Functional analysis and structural modeling of human APOBEC3G reveal the role of evolutionarily conserved elements in the inhibition of human immunodeficiency virus type 1 infection and Alu transposition.

Retroelements are important evolutionary forces but can be deleterious if left uncontrolled. Members of the human APOBEC3 family of cytidine deaminases can inhibit a wide range of endogenous, as well as exogenous, retroelements. These enzymes are structurally organized in one or two domains comprising a zinc-coordinating motif. APOBEC3G contains two such domains, only the C terminal of which is endowed with editing activity, while its N-terminal counterpart binds RNA, promotes homo-oligomerization, and is necessary for packaging into human immunodeficiency virus type 1 (HIV-1) virions. Here, we performed a large-scale mutagenesis-based analysis of the APOBEC3G N terminus, testing mutants for (i) inhibition of vif-defective HIV-1 infection and Alu retrotransposition, (ii) RNA binding, and (iii) oligomerization. Furthermore, in the absence of structural information on this domain, we used homology modeling to examine the positions of functionally important residues and of residues found to be under positive selection by phylogenetic analyses of primate APOBEC3G genes. Our results reveal the importance of a predicted RNA binding dimerization interface both for packaging into HIV-1 virions and inhibition of both HIV-1 infection and Alu transposition. We further found that the HIV-1-blocking activity of APOBEC3G N-terminal mutants defective for packaging can be almost entirely rescued if their virion incorporation is forced by fusion with Vpr, indicating that the corresponding region of APOBEC3G plays little role in other aspects of its action against this pathogen. Interestingly, residues forming the APOBEC3G dimer interface are highly conserved, contrasting with the rapid evolution of two neighboring surface-exposed amino acid patches, one targeted by the Vif protein of primate lentiviruses and the other of yet-undefined function.

Characterization of two receptors for TRAIL.

Two receptors for TRAIL, designated TRAIL-R2 and TRAIL-R3, have been identified. Both are members of the tumor necrosis factor receptor family. TRAIL-R2 is structurally similar to the death-domain-containing receptor TRAIL-R1 (DR-4), and is capable of inducing apoptosis. In contrast, TRAIL-R3 does not promote cell death. TRAIL-R3 is highly glycosylated and is membrane bound via a putative phosphatidylinositol anchor. The extended structure of TRAIL-R3 is due to the presence of multiple threonine-, alanine-, proline- and glutamine-rich repeats (TAPE repeats). TRAIL-R2 shows a broad tissue distribution, whereas the expression of TRAIL-R3 is restricted to peripheral blood lymphocytes (PBLs) and skeletal muscle. All three TRAIL receptors bind TRAIL with similar affinity, suggesting a complex regulation of TRAIL-mediated signals.

HLA photoaffinity labeling reveals overlapping binding of homologous melanoma-associated gene peptides by HLA-A1, HLA-A29, and HLA-B44.

Melanoma-associated genes (MAGEs) encode tumor-specific antigens that can be recognized by CD8+ cytotoxic T lymphocytes. To investigate the interaction of the HLA-A1-restricted MAGE-1 peptide 161-169 (EADPT-GHSY) with HLA class I molecules, photoreactive derivatives were prepared by single amino acid substitution with N beta-[iodo-4-azidosalicyloyl]-L-2,3-diaminopropionic acid. These derivatives were tested for their ability to bind to, and to photoaffinity-label, HLA-A1 on C1R.A1 cells. Only the derivatives containing the photoreactive amino acid in position 1 or 7 fulfilled both criteria. Testing the former derivative on 14 lymphoid cell lines expressing over 44 different HLA class I molecules indicated that it efficiently photoaffinity-labeled not only HLA-A1, but possibility also HLA-A29 and HLA-B44. MAGE peptide binding by HLA-A29 and HLA-B44 was confirmed by photoaffinity labeling with photoreactive MAGE-3 peptide derivatives on C1R.A29 and C1R.B44 cells, respectively. The different photoaffinity labeling systems were used to access the ability of the homologous peptides derived from MAGE-1, -2, -3, -4a, -4b, -6, and -12 to bind to HLA-A1, HLA-A29, and HLA-B44. All but the MAGE-2 and MAGE-12 nonapeptides efficiently inhibited photoaffinity labeling of HLA-A1, which is in agreement with the known HLA-A1 peptide-binding motif (acidic residue in P3 and C-terminal tyrosine). In contrast, photoaffinity labeling of HLA-A29 was efficiently inhibited by these as well as by the MAGE-3 and MAGE-6 nonapeptides. Finally, the HLA-B44 photoaffinity labeling, unlike the HLA-A1 and HLA-A29 labeling, was inhibited more efficiently by the corresponding MAGE decapeptides, which is consistent with the reported HLA-B44 peptide-binding motif (glutamic acid in P2, and C-terminal tyrosine or phenylalanine). The overlapping binding of homologous MAGE peptides by HLA-A1, A29, and B44 is based on different binding principles and may have implications for immunotherapy of MAGE-positive tumors.

