Tetrameric Orai1 Is a Teardrop-shaped Molecule with a Long, Tapered Cytoplasmic Domain

The Ca²⁺ release-activated Ca²⁺ channel is a principal regulator of intracellular Ca²⁺ rise, which conducts various biological functions, including immune responses. This channel, involved in store-operated Ca²⁺ influx, is believed to be composed of at least two major components. Orai1 has a putative channel pore and locates in the plasma membrane, and STIM1 is a sensor for luminal Ca²⁺ store depletion in the endoplasmic reticulum membrane. Here we have purified the FLAG-fused Orai1 protein, determined its tetrameric stoichiometry, and reconstructed its three-dimensional structure at 21-Å resolution from 3681 automatically selected particle images, taken with an electron microscope. This first structural depiction of a member of the Orai family shows an elongated teardrop-shape 150Å in height and 95Å in width. Antibody decoration and volume estimation from the amino acid sequence indicate that the widest transmembrane domain is located between the round extracellular domain and the tapered cytoplasmic domain. The cytoplasmic length of 100Å is sufficient for direct association with STIM1. Orifices close to the extracellular and intracellular membrane surfaces of Orai1 seem to connect outside the molecule to large internal cavities.Ca²⁺ is an intracellular second messenger that plays important roles in various physiological functions such as immune response, muscle contraction, neurotransmitter release, and cell proliferation. Intracellular Ca²⁺ is mainly stored in the endoplasmic reticulum (ER).² This ER system is distributed through the cytoplasm from around the nucleus to the cell periphery close to the plasma membrane. In non-excitable cells, the ER releases Ca²⁺ through the inositol 1,4,5-trisphosphate (IP₃) receptor channel in response to various signals, and the Ca²⁺ store is depleted. Depletion of Ca²⁺ then induces Ca²⁺ influx from outside the cell to help in refilling the Ca²⁺ stores and to continue Ca²⁺ rise for several minutes in the cytoplasm (1, 2). This Ca²⁺ influx was first proposed by Putney (3) and was named store-operated Ca²⁺ influx. In the immune system, store-operated Ca²⁺ influx is mainly mediated by the Ca²⁺ release-activated Ca²⁺ (CRAC) current, which is a highly Ca²⁺-selective inwardly rectified current with low conductance (4, 5). Pathologically, the loss of CRAC current in T cells causes severe combined immunodeficiency (6) where many Ca²⁺ signal-dependent gene expressions, including cytokines, are interrupted (7). Therefore, CRAC current is necessary for T cell functions.Recently, Orai1 (also called CRACM1) and STIM1 have been physiologically characterized as essential components of the CRAC channel (8–12). They are separately located in the plasma membrane and in the ER membrane; co-expression of these proteins presents heterologous CRAC-like currents in various types of cells (10, 13–15). Both of them are shown to be expressed ubiquitously in various tissues (16–18). STIM1 senses Ca²⁺ depletion in the ER through its EF hand motif (19) and transmits a signal to Orai1 in the plasma membrane. Although Orai1 is proposed as a regulatory component for some transient receptor potential canonical channels (20, 21), it is believed from the mutation analyses to be the pore-forming subunit of the CRAC channel (8, 22–24). In the steady state, both Orai1 and STIM1 molecules are dispersed in each membrane. When store depletion occurs, STIM1 proteins gather into clusters to form puncta in the ER membrane near the plasma membrane (11, 19). These clusters then trigger the clustering of Orai1 in the plasma membrane sites opposite the puncta (25, 26), and CRAC channels are activated (27).Orai1 has two homologous genes, Orai2 and Orai3 (8). They form the Orai family and have in common the four transmembrane (TM) segments with relatively large N and C termini. These termini are demonstrated to be in the cytoplasm, because both N- and C-terminally introduced tags are immunologically detected only in the membrane-permeabilized cells (8, 9). The subunit stoichiometry of Orai1 is as yet controversial: it is believed to be an oligomer, presumably a dimer or tetramer even in the steady state (16, 28–30).Despite the accumulation of biochemical and electrophysiological data, structural information about Orai1 is limited due to difficulties in purification and crystallization. In this study, we have purified Orai1 in its tetrameric form and have reconstructed the three-dimensional structure from negatively stained electron microscopic (EM) images.     

Mapping Daunorubicin-binding Sites in the ATP-binding Cassette Transporter MsbA Using Site-specific Quenching by Spin Labels

ATP-binding cassette (ABC) transporters transduce the free energy of ATP hydrolysis to power the mechanical work of substrate translocation across cell membranes. MsbA is an ABC transporter implicated in trafficking lipid A across the inner membrane of Escherichia coli. It has sequence similarity and overlapping substrate specificity with multidrug ABC transporters that export cytotoxic molecules in humans and prokaryotes. Despite rapid advances in structure determination of ABC efflux transporters, little is known regarding the location of substrate-binding sites in the transmembrane segment and the translocation pathway across the membrane. In this study, we have mapped residues proximal to the daunorubicin (DNR)-binding site in MsbA using site-specific, ATP-dependent quenching of DNR intrinsic fluorescence by spin labels. In the nucleotide-free MsbA intermediate, DNR-binding residues cluster at the cytoplasmic end of helices 3 and 6 at a site accessible from the membrane/water interface and extending into an aqueous chamber formed at the interface between the two transmembrane domains. Binding of a nonhydrolyzable ATP analog inverts the transporter to an outward-facing conformation and relieves DNR quenching by spin labels suggesting DNR exclusion from proximity to the spin labels. The simplest model consistent with our data has DNR entering near an elbow helix parallel to the water/membrane interface, partitioning into the open chamber, and then translocating toward the periplasm upon ATP binding.ATP-binding cassette (ABC)² transporters transduce the energy of ATP hydrolysis to power the movement of a wide range of substrates across the cell membranes (1, 2). They constitute the largest family of prokaryotic transporters, import essential cell nutrients, flip lipids, and export toxic molecules (3). Forty eight human ABC transporters have been identified, including ABCB1, or P-glycoprotein, which is implicated in cross-resistance to drugs and cytotoxic molecules (4, 5). Inherited mutations in these proteins are linked to diseases such as cystic fibrosis, persistent hypoglycemia of infancy, and immune deficiency (6).The functional unit of an ABC transporter consists of four modules. Two highly conserved ABCs or nucleotide-binding domains (NBDs) bind and hydrolyze ATP to supply the active energy for transport (7). ABCs drive the mechanical work of proteins with diverse functions ranging from membrane transport to DNA repair (3, 5). Substrate specificity is determined by two transmembrane domains (TMDs) that also provide the translocation pathway across the bilayer (7). Bacterial ABC exporters are expressed as monomers, each consisting of one NBD and one TMD, that dimerize to form the active transporter (3). The number of transmembrane helices and their organization differ significantly between ABC importers and exporters reflecting the divergent structural and chemical nature of their substrates (1, 8, 9). Furthermore, ABC exporters bind substrates directly from the cytoplasm or bilayer inner leaflet and release them to the periplasm or bilayer outer leaflet (10, 11). In contrast, bacterial importers have their substrates delivered to the TMD by a dedicated high affinity substrate-binding protein (12).In Gram-negative bacteria, lipid A trafficking from its synthesis site on the inner membrane to its final destination in the outer membrane requires the ABC transporter MsbA (13). Although MsbA has not been directly shown to transport lipid A, suppression of MsbA activity leads to cytoplasmic accumulation of lipid A and inhibits bacterial growth strongly suggesting a role in translocation (14-16). In addition to this role in lipid A transport, MsbA shares sequence similarity with multidrug ABC transporters such as human ABCB1, LmrA of Lactococcus lactis, and Sav1866 of Staphylococcus aureus (16-19). ABCB1, a prototype of the ABC family, is a plasma membrane protein whose overexpression provides resistance to chemotherapeutic agents in cancer cells (1). LmrA and MsbA have overlapping substrate specificity with ABCB1 suggesting that both proteins can function as drug exporters (18, 20). Indeed, cells expressing MsbA confer resistance to erythromycin and ethidium bromide (21). MsbA can be photolabeled with the ABCB1/LmrA substrate azidopine and can transport Hoechst 33342 ({"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342) across membrane vesicles in an energy-dependent manner (21).The structural mechanics of ABC exporters was revealed from comparison of the MsbA crystal structures in the apo- and nucleotide-bound states as well as from analysis by spin labeling EPR spectroscopy in liposomes (17, 19, 22, 23). The energy harnessed from ATP binding and hydrolysis drives a cycle of NBD association and dissociation that is transmitted to induce reorientation of the TMD from an inward- to outward-facing conformation (17, 19, 22). Large amplitude motion closes the cytoplasmic end of a chamber found at the interface between the two TMDs and opens it to the periplasm (23). These rearrangements lead to significant changes in chamber hydration, which may drive substrate translocation (22).Substrate binding must precede energy input, otherwise the cycle is futile, wasting the energy of ATP hydrolysis without substrate extrusion (7). Consistent with this model, ATP binding reduces ABCB1 substrate affinity, potentially through binding site occlusion (24-26). Furthermore, the TMD substrate-binding event signals the NBD to stimulate ATP hydrolysis increasing transport efficiency (1, 27, 28). However, there is a paucity of information regarding the location of substrate binding, the transport pathway, and the structural basis of substrate recognition by ABC exporters. In vitro studies of MsbA substrate specificity identify a broad range of substrates that stimulate ATPase activity (29). In addition to the putative physiological substrates lipid A and lipopolysaccharide (LPS), the ABCB1 substrates Ilmofosine, {"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342, and verapamil differentially enhance ATP hydrolysis of MsbA (29, 30). Intrinsic MsbA tryptophan (Trp) fluorescence quenching by these putative substrate molecules provides further support of interaction (29).Extensive biochemical analysis of ABCB1 and LmrA provides a general model of substrate binding to ABC efflux exporters. This so-called “hydrophobic cleaner model” describes substrates binding from the inner leaflet of the bilayer and then translocating through the TMD (10, 31, 32). These studies also identified a large number of residues involved in substrate binding and selectivity (33). When these crucial residues are mapped onto the crystal structures of MsbA, a subset of homologous residues clusters to helices 3 and 6 lining the putative substrate pathway (34). Consistent with a role in substrate binding and specificity, simultaneous replacement of two serines (Ser-289 and Ser-290) in helix 6 of MsbA reduces binding and transport of ethidium and taxol, although {"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342 and erythromycin interactions remain unaffected (34).The tendency of lipophilic substrates to partition into membranes confounds direct analysis of substrate interactions with ABC exporters (35, 36). Such partitioning may promote dynamic collisions with exposed Trp residues and nonspecific cross-linking in photo-affinity labeling experiments. In this study, we utilize a site-specific quenching approach to identify residues in the vicinity of the daunorubicin (DNR)-binding site (37). Although the data on DNR stimulation of ATP hydrolysis is inconclusive (20, 29, 30), the quenching of MsbA Trp fluorescence suggests a specific interaction. Spin labels were introduced along transmembrane helices 3, 4, and 6 of MsbA to assess their ATP-dependent quenching of DNR fluorescence. Residues that quench DNR cluster along the cytoplasmic end of helices 3 and 6 consistent with specific binding of DNR. Furthermore, many of these residues are not lipid-exposed but face the putative substrate chamber formed between the two TMDs. These residues are proximal to two Trps, which likely explains the previously reported quenching (29). Our results suggest DNR partitions to the membrane and then binds MsbA in a manner consistent with the hydrophobic cleaner model. Interpretation in the context of the crystal structures of MsbA identifies a putative translocation pathway through the transmembrane segment.     

