Low Endocytic pH and Capsid Protein Autocleavage Are Critical Components of Flock House Virus Cell Entry

The process by which nonenveloped viruses cross cell membranes during host cell entry remains poorly defined; however, common themes are emerging. Here, we use correlated in vivo and in vitro studies to understand the mechanism of Flock House virus (FHV) entry and membrane penetration. We demonstrate that low endocytic pH is required for FHV infection, that exposure to acidic pH promotes FHV-mediated disruption of model membranes (liposomes), and particles exposed to low pH in vitro exhibit increased hydrophobicity. In addition, FHV particles perturbed by heating displayed a marked increase in liposome disruption, indicating that membrane-active regions of the capsid are exposed or released under these conditions. We also provide evidence that autoproteolytic cleavage, to generate the lipophilic γ peptide (4.4 kDa), is required for membrane penetration. Mutant, cleavage-defective particles failed to mediate liposome lysis, regardless of pH or heat treatment, suggesting that these particles are not able to expose or release the requisite membrane-active regions of the capsid, namely, the γ peptides. Based on these results, we propose an updated model for FHV entry in which (i) the virus enters the host cell by endocytosis, (ii) low pH within the endocytic pathway triggers the irreversible exposure or release of γ peptides from the virus particle, and (iii) the exposed/released γ peptides disrupt the endosomal membrane, facilitating translocation of viral RNA into the cytoplasm.Flock House virus (FHV), a nonenveloped, positive-sense RNA virus, has been employed as a model system in several important studies to address a wide range of biological questions (reviewed in reference 55). FHV has been instrumental in understanding virus structure and assembly (17, 19, 45), RNA replication (2, 3, 37), and specific packaging of the genome (33, 44, 53, 54). Studies of FHV infection in Drosophila melanogaster flies have provided valuable information about the antiviral innate immune response in invertebrate hosts (29, 57). FHV is also used in nanotechnology applications as an epitope-presenting platform to develop novel vaccines and medical therapies (31, 48). In this report, we use FHV as a model system to further elucidate the means by which nonenveloped viruses enter host cells and traverse cellular membranes.During cell entry enveloped and nonenveloped viral capsid proteins undergo structural rearrangements that enable the virus to breach the membrane bilayer, ultimately releasing the viral genome or nucleocapsid into the cytoplasm. These entry-related conformational changes have been well characterized for enveloped viruses, which use membrane fusion to cross membrane bilayers (reviewed in reference 59). However, the mechanisms nonenveloped viruses employ to breach cellular membranes are poorly defined. Recently, significant parallels in the mechanisms of cell entry have emerged for a diverse group of nonenveloped viruses. Specifically, programmed capsid disassembly and release of small membrane-interacting peptides appear to be a common theme (reviewed in references 4 and 50).The site of membrane penetration depends upon the route of virus entry into the cell. Viruses can enter host cells via several distinct pathways, including clathrin-mediated endocytosis, caveolae-mediated endocytosis, lipid raft-mediated endocytosis, and macropinocytosis (reviewed in reference 40). The two primary routes of virus entry are clathrin-mediated endocytosis, where viruses encounter an acidic environment, and caveolae-mediated endocytosis, which is pH neutral. Many nonenveloped viruses, including adenovirus (24, 52), parvovirus (6), and reovirus (34, 49), require acidic pH during entry. However, numerous nonenveloped viruses have acid-independent entry mechanisms, including rotavirus (28), polyomavirus (43), simian virus 40 (41, 51), and several members of the picornavirus family (7, 14, 32, 42).Upon reaching the appropriate site of membrane penetration, nonenveloped virus capsid proteins are triggered by cellular factors, such as receptor binding and/or exposure to low pH within endosomes, to undergo conformational changes necessary for membrane interactions. These tightly regulated structural rearrangements may include capsid disassembly, exposure of hydrophobic regions, and/or release of membrane-lytic factors. For example, low pH within endosomes triggers adenovirus capsid disassembly, leading to the release of the membrane lytic protein VI (24, 60). In contrast, poliovirus is activated for membrane penetration by a pH-independent mechanism. Receptor binding triggers the poliovirus capsid to undergo a conformational change, resulting in the exposure of the N terminus of VP1 and the release of VP4 (18, 23), both of which facilitate membrane interactions (20). Notably, even though some viruses, such as reovirus, enter cells via an acidic endocytic pathway, membrane penetration is not acid activated (16), indicating that exposure to low pH and membrane penetration are not always mutual events.The overall simplicity of the FHV capsid, composed of a single gene product, along with the wealth of available high-resolution structural information (reviewed in reference 45) make FHV an ideal candidate for understanding nonenveloped virus entry and infection. FHV, a member of the family Nodaviridae, is a nonenveloped insect virus with a bipartite RNA genome surrounded by an icosahedral protein capsid. The quasi-equivalent T=3 virion (∼300-Å diameter) is initially composed of 180 copies of a single coat precursor protein α (44 kDa). Following capsid assembly the α protein undergoes autocatalytic cleavage to generate two particle-associated cleavage products, a large N-terminal fragment, β (39 kDa), and a small C-terminal fragment, γ (4.4 kDa) (22), creating the infectious virion (46). Mutant FHV particles that do not undergo autocatalytic cleavage, and therefore cannot release the γ peptide, are not infectious (46). It has been hypothesized that these particles are noninfectious because they cannot mediate membrane penetration, but this has never been shown directly.The FHV X-ray structure revealed that the γ peptides were located inside the capsid shell with residues 364 to 385 forming amphipathic helices (19). Subsequent studies showed that the FHV capsid is dynamic, with γ transiently exposed to the exterior of the capsid (11). These findings led to a structure-based model of FHV membrane disruption in which the dynamic γ peptides are reversibly exposed to the surface of the capsid (11), “sampling” the environment until they encounter the appropriate cellular trigger. The virus is then activated to undergo an irreversible conformational change in which the γ helical bundles located at each fivefold axis are externalized and released from the virus particle (17, 19). Upon release, the γ pentameric helical bundles are predicted to insert into and create a local disruption of the membrane bilayer to allow the RNA to enter the cytoplasm (10).While biochemical and structural studies have provided the foundation for a model of FHV cell entry, more rigorous in vivo and in vitro studies are necessary to confirm the ideas put forth in this model. Here, we clarify the route of FHV entry and characterize the tightly regulated events required for FHV membrane penetration. We demonstrate for the first time that low endocytic pH is required for FHV infection, that acidic pH promotes FHV membrane penetration, and that particles exposed to low pH exhibit increased hydrophobicity. In addition, we provide evidence that mutant, cleavage-defective particles are blocked specifically at the membrane penetration step during cell entry. Taken together, these findings offer an experimentally supported model of FHV entry into host cells. In addition, these results add to the accumulating evidence that nonenveloped viruses employ common mechanisms to traverse cellular membranes.     

