Neuroprotective Secreted Amyloid Precursor Protein Acts by Disrupting Amyloid Precursor Protein Dimers

The amyloid precursor protein (APP) is implied both in cell growth and differentiation and in neurodegenerative processes in Alzheimer disease. Regulated proteolysis of APP generates biologically active fragments such as the neuroprotective secreted ectodomain sAPPα and the neurotoxic β-amyloid peptide. Furthermore, it has been suggested that the intact transmembrane APP plays a signaling role, which might be important for both normal synaptic plasticity and neuronal dysfunction in dementia. To understand APP signaling, we tracked single molecules of APP using quantum dots and quantitated APP homodimerization using fluorescence lifetime imaging microscopy for the detection of Förster resonance energy transfer in living neuroblastoma cells. Using selective labeling with synthetic fluorophores, we show that the dimerization of APP is considerably higher at the plasma membrane than in intracellular membranes. Heparan sulfate significantly contributes to the almost complete dimerization of APP at the plasma membrane. Importantly, this technique for the first time structurally defines the initiation of APP signaling by binding of a relevant physiological extracellular ligand; our results indicate APP as receptor for neuroprotective sAPPα, as sAPPα binding disrupts APP dimers, and this disruption of APP dimers by sAPPα is necessary for the protection of neuroblastoma cells against starvation-induced cell death. Only cells expressing reversibly dimerized wild-type, but not covalently dimerized mutant APP are protected by sAPPα. These findings suggest a potentially beneficial effect of increasing sAPPα production or disrupting APP dimers for neuronal survival.The amyloid precursor protein (APP)⁴ is known both for its important role in the development and plasticity of the nervous system (1–6) and for its involvement in Alzheimer disease (AD) (7, 8). Despite intensive research efforts, the initial events that lead to the prevalent sporadic, i.e. non-familial, forms of AD are still unclear. Furthermore, although a higher gene dose of APP (9) or the presence of pathological APP mutations is sufficient to induce familial AD (for review, see Ref. 10), the exact pathological mechanism that is triggered by APP is still under debate.Some fragments of APP, such as the β-amyloid peptide (Aβ), are thought to contribute to synaptic dysfunction and neurotoxicity (11, 12). On the other hand, the α-secretase-derived extracellular fragment of APP (sAPPα), which is present at lower levels in AD patients than in controls (13), has been shown to be beneficial for memory function, to possess neuroprotective properties, and to counteract the effects of Aβ (14–18).Signaling by transmembrane APP may directly contribute to neurodegeneration in AD (19–24); however, the signal transduction pathway for transmembrane APP remains unknown, although several potential regulatory proteins, glycosaminoglycans, and metal ions are known to bind with high affinity to APP and sAPPα (25, 26). The most common form of signal transduction for single-pass transmembrane proteins is the ligand-induced perturbation of a monomer/dimer equilibrium. Indeed, the dimerization of transmembrane APP has been implied several times in the past. Several studies have investigated the effects of presumed dimer-breaking perturbations on biological read-outs, such as the production of Aβ (27, 28), but without directly measuring the APP aggregation state, or have investigated the aggregation state of APP subdomains, often reconstituted in cell-free systems (27–32). Dimerization interfaces in both the extracellular and the transmembrane domain have been suggested.In the studies investigating the aggregation state of full-length APP, most of the employed methods, such as chemical cross-linking and co-immunoprecipitation, do not lend themselves readily to a rigorous quantitative analysis of the abundance of potentially instable dimers (31, 33), whereas in other cases the use of chimeras may have influenced the dimerization potential or precluded the search for a natural stimulus (23, 34). The only previously reported direct observation of APP dimerization by Förster resonance energy transfer (FRET) microscopy uses an assay in which the FRET efficiency varies with the level of overexpression (35). Therefore, a concentration-dependent FRET component due to nonspecific stochastic encounters cannot be excluded in this study.Most importantly, as none of the published procedures permitted the selective detection of APP dimers on the surface of live cells, where they would encounter ligands, they could not differentiate between subpopulations of APP. This may be one reason why no natural ligand of APP has ever been shown to signal via modulation of its monomer/dimer equilibrium.Another elusive goal is the identity of the receptor for neuroprotective sAPPα (36–39). The ligand-dependent dimerization of sAPPα in solution (40) and its origination from transmembrane APP suggest that APP might serve as receptor for sAPPα, but this binding has never been experimentally shown.     

The Structure of an Interdomain Complex That Regulates Talin Activity

Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. It exists in both globular and extended conformations, and an intramolecular interaction between the N-terminal F3 FERM subdomain and the C-terminal part of the talin rod contributes to an autoinhibited form of the molecule. Here, we report the solution structure of the primary F3 binding domain within the C-terminal region of the talin rod and use intermolecular nuclear Overhauser effects to determine the structure of the complex. The rod domain (residues 1655–1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3 (residues 316–326) interacts with a cluster of acidic residues in the middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain competes with β3-integrin tails for binding to F3, and the structure of the complex suggests that the rod is also likely to sterically inhibit binding of the FERM domain to the membrane.The cytoskeletal protein talin has emerged as a key player, both in regulating the affinity of the integrin family of cell adhesion molecules for ligand (1) and in coupling integrins to the actin cytoskeleton (2). Thus, depletion of talin results in defects in integrin activation (3), integrin signaling through focal adhesion kinase, the maintenance of cell spreading, and the assembly of focal adhesions in cultured cells (4). In the whole organism, studies on the single talin gene in worms (5) and flies (6) show that talin is essential for a variety of integrin-mediated events that are crucial for normal embryonic development. In vertebrates, there are two talin genes, and mice carrying a talin1 null allele fail to complete gastrulation (7). Tissue-specific inactivation of talin1 results in an inability to activate integrins in platelets (8, 9), defects in the membrane-cytoskeletal interface in megakaryocytes (10), and disruption of the myotendinous junction in skeletal muscle (11). In contrast, mice homozygous for a talin2 gene trap allele have no phenotype, although the allele may be hypomorphic (12).Recent structural studies have provided substantial insights into the molecular basis of talin action. Talin is composed of an N-terminal globular head (∼50 kDa) linked to an extended flexible rod (∼220 kDa). The talin head contains a FERM² domain (made up of F1, F2, and F3 subdomains) preceded by a domain referred to here as F0 (2). Studies by Wegener et al. (30) have shown how the F3 FERM subdomain, which has a phosphotyrosine binding domain fold, interacts with both the canonical NPXY motif and the membrane-proximal helical region of the cytoplasmic tails of integrin β-subunits (13). The latter interaction apparently activates the integrin by disrupting the salt bridge between the integrin α- and β-subunit tails that normally keeps integrins locked in a low affinity state. The observation that the F0 region is also important in integrin activation (14) may be explained by our recent finding that F0 binds, albeit with low affinity, Rap1-GTP,³ a known activator of integrins (15, 16). The talin rod is made up of a series of amphipathic α-helical bundles (17–20) and contains a second integrin binding site (IBS2) (21), numerous binding sites for the cytoskeletal protein vinculin (22), at least two actin binding sites (23), and a C-terminal helix that is required for assembly of talin dimers (20, 24).Both biochemical (25) and cellular studies (16) suggest that the integrin binding sites in full-length talin are masked, and both phosphatidylinositol 4,5-bisphosphate (PIP2) and Rap1 have been implicated in exposing these sites. It is well established that some members of the FERM domain family of proteins are regulated by a head-tail interaction (26); gel filtration, sedimentation velocity, and electron microscopy studies all show that talin is globular in low salt buffers, although it is more elongated (∼60 nm in length) in high salt (27). By contrast, the talin rod liberated from full-length talin by calpain-II cleavage is elongated in both buffers, indicating that the head is required for talin to adopt a more compact state. Direct evidence for an interaction between the talin head and rod has recently emerged from NMR studies by Goksoy et al. (28), who demonstrated binding of ¹⁵N-labeled talin F3 to a talin rod fragment spanning residues 1654–2344, an interaction that was confirmed by surface plasmon resonance (K_d = 0.57 μm) (28). Chemical shift data also showed that this segment of the talin rod partially masked the binding site in F3 for the membraneproximal helix of the β3-integrin tail (28), directly implicating the talin head-rod interaction in regulating the integrin binding activity of talin. Goksoy et al. (28) subdivided the F3 binding site in this rod fragment into two sites with higher affinity (K_d ∼3.6 μm; residues 1654–1848) and lower affinity (K_d ∼78 μm; residues 1984–2344). Here, we define the rod domain boundaries and determine the NMR structure of residues 1655–1822, a five-helix bundle. We further show that this domain binds F3 predominantly via surface-exposed residues on helix 4, with an affinity similar to the high affinity site reported by Goksoy et al. (28). We also report the structure of the complex between F3 and the rod domain and show that the latter masks the known binding site in F3 for the β3-integrin tail and is expected to inhibit the association of the talin FERM domain with the membrane.     