The Endoplasmic Reticulum Enzyme DGAT2 Is Found in Mitochondria-associated Membranes and Has a Mitochondrial Targeting Signal That Promotes Its Association with Mitochondria

The synthesis and storage of neutral lipids in lipid droplets is a fundamental property of eukaryotic cells, but the spatial organization of this process is poorly understood. Here we examined the intracellular localization of acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2), an enzyme that catalyzes the final step of triacylglycerol (TG) synthesis in eukaryotes. We found that DGAT2 expressed in cultured cells localizes to the endoplasmic reticulum (ER) under basal conditions. After providing oleate to drive TG synthesis, DGAT2 also localized to near the surface of lipid droplets, where it co-localized with mitochondria. Biochemical fractionation revealed that DGAT2 is present in mitochondria-associated membranes, specialized domains of the ER that are highly enriched in lipid synthetic enzymes and interact tightly with mitochondria. The interaction of DGAT2 with mitochondria depended on 67 N-terminal amino acids of DGAT2, which are not conserved in family members that have different catalytic functions. This targeting signal was sufficient to localize a red fluorescent protein to mitochondria. A highly conserved, positively charged, putative mitochondrial targeting signal was identified in murine DGAT2 between amino acids 61 and 66. Thus, DGAT2, an ER-resident transmembrane domain-containing enzyme, is also found in mitochondria-associated membranes, where its N terminus may promote its association with mitochondria.Most eukaryotic cells can synthesize neutral lipids, such as triacylglycerols (TGs)² and sterol esters, and store them in cytosolic lipid droplets. Yet, a molecular understanding of this process and how it is spatially organized is lacking. For example, lipid substrates for TG synthesis (fatty acids and glycerolipid precursors) are found in the cytoplasm and membranes, energy for activating fatty acids (by converting to fatty acyl-CoA) comes from mitochondria, and the enzymes that catalyze TG formation are primarily found in the mitochondria and endoplasmic reticulum (ER). How the cell orchestrates this complex anabolic process to maximize lipid synthesis and storage during times of substrate excess is poorly understood.In most cells, TG synthesis occurs via the glycerol 3-phosphate (Kennedy) pathway and involves multiple enzymatic reactions in different subcellular compartments (1). The fatty acids for TG synthesis must first be “activated” by acyl-CoA synthases, a family of enzymes that localize to membranes of different compartments, including the ER, mitochondria, and plasma membrane (2), and utilize ATP to ligate CoA to the fatty acyl chain. Next, these fatty acids enter the Kennedy pathway of glycerolipid synthesis, in which the first two reactions occur in both the ER and mitochondria. In the first reaction, glycerol 3-phosphate and a fatty acyl-CoA are combined to yield lysophosphatidic acid through the actions of glycerol-3-phosphate acyltransferase enzymes (1, 3). In the second reaction, 1-acylglycerol-3-phosphate O-acyltransferase enzymes catalyze the esterification of lysophosphatidic acid with fatty acyl-CoA to form phosphatidic acid (1, 4). Next, phosphatidic acid is dephosphorylated at membrane surfaces by phosphatidate phosphatase to yield diacylglycerol (1, 5, 6). All these steps are highly organized spatially, which is likely to be important for the efficiency of the pathway.The final reaction of TG synthesis is catalyzed by acyl-CoA: diacylglycerol acyltransferase (DGAT) enzymes (7-9). The two mammalian DGATs, DGAT1 and DGAT2 (10, 11), which are encoded by genes of different families, have distinct roles in TG synthesis (12). DGAT2 is the major TG biosynthetic enzyme in eukaryotes. Dgat2-deficient mice die shortly after birth and are almost completely devoid of TG (13), indicating an essential requirement for DGAT2. Catalysis of TG synthesis is conserved in the DGAT2 gene family, with functional orthologs in many species, including Dga1p in Saccharomyces cerevisiae, which contributes to a major portion of TG synthesis (14-16).Little is known about the intracellular localization of DGAT enzymes. DGAT activity is present in microsomes (7, 17, 18), but in vitro assays do not distinguish between DGAT1 and DGAT2. A DGAT2-green fluorescent fusion protein expressed in HeLa cells localized to the ER (19), and Dga1p activity in S. cerevisiae localizes to the ER and lipid droplets (16). DGAT1 and DGAT2 expressed in COS-7 cells localized primarily to the ER (20). A recent study of the subcellular localizations of tung tree DGAT1 and DGAT2 in tobacco BY-2 cells revealed that the enzymes are located in distinct, non-overlapping regions of the ER (21). Most recently, DGAT2 was reported to co-localize with lipid droplets in cultured adipocytes (22). As a step toward a better understanding of the cellular organization of processes that contribute to TG synthesis and storage, we determined the subcellular localization of murine DGAT2 in mammalian cells.     

Loss of PINK1 Function Promotes Mitophagy through Effects on Oxidative Stress and Mitochondrial Fission

