon geranylgeranyl pyrophosphate is attached to the C-terminus of target proteins, catalyzed by farnesyl transferase or geranylgeranyl transferases, respectively. Protein prenylation facilitates the anchoring of proteins into the cell membrane and mediates protein–protein interactions. Among numerous proteins that undergo prenylation, small GTPases represent the largest group of prenylated proteins. Small GTPases are involved in regulating a plethora of cellular functions including synaptic plasticity. The prenylation status of small GTPases determines the subcellular locations and functions of the proteins. Dysregulation or dysfunction of small GTPases leads to the development of different types of disorders. Emerging evidence indicates that prenylated proteins, in particular small GTPases, may play important roles in the pathogenesis of Alzheimer’s disease. This review focuses on the prenylation of Ras and Rho subfamilies of small GTPases and its relation to synaptic plasticity and Alzheimer’s disease.     

Biochemical and structural studies with prenyl diphosphate analogues provide insights into isoprenoid recognition by protein farnesyl transferase

Protein farnesyl transferase (PFTase) catalyzes the reaction between farnesyl diphosphate and a protein substrate to form a thioether-linked prenylated protein. The fact that many prenylated proteins are involved in signaling processes has generated considerable interest in protein prenyl transferases as possible anticancer targets. While considerable progress has been made in understanding how prenyl transferases distinguish between related target proteins, the rules for isoprenoid discrimination by these enzymes are less well understood. To clarify how PFTase discriminates between FPP and larger prenyl diphosphates, we have examined the interactions between the enzyme and several isoprenoid analogues, GGPP, and the farnesylated peptide product using a combination of biochemical and structural methods. Two photoactive isoprenoid analogues were shown to inhibit yeast PFTase with K(I) values as low as 45 nM. Crystallographic analysis of one of these analogues bound to PFTase reveals that the diphosphate moiety and the two isoprene units bind in the same positions occupied by the corresponding atoms in FPP when bound to PFTase. However, the benzophenone group protrudes into the acceptor protein binding site and prevents the binding of the second (protein) substrate. Crystallographic analysis of geranylgeranyl diphosphate bound to PFTase shows that the terminal two isoprene units and diphosphate group of the molecule map to the corresponding atoms in FPP; however, the first and second isoprene units bulge away from the acceptor protein binding site. Comparison of the GGPP binding mode with the binding of the farnesylated peptide product suggests that the bulkier isoprenoid cannot rearrange to convert to product without unfavorable steric interactions with the acceptor protein. Taken together, these data do not support the "molecular ruler hypotheses". Instead, we propose a "second site exclusion model" in which PFTase binds larger isoprenoids in a fashion that prevents the subsequent productive binding of the acceptor protein or its conversion to product.     

Effects of prenyl pyrophosphates on the binding of PKCgamma with RACK1

Receptors for activated C kinase (RACKs) are a group of PKC binding proteins that have been shown to mediate isoform-selective functions of PKC and to be crucial in the translocation and subsequent functioning of the PKC isoenzymes on activation. RACK1 cDNA from the shrimp Penaeus japonicus was isolated by homology cloning. The hepatopancreas cDNA from this shrimp was found to encode a 318-residue polypeptide whose predicted amino acid sequence shared 91% homology with human G(beta2)-like proteins. Expression of the cDNA of shrimp RACK1 in vitro yielded a 45-kDa polypeptide with positive reactivity toward the monoclonal antibodies against RACK1 of mammals. The shrimp RACK1 was biotinylated and used to compare the effects of geranylgeranyl pyrophosphate and farnesyl pyrophosphate on its binding with PKCgamma in anti-biotin-IgG precipitates. PKCgammas were isolated from shrimp eyes and mouse brains. Both enzyme preparations were able to inhibit taxol-induced tubulin polymerization. Interestingly, when either geranylgeranyl pyrophosphate or farnesyl pyrophosphate was reduced to the submicrogram level, the recruitment activity of RACK1 with purified PKCgamma was found to increase dramatically. The activation is especially significant for RACK1 and PKCgamma from different species. The observation implies that the deprivation of prenyl pyrophosphate might function as a signal for RACK1 to switch the binding from the conventional isoenzymes of PKC (cPKC) to the novel isoenzymes of PKC (nPKC). A hydrophobic binding pocket for geranylgeranyl pyrophosphate in RACK1 is further revealed via prenylation with protein geranylgeranyl transferase I of shrimp P. japonicus.     