RIAM Activates Integrins by Linking Talin to Ras GTPase Membrane-targeting Sequences

Rap1 small GTPases interact with Rap1-GTP-interacting adaptor molecule (RIAM), a member of the MRL (Mig-10/RIAM/Lamellipodin) protein family, to promote talin-dependent integrin activation. Here, we show that MRL proteins function as scaffolds that connect the membrane targeting sequences in Ras GTPases to talin, thereby recruiting talin to the plasma membrane and activating integrins. The MRL proteins bound directly to talin via short, N-terminal sequences predicted to form amphipathic helices. RIAM-induced integrin activation required both its capacity to bind to Rap1 and to talin. Moreover, we constructed a minimized 50-residue Rap-RIAM module containing the talin binding site of RIAM joined to the membrane-targeting sequence of Rap1A. This minimized Rap-RIAM module was sufficient to target talin to the plasma membrane and to mediate integrin activation, even in the absence of Rap1 activity. We identified a short talin binding sequence in Lamellipodin (Lpd), another MRL protein; talin binding Lpd sequence joined to a Rap1 membrane-targeting sequence is sufficient to recruit talin and activate integrins. These data establish the mechanism whereby MRL proteins interact with both talin and Ras GTPases to activate integrins.Increased affinity (“activation”) of cellular integrins is central to physiological events such as cell migration, assembly of the extracellular matrix, the immune response, and hemostasis (1). Each integrin comprises a type I transmembrane α and β subunit, each of which has a large extracellular domain, a single transmembrane domain, and a cytoplasmic domain (tail). Talin binds to most integrin β cytoplasmic domains and the binding of talin to the integrin β tail initiates integrin activation (2–4). A small, PTB-like domain of talin mediates activation via a two-site interaction with integrin β tails (5), and this PTB domain is functionally masked in the intact talin molecule (6). A central question in integrin biology is how the talin-integrin interaction is regulated to control integrin activation; recent work has implicated Ras GTPases as critical signaling modules in this process (7).Ras proteins are small monomeric GTPases that cycle between the GTP-bound active form and the GDP-bound inactive form. Guanine nucleotide exchange factors (GEFs) promote Ras activity by exchanging bound GDP for GTP, whereas GTPase-activating proteins (GAPs)³ enhance the hydrolysis of Ras-bound GTP to GDP (for review, see Ref. 8). The Ras subfamily members Rap1A and Rap1B stimulate integrin activation (9, 10). For example, expression of constitutively active Rap1 activates integrin αMβ2 in macrophage, and inhibition of Rap1 abrogated integrin activation induced by inflammatory agonists (11–13). Murine T-cells expressing constitutively active Rap1 manifest enhanced integrin dependent cell adhesion (14). In platelets, Rap1 is rapidly activated by platelet agonists (15, 16). A knock-out of Rap1B (17) or of the Rap1GEF, RasGRP2 (18), resulted in impairment of αIIbβ3-dependent platelet aggregation, highlighting the importance of Rap1 in platelet aggregation in vivo. Thus, Rap1 GTPases play important roles in the activation of several integrins in multiple biological contexts.Several Rap1 effectors have been implicated in integrin activation (19–21). Rap1-GTP-interacting adaptor molecule (RIAM) is a Rap1 effector that is a member of the MRL (Mig-10/RIAM/Lamellipodin) family of adaptor proteins (20). RIAM contains Ras association (RA) and pleckstrin homology (PH) domains and proline-rich regions, which are defining features of the MRL protein family. In Jurkat cells, RIAM overexpression induces β1 and β2 integrin-mediated cell adhesion, and RIAM knockdown abolishes Rap1-dependent cell adhesion (20), indicating RIAM is a downstream regulator of Rap1-dependent signaling. RIAM regulates actin dynamics as RIAM expression induces cell spreading; conversely, its depletion reduces cellular F-actin content (20). Whereas RIAM is greatly enriched in hematopoietic cells, Lamellipodin (Lpd) is a paralogue present in fibroblasts and other somatic cells (22).Recently we used forward, reverse, and synthetic genetics to engineer and order an integrin activation pathway in Chinese hamster ovary cells expressing a prototype activable integrin, platelet αIIbβ3. We found that Rap1 induced formation of an “integrin activation complex” containing RIAM and talin (23). Here, we have established the mechanism whereby Ras GTPases cooperate with MRL family proteins, RIAM and Lpd, to regulate integrin activation. We find that MRL proteins function as scaffolds that connect the membrane targeting sequences in Ras GTPases to talin, thereby recruiting talin to integrins at the plasma membrane.     

Disulfide Bond Formation Significantly Accelerates the Assembly of Ure2p Fibrils because of the Proximity of a Potential Amyloid Stretch