Mitochondrial dysregulation is strongly implicated in Parkinson disease. Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is neuroprotective, less is known about neuronal responses to loss of PINK1 function. We found that stable knockdown of PINK1 induced mitochondrial fragmentation and autophagy in SH-SY5Y cells, which was reversed by the reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1. Moreover, stable or transient overexpression of wild-type PINK1 increased mitochondrial interconnectivity and suppressed toxin-induced autophagy/mitophagy. Mitochondrial oxidant production played an essential role in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines. Autophagy/mitophagy served a protective role in limiting cell death, and overexpressing Parkin further enhanced this protective mitophagic response. The dominant negative Drp1 mutant inhibited both fission and mitophagy in PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting oxidative stress, suggesting active involvement of autophagy in morphologic remodeling of mitochondria for clearance. To summarize, loss of PINK1 function elicits oxidative stress and mitochondrial turnover coordinated by the autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may cooperate through different mechanisms to maintain mitochondrial homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects ∼1% of the population worldwide. The causes of sporadic cases are unknown, although mitochondrial or oxidative toxins such as 1-methyl-4-phenylpyridinium, 6-hydroxydopamine (6-OHDA),³ and rotenone reproduce features of the disease in animal and cell culture models (1). Abnormalities in mitochondrial respiration and increased oxidative stress are observed in cells and tissues from parkinsonian patients (2, 3), which also exhibit increased mitochondrial autophagy (4). Furthermore, mutations in parkinsonian genes affect oxidative stress response pathways and mitochondrial homeostasis (5). Thus, disruption of mitochondrial homeostasis represents a major factor implicated in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD encodes for PTEN-induced kinase 1 (PINK1) (6, 7). PINK1 is a cytosolic and mitochondrially localized 581-amino acid serine/threonine kinase that possesses an N-terminal mitochondrial targeting sequence (6, 8). The primary sequence also includes a putative transmembrane domain important for orientation of the PINK1 domain (8), a conserved kinase domain homologous to calcium calmodulin kinases, and a C-terminal domain that regulates autophosphorylation activity (9, 10). Overexpression of wild-type PINK1, but not its PD-associated mutants, protects against several toxic insults in neuronal cells (6, 11, 12). Mitochondrial targeting is necessary for some (13) but not all of the neuroprotective effects of PINK1 (14), implicating involvement of cytoplasmic targets that modulate mitochondrial pathobiology (8). PINK1 catalytic activity is necessary for its neuroprotective role, because a kinase-deficient K219M substitution in the ATP binding pocket of PINK1 abrogates its ability to protect neurons (14). Although PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated mutations differentially destabilize the protein, resulting in loss of neuroprotective activities (13, 15).Recent studies indicate that PINK1 and Parkin interact genetically (3, 16-18) to prevent oxidative stress (19, 20) and regulate mitochondrial morphology (21). Primary cells derived from PINK1 mutant patients exhibit mitochondrial fragmentation with disorganized cristae, recapitulated by RNA interference studies in HeLa cells (3).Mitochondria are degraded by macroautophagy, a process involving sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs) for delivery to lysosomes (22, 23). Interestingly, mitochondrial fission accompanies autophagic neurodegeneration elicited by the PD neurotoxin 6-OHDA (24, 25). Moreover, mitochondrial fragmentation and increased autophagy are observed in neurodegenerative diseases including Alzheimer and Parkinson diseases (4, 26-28). Although inclusion of mitochondria in autophagosomes was once believed to be a random process, as observed during starvation, studies involving hypoxia, mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic substrates in facultative anaerobes support the concept of selective mitochondrial autophagy (mitophagy) (29, 30). In particular, mitochondrially localized kinases may play an important role in models involving oxidative mitochondrial injury (25, 31, 32).Autophagy is involved in the clearance of protein aggregates (33-35) and normal regulation of axonal-synaptic morphology (36). Chronic disruption of lysosomal function results in accumulation of subtly impaired mitochondria with decreased calcium buffering capacity (37), implicating an important role for autophagy in mitochondrial homeostasis (37, 38). Recently, Parkin, which complements the effects of PINK1 deficiency on mitochondrial morphology (3), was found to promote autophagy of depolarized mitochondria (39). Conversely, Beclin 1-independent autophagy/mitophagy contributes to cell death elicited by the PD toxins 1-methyl-4-phenylpyridinium and 6-OHDA (25, 28, 31, 32), causing neurite retraction in cells expressing a PD-linked mutation in leucine-rich repeat kinase 2 (40). Whereas properly regulated autophagy plays a homeostatic and neuroprotective role, excessive or incomplete autophagy creates a condition of “autophagic stress” that can contribute to neurodegeneration (28).As mitochondrial fragmentation (3) and increased mitochondrial autophagy (4) have been described in human cells or tissues of PD patients, we investigated whether or not the engineered loss of PINK1 function could recapitulate these observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous PINK1 gave rise to mitochondrial fragmentation and increased autophagy and mitophagy, whereas stable or transient overexpression of PINK1 had the opposite effect. Autophagy/mitophagy was dependent upon increased mitochondrial oxidant production and activation of fission. The data indicate that PINK1 is important for the maintenance of mitochondrial networks, suggesting that coordinated regulation of mitochondrial dynamics and autophagy limits cell death associated with loss of PINK1 function.     

TRAM1 Participates in Human Cytomegalovirus US2- and US11-mediated Dislocation of an Endoplasmic Reticulum Membrane Glycoprotein

The human cytomegalovirus proteins US2 and US11 have co-opted endoplasmic reticulum (ER) quality control to facilitate the destruction of major histocompatibility complex class I heavy chains. The class I heavy chains are dislocated from the ER to the cytosol, where they are deglycosylated and subsequently degraded by the proteasome. We examined the role of TRAM1 (translocating chain-associated membrane protein-1) in the dislocation of class I molecules using US2- and US11-expressing cells. TRAM1 is an ER protein initially characterized for its role in processing nascent polypeptides. Co-immunoprecipitation studies demonstrated that TRAM1 can complex with the wild type US2 and US11 proteins as well as deglycosylated and polyubiquitinated class I degradation intermediates. In studies using US2- and US11-TRAM1 knockdown cells, we observed an increase in levels of class I heavy chains. Strikingly, increased levels of glycosylated heavy chains were observed in TRAM1 knockdown cells when compared with control cells in a pulse-chase experiment. In fact, US11-mediated class I dislocation was more sensitive to the lack of TRAM1 than US2. These results provide further evidence that these viral proteins may utilize distinct complexes to facilitate class I dislocation. For example, US11-mediated class I heavy chain degradation requires Derlin-1 and SEL1L, whereas signal peptide peptidase is critical for US2-induced class I destabilization. In addition, TRAM1 can complex with the dislocation factors Derlin-1 and signal peptide peptidase. Collectively, the data support a model in which TRAM1 functions as a cofactor to promote efficient US2- and US11-dependent dislocation of major histocompatibility complex class I heavy chains.HCMV² can down-regulate cell surface expression of the immunologically important molecule major histocompatibility complex class I to avoid immune detection by cytotoxic T cells (1, 2). More specifically, the HCMV US2 and US11 gene products alone can target the ER-localized major histocompatibility complex class I heavy chains for extraction across the ER membrane by a process referred to as dislocation or retrograde translocation. The N-linked glycan is then removed upon exposure to the cytosol by N-glycanase (3), followed by proteasomal destruction (4, 5). The HCMV US2 and US11 proteins utilize the ER quality control process to eliminate class I heavy cells in a similar manner as misfolded or damaged ER proteins (e.g. genetic mutants of α₁-antitrypsin (6) and the cystic fibrosis transmembrane conductance regulator protein (7)) are targeted for degradation (8). Hence, analysis of US2- and US11-mediated destruction of class I heavy chains provides an excellent system to delineate viral protein function as well as the ER quality control process.ER and cytosolic proteins are required for US2- and US11-mediated dislocation/degradation of class I heavy chains. Some of these proteins have also been identified in the processing of aberrant ER polypeptides. The ER chaperones calnexin, calreticulin, and BiP have been implicated in US2-mediated class I destruction (9) as well as in the removal of some misfolded ER proteins (10). The ubiquitination machinery also participates in the extraction of class I heavy chains as ubiquitinated heavy chains are observed prior to dislocation (11, 12). For misfolded ER degradation substrates, ubiquitin conjugation enzymes (e.g. Ubc6p and Ubc7p/Cue1p) and ubiquitin ligases Hrd1p/Der3p, Doa10p, and Ubc1p have been implicated in the dislocation reaction (8). Interestingly, the ER membrane protein Derlin-1 along with SEL1L are involved in US11-mediated class I heavy chain degradation (13-15), whereas SPP is critical for US2-induced class I destabilization (16). The ubiquitinated substrates are dislocated by the AAA-ATPase complex composed of p97-Ufd1-Npl4 (17) while docked to the ER through its interaction with VIMP (14) followed by proteasome destruction. The inhibition of the proteasome causes the accumulation of deglycosylated class I heavy chain intermediate in US2 and US11 cells, allowing the dislocation and degradation reactions to be studied as separate processes (4, 5).Despite the identification of some cellular proteins that assist US2- and US11-mediated class I dislocation, the dislocation pore and accessory factors that mediate the efficient extraction of class I through the bilayer have yet to be completely defined. The current study explores the role of TRAM1 (translocating chain-associated membrane protein-1) in US2- and US11-mediated class I dislocation. TRAM1 is an ER-resident multispanning membrane protein that can mediate the lateral movement of select signal peptides and transmembrane segments from the translocon into the membrane bilayer (18), a property that makes it uniquely qualified to participate in the dislocation of a membrane protein. TRAM1 has been cross-linked to signal peptides as well as transmembrane domains of nascent polypeptides during the early stages of protein processing (19-25). Interestingly, unlike the Sec61 complex and the signal recognition particle receptor, TRAM1 is not essential for the translocation of all membrane proteins into the ER (20, 21). Hence, TRAM1 may utilize its ability to engage hydrophobic domains to assist in the efficient dislocation of membrane proteins. In fact, association and TRAM1 knockdown studies demonstrate that TRAM1 participates in US2- and US11-mediated dislocation of class I heavy chains. Collectively, our data suggest for the first time that TRAM1 plays a role in the dislocation of a membrane glycoprotein.     

Membrane Permeabilization by Trypanosome Lytic Factor, a Cytolytic Human High Density Lipoprotein