Processing and Topology of the Yeast Mitochondrial Phosphatidylserine Decarboxylase 1

The inner mitochondrial membrane plays a crucial role in cellular lipid homeostasis through biosynthesis of the non-bilayer-forming lipids phosphatidylethanolamine and cardiolipin. In the yeast Saccharomyces cerevisiae, the majority of cellular phosphatidylethanolamine is synthesized by the mitochondrial phosphatidylserine decarboxylase 1 (Psd1). The biogenesis of Psd1 involves several processing steps. It was speculated that the Psd1 precursor is sorted into the inner membrane and is subsequently released into the intermembrane space by proteolytic removal of a hydrophobic sorting signal. However, components involved in the maturation of the Psd1 precursor have not been identified. We show that processing of Psd1 involves the action of the mitochondrial processing peptidase and Oct1 and an autocatalytic cleavage at a highly conserved LGST motif yielding the α- and β-subunit of the enzyme. The Psd1 β-subunit (Psd1β) forms the membrane anchor, which binds the intermembrane space-localized α-subunit (Psd1α). Deletion of a transmembrane segment in the β-subunit results in mislocalization of Psd1 and reduced enzymatic activity. Surprisingly, autocatalytic cleavage does not depend on proper localization to the inner mitochondrial membrane. In summary, membrane integration of Psd1 is crucial for its functionality and for maintenance of mitochondrial lipid homeostasis.     

Dissecting the Functional Domains of a Nonenveloped Virus Membrane Penetration Peptide