Aggregation of the Ure2 protein is at the origin of the [URE3] prion trait in the yeast Saccharomyces cerevisiae. The N-terminal region of Ure2p is necessary and sufficient to induce the [URE3] phenotype in vivo and to polymerize into amyloid-like fibrils in vitro. However, as the N-terminal region is poorly ordered in the native state, making it difficult to detect structural changes in this region by spectroscopic methods, detailed information about the fibril assembly process is therefore lacking. Short fibril-forming peptide regions (4–7 residues) have been identified in a number of prion and other amyloid-related proteins, but such short regions have not yet been identified in Ure2p. In this study, we identify a unique cysteine mutant (R17C) that can greatly accelerate the fibril assembly kinetics of Ure2p under oxidizing conditions. We found that the segment QVNI, corresponding to residues 18–21 in Ure2p, plays a critical role in the fast assembly properties of R17C, suggesting that this segment represents a potential amyloid-forming region. A series of peptides containing the QVNI segment were found to form fibrils in vitro. Furthermore, the peptide fibrils could seed fibril formation for wild-type Ure2p. Preceding the QVNI segment with a cysteine or a hydrophobic residue, instead of a charged residue, caused the rate of assembly into fibrils to increase greatly for both peptides and full-length Ure2p. Our results indicate that the potential amyloid stretch and its preceding residue can modulate the fibril assembly of Ure2p to control the initiation of prion formation.The [URE3] phenotype of Saccharomyces cerevisiae arises because of conversion of the Ure2 protein to an aggregated propagatable prion state (1, 2). Ure2p contains two regions: a poorly structured N-terminal region and a compactly folded C-terminal region (3, 4). The N-terminal region is rich in Asn and Gln residues, is highly flexible, and is without any detectable ordered secondary structure (4–6). This region is necessary and sufficient for prion behavior in vivo (2) and amyloid-forming capacity in vitro (5, 7), so it is referred to as the prion domain (PrD).² The C-terminal region has a fold similar to the glutathione S-transferase superfamily (8, 9) and possesses glutathione-dependent peroxidase activity (10). Upon fibril formation, the N-terminal region undergoes a significant conformational change from an unfolded to a thermally resistant conformation (11), whereas the glutathione S-transferase-like C-terminal domain retains its enzymatic activity, suggesting that little conformational change occurs (10, 12). Ure2p fibrils show various morphologies, including variations in thickness and the presence or absence of a periodic twist (13–16). The overall structure of the fibrils imaged by cryoelectron microscopy suggests that the intact fibrils contain a 4-nm amyloid filament backbone surrounded by C-terminal globular domains (17).It is widely accepted that disulfide bonds play a critical role in maintaining protein stability (18–21) and also affect the process of protein folding by influencing the folding pathway (22–25). A recent study shows that the presence of a disulfide bond in a protein can markedly accelerate the folding process (26). Therefore, a disulfide bond is a useful tool to study protein folding. In the study of prion and other amyloid-related proteins, cysteine scanning has been widely used to study the structure of amyloid fibrils, the driving force of amyloid formation, and the plasticity of amyloid fibrils (13, 27–31).Short segments from amyloid-related proteins, including IAPP (islet amyloid polypeptide), β₂-microglobulin, insulin, and the amyloid-β peptide, show amyloid-forming capacity (32–34). Hence, the amyloid stretch hypothesis has been proposed, which suggests that a short amino acid stretch bearing a highly amyloidogenic motif might supply most of the driving force needed to trigger the self-catalytic assembly process of a protein to form fibrils (35, 36). In support of this hypothesis, it was found that the insertion of an amyloidogenic stretch into a non-amyloid-related protein can trigger the amyloidosis of the protein (36). At the same time, the structural information obtained from microcrystals formed by amyloidogenic stretches and bearing cross-β-structure has contributed significantly to our understanding of the structure of intact fibrils at the atomic level (34, 37). However, no amyloidogenic stretches <10 amino acids have so far been identified in the yeast prion protein Ure2.In this study, we performed a cysteine scan within the N-terminal PrD of Ure2p and found a unique cysteine mutant (R17C) that eliminates the lag phase of the Ure2p fibril assembly reaction upon the addition of oxidizing agents. Furthermore, we identified a 4-residue region adjacent to Arg¹⁷ as a potential amyloid stretch in Ure2p.     

Noonan Syndrome/Leukemia-associated Gain-of-function Mutations in SHP-2 Phosphatase (PTPN11) Enhance Cell Migration and Angiogenesis

Mutations in SHP-2 phosphatase (PTPN11) that cause hyperactivation of its catalytic activity have been identified in Noonan syndrome and various childhood leukemias. Recent studies suggest that the gain-of-function (GOF) mutations of SHP-2 play a causal role in the pathogenesis of these diseases. However, the molecular mechanisms by which GOF mutations of SHP-2 induce these phenotypes are not fully understood. Here, we show that GOF mutations in SHP-2, such as E76K and D61G, drastically increase spreading and migration of various cell types, including hematopoietic cells, endothelial cells, and fibroblasts. More importantly, in vivo angiogenesis in SHP-2 D61G knock-in mice is also enhanced. Mechanistic studies suggest that the increased cell migration is attributed to the enhanced β1 integrin outside-in signaling. In response to β1 integrin cross-linking or fibronectin stimulation, activation of ERK and Akt kinases is greatly increased by SHP-2 GOF mutations. Also, integrin-induced activation of RhoA and Rac1 GTPases is elevated. Interestingly, mutant cells with the SHP-2 GOF mutation (D61G) are more sensitive than wild-type cells to the suppression of cell motility by inhibition of these pathways. Collectively, these studies reaffirm the positive role of SHP-2 phosphatase in cell motility and suggest a new mechanism by which SHP-2 GOF mutations contribute to diseases.SHP-2, a multifunctional SH2 domain-containing protein-tyrosine phosphatase implicated in diverse cell signaling processes (1–3), plays a critical role in cellular function. Homozygous deletion of Exon 2 (4) or Exon 3 (5) of the SHP-2 gene (PTPN11) in mice leads to early embryonic lethality prior to and at midgestation, respectively. SHP-2 null mutant mice die much earlier, at peri-implantation (4). Exon 3 deletion mutation of SHP-2 blocks hematopoietic potential of embryonic stem cells both in vitro and in vivo (6–8), whereas SHP-2 null mutation causes inner cell mass death and diminished trophoblast stem cell survival (4). Recent studies on SHP-2 conditional knock-out or tissue-specific knock-out mice have further revealed an array of important functions of this phosphatase in various physiological processes (9–12). The phenotypes demonstrated by loss of SHP-2 function are apparently attributed to the role of SHP-2 in the cell signaling pathways induced by growth factors/cytokines. SHP-2 generally promotes signal transmission in growth factor/cytokine signaling in both catalytic-dependent and -independent fashion (1–3). The positive role of SHP-2 in the intracellular signaling processes, in particular, the ERK³ and PI3K/Akt kinase pathways, has been well established, although the underlying mechanism remains elusive, in particular, the signaling function of the catalytic activity of SHP-2 in these pathways is poorly understood.In addition to the role of SHP-2 in cell proliferation and differentiation, the phenotypes induced by loss of SHP-2 function may be associated with its role in cell migration. Indeed, dominant negative SHP-2 disrupts Xenopus gastrulation, causing tail truncations (13, 14). Targeted Exon 3 deletion mutation in SHP-2 results in decreased cell spreading, migration (15, 16), and impaired limb development in the chimeric mice (7). The role of SHP-2 in cell adhesion and migration has also been demonstrated by catalytically inactive mutant SHP-2-overexpressing cells (17–20). The molecular mechanisms by which SHP-2 regulates these cellular processes, however, have not been well defined. For example, the role of SHP-2 in the activation of the Rho family small GTPases that is critical for cell motility is still controversial. Both positive (19, 21, 22) and negative roles (18, 23) for SHP-2 in this context have been reported. Part of the reason for this discrepancy might be due to the difference in the cell models used. Catalytically inactive mutant SHP-2 was often used to determine the role of SHP-2 in cell signaling. In the catalytically inactive mutant SHP-2-overexpressing cells, the catalytic activity of endogenous SHP-2 is inhibited. However, as SHP-2 also functions independent of its catalytic activity, overexpression of catalytically deficient SHP-2 may also increase its scaffolding function, generating complex effects.The critical role of SHP-2 in cellular function is further underscored by the identification of SHP-2 mutations in human diseases. Genetic lesions in PTPN11 that cause hyperactivation of SHP-2 catalytic activity have been identified in the developmental disorder Noonan syndrome (24) and various childhood leukemias, including juvenile myelomonocytic leukemia (JMML), B cell acute lymphoblastic leukemia, and acute myeloid leukemia (25, 26). In addition, activating mutations in SHP-2 have been identified in sporadic solid tumors (27). The SHP-2 mutations appear to play a causal role in the development of these diseases as SHP-2 mutations and other JMML-associated Ras or Neurofibromatosis 1 mutations are mutually exclusive in the patients (24–27). Moreover, single SHP-2 gain-of-function (GOF) mutations are sufficient to induce Noonan syndrome, cytokine hypersensitivity in hematopoietic progenitor cells, and JMML-like myeloproliferative disease in mice (28–32). Gain-of-function cell models derived from the newly available SHP-2 GOF mutation (D61G) knock-in mice (28) now provide us with a good opportunity to clarify the role of SHP-2 in cell motility. Unlike the dominant negative approach in which overexpression of mutant forms of SHP-2 generates complex effects, the SHP-2 D61G knock-in model eliminates this possibility as the mutant SHP-2 is expressed at the physiological level (28). Additionally, defining signaling functions of GOF mutant SHP-2 in cell movement can also help elucidate the molecular mechanisms by which SHP-2 mutations contribute to the relevant diseases.     