Trypanosome lytic factor (TLF) is a subclass of human high density lipoprotein (HDL) that mediates an innate immune killing of certain mammalian trypanosomes, most notably Trypanosoma brucei brucei, the causative agent of a wasting disease in cattle. Mechanistically, killing is initiated in the lysosome of the target trypanosome where the acidic pH facilitates a membrane-disrupting activity by TLF. Here we utilize a model liposome system to characterize the membrane binding and permeabilizing activity of TLF and its protein constituents, haptoglobin-related protein (Hpr), apolipoprotein L-1 (apoL-1), and apolipoprotein A-1 (apoA-1). We show that TLF efficiently binds and permeabilizes unilamellar liposomes at lysosomal pH, whereas non-lytic human HDL exhibits inefficient permeabilizing activity. Purified, delipidated Hpr and apoL-1 both efficiently permeabilize lipid bilayers at low pH. Trypanosome lytic factor, apoL-1, and apoA-1 exhibit specificity for anionic membranes, whereas Hpr permeabilizes both anionic and zwitterionic membranes. Analysis of the relative particle sizes of susceptible liposomes reveals distinctly different membrane-active behavior for native TLF and the delipidated protein components. We propose that lysosomal membrane damage in TLF-susceptible trypanosomes is initiated by the stable association of the TLF particle with the lysosomal membrane and that this is a property unique to this subclass of human HDL.High density lipoproteins (HDL)² are complex yet ordered macromolecules consisting of characteristic proteins embedded in a phospholipid monolayer that surrounds a hydrophobic core of esterified cholesterol and triglycerides. A subclass of HDL is responsible for an innate immune killing of the African blood stream parasite Trypanosoma brucei brucei (1–3), and very recently, has been shown to be cytotoxic to intracellular Leishmania promastigotes (4). The trypanolytic HDL particle, termed trypanosome lytic factor (TLF), is characterized by the presence of two proteins, apolipoprotein L-1 (apoL-1) and haptoglobin-related protein (Hpr), as well as the HDL ubiquitous apolipoprotein A-1 (apoA-1) (1, 5–7). Killing of the susceptible parasite involves high affinity binding to a cell-surface receptor, endocytosis, and trafficking of the TLF particle to the lysosome (8–12). The acidic lysosomal environment facilitates a membrane-disrupting activity by the TLF particle and subsequent cell death (9, 13). It has been shown that purified, delipidated apoL-1 or Hpr are sufficient for trypanosome killing. When these proteins are incorporated into the same lipoprotein particle, a several hundredfold increase in killing activity is exhibited (5). In addition, Molina-Portela et al. (14) show that maximal protection against T. b. brucei in a transgenic mouse model requires the expression of human Hpr, apoL-1, and apoA-1, supporting a synergistic mode of action.Haptoglobin-related protein evolved during primate evolution and is restricted to apes, old world monkeys, and humans (15). Haptoglobin-related protein is highly similar (92%) to the acute phase serum protein haptoglobin (Hp) (16). All mammals use Hp as a scavenger of hemoglobin (Hb) released during hemolysis associated with infection or trauma. Haptoglobin binds cell-free Hb with high affinity and facilitates its removal from the circulation through a receptor-mediated process in the liver (17). Like Hp, Hpr binds free Hb, yet this Hpr·Hb complex is not recognized by the requisite receptors in mammals and is thus not removed from the circulation (18). TLF uptake by susceptible trypanosomes requires specific binding to an Hpr·Hb complex that facilitates trafficking of the TLF particle to the lysosome (10). It has been proposed that once inside the lysosomal compartment, Hpr·Hb contributes directly to membrane disruption through the generation of oxygen radicals with the bound Hb providing the iron necessary for Fenton chemistry (7, 10, 19).Apolipoprotein L-1 is a unique member of the apolipoprotein L protein family in that, unlike the remaining apoL proteins, it possesses an N-terminal signal sequence and is thus secreted from cells. As is the case for Hpr, apoL-1 appeared during primate evolution (20–22). Within the circulation of primates, apoL-1 is exclusively associated with HDL, and the majority of the protein is in the TLF subclass (5). The apoL family members are all predicted to adopt amphipathic α-helical conformations, suggesting that their physiological role involves membrane interaction (20). Apolipoprotein L-1 shares limited homology with channel-forming colicins and, consistent with this observation, has been shown to function as an ion channel when incorporated into lipid bilayers (23).The ultimate fate of TLF-targeted lysosomal membranes is not firmly established. Several studies employing both in vivo cellular analysis and artificial membrane systems address this point with conflicting results. Electron microscopy studies with gold-conjugated TLF revealed accumulation of TLF in intracellular vesicles and subsequent vesicle membrane breakdown and appearance of gold particles in the cytoplasm (9). Widener et al. (10) observed efflux of lysosomally localized large molecular mass dextrans (500 kDa) in TLF-treated T. b. brucei. These data suggest that the lysosomal membrane experiences large scale disruption. In contrast, Perez-Morga et al. (23) and Vanhollebeke et al. (24) report uncontrollable lysosomal swelling in susceptible trypanosomes treated with normal human serum, suggesting stability of the lamellar structure of the lysosomal membrane after TLF attack. Swelling is attributed to apoL-1-mediated influx of Cl^– ions and concomitant osmotic flow of water into the lysosome. However, Molina-Portela et al. (25) observed the formation of cation-selective pores in TLF-treated planar lipid bilayers composed of trypanosome lipids. The diversity of activities reported for TLF and normal human serum may reflect the packaging of multiple toxins within the same complex that can act synergistically to provide optimal killing activity (5, 14).Here we utilize model liposomes to monitor the membrane activity of TLF and its protein constituents. We describe the effects of TLF, delipidated Hpr, apoL-1, and apoA-1 on the permeability of unilamellar liposomes. Additionally, we show that TLF, apoL-1, and apoA-1 exhibit lipid specificity and that Hpr, apoL-1, and apoA-1 induce large scale changes in the geometry of liposomes. These results provide a molecular basis for the recognition of lysosomal membranes by this toxic HDL and support a multicomponent mechanism for trypanosome killing.     

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.     

Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5

NHE5 is a brain-enriched Na⁺/H⁺ exchanger that dynamically shuttles between the plasma membrane and recycling endosomes, serving as a mechanism that acutely controls the local pH environment. In the current study we show that secretory carrier membrane proteins (SCAMPs), a group of tetraspanning integral membrane proteins that reside in multiple secretory and endocytic organelles, bind to NHE5 and co-localize predominantly in the recycling endosomes. In vitro protein-protein interaction assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5 increased cell-surface abundance as well as transporter activity of NHE5 across the plasma membrane. Expression of a deletion mutant lacking the SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the N-terminal extension, reduced the transporter activity. Although both Arf6 and Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by dominant-negative Arf6 but not by dominant-negative Rab11. Together, these results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes and promotes its surface targeting in an Arf6-dependent manner.Neurons and glial cells in the central and peripheral nervous systems are especially sensitive to perturbations of pH (1). Many voltage- and ligand-gated ion channels that control membrane excitability are sensitive to changes in cellular pH (1-3). Neurotransmitter release and uptake are also influenced by cellular and organellar pH (4, 5). Moreover, the intra- and extracellular pH of both neurons and glia are modulated in a highly transient and localized manner by neuronal activity (6, 7). Thus, neurons and glia require sophisticated mechanisms to finely tune ion and pH homeostasis to maintain their normal functions.Na⁺/H⁺ exchangers (NHEs)³ were originally identified as a class of plasma membrane-bound ion transporters that exchange extracellular Na⁺ for intracellular H⁺, and thereby regulate cellular pH and volume. Since the discovery of NHE1 as the first mammalian NHE (8), eight additional isoforms (NHE2-9) that share 25-70% amino acid identity have been isolated in mammals (9, 10). NHE1-5 commonly exhibit transporter activity across the plasma membrane, whereas NHE6-9 are mostly found in organelle membranes and are believed to regulate organellar pH in most cell types at steady state (11). More recently, NHE10 was identified in human and mouse osteoclasts (12, 13). However, the cDNA encoding NHE10 shares only a low degree of sequence similarity with other known members of the NHE gene family, raising the possibility that this sodium-proton exchanger may belong to a separate gene family distantly related to NHE1-9 (see Ref. 9).NHE gene family members contain 12 putative transmembrane domains at the N terminus followed by a C-terminal cytosolic extension that plays a role in regulation of the transporter activity by protein-protein interactions and phosphorylation. NHEs have been shown to regulate the pH environment of synaptic nerve terminals and to regulate the release of neurotransmitters from multiple neuronal populations (14-16). The importance of NHEs in brain function is further exemplified by the findings that spontaneous or directed mutations of the ubiquitously expressed NHE1 gene lead to the progression of epileptic seizures, ataxia, and increased mortality in mice (17, 18). The progression of the disease phenotype is associated with loss of specific neuron populations and increased neuronal excitability. However, NHE1-null mice appear to develop normally until 2 weeks after birth when symptoms begin to appear. Therefore, other mechanisms may compensate for the loss of NHE1 during early development and play a protective role in the surviving neurons after the onset of the disease phenotype.NHE5 was identified as a unique member of the NHE gene family whose mRNA is expressed almost exclusively in the brain (19, 20), although more recent studies have suggested that NHE5 might be functional in other cell types such as sperm (21, 22) and osteosarcoma cells (23). Curiously, mutations found in several forms of congenital neurological disorders such as spinocerebellar ataxia type 4 (24-26) and autosomal dominant cerebellar ataxia (27-29) have been mapped to chromosome 16q22.1, a region containing NHE5. However, much remains unknown as to the molecular regulation of NHE5 and its role in brain function.Very few if any proteins work in isolation. Therefore identification and characterization of binding proteins often reveal novel functions and regulation mechanisms of the protein of interest. To begin to elucidate the biological role of NHE5, we have started to explore NHE5-binding proteins. Previously, β-arrestins, multifunctional scaffold proteins that play a key role in desensitization of G-protein-coupled receptors, were shown to directly bind to NHE5 and promote its endocytosis (30). This study demonstrated that NHE5 trafficking between endosomes and the plasma membrane is regulated by protein-protein interactions with scaffold proteins. More recently, we demonstrated that receptor for activated C-kinase 1 (RACK1), a scaffold protein that links signaling molecules such as activated protein kinase C, integrins, and Src kinase (31), directly interacts with and activates NHE5 via integrin-dependent and independent pathways (32). These results further indicate that NHE5 is partly associated with focal adhesions and that its targeting to the specialized microdomain of the plasma membrane may be regulated by various signaling pathways.Secretory carrier membrane proteins (SCAMPs) are a family of evolutionarily conserved tetra-spanning integral membrane proteins. SCAMPs are found in multiple organelles such as the Golgi apparatus, trans-Golgi network, recycling endosomes, synaptic vesicles, and the plasma membrane (33, 34) and have been shown to play a role in exocytosis (35-38) and endocytosis (39). Currently, five isoforms of SCAMP have been identified in mammals. The extended N terminus of SCAMP1-3 contain multiple Asn-Pro-Phe (NPF) repeats, which may allow these isoforms to participate in clathrin coat assembly and vesicle budding by binding to Eps15 homology (EH)-domain proteins (40, 41). Further, SCAMP2 was shown recently to bind to the small GTPase Arf6 (38), which is believed to participate in traffic between the recycling endosomes and the cell surface (42, 43). More recent studies have suggested that SCAMPs bind to organellar membrane type NHE7 (44) and the serotonin transporter SERT (45) and facilitate targeting of these integral membrane proteins to specific intracellular compartments. We show in the current study that SCAMP2 binds to NHE5, facilitates the cell-surface targeting of NHE5, and elevates Na⁺/H⁺ exchange activity at the plasma membrane, whereas expression of a SCAMP2 deletion mutant lacking the N-terminal domain containing the NPF repeats suppresses the effect. Further we show that this activity of SCAMP2 requires an active form of a small GTPase Arf6, but not Rab11. We propose a model in which SCAMPs bind to NHE5 in the endosomal compartment and control its cell-surface abundance via an Arf6-dependent pathway.     

