Context-Specific Function of the Engineered Peptide Domain of PHP.B.

One approach to improve the utility of adeno-associated virus (AAV)-based gene therapy is to engineer the AAV capsid to (i) overcome poor transport through tissue barriers and (ii) redirect the broadly tropic AAV to disease-relevant cell types. Peptide- or protein-domain insertions into AAV surface loops can achieve both engineering goals by introducing a new interaction surface on the AAV capsid. However, we understand little about the impact of insertions on capsid structure and the extent to which engineered inserts depend on a specific capsid context to function. Here, we examine insert-capsid interactions for the engineered variant AAV9-PHP.B. The 7-amino-acid peptide insert in AAV9-PHP.B facilitates transport across the murine blood-brain barrier via binding to the receptor Ly6a. When transferred to AAV1, the engineered peptide does not bind Ly6a. Comparative structural analysis of AAV1-PHP.B and AAV9-PHP.B revealed that the inserted 7-amino-acid loop is highly flexible and has remarkably little impact on the surrounding capsid conformation. Our work demonstrates that Ly6a binding requires interactions with both the PHP.B peptide and specific residues from the AAV9 HVR VIII region. An AAV1-based vector that incorporates a larger region of AAV9-PHP.B-including the 7-amino-acid loop and adjacent HVR VIII amino acids-can bind to Ly6a and localize to brain tissue. However, unlike AAV9-PHP.B, this AAV1-based vector does not penetrate the blood-brain barrier. Here we discuss the implications for AAV capsid engineering and the transfer of engineered activities between serotypes. IMPORTANCE Targeting AAV vectors to specific cellular receptors is a promising strategy for enhancing expression in target cells or tissues while reducing off-target transgene expression. The AAV9-PHP.B/Ly6a interaction provides a model system with a robust biological readout that can be interrogated to better understand the biology of AAV vectors' interactions with target receptors. In this work, we analyzed the sequence and structural features required to successfully transfer the Ly6a receptor-binding epitope from AAV9-PHP.B to another capsid of clinical interest, AAV1. We found that AAV1- and AAV9-based vectors targeted to the same receptor exhibited different brain-transduction profiles. Our work suggests that, in addition to attachment-receptor binding, the capsid context in which this binding occurs is important for a vector's performance.

CALHM3 Is Essential for Rapid Ion Channel-Mediated Purinergic Neurotransmission of GPCR-Mediated Tastes.

Binding of sweet, umami, and bitter tastants to G protein-coupled receptors (GPCRs) in apical membranes of type II taste bud cells (TBCs) triggers action potentials that activate a voltage-gated nonselective ion channel to release ATP to gustatory nerves mediating taste perception. Although calcium homeostasis modulator 1 (CALHM1) is necessary for ATP release, the molecular identification of the channel complex that provides the conductive ATP-release mechanism suitable for action potential-dependent neurotransmission remains to be determined. Here we show that CALHM3 interacts with CALHM1 as a pore-forming subunit in a CALHM1/CALHM3 hexameric channel, endowing it with fast voltage-activated gating identical to that of the ATP-release channel in vivo. Calhm3 is co-expressed with Calhm1 exclusively in type II TBCs, and its genetic deletion abolishes taste-evoked ATP release from taste buds and GPCR-mediated taste perception. Thus, CALHM3, together with CALHM1, is essential to form the fast voltage-gated ATP-release channel in type II TBCs required for GPCR-mediated tastes.

MICU2 Restricts Spatial Crosstalk between InsP3R and MCU Channels by Regulating Threshold and Gain of MICU1-Mediated Inhibition and Activation of MCU.