The Cholinesterase-like Domain, Essential in Thyroglobulin Trafficking for Thyroid Hormone Synthesis, Is Required for Protein Dimerization

The carboxyl-terminal cholinesterase-like (ChEL) domain of thyroglobulin (Tg) has been identified as critically important in Tg export from the endoplasmic reticulum. In a number of human kindreds suffering from congenital hypothyroidism, and in the cog congenital goiter mouse and rdw rat dwarf models, thyroid hormone synthesis is inhibited because of mutations in the ChEL domain that block protein export from the endoplasmic reticulum. We hypothesize that Tg forms homodimers through noncovalent interactions involving two predicted α-helices in each ChEL domain that are homologous to the dimerization helices of acetylcholinesterase. This has been explored through selective epitope tagging of dimerization partners and by inserting an extra, unpaired Cys residue to create an opportunity for intermolecular disulfide pairing. We show that the ChEL domain is necessary and sufficient for Tg dimerization; specifically, the isolated ChEL domain can dimerize with full-length Tg or with itself. Insertion of an N-linked glycan into the putative upstream dimerization helix inhibits homodimerization of the isolated ChEL domain. However, interestingly, co-expression of upstream Tg domains, either in cis or in trans, overrides the dimerization defect of such a mutant. Thus, although the ChEL domain provides a nidus for Tg dimerization, interactions of upstream Tg regions with the ChEL domain actively stabilizes the Tg dimer complex for intracellular transport.The synthesis of thyroid hormone in the thyroid gland requires secretion of thyroglobulin (Tg)² to the apical luminal cavity of thyroid follicles (1). Once secreted, Tg is iodinated via the activity of thyroid peroxidase (2). A coupling reaction involving a quinol-ether linkage especially engages di-iodinated tyrosyl residues 5 and 130 to form thyroxine within the amino-terminal portion of the Tg polypeptide (3, 4). Preferential iodination of Tg hormonogenic sites is dependent not on the specificity of the peroxidase (5) but upon the native structure of Tg (6, 7). To date, no other thyroidal proteins have been shown to effectively substitute in this role for Tg.The first 80% of the primary structure of Tg (full-length murine Tg: 2,746 amino acids) involves three regions called I-II-III comprised of disulfide-rich repeat domains held together by intradomain disulfide bonds (8, 9). The final 581 amino acids of Tg are strongly homologous to acetylcholinesterase (10–12). Rate-limiting steps in the overall process of Tg secretion involve its structural maturation within the endoplasmic reticulum (ER) (13). Interactions between regions I-II-III and the cholinesterase-like (ChEL) domain have recently been suggested to be important in this process, with ChEL functioning as an intramolecular chaperone and escort for I-II-III (14). In addition, Tg conformational maturation culminates in Tg homodimerization (15, 16) with progression to a cylindrical, and ultimately, a compact ovoid structure (17–19).In human congenital hypothyroidism with deficient Tg, the ChEL domain is a commonly affected site of mutation, including the recently described A2215D (20, 21), R2223H (22), G2300D, R2317Q (23), G2355V, G2356R, and the skipping of exon 45 (which normally encodes 36 amino acids), as well as the Q2638stop mutant (24) (in addition to polymorphisms including P2213L, W2482R, and R2511Q that may be associated with thyroid overgrowth (25)). As best as is currently known, all of the congenital hypothyroidism-inducing Tg mutants are defective for intracellular transport (26). A homozygous G2300R mutation (equivalent to residue 2,298 of mouse Tg) in the ChEL domain is responsible for congenital hypothyroidism in rdw rats (27, 28), whereas we identified the Tg-L2263P point mutation as the cause of hypothyroidism in the cog mouse (29). Such mutations perturb intradomain structure (30), and interestingly, block homodimerization (31). Acquisition of quaternary structure has long been thought to be required for efficient export from the ER (32) as exemplified by authentic acetylcholinesterase (33, 34) in which dimerization enhances protein stability and export (35).Tg comprised only of regions I-II-III (truncated to lack the ChEL domain) is blocked within the ER (30), whereas a secretory version of the isolated ChEL domain of Tg devoid of I-II-III undergoes rapid and efficient intracellular transport and secretion (14). A striking homology positions two predicted α-helices of the ChEL domain to the identical relative positions of the dimerization helices in acetylcholinesterase. This raises the possibility that ChEL may serve as a homodimerization domain for Tg, providing a critical function in maturation for Tg transport to the site of thyroid hormone synthesis (1).In this study, we provide unequivocal evidence for homodimerization of the ChEL domain and “hetero”-dimerization of that domain with full-length Tg, and we provide significant evidence that the predicted ChEL dimerization helices provide a nidus for Tg assembly. On the other hand, our data also suggest that upstream Tg regions known to interact with ChEL (14) actively stabilize the Tg dimer complex. Together, I-II-III and ChEL provide unique contributions to the process of intracellular transport of Tg through the secretory pathway.     

A Role for the Proton-coupled Folate Transporter (PCFT-SLC46A1) in Folate Receptor-mediated Endocytosis