Recent studies have established that several nonenveloped viruses utilize virus-encoded lytic peptides for host membrane disruption. We investigated this mechanism with the “gamma” peptide of the insect virus Flock House virus (FHV). We demonstrate that the C terminus of gamma is essential for membrane disruption in vitro and the rescue of immature virus infectivity in vivo, and the amphipathic N terminus of gamma alone is not sufficient. We also show that deletion of the C-terminal domain disrupts icosahedral ordering of the amphipathic helices of gamma in the virus. Our results have broad implications for understanding membrane lysis during nonenveloped virus entry.The presence of membrane lytic peptides in many nonenveloped viruses is well established (3, 16), but how these peptides are deployed from the virus capsid during host cell entry and disrupt membranes remains unclear. These peptides are typically generated by a postassembly proteolytic processing event (1, 11) and are exposed from a previously buried position during conformational alterations in the capsid triggered by host cell conditions (2, 18). Flock House virus (FHV), an insect nodavirus, contains a 4-kDa peptide called “gamma” (γ), which shares many of the characteristics of other nonenveloped virus lytic peptides (3). The FHV capsid is made from 180 copies of a single-coat protein (α) enclosing a single-stranded bipartite RNA genome (9). Gamma is generated by the autocatalytic cleavage of α during virus maturation (α → β + γ) (15), remains localized in the capsid interior (9) with occasional externalization or “breathing” (6), and is exposed under low-pH conditions in the endosomes during entry (Odegard et al., submitted for publication). Covalently independent gamma is necessary for virus infection, since maturation-defective FHV (D75N/N363T FHV), which does not undergo the autocleavage of α, is not infectious (15, 17). The N-terminal ∼21 residues of gamma (corresponding to residues 364 to 384 of α) constitute an amphipathic helix which can disrupt membranes in vitro when synthetically produced (4, 5) and is recognized as the host membrane-interacting region of FHV during entry. The hydrophobic, ∼23-residue-long C terminus of gamma, especially certain phenylalanine residues (at positions 402, 405, and 407), is responsible for specifically packaging viral RNA into capsids during assembly (14).It was recently demonstrated that a supply of full-length gamma from noninfectious virus-like particles (VLPs) of FHV (13) during entry can restore infectivity to maturation-defective FHV (17), suggesting that gamma can function in trans to mediate access into host cells. This trans-complementation assay (17) was utilized to provide a quantitative readout of the effect of gamma mutations specifically on virus entry and to thus assess the region(s) of gamma required during virus entry. To determine the minimal sequence of gamma required for trans-complementation, FHV VLPs were produced that included the first 384, 390, or 395 amino acids of capsid protein α (designated Δγ384, Δγ390, and Δγ395, respectively). These VLPs contained only the amphipathic region of gamma, or that and additional parts of the C-terminal region (Fig. ​(Fig.1A),1A), and underwent normal assembly and maturation cleavage (Fig. ​(Fig.1B).1B). The amount of progeny virus produced by trans-complementing maturation-defective D75N/N363T FHV with these mutated VLPs was negligible (∼5%) compared to that produced by the wild-type (WT) VLPs (considered 100%) (Fig. ​(Fig.1C).1C). Thus, the amphipathic region of gamma was not sufficient by itself to restore infectivity to maturation-defective FHV, and the C terminus of gamma, beyond residue 395, was essential for rescue. Three phenylalanines located beyond residue 395 in gamma were separately mutated to alanines, and mature VLPs were generated (Fig. 1A and B) and tested in the trans-complementation assay. The relative efficiencies of rescue demonstrated by the F402A, F405A, and F407A VLPs were 15%, 29%, and 43%, respectively (Fig. ​(Fig.1C),1C), compared to that by the WT VLPs (100%). This rescue efficiency was similar to the relative infectivity previously determined for FHV containing the same point mutations (14). This suggests that the phenylalanines at the gamma C terminus are not only involved in the specific packaging of genomic RNA during FHV assembly (14) but are also required during virus entry.Open in a separate windowFIG. 1.C-terminal region of gamma required for trans-complementation during the entry of maturation-defective FHV. (A) Schematic of truncations or single mutations in the gamma region of FHV capsid protein α in VLPs. The sequence of gamma in each of the mutated VLPs is shown, with the N-terminal amphipathic region boxed. The single F→A mutations in F402A, F405A, and F407A are indicated in boldface. (B) A total of 5 μl of WT, Δγ384, phenylalanine mutant, or D75N VLPs, at a concentration of 5 mg/ml, was subjected to SDS-PAGE on a 4 to 20% Tris-glycine gel (Invitrogen) and stained with Coomassie brilliant blue. The position of gamma is indicated. The cleavage-defective D75N VLPs do not have gamma. (C) Drosophila DL-1 cells (1 × 10⁸) were coinfected with 1.5 × 10³ particles/cell of D75N/N363T FHV and 9 × 10³ particles/cell of WT, Δγ384, Δγ390, Δγ395, F402A, F405A, or F407A VLPs. [³⁵S]methionine-cysteine-labeled progeny virus was quantified, with the amount of progeny produced during coinfection with D75N/N363T FHV and WT VLPs normalized at 100%. The standard deviation was calculated from three replicates.Since the primary function of gamma during FHV entry is expected to be host membrane disruption, the ability of the mutated VLPs to disrupt DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)-treated liposomes and release enclosed fluorescent dye was determined in comparison to the WT VLPs. We found that the in vivo rescue behavior of VLPs containing truncated gamma correlated with their in vitro membrane disruption activities. WT VLPs, at a concentration of 0.1 mg/ml (6.37 × 10¹¹ particles), released dye from liposomes at pH 7.0 (Fig. ​(Fig.2A)2A) and at a much higher rate at pH 6.0 (Fig. ​(Fig.2B),2B), which mimics the acidic endosomal environment. In contrast, the Δγ384, Δγ390, and Δγ395 VLPs were severely impaired in disrupting liposomes at both pH conditions (Fig. 2A and B), indicating that diminished endosomal membrane lysis by truncated gamma peptides could be responsible for the inability of mutated VLPs to rescue the infectivity of maturation-defective particles. The maturation-defective D75N VLP, which is unable to rescue infection (17), was also inefficient in liposome disruption at a neutral or low pH (Fig. 2A and B), indicating that in vitro membrane disruption by VLPs is a reliable indicator of their in vivo entry behavior.Open in a separate windowFIG. 