Ca2+ entry into mitochondria is mediated by the Ca2+ uniporter-channel complex containing MCU, the Ca2+-selective pore, and associated regulatory proteins. The roles of MICU proteins are controversial. MICU1 was proposed to be necessary for MCU activity, whereas subsequent studies suggested it inhibits the channel in the low-cytoplasmic Ca2+ ([Ca2+]c) regime, a mechanism referred to as "gatekeeping," that imposes a [Ca2+]c threshold for channel activation at ∼1-3 µM. Here, we measured MCU activity over a wide range of quantitatively controlled and recorded [Ca2+]c. MICU1 alone can mediate gatekeeping as well as highly cooperative activation of MCU activity, whereas the fundamental role of MICU2 is to regulate the threshold and gain of MICU1-mediated inhibition and activation of MCU. Our results provide a unifying model for the roles of the MICU1/2 heterodimer in MCU-channel regulation and suggest an evolutionary role for MICU2 in spatially restricting Ca2+ crosstalk between single inositol 1,4,5-trisphosphate receptor (InsP3R) and MCU channels.

Influenza A virus infection engenders a poor antibody response against the ectodomain of matrix protein 2.

BACKGROUND: Matrix protein 2 (M2) is an integral tetrameric membrane protein of influenza A virus (IAV). Its ectodomain (M2e) shows remarkably little diversity amongst human IAV strains. As M2e-specific antibodies (Abs) have been shown to reduce the severity of infection in animals, M2e is being studied for its capability of providing protection against a broad range of IAV strains. Presently, there is little information about the concentration of M2e-specific Abs in humans. Two previous studies made use of ELISA and Western blot against M2e peptides and recombinant M2 protein as immunosorbents, respectively, and reported Ab titers to be low or undetectable. An important caveat is that these assays may not have detected all Abs capable of binding to native tetrameric M2e. Therefore, we developed an assay likely to detect all M2e tetramer-specific Abs. RESULTS: We generated a HeLa cell line that expressed full length tetrameric M2 (HeLa-M2) or empty vector (HeLa-C10) under the control of the tetracycline response element. These cell lines were then used in parallel as immunosorbents in ELISA. The assay was standardized and M2e-specific Ab titers quantified by means of purified murine or chimeric (mouse variable regions, human constant regions) M2e-specific Abs in the analysis of mouse and human sera, respectively. We found that the cell-based ELISA was substantially more effective than immobilized M2e peptide in detecting M2e-specific Abs in sera of mice that had recovered from repetitive IAV infections. Still, titers remained low (< 5 microg/ml) even after two consecutive infections but increased to approximately 50 microg/ml after the third infection. Competition with free M2e peptide indicated that approximately 20% of M2e-specific Abs engendered by infection reacted with M2e peptide. In humans presenting with naturally acquired influenza virus infection, 11 of 24 paired sera showed a > or = 4-fold increase in M2e-specific Ab titer. The Ab response appeared to be of short duration as titers were very low (average 0.2 mug/ml) in all patients at onset of infection and in controls, in spite of evidence for previous exposure to IAV. CONCLUSION: The results provide convincing evidence that M2e-specific Ab-mediated protection is currently lacking or suboptimal in humans.

EMRE Is a Matrix Ca(2+) Sensor that Governs Gatekeeping of the Mitochondrial Ca(2+) Uniporter.

The mitochondrial uniporter (MCU) is an ion channel that mediates Ca(2+) uptake into the matrix to regulate metabolism, cell death, and cytoplasmic Ca(2+) signaling. Matrix Ca(2+) concentration is similar to that in cytoplasm, despite an enormous driving force for entry, but the mechanisms that prevent mitochondrial Ca(2+) overload are unclear. Here, we show that MCU channel activity is governed by matrix Ca(2+) concentration through EMRE. Deletion or charge neutralization of its matrix-localized acidic C terminus abolishes matrix Ca(2+) inhibition of MCU Ca(2+) currents, resulting in MCU channel activation, enhanced mitochondrial Ca(2+) uptake, and constitutively elevated matrix Ca(2+) concentration. EMRE-dependent regulation of MCU channel activity requires intermembrane space-localized MICU1, MICU2, and cytoplasmic Ca(2+). Thus, mitochondria are protected from Ca(2+) depletion and Ca(2+) overload by a unique molecular complex that involves Ca(2+) sensors on both sides of the inner mitochondrial membrane, coupled through EMRE.