Recently, this laboratory identified a proton-coupled folate transporter (PCFT), with optimal activity at low pH. PCFT is critical to intestinal folate absorption and transport into the central nervous system because there are loss-of-function mutations in this gene in the autosomal recessive disorder, hereditary folate malabsorption. The current study addresses the role PCFT might play in another transport pathway, folate receptor (FR)-mediated endocytosis. FRα cDNA was transfected into novel PCFT⁺ and PCFT^– HeLa sublines. FRα was shown to bind and trap folates in vesicles but with minimal export into the cytosol in PCFT^– cells. Cotransfection of FRα and PCFT resulted in enhanced folate transport into cytosol as compared with transfection of FRα alone. Probenecid did not inhibit folate binding to FR, but inhibited PCFT-mediated transport at endosomal pH, and blocked FRα-mediated transport into the cytosol. FRα and PCFT co-localized to the endosomal compartment. These observations (i) indicate that PCFT plays a role in FRα-mediated endocytosis by serving as a route of export of folates from acidified endosomes and (ii) provide a functional role for PCFT in tissues in which it is expressed, such as the choroid plexus, where the extracellular milieu is at neutral pH.Loss of function mutations of the proton-coupled folate transporter (PCFT),² which functions optimally at low pH, are the molecular basis for the autosomal recessive disorder, hereditary folate malabsorption (HFM) (1–4). Infants present with this disorder several months after birth with marked folate deficiency anemia, hypogammaglobulinemia with immune deficiency and infections, neurological deficits, and often seizures (5). PCFT is highly expressed at the apical brush-border membrane of the duodenum and proximal jejunum (6–9) where the pH at the microclimate of the surface of this epithelium is low (pH 5.8–6.0), and folates are absorbed (1, 7, 10, 11). Hence, the failure to absorb folates in the absence of this transporter in HFM is expected. However, PCFT expression, and its associated folate transport activity at low pH, is observed in many tissues where the transport interface is presumed to be at pH 7.4 (12). Of particular interest is the mechanism by which PCFT mediates transport of folates into the central nervous system (CNS) where this transporter is expressed in brain and choroid plexus (1, 7, 13). Transport into the CNS is impaired in patients with HFM who have very low cerebrospinal fluid (CSF) folate levels and marked reversal of the blood:CSF folate gradient which is normally 2–3:1 (5).Folates are also transported into cells by a receptor-mediated process. Folate receptor-α (FRα) is anchored to cell membranes via a glycosylphosphatidylinositol domain. Uptake begins with folate binding to receptor at the cell surface followed by invagination of the membrane and the formation of endosomes that traffic along microtubules to a perinuclear compartment before returning to the plasma membrane (14–16). During transit in the cytoplasm, endosomes acidify to a pH of ∼6.0–6.5 (17), folate is released from the receptor and exported from the intact endosome into the cytoplasm. This putative exporter was shown to require a trans-endosomal pH gradient (18–20).The current report addresses the hypothesis that PCFT is an endosomal folate exporter and thereby plays a role in FRα-mediated endocytosis (1, 2, 21, 22), that the ubiquitous expression of PCFT in mammalian tissues may be related to this function, and that loss of this function may be a basis for the low CSF folate levels in HFM. The experimental approach utilized a series of HeLa sublines, developed in this laboratory, in which constitutive expression of FRα is negligible. HeLa R5 cells lack reduced folate carrier (RFC) function due to a genomic deletion of this gene (23). A derivative of R5 cells, HeLa R1-11 cells lack, in addition, PCFT expression, while an R1-11 revertant re-expresses PCFT (24). The impact of PCFT on FRα-mediated endocytosis, achieved by transfection of the receptor into these cell lines, was assessed under conditions in which there was negligible PCFT-mediated transport directly across the plasma membrane into cells.     

Structural and Mechanistic Insights into Lunatic Fringe from a Kinetic Analysis of Enzyme Mutants

The Notch receptor is critical for proper development where it orchestrates numerous cell fate decisions. The Fringe family of β1,3-N-acetylglucosaminyltransferases are regulators of this pathway. Fringe enzymes add N-acetylglucosamine to O-linked fucose on the epidermal growth factor repeats of Notch. Here we have analyzed the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis strategy for Lfng was guided by a multiple sequence alignment of Fringe proteins and solutions from docking an epidermal growth factor-like O-fucose acceptor substrate onto a homology model of Lfng. We targeted three main areas as follows: residues that could help resolve where the fucose binds, residues in two conserved loops not observed in the published structure of Manic Fringe, and residues predicted to be involved in UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a kinetic analysis of mutant enzyme activity toward the small molecule acceptor substrate 4-nitrophenyl-α-l-fucopyranoside to judge their effect on Lfng activity. Our results support the positioning of O-fucose in a specific orientation to the catalytic residue. We also found evidence that one loop closes off the active site coincident with, or subsequent to, substrate binding. We propose a mechanism whereby the ordering of this short loop may alter the conformation of the catalytic aspartate. Finally, we identify several residues near the UDP-GlcNAc-binding site, which are specifically permissive toward UDP-GlcNAc utilization.Defects in Notch signaling have been implicated in numerous human diseases, including multiple sclerosis (1), several forms of cancer (2-4), cerebral autosomal dominant arteriopathy with sub-cortical infarcts and leukoencephalopathy (5), and spondylocostal dysostosis (SCD)³ (6-8). The transmembrane Notch signaling receptor is activated by members of the DSL (Delta, Serrate, Lag2) family of ligands (9, 10). In the endoplasmic reticulum, O-linked fucose glycans are added to the epidermal growth factor-like (EGF) repeats of the Notch extracellular domain by protein O-fucosyltransferase 1 (11-13). These O-fucose monosaccharides can be elongated in the Golgi apparatus by three highly conserved β1,3-N-acetylglucosaminyltransferases of the Fringe family (Lunatic (Lfng), Manic (Mfng), and Radical Fringe (Rfng) in mammals) (14-16). The formation of this GlcNAc-β1,3-Fuc-α1, O-serine/threonine disaccharide is necessary and sufficient for subsequent elongation to a tetrasaccharide (15, 19), although elongation past the disaccharide in Drosophila is not yet clear (20, 21). Elongation of O-fucose by Fringe is known to potentiate Notch signaling from Delta ligands and inhibit signaling from Serrate ligands (22). Delta ligands are termed Delta-like (Delta-like1, -2, and -4) in mammals, and the homologs of Serrate are known as Jagged (Jagged1 and -2) in mammals. The effects of Fringe on Drosophila Notch can be recapitulated in Notch ligand in vitro binding assays using purified components, suggesting that the elongation of O-fucose by Fringe alters the binding of Notch to its ligands (21). Although Fringe also appears to alter Notch-ligand interactions in mammals, the effects of elongation of the glycan past the O-fucose monosaccharide is more complicated and appears to be cell type-, receptor-, and ligand-dependent (for a recent review see Ref. 23).The Fringe enzymes catalyze the transfer of GlcNAc from the donor substrate UDP-α-GlcNAc to the acceptor fucose, forming the GlcNAc-β1,3-Fuc disaccharide (14-16). They belong to the GT-A-fold of inverting glycosyltransferases, which includes N-acetylglucosaminyltransferase I and β1,4-galactosyltransferase I (17, 18). The mechanism is presumed to proceed through the abstraction of a proton from the acceptor substrate by a catalytic base (Asp or Glu) in the active site. This creates a nucleophile that attacks the anomeric carbon of the nucleotide-sugar donor, inverting its configuration from α (on the nucleotide sugar) to β (in the product) (24, 25). The enzyme then releases the acceptor substrate modified with a disaccharide and UDP. The Mfng structure (26) leaves little doubt as to the identity of the catalytic residue, which in all likelihood is aspartate 289 in mouse Lfng (we will use numbering for mouse Lunatic Fringe throughout, unless otherwise stated). The structure of Mfng with UDP-GlcNAc soaked into the crystals (26) showed density only for the UDP portion of the nucleotide-sugar donor and no density for two loops flanking either side of the active site. The presence of flexible loops that become ordered upon substrate binding is a common observation with glycosyltransferases in the GT-A fold family (18, 25). Density for the entire donor was observed in the structure of rabbit N-acetylglucosaminyltransferase I (27). In this case, ordering of a previously disordered loop upon UDP-GlcNAc binding may have contributed to increased stability of the donor. In the case of bovine β1,4-galactosyltransferase I, a section of flexible random coil from the apo-structure was observed to change its conformation to α-helical upon donor substrate binding (28). Both loops in Lfng are highly conserved, and we have mutated a number of residues in each to test the hypothesis that they interact with the substrates. The mutagenesis strategy was also guided by docking of an EGF-O-fucose acceptor substrate into the active site of the Lfng model as well as comparison of the Lfng model with a homology model of the β1,3-glucosyltransferase (β3GlcT) that modifies O-fucose on thrombospondin type 1 repeats (29, 30). The β3GlcT is predicted to be a GT-A fold enzyme related to the Fringe family (17, 18, 29).     