2.Disruption of liposomes and fluorescent dye release by VLPs in vitro. In each case, total fluorescence is normalized to dye release achieved by the addition of 0.1% Triton X-100 to liposomes under the same conditions. In the case of panels A, B, and D, closely similar results were obtained in three different experiments, whereas the standard deviations in panel C were calculated from three replicates. Fluorescence measurements were carried out at excitation/emission maxima of 492/514 nm for 6-carboxyfluorescein and 535/585 nm for SulfoB. (A) Kinetic study of 6-carboxyfluorescein release from DOPC-treated liposomes upon the addition of 6.37 × 10¹¹ particles (lipid/particle molar ratio, 481:1) of WT, gamma-truncated, or maturation-defective D75N VLPs to liposomes in 50 mM HEPES (pH 7.0). (B) SulfoB release from DOPC-treated liposomes in 50 mM Bis-Tris (pH 6.0) upon the addition of 6.37 × 10¹¹ particles of VLPs. (C) SulfoB release from DOPC-treated liposomes by 6.37 × 10¹¹ particles of WT, F402A, F405A, or F407A VLPs after 1 h at pH 7.0 (black) or by 2 × 10¹¹ particles (lipid/particle molar ratio, 1,387:1) of each of the VLPs after 15 min at pH 6.0 (gray). (D) SulfoB fluorescence upon the addition of heat-released gamma peptide with the WT sequence or with phenylalanine mutations at F402, F405, and F407 to dye-filled liposomes.VLPs containing single phenylalanine mutations in gamma were able to disrupt liposomes but were overall less effective than the WT. At a concentration of 0.1 mg/ml at pH 7.0, F402A and F405A VLPs displayed ∼60% liposome disruption (1 h postincubation) compared to that of the WT (Fig. ​(Fig.2C).2C). At pH 6.0, these VLPs caused significantly less liposome disruption than the WT at an early time point (15 min postincubation) at a concentration of 0.035 mg/ml (2 × 10¹¹ particles) (Fig. ​(Fig.2C),2C), although after 45 min of incubation, the extent of the liposome disruption approached the WT levels (data not shown). The F407A mutant VLPs, approximately half as competent as the WT VLPs in rescuing infection (Fig. ​(Fig.1C),1C), were nonetheless as efficient as the WT VLPs in membrane disruption at low pH, although they were ∼80% as competent at a neutral pH (Fig. ​(Fig.2C).2C). When gamma was isolated from the WT and phenylalanine mutant VLPs, by heating equal amounts (80 μg) at 65°C in Bis-Tris (pH 6.0) and centrifuging the heated material on a 30% sucrose cushion, the top fraction, which contained WT or mutated gamma peptides, caused the immediate release of fluorescent dye from liposomes (Fig. ​(Fig.2D).2D). Given the dissimilar behavior of particle-associated phenylalanine mutant gamma and free mutant gamma, we hypothesized that the mutations in the C terminus could affect the organization of gamma within the particle.To test this hypothesis, cryoelectron microscopy (CryoEM) and image reconstruction were carried out to locate and compare the gamma peptides of WT FHV VLPs and Δγ384 VLPs, which had the most drastic truncation in gamma and could provide robust structural evidence. Data were collected on frozen hydrated samples of the VLPs (concentrated to 11 mg/ml in 50 mM HEPES [pH 7.0]) at the National Resource for Automated Molecular Microscopy on an FEI Tecnai F20 electron microscope operating at 120 kV. Images were processed using the EMAN suite (10), with a map calculated from the protein atomic coordinates of FHV (PDB entry no. 2Z2Q) as the starting model for image reconstructions. Real space and rigid body refinement was carried out using Chimera (12) and CNS (7), respectively. The estimated resolution for each image reconstruction was 8.8 Å, and the final R/R_free were 28.4%/28.3% for WT and 29.5%/29.3% for Δγ384 VLPs. While the WT and Δγ384 reconstructions were nearly indistinguishable at the surface, striking changes were detected in the gamma region (Fig. ​(Fig.3B).3B). To quantify these changes, the electron densities for the pseudoatomic models of FHV, lacking the interior RNA, the gamma helices, and the capsid protein N termini, and calculated at the resolution of the CryoEM image reconstructions, were subtracted from the WT and Δγ384 VLP image reconstructions. The volume of the density associated with the gamma helices and the capsid protein N termini was calculated above a threshold of 0.4σ. While the densities of the capsid protein N termini (residues 59 to 72), calculated as controls, appeared similar for the A, B, and C subunits in the icosahedral asymmetric unit (iASU) between the two reconstructions, the densities of the gamma helices (residues 364 to 381) in the A and B subunits were increased approximately fourfold and twofold, respectively, in the WT reconstruction, whereas the density of C-subunit gamma was fourfold stronger in the Δγ384 reconstruction (Fig. ​(Fig.3C).3C). The magnitude of the difference in density represents a clear structural dissimilarity between the WT and Δγ384 VLPs, with the gamma amphipathic helices becoming less icosahedrally ordered overall in the Δγ384 reconstruction. Interestingly, the pentameric helical bundles formed by gamma N termini at the fivefold axis of symmetry of the FHV capsid (Fig. ​(Fig.3A),3A), and thought to be primarily involved in membrane interaction (8), were visible in the WT VLP reconstruction, but the corresponding density weakened significantly in the Δγ384 VLP (Fig. ​(Fig.3B),3B), indicating significant disorder.Open in a separate windowFIG. 3.CryoEM image reconstructions of WT and Δγ384 VLPs. (A) Model of WT FHV showing the iASU consisting of the A, B, and C subunits, as well as the A subunits at the fivefold axis of symmetry. An expanded view of the fivefold axis shows the amphipathic region of the gamma peptides (red) forming a pentameric helical bundle. (B) Panel I, inner surface of WT VLP and Δγ384 VLP image reconstructions contoured at 0.4σ. The coloring is as follows: density for the capsid protein N termini (residues 59 to 72) in the iASU is in yellow, density for the gamma amphipathic helices (residues 364 to 381) is in cyan, and a potential pocket factor is in purple. Modeled into the density for the gamma amphipathic helices are the corresponding residues from the FHV crystal structure (PDB entry 2Z2Q) in red. Panel II, inner view, looking down on the fivefold axis of symmetry of WT and Δγ384 VLP image reconstructions. The density for the gamma amphipathic helices from the A subunits is in cyan, with the corresponding residues from the FHV crystal structure modeled in red. A pocket factor is in purple. (C) Volume ratios corresponding to the gamma amphipathic helices and the capsid protein N termini for subunits A, B, and C in the iASU. The reported ratio for each volume pair is the WT VLP density to the corresponding Δγ384 VLP density.Our data show that the N-terminal amphipathic helix of gamma, previously thought to be the key to infection, is not sufficient for membrane permeabilization during FHV entry and is coincidentally less icosahedrally ordered in the capsid interior in the absence of the C terminus. Although the role of the gamma C terminus in entry is not clear, one interesting possibility is that the phenylalanine residues in this region interact with other capsid elements or packaged RNA to maintain the N-terminal helices in a structural conformation essential for biological activity. We demonstrate that other regions of nonenveloped virus lytic peptides, in addition to the membrane-interacting domains, might be essential in order to gain maximum leverage during entry.     