Products of lipid peroxidation induce missorting of the principal lysosomal protease in retinal pigment epithelium.

Phagocytosis of photoreceptor outer segments (OS) by retinal pigment epithelium (RPE) is essential for OS renewal and survival of photoreceptors. Internalized, oxidatively modified macromolecules perturb the lysosomal function of the RPE and can lead to impaired processing of photoreceptor outer segments. In this study, we sought to investigate the impact of intracellular accumulation of oxidatively damaged lipid-protein complexes on maturation and distribution of cathepsin D, the major lysosomal protease in the RPE. Primary cultures of human RPE cells were treated with copper-oxidized low density lipoprotein (LDL) and then challenged with serum-coated latex beads to stimulate phagocytosis. Three observations were noted to occur in this experimental system. First, immature forms of cathepsin D (52 and 46 kDa) were exclusively associated with latex-containing phagosomes. Second, maturation of cathepsin D was severely impaired in RPE cells loaded with oxidized LDL (oxLDL) prior to the phagocytic challenge. Third, pre-treatment with oxLDL caused sustained secretion of pro-cathepsin D and the latent form of gelatinase A into the extracellular space in a dose-dependent manner. These data stimulate the hypothesis that intracellular accumulation of poorly degradable, oxidized lipid-protein cross-links, may alter the turnover of cathepsin D, causing its mistargeting into the extracellular space together with the enhanced secretion of a gelatinase.

Phospholipid hydroxyalkenals: biological and chemical properties of specific oxidized lipids present in atherosclerotic lesions.

OBJECTIVE: Phosphatidylcholine hydroxyalkenals (PC-HAs) are a class of oxidized PCs derived from lipid peroxidation of arachidonate or linoleate at the sn-2 position to form terminal gamma-hydroxy, alpha-, and beta-unsaturated aldehydes. The aim of this study was to characterize some of their biological properties, ascertain the mechanism of their action, and assess whether they have in vivo relevance. METHODS AND RESULTS: Combinations of cell biological approaches with radiolabels, mass spectroscopy, and immunochemical as well as immunohistochemical techniques were used to show that PC-HAs reduce the proteolytic degradation by mouse peritoneal macrophages (MPMs) of internalized macromolecules, such as maleylated bovine serum albumin, and that the activity of the lysosomal protease, cathepsin B, in MPMs form Michael adducts with MPM proteins and with N-acetylated cysteine in vitro form pyrrole adducts with MPM proteins and reduce the maturation of Rab5a, thereby impairing phagosome-lysosome fusion (maturation) in phagocytes; they are present unbound and as pyrrole adducts in human atherosclerotic lesions. CONCLUSIONS: PC-HAs are present in vivo and possess multiple functions characteristic of oxidized LDL and 4-hydroxynonenal.

Ubinuclein-1 confers histone H3.3-specific-binding by the HIRA histone chaperone complex.

Histone chaperones bind specific histones to mediate their storage, eviction or deposition from/or into chromatin. The HIRA histone chaperone complex, composed of HIRA, ubinuclein-1 (UBN1) and CABIN1, cooperates with the histone chaperone ASF1a to mediate H3.3-specific binding and chromatin deposition. Here we demonstrate that the conserved UBN1 Hpc2-related domain (HRD) is a novel H3.3-specific-binding domain. Biochemical and biophysical studies show the UBN1-HRD preferentially binds H3.3/H4 over H3.1/H4. X-ray crystallographic and mutational studies reveal that conserved residues within the UBN1-HRD and H3.3 G90 as key determinants of UBN1-H3.3-binding specificity. Comparison of the structure with the unrelated H3.3-specific chaperone DAXX reveals nearly identical points of contact between the chaperone and histone in the proximity of H3.3 G90, although the mechanism for H3.3 G90 recognition appears to be distinct. This study points to UBN1 as the determinant of H3.3-specific binding and deposition by the HIRA complex.