Inhibition of geranylgeranylation mediates sensitivity to CHOP-induced cell death of DLBCL cell lines

Prenylation is a post-translational hydrophobic modification of proteins, important for their membrane localization and biological function. The use of inhibitors of prenylation has proven to be a useful tool in the activation of apoptotic pathways in tumor cell lines. Rab geranylgeranyl transferase (Rab GGT) is responsible for the prenylation of the Rab family. Overexpression of Rab GGTbeta has been identified in CHOP refractory diffuse large B cell lymphoma (DLBCL). Using a cell line-based model for CHOP resistant DLBCL, we show that treatment with simvastatin, which inhibits protein farnesylation and geranylgeranylation, sensitizes DLBCL cells to cytotoxic treatment. Treatment with the farnesyl transferase inhibitor FTI-277 or the geranylgeranyl transferase I inhibitor GGTI-298 indicates that the reduction in cell viability was restricted to inhibition of geranylgeranylation. In addition, treatment with BMS1, a combined inhibitor of farnesyl transferase and Rab GGT, resulted in a high cytostatic effect in WSU-NHL cells, demonstrated by reduced cell viability and decreased proliferation. Co-treatment of BMS1 or GGTI-298 with CHOP showed synergistic effects with regard to markers of apoptosis. We propose that inhibition of protein geranylgeranylation together with conventional cytostatic therapy is a potential novel strategy for treating patients with CHOP refractory DLBCL.     

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : The Purification and Properties of Farnesyl Transferase from Elicited Seedlings

Farnesyl transferase (farnesyl pyrophosphate: isopentenyl pyrophosphate farnesyl transferase; geranylgeranyl pyrophosphate synthetase) was purified at least 400-fold from extracts of castor bean (Ricinus communis L.) seedlings that were elicited by exposure for 10 h to Rhizopus stolonifer spores. The purified enzyme was free of isopentenyl pyrophosphate isomerase and phosphatase activities which interfere with prenyl transferase assays. The purified enzyme showed a broad optimum for farnesyl transfer between pH 8 and 9. The molecular weight of the enzyme was estimated to be 72,000 ± 3,000 from its behavior on a calibrated G-100 Sephadex molecular sieving column. Mg²⁺ ion at 4 millimolar gave the greatest stimulation of activity; Mn²⁺ ion gave a small stimulation at 0.5 millimolar, but was inhibitory at higher concentrations. Farnesyl pyrophosphate (K_m = 0.5 micromolar) in combination with isopentenyl pyrophosphate (K_m = 3.5 micromolar) was the most effective substrate for the production of geranylgeranyl pyrophosphate. Geranyl pyrophosphate (K_m = 24 micromolar) could replace farnesyl pyrophosphate as the allylic pyrophosphate substrate, but dimethylallyl pyrophosphate was not utilized by the enzyme. One peak of farnesyl transferase activity (geranylgeranyl pyrophosphate synthetase) and two peaks of geranyl transferase activity (farnesyl pyrophosphate synthetases) from extracts of whole elicited seedlings were resolved by DEAE A-25 Sephadex sievorptive ion exchange chromatography. These results suggest that the pathway for geranylgeranyl pyrophosphate synthesis in elicited castor bean seedlings involves the successive actions of two enzymes—a geranyl transferase which utilizes dimethylallypyrophosphate and isopentenyl pyrophosphate as substrates and a farnesyl transferase which utilizes the farnesyl pyrophosphate produced in the first step and isopentenyl pyrophosphate as substrates.     

Molecular and structural basis of metabolic diversity mediated by prenyldiphosphate converting enzymes

General thermodynamic calculations using the semiempiric PM3 method have led to the conclusion that prenyldiphosphate converting enzymes require at least one divalent metal cation for the activation and cleavage of the diphosphate–prenyl ester bond, or they must provide structural elements for the efficient stabilization of the intermediate prenyl cation. The most important common structural features, which guide the product specificity in both terpene synthases and aromatic prenyl transferases are aromatic amino acid side chains, which stabilize prenyl cations by cation–π interactions. In the case of aromatic prenyl transferases, a proton abstraction from the phenolic hydroxyl group of the second substrate will enhance the electron density in the phenolic ortho-position at which initial prenylation of the aromatic compound usually occurs.A model of the structure of the integral transmembrane-bound aromatic prenyl transferase UbiA was developed, which currently represents the first structural insight into this group of prenylating enzymes with a fold different from most other aromatic prenyl transferases. Based on this model, the structure–activity relationships and mechanistic aspects of related proteins, for example those of Lithospermum erythrorhizon or the enzyme AuaA from Stigmatella aurantiaca involved in the aurachin biosynthesis, were elucidated. The high similarity of this group of aromatic prenyltransferases to 5-epi-aristolochene synthase is an indication of an evolutionary relationship with terpene synthases (cyclases). This is further supported by the conserved DxxxD motif found in both protein families. In contrast, there is no such relationship to the aromatic prenyl transferases with an ABBA-fold, such as NphB, or to any other known family of prenyl converting enzymes. Therefore, it is possible that these two groups might have different evolutionary ancestors.     

Novel benzimidazole phosphonates as potential inhibitors of protein prenylation

Benzimidazole carboxyphosphonates and bisphosphonates have been prepared and evaluated for their activity as inhibitors of protein prenylation or isoprenoid biosynthesis. The nature of the phosphonate head group was found to dictate enzyme specificity. The lead carboxyphosphonate inhibits geranylgeranyl transferase II while its corresponding bisphosphonate analogue potently inhibits farnesyl diphosphate synthase. The most active inhibitors effectively disrupted protein prenylation in human multiple myeloma cells.     