Tiam1 and Rac1 Are Required for Platelet-activating Factor-induced Endothelial Junctional Disassembly and Increase in Vascular Permeability

It is known that platelet-activating factor (PAF) induces severe endothelial barrier leakiness, but the signaling mechanisms remain unclear. Here, using a wide range of biochemical and morphological approaches applied in both mouse models and cultured endothelial cells, we addressed the mechanisms of PAF-induced disruption of interendothelial junctions (IEJs) and of increased endothelial permeability. The formation of interendothelial gaps filled with filopodia and lamellipodia is the cellular event responsible for the disruption of endothelial barrier. We observed that PAF ligation of its receptor induced the activation of the Rho GTPase Rac1. Following PAF exposure, both Rac1 and its guanine nucleotide exchange factor Tiam1 were found associated with a membrane fraction from which they co-immunoprecipitated with PAF receptor. In the same time frame with Tiam1-Rac1 translocation, the junctional proteins ZO-1 and VE-cadherin were relocated from the IEJs, and formation of numerous interendothelial gaps was recorded. Notably, the response was independent of myosin light chain phosphorylation and thus distinct from other mediators, such as histamine and thrombin. The changes in actin status are driven by the PAF-induced localized actin polymerization as a consequence of Rac1 translocation and activation. Tiam1 was required for the activation of Rac1, actin polymerization, relocation of junctional associated proteins, and disruption of IEJs. Thus, PAF-induced IEJ disruption and increased endothelial permeability requires the activation of a Tiam1-Rac1 signaling module, suggesting a novel therapeutic target against increased vascular permeability associated with inflammatory diseases.The endothelial barrier is made up of endothelial cells (ECs)⁴ connected to each other by interendothelial junctions (IEJs) consisting of protein complexes organized as tight junctions (TJs) and adherens junctions (AJs). In addition, the focal adhesion complex located at the basal plasma membrane enables firm contact of ECs with the underlying basement membrane and also contributes to the barrier function (1-3). The glycocalyx, the endothelial monolayer, and the basement membrane all together constitute the vascular barrier.The structural integrity of the ECs along with their proper functionality are the two most important factors controlling the tightness of the endothelial barrier. Changes affecting these factors cause loss of barrier restrictiveness and leakiness. Therefore, defining and understanding the cellular and molecular mechanisms controlling these processes is of paramount importance. Increased width of IEJs in response to permeability-increasing mediators (4) regulates the magnitude of transendothelial exchange of fluid and solutes. Disruption of IEJs and the resultant barrier leakiness contribute to the genesis of diverse pathological conditions, such as inflammation (5), metastasis (6, 7), and uncontrolled angiogenesis (8, 9).Accumulated evidence demonstrated that IEJs changes are responsible for increased or decreased vascular permeability, and the generally accepted mechanism responsible for them was the myosin light chain (MLC)-mediated contraction of ECs (5, 10). However, published evidence showed that an increase in vascular permeability could be obtained without a direct involvement of any contractile mechanism (11-16).The main component of the vascular barrier, the ECs, has more than 10% of their total protein represented by actin (17), which under physiological salt concentrations subsists as monomers (G-actin) and assembled into filaments (F-actin). A large number of actin-interacting proteins may modulate the assembly, disassembly, and organization of G-actin and of actin filaments within a given cell type. Similar to the complexity of actin-interacting proteins found in other cell types, the ECs utilize their actin binding proteins to stabilize the endothelial monolayer in order to efficiently function as a selective barrier (11). In undisturbed ECs, the actin microfilaments are organized as different networks with distinctive functional and morphological characteristics: the peripheral filaments also known as peripheral dense band (PDB), the cytoplasmic fibers identified as stress fibers (SF), and the actin from the membrane cytoskeleton (18). The peripheral web, localized immediately under the membrane, is associated with (i) the luminal plasmalemma (on the apical side), (ii) the IEJ complexes on the lateral surfaces, and (iii) the focal adhesion complexes on the abluminal side (the basal part) of polarized ECs. The SF reside inside the endothelial cytoplasm and are believed to be directly connected with the plasmalemma proper on the luminal as well as on the abluminal side of the cell. As described, the endothelial actin cytoskeleton (specifically the SF) seems to be a stable structure helping the cells to remain flat under flow (19). It is also established that the actin fibers participate in correct localization of different junctional complexes while keeping them in place (20). However, it was suggested that the dynamic equilibrium between F- and G-actin might modulate the tightness of endothelial barrier in response to different challenges (13).Mediators effective at nanomolar concentrations or less that disrupt the endothelial barrier and increase vascular permeability include C2 toxin of Clostridium botulinum, vascular permeability factor, better known as vascular endothelial growth factor, and PAF (21). C2 toxin increases endothelial permeability by ribosylating monomeric G-actin at Arg-177 (22). This results in the impairment of actin polymerization (23), followed by rounding of ECs (16) and the disruption of junctional integrity. Vascular permeability factor was shown to open IEJs by redistribution of junctional proteins (24, 25) and by interfering with the equilibrium of actin pools (26). PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocoline), a naturally synthesized phospholipid is active at 10^-10 m or less (27). PAF is synthesized by and acts on a variety of cell types, including platelets (28), neutrophils (29), monocytes (30), and ECs (31). PAF-mediated activation of ECs induced cell migration (32), angiogenesis (7), and vascular hyperpermeability (33) secondary to disassembly of IEJs (34). The effects of PAF on the endothelium are initiated through a G protein-coupled receptor (PAF-R) localized at the plasmalemma, in a large endosomal compartment inside the cell (34), and also in the nuclear membrane (35). In ECs, PAF-R was shown to signal through Gα_q and downstream activation of phospholipase C isozymes (PLCβ₃ and PLCγ₁), and via cSrc (32, 36). Studies have shown that PAF challenge induced endothelial actin cytoskeletal rearrangement (37) and marked vascular leakiness (38); however, the signaling pathways have not been elucidated.Therefore, in the present study, we carried out a systematic analysis of PAF-induced morphological and biochemical changes of endothelial barrier in vivo and in cultured ECs. We found that the opening of endothelial barrier and the increased vascular leakiness induced by PAF are the result of a shift in actin pools without involvement of EC contraction, followed by a redistribution of tight junctional associated protein ZO-1 and adherens junctional protein VE-cadherin.     

SCAMP5 Links Endoplasmic Reticulum Stress to the Accumulation of Expanded Polyglutamine Protein Aggregates via Endocytosis Inhibition

Accumulation of expanded polyglutamine proteins is considered to be a major pathogenic biomarker of Huntington disease. We isolated SCAMP5 as a novel regulator of cellular accumulation of expanded polyglutamine track protein using cell-based aggregation assays. Ectopic expression of SCAMP5 augments the formation of ubiquitin-positive and detergent-resistant aggregates of mutant huntingtin (mtHTT). Expression of SCAMP5 is markedly increased in the striatum of Huntington disease patients and is induced in cultured striatal neurons by endoplasmic reticulum (ER) stress or by mtHTT. The increase of SCAMP5 impairs endocytosis, which in turn enhances mtHTT aggregation. On the contrary, down-regulation of SCAMP5 alleviates ER stress-induced mtHTT aggregation and endocytosis inhibition. Moreover, stereotactic injection into the striatum and intraperitoneal injection of tunicamycin significantly increase mtHTT aggregation in the striatum of R6/2 mice and in the cortex of N171-82Q mice, respectively. Taken together, these results suggest that exposure to ER stress increases SCAMP5 in the striatum, which positively regulates mtHTT aggregation via the endocytosis pathway.The expansion of CAG repeats (usually beyond a critical threshold of ∼37 glutamine repeats) encoding polyglutamine (polyQ)³ causes, to date, nine late-onset progressive neurodegenerative disorders (1, 2). Expanded polyQ-containing huntingtin is the main aggregate component in the affected neurons (3). Also, molecular chaperones, such as Hsp70, Hsp40/HDJ1 (dHDJ1), and chaperonin TRiC, perturb the aggregation of polyQ track protein and reduce polyQ track cytotoxicity in yeast and cell lines (4–6) and in Drosophila and mouse models (4, 7). Thus, it seems that HD pathology is closely correlated with the accumulation of insoluble aggregates of mutant huntingtin (mtHTT) containing expanded polyQ (2, 3, 8, 9).Endoplasmic reticulum (ER) stress is crucial in many biological responses and is generated by various signals, such as unfolded protein response, aberrant calcium regulation, oxidative stress, and inflammation (10, 11). ER stress response is generally considered an adaptive reaction of cells to environmental stress, serving as a survival signal (10). On the other hand, increasing evidence also strengthens the importance of ER stress in human diseases. A malfunction or excess of ER stress response caused by aging, genetic mutations, and environmental insults is implicated in human diseases, such as Alzheimer disease, Parkinson disease, diabetes mellitus, and inflammation (12–16). mtHTT also induces ER stress at the early stage of HD, and pathogenic ER stress from an aging or stressful environment is severe at the late stage of HD (17–19). However, the molecular event linking the aggregation of polyQ track protein to ER stress response is unknown.The ubiquitin/proteasome pathway, a major protein degradation system, is altered or impaired in the cell culture model of HD (20–22). On the contrary, autophagy employing lysosomal degradation has been recently considered as a major clearance pathway of insoluble aggregates of polyQ track protein. Thus, inhibition of autophagy has been suggested to modulate the aggregate formation of mtHTT and to affect the toxicity of polyglutamine expansions in fly and mouse models of HD (23–25). However, a key molecule controlling the aggregation and clearance of polyQ track proteins needs to be identified.To further our understanding of the regulation of polyQ track protein aggregation, we screened human full-length cDNAs and isolated SCAMP5 (secretory carrier membrane protein 5) as a modulator of polyQ track protein aggregation. SCAMP5 is up-regulated by mtHTT and ER stress and functions to inhibit endocytosis to increase mtHTT aggregation.     