Matrix Metalloproteinase-2-deficient Fibroblasts Exhibit an Alteration in the Fibrotic Response to Connective Tissue Growth Factor/CCN2 because of an Increase in the Levels of Endogenous Fibronectin

Matrix metalloproteinase-2 (MMP-2) is an important extracellular matrix remodeling enzyme, and it has been involved in different fibrotic disorders. The connective tissue growth factor (CTGF/CCN2), which is increased in these pathologies, induces the production of extracellular matrix proteins. To understand the fibrotic process observed in diverse pathologies, we analyzed the fibroblast response to CTGF when MMP-2 activity is inhibited. CTGF increased fibronectin (FN) amount, MMP-2 mRNA expression, and gelatinase activity in 3T3 cells. When MMP-2 activity was inhibited either by the metalloproteinase inhibitor GM-6001 or in MMP-2-deficient fibroblasts, an increase in the basal amount of FN together with a decrease of its levels in response to CTGF was observed. This paradoxical effect could be explained by the fact that the excess of FN could block the access to other ligands, such as CTGF, to integrins. This effect was emulated in fibroblasts by adding exogenous FN or RGDS peptides or using anti-integrin α_V subunit-blocking antibodies. Additionally, in MMP-2-deficient cells CTGF did not induce the formation of stress fibers, focal adhesion sites, and ERK phosphorylation. Anti-integrin α_V subunit-blocking antibodies inhibited ERK phosphorylation in control cells. Finally, in MMP-2-deficient cells, FN mRNA expression was not affected by CTGF, but degradation of ¹²⁵I-FN was increased. These results suggest that expression, regulation, and activity of MMP-2 can play an important role in the initial steps of fibrosis and shows that FN levels can regulate the cellular response to CTGF.Extracellular proteolysis is an essential physiological process that controls the immediate cellular environment and thus plays a key role in cellular behavior and survival (1). The members of the matrix metalloproteinase (MMP)² family of zinc-dependent endopeptidases are major mediators of extracellular proteolysis by promoting the degradation of extracellular matrix (ECM) components and cell surface-associated proteins (2, 3). Each one of these enzymes is negatively regulated by tissue inhibitors of metalloproteinases (TIMPs) (4) and is secreted as a zymogen (pro-MMPs) that is activated in the extracellular space (5–7). This mechanism is an important form of regulation of gelatinase activity and in consequence, highly significant for ECM homeostasis. Among the members of the MMP family, the metalloproteinase type 2 (MMP-2 or gelatinase A) is known to be a key player in many physiological and pathological processes, such as cell migration, inflammation, angiogenesis, and fibrosis (8–11).Fibrotic disorders are typified by excessive connective tissue and ECM deposition that precludes normal healing of different tissues. ECM accumulation can be explained in two ways: increasing expression and deposition of connective tissue proteins and/or decreasing degradation of ECM proteins (12). Transforming growth factor type β, a multifunctional cytokine, is strongly overexpressed, and it is associated to the pathogenesis of these diseases (13, 14). It stimulates the expression of connective tissue growth factor (CTGF/CCN2) (15), a cytokine that is responsible for transforming growth factor type β fibrotic activity (16, 17). The role of CTGF in fibrosis has gained attention in recent years (16, 18–22). CTGF overexpression is known to occur in a variety of fibrotic skin disorders (23, 24), renal (25), hepatic (26), and pulmonary fibrosis (27) and in muscles from patients with Duchenne muscular dystrophy (28).On the other hand, several pathologies involving fibrosis show an increase in MMP expression, including gelatinase A. Augmented expression of MMP-2 was found in submucous (29), skin (30), liver (31), and lung fibrosis (32, 33) and dystrophic myotubes from fibrotic muscles of Duchenne muscular dystrophy (34). It has been shown that transforming growth factor type β induces an increase in the amount of MMP-2 in fibroblasts (35) and that CTGF induces MMP-2 expression in cultured renal interstitial fibroblasts (36). The putative role assigned to MMP-2 in fibrotic disorders is related to tissue regeneration because of the capacity of this enzyme to degrade basal lamina (37–39). Because MMP-2 expression is up-regulated in these pathologies but still a high ECM deposition is observed, we propose that this accumulation could be explained by a diminution of the MMP-2 enzymatic activity.In this article, we demonstrate that CTGF increases fibronectin (FN) amount, MMP-2 expression, and gelatinase activity in 3T3 fibroblasts. More significantly, we show that MMP-2-deficient cells have an increased basal amount of FN and show a response to CTGF that is opposite to that of control cells. This paradoxical effect could be explained by the increase in the FN amount that blocks the integrins (at least integrins with α_V subunit), which can act like CTGF receptors.     

A Highly Conserved Motif within the NH2-terminal Coiled-coil Domain of Angiopoietin-like Protein 4 Confers Its Inhibitory Effects on Lipoprotein Lipase by Disrupting the Enzyme Dimerization