A TAG on to the neurogenic functions of APP

The proteolytic processing of amyloid β precursor protein (APP) has long been studied because of its association with the pathology of Alzheimer''s disease (AD). The ectodomain of APP is shed by α- or β-secretase cleavage. The remaining membrane bound stub can then undergo regulated intramembrane proteolysis (RIP) by γ-secretase. This cleavage can release amyloid β (Aβ) from the stub left by β-secretase cleavage but also releases the APP intracellular domain (AICD) after α- or β-secretase cleavage. The physiological functions of this proteolytic processing are not well understood. We compare the proteolytic processing of APP to the ligand-dependent RIP of Notch. In this review, we discuss recent evidence suggesting that TAG1 is a functional ligand for APP. The interaction between TAG1 and APP triggers γ-secretase-dependent release of AICD. TAG1, APP and Fe65 colocalise in the neurogenic ventricular zone and in fetal neural progenitor cells in vitro. Experiments in TAG1, APP and Fe65 null mice as well as TAG1 and APP double-null mice demonstrate that TAG1 induces a γ-secretase- and Fe65-dependent suppression of neurogenesis.Key words: Amyloid β precursor protein, APP, TAG1, AICD, Fe65, neurogenesis, Alzheimer''s disease     

Cleavage Susceptibility of Reovirus Attachment Protein ς1 during Proteolytic Disassembly of Virions Is Determined by a Sequence Polymorphism in the ς1 Neck

A requisite step in reovirus infection of the murine intestine is proteolysis of outer-capsid proteins to yield infectious subvirion particles (ISVPs). When converted to ISVPs by intestinal proteases, virions of reovirus strain type 3 Dearing (T3D) lose 90% of their original infectivity due to cleavage of viral attachment protein ς1. In an analysis of eight field isolate strains of type 3 reovirus, we identified one additional strain, type 3 clone 31 (T3C31), that loses infectivity and undergoes ς1 cleavage upon conversion of virions to ISVPs. We examined the ς1 deduced amino acid sequences of T3D and the eight field isolate strains for a correlation between sequence variability and ς1 cleavage. The ς1 proteins of T3D and T3C31 contain a threonine at amino acid position 249, whereas an isoleucine occurs at this position in the ς1 proteins of the remaining strains. Thr²⁴⁹ occupies the d position of a heptad repeat motif predicted to stabilize ς1 oligomers through α-helical coiled-coil interactions. This region of sequence comprises a portion of the fibrous tail domain of ς1 known as the neck. Substitution of Thr²⁴⁹ with isoleucine or leucine resulted in resistance to cleavage by trypsin, whereas replacement with asparagine did not affect cleavage susceptibility. These results demonstrate that amino acid position 249 is an independent determinant of T3D ς1 cleavage susceptibility and that an intact heptad repeat is required to confer cleavage resistance. We performed amino-terminal sequence analysis on the ς1 cleavage product released during trypsin treatment of T3D virions to generate ISVPs and found that trypsin cleaves ς1 after Arg²⁴⁵. Thus, the sequence polymorphism at position 249 controls cleavage at a nearby site in the neck region. The relevance of these results to reovirus infection in vivo was assessed by treating virions with the contents of a murine intestinal wash under conditions that result in generation of ISVPs. The pattern of ς1 cleavage susceptibility generated by using purified protease was reproduced in assays using the intestinal wash. These results provide a mechanistic explanation for ς1 cleavage during exposure of virions to intestinal proteases and may account for certain strain-dependent patterns of reovirus pathogenesis.     

Cleavage of Signal Regulatory Protein α (SIRPα) Enhances Inflammatory Signaling

Signal regulatory protein α (SIRPα) is a membrane glycoprotein immunoreceptor abundant in cells of monocyte lineage. SIRPα ligation by a broadly expressed transmembrane protein, CD47, results in phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, resulting in the inhibition of NF-κB signaling in macrophages. Here we observed that proteolysis of SIRPα during inflammation is regulated by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), resulting in the generation of a membrane-associated cleavage fragment in both THP-1 monocytes and human lung epithelia. We mapped a charge-dependent putative cleavage site near the membrane-proximal domain necessary for ADAM10-mediated cleavage. In addition, a secondary proteolytic cleavage within the membrane-associated SIRPα fragment by γ-secretase was identified. Ectopic expression of a SIRPα mutant plasmid encoding a proteolytically resistant form in HeLa cells inhibited activation of the NF-κB pathway and suppressed STAT1 phosphorylation in response to TNFα to a greater extent than expression of wild-type SIRPα. Conversely, overexpression of plasmids encoding the proteolytically cleaved SIRPα fragments in cells resulted in enhanced STAT-1 and NF-κB pathway activation. Thus, the data suggest that combinatorial actions of ADAM10 and γ-secretase on SIRPα cleavage promote inflammatory signaling.     