Thy-1 binds to integrin beta(3) on astrocytes and triggers formation of focal contact sites.

BACKGROUND: Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Astrocytes, ubiquitous cells of the brain, express a putative Thy-1 ligand that prevents neurite outgrowth. In this paper, a ligand molecule for Thy-1 was identified, and the consequences of Thy-1 binding for astrocyte function were investigated. RESULTS: Thy-1 has been implicated in cell adhesion and, indeed, all known Thy-1 sequences were found to contain an integrin binding, RGD-like sequence. Thy-1 interaction with beta3 integrin on astrocytes was demonstrated in an adhesion assay using a thymoma line (EL-4) expressing high levels of Thy-1. EL-4 cells bound to astrocytes five times more readily than EL-4(-f), control cells lacking Thy-1. Binding was blocked by either anti-Thy-1 or anti-beta3 antibodies, by RGD-related peptides, or by soluble Thy-1-Fc chimeras. However, neither RGE/RLE peptides nor Thy-1(RLE)-Fc fusion protein inhibited the interaction. Immobilized Thy-1-Fc, but not Thy-1(RLE)-Fc fusion protein supported the attachment and spreading of astrocytes in a Mn(2+)-dependent manner. Binding to Thy-1-Fc was inhibited by RGD peptides. Moreover, vitronectin, fibrinogen, denatured collagen (dcollagen), and a kistrin-derived peptide, but not fibronectin, also mediated Mn(2+)-dependent adhesion, suggesting the involvement of beta3 integrin. The addition of Thy-1 to matrix-bound astrocytes induced recruitment of paxillin, vinculin, and focal adhesion kinase (FAK) to focal contacts and increased tyrosine phosphorylation of proteins such as p130(Cas) and FAK. Furthermore, astrocyte binding to immobilized Thy-1-Fc alone was sufficient to promote focal adhesion formation and phosphorylation on tyrosine. CONCLUSIONS: Thy-1 binds to beta3 integrin and triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment, and spreading.

Functional and structural basis for a bacteriophage homolog of human RAD52.

In eukaryotes, homologous recombination proteins such as RAD51 and RAD52 play crucial roles in DNA repair and genome stability. Human RAD52 is a member of a large single-strand annealing protein (SSAP) family [1] and stimulates Rad51-dependent recombination [2, 3]. In prokaryotes and phages, it has been difficult to establish the presence of RAD52 homologs with conserved sequences. Putative SSAPs were recently found in several phages that infect strains of Lactococcus lactis[4]. One of these SSAPs was identified as Sak and was found in the virulent L. lactis phage ul36, which belongs to the Siphoviridae family [4, 5]. In this study, we show that Sak is homologous to the N terminus of human RAD52. Purified Sak binds single-stranded DNA (ssDNA) preferentially over double-stranded DNA (dsDNA) and promotes the renaturation of long complementary ssDNAs. Sak also binds RecA and stimulates homologous recombination reactions. Mutations shown to modulate RAD52 DNA binding [6] affect Sak similarly. Remarkably, electron-microscopic reconstruction of Sak reveals an undecameric (11) subunit ring, similar to the crystal structure of the N-terminal fragment of human RAD52 [7, 8]. For the first time, we propose a viral homolog of RAD52 at the amino acid, phylogenic, functional, and structural levels.