Lipoprotein lipase (LPL) is a principal enzyme responsible for the clearance of chylomicrons and very low density lipoproteins from the bloodstream. Two members of the Angptl (angiopoietin-like protein) family, namely Angptl3 and Angptl4, have been shown to inhibit LPL activity in vitro and in vivo. Here, we further investigated the structural basis underlying the LPL inhibition by Angptl3 and Angptl4. By multiple sequence alignment analysis, we have identified a highly conserved 12-amino acid consensus motif that is present within the coiled-coil domain (CCD) of both Angptl3 and Angptl4, but not other members of the Angptl family. Substitution of the three polar amino acid residues (His⁴⁶, Gln⁵⁰, and Gln⁵³) within this motif with alanine abolishes the inhibitory effect of Angptl4 on LPL in vitro and also abrogates the ability of Angptl4 to elevate plasma triglyceride levels in mice. The CCD of Angptl4 interacts with LPL and converts the catalytically active dimers of LPL to its inactive monomers, whereas the mutant protein with the three polar amino acids being replaced by alanine loses such a property. Furthermore, a synthetic peptide consisting of the 12-amino acid consensus motif is sufficient to inhibit LPL activity, although the potency is much lower than the recombinant CCD of Angptl4. In summary, our data suggest that the 12-amino acid consensus motif within the CCD of Angptl4, especially the three polar residues within this motif, is responsible for its interaction with and inhibition of LPL by blocking the enzyme dimerization.Lipoprotein lipase (LPL)³ is an endothelium-bound enzyme that catalyzes the hydrolysis of plasma triglyceride (TG) associated with chylomicrons and very low density lipoproteins (1, 2). This enzyme plays a major role in maintaining lipid homeostasis by promoting the clearance of TG-rich lipoproteins from the bloodstream. Abnormality in LPL functions has been associated with a number of pathological conditions, including atherosclerosis, dyslipidemia associated with diabetes, and Alzheimer disease (1).LPL is expressed in a wide variety of cell types, particularly in adipocytes and myocytes (2). As a rate-limiting enzyme for clearance of TG-rich lipoproteins, the activity of LPL is tightly modulated by multiple mechanisms in a tissue-specific manner in response to nutritional changes (3, 4). The enzymatic activity of LPL in adipose tissue is enhanced after feeding to facilitate the storage of TG, whereas it is down-regulated during fasting to increase the utilization of TG by other tissues (5). The active form of LPL is a noncovalent homodimer with the subunits associated in a head-to-tail manner, and the dissociation of its dimeric form leads to the formation of a stable inactive monomeric conformation and irreversible enzyme inactivation (6). At the post-translational level, the LPL activity is regulated by numerous apolipoprotein co-factors. For instance, apoCII, a small apolipoprotein consisting of 79 amino acid residues in human, activates LPL by directly binding to the enzyme (7, 8). By contrast, several other apolipoproteins such as apoCI, apo-CIII, and apoE have been shown to inhibit the LPL activity in vitro (3).Angiopoietin-like proteins (Angptl) are a family of secreted proteins consisting of seven members, Angptl1 to Angptl7 (9, 10). All the members of the Angptl family share a similar domain organization to those of angiopoietins, with an NH₂-terminal coiled-coil domain (CCD) and a COOH-terminal fibrinogen-like domain. Among the seven family members, only Angptl3 and Angptl4 have been shown to be involved in regulating triglyceride metabolism (10, 11). The biological functions of Angptl3 in lipid metabolism were first discovered by Koishi et al. (12) in their positional cloning of the recessive mutation gene responsible for the hypolipidemia phenotype in a strain of obese mouse KK/snk. Subsequent studies have demonstrated that Angptl3 increases plasma TG levels by inhibiting the LPL enzymatic activity (13–15). Angptl4, also known as fasting-induced adipocyte factor, hepatic fibrinogen/angiopoietin-related protein, or peroxisome proliferator-activated receptor-γ angiopoietin-related, is a secreted glycoprotein abundantly expressed in adipocyte, liver, and placenta (16–18). In addition to its role in regulating angiogenesis, a growing body of evidence demonstrated that Angptl4 is an important player of lipid metabolism (10, 11). Elevation of circulating Angptl4 by transgenic or adenoviral overexpression, or by direct supplementation of recombinant protein, leads to a marked elevation in the levels of plasma TG and low density lipoprotein cholesterol in mice (19–22). By contrast, Angptl4 knock-out mice exhibit much lower plasma TG and cholesterol levels compared with the wild type littermates (19, 20). Notably, treatment of several mouse models (such as C57BL/6J, ApoE^–/–, LDLR^–/–, and db/db obese/diabetic mice) with a neutralizing antibody against Angptl4 recapitulate the lipid phenotype found in Angptl4 knock-out mice (19). The role of Angptl4 as a physiological inhibitor of LPL is also supported by the finding that its expression levels in adipose tissue change rapidly during the fed-to-fasting transitions and correlate inversely with LPL activity (23). In humans, a genetic variant of the ANGPTL4 gene (E40K) has been found to be associated with significantly lower plasma TG levels and higher high density lipoprotein cholesterol concentrations in several ethnic groups (24–26).Angptl3 and Angptl4 share many common biochemical and functional properties (10). In both humans and rodents, Angptl3 and Angptl4 are proteolytically cleaved at the linker region and circulate in plasma as two truncated fragments, including NH₂-terminal CCD and COOH-terminal fibrinogen-like domain (14, 27–29). The effects of both Angptl3 and Angptl4 on elevating plasma TG levels are mediated exclusively by their NH₂-terminal CCDs (15, 22, 23, 27, 30). The CCDs of Angptl3 and Angptl4 have been shown to inhibit the LPL activity in vitro as well as in mice (23,30,31). Angptl4 inhibits LPL by promoting the conversion of the catalytically active LPL dimers into catalytically inactive LPL monomers, thereby leading to the inactivation of LPL (23, 31). However, the detailed structural and molecular basis underlying the LPL inhibition by Angptl3 and Angptl4 remain poorly characterized at this stage.In this study, we analyzed all known amino acid sequences of Angptl3 and Angptl4 from various species and found a short motif, LAXGLLXLGXGL (where X represents polar amino acid residues), which corresponds to amino acid residues 46–57 and 44–55 of human Angptl3 and Angptl4, respectively, is highly conserved despite the low degree of their overall homology (∼30%). Using both in vitro and in vivo approaches, we demonstrated that this 12-amino acid sequence motif, in particular the three polar amino acid residue within this motif, is essential for mediating the interactions between LPL and Angpt4, which in turn disrupts the dimerization of the enzyme.     

The Activity of the Epithelial Sodium Channels Is Regulated by Caveolin-1 via a Nedd4-2-dependent Mechanism