Intersectin Regulates Dendritic Spine Development and Somatodendritic Endocytosis but Not Synaptic Vesicle Recycling in Hippocampal Neurons

Intersectin-short (intersectin-s) is a multimodule scaffolding protein functioning in constitutive and regulated forms of endocytosis in non-neuronal cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of Drosophila and Caenorhabditis elegans. In vertebrates, alternative splicing generates a second isoform, intersectin-long (intersectin-l), that contains additional modular domains providing a guanine nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is expressed in multiple tissues and cells, including glia, but excluded from neurons, whereas intersectin-l is a neuron-specific isoform. Thus, intersectin-I may regulate multiple forms of endocytosis in mammalian neurons, including SV endocytosis. We now report, however, that intersectin-l is localized to somatodendritic regions of cultured hippocampal neurons, with some juxtanuclear accumulation, but is excluded from synaptophysin-labeled axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV recycling. Instead intersectin-l co-localizes with clathrin heavy chain and adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces the rate of transferrin endocytosis. The protein also co-localizes with F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation during development. Our data indicate that intersectin-l is indeed an important regulator of constitutive endocytosis and neuronal development but that it is not a prominent player in the regulated endocytosis of SVs.Clathrin-mediated endocytosis (CME)⁴ is a major mechanism by which cells take up nutrients, control the surface levels of multiple proteins, including ion channels and transporters, and regulate the coupling of signaling receptors to downstream signaling cascades (1-5). In neurons, CME takes on additional specialized roles; it is an important process regulating synaptic vesicle (SV) availability through endocytosis and recycling of SV membranes (6, 7), it shapes synaptic plasticity (8-10), and it is crucial in maintaining synaptic membranes and membrane structure (11).Numerous endocytic accessory proteins participate in CME, interacting with each other and with core components of the endocytic machinery such as clathrin heavy chain (CHC) and adaptor protein-2 (AP-2) through specific modules and peptide motifs (12). One such module is the Eps15 homology domain that binds to proteins bearing NPF motifs (13, 14). Another is the Src homology 3 (SH3) domain, which binds to proline-rich domains in protein partners (15). Intersectin is a multimodule scaffolding protein that interacts with a wide range of proteins, including several involved in CME (16). Intersectin has two N-terminal Eps15 homology domains that are responsible for binding to epsin, SCAMP1, and numb (17-19), a central coil-coiled domain that interacts with Eps15 and SNAP-23 and -25 (17, 20, 21), and five SH3 domains in its C-terminal region that interact with multiple proline-rich domain proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS (16, 22-25). The rich binding capability of intersectin has linked it to various functions from CME (17, 26, 27) and signaling (22, 28, 29) to mitogenesis (30, 31) and regulation of the actin cytoskeleton (23).Intersectin functions in SV recycling at the neuromuscular junction of Drosophila and C. elegans where it acts as a scaffold, regulating the synaptic levels of endocytic accessory proteins (21, 32-34). In vertebrates, the intersectin gene is subject to alternative splicing, and a longer isoform (intersectin-l) is generated that is expressed exclusively in neurons (26, 28, 35, 36). This isoform has all the binding modules of its short (intersectin-s) counterpart but also has additional domains: a DH and a PH domain that provide guanine nucleotide exchange factor (GEF) activity specific for Cdc42 (23, 37) and a C2 domain at the C terminus. Through its GEF activity and binding to actin regulatory proteins, including N-WASP, intersectin-l has been implicated in actin regulation and the development of dendritic spines (19, 23, 24). In addition, because the rest of the binding modules are shared between intersectin-s and -l, it is generally thought that the two intersectin isoforms have the same endocytic functions. In particular, given the well defined role for the invertebrate orthologs of intersectin-s in SV endocytosis, it is thought that intersectin-l performs this role in mammalian neurons, which lack intersectin-s. Defining the complement of intersectin functional activities in mammalian neurons is particularly relevant given that the protein is involved in the pathophysiology of Down syndrome (DS). Specifically, the intersectin gene is localized on chromosome 21q22.2 and is overexpressed in DS brains (38). Interestingly, alterations in endosomal pathways are a hallmark of DS neurons and neurons from the partial trisomy 16 mouse, Ts65Dn, a model for DS (39, 40). Thus, an endocytic trafficking defect may contribute to the DS disease process.Here, the functional roles of intersectin-l were studied in cultured hippocampal neurons. We find that intersectin-l is localized to the somatodendritic regions of neurons, where it co-localizes with CHC and AP-2 and regulates the uptake of transferrin. Intersectin-l also co-localizes with actin at dendritic spines and disrupting intersectin-l function alters dendritic spine development. In contrast, intersectin-l is absent from presynaptic terminals and has little or no role in SV recycling.     

Vesicular Stomatitis Virus Matrix Protein Mutations That Affect Association with Host Membranes and Viral Nucleocapsids

Viral matrix (M) proteins bind the nucleoprotein core (nucleocapsid) to host membranes during the process of virus assembly by budding. Previous studies using truncated M proteins had implicated the N-terminal 50 amino acids of the vesicular stomatitis virus M protein in binding both membranes and nucleocapsids and a sequence from amino acids 75-106 as an additional membrane binding region. Structure-based mutations were introduced into these two regions, and their effects on membrane association and incorporation into nucleocapsid-M protein complexes were determined using quantitative assays. The results confirmed that the N terminus of M protein is involved in association with plasma membranes as well as nucleocapsids, although these two activities were differentially affected by individual mutations. Mutations in the 75-106 region affected incorporation into nucleocapsid-M complexes but had only minor effects on association with membranes. The ability of site-specific mutant M proteins to complement growth of temperature-sensitive M mutant virus did not correlate well with the ability to associate with membranes or nucleocapsids, suggesting that complementation involves an additional activity of M protein. Mutants with similar abilities to associate with membranes and nucleocapsids but differing in complementation activity were incorporated into infectious cDNA clones. Infectious virus was repeatedly recovered containing mutant M proteins capable of complementation but was never recovered with mutant M proteins that lacked complementation activity, providing further evidence for a separate activity of M protein that is essential for virus replication.Most viruses that have a membrane or envelope as part of their structure acquire their envelopes by budding from the plasma membrane of the host cell. For budding to occur, the nucleoprotein core of the virus (nucleocapsid) must interact with the cytoplasmic surface of the host membrane. For many viruses this interaction is mediated by a matrix (M)² protein that binds to both the nucleocapsid and the host membrane (1, 2). Despite the similarity in the functions of viral M proteins, there is little structural or sequence similarity among the M proteins of different virus families (3). Thus, understanding the relationship of structure to function must be undertaken for individual M proteins before the general principles involved in virus budding can be understood. The goal of the experiments described here was to determine sequences in the M protein of vesicular stomatitis virus (VSV) involved in binding to membranes and nucleocapsids.VSV is the prototype member of the Rhabdoviridae family and has been widely studied to determine mechanisms involved in virus budding (2). The core of the virus contains an ∼11-kilobase negative-stranded RNA genome covered by 1300 copies of a single nucleocapsid protein (4). The nucleocapsid also contains lesser amounts of two proteins, P and L, which constitute the viral RNA-dependent RNA polymerase. The envelope contains a single species of transmembrane glycoprotein (G protein) that mediates virus attachment and entry into host cells. The virion contains ∼2000 copies of the M protein (4), which binds the nucleocapsid to the envelope and condenses the nucleocapsid into a tightly coiled helical nucleocapsid-M protein (NCM) complex that gives the virion its bullet-like shape (5-8). In cells infected with VSV and in transfected cells that express M protein in the absence of other VSV components, M protein is present both in a soluble form and bound to the cytoplasmic surface of the host plasma membrane (9-18). Mutagenesis studies, affinity labeling, and membrane reconstitution experiments have suggested that a combination of hydrophobic and ionic interactions mediate M protein binding to membranes by binding acidic phospholipids on the inner surface of the host plasma membrane (for review, see Ref. 19). Binding of M protein to nucleocapsids is less well understood than its binding to membranes. Most of the M protein in isolated NCM complexes is bound in a rapidly reversible equilibrium (20). However, M protein does not bind to nucleocapsids from which all of the M protein has been dissociated or to intracellular nucleocapsids that have never been assembled with M protein (11, 20). This suggests that binding of M protein to nucleocapsids in infected cells must be initiated in a separate step, after which most of the M protein is recruited into the NCM complex through the reversible binding step.M protein does not have separately folded domains that mediate binding to membranes versus nucleocapsids. The 229-amino acid (aa) M protein contains a positively charged N terminus (aa 1-50) that is highly exposed to proteolysis. The remainder of M protein (aa 51-229) is compactly folded to form a protease-resistant core (16, 21-23). The ability to obtain crystals of M protein required proteolytic removal of both the N-terminal sequence (aa 1-47) and a hydrophobic sequence (aa 121-124) to prevent M protein self-association (21, 22); however, the resulting structure showed a single-domain fold for the crystallized portion of M. In the present study we focused on two regions of the M protein structure that had been suggested to be involved in binding to either membranes or nucleocapsids; 1) previous data had implicated the N-terminal sequence in binding to both nucleocapsids and membranes (9, 10, 16, 22-25) and 2) deletion mutagenesis studies had implicated an additional region from aa 75-106 in membrane binding (16).In the experiments described here, M protein sequence substitutions were made using a scanning approach in the N-terminal sequence, and substitutions were based on the crystal structure in the 75-106-aa region. These mutants were used to determine the specific amino acids involved in these interactions. The results confirm that the N terminus of M protein is involved in association with plasma membranes as well as nucleocapsids, although these two activities are differentially affected by individual mutations. Mutations in the 75-106-aa region affected incorporation into NCM complexes but had only minor effects on association with membranes. Furthermore, the ability of mutant M proteins to function in the context of virus infection suggested that a new activity of M protein that is separate from its ability to associate with membranes or NCM complexes is critical for virus assembly.     