Hydroxynonenal inactivates cathepsin B by forming Michael adducts with active site residues.

Oxidation of plasma low-density lipoprotein (oxLDL) generates the lipid peroxidation product 4-hydroxy-2 nonenal (HNE) and also reduces proteolytic degradation of oxLDL and other proteins internalized by mouse peritoneal macrophages in culture. This leads to accumulation of undegraded material in lysosomes and formation of ceroid, a component of foam cells in atherosclerotic lesions. To explore the possibility that HNE contributes directly to the inactivation of proteases, structure-function studies of the lysosomal protease cathepsin B have been pursued. We found that treatment of mouse macrophages with HNE reduces degradation of internalized maleyl bovine serine albumin and cathepsin B activity. Purified bovine cathepsin B treated briefly with 15 microM HNE lost approximately 76% of its protease activity and also developed immunoreactivity with antibodies to HNE adducts in Western blot analysis. After stabilization of the potential Michael adducts by sodium borohydride reduction, modified amino acids were localized within the bovine cathepsin B protein structure by mass spectrometric analysis of tryptic peptides. Michael adducts were identified by tandem mass spectrometry at cathepsin B active site residues Cys 29 (mature A chain) and His 150 (mature B chain). Thus, covalent interaction between HNE and critical active site residues inactivates cathepsin B. These results support the hypothesis that the accumulation of undegraded macromolecules in lysosomes after oxidative damage are caused in part by direct protease inactivation by adduct formation with lipid peroxidation products such as HNE.

Phospholipids in oxidized LDL not adducted to apoB are recognized by the CD36 scavenger receptor.

Previous studies have shown that oxidation of low-density lipoprotein (oxLDL) results in its recognition by scavenger receptors on macrophages. Whereas blockage of lysyl residues on apoB-100 of oxLDL by lipid peroxidation products appears to be critical for recognition by the scavenger receptor class A (SR-A), modification of the lipid moiety has been suggested to be responsible for recognition by the scavenger class B receptor, CD36. We studied the recognition by scavenger receptors of oxidized LDL in which lysyl residues are blocked prior to oxidation through methylation [ox(m)LDL]. This permits us to minimize any contribution of modified apoB-100 to the recognition of oxLDL, but does not disrupt the native configuration of lipids in the particle. We found that ox(m)LDL was recognized by receptors on mouse peritoneal macrophages (MPM) almost as well as oxLDL. Ox(m)LDL was recognized by CD36-transfected cells but not by SR-A-transfected cells. Oxidized phospholipids (oxPC) transferred from oxLDL or directly from oxPC to LDL, conveyed recognition by CD36-transfected cells, confirming that CD36 recognized unbound oxidized phospholipids in ox(m)LDL. Collectively, these results suggest that oxPC not adducted to apoB within the intact oxLDL particle are recognized by the macrophage scavenger receptor CD36, that these lipids are not recognized by SR-A, and that they can transfer from oxidized to unoxidized LDL and induce CD36 recognition.

Phospholipids in oxidized low density lipoproteins perturb the ability of macrophages to degrade internalized macromolecules and reduce intracellular cathepsin B activity.

Previous studies showed that pre-treatment of mouse peritoneal macrophages (MPM) with oxidized low density lipoprotein (oxLDL) repressed subsequent degradation of oxLDL following uptake. Parallel studies on the activity of the lysosomal protease, cathepsin B in MPM and in vitro indicate that oxLDL also induces a reduction in this activity. We now report that pre-treatment of MPM with the lipid portion of oxLDL induced a reduction both in the degradation of internalized small macromolecules such as maleylated (mal) BSA (30%) or larger ones such as aggregated LDL (100%), and in cellular cathepsin B activity (42%). Binding and uptake of malBSA were not affected. Pre-treatment of MPM for 2 h with oxidized phosphatidylcholine (oxPC) isolated from oxLDL or generated from Cu2+-treated 1-palmitoyl-2-linoleoyl phosphatidylcholine (oxPLPC), also inhibited 125I-malBSA degradation and reduced cathepsin B activity in MPM and in vitro. Further separation of oxPLPC and oxPC from oxLDL by thin layer chromatography led to the isolation of a polar lipid fraction possessing most of the biological activity in oxPC. Partial characterization of this fraction from oxPLPC using liquid chromatography/electrospray ionization/mass spectrometry indicated that this polar fraction containing fragmentation products of linoleate, was still comprised of multiple bioactive molecular ions. Collectively, these results suggest that specific oxPC fractions in oxLDL are partially responsible for the alterations in MPM metabolism under study induced by oxLDL.