It has recently been shown that the epithelial Na⁺ channel (ENaC) is compartmentalized in caveolin-rich lipid rafts and that pharmacological depletion of membrane cholesterol, which disrupts lipid raft formation, decreases the activity of ENaC. Here we show, for the first time, that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the activity and membrane surface expression of ENaC. Physical interaction between ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin protein ligase of the Nedd4 family, which is known to induce ubiquitination and internalization of ENaC. The effect of Cav-1 on ENaC requires the proline-rich motifs at the C termini of the β- and γ-subunits of ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken together, our data suggest that Cav-1 inhibits the activity of ENaC by decreasing expression of ENaC at the cell membrane via a mechanism that involves the promotion of Nedd4-2-dependent internalization of the channel.Amiloride-sensitive epithelial Na⁺ channels (ENaC)³ are membrane proteins that are expressed in salt-absorptive epithelia, including the distal collecting tubules of the kidney, the mucosa of the distal colon, the respiratory epithelium, and the excretory ducts of sweat and salivary glands (1–4). Na⁺ absorption via ENaC is critical to the normal regulation of Na⁺ and fluid homeostasis and is important for maintaining blood pressure (5) and the volume of fluid in the respiratory passages (6). Increased ENaC activity has been implicated in the salt-sensitive inherited form of hypertension, Liddle''s syndrome (7), and dehydration of the surface of the airway epithelium in the pathology associated with cystic fibrosis lung disease (8).Expression of ENaC at the cell membrane surface is regulated by the E3 ubiquitin protein ligase, Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4) (9). Interaction between the WW domains of Nedd4-2 and the proline-rich PY motifs (PPPXY) on ENaC is essential for Nedd4-2 to exert a negative effect on the channel (10, 11). This interaction leads to ubiquitination-dependent internalization of ENaC (12, 13). Several regulators of ENaC exert their effects on the channel by modulating the action of Nedd4-2. For instance, serum and glucocorticoid-dependent protein kinase (14), protein kinase B (15), and G protein-coupled receptor kinase (16) up-regulate activity of ENaC by inhibiting Nedd4-2. Although the details of cellular mechanisms that underlie internalization of ENaC remain to be elucidated, the physiological significance of Nedd4-dependent internalization of the channel has been well established. For instance, heritable mutations that delete the cytosolic termini of the β-or γ-subunit of ENaC, which contain the proline-rich motifs, are known to cause hyperactivity of ENaC in the kidney (17) and increase cell surface expression of the channel (7, 18).The plasma membranes of most cell types contain lipid raft microdomains that are enriched with glycosphingolipid and cholesterol (19), that have distinctive biophysical properties, and that selectively include or exclude signaling molecules (20). These microdomains promote clustering of an array of integral membrane proteins in the membrane leaflets (21) and may be important for organizing cascades of signaling molecules (22, 23). Processes in which raft microdomains are involved include the intracellular transport of proteins and lipids to the cell membrane (24), the endocytotic retrieval of membrane proteins (25, 26), and signal transduction (27, 28). In addition, segregation of signaling molecules within lipid rafts may facilitate cross-talk between signal transduction pathways (29), a phenomenon that may be important in ensuring rapid and efficient integration of multiple cellular signaling events (30, 31). Of particular interest is the subpopulation of lipid rafts enriched with caveolin proteins. Caveolin-1 (Cav-1), a major caveolin isoform expressed in nonmuscle cells, has been identified as being involved in diverse cellular functions, such as vesicular transport, cholesterol homeostasis, and signal transduction (32). Cav-1 also regulates the activity and membrane expression of ion channels and transporters (28).In epithelia, the majority of lipid rafts exist at the apical membrane surface (22). Pools of ENaC (33–36) and several proteins that regulate activity of ENaC, such as Nedd4 (37), protein kinase B (38), protein kinase C (39), G_o (40), and the G protein-coupled receptor kinase (41), have been identified in detergent-insoluble and cholesterol-rich membrane fractions from a variety of cell types, consistent with localization of these proteins in lipid rafts. Furthermore, detergent-free buoyant density separation of lipid rafts has revealed the presence of Cav-1 with ENaC in the lipid raft-rich membrane fraction (35). The physiological role of lipid rafts in the regulation of ENaC has been the subject of many recent investigations. Most of these studies used a pharmacological agent, methyl-β-cyclodextrin (MβCD), to promote redistribution of proteins away from the cholesterol-enriched membrane domains. The results were, however, inconclusive. In some studies, MβCD treatment was found to inhibit open probability (42) or cell surface expression of ENaC (35), whereas others found no direct effect of MβCD on the channel (33, 43).Despite a number of studies into the role of lipid rafts on the regulation of ENaC, little is known about the physiological relevance of caveolins to the function of this ion channel. In the present study, we use gene interference and gene expression techniques to determine the role of Cav-1 in the regulation of ENaC activity. We provide evidence of the association of Cav-1 with ENaC and evidence that Cav-1 negatively regulates both activity and abundance of ENaC at the surface of epithelial cells. Importantly, we demonstrate, for the first time, that the mechanism by which Cav-1 regulates activity of ENaC involves the E3 ubiquitin protein ligase, Nedd4-2.     

Rab1 Guanine Nucleotide Exchange Factor SidM Is a Major Phosphatidylinositol 4-Phosphate-binding Effector Protein of Legionella pneumophila

The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a nonbiased screen for novel L. pneumophila PI-binding proteins, we identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant PtdIns(4)P-binding protein. Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed “P4M” (PtdIns4P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher PtdIns(4)P binding activity in the context of the α-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by Icm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via its P4M domain. An L. pneumophila ΔsidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally, RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by host PtdIns 4-kinase IIIβ. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIβ to anchor the effectors SidC and SidM to LCVs.The Gram-negative pathogen Legionella pneumophila is the causative agent of Legionnaires disease, but it evolved as a parasite of various species of environmental predatory protozoa, including the social amoeba Dictyostelium discoideum (1, 2). The human disease is linked to the inhalation of contaminated aerosols, followed by replication in alveolar macrophages. To accommodate the transfer between host cells, L. pneumophila alternates between replicative and transmissive phases, the regulation of which includes an apparent quorum-sensing system (3–5).In macrophages and amoebae, L. pneumophila forms a replicative compartment, the Legionella-containing vacuole (LCV).³ LCVs avoid fusion with lysosomes (6), intercept vesicular traffic at endoplasmic reticulum (ER) exit sites (7), and fuse with the ER (8–10). The uptake of L. pneumophila and formation of LCVs in macrophages and amoebae depends on the Icm/Dot type IV secretion system (T4SS) (11–14). Although more than 100 Icm/Dot substrates (“effector” proteins) have been identified to date, only few are functionally characterized, including effectors that interfere with host cell signal transduction, vesicle trafficking, or apoptotic pathways (15–18).Two Icm/Dot-translocated substrates, SidM/DrrA (19, 20) and RalF (21), have been characterized as guanine nucleotide exchange factors (GEFs) for the Rho subfamily of small GTPases. These bacterial GEFs are recruited to and activate their targets on LCVs. Small GTPases of the Rho subfamily are involved in many eukaryotic signal transduction pathways and in actin cytoskeleton regulation (22). Inactive Rho GTPases bind GDP and a guanine nucleotide dissociation inhibitor (GDI). The GTPases are activated by removal of the GDI and the exchange of GDP with GTP by GEFs, which promotes the interaction with downstream effector proteins, such as protein or lipid kinases and various adaptor proteins. The cycle is closed by hydrolysis of the bound GTP, which is mediated by GTPase-activating proteins.SidM is a GEF for Rab1, which is essential for ER to Golgi vesicle transport, and additionally, SidM acts as a GDI displacement factor (GDF) to activate Rab1 (23, 24). The function of SidM is assisted by the Icm/Dot substrate LidA, which also localizes to LCVs. LidA preferentially binds to activated Rab1, thus supporting the recruitment of early secretory vesicles by SidM (19, 20, 23, 25, 26). Another Icm/Dot substrate, LepB (27), contributes to Rab1-mediated membrane cycling by inactivating Rab1 through its GTPase-activating protein function, thus acting as an antagonist of SidM (24).The Icm/Dot substrate RalF recruits and activates the small GTPase ADP-ribosylation factor 1 (Arf1), which is involved in retrograde vesicle transport from Golgi to ER (21). Dominant negative Arf1 (7, 28) or knockdown of Arf1 by RNA interference (29) impairs the formation of LCVs, as well as the recruitment of the Icm/Dot substrate SidC to the LCV (30).SidC and its paralogue SdcA localize to the LCV membrane (31), where the proteins specifically bind to the host cell lipid phosphatidylinositol 4-phosphate (PtdIns(4)P) (32, 33). Phosphoinositides (PIs) regulate eukaryotic receptor-mediated signal transduction, actin remodeling, and membrane dynamics (34, 35). PtdIns(4)P is present on the cytoplasmic membrane, but localizes preferentially to the trans-Golgi network (TGN), where this PI is produced by an Arf-dependent recruitment of PtdIns(4)P kinase IIIβ (PI4K IIIβ) (36) to promote trafficking along the secretory pathway. Recently, PtdIns(4)P was found to also mediate the export of early secretory vesicles from ER exit sites (37). At present, the L. pneumophila effector proteins that mediate exploitation of host PI signaling remain ill defined.In a nonbiased screen for L. pneumophila PI-binding proteins using different PIs coupled to agarose beads, we identified SidM as a major PtdIns(4)P-binding effector. We mapped its PtdIns(4)P binding activity to a novel P4M domain within a 12-kDa C-terminal sequence. SidM constructs, including the P4M domain, were found to be translocated and bind the LCV membrane, where the levels of PtdIns(4)P are controlled by PI4K IIIβ.