Dual Role of α-Secretase Cleavage in the Regulation of γ-Secretase Activity for Amyloid Production

Processing of the amyloid precursor protein (APP) by β- and γ-secretases generates pathogenic β-amyloid (Aβ) peptides associated with Alzheimer disease (AD), whereas cleavage of APP by α-secretases precludes Aβ formation. Little is known about the role of α-secretase cleavage in γ-secretase regulation. Here, we show that α-secretase-cleaved APP C-terminal product (αCTF) functions as an inhibitor of γ-secretase. We demonstrate that the substrate inhibitory domain (ASID) within αCTF, which is bisected by the α-secretase cleavage site, contributes to this negative regulation because deleting or masking this domain turns αCTF into a better substrate for γ-secretase. Moreover, α-secretase cleavage can potentiate the inhibitory effect of ASID. Inhibition of γ-secretase activity by αCTF is observed in both in vitro and cellular systems. This work reveals an unforeseen role for α-secretase in generating an endogenous γ-secretase inhibitor that down-regulates the production of Aβ. Deregulation of this feedback mechanism may contribute to the pathogenesis of AD.     

Presenilin-dependent gamma-secretase processing of beta-amyloid precursor protein at a site corresponding to the S3 cleavage of Notch

The presenilin (PS)-dependent site 3 (S3) cleavage of Notch liberates its intracellular domain (NICD), which is required for Notch signaling. The similar γ-secretase cleavage of the β-amyloid precursor protein (βAPP) results in the secretion of amyloid β-peptide (Aβ). However, little is known about the corresponding C-terminal cleavage product (CTFγ). We have now identified CTFγ in brain tissue, in living cells, as well as in an in vitro system. Generation of CTFγ is facilitated by PSs, since a dominant-negative mutation of PS as well as a PS gene knock out prevents its production. Moreover, γ-secretase inhibitors, including one that is known to bind to PS, also block CTFγ generation. Sequence analysis revealed that CTFγ is produced by a novel γ-secretase cut, which occurs at a site corresponding to the S3 cleavage of Notch.     

Octyl Gallate Markedly Promotes Anti-Amyloidogenic Processing of APP through Estrogen Receptor-Mediated ADAM10 Activation

Our previous studies showed that the green tea-derived polyphenolic compound (−)-epigallocatechin-3 gallate (EGCG) reduces amyloid-β (Aβ) production in both neuronal and mouse Alzheimer’s disease (AD) models in concert with activation of estrogen receptor-α/phosphatidylinositide 3-kinase/protein kinase B (ERα/PI3K/Akt) signaling and anti-amyloidogenic amyloid precursor protein (APP) α-secretase (a disintegrin and metallopeptidase domain-10, ADAM10) processing. Since the gallate moiety in EGCG may correspond to the 7α position of estrogen, thereby facilitating ER binding, we extensively screened the effect of other gallate containing phenolic compounds on APP anti-amyloidogenic processing. Octyl gallate (OG; 10 µM), drastically decreased Aβ generation, in concert with increased APP α-proteolysis, in murine neuron-like cells transfected with human wild-type APP or “Swedish” mutant APP. OG markedly increased production of the neuroprotective amino-terminal APP cleavage product, soluble APP-α (sAPPα). In accord with our previous study, these cleavage events were associated with increased ADAM10 maturation and reduced by blockade of ERα/PI3k/Akt signaling. To validate these findings in vivo, we treated Aβ-overproducing Tg2576 mice with OG daily for one week by intracerebroventricular injection and found decreased Aβ levels associated with increased sAPPα. These data indicate that OG increases anti-amyloidogenic APP α-secretase processing by activation of ERα/PI3k/Akt signaling and ADAM10, suggesting that this compound may be an effective treatment for AD.     

Laminins promote postsynaptic maturation by an autocrine mechanism at the neuromuscular junction

A prominent feature of synaptic maturation at the neuromuscular junction (NMJ) is the topological transformation of the acetylcholine receptor (AChR)-rich postsynaptic membrane from an ovoid plaque into a complex array of branches. We show here that laminins play an autocrine role in promoting this transformation. Laminins containing the α4, α5, and β2 subunits are synthesized by muscle fibers and concentrated in the small portion of the basal lamina that passes through the synaptic cleft at the NMJ. Topological maturation of AChR clusters was delayed in targeted mutant mice lacking laminin α5 and arrested in mutants lacking both α4 and α5. Analysis of chimeric laminins in vivo and of mutant myotubes cultured aneurally demonstrated that the laminins act directly on muscle cells to promote postsynaptic maturation. Immunohistochemical studies in vivo and in vitro along with analysis of targeted mutants provide evidence that laminin-dependent aggregation of dystroglycan in the postsynaptic membrane is a key step in synaptic maturation. Another synaptically concentrated laminin receptor, Bcam, is dispensable. Together with previous studies implicating laminins as organizers of presynaptic differentiation, these results show that laminins coordinate post- with presynaptic maturation.     

γ-Secretase Dependent Production of Intracellular Domains Is Reduced in Adult Compared to Embryonic Rat Brain Membranes

Background

γ-Secretase is an intramembrane aspartyl protease whose cleavage of the amyloid precursor protein (APP) generates the amyloid β-peptide (Aβ) and the APP intracellular domain. Aβ is widely believed to have a causative role in Alzheimer''s disease pathogenesis, and therefore modulation of γ-secretase activity has become a therapeutic goal. Besides APP, more than 50 substrates of γ-secretase with different cellular functions during embryogenesis as well as adulthood have been revealed. Prior to γ-secretase cleavage, substrates are ectodomain shedded, producing membrane bound C-terminal fragments (CTFs).