Cloning and expression of rat pancreatic beta-cell malonyl-CoA decarboxylase.

To gain insight into the function and regulation of malonyl-CoA decarboxylase (MCD) we have cloned rat MCD cDNA from a differentiated insulin-secreting pancreatic beta-cell-line cDNA library. The full-length cDNA sequence shows 69% identity with the cDNA cloned previously from the goose uropygial gland, and predicts a 492 amino acid protein of 54.7 kDa. The open reading frame contains an N-terminal mitochondrial targeting sequence and the C-terminal part of the enzyme ends with a peroxisomal (Ser-Lys-Leu) targeting motif. Since the sequence does not reveal hydrophobic domains, MCD is most likely expressed in the mitochondrial matrix and inside the peroxisomes. A second methionine residue, located 3' of the mitochondrial presequence, might be the first amino acid of a putative cytosolic MCD, since the nucleotide sequence around it fits fairly well with a consensus Kozak site for translation initiation. However, primer extension detects the presence of only one transcript initiating upstream of the first ATG, indicating that the major, if not exclusive, transcript expressed in the pancreatic beta-cell encodes MCD with its mitochondrial presequence. The sequence also shows multiple possible sites of phosphorylation by casein kinase II and protein kinase C. mRNA tissue-distribution analysis indicates a transcript of 2.2 kb, and that the MCD gene is expressed over a wide range of rat tissues. The distribution of the enzyme shows a broad range of activities from very low in the brain to elevated in the liver and heart. The results provide the foundations for further studies of the role of MCD in lipid metabolism and metabolic signalling in various tissues.

Specific mutations in the estrogen receptor change the properties of antiestrogens to full agonists.

The estrogen receptor (ER) stimulates transcription of target genes by means of its two transcriptional activation domains, AF-1 in the N-terminal part of the receptor and AF-2 in its ligand-binding domain. AF-2 activity is dependent upon a putative amphipathic alpha-helix between residues 538 and 552 in the mouse ER. Point mutagenesis of conserved hydrophobic residues within this region reduces estrogen-dependent transcriptional activation without affecting hormone and DNA binding significantly. Here we show that these mutations dramatically alter the pharmacology of estrogen antagonists. Both tamoxifen and ICI 164,384 behave as strong agonists in HeLa cells expressing the ER mutants. In contrast to the wild-type ER, the mutant receptors maintain nuclear localization and DNA-binding activity after ICI 164,384 treatment. Structural alterations in AF-2 caused by gene mutations such as those described herein or by estrogen-independent signaling pathways may account for the insensitivity of some breast cancers to tamoxifen treatment.

Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHA0.

The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis in P. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2. The hcnA promoter was mapped by primer extension; the -40 sequence (TTGGC ... ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT ... ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion. Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.

Junctional adhesion molecule-2 (JAM-2) promotes lymphocyte transendothelial migration.

The molecular mechanisms underlying lymphocyte extravasation remain poorly characterized. We have recently identified junctional adhesion molecule-2 (JAM-2), and have shown that antibodies to JAM-2 stain high endothelial venules (HEVs) within lymph nodes and Peyer patches of adult mice. Here we show that mouse lymphocytes migrate in greater numbers across monolayers of endothelioma cells transfected with JAM-2. The significance of these findings to an understanding of both normal and pathologic lymphocyte extravasation prompted us to clone the human homologue of JAM-2. We herein demonstrate that an anti-JAM-2 antibody, or a soluble JAM-2 molecule, blocks the transmigration of primary human peripheral blood leukocytes across human umbilical vein endothelial cells expressing endogenous JAM-2. Furthermore, we show that JAM-2 is expressed on HEVs in human tonsil and on a subset of human leukocytes, suggesting that JAM-2 plays a central role in the regulation of transendothelial migration.