Phospholipids and oxophospholipids in atherosclerotic plaques at different stages of plaque development.

We identified and quantified the hydroperoxides, hydroxides, epoxides, isoprostanes, and core aldehydes of the major phospholipids as the main components of the oxophospholipids (a total of 5-25 pmol/micromol phosphatidylcholine) in a comparative study of human atheroma from selected stages of lesion development. The developmental stages examined included fatty streak, fibrous plaque, necrotic core, and calcified tissue. The lipid analyses were performed by normal-phase HPLC with on-line electrospray MS using conventional total lipid extracts. There was great variability in the proportions of the various oxidation products and a lack of a general trend. Specifically, the early oxidation products (hydroperoxides and epoxides) of the glycerophosphocholines were found at the advanced stages of the plaques in nearly the same relative abundance as the more advanced oxidation products (core aldehydes and acids). The anticipated linear accumulation of the more stable oxidation products with progressive development of the atherosclerotic plaque was not apparent. It is therefore suggested that lipid infiltration and/or local peroxidation is a continuous process characterized by the formation and destruction of both early and advanced products of lipid oxidation at all times. The process of lipid deposition appears to have been subject to both enzymatic and chemical modification of the normal tissue lipids. Clearly, the appearance of new and disproportionate old lipid species excludes randomness in any accumulation of oxidized LDL lipids in atheroma.

Downregulation of IRS-1 protein in thapsigargin-treated human prostate epithelial cells.

Thapsigargin treatment of cultured cells leads to an increase in the intracellular calcium concentration, activation of calpain, and, in some cell types, apoptosis. Using a human prostate epithelial cell line that undergoes apoptosis in the presence of thapsigargin, we find decreased levels of IRS-1 protein levels during apoptosis. Inhibition of calpain prevents this decrease in IRS-1 protein; however, inhibitors of caspases or the proteasome are ineffective in maintaining IRS-1 levels. In terms of IGF-I-related second messenger proteins, the effect of thapsigargin is specific for IRS-1 since the protein levels of IGF-I receptor beta-subunit, Akt, Erk, and Shc are not affected. In addition to preventing the reduction in IRS-1, treatment of cells with calpain inhibitor II prevents apoptosis in response to thapsigargin. Finally, IRS-1 and calpain can be identified in protein complexes isolated using IRS-1-specific antibodies, indicating that calpain can associate with either IRS-1 or one of the proteins present in protein complexes that contain IRS-1. In total, these results suggest that IRS-1 may be targeted for degradation by calpain during apoptosis.

Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages.

Modification of low density lipoprotein (LDL) can result in the avid uptake of these lipoproteins via a family of macrophage transmembrane proteins referred to as scavenger receptors (SRs). The genetic inactivation of either of two SR family members, SR-A or CD36, has been shown previously to reduce oxidized LDL uptake in vitro and atherosclerotic lesions in mice. Several other SRs are reported to bind modified LDL, but their contribution to macrophage lipid accumulation is uncertain. We generated mice lacking both SR-A and CD36 to determine their combined impact on macrophage lipid uptake and to assess the contribution of other SRs to this process. We show that SR-A and CD36 account for 75-90% of degradation of LDL modified by acetylation or oxidation. Cholesteryl ester derived from modified lipoproteins fails to accumulate in macrophages taken from the double null mice, as assessed by histochemistry and gas chromatography-mass spectrometry. These results demonstrate that SR-A and CD36 are responsible for the preponderance of modified LDL uptake in macrophages and that other scavenger receptors do not compensate for their absence.