Role of isoprenoid lipids on the heterotrimeric G protein gamma subunit in determining effector activation.

Post-translational prenylation of heterotrimeric G protein gamma subunits is essential for high affinity alpha-beta gamma and alpha-beta gamma-receptor interactions, suggesting that the prenyl group is an important domain in the beta gamma dimer. To determine the role of the prenyl modification in the interaction of beta gamma dimers with effectors, the CAAX (where A indicates alipathic amino acid) motifs in the gamma1, gamma2, and gamma11 subunits were altered to direct modification with different prenyl groups. Six recombinant beta gamma dimers were overexpressed in baculovirus-infected Sf9 insect cells, purified, and examined for their ability to stimulate three phospholipase C-beta isozymes and type II adenylyl cyclase. The native beta1 gamma2 dimer (gamma subunit modified with geranylgeranyl) is more potent and effective in activating phospholipase C-beta than either the beta1 gamma1 (farnesyl) or the beta1 gamma11 (farnesyl) dimers. However, farnesyl modification of the gamma subunit in the beta1 gamma2 dimer (beta1 gamma2-L71S) caused a decrement in its ability to activate phospholipase C-beta. In contrast, both the beta1 gamma1-S74L (geranylgeranyl) and the beta1 gamma11-S73L (geranylgeranyl) dimers were more active than the native forms. The beta1 gamma2 dimer activates type II adenylyl cyclase about 12-fold; however, neither the beta1 gamma1 nor the beta1 gamma11 dimers activate the enzyme. As was the case with phospholipase C-beta, the beta1gamma2-L71S dimer was less able to activate adenylyl cyclase than the native beta1 gamma2 dimer. Interestingly, neither the beta1 gamma1-S74L nor the beta1 gamma11-S73L dimers stimulated adenylyl cyclase. The results suggest that both the amino acid sequence of the gamma subunit and its prenyl group play a role in determining the activity of the beta gamma-effector complex.     

Prenylation of Rab GTPases: molecular mechanisms and involvement in genetic disease.

Small GTPases of the Rab family regulate membrane transport pathways. More than 50 mammalian Rab proteins are known, many with transport step-specific localisation. Rabs must associate with cellular membranes for activity and membrane attachment is mediated by prenyl (geranylgeranyl) post-translational modification. Mutations in genes encoding proteins essential for the geranylgeranylation reaction, Rab escort protein and Rab geranylgeranyl transferase, underlie genetic diseases. Choroideremia patients have loss of function mutations in REP1 and the murine Hermansky-Pudlak syndrome model gunmetal possesses a splice-site mutation in the alpha-subunit of RGGT. Here we discuss recent insights into Rab prenylation and advances towards our understanding of both diseases.     

Isoprenoids and protein prenylation: implications in the pathogenesis and therapeutic intervention of Alzheimer’s disease

The mevalonate–isoprenoid–cholesterol biosynthesis pathway plays a key role in human health and disease. The importance of this pathway is underscored by the discovery that two major isoprenoids, farnesyl and geranylgeranyl pyrophosphate, are required to modify an array of proteins through a process known as protein prenylation, catalyzed by prenyltransferases. The lipophilic prenyl group facilitates the anchoring of proteins in cell membranes, mediating protein–protein interactions and signal transduction. Numerous essential intracellular proteins undergo prenylation, including most members of the small GTPase superfamily as well as heterotrimeric G proteins and nuclear lamins, and are involved in regulating a plethora of cellular processes and functions. Dysregulation of isoprenoids and protein prenylation is implicated in various disorders, including cardiovascular and cerebrovascular diseases, cancers, bone diseases, infectious diseases, progeria, and neurodegenerative diseases including Alzheimer’s disease (AD). Therefore, isoprenoids and/or prenyltransferases have emerged as attractive targets for developing therapeutic agents. Here, we provide a general overview of isoprenoid synthesis, the process of protein prenylation and the complexity of prenylated proteins, and pharmacological agents that regulate isoprenoids and protein prenylation. Recent findings that connect isoprenoids/protein prenylation with AD are summarized and potential applications of new prenylomic technologies for uncovering the role of prenylated proteins in the pathogenesis of AD are discussed.     

Therapeutic intervention based on protein prenylation and associated modifications

In eukaryotic cells, a specific set of proteins are modified by C-terminal attachment of 15-carbon farnesyl groups or 20-carbon geranylgeranyl groups that function both as anchors for fixing proteins to membranes and as molecular handles for facilitating binding of these lipidated proteins to other proteins. Additional modification of these prenylated proteins includes C-terminal proteolysis and methylation, and attachment of a 16-carbon palmitoyl group; these modifications augment membrane anchoring and alter the dynamics of movement of proteins between different cellular membrane compartments. The enzymes in the protein prenylation pathway have been isolated and characterized. Blocking protein prenylation is proving to be therapeutically useful for the treatment of certain cancers, infection by protozoan parasites and the rare genetic disease Hutchinson-Gilford progeria syndrome.