Identification and functional characterization of a monofunctional peroxisomal enoyl-CoA hydratase 2 that participates in the degradation of even cis-unsaturated fatty acids in Arabidopsis thaliana.

A gene, named AtECH2, has been identified in Arabidopsis thaliana to encode a monofunctional peroxisomal enoyl-CoA hydratase 2. Homologues of AtECH2 are present in several angiosperms belonging to the Monocotyledon and Dicotyledon classes, as well as in a gymnosperm. In vitro enzyme assays demonstrated that AtECH2 catalyzed the reversible conversion of 2E-enoyl-CoA to 3R-hydroxyacyl-CoA. AtECH2 was also demonstrated to have enoyl-CoA hydratase 2 activity in an in vivo assay relying on the synthesis of polyhydroxyalkanoate from the polymerization of 3R-hydroxyacyl-CoA in the peroxisomes of Saccharomyces cerevisiae. AtECH2 contained a peroxisome targeting signal at the C-terminal end, was addressed to the peroxisome in S. cerevisiae, and a fusion protein between AtECH2 and a fluorescent protein was targeted to peroxisomes in onion cells. AtECH2 gene expression was strongest in tissues with high beta-oxidation activity, such as germinating seedlings and senescing leaves. The contribution of AtECH2 to the degradation of unsaturated fatty acids was assessed by analyzing the carbon flux through the beta-oxidation cycle in plants that synthesize peroxisomal polyhydroxyalkanoate and that were over- or underexpressing the AtECH2 gene. These studies revealed that AtECH2 participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2E-enoyl-CoA for further degradation through the core beta-oxidation cycle.

An isoform of microtubule-associated protein 2 (MAP2) containing four repeats of the tubulin-binding motif.

Microtubule-associated protein 2 (MAP2) exists in both high- and low-molecular mass isoforms, each of which has a tubulin-binding domain consisting of 3 imperfect tandem repeats of 31 amino acids containing a more highly conserved 18 amino acid 'core' sequence. We describe here a novel form of low molecular mass MAP2 (MAP2c) that contains an additional 4th repeat of this tubulin-binding motif. Like the 3 previously known repeat sequences, this 4th copy is highly conserved between MAP2 and the two other known members of the same gene family, tau and MAP4. In each of these three genes the additional 4th repeat is inserted between the 1st and 2nd repeats of the 3-repeat form of the molecule. Experiments with brain cell cultures, in which the relative proportions of neurons and glia had been manipulated by drug treatment, showed that 4-repeat MAP2c is associated with glial cells whereas 3-repeat MAP2c is expressed in neurons. Whereas 3-repeat MAP2c is expressed early in development and then declines, the level of 4-repeat MAP2c increases later in development, corresponding to the relatively late differentiation of glial cells compared to neurons. When transfected into non-neuronal cells, the 4-repeat version of MAP2c behaved indistinguishably from the 3-repeat form in stabilising and rearranging cellular microtubules. The presence of an additional 4th repeat of the tubulin-binding motif in all three members of the MAP2 gene family suggests that this variant arose prior to their differentiation from an ancestral gene.

Orthologues of the Drosophila melanogaster E75 molting control gene in the filarial parasites Brugia malayi and Dirofilaria immitis.

Filarial parasites cause debilitating diseases in humans and domesticated animals. Brugia malayi and Dirofilaria immitis are transmitted by mosquitoes and infect humans and dogs, respectively. Their life cycle is punctuated by a series of cuticular molts as they move between different hosts and tissues. An understanding of the genetic basis for these developmental transitions may suggest potential targets for vaccines or chemotherapeutics. Nuclear receptor (NR) proteins have been implicated in molting in the free-living nematode Caenorhabditis elegans and have well characterized roles in molting during larval development of Drosophila melanogaster. For example, the D. melanogaster E75 (NR1D3) NR gene is required for molting and metamorphosis, as well as egg chamber development in adult females. We have identified Bm-nhr-11and Di-nhr-6, B. malayi and D. immitis orthologues of E75. Both genes encode canonical nuclear receptor proteins, are developmentally regulated, and are expressed in a sex-specific manner in adults.