A novel family of atherogenic oxidized phospholipids promotes macrophage foam cell formation via the scavenger receptor CD36 and is enriched in atherosclerotic lesions.

The macrophage scavenger receptor CD36 plays an important role in binding and uptake of oxidized forms of low-density lipoprotein (LDL), foam cell formation, and lesion development during atherosclerosis. The structural basis of CD36-lipoprotein ligand recognition is an area of intense interest. In a companion article we reported the characterization of a structurally conserved family of oxidized choline glycerophospholipids (oxPC(CD36)) that serve as novel high affinity ligands for cells stably transfected with CD36, mediating recognition of multiple oxidized forms of LDL (Podrez, E. A., Poliakov, E., Shen, Z., Zhang, R., Deng, Y., Sun, M., Finton, P., Shan, L., Gugiu, B., Fox, P. L., Hoff, H. F., Salomon, R. G., and Hazen, S. L. (July 8, 2002) J. Biol. Chem. 277, 10.1074/jbc.M203318200). Here we use macrophages from wild-type and CD36 null mice to demonstrate that CD36 is the major receptor on macrophages mediating recognition of oxPC(CD36) species when presented (+/- plasma) in pure form, within PC bilayers in small unilamellar vesicles, and within liposomes generated from lipid extracts of native LDL. We also show that oxPC(CD36) promote CD36-dependent recognition when present at only a few molecules per particle, resulting in macrophage binding, uptake, metabolism, cholesterol accumulation, and foam cell formation. Finally, using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS), we demonstrate that oxPC(CD36) are generated in vivo and are enriched in atherosclerotic lesions. Collectively, our data suggest that formation of this novel family of oxidized phospholipids participates in CD36-mediated recognition of oxidized lipoproteins and foam cell formation in vivo.

Identification of a novel family of oxidized phospholipids that serve as ligands for the macrophage scavenger receptor CD36.

The macrophage scavenger receptor CD36 plays an important role in the uptake of oxidized forms of low density lipoprotein (LDL) and contributes to lesion development in murine models of atherosclerosis. However, the structural basis of CD36 lipoprotein ligand recognition is unknown. We now identify a novel class of oxidized phospholipids that serve as high affinity ligands for CD36 and mediate recognition of oxidized forms of LDL by CD36 on macrophages. Small unilamellar vesicles of homogeneous phosphatidylcholine (PC) molecular species were oxidized by the myeloperoxidase (MPO)-H(2)O(2)-NO(2)(-) system, and products were separated by sequential LC/ESI/MS/MS. In parallel, fractions were tested for their ability to bind to CD36. Four major structurally related phospholipids with CD36 binding activity were identified from oxidized 1-palmitoyl-2-arachidonyl-PC, and four corresponding structural analogs with CD36 binding activity were identified from oxidized 1-palmitoyl-2-linoleoyl-PC. Each was then synthetically prepared, its structure confirmed by multinuclear NMR and high resolution mass spectrometry, and shown to possess identical CD36 binding activity and LC/ESI/MS/MS characteristics in both native and derivatized forms. Based upon the structures of the active compounds identified, and structure-function studies with a variety of synthetic analogs, we conclude that the structural characteristics required for high affinity binding of oxidized PC species to CD36 are a phospholipid with an sn-2 acyl group that incorporates a terminal gamma-hydroxy(or oxo)-alpha,beta-unsaturated carbonyl (oxPC(CD36)). LC/ESI/MS/MS studies demonstrate that oxPC(CD36) are formed during LDL oxidation by multiple distinct pathways. Formation of this novel class of oxidized PC species contributes to CD36-mediated recognition of LDL oxidized by MPO and other biologically relevant mechanisms. The present results offer structural insights into the molecular patterns recognized by the scavenger receptor CD36 and provide a platform for the development of potential therapeutic inhibitory agents.