VCP Mutations Causing Frontotemporal Lobar Degeneration Disrupt Localization of TDP-43 and Induce Cell Death

Frontotemporal lobar degeneration (FTLD) with inclusion body myopathy and Paget disease of bone is a rare, autosomal dominant disorder caused by mutations in the VCP (valosin-containing protein) gene. The disease is characterized neuropathologically by frontal and temporal lobar atrophy, neuron loss and gliosis, and ubiquitin-positive inclusions (FTLD-U), which are distinct from those seen in other sporadic and familial FTLD-U entities. The major component of the ubiquitinated inclusions of FTLD with VCP mutation is TDP-43 (TAR DNA-binding protein of 43 kDa). TDP-43 proteinopathy links sporadic amyotrophic lateral sclerosis, sporadic FTLD-U, and most familial forms of FTLD-U. Understanding the relationship between individual gene defects and pathologic TDP-43 will facilitate the characterization of the mechanisms leading to neurodegeneration. Using cell culture models, we have investigated the role of mutant VCP in intracellular trafficking, proteasomal function, and cell death and demonstrate that mutations in the VCP gene 1) alter localization of TDP-43 between the nucleus and cytosol, 2) decrease proteasome activity, 3) induce endoplasmic reticulum stress, 4) increase markers of apoptosis, and 5) impair cell viability. These results suggest that VCP mutation-induced neurodegeneration is mediated by several mechanisms.Frontotemporal lobar degeneration (FTLD)² accounts for 10% of all late onset dementias and is the third most frequent neurodegenerative disease after Alzheimer disease and dementia with Lewy bodies (1). FTLD with ubiquitin-immunoreactive inclusions is genetically, clinically, and neuropathologically heterogeneous (2, 3). FTLD-U comprises several distinct entities, including sporadic forms and familial cases caused by mutations in the genes encoding VCP (valosin-containing protein), GRN (progranulin), CHMP2B (charged multivesicular body protein 2B), TDP-43 (TAR DNA-binding protein of 43 kDa) and an unknown gene linked to chromosome 9 (2, 3). Frontotemporal dementia with inclusion body myopathy and Paget disease of bone is a rare, autosomal dominant disorder caused by mutations in the VCP gene located on chromosome 9p13-p12 (4-10) (Fig. 1). This multisystem disease is characterized by progressive muscle weakness and atrophy, increased osteoclastic bone resorption, and early onset frontotemporal dementia, also called FTLD (9, 11). Mutations in VCP are also associated with dilatative cardiomyopathy with ubiquitin-positive inclusions (12). Neuropathologic features of FTLD with VCP mutation include frontal and temporal lobar atrophy, neuron loss and gliosis, and ubiquitin-positive inclusions (FTLD-U). The majority of aggregates are ubiquitin- and TDP-43-positive neuronal intranuclear inclusions (NIIs); a smaller proportion is made up of TDP-43-immunoreactive dystrophic neurites (DNs) and neuronal cytoplasmic inclusions (NCIs). A small number of inclusions are VCP-immunoreactive (5, 13). Pathologic TDP-43 in inclusions links a spectrum of diseases in which TDP-43 pathology is a primary feature, including FTLD-U, motor neuron disease, including amyotrophic lateral sclerosis, FTLD with motor neuron disease, and inclusion body myopathy and Paget disease of bone, as well as an expanding spectrum of other disorders in which TDP-43 pathology is secondary (14, 15).Open in a separate windowFIGURE 1.Model of pathogenic mutations and domains in valosin-containing protein. CDC48 (magenta), located within the N terminus (residues 22-108), binds the following cofactors: p47, gp78, and Npl4-Ufd1 (23-25, 28). There are two AAA-ATPase domains (AAA; blue) at residues 240-283 and 516-569, which are joined by two linker regions (L1 and L2; red).TDP-43 proteinopathy in FTLD with VCP mutation has a biochemical signature similar to that seen in other sporadic and familial cases of FTLD-U, including sporadic amyotrophic lateral sclerosis, FTLD-motor neuron disease, FTLD with progranulin (GRN) mutation, and FTLD linked to chromosome 9p (3, 16). TDP-43 proteinopathy in these disorders is characterized by hyperphosphorylation of TDP-43, ubiquitination, and cleavage to form C-terminal fragments detected only in insoluble brain extracts from affected brain regions (16). Identification of TDP-43 as the major component of the ubiquitin-immunoreactive inclusions of FTLD with VCP mutation supports the hypothesis that VCP gene mutations cause an alteration of VCP function, leading to TDP-43 proteinopathy.VCP/p97 (valosin-containing protein) is a member of the AAA (ATPase associated with diverse cellular activities) superfamily. The N-terminal domain of VCP has been shown to be involved in cofactor binding (CDC48 (cell division cycle protein 48)) and two AAA-ATPase domains that form a hexameric complex (Fig. 1) (17). Recently, it has been shown that the N-terminal domain of VCP binds phosphoinositides (18, 19). AKT (activated serine-threonine protein kinase) phosphorylates VCP and is required for constitutive VCP function (20, 21). AKT is activated through phospholipid binding and phosphorylation via the phosphoinositide 3-kinase signaling pathway, which is involved in cell survival (22). The lipid binding domain may recruit VCP to the cell membrane where it is phosphorylated by AKT (19).The diversity of VCP functions is modulated, in part, by a variety of intracellular cofactors, including p47, gp78, and Npl4-Ufd1 (23). Cofactor p47 has been shown to play a role in the maintenance and biogenesis of both the endoplasmic reticulum (ER) and Golgi apparatus (24). The structure of p47 contains a ubiquitin regulatory X domain that binds the N-terminus of VCP, and together they act as a chaperone to deliver membrane fusion machinery to the site of adjacent membranes (25). The function of the p47-VCP complex is dependent upon cell division cycle 2 (CDC2) serine-threonine kinase phosphorylation of p47 (26, 27). Also, VCP has been found to interact with the cytosolic tail of gp78, an ER membrane-spanning E3 ubiquitin ligase that exclusively binds VCP and enhances ER-associated degradation (ERAD) (28). The Npl4-Ufd1-VCP complex is involved in nuclear envelope assembly and targeting of proteins through the ubiquitin-proteasome system (29, 30). The cell survival response of this complex has been found to be important in DNA damage repair though activation by phosphorylation and its recruitment to double-stranded breaks (20, 31). The Npl4-Ufd1-VCP cytosolic complex is also recruited to the ER membrane, interacting with Derlin 1, VCP-interacting membrane proteins (VIMP), and other complexes. At the ER membrane, these misfolded proteins are targeted to the proteasome via ERAD (32-34). VCP also targets IKKβ for ubiquitination to the ubiquitin-proteasome system, implicating VCP in the cell survival pathway and neuroprotection (21, 35-37).To investigate the mechanism of neurodegeneration caused by VCP mutations, we first tested the hypothesis that VCP mutations decrease cell viability in vitro using a neuroblastoma SHSY-5Y cell line and then investigated cellular pathways that are known to lead to neurodegeneration, including decrease in proteasome activity, caspase-mediated degeneration, and a change in cellular localization of TDP-43.     