Principal Findings

Here, we investigated γ-secretase cleavage of five substrates; APP, Notch1, N-cadherin, ephrinB and p75 neurotrophin receptor (p75-NTR) in membranes isolated from embryonic, young or old adult rat brain by analyzing the release of the corresponding intracellular domains (ICDs) or Aβ40 by western blot analysis and ELISA respectively. The highest levels of all ICDs and Aβ were produced by embryonic membranes. In adult rat brain only cleavage of APP and Notch1 could be detected and the Aβ40 and ICD production from these substrates was similar in young and old adult rat brain. The CTF levels of Notch1, N-cadherin, ephrinB and p75-NTR were also clearly decreased in the adult brain compared to embryonic brain, whereas the APP CTF levels were only slightly decreased.

Conclusions

In summary our data suggests that γ-secretase dependent ICD production is down-regulated in the adult brain compared to embryonic brain. In addition, the present approach may be useful for evaluating the specificity of γ-secretase inhibitors.     

The N-terminal Domain Allosterically Regulates Cleavage and Activation of the Epithelial Sodium Channel

The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP₂), we found that PIP₂ increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP₂ or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr³⁷⁰ in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation.     

Verteporfin is a substrate-selective γ-secretase inhibitor that binds the amyloid precursor protein transmembrane domain

This work reports substrate-selective inhibition of a protease with broad substrate specificity based on direct binding of a small-molecule inhibitor to the substrate. The target for these studies was γ-secretase protease, which cleaves dozens of different single-span membrane protein substrates, including both the C99 domain of the human amyloid precursor protein and the Notch receptor. Substrate-specific inhibition of C99 cleavage is desirable to reduce production of the amyloid-β polypeptide without inhibiting Notch cleavage, a major source of toxicity associated with broad specificity γ-secretase inhibitors. In order to identify a C99-selective inhibitors of the human γ-secretase, we conducted an NMR-based screen of FDA-approved drugs against C99 in model membranes. From this screen, we identified the small-molecule verteporfin with these properties. We observed that verteporfin formed a direct 1:1 complex with C99, with a K_D of 15–47 μM (depending on the membrane mimetic used), and that it did not bind the transmembrane domain of the Notch-1 receptor. Biochemical assays showed that direct binding of verteporfin to C99 inhibits γ-secretase cleavage of C99 with IC₅₀ values in the range of 15–164 μM, while Notch-1 cleavage was inhibited only at higher concentrations, and likely via a mechanism that does not involve binding to Notch-1. This work documents a robust NMR-based approach to discovery of small-molecule binders to single-span membrane proteins and confirmed that it is possible to inhibit γ-secretase in a substrate-specific manner.     

γ-Secretase Associated with Lipid Rafts: MULTIPLE INTERACTIVE PATHWAYS IN THE STEPWISE PROCESSING OF β-CARBOXYL-TERMINAL FRAGMENT*

γ-Secretase generates amyloid β-protein (Aβ), a pathogenic molecule in Alzheimer disease, through the intramembrane cleavage of the β-carboxyl-terminal fragment (βCTF) of β-amyloid precursor protein. We previously showed the framework of the γ-secretase cleavage, i.e. the stepwise successive processing of βCTF at every three (or four) amino acids. However, the membrane integrity of γ-secretase was not taken into consideration because of the use of the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized reconstituted γ-secretase system. Here, we sought to address how the membrane-integrated γ-secretase cleaves βCTF by using γ-secretase associated with lipid rafts. Quantitative analyses using liquid chromatography-tandem mass spectrometry of the βCTF transmembrane domain-derived peptides released along with Aβ generation revealed that the raft-associated γ-secretase cleaves βCTF in a stepwise sequential manner, but novel penta- and hexapeptides as well as tri- and tetrapeptides are released. The cropping of these peptides links the two major tripeptide-cleaving pathways generating Aβ40 and Aβ42 at several points, implying that there are multiple interactive pathways for the stepwise cleavages of βCTF. It should be noted that Aβ38 and Aβ43 are generated through three routes, and γ-secretase modulator 1 enhances all the three routes generating Aβ38, which results in decreases in Aβ42 and Aβ43 and an increase in Aβ38. These observations indicate that multiple interactive pathways for stepwise successive processing by γ-secretase define the species and quantity of Aβ produced.     

In vitro reconstitution reveals cooperative mechanisms of adapter protein-mediated activation of phospholipase C-γ1 in T cells

Activation of T cells upon engagement of the T cell antigen receptor rapidly leads to a number of phosphorylation and plasma membrane recruitment events. For example, translocation of phospholipase-Cγ1 (PLC−γ1) to the plasma membrane and its association with the transmembrane adapter protein LAT and two other adapter proteins, Gads and SLP-76, are critical events in the early T cell activation process. We have previously characterized the formation of a tetrameric LAT-Gads-SLP-76-PLC−γ1 complex by reconstitution in vitro and have also characterized the thermodynamics of tetramer formation. In the current study, we define how PLC−γ1 recruitment to liposomes, which serve as a plasma membrane surrogate, and PLC−γ1 activation are regulated both independently and additively by recruitment of PLC−γ1 to phosphorylated LAT, by formation of the LAT-Gads-SLP-76-PLC−γ1 tetramer, and by tyrosine phosphorylation of PLC−γ1. The recently solved structure of PLC−γ1 indicates that, in the resting state, several PLC−γ1 domains inhibit its enzymatic activity and contact with the plasma membrane. We propose the multiple cooperative steps that we observed likely lead to conformational alterations in the regulatory domains of PLC−γ1, enabling contact with its membrane substrate, disinhibition of PLC−γ1 enzymatic activity, and production of the phosphoinositide cleavage products necessary for T cell activation.     