Molecular evidence for a functional ecdysone signaling system in Brugia malayi.

BACKGROUND: Filarial nematodes, including Brugia malayi, the causative agent of lymphatic filariasis, undergo molting in both arthropod and mammalian hosts to complete their life cycles. An understanding of how these parasites cross developmental checkpoints may reveal potential targets for intervention. Pharmacological evidence suggests that ecdysteroids play a role in parasitic nematode molting and fertility although their specific function remains unknown. In insects, ecdysone triggers molting through the activation of the ecdysone receptor: a heterodimer of EcR (ecdysone receptor) and USP (Ultraspiracle). METHODS AND FINDINGS: We report the cloning and characterization of a B. malayi EcR homologue (Bma-EcR). Bma-EcR dimerizes with insect and nematode USP/RXRs and binds to DNA encoding a canonical ecdysone response element (EcRE). In support of the existence of an active ecdysone receptor in Brugia we also cloned a Brugia rxr (retinoid X receptor) homolog (Bma-RXR) and demonstrate that Bma-EcR and Bma-RXR interact to form an active heterodimer using a mammalian two-hybrid activation assay. The Bma-EcR ligand-binding domain (LBD) exhibits ligand-dependent transactivation via a GAL4 fusion protein combined with a chimeric RXR in mammalian cells treated with Ponasterone-A or a synthetic ecdysone agonist. Furthermore, we demonstrate specific up-regulation of reporter gene activity in transgenic B. malayi embryos transfected with a luciferase construct controlled by an EcRE engineered in a B. malayi promoter, in the presence of 20-hydroxy-ecdysone. CONCLUSIONS: Our study identifies and characterizes the two components (Bma-EcR and Bma-RXR) necessary for constituting a functional ecdysteroid receptor in B. malayi. Importantly, the ligand binding domain of BmaEcR is shown to be capable of responding to ecdysteroid ligands, and conversely, ecdysteroids can activate transcription of genes downstream of an EcRE in live B. malayi embryos. These results together confirm that an ecdysone signaling system operates in B. malayi and strongly suggest that Bma-EcR plays a central role in it. Furthermore, our study proposes that existing compounds targeting the insect ecdysone signaling pathway should be considered as potential pharmacological agents against filarial parasites.

Identification of CARDIAK, a RIP-like kinase that associates with caspase-1.

Members of the tumor necrosis factor receptor (TNFR) superfamily have an important role in the induction of cellular signals resulting in cell growth, differentiation and death. TNFR-1 recruits and assembles a signaling complex containing a number of death domain (DD)-containing proteins, including the adaptor protein TRADD and the serine/threonine kinase RIP, which mediates TNF-induced NF-kappa B activation. RIP also recruits caspase-2 to the TNFR-1 signaling complex via the adaptor protein RAIDD, which contains a DD and a caspase-recruiting domain (CARD). Here, we have identified a RIP-like kinase, termed CARDIAK (for CARD-containing interleukin (IL)-1 beta converting enzyme (ICE) associated kinase), which contains a serine/threonine kinase domain and a carboxy-terminal CARD. Overexpression of CARDIAK induced the activation of both NF-kappa B and Jun N-terminal kinase (JNK). CARDIAK interacted with the TNFR-associated factors TRAF-1 and TRAF-2, and a dominant-negative form of TRAF-2 inhibited CARDIAK-induced NF-kappa B activation. Interestingly, CARDIAK specifically interacted with the CARD of caspase-1 (previously known as ICE), and this interaction correlated with the processing of pro-caspase-1 and the formation of the active p20 subunit of caspase-1. Together, these data suggest that CARDIAK may be involved in NF-kappa B/JNK signaling and in the generation of the proinflammatory cytokine IL-1 beta through activation of caspase-1.