Engineered Protein Connectivity to Actin Mimics PDZ-dependent Recycling of G Protein-coupled Receptors but Not Its Regulation by Hrs

Many G protein-coupled receptors (GPCRs) recycle after agonist-induced endocytosis by a sequence-dependent mechanism, which is distinct from default membrane flow and remains poorly understood. Efficient recycling of the β2-adrenergic receptor (β2AR) requires a C-terminal PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (PDZbd), an intact actin cytoskeleton, and is regulated by the endosomal protein Hrs (hepatocyte growth factor-regulated substrate). The PDZbd is thought to link receptors to actin through a series of protein interaction modules present in NHERF/EBP50 (Na⁺/H+ exchanger 3 regulatory factor/ezrin-binding phosphoprotein of 50 kDa) family and ERM (ezrin/radixin/moesin) family proteins. It is not known, however, if such actin connectivity is sufficient to recapitulate the natural features of sequence-dependent recycling. We addressed this question using a receptor fusion approach based on the sufficiency of the PDZbd to promote recycling when fused to a distinct GPCR, the δ-opioid receptor, which normally recycles inefficiently in HEK293 cells. Modular domains mediating actin connectivity promoted receptor recycling with similarly high efficiency as the PDZbd itself, and recycling promoted by all of the domains was actin-dependent. Regulation of receptor recycling by Hrs, however, was conferred only by the PDZbd and not by downstream interaction modules. These results suggest that actin connectivity is sufficient to mimic the core recycling activity of a GPCR-linked PDZbd but not its cellular regulation.G protein-coupled receptors (GPCRs)² comprise the largest family of transmembrane signaling receptors expressed in animals and transduce a wide variety of physiological and pharmacological information. While these receptors share a common 7-transmembrane-spanning topology, structural differences between individual GPCR family members confer diverse functional and regulatory properties (1-4). A fundamental mechanism of GPCR regulation involves agonist-induced endocytosis of receptors via clathrin-coated pits (4). Regulated endocytosis can have multiple functional consequences, which are determined in part by the specificity with which internalized receptors traffic via divergent downstream membrane pathways (5-7).Trafficking of internalized GPCRs to lysosomes, a major pathway traversed by the δ-opioid receptor (δOR), contributes to proteolytic down-regulation of receptor number and produces a prolonged attenuation of subsequent cellular responsiveness to agonist (8, 9). Trafficking of internalized GPCRs via a rapid recycling pathway, a major route traversed by the β2-adrenergic receptor (β2AR), restores the complement of functional receptors present on the cell surface and promotes rapid recovery of cellular signaling responsiveness (6, 10, 11). When co-expressed in the same cells, the δOR and β2AR are efficiently sorted between these divergent downstream membrane pathways, highlighting the occurrence of specific molecular sorting of GPCRs after endocytosis (12).Recycling of various integral membrane proteins can occur by default, essentially by bulk membrane flow in the absence of lysosomal sorting determinants (13). There is increasing evidence that various GPCRs, such as the β2AR, require distinct cytoplasmic determinants to recycle efficiently (14). In addition to requiring a cytoplasmic sorting determinant, sequence-dependent recycling of the β2AR differs from default recycling in its dependence on an intact actin cytoskeleton and its regulation by the conserved endosomal sorting protein Hrs (hepatocyte growth factor receptor substrate) (11, 14). Compared with the present knowledge regarding protein complexes that mediate sorting of GPCRs to lysosomes (15, 16), however, relatively little is known about the biochemical basis of sequence-directed recycling or its regulation.The β2AR-derived recycling sequence conforms to a canonical PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (henceforth called PDZbd), and PDZ-mediated protein association(s) with this sequence appear to be primarily responsible for its endocytic sorting activity (17-20). Fusion of this sequence to the cytoplasmic tail of the δOR effectively re-routes endocytic trafficking of engineered receptors from lysosomal to recycling pathways, establishing the sufficiency of the PDZbd to function as a transplantable sorting determinant (18). The β2AR-derived PDZbd binds with relatively high specificity to the NHERF/EBP50 family of PDZ proteins (21, 22). A well-established biochemical function of NHERF/EBP50 family proteins is to associate integral membrane proteins with actin-associated cytoskeletal elements. This is achieved through a series of protein-interaction modules linking NHERF/EBP50 family proteins to ERM (ezrin-radixin-moesin) family proteins and, in turn, to actin filaments (23-26). Such indirect actin connectivity is known to mediate other effects on plasma membrane organization and function (23), however, and NHERF/EBP50 family proteins can bind to additional proteins potentially important for endocytic trafficking of receptors (23, 25). Thus it remains unclear if actin connectivity is itself sufficient to promote sequence-directed recycling of GPCRs and, if so, if such connectivity recapitulates the normal cellular regulation of sequence-dependent recycling. In the present study, we took advantage of the modular nature of protein connectivity proposed to mediate β2AR recycling (24, 26), and extended the opioid receptor fusion strategy used successfully for identifying diverse recycling sequences in GPCRs (27-29), to address these fundamental questions.Here we show that the recycling activity of the β2AR-derived PDZbd can be effectively bypassed by linking receptors to ERM family proteins in the absence of the PDZbd itself. Further, we establish that the protein connectivity network can be further simplified by fusing receptors to an interaction module that binds directly to actin filaments. We found that bypassing the PDZ-mediated interaction using either domain is sufficient to mimic the ability of the PDZbd to promote efficient, actin-dependent recycling of receptors. Hrs-dependent regulation, however, which is characteristic of sequence-dependent recycling of wild-type receptors, was recapitulated only by the fused PDZbd and not by the proposed downstream interaction modules. These results support a relatively simple architecture of protein connectivity that is sufficient to mimic the core recycling activity of the β2AR-derived PDZbd, but not its characteristic cellular regulation. Given that an increasing number of GPCRs have been shown to bind PDZ proteins that typically link directly or indirectly to cytoskeletal elements (17, 27, 30-32), the present results also suggest that actin connectivity may represent a common biochemical principle underlying sequence-dependent recycling of various GPCRs.     

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.     

Bortezomib Overcomes Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance in Hepatocellular Carcinoma Cells in Part through the Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However, many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis. Comparing the molecular change in HCC cells treated with these agents, we found that down-regulation of phospho-Akt (P-Akt) played a key role in mediating TRAIL sensitization of bortezomib. The first evidence was that bortezomib down-regulated P-Akt in a dose- and time-dependent manner in TRAIL-treated HCC cells. Second, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, also sensitized resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells. Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in bortezomib-treated cells, and PP2A knockdown by small interference RNA also reduced apoptosis induced by the combination of TRAIL and bortezomib, indicating that PP2A may be important in mediating the effect of bortezomib on TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at clinically achievable concentrations in hepatocellular carcinoma cells, and this effect is mediated at least partly via inhibition of the PI3K/Akt pathway.Hepatocellular carcinoma (HCC)² is currently the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death. To date, surgery is still the only curative treatment but is only feasible in a small portion of patients (1). Drug treatment is the major therapy for patients with advanced stage disease. Unfortunately, the response rate to traditional chemotherapy for HCC patients is unsatisfactory (1). Novel pharmacological therapy is urgently needed for patients with advanced HCC. In this regard, the approval of sorafenib might open a new era of molecularly targeted therapy in the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a type II transmembrane protein and a member of the TNF family, is a promising anti-tumor agent under clinical investigation (2). TRAIL functions by engaging its receptors expressed on the surface of target cells. Five receptors specific for TRAIL have been identified, including DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4 and DR5 contain an effective death domain that is essential to formation of death-inducing signaling complex (DISC), a critical step for TRAIL-induced apoptosis. Notably, the trimerization of the death domains recruits an adaptor molecule, Fas-associated protein with death domain (FADD), which subsequently recruits and activates caspase-8. In type I cells, activation of caspase-8 is sufficient to activate caspase-3 to induce apoptosis; however, in another type of cells (type II), the intrinsic mitochondrial pathway is essential for apoptosis characterized by cleavage of Bid and release of cytochrome c from mitochondria, which subsequently activates caspase-9 and caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms responsible for the resistance include receptors and intracellular resistance. Although the cell surface expression of DR4 or DR5 is absolutely required for TRAIL-induced apoptosis, tumor cells expressing these death receptors are not always sensitive to TRAIL due to intracellular mechanisms. For example, the cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but without protease activity, has been linked to TRAIL resistance in several studies (4, 5). In addition, inactivation of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL in MMR-deficient tumors (6, 7), and reintroduction of Bax into Bax-deficient cells restored TRAIL sensitivity (8), indicating that the Bcl-2 family plays a critical role in intracellular mechanisms for resistance of TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma and mantle cell lymphoma, has been investigated intensively for many types of cancer (9). Accumulating studies indicate that the combination of bortezomib and TRAIL overcomes the resistance to TRAIL in various types of cancer, including acute myeloid leukemia (4), lymphoma (10–13), prostate (14–17), colon (15, 18, 19), bladder (14, 16), renal cell carcinoma (20), thyroid (21), ovary (22), non-small cell lung (23, 24), sarcoma (25), and HCC (26, 27). Molecular targets responsible for the sensitizing effect of bortezomib on TRAIL-induced cell death include DR4 (14, 27), DR5 (14, 20, 22–23, 28), c-FLIP (4, 11, 21–23, 29), NF-κB (12, 24, 30), p21 (16, 21, 25), and p27 (25). In addition, Bcl-2 family also plays a role in the combinational effect of bortezomib and TRAIL, including Bcl-2 (10, 21), Bax (13, 22), Bak (27), Bcl-xL (21), Bik (18), and Bim (15).Recently, we have reported that Akt signaling is a major molecular determinant in bortezomib-induced apoptosis in HCC cells (31). In this study, we demonstrated that bortezomib overcame TRAIL resistance in HCC cells through inhibition of the PI3K/Akt pathway.