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel’s γ-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (ΔI_ami) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of γENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of proteolytic activation of ENaC-mediated whole-cell currents correlates with the appearance of a γENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, they confirm the concept that a cleavage event in γENaC is required as a final step in proteolytic channel activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins.     

A Role for the Disintegrin Domain of Cyritestin, a Sperm Surface Protein Belonging to the ADAM Family, in Mouse Sperm–Egg Plasma Membrane Adhesion and Fusion

Sperm–egg plasma membrane fusion is preceded by sperm adhesion to the egg plasma membrane. Cell–cell adhesion frequently involves multiple adhesion molecules on the adhering cells. One sperm surface protein with a role in sperm–egg plasma membrane adhesion is fertilin, a transmembrane heterodimer (α and β subunits). Fertilin α and β are the first identified members of a new family of membrane proteins that each has the following domains: pro-, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic domain. This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain. Previous studies indicate that the disintegrin domain of fertilin β functions in sperm–egg adhesion leading to fusion. Full length cDNA clones have been isolated for five ADAMs expressed in mouse testis: fertilin α, fertilin β, cyritestin, ADAM 4, and ADAM 5. The presence of the disintegrin domain, a known integrin ligand, suggests that like fertilin β, other testis ADAMs could be involved in sperm adhesion to the egg membrane. We tested peptide mimetics from the predicted binding sites in the disintegrin domains of the five testis-expressed ADAMs in a sperm–egg plasma membrane adhesion and fusion assay. The active site peptide from cyritestin strongly inhibited (80–90%) sperm adhesion and fusion and was a more potent inhibitor than the fertilin β active site peptide. Antibodies generated against the active site region of either cyritestin or fertilin β also strongly inhibited (80–90%) both sperm–egg adhesion and fusion. Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm. Indirect immunofluorescence on live, acrosome-reacted sperm using antibodies against either cyritestin or fertilin β showed staining of the equatorial region, a region of the sperm membrane that participates in the early steps of membrane fusion. Collectively, these data indicate that a second ADAM family member, cyritestin, functions with fertilin β in sperm–egg plasma membrane adhesion leading to fusion.     

Activity-dependent α-Cleavage of Nectin-1 Is Mediated by A Disintegrin and Metalloprotease 10 (ADAM10)

Nectin-1 is known to undergo ectodomain shedding by α-secretase and subsequent proteolytic processing by γ-secretase. How secretase-mediated cleavage of nectin-1 is regulated in neuronal cells and how nectin-1 cleavage affects synaptic adhesion is poorly understood. We have investigated α-and γ-secretase-mediated processing of nectin-1 in primary cortical neurons and identified which protease acts as a α-secretase. We report here that NMDA receptor activation, but not stimulation of AMPA or metabotropic glutamate receptors, resulted in robust α- and γ-secretase cleavage of nectin-1 in mature cortical neurons. Cleavage of nectin-1 required influx of Ca²⁺ through the NMDA receptor, and activation of calmodulin, but was not dependent on calcium/calmodulin-dependent protein kinase II (CaMKII) activation. We found that ADAM10 is the major secretase responsible for nectin-1 ectodomain cleavage in neurons and the brain. These observations suggest that α- and γ-secretase processing of nectin-1 is a Ca²⁺/calmodulin-regulated event that occurs under conditions of activity-dependent synaptic plasticity and ADAM10 and γ-secretase are responsible for these cleavage events.     

Highly Conserved Residues in the Helical Domain of Dengue Virus Type 1 Precursor Membrane Protein Are Involved in Assembly,Precursor Membrane (prM) Protein Cleavage,and Entry

The envelope and precursor membrane (prM) proteins of dengue virus (DENV) are present on the surface of immature virions. During maturation, prM protein is cleaved by furin protease into pr peptide and membrane (M) protein. Although previous studies mainly focusing on the pr region have identified several residues important for DENV replication, the functional role of M protein, particularly the α-helical domain (MH), which is predicted to undergo a large conformational change during maturation, remains largely unknown. In this study, we investigated the role of nine highly conserved MH domain residues in the replication cycle of DENV by site-directed mutagenesis in a DENV1 prME expression construct and found that alanine substitutions introduced to four highly conserved residues at the C terminus and one at the N terminus of the MH domain greatly affect the production of both virus-like particles and replicon particles. Eight of the nine alanine mutants affected the entry of replicon particles, which correlated with the impairment in prM cleavage. Moreover, seven mutants were found to have reduced prM-E interaction at low pH, which may inhibit the formation of smooth immature particles and exposure of prM cleavage site during maturation, thus contributing to inefficient prM cleavage. Taken together, these results are the first report showing that highly conserved MH domain residues, located at 20–38 amino acids downstream from the prM cleavage site, can modulate the prM cleavage, maturation of particles, and virus entry. The highly conserved nature of these residues suggests potential targets of antiviral strategy.     

Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α1-Antitrypsin Variants

Background

Proteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P) is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication.

Methodology/Principal Finding

We demonstrate that stable cell lines inducibly expressing S1P-adapted α₁-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of α₁-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific α₁-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different α₁-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor.

Conclusions/Significance

Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.